potential transmission routes of campylobacter …

POTENTIAL TRANSMISSIONROUTES OF CAMPYLOBACTER

FROM ENVIRONMENTTO HUMANS

Water & Faecal Routes (funded by MoH Water)Food Route (funded by MoH Food)

Objective One

Prepared as part of a Ministry of Healthcontract for scientific services

ByMichael BakerAndrew BallMeg DevaneNick GarrettBrent Gilpin

Andrew HudsonJohn Klena

Carolyn NicolMarion SavillPaula Scholes

Daniel Williams

(Authors arranged in alphabetical order)

August 2002

Client ReportFW0246

POTENTIAL TRANSMISSION ROUTES OFCAMPYLOBACTER FROM

ENVIRONMENT TO HUMANS

Water & Faecal RoutesFood Route

Alistair SheatWater Programme Manager

Peter DaviesExternal ReviewerMassey University

Project LeaderMarion Savill

Peer ReviewerCraig Thornley

Peer ReviewerFiona Thomson-Carter

Potential Transmission Routes of Campylobacter i August 2002From Environment To Humans

DISCLAIMER

This report or document ("the Report") is given by the Institute of Environmental Scienceand Research Limited ("ESR") solely for the benefit of the Ministry of Health, PublicHealth Services Providers and other Third Party Beneficiaries as defined in the Contractbetween ESR and the Ministry of Health, and is strictly subject to the conditions laid out inthat Contract.

Neither ESR nor any of its employees makes any warranty, express or implied, or assumesany legal liability or responsibility for use of the Report or its contents by any other personor organisation.

Potential Transmission Routes of Campylobacter ii August 2002From Environment To Humans

ACKNOWLEDGMENTS

ESR thanks the following groups and individuals for their support and advice: CrownPublic Health: CPH Timaru, including Monika Hansen and Chris Ambrose; Public HealthLaboratory personnel for their dedication in the processing of samples; Ashburton DistrictCouncil: Richard Durie, Dennis Burridge and Peter Thompson whose continual hard workhas supplied us with samples and information, without which the study could not havebeen undertaken; Sally Harrow (University of Canterbury) and Sue Walker (KSC, ESR)for the PFGE subtyping of isolates; Jenny Bennett (KSC,ESR) for the serotyping ofisolates; Liza Lopez and Kylie Gilmour (KSC, ESR) for support with the Episurv data:Ruth Pirie (ESR) for GIS information and maps; Els Maas for help with methodology; PhilCarter (ESR) for PFGE expertise and Margaret Tanner (CSC) for her patience and support.We also thank all the farmers and retailers of the Ashburton District for their cooperationin the collection of samples.

Potential Transmission Routes of Campylobacter iii August 2002From Environment To Humans

CONTENTS

LIST OF TABLES .............................................................................................................. V

LIST OF FIGURES ....................................................................................................... VIII

LIST OF ABBREVIATIONS ...........................................................................................IX

EXECUTIVE SUMMARY................................................................................................. X

1. INTRODUCTION .................................................................................................11.1 Background..............................................................................................................11.2 Serious Sequelae......................................................................................................31.3 Economic Cost.........................................................................................................41.4 Overview of the Study .............................................................................................4

1.4.1 Study Area .................................................................................................41.4.2 Reservoirs ..................................................................................................51.4.3 Sampling and Analysis ..............................................................................5

1.5 Aim ...................................................................................................................61.6 Hypotheses...............................................................................................................61.7 Objectives ................................................................................................................7

1.7.1 Objective 1 Transmission Routes ..............................................................7

2. LITERATURE REVIEW .....................................................................................82.1 Epidemiological Studies ..........................................................................................92.2 Potential Transmission Routes ..............................................................................102.3 Survival in Transmission Routes ...........................................................................12

2.3.1 General Survival ......................................................................................122.3.2 Survival in Faeces and Slurry..................................................................132.3.3 Survival in Food ......................................................................................142.3.4 Water .......................................................................................................172.3.5 Sediment ..................................................................................................18

2.4 Correlation Between Survival and Pathogenicity..................................................182.5 Transmission Routes Considered in the Present Study .........................................20

2.5.1 Human Faeces..........................................................................................202.5.2 Raw Poultry .............................................................................................212.5.3 Ruminant Animals...................................................................................222.5.4 Meat Products ..........................................................................................232.5.5 Ducks .......................................................................................................242.5.6 Water .......................................................................................................25

2.6 Direction of Transmission Between Reservoirs of Campylobacter ......................262.6.1 The Selection of Subtyping Methods for the Discrimination of

Campylobacter Isolates ...........................................................................262.6.2 The Stability of Genotypic Methods .......................................................31

2.7 Conclusions............................................................................................................332.7.1 Aspects of the Microbial Ecology of Campylobacter .............................332.7.2 Subtyping Methods..................................................................................35

3. MATERIALS AND METHODS ........................................................................373.1 Identification and Interviewing of Human Cases and Data Analysis....................373.2 Sample Sites...........................................................................................................39

3.2.1 Water sample sites ...................................................................................393.2.2 Farm sites for collection of ruminant animal faeces ...............................40

Potential Transmission Routes of Campylobacter iv August 2002From Environment To Humans

3.2.3 Retail outlets for meat products...............................................................403.3 Sample Collection..................................................................................................41

3.3.1 Human faecal sample collection..............................................................433.3.2 Collection of samples from each environmental matrix..........................443.3.3 Collection of meat products from retailers ..............................................443.3.4 Initial sampling plan numbers based on January projected prevalence

of Campylobacter ....................................................................................453.4 Isolation and Detection ..........................................................................................46

3.4.1 Methods for isolation and detection of Campylobacter species..............463.4.2 Subtyping Methods..................................................................................473.4.3 Pulsed Field Gel Electrophoresis (PFGE) ...............................................48

3.5 Analysis of Campylobacter Subtypes....................................................................513.6 Statistical Analysis ................................................................................................51

3.6.1 Czekanowski Index (Proportional Similarity Index)...............................523.7 Survival of Campylobacter in Environmental Reservoirs.....................................52

3.7.1 Unknowns................................................................................................57

4. RESULTS .............................................................................................................584.1 Sampling Overview ...............................................................................................584.2 Human Cases of Campylobacteriosis ....................................................................58

4.2.1 Demographics of Human Cases ..............................................................614.3 Crude Prevalence of Campylobacter .....................................................................62

4.3.1 Seasonality of Campylobacter Prevalence ..............................................644.4 Distribution ............................................................................................................70

4.4.1 General Matrix Distribution ....................................................................704.4.2 Distribution of Campylobacter spp. in Matrices within Different

Regions ....................................................................................................774.5 Prevalence of Campylobacter from Regions A, B and C......................................774.6 Serotype Distribution of C. jejuni Isolates ............................................................794.7 Distribution of Campylobacter PFGE subtypes ....................................................81

4.7.1 Distribution of C. coli PFGE subtypes ....................................................814.7.2 Distribution of C. jejuni PFGE subtypes.................................................83

4.8 Temporal and spatial clustering of subtypes .........................................................894.9 Czekanowski Index................................................................................................904.10 Association between C. jejuni “Subtypes” from Human Cases and Risk

Factors identified from Questionnaire ...................................................................964.10.1 Analysis of water supplies.....................................................................101

4.11 Potential Linkages Identified for Campylobacter ...............................................102

5. DISCUSSION.....................................................................................................1135.1 Isolation of Campylobacter from the Matrices Tested ........................................1135.2 Temporal and spatial clustering of subtypes .......................................................1155.3 Penner Serotypes of C. jejuni ..............................................................................117

5.3.1 Subtypes Identified in the CTR Study...................................................1175.3.2 Comparison with Prior New Zealand Data ...........................................1175.3.3 Comparison with Overseas Data ...........................................................119

5.4 Pulsed Field Gel Electrophoresis Subtypes of C. jejuni ......................................1215.4.1 CTR Data...............................................................................................1225.4.2 Comparisons with Previous New Zealand Data ....................................122

5.5 C. jejuni PFGE and Penner Subtypes ..................................................................1235.5.1 CTR Data...............................................................................................124

Potential Transmission Routes of Campylobacter v August 2002From Environment To Humans

5.6 Comparisons with Prior New Zealand Data ........................................................1255.7 Pulsed Field Gel Electrophoresis Subtypes of C. coli .........................................1275.8 Czekanowski Similarity Indices ..........................................................................1275.9 Potential Linkages of Campylobacter between matrices.....................................1295.10 Characteristics of human cases............................................................................1315.11 Exposure histories of human cases ......................................................................1325.12 Characteristics of Campylobacter infecting humans...........................................1325.13 Conclusions about Linkages ................................................................................1345.14 Limitations of this analysis..................................................................................1355.15 Implications for public health..............................................................................139

RECOMMENDATIONS.................................................................................................140

6. CONCLUSIONS ................................................................................................141

REFERENCES ...............................................................................................................144

GLOSSARY ...............................................................................................................152

APPENDIX 1: DESCRIPTIONS OF SUBTYPING SYSTEMS...............................156

APPENDIX 2: MODIFIED CROWN PUBLIC HEALTH QUESTIONNAIRE ....158

APPENDIX 3: TABLE OF MEAT PRODUCT SALES IN ASHBURTON............174

APPENDIX 4: LABORATORY PROTOCOLS FOR DETECTION OF

CAMPYLOBACTER FROM ENVIRONMENTAL MATRICES .................175SECTION A: LABORATORY PROTOCOLS FOR ENRICHMENT OF

CAMPYLOBACTER FROM ENVIRONMENTAL MATRICES .........175SECTION B: CONTROLS ..........................................................................................179SECTION C: PREPARATION OF ENRICHMENT BROTH CELLS FOR

TESTING BY PCR ...............................................................................182SECTION D: STANDARD PROTOCOL FOR THE DETECTION OF

CAMPYLOBACTER JEJUNI AND CAMPYLOBACTER COLI BYTHE POLYMERASE CHAIN REACTION.........................................184

SECTION E: PROCEDURE FOR ISOLATION AND RESUSCITATION OFC. JEJUNI AND/OR C. COLI...............................................................190

SECTION F: MEDIA AND REAGENTS...................................................................192

APPENDIX 5: PULSED FIELD GEL ELECTROPHORESIS ................................195

APPENDIX 6: RELATIONSHIPS BETWEEN C. JEJUNI PFGE SUBTYPES ...199

APPENDIX 7: DISTRIBUTION OF C. JEJUNI SUBTYPES IN INDIVIDUAL

MATRICES........................................................................................................202

APPENDIX 8: DISTRIBUTION OF C. JEJUNI SUBTYPES ISOLATED

FROM MEAT PRODUCTS .............................................................................206

APPENDIX 9: POTENTIAL RISK FACTOR ASSOCIATIONS............................211

APPENDIX 10: INDIVIDUAL LEVEL ANALYSIS FOR C. COLI AND

C. JEJUNI ISOLATED FROM HUMAN CASES .........................................216

LIST OF TABLES

Potential Transmission Routes of Campylobacter vi August 2002From Environment To Humans

Table 1 Comparison of Campylobacteriosis Incidence Between Countries.................... 3Table 2 Risk Factors for Campylobacteriosis Identified by Eberhart-Phillips

et al. (1997) ......................................................................................................... 9Table 3 Carriage Rates in Ruminant Animals................................................................ 23Table 4 Prevalence of Campylobacter Contamination in Offal (Kramer et al., 2000)..24Table 5 Prevalence of Campylobacter in Surface Water ............................................... 25Table 6 Collection Routine for all Samples ................................................................... 43Table 7 Plan A for Meat Sampling ................................................................................ 45Table 8 Plan B for Meat Sampling................................................................................. 45Table 9 Survival of Campylobacter in Various Matrices .............................................. 54Table 10 Maximum Time Assumed for Determination of a Transmission Route........... 55Table 11 Human Cases of Campylobacterosis................................................................. 59Table 12 Age Distribution of Human Cases .................................................................... 61Table 13 Ethnicity Distribution of Human Cases ............................................................ 61Table 14 Sex Distribution of Human Cases..................................................................... 61Table 15 Hospitalisation of Human Cases ....................................................................... 61Table 16 Prevalence of C. coli and C. jejuni in the Matrices and Diversity of

PFGE Subtypes ................................................................................................. 63Table 17 Samples Containing a Mixed Population of C. jejuni and C. coli .................... 74Table 18 Regional Distribution of Campylobacter Isolation........................................... 77Table 19 Similarity Matrix of C. jejuni Penner Serotypes............................................... 93Table 20 Similarity Matrix of C. jejuni PFGE Subtypes ................................................. 94Table 21 Similarity Matrix of C. jejuni Serotype and PFGE Subtypes ........................... 95Table 22 Association between C. jejuni “Subtypes” from Human Cases and Risk

Factors............................................................................................................... 97Table 23 Potential Linkages Identified for C. coli as isolated in Ashburton

District during the Sampling Period of 2001 .................................................. 104Table 24 Potential Linkages Identified for C. jejuni as isolated in Ashburton

District during the Sampling Period of 2001. ................................................. 106Table 25 Description of Phenotypic Subtyping Systems............................................... 156Table 26 Description of Genotypic Subtyping Systems ................................................ 156Table 27 A Comparison of Meat Volumes sold by Retailers in Ashburton and

Tinwald Townships......................................................................................... 174Table 28 Template of the Premix for C. jejuni and C. coli specific PCR...................... 186Table 29 Comparison of Detection limits of Campylobacter for the Enrichment

PCR Method and the Conventional Plating Method....................................... 189Table 30 Related PFGE Subtypes of C. jejuni ............................................................... 199Table 31 Determination of spatial/temporal distribution of C. jejuni subtypes

isolated from meat products............................................................................ 206Table 32 Humans who had animal contact – Cattle (dairy cows, calves or

non-dairy cattle) in the last 10 days ................................................................ 211Table 33 Humans who had animal contact – chickens (last 10 days)............................ 211Table 34 Humans who consumed chicken at other home (last 10 days) ....................... 212Table 35 Humans who consumed beef at home (last 10 days) ...................................... 212Table 36 Humans who consumed untreated water (last 10 days).................................. 213Table 37 Humans who consumed Well/Bore Water Supply (within last 10 days)........ 213Table 38 Humans who consumed Town Water Supply (last 10 days) .......................... 214Table 39 Humans who had contact with dogs (last 10 days)......................................... 214Table 40 Humans who had contact with dairy cattle (last 10 days) .............................. 215

Potential Transmission Routes of Campylobacter vii August 2002From Environment To Humans

Table 41 Risk factors associated with Cases of Subtype HS2:P18................................ 220

Potential Transmission Routes of Campylobacter viii August 2002From Environment To Humans

LIST OF FIGURES

Figure 1 Incidence of Notified Campylobacteriosis by Year, 1980-2001......................... 1Figure 2 Campylobacteriosis Notifications by Month, June 1996 - January 2002............ 2Figure 3 The Campylobacter Conceptual Model ............................................................ 12Figure 4 Flow of Information and Samples relating to the Human Clinical Isolates ...... 38Figure 5 Map of the Farm and Water Sampling Regions A, B and C ............................. 41Figure 6 Gel image of related PFGE subtypes................................................................. 50Figure 7 Potential Reservoirs and Transmission Routes for Campylobacter .................. 53Figure 8 Map of Sampling Locations and Human Cases ................................................ 60Figure 9 Seasonality of C. jejuni Isolation from Meat Products ..................................... 65Figure 10 Seasonality of C. jejuni Isolated from Matrices with Composite

Sampling Regimes............................................................................................. 66Figure 11 Seasonality of C. coli Isolated from Matrices with Composite Sampling ...........

Regimes ............................................................................................................. 67Figure 12 Seasonality of C. coli Isolated from Meat Products ......................................... 68Figure 13 Seasonality of C. jejuni and C. coli Isolated from Human Faeces .................... 69Figure 14 Seasonal Variation in Temperature of the Ashburton River ............................. 70Figure 15 Prevalence of C. jejuni on Farms and Water Sites ............................................ 72Figure 16 Prevalence of C. jejuni in Duck Ponds, Meat Retailers and Human Cases in

Ashburton Township ......................................................................................... 73Figure 17 Prevalence of C. coli on Farms and Water Sites ............................................... 75Figure 18 Prevalence of C. coli in Duck Ponds and Meat Retailers and Human Cases in

Ashburton Township ......................................................................................... 76Figure 19 Distribution of C. jejuni Serotypes in the Environmental Matrices of the

Ashburton District ............................................................................................. 79Figure 20 Detail of the Distribution of Selected C. jejuni Serotypes in the

Environmental Matrices of the Ashburton District ........................................... 80Figure 21 Distribution of C. coli PFGE subtypes in the Environmental Matrices of the

Ashburton District ............................................................................................. 82Figure 22 Comparison of C. jejuni Subtypes (combined serotype and PFGE)

between Matrices............................................................................................... 84Figure 23 Genetic Relationships among the Clonal Group P18...................................... 126Figure 24 Controls for Campylobacter Enrichment Process ........................................... 180Figure 25 Procedure for Enrichment of Campylobacter cells ......................................... 181Figure 26 Bacterial Cell Harvest and Washing ............................................................... 183Figure 27 Campylobacter Isolation and Resuscitation .................................................... 190Figure 28 Distribution of C. jejuni Subtypes (combined serotype and PFGE) in

individual matrices .......................................................................................... 202

Potential Transmission Routes of Campylobacter ix August 2002From Environment To Humans

List of Abbreviations

AFLP Amplified Fragment Length PolymorphismCTR Campylobacter Transmission Routes StudyDGGE Denaturing Gradient Gel ElectrophoresisDNA Deoxyribonucleic acidHS SerotypeHS:P Subtype of combined serotype and PFGE subtyping dataLEP Laboratory of Enteric PathogensMLEE Multi Locus Enzyme ElectrophoresisMLST Multi Locus Sequence TypingPCR Polymerase Chain ReactionPFGE Pulsed Field Gel ElectrophoresisRE Restriction enzymeRAPD Random Amplified Polymorphic DNARFLP Restriction Fragment Length Polymorphismχ2 chi-square (statistical test)

Potential Transmission Routes of Campylobacter x August 2002From Environment To Humans

EXECUTIVE SUMMARY

Introduction

This report describes the results of a three-year investigation of the transmission routes of

human campylobacteriosis. This was achieved by investigating the prevalence of

Campylobacter subtypes in environmental reservoirs. Data collected from the subtyping of

Campylobacter isolates were combined with epidemiological information from human

cases to test hypotheses about the relationships between Campylobacter subtypes in the

environment and those associated with human campylobacteriosis. The aim of this pilot

study was to advance the understanding of potential reservoirs and transmission routes to

help prioritise the development of risk management strategies. In this way resources could

be best allocated to achieve the goal of reducing the health burden imposed by pathogenic

Campylobacter. This project falls under the umbrella of the MoH Zoonoses programme

and is funded by the Ministry of Health.

Procedure

The investigation was unusual in that it used combined microbiological data on the

prevalence of Campylobacter subtypes in environmental reservoirs and human cases along

with epidemiological information from these cases.

In the first part of this project, new methods were developed and established to optimise

the detection of Campylobacter spp. in a range of sample subtypes, from faeces to water to

food products. Once a positive sample was detected by these new methods the organism

was isolated from the sample and then subjected to a combination of subtyping by Penner

serotyping and pulsed field gel electrophoresis (PFGE) for C. jejuni isolates and PFGE

subtyping for C. coli. Subtyping allowed discrimination among isolates of the same species

to enable the tracking of specific subtypes in the environment.

The Ashburton District was selected for study because the South Canterbury Health

District is consistently among those health districts with higher than average rates of

Potential Transmission Routes of Campylobacter xi August 2002From Environment To Humans

campylobacteriosis. The Ashburton District is relatively geographically contained and its

remoteness makes it likely that most of its inhabitants live, work and buy food from local

sources. The largest township in this district is Ashburton and it is serviced by one primary

reticulated water source which is derived from disinfected river water and untreated bore

water. There are 42 registered public water supplies within this district and many private

supplies.

The sample collection period was for the calendar year of 2001, although human clinical

samples were collected until the end of January 2002, to account for the incubation period

of the organism. Environmental matrices included in the study were: river water, duck

faeces, ruminant animal faeces (beef, dairy cattle and sheep) and meat products. All of

these matrices are known to harbour Campylobacter spp. to varying degrees. The subtypes

isolated from these sources were compared with the subtypes isolated from human cases of

campylobacteriosis in the study area. When human cases were notified, samples were

collected and a questionnaire administered to attempt to identify risk factors that may have

been responsible for the infection.

Results

• The prevalences from the various samples were similar to previous reports. The

exception being fresh chicken where the prevalence (27.5%) was around half that

determined previously. This might be because whole chicken carcasses, which were

tested in this study, are less frequently contaminated than portions (tested in previous

studies). The composite sampling regime used for animal faecal samples and water

generated a high proportion of isolates from these matrices. Therefore the data

produced by the Campylobacter Transmission Routes (CTR) study from ruminant

animals and ducks do not represent isolation rates for individual animals.

• C. jejuni was the predominant species identified in human faecal samples (82.6%) and

in all other samples, except pork offal which had equal numbers of C. jejuni and

C. coli.

Potential Transmission Routes of Campylobacter xii August 2002From Environment To Humans

• The percentage of C. jejuni in sheep faeces was the lowest for the animals but sheep

faeces yielded the highest proportion of C. coli of all of the matrices tested. This high

proportion of C. coli was not reflected in the proportion of positive sheep offal

samples. Sheep offal produced the highest prevalence of C. jejuni for the meat

products. Pork and beef offal had significantly lower prevalences for C. jejuni in

comparison to sheep offal and chicken carcasses. However pork offal had the highest

prevalence of C. coli compared to the other meat products. It is of particular interest

that prevalence of C. jejuni in beef faeces is much higher than sheep faeces, but that

beef offal prevalence is much lower than sheep offal.

• All human cases appear to have been sporadic infections. There was no evidence of

common source outbreaks in this population. Person-to-person contact with another

case was only reported by eight cases (14%). None of these eight cases was able to be

definitively identified as a secondary case, due to the limited information recorded on

the timing and nature of the contact, plus the fact that very few of the related cases had

provided a faecal sample for testing.

• There is little information available from other New Zealand studies for comparison

between PFGE subtypes of C. jejuni and between PFGE subtypes of C. coli. However,

a reasonable quantity of Penner serotyping data is available for C. jejuni, and

comparison of the historical data and those from the CTR isolates tend to indicate that

the serotypes isolated from the CTR study were not unusual. Therefore there is no

reason to believe that the pattern of Campylobacter species and strains in the

Ashburton area is unusual or markedly different from the overall New Zealand

situation.

Analysis of Campylobacter spp. isolates revealed a high diversity of subtypes of C. coli

and C. jejuni within each matrix. There were overlaps of subtypes between matrices, which

have been informative in demonstrating potential linkages.

• A total of 250 Serotype:PFGE subtypes of C. jejuni were isolated from matrices in the

CTR study. Of these, 44 (19%) were isolated from humans.

Potential Transmission Routes of Campylobacter xiii August 2002From Environment To Humans

• A total of 39 PFGE subtypes of C. coli were isolated from matrices sampled in the

CTR study. Of these, 5 (13%) were isolated from humans.

• The range of subtypes infecting humans was diverse. There were 44 subtypes of

C. jejuni found in the 56 human isolates (diversity of 78.5%) and 5 subtypes of C. coli

for 6 human isolates (diversity of 83%).

• Twenty-one subtypes of C. jejuni were unique to humans in this study, and these

subtypes accounted for 46 % of cases.

• There were 27 human C. jejuni cases (48%), infected by subtypes found in other

matrices. These 27 cases were used to explore potential relationships with subtyping

information obtained from samples collected from other matrices.

• For C. coli all of the PFGE subtypes found in humans were also found in other

matrices.

Analysis of the CTR data employed three major approaches:

1) use of the Czekanowski Index to estimate the similarity in the spectrum of isolates

obtained from each of the matrices in a pairwise analysis

2) analysis of the subtypes in cases exposed to a potential risk factor compared to those

cases who were not

3) descriptive analysis of potential linkages based on the collation of data derived from

subtyping (Penner/PFGE), spatial, temporal and epidemiological analyses

The results produced by these three approaches were largely consistent, however, the three

analyses, in particular, human risk factor analysis can only be considered to be indicative

due to the small sample size, level of diversity and multiple univariate tests or comparisons

undertaken.

Subtypes of C. jejuni isolated from ruminant animal sources, whether faeces or meat, were

the most similar to one another according to the Czekanowski Index. They were also the

most similar to those isolated from human cases.

Potential Transmission Routes of Campylobacter xiv August 2002From Environment To Humans

The data was too sparse in that there were too many Campylobacter subtypes distributed

among the small number of human cases for firm conclusions to be made from risk factor

analysis. However, indicative results are that contact with bovine animals and live

chickens are the more important risk factors for this study population.

Analysis on a case-by-case basis largely failed to provide compelling evidence to identify

definitive transmission routes/linkages (third approach) by use of bacterial subtyping,

temporal and geographical data. Any analyses of this nature were necessarily complicated

by the numerous potential exposures reported by the cases. The linkages identified

indistinguishable Campylobacter subtypes common to ruminant animals (faeces and meat)

and humans. This linkage data supported the findings of the Czekanowski analysis.

The main conclusion that can be drawn from the three analyses is that, for the population

sampled, bovine animal contact, direct or indirect, was the highest risk factor identified in

the CTR study.

Conclusion

This project has provided a useful pilot investigation that has identified the most likely

causes of campylobacteriosis in semi-rural populations. Due to the limitations of the pilot

study, we cannot conclusively define transmission routes in this semi-rural population. The

main conclusion that can be drawn from these data is that, for the population sampled,

bovine animal contact, direct or indirect, was the highest risk factor identified in the CTR

study. This finding would warrant further investigation to establish its significance as an

important risk factor.

Observations from this study are likely to be characteristic of other rural towns in New

Zealand. It is likely that the epidemiology of campylobacteriosis in New Zealand differs

between “rural” and “urban” populations. It was not possible to carry out this analysis in

the Ashburton data as by far the largest proportion of cases had some “rural” exposure (as

illustrated by the questionnaire responses).

Potential Transmission Routes of Campylobacter xv August 2002From Environment To Humans

The results of the CTR study are extremely useful in identifying risk management options

for rural communities. Farmers, farm workers, people living on farms, people visiting

farms and others with occupational exposure to animals may not be aware that ruminant

faeces commonly contain Campylobacter. If this were known then such contact might be

avoided. For example, people in direct contact with animals need to wash their hands

thoroughly prior to activities such as eating and smoking, where cross contamination and

inadvertent consumption of Campylobacter could occur. Intervention messages, such as,

educational messages counseling against the consumption of raw milk and avoiding the

consumption of untreated water could be conveyed to the general public.

From the number of cases with no apparent link to an environmental matrix sampled in this

study, it is apparent that there are some environmental reservoirs not identified. It was not

possible to sample all potential reservoirs during the course of this study.

Campylobacter is the most commonly notified disease in New Zealand accounting for

almost 50% of notifications in 2001. Results of the CTR study suggest that bovine animals

may be an important reservoir and source of infection for rural New Zealanders. Although

this link has been observed in international studies it could have greater significance for

the New Zealand setting. A high proportion of New Zealanders live in or have contact with

rural environments. The role of bovine animals as a source of human Campylobacter

infection needs to be confirmed and quantified. It would also be useful to investigate the

role of this animal source for other important enteric diseases, notable salmonellosis,

giardiasis, cryptosporidiosis, and STEC. Such work would support the development of

effective 0interventions.

Limitations of the Study

The potential for this study to identify transmission pathways/routes/linkages was limited

by the following:

Potential Transmission Routes of Campylobacter xvi August 2002From Environment To Humans

Small size of the Pilot Study

This limitation was particularly important for human cases, where both epidemiological

information and typable isolates were only obtained for 61 people.

Lack of dominant micro-organism subtypes

A striking feature of these results, at least with the subtyping systems being used here, is

the absence of dominant Campylobacter subtypes in the matrices examined. This feature of

the biological system inevitably limits the power of the study to propose definitive

transmission pathways and is also exacerbated by the resultant small sample size. The

analysis of human exposures was hampered by a combination of a relatively small sample

size and a large diversity in the number of subtypes therefore only very simple analyses

were undertaken which were only able to provide indicative results.

Sampling issues in food, water, animal and environmental

This study suggests that food, water, animals and the environment are being contaminated

with a wide range of Campylobacter subtypes. It will therefore be difficult for such a study

design to sample from these matrices in a way that conclusively establishes infection

sources for human cases, or transmission pathways/routes/linkages within the environment.

There are no data on whether subtypes can be isolated on a continuing basis from ruminant

faeces or whether subtypes turn over rapidly in relation to their host. It is also likely that

some of the samples tested contained a number of subtypes, only one of which was

isolated and identified.

Genomic Stability

The genome of Campylobacter undergoes recombinational events quite readily, therefore

genotypic subtyping results need to be interpreted with caution when proposing definitive

transmission pathways/routes/linkages.

Potential Transmission Routes of Campylobacter xvii August 2002From Environment To Humans

General Application of CTR study conclusions to other regions

A further limitation of this study is the general application of the conclusions from the

CTR study to other regions. For good reasons it has focused on a single geographical area.

Inevitably this area is not representative of New Zealand as a whole. Obvious differences

include the low proportion of Maori and Pacific People, and the relatively high proportion

of people living in rural areas. Some of the foods available in this area, such as chicken,

came from a single supplier, which again is not a typical situation. Findings from this study

therefore need to be interpreted with caution when applying them to the New Zealand

population as a whole.

Implications for public health

Findings from this study support public health advice in the following areas:

• Farmers and their families should take precautions to avoid being infected following

contact with farm animals and birds. Such precautions include careful handwashing

after contact with animals and the farm environment, and especially prior to eating or

smoking where ingestion of the organism might occur.

General points to be reiterated include:

• The public should avoid drinking untreated water and unpasteurised milk.

• The public should thoroughly cook chicken and offal derived from cattle, sheep and

pigs, and avoid cross-contamination of other foods through contact with raw chicken

and red meat products.

Potential Transmission Routes of Campylobacter xviii August 2002From Environment To Humans

RECOMMENDATIONS

1. Conduct an enteric disease (campylobacteriosis) intervention study in a rural area,

based on the findings of the CTR study. This study could be carried out in the

Ashburton area to build on data from this present research project.

2. Include other potential reservoirs in additional future studies, notably companion

animals and asymptomatic household members.

3. Further investigate potential transmission routes to humans on farms, particularly the

role of direct animal contact, consumption of unpasteurised milk and untreated water

and the effects of farming practices.

4. Carry out an investigation of potential Campylobacter linkages in an urban population

by focusing on a larger number of samples in a smaller number of reservoirs and/or

transmission routes.

Potential Transmission Routes of Campylobacter 1 August 2002From Environment To Humans

1. INTRODUCTION

1.1 Background

Campylobacteriosis is New Zealand’s most frequently notified disease with an incidence in

2001 of 10 148 cases (271.5 per 100 000) (ESR website). Data on the incidence of

campylobacteriosis in New Zealand have been kept since the disease became notifiable in

1980 (Figure 1). Since then, there has been an increasing trend in the number of reported

cases.

Figure 1 Incidence of Notified Campylobacteriosis by Year, 1980-2001

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80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 00 01Y ear

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Campylobacteriosis is highly seasonal with a marked peak in most summers (Figure 2) and

declining incidence over winter. This seasonal decline was less apparent in 1998, which

contributed to that year recording the highest rate of disease, with more than 300 cases per

100 000. It is of note that the number of cases recorded in January 2002 is the highest

reported for any month since the disease became notifiable, and the 2001-2002 summer peak

is also the largest reported. Whether this trend continues for the rest of 2002 cannot be

predicted, but the summer peak has meant that the incidence for 2001 was 279.8, compared to

233.0 in the previous year.


Figure 2 Campylobacteriosis Notifications by Month, June 1996 - January 2002

Source: ESR

The incidence of reported campylobacteriosis in New Zealand is markedly greater than

observed in comparable developed countries (Table 1). New Zealand generally has rates two

to three times higher than other developed countries and more than ten times higher than the

United States. Differences in respective reporting systems operating at different levels of

efficiency might partially explain this observation but in a previous analysis, the increase in

the number of cases was not considered to be an artefact of reporting or improved

methodology (Lane and Baker, 1993).

While the consumption of undercooked chicken has been regarded as an important source of

disease there is little doubt that some other exposure(s) must also contribute significantly to

the disease burden (Ikram et al., 1994).

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Table 1 Comparison of Campylobacteriosis Incidence Between Countries

Country Period Rate /100,000 ReferenceNew Zealand 12 months to

December 2001279.8 Anonymous, 2001a,

ESR websiteUSA 2000 20.1 Anonymous, 2001bEngland and Wales 1998 111 Tam, 2001Canada 1986-1998 39-54 Health Canada, 2001Denmark 1999 78 Dansk Zoonosecenter, 2000Australia* 2000 107 Communicable Diseases

Australia, 2001*Excludes New South Wales which does not report campylobacteriosis

The reasons why New Zealand routinely reports elevated rates compared with other

developed countries are not known.

Questions as to which transmission routes are the most important, and so warrant

intervention, remain largely unanswered. This lack of information is due to three primary

reasons:

1) Campylobacter transmission routes are complex;

2) Studies reported in the scientific literature tend to deal with small aspects of transmission

in isolation and have rarely involved a cross disciplinary approach; and

3) Until recently, little research has been conducted into campylobacteriosis in New Zealand.

1.2 Serious Sequelae

Chronic sequelae of infection with Campylobacter spp. are recognised worldwide and include

Guillain-Barré syndrome (GBS) and reactive arthritis. The frequency of GBS resulting from

campylobacteriosis has been estimated as 0.1% (Altekruse et al., 1999). Approximately 20%

of patients with GBS are permanently disabled and approximately 5% die.

Campylobacteriosis is also associated with Reiters syndrome, a reactive arthropathy. The

frequency of this illness has been estimated as 1% of all cases of campylobacteriosis

(Altekruse et al., 1999).


1.3 Economic Cost

Cases of campylobacteriosis caused by foodborne transmission have been estimated to cost

$40,136,000 annually, 73% of the total economic cost of foodborne infectious intestinal

disease in New Zealand (Scott et al., 2000). This is by far the majority of the cost of

foodborne illness; all the other nine foodborne enteric diseases included in the study each

represented costs of less than 10% of the total. The number of cases and outcomes used for

this estimate were based on an average of notification and hospitalisation data from 1991 to

1998 (Lake et al., 2000). This estimate was based on several assumptions, the most important

being that 65% of all cases of campylobacteriosis were caused by foodborne transmission.

The estimated dollar value includes direct and indirect medical costs, the value of productive

days lost, and the statistical value of mortality, but not the value of lost quality of life.

The estimate assumed that the ratio of notified (visit a GP) to unreported (community) cases

of campylobacteriosis was 1:7.6, based on data from a prospective English study (Wheeler et

al., 1999). The notification figure for this estimate was taken from the most up to date figure

at the time, i.e. 1998. Consequently the estimated cost will have declined as the notification

rate has declined. However campylobacteriosis will still represent the majority of infectious

intestinal disease costs.

1.4 Overview of the Study

1.4.1 Study Area

The Ashburton District was selected for study, because the South Canterbury Health District

is consistently among those health districts with higher than average rates of

campylobacteriosis. Ashburton Township has one primary reticulated water source and its

geographical remoteness makes it likely that most of its inhabitants live and work in the area.

Consequently exposure to contaminated foods is likely to be from foods bought locally.


1.4.2 Reservoirs

Potential reservoirs of infection, which were examined, were dairy and beef cattle, sheep and

ducks. The literature review demonstrates that these carry Campylobacter spp. Potential

reservoirs were sampled by testing faeces collected from farms adjacent to the river system.

Transmission routes studied included foods derived from these animals with the exceptions

that pork products were included and duck excluded. Isolates of Campylobacter were

obtained from whole chickens, but for beef, sheep and pigs, offal was the source of isolates.

This approach was taken as Campylobacter is rarely isolated from red meats but is known to

be present in offal at significant prevalences. Offal isolates were taken as surrogates of

Campylobacter subtypes infrequently present in red meat.

Another reservoir investigated was river water, as Campylobacter is known from both

overseas and New Zealand studies to be present in river water at high prevalences. In

addition, while outbreaks of campylobacteriosis are rare they usually occur through

contaminated drinking water. The contribution of recreational exposure during swimming for

example is unknown, but outbreaks of disease caused by Escherichia coli O157:H7 have

occurred in people swimming in contaminated water in the United States.

1.4.3 Sampling and Analysis

Campylobacter spp. were isolated from water sampled at two different points along the

Ashburton River that receive drainage from different land areas. In addition samples were

collected at the Ashburton drinking water plant intake. This was again surrogate sampling to

identify isolates that could have contaminated the drinking water supply. It was felt that

attempting to isolate the organism from drinking water directly would not result in a sufficient

number of organisms being isolated, if any.

Concurrently, laboratories provided faecal samples from laboratory confirmed cases of

campylobacteriosis residing in the Ashburton area. Follow up visits were made to these

people and an enhanced case questionnaire administered. Data from this questionnaire were

used to assess factors that might have significance e.g. recent overseas travel and/or direct


contact with farm animals. The possibilities that cases comprised part of an outbreak or

represented secondary household transmission were also considered.

All samples were screened for the presence of C. jejuni and C. coli, which are the two species

causing the majority of cases of campylobacteriosis in New Zealand. C. jejuni isolates were

subtyped by Penner serotyping and pulsed field gel electrophoresis (PFGE), a combination of

methods that allows comparison with existing subtyping data and that offers good

discrimination and reproducibility. C. coli isolates were typed by PFGE only as as there is a

lack of suitable reference strains characterised for this Campylobacter species.

1.5 Aim

This report describes the results of a one-year preliminary investigation into

campylobacteriosis transmission routes. Campylobacter is the first zoonotic organism to be

studied in New Zealand in a cross-disciplinary co-ordinated approach.

The aim of this work was to identify routes of Campylobacter transmission to humans.

Further study of these transmission routes will help to prioritise development of risk

management strategies. In this way resources can be best allocated to achieve the goal of

reducing the health burden imposed by pathogenic Campylobacter.

1.6 Hypotheses

• That there is a relationship in time between the subtypes of Campylobacter affecting

people in the Ashburton district and the subtypes of Campylobacter found in the drinking

water source (river) along the river and at the point of entry into the water treatment plant.

• That there is a relationship in time and place between the subtypes of Campylobacter

found in the drinking water source (river) and subtypes of Campylobacter found in the

animals in the farms along the river.

• That there is a relationship in time between the subtypes of Campylobacter affecting

people in the Ashburton district and the subtypes of Campylobacter found in specific

foods supplied to local shops (notably raw pork, beef, lamb, chicken).


• That the subtypes of Campylobacter affecting people with exposures to defined sources

are more similar to those isolated from those sources than those isolated from people who

do not report such exposures.

1.7 Objectives

There were three objectives for this study. The main objective was transmission routes and

reservoirs. Additional objectives relate to the preliminary work investigating the river

sediment as a potential reservoir and investigation of the viability of Campylobacter. The

latter two objectives can be reviewed in Report Two.

1.7.1 Objective 1 Transmission Routes

The objectives are:

• To determine the prevalence and seasonal distribution of species and subtypes of

Campylobacter in associated receiving waters over a one-year period.

• To investigate the overall association of the animal reservoirs with receiving waters and

final human contact in terms of the prevalence of species and the subtypes of

Campylobacter identified.

• To determine the prevalence and seasonal distribution of species and subtypes of

Campylobacter in food produced from such animals over a one-year period.

• To investigate the overall association of the food products with human contact in terms of

the prevalence of species and the subtypes of Campylobacter identified.

• To analyse the information with regard to spatial and temporal distribution of subtypes in

order to identify transmission routes associated with human disease.


2. LITERATURE REVIEW

The aim of the literature review is to provide a background for the work undertaken in the

CTR project. It provides information on the particular transmission routes and reservoirs

selected, highlights some of the relevant properties of the survival of the organism in the

environment, and discusses the benefits and pitfalls of the methods used. At the end of the

literature review the conclusion condenses the information presented and summarises the

important factors in selecting methods.

The aim of this review was to identify the following:

• important reservoirs with respect to infection of humans

• relevant survival and pathogenicity characteristics

• potential transmission routes to humans (food, water, sediment, animals)

• the most appropriate methodologies for detection and classification of Campylobacter

spp. to determine potential transmission routes.

The databases: Evaluated Medline, Cambridge Scientific Abstracts and Scirus were searched

to obtain information to compile the literature review. The keywords identified and searched

in various combinations with Campylobacter were: survival, faeces, transmission routes,

chicken, offal, meat, water, sediments, ducks, birds, gulls, dairy cows, cattle, sheep, ruminant,

farm animals, human faeces.

For the review of subtyping methods the keywords identified and searched in combination

with Campylobacter were: serotyping, pulsed field gel electrophoresis, PFGE, Bionumerics,

DICE, computer assisted analysis, subtyping, genotypic subtyping, AFLP (Amplified

Fragment Length Polymorphism), MLST, (Multi Locus Sequence Typing), genetic stability,

stability/instability and recombination events.


2.1 Epidemiological Studies

Some information from case control studies is available for New Zealand. Brieseman (1990)

observed the following; a peak in cases in the 0-4 age group, a high incidence in young males

in rural areas, peaks in the spring and summer, chicken consumption was high in sufferers

(although not statistically significant). The main finding was an association of disease and

household contact with a dog.

A case-control study in Christchurch was carried out in the summer of 1992-1993 (Ikram et

al., 1994). Eating chicken at home was protective, while eating chicken at a friend’s house

introduced a risk factor. Statistical significance was almost reached for eating barbecued

chicken as a risk factor. Contrary to the study of Brieseman (1990) there was no risk

associated with pet ownership.

The only national study identified in this review was undertaken between 1994 and 1995

(Eberhart-Phillips et al., 1997). The risk factors identified in that study are detailed in Table

2.

Table 2 Risk Factors for Campylobacteriosis Identified by Eberhart-Phillips et al. (1997)

Risk Factor Adjusted OddsRatio*

95%confidenceinterval

Rainwater source for home water supply 3.11 1.30, 7.41Preference for chicken liver ≥ 1/month 2.47 1.22, 4.98Preference for chicken pieces ≥ 1/week 1.44 1.10, 1.89Puppy ownership 3.94 1.57, 9.88Eating chicken raw or undercooked within the last 10 days 3.71 2.24, 6.13Eating any chicken prepared at a sit down restaurant within the last 10days

3.53 2.17, 5.72

Eating chicken prepared at someone else’s house within the last 10 days 1.77 1.12, 2.80Not eating baked/roast chicken within the last 10 days 1.75 1.33, 2.32Eating barbecued chicken within the last 10 days 1.88 1.05, 3.36Drinking unpasteurised milk within the last 10 days 3.92 1.66, 9.27Handling calf faeces within the last 10 days 4.40 1.34, 14.39Sewerage problems at home within the last 10 days 4.35 1.55, 12.18Eating other raw or undercooked meat or fish within the last 10 days 3.67 2.07, 6.50* Adjusted for age, sex and region.

The study concluded that factors concerning the consumption of chicken accounted for more

cases of campylobacteriosis in New Zealand than all other risk factors combined. This

conclusion is plausible given the prevalence of Campylobacter in raw poultry (see below).


Other work has shown an extremely low prevalence (0.07%) in New Zealand cooked poultry

(Campbell and Gilbert, 1995). The question as to what contribution to disease the

consumption of raw or undercooked chicken, as opposed to cross contamination from raw

chicken makes remains unresolved (Lake et al. 2002).

Consumption of water from a roof-collected supply is a plausible risk factor for

campylobacteriosis, as birds can carry Campylobacter. Faecal material from birds on roofs

used to collect water, or in storage tanks, may contaminate this drinking water source. Roof-

collected drinking water has been shown to be contaminated by Campylobacter in a New

Zealand study, although C. jejuni was not detected (Savill et al., 2001a).

2.2 Potential Transmission Routes

Recent changes in approach to food safety have meant that an understanding of the hazards

and control points at all stages of the food production chain need to be taken into account

using a “farm to fork” approach. An understanding of the epidemiology of this disease

therefore requires a good understanding of the microbial ecology of Campylobacter in

reservoirs and transmission routes.

Few New Zealand data exist to describe potential transmission routes. A small, now five-

year-old, study was carried out in the Christchurch area using the same subtyping

methodology as was used in the work described in the CTR study. Isolates were obtained

from raw chicken, milk, water, and human and veterinary cases in the summer and winter

(Hudson et al., 1999). Five subtypes of Campylobacter represented by more than two isolates

were identified at the two different times of the year. Three of those subtypes were only found

in humans, indicating that either these may be human-specific subtypes, or that transmission

routes for these subtypes have not yet been identified. In summer one dominant subtype

emerged and this contained isolates from human cases and chicken. Only one subtype was

common to both summer and winter, and this comprised isolates from human and veterinary

cases, along with two from chickens. Such studies can show links between transmission

routes and reservoirs, but do not show the direction, if any, of transmission. Other

unrecognised transmission routes may also confound apparent links.


The Ministry of Health engaged the National Institute of Water and Atmospheric Research

(NIWA) to produce a model that would represent reservoirs and transmission routes with a

view to adding data to allow modeling of Campylobacter in the environment. While this level

of data is not yet available, the “Campylobacter conceptual model” is a useful representation

of reservoirs and transmission routes. Figure 3 shows this model. While this model is not

entirely comprehensive, for example there is no link between water and food processing, it

represents a very useful model on which to base a description of the behaviour of

Campylobacter in the various reservoirs and transmission routes. For the purposes of this

review the definitions1 of reservoir and transmission route are:

RESERVOIR: The habitat, in which an infectious agent normally lives, grows and

multiplies. Reservoirs include human reservoirs, animal reservoirs, and environmental

reservoirs.

TRANSMISSION OF INFECTION: Any mode or mechanism by which an infectious agent

is spread through the environment or to another person.

The range of Campylobacter reservoirs is very limited, because the minimum growth

temperature for the species C. jejuni and C. coli is around 30oC. Therefore Campylobacter

reservoirs are solely warm-blooded animals such as mammals and birds. Reservoirs are

shown in the rectangular boxes in Figure 3.

1 (CDC’s glossary of epidemiology terms: www.cdc.gov/nccdphp/drh/epi_gloss2.htm).


Figure 3 The Campylobacter Conceptual Model

When Campylobacter is being transmitted it is not growing; i.e. numbers are static or

decreasing. Therefore a large part of the model depicted in Figure 3 depends on the ability of

Campylobacter to survive while in transit between hosts. The ability of Campylobacter to

survive under these circumstances is discussed in the following section.

2.3 Survival in Transmission Routes

2.3.1 General Survival

It is acknowledged that the minimum growth temperature for C. jejuni and C. coli is around

30oC. However, this does not mean that metabolic activity ceases at lower temperatures,

implying that there is a potential for the organisms to adapt to environmental stresses even

when they are unable to grow. Hazeleger et al. (1998) showed metabolic activity (ATP

production, catalase activity and respiration by oxygen uptake) in C. jejuni at temperatures as

low as 4oC. Chemotaxis toward formate and aerotaxis toward microaerophilic conditions has

also been demonstrated at temperatures down to 4oC, illustrating the potential for the

organism to migrate to conditions that might extend survival in the environment.

Human Population

consumption

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food distribution

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consumption

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treateddrinking-water

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The physiology of C. jejuni is complex (Kelly, 2001) and there are a number of chemicals in

the environment that the organism can use as terminal electron acceptors. This comparative

versatility may enable the organism to metabolise in diverse anaerobic environments outside

the host.

C. jejuni is thought to die rapidly in the presence of oxygen and under dry conditions.

However, studies show that C. jejuni can survive much better in vivo than in vitro. For

example C. jejuni has been isolated from dry beach sand (Bolton et al., 1999), which

contradicts laboratory studies on survival in drying liquid droplets (Humphrey et al., 1995).

One proposed explanation for this unexplained resilience is that Campylobacter may form

viable but non-culturable (VNC) cells. In an original concept of this state, cells change from a

spiral morphology to a coccoid form and become undetectable by normal culture techniques,

but retain the potential to be resuscitated to an infectious form. While the evidence for VNC

formation by C. jejuni remains equivocal, the ability for Vibrio to become VNC has been well

characterised (Oliver, 1996), and several other pathogens have also been described as

undergoing a VNC transformation. A more recent theory suggests that only a few strains of

Campylobacter transform to VNC cells. Those that retain their spiral morphology (Federighi

et al., 1998) undergo a gradual loss of ability to maintain homeostasis (Tholozan et al., 1999),

and so presumably there is a period where cells are VNC followed by death.

This latter theory is independently supported by studies where vibrioid cells were observed in

chicken shed water supplies but could not be cultured (Pearson et al., 1993), and have also

been observed in a continuous culture microcosm where spiral cells persisted in biofilms

(Buswell, et al., 1998). In studies on the survival of clinical and poultry isolates at 4oC, four

from six poultry isolates became coccoid after ten days incubation, while only two from seven

clinical isolates became coccoid. If a functional VNC state is found to be real, then this has

significant implications for our knowledge about the survival of C. jejuni in the environment.

2.3.2 Survival in Faeces and Slurry

The understanding of survival of Campylobacter in faeces is pivotal in understanding the

transmission of the organism from the environment to humans, since this is the link between

the reservoirs and the transmission routes. Faeces are the ultimate source of this organism,

whether it reaches humans via food, water or any other mode of transmission that is known or


suspected. Robust survival in faeces will therefore greatly assist in the transmission of this

organism. However, little information has been published concerning this aspect of the

organism’s survival.

Jones et al. (1999) reported that Campylobacter were present at levels between 35,000-

56,000/g in sheep faeces and could be isolated from the faeces for 3-4 days when stored

outside at ambient temperatures. In experiments with Campylobacter-positive human faeces

stored at 4oC, it was found that 10 from 20 samples were positive for Campylobacter after 24

hours, eight were positive after two to seven days storage, and two were positive after 12 to

20 days storage (Valdas-Dapena et al., 1983).

C. jejuni has been shown to survive well in an anaerobic digestor operating at 28oC treating

farm waste. A decimal reduction time of approximately 440 days was found and the effluent

contained in excess of 104 colony forming units (cfu) Campylobacter/ml (Kearney et al.,

1993).

Unpublished New Zealand data shows that Campylobacter have good survival times (1

month) if present in bovine faeces under moist, cool conditions.

2.3.3 Survival in Food

Campylobacter has been reported as being a contaminant in a limited range of foods. Most

work has focused on the prevalence in poultry, where the proportion of contaminated product

may be very high. For example, in a British study Kramer et al. (2000) found a prevalence of

83.3% in poultry samples. Other foods found to be contaminated by this organism include

shrimp (Adesiyun 1993), vegetables (Kumar et al., 2001; Park and Sanders, 1992) offal

(Kramer et al., 2000), shellfish (Wilson and Moore, 1996), crabs (Reinhard et al., 1995),

garlic butter (Zhao et al., 2000) and mushrooms (Doyle and Schoeni, 1986). The prevalence

in offal is moderate, with a much lower prevalence in all of these other foods (however, it

must be remembered that a rare occurrence in a food that is consumed frequently may still

result in a significant number of cases).


The overriding observation concerning the survival of Campylobacter in foods is that, as long

as they are not frozen, then the colder the food is kept the better the organism survives.

Conditions designed to prevent the growth of other foodborne pathogens can therefore

enhance survival of this pathogen. Curtis et al. (1995) demonstrated that survival of

Campylobacter at 2oC in a range of foods was between 1.5 and 15 times as long as survival at

20oC.

On raw sterile liver slices Campylobacter survived for four days, with no reduction in

numbers at 4°C or 15°C, and only a 1.5 log10 reduction in numbers at 37°C (Moore and

Madden, 2001). The reduction in numbers was more rapid in liver homogenate, but even then

the reduction in numbers was <1 log10 at 4oC.

Following an outbreak of campylobacteriosis that implicated garlic butter as the transmission

vehicle, experiments were carried out to examine the survival of Campylobacter in butter

(Zhao et al., 2000). Survival was found to be reasonable in butter stored at 5°C or 21°C.

Repeated experiments with garlic butter showed variability in results, with survival of a

modest inoculum (104/g) for several hours at 5°C.

On watermelon, Campylobacter reduced in numbers by 38-87% over six hours at ambient

temperature. A reduction under the same conditions on papaya was in excess of 90% (Castillo

and Escartin, 1994).

In experiments with sterile minced chicken meat Blankenship and Craven (1982) showed that

two from three Campylobacter isolates incubated at 4°C survived for 18 days with less than a

1 log10 reduction in numbers. At 23°C the reduction in numbers ranged from approximately

1.5 to 5 log10 in 18 days while at 43°C, inocula added at approximately 106-107/g were at, or

below, the limit of detection between 10 and 16 days. At 37°C growth followed by survival

up to 18 days was observed. In raw drumsticks stored at 4°C numbers reduced by

approximately one log10 cycle under a CO2 atmosphere, and 4 log10 under air after 24 days of

storage.

A comparison of isolates from clinical cases and raw chicken indicated that the clinical

isolates survived better than their poultry-derived counterparts at 4°C (Chan et al 2001). In


some isolates there was a reduction of less than 1 log10 after 14 days incubation at 4°C, while

others reduced to below the limit of detection in 12 days.

In New Zealand the high prevalence of Campylobacter in chicken has been found in repeated

studies (e.g. Campbell & Gilbert, 1995; Hudson et al., 1999), and all studies find a prevalence

in the range of 50-70%.

In experiments investigating the effects of freezing on Campylobacter survival was better on

high pH beef with approximately a 1 log10 decrease in numbers at –18°C up to 30 days of

storage, with no appreciable reduction from day 30 to day 40 (Gill and Harris, 1982). On

normal pH meat the initial reduction in numbers was somewhat greater (approximately 2 log10

units), but survival after day 30 the same. A similar initial reduction followed by a

stabilisation of numbers was shown on sterile raw liver slices at the same temperature, albeit

over a shorter time period (Moore and Madden, 2001).

At –20°C Chan et al (2001) showed that six isolates reduced in numbers by at least three log10

cycles when incubated at –20°C. In further experiments survival was better in chicken rinse

than it was in Mueller-Hinton broth. Survival was characterised by an initial four log10

reduction in numbers after 12 days followed by a period where less than a further two log10

reduction was measured up to 32 days of storage. This pattern of an initial reduction in

numbers followed by greater persistence is consistent throughout the studies of

Campylobacter survival at freezing temperatures.

Gill and Harris (1982) investigated the survival of one isolate of C. jejuni on beef at normal

pH (5.8) and high pH (6.4). At the higher pH, inactivation at –1°C involved an initial

reduction of 1 log10 unit followed by a slow decline of approximately 0.5 log10 unit over the

following 30 days. At normal pH, inactivation was much more rapid, with a reduction of

approximately 3.5 log10 units in 10 days. The pH of the meat was therefore critical to the

survival of the organism at refrigeration temperatures, and there was significant survival at

the higher pH. At 25oC inactivation was even faster for meat at both pH with a 3.5 log10 unit

reduction in seven days.


2.3.4 Water

The survival of Campylobacter in water can be summarised as poor at temperatures above

10°C and when exposed to direct sunlight. These observations seem to run counter to the fact

that the organism can routinely be isolated from river waters, and sometimes at high numbers

(Savill et al., 2001a).

The work of Obiri-Danso et al (2001) encapsulates most of the relevant information about

survival of Campylobacter in water. This work showed that pathogenic Campylobacter spp.

survived less well than other Campylobacter species of ambiguous pathogenic potential.

Survival was somewhat better in seawater than river water, with natural populations

disappearing in 12-24 h at 20°C and 37°C, but persisting up to 120 h at 4°C and 10°C. A one

log10 reduction was reported in approx. 100 hours at 4°C in natural populations of river water

incubated in the dark. For river water temperatures of 10 °C and 20 °C the time taken for a 1

log reduction was 90 hours and less than 12 hours respectively. Natural populations became

undetectable within 30 minutes when exposed to sunlight (equivalent to an English June day)

and held at 17-20oC. This work showed that survival of C. jejuni and C. coli was comparable

under the conditions used.

The response to temperature and survival are consistent with observations that

Campylobacter tend to be more frequently isolated from water in the winter months, as this is

the time when the water temperature and exposure to UV will be lower.

Campylobacter has been shown to not survive very well in low osmolality liquid media at all

temperatures tested (Reezal et al., 1998) indicating poor osmoadaptability. However, these

experiments were carried out under microaerophilic conditions and the situation might be

different under aerobic conditions, which would be more like the conditions found in the

environment. In addition, even the low osmolality medium used would still have contained

significantly more dissolved solids than river water.


2.3.5 Sediment

The seasonal distribution described for Campylobacter in water has not been observed for

Campylobacter in sediments (Obiri-Danso and Jones 1999). Obiri-Danso and Jones (1999)

found that the Campylobacter present in sediments did not follow a seasonal trend, as they

could be isolated from the sediments at all times of the year. This suggests either that there is

a continuous input from agricultural run-off and other sources and/or that Campylobacter

survive for longer periods in sediments compared to water. Numbers in the sediments were

less than 1 log10 /g dry weight and most of the isolates identified were either C. jejuni or

C. coli.

2.4 Correlation Between Survival and Pathogenicity

A number of activities, which are important to survival of Campylobacter in the environment,

are also important to pathogenesis. Three aspects are discussed in this section, which show

links between environmental survival and virulence mechanisms.

It is recognised that the acquisition of iron in the animal host is an important pre-requisite for

infection. There is essentially no free iron in humans and so iron must be obtained by the

bacterium from one or more of the iron-containing compounds available, such as ferritin and

haemoglobin. In bacteria, siderophores and/or the production of haemolysin usually mediate

iron acquisition, and aspects of these activities may reside in the one protein (Park and

Richardson, 1995).

Campylobacter have been shown to use haemin and haemoglobin as sources of iron (Pickett

et al., 1992). It was also demonstrated that of five isolates tested, only three produced

haemolysin. C. coli has been shown to possess a phospholipase, which contributes to the

organism’s overall haemolytic activity (Grant et al., 1997), and these enzymes are often

associated with pathogenicity. The production of siderophores has been demonstrated in

seven of 26 isolates of C. jejuni (Field et al., 1986).


Acquisition and safe storage of iron is also important for environmental survival under

aerobic conditions (Wai et al., 1996). These authors concluded that ferritin is essential for the

survival of Campylobacter between or within animal hosts.

Pathogenic Campylobacter spp. are regarded as being microaerophilic. However the growth

of C. jejuni under aerobic conditions after a period of adaptation (Jones et al., 1993) has been

reported. Aerobically adapted cells do not show enhanced survival in foods compared to

microaerophilically-grown cells when incubated under aerobic conditions (Chynoweth et al.,

1998). Continuous culture experiments have demonstrated that high oxygen tension results in

a change from spiral to coccoid form. This may be a transitional state as subsequently there is

a selection of cells, which are more oxygen tolerant with the typical spiral morphology

(Harvey and Leach, 1998).

`

Part of the adaptation to high oxygen environments is the production of catalase and

superoxide dismutase (SOD), but it appears that in Campylobacter the SOD activity has the

main role of protecting against toxic by-products of oxygen (Purdy et al., 1999). C. jejuni is

thought to possess only one SOD encoding gene, from which the derived protein incorporates

iron into its structure, i.e. it is an FeSOD (Pesci et al., 1994). Further experiments with the

FeSOD of C. coli have shown that the inactivation of the gene results in reduced survival

capability of the organism in food (milk and chicken skin), and a reduced ability to colonise

the intestine of one day old chicks (Purdy et al., 1999). In addition SOD deficient mutants of

C. coli are sensitive to freezing and thawing (Stead and Park, 2000) and similar mutants in

C. jejuni showed a decreased ability to invade INT 407 cells compared to the wild type (Pesci

et al., 1994). This enzyme activity is therefore clearly of great importance in both survival

and the ability of the organism to invade intestinal cells and then, presumably, to cause

disease.

In response to heat, alkaline pH (Wu et al., 1994) or oxygen (Takata et al., 1995)

Campylobacter produces GroEL and GroES “heat stress” proteins. These proteins are also

known as “chaperonins” and are involved in trans-membrane protein transport, as well as

secretion and post-translational assembly/disassembly of protein oligomers. In addition Hsp-

40 (Konkel et al., 1998) and Hsp-70 like proteins (Thies et al., 1999) have been shown in

Campylobacter. It is believed that the expression of heat shock proteins serves to protect cells

from adverse environmental factors. These proteins have also been demonstrated to have


likely roles in the virulence of Helicobacter and Salmonella (Ensgraber and Loos, 1992), and

so a further link between virulence and environmental survival of Campylobacter is suggested

by these observations.

2.5 Transmission Routes Considered in the Present Study

From this review, the matrices identified as important for investigation as environmental

reservoirs of Campylobacter were human, animal and bird faeces, offal and chicken products,

and water. These matrices and their potential as reservoirs or transmission routes for human

campylobacteriosis are discussed in the following section.

2.5.1 Human Faeces

A United Kingdom (UK) survey of isolates from 3,378 human faecal samples used PCR

detection to identify 493 (14.5%) samples as positive for Campylobacter (Lawson et al.,

1999). When identified to the species level by PCR, 89% of isolates were C. jejuni and 18%

were C. coli. This data included 19 samples that were positive for a mixed infection of

C. jejuni and C. coli. The other Campylobacter species present were C. upsaliensis (2%), C.

hyointestinalis (0.6%) and C. lari (0.2%). Figures for New Zealand clinical cases of

campylobacteriosis are difficult to obtain, as most clinical laboratories do not identify

Campylobacter to the species level.

The incubation period of Campylobacter infection is usually between 1-3 days but can be as

long as 10 days (Faoagali, 1984; Koenraad et al., 1997). The symptoms of human

campylobacteriosis include an initial period of fever, headaches and malaise which lasts for

up to 24 hours. This is then followed by diarrhoea and in most cases severe abdominal pain.

The fever persists, but nausea and vomiting are less common features of the infection

(Koenraad et al., 1997). The patient may excrete Campylobacter organisms for up to 3 weeks

and the Campylobacter count in human faeces is in the range of 106 to 108 bacteria/g faeces

(Taylor et al., 1993). Most cases of campylobacteriosis are self-limiting, however

complications arising from an infection by C. jejuni include Guillain–Barré syndrome. This is

a neuroparalytic syndrome which can lead to fatal respiratory paralysis (Endtz et al., 2000;

Hadden et al., 2001).


2.5.2 Raw Poultry

Comparison of Campylobacter that infect humans with those present in poultry indicates that

chickens are implicated as the vehicle in a significant number of human Campylobacter

infections. The undercooking of Campylobacter contaminated chicken products is believed to

be an important transmission route for sporadic cases of human campylobacteriosis (Tauxe,

1992). A study which obtained Campylobacter isolates from random clinical human faecal

samples and poultry products in the Netherlands showed the same genotypes present in both

matrices (Duim et al., 1999). A New Zealand study by Kakoyiannis et al. (1988) using

genotyping found that nearly half (49.7%) of human isolates typed were indistinguishable

from poultry isolates. This was supported by another New Zealand study (Hudson et al.,

1999), which used Penner serotyping and PFGE to show common strains of C. jejuni present

in chicken portions and human cases of campylobacteriosis. A case control study undertaken

in Christchurch to determine the risk factors for human campylobacteriosis identified the

recent consumption of chicken in 80 % of clinical cases (Ikram et al., 1994). Stern (1992)

suggests that the literature indicates 50 to 70% of cases of human campylobacteriosis can be

attributed to contamination of poultry by C. jejuni.

Surveys have shown that 30-100% of poultry harbour Campylobacter as normal commensal

flora of their intestinal tract (O’Sullivan et al., 2000). Campylobacter in the intestinal

contents of chickens at the time of slaughter are reported to be present in numbers up to 107

cfu/g (Stern, 1984). This high number is reflected in the prevalence of C. jejuni, which is

present in 50–85% of commercial broiler carcasses (Bryan and Doyle, 1995; Park et al.,

1991). In studies discussed by Stern (1992) C. jejuni was isolated from chicken carcasses at

rates from 48 to 98%, although one study from the Netherlands yielded only 16 % C. jejuni.

A survey in the UK which tested 198 chicken portions isolated C. jejuni from 77% and C. coli

from 6.6% of the samples (Kramer et al., 2000).

While poultry products seem to be responsible for a high proportion of sporadic cases of

campylobacteriosis (Federighi, 1999; Hanninen et al., 2001), data from many studies suggest

the implication of other animal reservoirs (Corry and Atabay, 2001; Nielsen et al., 2000; On

et al., 1998; Petersen et al., 2001). An outbreak of campylobacteriosis in Ontario, Canada was

investigated by serotyping and molecular genotyping of the human clinical isolates and 20

cattle implicated in the outbreak. The subtyping results showed that all of the human isolates


and ten of the cattle isolates had restriction patterns that were indistinguishable, suggesting a

single infecting strain (Bradbury, et al., 1984).

2.5.3 Ruminant Animals

In a UK study of thermotolerant Campylobacter isolates from sheep, the main species isolated

was C. jejuni (90%), followed by C. coli (8%) and C. lari (2%) (Jones et al., 1999). This

same pattern was found with the isolation of Campylobacter from sheep intestines at the time

of slaughter (Stanley et al., 1998a). Over a one-year sampling period, there were consistently

high carriage rates of Campylobacter detected in the intestines of sheep at slaughter.

However, shedding of Campylobacter in faeces was found to vary depending on feed and the

season, with high numbers of Campylobacter isolated during the lambing season and low

prevalence during the winter period. C. jejuni was found to survive in sheep faeces left in the

outside environment for up to four days. The numbers of campylobacters found in sheep

faeces were consistently lower than the numbers found in their intestines. Jones et al. (1999)

postulated that a sheep may shed up to 7 x 107 Campylobacter per day (figures from late

summer sampling), which would contribute to the bacterial loading of runoff in to streams

and rivers.

The seasonal variation of thermotolerant campylobacters observed in sheep also seems to be

the case for beef and dairy cattle in the UK (Stanley et al., 1998c). The peak periods for both

beef and dairy cattle occurred in spring and autumn. A New Zealand study by Meanger and

Marshall (1989) found the peak period of infection to be autumn (31%) closely followed by

summer (24 %). It also demonstrated that the same genotypes of C. jejuni and C. coli were

found in sheep and dairy cows on the same farm. This suggests cross infection between the

two animal species and was the basis for sampling sheep and cattle faeces from

geographically separated farms in the CTR study. Some caution must be exercised when

attempting to compare New Zealand data concerning farmed animals with those from

overseas. This is because farming practices in New Zealand are characteristic of this country,

and their effects on the epidemiology of zoonotic organism within farmed animals is likely

also to be characteristic.

Although Meanger and Marshall (1989) found no correlation between farm animal and

human genotypes of C. jejuni, they suggested that this was due to the limited scale of the


study. Stanley et al. (1998b) explored the presence and survivability of Campylobacter in

dairy slurries. Thermotolerant Campylobacter were readily isolated from stored slurries and

from slurries disposed onto land during the winter. The campylobacters could be detected in

the slurry for up to 20 days after application.

In overseas studies the intestinal cell density of Campylobacter in beef cattle, as determined

by the Most Probable Number (MPN) technique at the time of slaughter, was 6.1 x102 MPN/g

fresh weight of intestinal contents (MPN/gfw) (Stanley et al., 1998c). In the same study the

average number of Campylobacter present in adult dairy cattle was found to be 70 MPN/gfw

and 3.3 x104 MPN/gfw in calves. A range of faecal carriage rates for C. jejuni in dairy cows

have been reported (Table 3).

Table 3 Carriage Rates in Ruminant Animals

Percentage carriage Number of animals Author37.7 2,085 Wesley et al., 20007 136 dairy cows and calves Atabay and Corry, 199854 24 calves Grau, 198812.5 96 adult dairy Grau, 1988

Although farm animals are born free of Campylobacter, various studies have demonstrated

the transfer of campylobacters from mothers and the immediate farm environment to lambs

(Jones et al., 1999), calves (Stanley et al., 1998c) and pigs (Weijtens et al., 1997). The higher

Campylobacter numbers found in the offspring of farm animals decreases as they reach

maturity as their intestinal tracts become fully developed. The Campylobacter species most

commonly isolated from pigs is C. coli (Christensen and Sorenson, 1999). The prevalence of

C. coli in pig faeces has been reported as 58% (n = 203) (Munroe et al., 1983). A study by

Weijtens et al. (1997) reported that Campylobacter counts in pig faeces ranged from 102 to

104 cfu/g. Overall there is a paucity of published data for New Zealand.

2.5.4 Meat Products

The ubiquitous presence of campylobacters in the intestines of cattle, sheep and pigs suggests

that they would be common on eviscerated carcasses. This has been found to be the case but

their numbers decline rapidly, presumably due to the sensitivity of campylobacters to drying

(Park et al., 1991). A survey of abattoirs in Northern Ireland revealed no isolates of

Campylobacter in 100 lamb and 100 beef carcasses (Madden et al., 1998). The same study


also detected no Campylobacter species in 50 retail packs of beef and 50 packs of pork. This

concurs with a Japanese study by Ono and Yamamoto (1999), which failed to detect C. jejuni

in beef and pork. Madden et al. (1998) suggested that this low prevalence could be taken as

an indicator of good slaughterhouse hygiene practices. A report for the Danish Meat Research

Institute (Christensen and Sorenson, 1999) discusses the problems of Campylobacter

contamination during the slaughter process. Campylobacter were found on 43-85% of pig

carcasses before the tunnel chilling process. After chilling the Campylobacter isolation had

dropped to 11-18% of the carcasses. Almost all of the Campylobacter isolates were C. coli.

Offal may be more highly contaminated by Campylobacter because of its moist nature,

which, given that the product is chilled, will enhance the survival of the organism (Park et al.,

1991). From a survey of 400 slaughterhouse pigs, Moore and Madden (1998) isolated

Campylobacter species from 6 % of the livers. Of the positive livers 67% contained C. coli,

30% C. jejuni and 3% C. lari. A UK study (Kramer et al., 2000) tested for Campylobacter in

lamb, ox and pig livers purchased over a two month period from retail outlets. The

prevalences were much higher for these samples (Table 4).

Table 4 Prevalence of Campylobacter Contamination in Offal (Kramer et al., 2000)

Matrix Campylobacter Total number of samples Percentage positiveLamb Liver C. jejuni 75.0

C. coli96

13.5Ox liver C. jejuni 49.0

C. coli96

2.1C .jejuni 34.3

Pig Liver C. coli99

42.4

2.5.5 Ducks

The mobility of wild birds and their internal temperature of 42°C makes them ideal

candidates for aiding the transmission of Campylobacter through the environment (Skirrow,

1990; Jones, 2001). Several studies have highlighted the potential role of birds as vectors for

Campylobacter transmission from farm animal faeces. Skirrow (1994) demonstrated the

transfer of campylobacters by birds pecking cowpats and Jones et al. (1999) suggested the

same transmission route for sheep. The prevalence of C. jejuni isolated from mallard ducks

has been reported as 34% (n = 243) (Luechtefeld et al., 1980) and 40% (n = 82) (Fallacara et

al., 2001).


2.5.6 Water

Thermotolerant Campylobacter are widespread in the environment and subsequently in

waterways (Savill et al., 2001a) where their presence is a sign of recent contamination with

animal and bird faeces, farm run-off or sewage (Jones, 2001). The Campylobacter count

prevalence found in various water systems during a New Zealand survey is displayed in Table

5. The prevalence of Campylobacter in water increases in winter when water temperatures are

lowest. During the summer when there is an increase in ultraviolet radiation and the water

temperature rises the prevalences of Campylobacter fall (Obiri-Danso et al., 2001).

Campylobacter do not multiply in water because of their high minimum growth temperature

(circa 30oC). Instead water acts as a transmission route between warm-blooded hosts.

Contamination of waterways by Campylobacter follows seasonal trends.

Table 5 Prevalence of Campylobacter in Surface Water

Water Type Campylobacter MPN 100/ml PercentageCampylobacter

numberstested

Minimum Maximum MedianSurface water <0.12 >11 0.18 60 30

Reported cases of campylobacteriosis in New Zealand attributed to water include an outbreak

in the township of Ashburton (Brieseman, 1987). In this incident contamination of the town

water supply after heavy rains was implicated as the likely source of infection. The water for

the town supply is derived from the Ashburton River, which is surrounded by sheep and beef

farms. Chlorination of the supply only occurs at times of heavy rainfall and the delay in

beginning chlorination after the onset of rain may have allowed Campylobacter into the town

supply. Other water related incidents of Campylobacter outbreak are a camp and convention

centre in Christchurch (Stehr-Green et al., 1991) and Te Aute College, where a

malfunctioning UV treatment light may have caused the influx of Campylobacter into the

water supply (Inkson, 2002). The Canadian Walkerton Inquiry (O’Connor, 2002) highlights

the dangers of waterborne transmission of pathogens. Seven people died and over 2,300

became ill when Walkerton Town’s water supply became contaminated with Campylobacter

and E. coli O157:H7. It was presumed that the contamination arose from farm animal run-off

into a shallow well, from which the water supply was taken.


2.6 Direction of Transmission Between Reservoirs of Campylobacter

Many of the studies cited above illustrate that some subtypes of C. jejuni and C. coli are

found in environmental reservoirs as are found in human cases of campylobacteriosis. As

stated by Petersen et al. (2001) identification of clonally related Campylobacter strains in

different animal reservoirs suggests an exchange of campylobacters may take place. However

this does not aid our understanding of the direction of flow (or if there is any flow) between

these varied reservoirs, humans and connecting transmission routes (Petersen et al., 2001).

The criteria for determining a potential transmission route are inferred from spatial, temporal

and human epidemiological data. The combination of these data may establish links between

varied environmental and human matrices. In the UK, Kramer et al. (2000) reported the first

study to apply an identical subtyping system to isolates of Campylobacter but only from two

matrices (meat and poultry) collected within the same geographical area and time period as

the human clinical isolates. However, the study by Kramer et al. (2000) did not investigate

epidemiological links.

2.6.1 The Selection of Subtyping Methods for the Discrimination of CampylobacterIsolates

The subtyping of bacterial isolates from various sources and a determination of their relative

contribution to human infection is a prerequisite for the investigation of the transmission

routes of a pathogen. It also allows the detection of changes in infectious disease aetiology.

Numerous methods for the subtyping of Campylobacter have been described. The

international literature was reviewed to determine the optimal approach for the CTR. No

single ideal method has been identified as suitable for all research studies (Nielsen et al.,

2000, McKay et al., 2001), therefore each method considered relevant was evaluated

according to the criteria of discrimination, typability, reproducibility and cost effectiveness

(Wassenaar and Newell, 2000). These techniques are discussed in detail below, with a

comparison of alternative phenotypic and genotypic methods. Table 25 and Table 26 and

(Appendix 1) have brief descriptions of each of the subtyping methods discussed in this

section.


This review determined that the combination of both Penner serotyping and pulsed field gel

electrophoresis (PFGE) using SmaI digestion were the most appropriate techniques for the

subtyping of isolates from the CTR study.

Penner Serotyping

Serotyping is a phenotypic subtyping method, which relies on the detection of antigens

present on the surface of microorganisms. Penner serotyping is a system developed by Penner

and Hennessy (1980). It uses passive haemagglutination to differentiate Campylobacter

strains on the basis of soluble heat-stable (HS) antigens. The Penner serotyping scheme has

world-wide recognition and data covering prevalent subtypes found in various countries is

useful for comparative purposes (Patton and Wachsmuth, 1992). A Penner serotyping facility

has been established at the Institute for Nachamkin Environmental Science and Research

Limited in Wellington, New Zealand and has built up a large databank of C. jejuni serotypes

present in the New Zealand environment.

Several Penner serotypes (e.g. O19) are over represented in the C. jejuni isolates which have

been associated with Guillain–Barré syndrome, a neural paralytic syndrome which can result

in severe respiratory failure (Endtz, et al,. 2000; Hadden, et al., 2001). Therefore

identification of the serotype of Campylobacter isolates is useful for tracking serotypes

implicated in some cases of complicated campylobacteriosis.

In a comparative study of ten subtyping systems used to distinguish isolates from four

outbreaks of Campylobacter, Penner serotyping proved to be as discriminatory as multi locus

enzyme electrophoresis (MLEE), restriction enzyme digestion analysis with various enzymes,

and ribotyping with various enzymes (Patton et al., 1991). It was slightly more discriminatory

than the Lior method of serotyping, which detects heat labile antigens on the surface of the

cell. The Penner method detected eight different serotypes compared to seven different

serotypes by Lior serotyping. The Penner method has gained greater international recognition

than the Lior method (Klena, 2001). Consequently there is a larger worldwide database of

strains typed by the Penner method, which can be accessed for epidemiological studies. The

phenotypic methods of Lior biotyping, Lior phagetyping and plasmid profile analysis used in

the study of Patton et al., (1991) were less discriminatory than the other methods.


The Penner scheme reportedly has high cross reactivity (Oza et al., 2000). A study by McKay

et al. (2001) compared the Penner method and a modified method of Penner termed the

Laboratory of Enteric Pathogens (LEP) method. LEP uses absorbed antisera in an effort to

overcome any cross reactivity associated with the Penner scheme. The LEP method was

shown to have only a marginal improvement in discriminatory power compared with the

Penner method, but more uptypable isolates, cross reactivity also still occurred (McKay et al.,

2001). The slight advantage in discrimination was negated by the LEP method having 36.6%

of isolates reported as untypable, compared with 8.2 % untypable by the Penner method.

Nielsen et al. (1999) identified Penner serotyping as a useful method for the subtyping of

non-outbreak isolates, as it is easy to compare strains obtained over long time periods. In a

comparative study of subtyping methods Penner serotyping was found to be less

discriminatory than genotypic methods but it was considered a stable method in that it did not

separate strains that had been grouped together using genotyping methods (Nielsen et al.,

2000). Therefore Penner serotyping is a useful primary method that has been employed to

obtain a broad grouping of isolates which can be further refined by more discriminatory

subtyping methods (On et al., 1998; Nielsen et al., 2000). Wassenaar and Newell (2000) and

Hanninen et al. (2001) concluded that C. jejuni serotypes can exhibit considerable genetic

diversity within each serogroup, and therefore it is a more powerful method for

epidemiological studies when serotyping is combined with a genotypic method.

Genotyping

The advantages and disadvantages of Amplified Fragment Length Polymorphism (AFLP) and

Multi Locus Sequence Typing (MLST), and the more commonly used Pulsed Field

Electrophoresis (PFGE) are considered below

The genotyping technique of AFLP generates between 50-80 bands. The high number of

multiple small bands makes it less likely that this technique is susceptible to genomic

instability (Wassenaar and Newell, 2000) and this complex method is highly discriminatory

between isolates (Lindstedt et al., 2000). A comparison of molecular genotyping methods

identified AFLP as the most discriminatory method, and subdivided 50 Campylobacter strains

derived from poultry into 41 distinct genotypes (de Boer et al., 2000). The next most


discriminatory method was PFGE using SmaI digestion, which identified 38 genotypes,

followed by flaA RFLP (31 genotypes) and ribotyping (26 genotypes).

Disadvantages of the AFLP method are that an automated DNA sequencer and computer

assisted analysis are essential for identification and therefore a major capital investment is

required (Duim et al., 1999).

MLST is also able to distinguish between closely related strains as it is based on sequence

polymorphisms within seven to ten selected conserved housekeeping genes. Population

studies of C. jejuni, by MLST, have revealed a low overall degree of sequence diversity

(Suerbaum et al., 2001). However C. jejuni has high rates of intraspecies recombination,

which creates many different combinations of alleles suitable for generating a large number of

discriminatory sequence subtypes (Suerbaum et al., 2001).

It is a highly reproducible method but its requirement for a high capital investment, similar to

that required for AFLP, and its complex data analysis requirements means that it is not

applicable as a routine subtyping method. These techniques may be useful as non-routine

methods for high resolution genotyping where further discrimination between isolates is

required for the determination of genetic lineages. In a comparative commentary on molecular

methods of genotyping, Olive and Bean (1999) described PFGE as being considered as the

“gold standard” for DNA-based subtyping. They describe PFGE as having high

discrimination power between strains but with moderate set up costs in comparison to AFLP

and DNA sequencing.

Pulsed field gel electrophoresis (PFGE) is the more widely used genotypic method for routine

subtyping. The entire bacterial genome is digested with restriction endonucleases that cut the

genome infrequently. The resulting DNA fragments are separated by size difference in an

agarose gel. The gel is run under special electrophoretic conditions that switch the orientation

of the electric field in a pulsed manner to separate the large (20 to 200 kb) DNA fragments.

An evaluation study of subtyping methods to distinguish between C. jejuni isolates associated

with a campylobacteriosis outbreak and other sporadic isolates (Fitzgerald et al. 2001)

concluded that PFGE was the most discriminatory subtyping method. The other methods

evaluated were Penner serotyping, restriction fragment length polymorphisms (RFLP) of the


flagellin (flaA) gene, sequencing a 582 base pair (bp) region of the flaA gene and sequencing

the entire flaA gene. The PFGE, serotyping and sequencing of the 582-bp region all separated

the outbreak cases from the sporadic cases. PFGE analysis employed two enzymes: SmaI and

SalI. The SmaI digest was shown to be more discriminatory than the SalI restriction enzyme

(RE) digestion. The discriminatory power of PFGE is attributed to its ability to determine

polymorphisms derived from the entire bacterial genome rather than relying on differences

within one or two genes (or gene products), as is the case with the other subtyping schemes

tested by Fitzgerald et al. (2001). Nielsen et al. (2000) supported these results and found that

the genotypic techniques of PFGE and random amplified polymorphic DNA (RAPD) were

highly discriminatory. In comparison, the methods of RFLP of the flagellin gene (RFLP

flaA), denaturing gradient gel electrophoresis of the flagellin gene (fla-DGGE) and

riboprinting were found to be less discriminatory. Smith et al. (2000) confirmed the benefits

of the use of the combination of Penner serotyping and PFGE by SmaI digestion in a study of

the different subtypes present in clinical and chicken isolates.

The utility of using SmaI as the restriction enzyme of choice for PFGE analysis was

investigated by On et al. (1998). They compared the profile groups obtained with SmaI and

three other restriction enzymes and concluded that SmaI is a “generally robust means of

accurately determining C. jejuni strain relationships”. However they also noted that some

isolates giving the same profile for SmaI digestion could be further subdivided by the use of a

second restriction enzyme. It is suggested that the use of two restriction enzymes is

potentially useful in cases where isolates are collected over an extended period of time

(Tenover et al., 1995). On et al. (1998) and Wassenaar and Newell (2000) describe the

combination of serotyping and a genotypic method such as PFGE, as an appropriate method

for the epidemiological investigation of strain relationships for sporadic cases of C. jejuni.

Pulsenet is a US based National Molecular Typing Network for Foodborne Disease

Surveillance, which was established by the National Centre for Infectious Diseases and the

Centres for Disease Control and Prevention (CDC). A recent paper published by Pulsenet

(Ribot et al., 2001) investigated the reproducibility of SmaI PFGE protocols for the subtyping

of C. jejuni. The aim was to determine standardised protocols, which would produce high

quality interlaboratory comparisons of data. These protocols allow for rapid comparison of

DNA fingerprints for C. jejuni isolates from geographically dispersed laboratories to enhance

the national surveillance of foodborne diseases. In this survey five independent laboratories


typed the same seven isolates and gel image results were compared using computer-assisted

analysis. In each case there was a perfect match between the PFGE patterns for each of the

isolates, indicating the reproducibility and utility of this method. Pulsenet use the restriction

endonuclease SmaI as their primary enzyme for PFGE, but acknowledge that a secondary

enzyme can be useful for further discriminatory power where the results with SmaI are

inconclusive. It is expected that the importance of data exchange between international

laboratories will increase as global epidemiological studies are undertaken to detect emerging

infectious diseases and changes in disease aetiology (Stephens and Farley, 1996; Olive and

Bean, 1999; Wassenaar and Newell, 2000; Woodward and Rodgers, 2002) and that this

important aspect must be taken into consideration in the adoption of a methodology.

2.6.2 The Stability of Genotypic Methods

An important aspect of a subtyping system is its stability over an extended period of time.

Campylobacter is a naturally competent bacterium (Duim et al., 1999), that is, it is able to

take up foreign DNA from its surrounding environment and incorporate it into its own

genome. This natural competence, as well as internal rearrangements of the genome may be

important for increasing an organism’s ability to survive within a changing environment

(Manning et al., 2001). This is relevant for CTR, as Campylobacter was isolated from a wide

range of diverse habitats where its passage through the environment was being investigated.

Genetic instability could undermine the applicability of genetic subtyping. For example,

RFLP subtyping of the flagellin gene locus has demonstrated hypervariable regions, that are

subject to recombination events (Harrington et al., 1997). Ideally, genetic subtyping methods

need to target highly conserved genes with a low frequency of recombination events. Methods

that utilise the entire genome are inherently more stable than those which focus on one or two

genes (Wassenaar and Newell, 2000).

Petersen et al., (2001) compared serotypes, flagellin RFLP (fla type) and PFGE subtypes of

C. jejuni isolates from broiler flocks, humans and wild animals and birds. The isolates were

collected over a three-year period and a wide geographical area within Denmark, where the

study was conducted. Comparison of the PFGE profiles produced by three different

restriction endonucleases identified clonal lineages that had been genetically stable over long

time periods (e.g. two and a half years) and wide geographical ranges (within Denmark).

Studies by Manning et al., (2001) have demonstrated longer-term genetic stability of


environmental isolates collected over a two month period in 1998, which clustered with

human isolates from an outbreak in 1981. The related human and environmental isolates had

the same PFGE subtype when cut with three different endonucleases. Relatedness of the same

isolates by AFLP subtyping was determined by demonstrating genetic homology of more than

90% when analysed by GelCompar software and the unweighted pair group method using

average linkage for cluster analysis. One of the isolates from the human waterborne outbreak

was subsequently used as an international standard strain for C. jejuni. It showed the same

genotypic stability even though it had been subcultured frequently and showed reduced

colonisation potential when used for infection studies of chickens. From these data Manning

et al. (2001) have proposed that “genome shuffling may not be as essential for Campylobacter

stress adaptation as previously thought” and that these mechanisms need further investigation

but do not undermine the usefulness of genotyping, at least for short term epidemiological

studies. Both these studies confirmed the use of genotyping techniques such as PFGE for the

investigation of complex epidemiologies.

Wassenaar et al. (1998) reported PFGE revealed genomic instability, where 21 isolates from a

single batch of poultry displayed 14 different C. jejuni PFGE subtypes when digested with

SmaI and another enzyme BssHII. This was unexpected as most batches of poultry are found

to have only one or two genotypes of C. jejuni present. The 14 different PFGE subtypes were

similar and further phenotypic and RFLP fla subtyping demonstrated that they were of clonal

origin. The variation was attributed to genomic variation due to recombination events. The

same study investigated the stability of the 14 PFGE subtypes in vitro and confirmed that,

after repeated subculturing (ten passages) of each isolate, the PFGE subtypes remained stable.

One of the isolates was also passaged in vivo in two 1-day old chicks. All 20 C. jejuni re-

isolated after five days from each chick’s caecal contents had the same PFGE genotype as the

original infective strain. This suggests no recombination events had occurred during in vivo

passage of C. jejuni. A similar study by Hanninen et al., (1999) however, did find phenotypic

and genomic changes detected by PFGE and serotyping in two of twelve C. jejuni isolates

inoculated into chicks. The occurrence of natural transformation within the chicken gut was

also investigated by the in vivo passage of isogenic mutants containing two different

antibiotic resistance markers (Wassenaar et al., 1998). There was no DNA exchange observed

between these two mutants.


A recent study by de Boer et al., (2002) provides substantial experimental evidence for

horizontal DNA transfer among heterologous C. jejuni strains during their colonisation of

chickens. Intragenomic alterations were also observed which added to the genetic diversity

detected by changes in PFGE subtypes. An important factor in regard to these in vivo

experiments was the absence of selective pressure therefore, recombination was occurring

within the natural habitat of Campylobacter. When these same strains were passaged more

than 300 times in the laboratory they revealed no genomic recombinations when typed by

PFGE. These findings concur with those mentioned above, indicating the stability of strains

cultured in the laboratory and that genetic differences are generated by in vivo environmental

differences.

From the results of this experiment de Boer et al. (2002) proposed that PFGE may be too

sensitive as a subtyping method for the determination of genetic relatedness of strains of

C. jejuni and that PFGE data need to be supported by another, preferably genotypic,

subtyping method. The genotypic methods suggested by de Boer et al. (2002) included RFLP

subtyping of the flagellin genes. This technique is, itself, inherently unstable as Harrington et

al., (1997) have described intra- and intergenomic recombination events between the flaA and

flaB genes. MLST and AFLP were also proposed.

From these studies it would appear that the PFGE subtype of C. jejuni isolates is stable during

routine laboratory passaging. Earlier reports (Manning et al., 2001, Petersen et al., 2001)

suggested that although genomic rearrangements can occur in a population it is a rare event.

In the CTR study a combination of Penner serotyping and pulsed field gel electrophoresis,

using SmaI cleavage, was used to subtype Campylobacter isolates. Reported genetic diversity

may play an important role in adapting the pathogen to a variety of host habitats.

2.7 Conclusions

2.7.1 Aspects of the Microbial Ecology of Campylobacter

Potential reservoirs of Campylobacter will be few as they will be restricted to warm blooded

animals, i.e. birds and mammals. However, while the diversity of such animals in New

Zealand is perhaps not as great as in other countries, they are present in large numbers given

that pastoral agricultural activity is a major feature of New Zealand’s economy and lifestyle.


From overseas work, the faeces of sheep, dairy and beef cattle are known to contain

Campylobacter at relatively high prevalences and in high numbers. In particular, the faeces of

young animals contain campylobacters more frequently and in greater numbers. Given this

information these animals must be considered as very important in the transmission of

Campylobacter.

Birds are also a reservoir, and this extends beyond carriage by chickens. Wild birds may also

contribute to the dispersal of Campylobacter. Ducks have been shown to carry this organism

at significant prevalences.

It is unlikely that growth of campylobacters occurs outside warm-blooded hosts although

some growth could occur in fresh faeces. Dispersal in the environment is influenced by the

organism’s ability to survive. For an organism that is considered to be “fragile” in the

environment, Campylobacter shows considerable robustness, especially under cool moist

conditions. Environmental survival may also be linked to the ability of the organism to cause

disease.

The relative importance of transmission routes is likely to be a function of the initial numbers

of the organism, the rate of introduction of the organism, the rate of die off or dilution, and

the eventual exposure to the human population. As described above the faeces of all of the

reservoirs studied in the CTR project contain Campylobacter. Therefore occupational or other

contact with animal faeces may result in exposure to the organism, e.g. in meat processing

workers or farmers.

Water is well established as a potential transmission route, whether the exposure is from

recreational activities such as swimming in rivers, or the consumption of contaminated

drinking water. River water is known to be contaminated quite frequently with

Campylobacter, albeit at (usually) quite low numbers. River water, which is treated for

consumption, must therefore be disinfected correctly or an outbreak of disease is likely to

ensue. Recreational exposure to river water may also result in disease if river water is

accidentally swallowed. Campylobacter is likely to survive best in cold river water that is

minimally exposed to UV light. The organism is also likely to survive in sediments, which

may become resuspended after heavy rainfall events.


Given that raw animal-derived foods are distributed under refrigeration, the survival of

Campylobacter in these foods is optimised by these conditions. Raw chicken and offal are

known to be contaminated by Campylobacter at high prevalences, although much less is

known about the level of contamination. These foods may therefore act as transmission routes

if they are improperly cooked or are the source of cross contamination in the domestic or food

service kitchen. The presence of Campylobacter on offal also acts as a surrogate for the

subtypes of Campylobacter that might occur on red meat. While it is acknowledged that the

contamination of red meat products is likely to be much less than the corresponding offal,

they are also likely to be eaten in much higher volumes. Therefore the exposure (a function of

contamination and consumption) to the population of these subtypes is also likely to occur via

red meat products although the data are not available to estimate the degree of this exposure.

The Campylobacter Transmission Routes Study (CTR) attempts to address the direction of

flow of transmission by confining all of the Campylobacter isolated during a one-year

sampling period to a defined geographical region. The choice of sampling sites reflects the

intention to detect the potential transmission of Campylobacter strains between environmental

matrices and ultimately, to a human host. The combination of spatial, temporal and human

epidemiological data may establish links between varied environmental and human matrices.

2.7.2 Subtyping Methods

Many genotyping methods have been developed over recent years. Each has its own

advantages and disadvantages and no single method is universally applicable. A problem with

Campylobacter is the apparent ability for its DNA to recombine quite frequently. Analysis of

single genes with a known propensity for recombination is not considered useful for long

term investigations, while other methods are more robust to these changes, yet are

prohibitively expensive to perform. It is almost universally acknowledged that two subtyping

methods should be used to give the required specificity and robustness. Comparability of data

with other studies is also desirable. A combination that has been used in studies in New

Zealand is to apply both Penner serotyping and pulsed field gel electrophoresis (PFGE).

Penner serotyping is the most robust serotyping system available and has the advantage that

data can be compared with overseas studies. PFGE represents a relatively cost effective,

whole genome subtyping method, but there is some discussion and much conflicting

information in the literature regarding the stability of PFGE subtypes over time. However, the


more recent papers tend to regard problems of this type to be minimal with this technique.

The combination of the two methods adds to the confidence that already exists in the two

individual techniques.

It is concluded that the combination of both Penner serotyping and pulsed field gel

electrophoresis (PFGE) using SmaI were the most appropriate techniques. The images

produced will be comparable with those generated by the Pulsenet group and other

international databases such as Campynet. This allows ESR the opportunity to share

information, which will assist global surveillance of Campylobacter strains important to

human infection.


3. MATERIALS AND METHODS

A brief description of the methodology for sample collection from the Ashburton District and

Campylobacter isolation, detection and subtyping is outlined in the following section. The

details of methodology protocols, the establishment of sampling procedures and identification

of sampling sites were described in the Ministry of Health Client Report FW0149 (2001).

Development of the enrichment PCR methodology was described in the Ministry of Health

Client Report FW0058 (2000) and can be viewed in Appendix 4.

3.1 Identification and Interviewing of Human Cases and Data Analysis

Cases were defined as persons resident in the Ashburton District and notified with

campylobacteriosis to the South Canterbury Medical Officer of Health between 1 January

2001 and 31 January 2002.

Cases were identified from notifications received by the South Canterbury public health unit.

Information was then passed on to the Ashburton District Council for interview. Each case

was interviewed face-to-face by a staff member of the Ashburton District Council (ADC)

using a standardised questionnaire.

The questionnaire (Appendix 2) was developed as an extension of the routine enteric disease

case report form. That form includes information on case demographics, basis for diagnosis

and the timing, course and outcome of the illness. The questionnaire included additional

questions on exposures considered potentially important for Campylobacter infection. These

exposures included contact with animals, foods from animal sources, various forms of

drinking water and potentially contaminated environments.

Information on the completed questionnaires was then entered onto a purpose-built Access

database, and data transferred to ESR Kenepuru Science Centre in Porirua. Data were

subsequently checked for completeness and internal consistency by a dedicated research

associate, and missing data were followed-up by the Ashburton interviewer.

A summary of the procedure is shown in

Figure 4.


Figure 4 Flow of Information and Samples relating to the Human Clinical Isolates

Case patient

visits GP with interviews case gastroenteritis

GP HPO Ashburton

sends faecal notifies case to HPOspecimen to lab CPH Timaru arranges interview&

reports the result return of to GP questionnaire

Clinical Labs CPH Timaru test positivefor Campylobacter

enters notificationfaecal specimen to ESR & questionnaire data

(when available)

Subtyping of isolateisolate subtyping weekly transferresults of EpiSurv data

ESR Wellington

combine isolate datawith EpiSurv quest data

Complete databasefor analysis


3.2 Sample Sites

3.2.1 Water sample sites

The effect of numerous water sampling sites on the spatial variable was minimised by using

three water sampling sites on a recurring basis. This water sampling regime maximised the

likelihood of establishing potential Campylobacter transmission routes/linkages.

1) Region A: The Ashburton intake

The Ashburton Intake site is in the Ashburton River, upstream of the Ashburton Township

and the position where the infiltration gallery for the township was sited. It was decided to

sample from this site to gain an understanding of the Campylobacter strains that have the

potential to pass into the township’s drinking water supplies.

Map coordinates E 240 2100

N 570 9100

2) Region B Above the Ashburton Forks on the South Branch

This site was chosen because it is downstream of a significant area of dairy, beef cattle and

sheep farming and therefore the water flowing through this site has been exposed to farm

runoff.

Map Coordinates E 239 2000

N 571 8000

3) Region C Above the Ashburton Forks but below the convergence of Bowyers and

Taylors Stream

This site was chosen because it is downstream of a significant area of dairy, beef cattle and

sheep farming and therefore the water flowing through this site has been exposed to farm

runoff.


Map Coordinates E 239 2500

N 571 8500

3.2.2 Farm sites for collection of ruminant animal faeces

Farms upstream of the water sites were chosen to ensure that there is a relationship in time

and place between the subtypes of Campylobacter found in the drinking water source (river)

and subtypes of Campylobacter found in the animals in the farms along the river.

Farms were chosen to reflect the diversity of farm subtypes (sheep, dairy, cattle) required by

the study. The logistics of traveling in one day to all farm and water sites was given careful

consideration in terms of cost effectiveness. It was important that all samples were sent to

ESR and analysed within 24 hours after collection.

Figure 5 is a map of the Ashburton District depicting the course of the Ashburton River as it

flows through the three farming regions from which water and animal faecal samples were

collected. These three regions and their corresponding water sites are upstream of the

Ashburton Township.

3.2.3 Retail outlets for meat products

Determination of the volume of sales of each particular meat type sold in Ashburton and

Tinwald Townships was conducted prior to establishing the sampling regime for meat

products.

To ensure the best epidemiological use of the data generated from food sampling, it was

necessary to establish the volume of sales of each particular meat type sold in Ashburton. The

meat subtypes targeted for sampling in this study were: whole, fresh chicken; sheep

liver/kidney; beef liver/kidney and pig liver/kidney. Offal was chosen as the red meat source,

because it yields a higher number of Campylobacter positives in comparison to other meat

subtypes (Kramer et al., 2000). A survey of butcheries and supermarkets in Tinwald and

Ashburton was conducted to determine the volumes of each meat type sold and who supplied

the products to the retailers (Table 27), Appendix 3).


Figure 5 Map of the Farm and Water Sampling Regions A, B and C

3.3 Sample Collection

Samples were collected each week as presented in Table 6. Further details of sampling

procedure can be found in the Ministry of Health Client Report FW0149 (2001). Note that

human faecal samples continued to be collected from January 2001 until the end of January

2002. This is in contrast to all other matrices, which were sampled from January 2001 to the

last week of December 2001.

A sampling plan involving the collection of all environmental samples at one time was

rejected for the following reasons. The sample numbers would be too large for processing all

samples within a 24 hour collection period. The time period of collection would restrict the

Ashburton Township

Ashburton RiverSouth Branch

AshburtonForks

Ashburton RiverNorth Branch

TaylorsStreamBowyers

Stream

SouthBranch

Mt Somers

Region AWater Site

Region CWater Site

Region BWater Site

Region A

Region B

Region C


analysis of results in finding a temporal relationship. The logistics of travel and time excluded

the option of collecting from all sample matrices on a weekly basis.

The alternating sampling plan involved collection of all farm samples, some of the water

samples and all of the duck faecal specimens in the first week of sampling. The second

sampling week combined the collection of all food samples with extra water samples from the

Ashburton Intake. This fortnightly plan had the advantages listed below:

• A fortnightly collection was preferable for the convenience of farmers and retailers.

• It led to consistency in the temporal variable, as the sampling was designed to obtain a

representative sample of subtypes present in a particular matrix on that day.

• An increase in the diversity of Campylobacter subtypes by minimising the likelihood of

routinely isolating the same subtypes from a matrix.

The environmental matrices chosen to be investigated as potential reservoirs of

Campylobacter were water; chicken carcasses; offal (kidney or liver) products from beef, pig

and sheep; animal faeces from beef and dairy cattle, sheep and mallard ducks.


Table 6 Collection Routine for all Samples

Matrix Samplingfrequency

Number ofsamplescollected

Sampling plan Samplemethod

Region A Weekly 4 Collected on sameday

2 in morning,2 in afternoon

composite

Region B Fortnightly 2 Collected on sameday


composite

Water

Region C Fortnightly 2 Collected on sameday


composite

Beef cattleDairy cattle

Sheep

Fortnightly 3One from each

region

8 week rotationbased on

3-4 farms withineach region

composite(of 5

animal faecesfrom each

farm)Ducks Fortnightly 3 Alternating

between ponds atAshburton and

Tinwald Domains

Composite(of 5

individualduck faeces)

Faecal samples

Human Weekly asnotified

single

*Meat Products Fortnightly Rotationalbetween retailersbased on volume

of sales

single

* Refer to Table 7 and Table 8 for meat product sampling plan

3.3.1 Human faecal sample collection

Human faecal samples were collected from cases of campylobacteriosis, notified to the

Crown Public Health from within the Ashburton District. The faecal samples were sent to

Christchurch clinical laboratories and transferred to ESR Christchurch Science Centre for

processing.

The Christchurch laboratories enrolled for the supply of Campylobacter clinical specimens

were: Southern Community Laboratories Ltd., Medlab and Canterbury Health Laboratories.

Each clinical laboratory was given a list of doctors from the Ashburton Region as a guide for

the Campylobacter specimens important to the CTR study. Clinical laboratories were asked to

store any clinical faecal samples from the Ashburton area, which had tested positive for


Campylobacter at 4°C. It was requested that these specimens be sent once a week to ESR in

the biohazard bottles provided by ESR.

3.3.2 Collection of samples from each environmental matrix

The environmental matrices chosen to be investigated as potential reservoirs of

Campylobacter were water; animal faeces from beef and dairy cattle, sheep and mallard

ducks.

Contact was established with Ashburton District Council (ADC) and a partnership was set up

for the collection of water, food and faecal samples. The ADC set up a travel route for

collection of animal faecal and water samples which was based on the data supplied.

Farm sampling occurred every fortnight and the collector rotated the rounds to increase the

diversity of farms and thus increase the probability of isolating a greater array of

Campylobacter strains. Three to four farms of each animal type were sampled from each of

the regions on a rotational basis. Therefore, each farm was sampled approximately once every

two months. Although some of the farms chosen for faecal sampling carried both sheep and

cattle at the same time, only one animal faecal type from each farm was collected throughout

the entire sampling period.

To increase the likelihood of obtaining positive Campylobacter samples, it was decided to

collect five different faecal samples for each animal matrix from each farm. The duck samples

were also composite samples of five duck faeces.

3.3.3 Collection of meat products from retailers

All meat product samples were collected from retailers in the Ashburton and adjoining

Tinwald Township.


The information from the survey of meat retailers was applied to construct a monthly

sampling plan based on a fortnightly rotation of Plan A and B (Table 7 and Table 8). This

plan ensured that a representative distribution of sampling numbers, between retailers, was

achieved. The plan was initially based on the collection of 12 (average) of each animal offal

type per fortnight (e.g. 12 x beef liver) and 6 whole chickens per fortnight.

Table 7 Plan A for Meat Sampling

Sample numbers per fortnightRetailer Sheep offal Beef offal Pig offal Chicken carcass

A 1 2 2 3B 1 0 2 2C 1 1 1 0D 1 1 1 0E 1 2 2 0F 4 3 3 2G 4 3 2 2

Totals 13 12 13 9

Table 8 Plan B for Meat Sampling

Sample numbers per fortnightRetailer Sheep offal Beef offal Pig offal Chicken carcass

A 1 2 1 3B 1 1 2 2D 1 1 1 0E 0 2 2 0F 4 3 3 2G 4 3 2 2

Totals 11 12 11 9

3.3.4 Initial sampling plan numbers based on January projected prevalence ofCampylobacter

Initial sampling plan:

Fortnightly sampling of each matrix i.e.

Week One: 6 beef cattle faeces Week Two: 12 beef liver (average)6 dairy cattle faeces 12 sheep’s liver6 sheep faeces 12 pig’s liver (average)6 duck faeces 6 whole chickens8 water samples 4 water samples32 samples 46 samples

Plus human clinical specimens (2-4 per week)


Financial constraints led to a reassessment of resources. After the completion of 4 months of

sampling an investigation of actual Campylobacter prevalence in each of the matrices was

used to adjust our sampling numbers. This led to an increase in numbers of chicken samples

and a decrease in the numbers of ruminant faecal and duck faecal samples. Fluctuations in

availability of offal subtypes from retailers led to a reduction in the offal samples, although a

representative distribution of sampling numbers was maintained based on previously

established meat volumes (Table 27), Appendix 3).

Fortnightly sampling of each matrix as at May 2001

The revised sampling plan as at May 2001:

Week One: 3 beef cattle faeces Week Two: 7 beef liver3 dairy cattle faeces 6 sheep’s liver3 sheep faeces 7 pig’s liver3 duck faeces 9 whole chickens8 water samples 4 water samples20 samples 33 samples

Plus human clinical specimens (2-4 per week)

3.4 Isolation and Detection

3.4.1 Methods for isolation and detection of Campylobacter species

Samples from each matrix were transferred to ESR Christchurch Science Centre for

processing to determine the presence of C. jejuni and C. coli. Samples were transported at

4°C and analysed within 24 hours of collection. A full description of the methods can be

found in the Ministry of Health Client Report FW0149 (2001).

In brief, samples were double enriched in Exeter medium to ensure detection of only viable

Campylobacter cells. Exeter medium is a blood-based broth that contains antibiotics and

growth supplements, which select for thermotolerant campylobacters. The resulting bacterial

cells were harvested and washed in preparation for detection of C. jejuni and C. coli by

multiplex Polymerase Chain Reaction (Enrichment PCR, Appendix 4). Electrophoresis of

PCR amplicons was carried out in agarose gels followed by visualisation of DNA by staining

with ethidium bromide. The detection limits of this method for both C. jejuni and C. coli in

the matrices tested in the CTR study are presented in Table 29 in Appendix 4.


Exeter broths that tested positive for C. jejuni and C. coli were plated out onto Exeter agar

medium and Campylobacter colonies purified. One colony from each positive sample was

purified and typed, unless the sample tested positive for both target Campylobacter species, in

which case, one isolate of each species was identified. Previous testing of multiple colonies

isolated from one water sample, had confirmed the presence of a dominant PFGE subtype in

the enrichment. Financial constraints made it prudent to increase the number of samples

collected from a matrix, rather than attempt to isolate more than one subtype of

Campylobacter from each sample.

The identity of isolates was confirmed by PCR amplification. Isolates were frozen at -80°C

for long term storage. C. jejuni isolates were sent to the Enteric Reference Laboratory (ESR-

KSC) for serotyping. C. jejuni and C. coli isolates were sent to the microbiology research

laboratory at the Plant and Microbial Sciences Department (PaMS) in the University of

Canterbury and to the Enteric Reference Laboratory (ESR-KSC) for PFGE analysis.

3.4.2 Subtyping Methods

3.4.2.1 Serotyping

Penner serotyping was performed by the passive haemagglutination technique described by

Penner and Hennessy (1980) to determine the heat stable serotypes of C. jejuni isolates.

Antisera were produced at the Enteric Reference Laboratory (ESR-KSC) by the methods

described by Penner and Hennessy (1980) using their reference strains for antisera

production. Penner serotyping was not performed on C. coli isolates as the requisite antisera

were not available.


3.4.2.2 Grouping of Serotypes

Serotypes can be grouped as follows:

• Serotype 1 includes the cross-reacting serotype 1,44.

• Serotype 4 complex includes strains expressing any combination of 4,13,16, 50 serotypes.

• Serotype 8 includes the cross-reacting serotype 17.

• Serotype 23 includes the cross reacting serotype 36.

3.4.3 Pulsed Field Gel Electrophoresis (PFGE)

PFGE by SmaI restriction endonuclease digestion was performed on all C. jejuni and C. coli

isolates by the method described in Appendix 5. The two laboratories involved in this

subtyping procedure were the Enteric Reference Laboratory (ESR-KSC) and the PaMS

microbiology research group at the University of Canterbury.

3.4.3.1 Computerized Gel Analysis of PFGE subtypes

To ensure accurate normalisation for inter-gel comparisons all agarose gels were run with 3

molecular weight ladders of Lambda Ladder PFG Marker (#N0340S, New England Biolabs

Inc., Beverly, USA). Two of the ladders were run in each of the outside lanes and one ladder

was run in the centre lane of each gel.

After electrophoresis the DNA gels were stained with ethidium bromide, scanned and saved

in a tagged image file format (TIFF). The images were analysed by Bionumerics software,

version 2.5 (Applied Maths, Sint-Martens-Latem, Belgium). After conversion and

normalisation of gels, the degrees of similarity of DNA profiles were determined by the Dice

coefficient and dendrograms were generated by the unweighted pair group method with an

optimisation setting of 1 % and 1.2% position tolerance. The PFGE subtype identity of new

strains was determined by matching their normalised DNA profiles against the constructed

computerised PFGE libraries.

3.4.3.2 Related PFGE subtypes

The following section describes the criteria employed in the CTR study to determine if two

isolates, with similar PFGE subtypes, were genetically related.


Definition of “genetically related”

Genetically related isolates are termed clones. Tenover et al. (1995) describes clones as

“isolates that are indistinguishable from each other by a variety of genetic tests (e.g., PFGE

and ribotyping) or that are so similar that they are presumed to be derived from a common

parent [Given the potential for cryptic genetic changes detectable only by DNA sequencing or

other specific analyses, evidence for clonality is best considered relative rather than absolute

(Eisentein, 1989)].”

Interpretative criteria for determining relatedness between isolates have been proposed by

Tenover et al., (1995) for outbreaks of pathogens. However it is more difficult to apply these

criteria over the time period of longer-term studies. Ribot (2002) suggests that studies which

collect samples over a period of more than one year require careful interpretation of results.

One of the future aims of Surveillance Networks is to establish interpretative criteria by

ongoing collection of data from varied geographies in the United States. This will allow a

more robust interpretation of numerical estimates of relatedness based on indistinguishable

patterns and a small number of differing bands. It is recognised that there are differences in

genome stability between pathogenic species. For example, Escherichia coli O157:H7, is

considered a highly clonal organism and has a stable genome and therefore single band

differences may signal unrelatedness (Ribot, 2002). This is in contrast to C. jejuni, which is

now regarded as genetically diverse with a high frequency of DNA recombination events

within and between organisms (de Boer et al., 2002). Therefore one to three PFGE band

differences may be interpreted as signaling a degree of relatedness. However Ribot (2002)

cautions against the over interpretation of results and stresses the importance of

epidemiological information to confirm linkages.

The CTR study collected isolates over a one-year sampling period and therefore analysis of

related PFGE subtypes was based on a conservative interpretation of Tenover et al., (1995).

PFGE subtypes were considered to be clonally related when:

i) The subtypes differed by one band shift, which indicated an event had occurred

resulting in a DNA fragment running as a larger or smaller band due to either an

insertion or deletion of DNA (respectively). For example Figure 6 lanes 2-4 represent


PFGE Subtype 3a and are clonally related to PFGE subtypes 3d (lane 5) and 3 (lane

6).

ii) A large molecular weight band was replaced by two smaller molecular weight bands,

the sum of whose DNA approximated the original larger molecular weight band. This

change in PFGE pattern is indicative of the gain of a new restriction site resulting in

the formation of 2 new bands, and the loss of the larger molecular weight band. This

was a rare event for this study.

A table showing the clonally related subtypes of C. jejuni is presented in Table 30, Appendix

6.

Figure 6 Gel image of related PFGE subtypes

Lanes 1 and 19: Molecular weight standards (Lambda concatamer; New England Biolabs)

9 16141 2 3 4 5 6 7 8 10 11 12 13 15 17 18 19

48.5

145.5

97.0

194.0

242.5

291.0

339.5

388.0436.5485.0 Kb


3.5 Analysis of Campylobacter Subtypes

Comparison of all C. coli isolates was based only on PFGE subtyping. The comparison of

C. jejuni subtypes was based on the clustering of isolates into the individual serotype

grouping which was further resolved by the PFGE subtype. Therefore subtyping results for

C. jejuni are presented as serotype (heat stable antigen, HS):PFGE subtype (HS:P).

Isolates untypable by serotyping but that produced a PFGE pattern were included in the

statistical analyses as HS serotype untypable (SUT): P, but were not included when

commenting on linkages between matrices. Conversely, isolates, which were identified by a

serotype but did not produce a PFGE pattern because their DNA was recalcitrant to restriction

by SmaI, were included in statistical analyses but were removed from the discussion of

linkages between matrices. These PFGE subtypes were described as non-cutting.

3.6 Statistical Analysis

Data analysis was carried out using SAS software. For the testing of association between

matrices or risk factors the chi-square test or, where appropriate, Fishers Exact test was used.

The chi-square (χ2) and Fishers exact test statistics both measure the association between two

categorical factors. The chi-square test is not valid when the sample size (number of samples

or cases) is small, which is defined when the number of expected cases in any cell (i.e. any

combination of categories) is less than 5, in these cases the Fishers exact test should be used.

Exposures to potential risk factor information were collected from cases after notification.

The examination of the association between Campylobacter subtypes and the potential risk

factors identified from the questionnaire for human cases used the fishers exact test to

measure any statistical significance of the relationships. These results were considered at both

the serotype and at the serotype:PFGE subtype levels by the use of the Fishers exact test. It is

recognised that with the small sample size, level of diversity and the multiple univariate

comparisons that the statistical results can not be seen as definitive, but as indicative results

that are then reviewed in conjunction with other results. Multivariate analysis of these results

was not undertaken as it was deemed unreliable due to the relatively small sample size in

conjunction with the level of diversity in subtypes.


3.6.1 Czekanowski Index (Proportional Similarity Index)

To estimate the similarity of the distributions of subtypes between each of the matrices the

data were analysed using an approach previously used for the same task. Rosef et al. (1985)

described the use of the proportional similarity index to compare serotype distributions

among Campylobacter isolates from poultry, wild birds, flies, pigs and others. The index

produces a numerical value between 0 and 1, where 1 indicates that the distribution of

subtypes between two sources is identical. Data concerning “untypable” (serotype) or “non-

cutting” (PFGE) isolates had special consideration in the analysis. In the analysis of data two

results were derived, the first case assumes that all of the “untypable” (serotype) or “non-

cutting” (PFGE) subtypes, occurring only once in one matrix, were identical to those

occurring in the other matrix. The second case assumes that none of them are common to both

matrices and all relate to a unique pattern, i.e. have no similarity. It is likely that the reality is

somewhere in between. In analyses involving PFGE data unique isolates from one matrix

were treated only as being dissimilar to unique isolates in the other.

3.7 Survival of Campylobacter in Environmental Reservoirs

The temporal linkage between isolates from matrices was one of the factors considered when

postulating a transmission route of Campylobacter through the environment to humans. This

section outlines the procedure to identify the criteria for establishing a temporal linkage.

Figure 7 shows the links between selected reservoirs and transmission routes for

Campylobacter. Potential transmission of Campylobacter to humans will depend on the

maximum survival time of Campylobacter in each matrix. Survival times are dependent on

seasonal variations. The “summertime” survival period was designated as October to March

and the “wintertime” period was designated as April to September. Survival times are

separated into summer and winter, where applicable.


Figure 7 Potential Reservoirs and Transmission Routes for Campylobacter

The survival time of Campylobacter in each matrix was considered individually (Table 9)

before compiling additive values for time passage from an environmental matrix to humans

(Table 10). The data in Table 9 were compiled based on data in the literature and are

discussed in detail below.

Table 10 presents the maximum time period assumed for establishing a direct transmission

route between matrices based on the temporal distribution of data. The figures in Table 10 are

based on figures presented inTable 9.

Animal/DuckFaeces

Soils/Sediments/ di / di

Water

Human Faeces

Food

Direct routeie. no incubation period

Pote

ntia

l Tra

nsm

issi

on R

oute

s of C

ampy

loba

cter

54Au

gust

200

2F

rom

Env

iron

men

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oval

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20 d

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∗ refe

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for d

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l


The incubation period for human cases of campylobacteriosis is reported as being two to

five days with a range of one to ten days (Chin, 2000). Therefore the maximum incubation

period presumed for the purposes of this study is ten days. Reporting time for the infection

i.e. going to the doctor and a faecal sample being submitted to the clinical laboratory was

presumed to be, at a maximum, seven days. Survival of campylobacters in human faeces is

not relevant here, because human faeces, in general, are voided directly into water and

therefore, Campylobacter survival is viewed in relation to survival in water.

Based on the literature review Campylobacter could be expected to survive in raw chicken

at 4°C for up to 18-24 days. However as chilled meat would show signs of spoilage after 10

days this period was used as the presumed length of survival.

Survival time and dilution must be considered in estimating the potential of water as a

reservoir. A study of Campylobacter survival in water by Obiri-Danso et al. (2001) found a

1 log reduction in number of campylobacters (colony forming units per ml, cfu/ml) in

natural populations from river water after approximately 100 hours incubated at 4°C in the

dark. For river water temperatures of 10°C and 20°C, the time taken for a 1 log reduction in

the number of campylobacters (cfu/ml) was 90 hours, and less than 12 hours, respectively.

The Ashburton River is comparatively short, with most water flowing quickly out to sea.

The Ashburton Forks, site of two water sampling sites is approximately 15 kilometres

upstream of the Ashburton Intake, the third sampling site. Using mean flow conditions for

low and normal flow (Graeme Horrell, Environment Canterbury, personal communication)

it would take nine hours under mean low flow conditions (0.3metres/second, m/s) and 4.5

hours under mean average flow (0.9m/s) to travel a distance of 15km between Ashburton

Forks, and the Ashburton Intake (5). This suggests that the survival of Campylobacter in

river water would only be secondary to its removal rate by the river flow.

With only limited data available on Campylobacter survival in sediments, it is difficult to

draw conclusions on their potential role as an environmental reservoir. Physical disturbance,

however, such as heavy rain or flood events are assumed to increase resuspension of

campylobacters residing in the sediments into the overhead water column.


Campylobacter has been reported to survive in sheep faeces for four days (Jones et al.,

1999). Campylobacter inside cow faecal pats would be protected from drying and UV

radiation and could be expected to survive for long periods. Unpublished New Zealand data

shows good survival (one month) under moist, cool conditions (Hudson et al., unpublished

data).

Data provided by Keith Jones (Lancaster University) shows a maximum of seven days

survival of Campylobacter in gull faeces deposited at a rubbish tip. As there is limited data

available on Campylobacter survival in duck faeces, it is assumed in this study to be similar

to the survival time of Campylobacter in gull faeces.

3.7.1 Unknowns

When calculating the maximum allowable time for the determination of a temporal linkage

between matrices the following factors were unknown:

• The persistence of each strain in an animal reservoir as a function of time, i.e. resident

campylobacters versus transient populations.

• The persistence of Campylobacter in sediments and the effects of resuspension of

Campylobacter from the sediments into the overhead water column.


4. RESULTS

4.1 Sampling Overview

Figure 8 depicts the locations of sampling locations; nine dairy farms, twelve beef (non-

dairy cattle) farms, twelve sheep farms, three water sites, two duck ponds, seven meat

wholesalers, and all cases residing in South Canterbury notified to the local public health

service. Note that cases with non-valid or rural postal addresses have been geographically

located at the nearest district or town midpoint. The region under study was bounded by the

Rakaia River to the north and the Rangitata River to the south as can be seen in the map.

4.2 Human Cases of Campylobacteriosis

For human faecal samples several steps were required to fulfill all conditions of the

sampling programme and these are detailed in Table 11. ESR received eight faecal samples

from cases which were later found to be either located outside the study region or were not

notified through the local Public Health Service. These were therefore excluded from further

data analysis. Unknown circumstances led to faecal samples from 24 cases of

campylobacteriosis not being forwarded to ESR, although a questionnaire was collected for

21 of these cases. These 24 cases were excluded from further analysis. From this point on,

therefore, analysis of human data will be limited to the 61 human cases with both

questionnaire and bacterial subtyping information. These cases comprise 55 cases of

C. jejuni infection only, five cases of C. coli infection only and one case of C. jejuni and

C. coli co-infection. In total, there were 56 isolates of C. jejuni and six isolates of C. coli

from human cases.


Table 11 Human Cases of Campylobacterosis

Resides in SouthCanterbury andnotified to localPublic Health

Service

QuestionnaireCompleted

Laboratory samplereceived

CampylobacterIsolated

Number of Cases

Yes 61YesNo 7

Yes

No N / A 21Yes 1YesNo 0

No

No N / A 3

Yes

Total 93Yes 4No YesNo 4

No

Total 8Total 101

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60Au

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2Fr

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onm

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o H

uman

s

Figu

re 8

Map

of S

ampl

ing

Loc

atio

ns a

nd H

uman

Cas

es


4.2.1 Demographics of Human Cases

The demographics of the human cases are presented in Table 12 to Table 15. There was a

predominance of cases aged 0-4 years old (Table 12). Table 13 shows that the majority of

cases are European. There were slightly more male cases than female (Table 14). Only four

cases were hospitalised resulting in a hospitalisation rate of 6.6% (Table 15).

Table 12 Age Distribution of Human Cases

Age Number Percent0-4 13 21.3%5-9 1 1.6%10-14 2 3.3%15-19 7 11.5%20-24 6 9.8%25-29 3 4.9%30-34 4 6.6%35-39 5 8.2%40-44 3 4.9%45-49 4 6.6%50-54 4 6.6%55-59 4 6.6%60+ 5 8.2%

Table 13 Ethnicity Distribution of Human Cases

Ethnicity Number PercentEuropean 57 93.4%Maori 0 0.0%Pacific Island 0 0.0%Other 3 4.9%Unknown 1 1.6%

Table 14 Sex Distribution of Human Cases

Sex Number PercentMale 33 54.1%Female 28 45.9%

Table 15 Hospitalisation of Human Cases

Hospitalised Number PercentYes 4 6.6%No 57 93.4%


4.3 Crude Prevalence of Campylobacter

The number and percentage of samples testing positive for C. coli and C. jejuni are

presented in Table 16.

Samples collected from the different environmental matrices differ in the nature of their

sampling frames. All human cases were laboratory confirmed at a primary diagnostic

laboratory before their faecal sample was forwarded to ESR. Therefore the prevalence∗ of

Campylobacter reported for human cases is largely dependent upon the faecal sample

handling, distribution of the pathogen within the faecal sample and viability of the isolate

after initial confirmation. Animal and duck faecal samples were composed of samples from

five individual animals, therefore, the prevalences were expected to be higher than those for

individual animals. Water samples, are by definition composite samples, potentially

including organisms from every source in the water catchment area. By contrast, the meat

samples represent the prevalence of Campylobacter for each individual sample of offal or

chicken carcass.

Results for each group of sample matrices are presented in Table 16. There were significant

differences between the prevalences for different animal faeces (sheep, dairy, and non-dairy

cattle) for both C. jejuni (χ2, p<0.0001) and C. coli (χ2, p<0.0001). Dairy faeces had the

highest percentage of samples positive for C. jejuni (97.8%), followed closely by beef

faeces (83.9%), while sheep faeces had the highest percentage of samples positive for

C. coli (47.1).

There are also statistically significant differences between different subtypes of meat

product (beef offal, sheep offal, pork offal, and chicken carcasses) for both C. jejuni (χ2,

p>0.001) and C. coli (χ2, p=0.02). Of the foods, sheep offal had the highest percentage of

samples positive for C. jejuni (38.9%), followed closely by chicken carcasses (27.5%),

while pork offal had the highest percentage of samples positive for C. coli (4.8%).

∗ specified as the proportion of a group of samples that are positive for C. jejuni or C. coli by either PCR orculture


Table 16 Prevalence of C. coli and C. jejuni in the Matrices and Diversity of PFGE Subtypes

C. coli C. jejuniMatrix Totalnumber ofsamples

Numberpositive

Percentagepositive

Diversity of PFGEsubtypes

Numberpositive

Percent-age

positive

Diversity ofcombined

serotype:PFGESubtypes

No. Percentagediversity†

No. Percentagediversity†

Human faeces∞ 69 6 8.7 5 83 57Ω 82.6 44 77

Water* 293 12 4.1 7 58 162 55.3 91 60Duck faeces* 92 1 1.1 1 100 60 65.2 42 72Dairy faeces* 91 9 9.9 5 56 89 97.8 33 38Beef faeces* 87 14 16.1 10 71 73 83.9 43 61Sheep faeces* 87 41 47.1 11 27 52 59.8 34 71

Beef Offal 178 1 0.6 1 100 15 9.0 12 80Sheep Offal 162 6 3.7 4 67 63 38.9 36 57Pork Offal 187 9 4.8 8 89 9 4.8 26 78Chicken Carcass 204 2 1.0 1 50 56 27.5 7 46

Total 1,450 101 7.0 39 39 637 43.9 250 40*Composite samples**The number of valid human faecal samples from laboratory-diagnosed campylobacteriosis received by ESRfrom the region of study.Table 11 details analysis of the breakdown of samples and questionnaires received by ESR and the numbers ofCampylobacter isolated from human samples.†Diversity is the percentage value of the number of subtypes divided by the number of positive samplescalculated for each matrix.N.B. Diversity for C. coli PFGE subtypes counts “non-cutting” as an individual subtype, and similarly forC. jejuni, any serotype in conjunction with “non-cutting” PFGE subtype also appears as an individual subtype.‡This number of human samples includes the single case who did not return a questionnaire and is notconsidered in later analyses.

Table 16 demonstrates that C. jejuni was the predominant species identified in human faecal

samples and in all other samples compared with C. coli, with the exception of pork offal

which had equal numbers of both species. Because of its structure, the composite sampling

regime used for animal faecal samples and water generated a high percentage of isolates.

The percentage of C. jejuni in sheep faeces was the lowest for animals but sheep faeces

yielded the highest percentage of C. coli of all of the matrices tested. This was not reflected

in the percentage of positive sheep offal samples, although sheep offal did produce the

highest prevalence of C. jejuni of all meat products tested. Pork and beef offal had

significantly lower prevalences for C. jejuni in comparison to sheep offal and chicken


carcasses. However pork offal had the highest prevalence of C. coli compared to the other

meat products. C. jejuni prevalence from beef faeces was much higher than sheep faeces,

but the beef offal prevalence was much lower than sheep offal.

The diversity of subtypes isolated from each matrix is presented in Table 16. Overall there

were 37 different PFGE subtypes of C. coli isolated from the Ashburton District. The

diversity of C. jejuni subtypes represents the combined serotype and PFGE subtypes (HS:P)

of which there were 250 different combinations.

Isolates from sheep and beef faecal matrices showed the highest number of different C. coli

subtypes. (for example, there were 11 PFGE subtypes from 41 isolates). This would be

expected from composite samples. In general the levels of diversity of both C. coli and

C. jejuni subtypes were fairly consistent, 60% or greater, across the different matrices, with

only two exceptions: dairy faeces and sheep faeces. The diversity of C. jejuni subtypes in

dairy faeces was 38%, whereas the diversity for C. coli subtypes in the same matrix was

56%. The diversity of C. coli subtypes in sheep faeces was 27%, whereas it was 71% for

C. jejuni subtypes in the same matrix.

Of the non-composite samples, beef offal had the highest overall diversity (80%) of

C. jejuni subtypes and chicken isolates the lowest diversity (46%). The human matrix had a

high percentage of isolates (77%) grouping into different C. jejuni subtypes.

4.3.1 Seasonality of Campylobacter Prevalence

To investigate the seasonality of Campylobacter isolation, the distribution of C. jejuni andC. coli was analysed by yearly quarter. Results were expressed as prevalences and arepresented in the following to Figure 13.


Figure 9 Seasonality of C. jejuni Isolation from Meat Products

The prevalence of C. jejuni was highest in sheep offal and chicken carcasses (Figure 9). In

both, the prevalences were highest in the summer and spring quarters, with lower prevalence

over the autumn and winter periods. Overall, prevalence of C. jejuni isolated from beef and

pork offal was low with no positives from pork offal in the last quarter of the year.

C. jejuni Meat

0%

10%

20%

30%

40%

50%

60%

Beef Offal Sheep Offal Chicken Carcass Pork Offal

Matrix

Posi

tive

Rate

Jan-Mar

Apr-Jun

Jul-Sep

Oct-Dec


Figure 10 Seasonality of C. jejuni Isolated from Matrices with Composite Sampling Regimes

Overall, prevalence of C. jejuni isolated from matrices where sampling was based on

composite samples was higher and more evenly distributed throughout the year in

comparison to prevalences from single samples (Figure 9). There was also a slight tendency

towards higher prevalence during the summer quarter in the samples derived from animals.

Water samples differed and showed slightly lower prevalences in the summer period.

Composite C. jejuni

0%

20%

40%

60%

80%

100%

120%

Water Duck Dairy Beef Sheep

Matrix

Posi

tive

Rat

eJan-Mar

Apr-Jun

Jul-SepOct-Dec


Figure 11 Seasonality of C. coli Isolated from Matrices with Composite Sampling Regimes

There was a high prevalence of C. coli from sheep faeces throughout the year, with the

highest prevalence occurring during the second and final quarters of the year (Figure 11).

C. coli was also isolated from beef and dairy faeces throughout the sampling period but at

much lower prevalences. C. coli was rarely isolated from water and duck faeces with the

highest prevalences for these two matrices occurring in the final quarter of the year.

Composite C. coli

0%

10%

20%

30%

40%

50%

60%

70%

Water Duck Dairy Beef Sheep

Matrix

Posi

tive

Rat

e

Jan-Mar

Apr-Jun

Jul-SepOct-Dec


Figure 12 Seasonality of C. coli Isolated from Meat Products

Prevalence of C. coli isolated from meat products was low at less than 11% for all matrices

(Figure 12). Pork offal, followed by sheep offal, demonstrated the most frequent isolations

of C. coli with highest prevalences during the winter months of the third quarter. Neither of

these matrices demonstrated isolation of C. coli during the first quarter of the year. C. coli

was isolated from beef offal only during the first quarter summer period. C. coli was

isolated from chicken carcasses only in the second quarter of the year.

C. coli meat

0%

2%

4%

6%

8%

10%

12%

Beef Of f al Sheep Of f al Chicken Carcass Pork Of f al

Matrix

Jan-Mar

Apr-Jun

Jul-Sep

Oct-Dec


Figure 13 Seasonality of C. jejuni and C. coli Isolated from Human Faeces

Figure 13 represents the seasonal isolation of C. coli and C. jejuni2 from human faecal

samples received by ESR during the period of January 2001 to January 2002, inclusive.

These data do not represent all cases of campylobacteriosis notified in Ashburton District

during the same period. Reasons for this discrepancy include: 27% of faecal samples from

cases of campylobacteriosis which were not sent to ESR for laboratory analysis and 6% of

human faecal samples in which Campylobacter was not detected (Table 11). C. jejuni was

isolated from human faeces throughout the year, whereas C. coli was isolated only in the

last two quarters of the year. It should be noted that sampling of human faeces for the first

quarter of 2002 ended at the beginning of February and therefore prevalence of

Campylobacter is not representative of the entire quarter. The C. coli prevalence in human

faeces is consistent with its prevalence in sheep and pork offal in the last two quarters of

2001. The higher summer prevalence of C. jejuni human isolates are consistent with the

high prevalence in sheep offal during the first quarter of 2001 and chicken carcasses in the

2 Rates per 100,000 of Ashburton population as per census data for the year 2001.

0

10

20

30

40

50

60

70

80

90

coli jejuni

Date

Jan-Mar 01Apr-Jun 01Jul-Sep 01Oct-Dec 01Jan-Mar 02


last quarter. The human prevalence of C. jejuni was also consistent with the high prevalence

in duck faeces in the first quarter and the higher summer prevalence in sheep faeces.

The annual temperature variations of the Ashburton River as measured at Region A water

sampling site, are presented in Figure 14. The temperatures vary from a summer high of

18°C in March to a wintertime low of 3°C in July.

Figure 14 Seasonal Variation in Temperature of the Ashburton River

4.4 Distribution

4.4.1 General Matrix Distribution

The following maps show the distribution of C. jejuni and C. coli isolated from the

Ashburton District. These results are presented as prevalences, which were based on the

proportion of all samples collected during 2001 that were positive for either species. Figure

15 demonstrates the spatial distribution of C. jejuni prevalence over the farms and water

sampling sites. In all instances these were composite samples. The geographic location of

human cases is also presented.

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

18.0

20.0

19-Feb

-01

5-Mar-

01

19-M

ar.-01

2-Apr-

01

17-A

pr-01

30-A

pr-01

14-M

ay-01

5-Jun

-01

18-Ju

n-01

2-Jul-

01

16-Ju

l-01

30-Ju

l-01

13-A

ug-01

27-A

ug-01

10-S

ep-01

24-S

ep-01

8-Oct-

01

23-O

ct-01

5-Nov

-01

19-N

ov-01

3-Dec

-01

17-D

ec-01

Date

Tem

pera

ture

(deg

rees

cel

sius

)


The spatial distribution of C. jejuni prevalence over the duck ponds and meat retailer sites is

shown in Figure 16. The locations of human C. jejuni cases in the Ashburton Township are

also shown for comparison. This map is a close up of the Ashburton township and shows all

the butcheries and duck ponds that were sampled. The most evident pattern in Figure 16 is

that the meat retailers in Ashburton East have a higher prevalence of C. jejuni compared

with those in Tinwald. The location of the township within the Ashburton region is

presented in Figure 8. The grey shading represents the area within the town and which will

encompass some semi-rural, industrial and/or reserve land.

Pote

ntia

l Tra

nsm

issi

on R

oute

s of C

ampy

loba

cter

72Au

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200

2Fr

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s

Figu

re 1

5Pr

eval

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of C

. jej

uni o

n Fa

rms a

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ater

Site

s

Pote

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73Au

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6Pr

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. jej

uni i

n D

uck

Pond

s, M

eat R

etai

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and

Hum

an C

ases

in A

shbu

rton

Tow

nshi

p


The spatial distribution of farms and water sampling sites, along with the prevalence of

C. coli at each site, is shown in Figure 17. The locations of human C. coli cases are also

shown in this region. There were no obvious regional patterns for farms or for the type of

farm.

The spatial distribution of duck pond and meat retailer sampling sites, along with the

prevalence of C. coli at each site, is shown in Figure 18. There was a very low prevalence

of C. coli from meat samples and duck ponds. There were no obvious spatial patterns

evident between meat retail outlets, nor between duck ponds.

Samples found to contain a mixed population of C. jejuni and C. coli are presented in

Table 17. Only one of the human cases was found to have a mixed infection of C. coli and

C. jejuni. The sheep faecal matrix contained the highest number of samples from which

both Campylobacter species were isolated. However data on mixed populations for animal

and bird faeces should be viewed in the context that all samples were composite faecal

samples derived from five different animals/birds and therefore are more likely to be

mixed. Meat products had a low proportion of mixed populations, ranging from 0 to 2.5%.

Table 17 Samples Containing a Mixed Population of C. jejuni and C. coli

Matrix Total Number ofPositive samples

Samples containingC. jejuni and C. coli

% of mixed populationsfor all samples

Human 61 1 1.6

Water* 162 12 7.4Duck Faeces* 60 1 1.7Dairy Faeces* 89 9 10.1Beef Faeces* 73 14 19.2Sheep Faeces* 66 27 40.9

Beef Offal 16 0 0Sheep Offal 65 4 6.2Chicken Carcass 56 2 3.6Pork Offal 15 3 20

Total 664 73 11.0*Composite samples

Pote

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75Au

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2Fr

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Figu

re 1

7Pr

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ence

of C

. col

i on

Farm

s and

Wat

er S

ites

Pote

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76Au

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Figu

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8Pr

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. col

i in

Duc

k Po

nds a

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and

Hum

an C

ases

in A

shbu

rton

Tow

nshi

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4.4.2 Distribution of Campylobacter spp. in Matrices within Different Regions

The rural region can be divided into three main areas (A, B and C) as shown in Figure 5.

4.5 Prevalence of Campylobacter from Regions A, B and C

The prevalence of C. jejuni and C. coli for each of the environmental matrices in each

region is shown in Table 18. Region B is the area adjacent to both banks of the South

Branch of the Ashburton River and Region C is the area surrounding the two tributaries of

the South Branch: Bowyers and Taylors Streams. Region A is below the confluence of the

rivers flowing through Regions B and C (Figure 5).

Table 18 Regional Distribution of Campylobacter Isolation

Matrix Area % Isolation ofC. jejuni

% Isolation ofC. coli

Total samplenumbers

Water* Region A 59 6 193Region B 16 0 50Region C 60 0 50

Duck* Ashburton Domain 74 2 47Tinwald Domain 51 0 45

Dairy Faeces* Region A 91 3 33Region B 97 10 31Region C 100 15 27

Beef Faeces* Region A 90 14 29Region B 72 3 29Region C 83 24 29

Sheep Faeces* Region A 48 24 29Region B 43 63 30Region C 75 54 28

*Composite samples

Samples of water from the river draining Region B demonstrate a lower prevalence of

C. jejuni compared with the other two water sampling sites (χ2, p<0.0001). C. coli was not

isolated from Region B and C water sampling sites, which are upstream of the Region A

water site. Differences between water prevalence between regions are not significant for

C. coli (Fisher’s exact test, p=0.46). The isolation of C. coli from Region A water sampling

site may be the result of a four fold higher sampling rate between this site and the other two

water sites.


All three regions had similar dairy cattle (Fisher’s exact test, p=1.0) and beef cattle (Fisher’s

exact test, p=0.67) prevalences of C. jejuni (Table 18). However, the C. jejuni prevalence of

sheep faeces was significantly different across the different regions (χ2, p=0.05), with higher

prevalence in Region C. The prevalence of Campylobacter by farm are presented in Figure

15 for C. jejuni and Figure 17 for C. coli. The prevalence of C. coli from sheep faeces was

significantly different across the different regions (χ2, p=0.008), with C. coli prevalence

lower for Region A compared with Regions B and C. There were no significant differences

in the prevalence of C. coli in dairy cattle (Fisher’s exact test, p=0.22). There were

significant differences in prevalence of C. coli in beef cattle faeces (Fisher’s exact test,

p=0.02), with Region C much higher than Region A, which was much higher than Region

B.

Duck faeces sampled from the Ashburton Domain had a higher prevalence of C. jejuni than

duck faeces from the Tinwald Domain. This relationship in duck faeces prevalences

(Ashburton vs. Tinwald domains) was significant for C. jejuni (χ2, p=0.02), but not

significant for C. coli (Fisher’s exact test, p=1.00).


4.6 Serotype Distribution of C. jejuni Isolates

The distribution of C. jejuni serotypes in the Ashburton district is presented in Figure 19

with further detail in Figure 20.

Figure 19 Distribution of C. jejuni Serotypes in the Environmental Matrices of the AshburtonDistrict

0%

5%

10%

15%

20%

25%

30%

35%

40%

unty

pabl

e 57

55

53

52

45

44

42

41

37

35

33

31

27

25

24

23,3

6 22

21

19

18

15

12

11

10

8,17

6 5

4 co

mpl

ex 3 2

1,44

Human Water Duck Dairy Beef Sheep Beef Offal Sheep Offal Chicken Carcass Pork Offal


Figure 20 Detail of the Distribution of Selected C. jejuni Serotypes in the EnvironmentalMatrices of the Ashburton District

Thirty-two serotypes of C. jejuni were identified in the Ashburton District. Water isolates

were the most diverse in regard to serotype; 27 serotypes were identified. Isolates from

human faecal samples were the next most diverse; 17 serotypes were identified.

Figure 20 is a detailed section of Figure 19 showing the serotypes 1,44; 2; 3; 4 complex and

5, which contain isolates from most of the matrices. A high number of the human serotypes

were represented by HS 4 complex (14%) and HS 2 (30%). The HS 4 complex was also

represented in the farm animal faecal isolates with prevalences ranging from 17-20 %. This

serotype was identified in the offal isolates in a range from 14-20%, but it did not feature

largely in either the duck or water isolates (both 5%). HS 2 had a high percentage of isolates

from pork and sheep offal, chicken and beef faeces. However, only 7% of beef offal isolates

were HS 2 compared with 24% of isolates from beef faeces. Isolates identified in duck

faeces and water were infrequently HS 2. Serotype 1,44 was represented in all matrices

5

4 c om plex

3

2

1,44

Hum

an

Wat

er

Duc

k

Dai

ry

Bee

f

She

ep

Bee

f Offa

l

Shee

p O

ffal

Chi

cken

Car

cass

Pork

Offa

l

0%

5%

10%

15%

20%

25%

30%

S erot ype

M atr ix


except pork offal. Most isolates of this serotype were from beef and sheep offal. Water and

human matrices yielded a similar prevalence of 1,44 at 10 and 9 % respectively. There was

only a low prevalence of HS 1, 44 in duck and farm animal faeces.

Isolates from other matrices serotyped in a smaller number of groups with HS 23,36 having

a high prevalence in dairy faeces (39 %) and lower prevalences in other farm animal faeces.

Sheep and pork offal also contained HS 23,36, but there was a low prevalence of human and

water isolates of this serotype. HS 21 had the highest prevalence in chicken carcasses but no

human isolates of this serotype were identified.

Samples collected from water, ducks and chicken carcasses had the largest numbers of

untypable serotypes. The overall percentage of untypable Campylobacter serotypes reported

in this study was 14.4 %.

4.7 Distribution of Campylobacter PFGE subtypes

4.7.1 Distribution of C. coli PFGE subtypes

The distribution of C. coli PFGE subtypes between the matrices is represented in Figure 21.

During the course of this study 37 different C. coli PFGE subtypes were identified. There

were only two C. coli isolates that were unable to be typed by PFGE and they were isolated

from water and beef faeces. The highest percentage of isolates of C. coli was found in sheep

faeces (47.1%), which also had the most PFGE subtypes. The PFGE subtype of two chicken

isolates was not isolated from any other matrix. Isolates of PFGE subtype 1 were isolated

from many farm animal faeces, sheep offal and beef offal isolates. This subtype however,

was not present in human faeces. C. coli isolates of PFGE subtype 3 were obtained from

human cases and sheep faeces. Two C. coli PFGE subtypes (11a and 17) were isolated only

from water and human cases. Isolates of PFGE subtype 11, clonally related to subtype 11a

were isolated from beef faeces, sheep faeces and sheep liver. The pork offal C. coli isolates

do not share any PFGE subtypes with the other matrices. Isolates of PFGE subtype 10 were

isolated from human, duck, dairy and sheep faeces. PFGE subtype 29, clonally related to

PFGE subtype 10, was isolated from water.


Figure 21 Distribution of C. coli PFGE subtypes in the Environmental Matrices of theAshburton District

0

2

4

6

8

10

12

Freq

uenc

y

not cu

tting

3433323130292827262524232221201918171615141312 11

a 1110987 6b

6 5a

54321

C. coli PFGE types

01 Human 02 Water 03 Duck 04 Dairy 05 Beef 06 Sheep 07 Beef 08 Sheep 09 Chick 10 Pork

C. coli PFGE subtypes


4.7.2 Distribution of C. jejuni PFGE subtypes

C. jejuni subtypes identified at less than 2% prevalence in a matrix were considered to be

rare subtypes for that matrix. For clarity these rare subtypes have not been included in

Figure 22, which represents combined serotype:PFGE (HS:P) C. jejuni subtypes. Subtypes

in Figure 22 were identified at a prevalence greater than 2% in each matrix and are

compared with the prevalence of the same subtypes in other matrices.

All combined serotype and PFGE subtypes (HS:P) isolated from each matrix are presented

in Figure 28 (Appendix 7). Those subtypes isolated at a prevalence of 2% or less have been

combined for each matrix. In some matrices these low prevalence subtypes form a high

percentage of total isolates, suggesting a high diversity of subtypes (e.g. water and sheep

faeces).

Prevalence of C. coli isolated from meat products was low at less than 11% for all matrices

(Figure 21). Pork offal, followed by sheep offal, demonstrated the most frequent isolations

of C. coli with highest prevalences during the winter months of the third quarter. Neither of

these matrices demonstrated isolation of C. coli during the first quarter of the year. C. coli

was isolated from beef offal only during the first quarter summer period. C. coli was

isolated from chicken carcasses only in the second quarter of the year.


Figure 22 Comparison of C. jejuni Subtypes (combined serotype and PFGE) betweenMatrices

Figure 22a: Human faeces. Water isolates shared five of the same subtypes as the human matrix,representing the largest overlap of subtypes between two matrices. Subtype HS23,36:19b is acommon subtype found in many matrices with its highest prevalence in dairy cow faeces.

0%5%

10%15%20%25%30%

Isol

atio

n fr

eque

ncy

Hum

an

Wat

er

Duc

k

Dai

ry

Beef

Shee

p

Beef

Offa

l

Shee

p O

ffal

Chi

cken

Car

cass

Pork

Offa

l HS1,44: P33

HS2: P16 HS2: P1c

HS2: P28 HS23,36: P19b

HS6: PNC HSU: P25

HS11: P35 HS2: P18a

Matrix

Sero

type

: PFG

E

a) Comparison of Subtypes in Humans with Other Matrices

0%

2%

4%

6%

8%

10%

12%

Isol

atio

n Fr

eque

ncy

Wat

er

Hum

an

Duc

k

Dai

ry

Beef

Shee

p

Beef

Offa

l

Shee

p O

ffal

Chi

cken

Car

cass

Pork

Offa

l

HS1

,44:

P16

H

S15:

P60

b H

S6: P

NC

H

S5: P

21

HS5

: P25

b H

SUT:

P25

H

SUT:

P25

b H

S8,1

7: P

236

HSU

T: P

221

Matrix

Serotype: PFGE

b) Comparison of Subtypes in Water with Other Matrices


Figure 22c Duck faeces: Apart from isolates from water, the subtypes isolated from ducks are notrepresented in the other matrices.

0%

2%

4%

6%

8%

10%

12%

Isol

atio

n Fr

eque

ncy

Duc

k

Hum

an

Wat

er

Dai

ry

Beef

Shee

p

Beef

Offa

l

Shee

p O

ffal

Chi

cken

Car

cass

Pork

Offa

l

HS19: P208HS4c: P221

HS5: P245HS52: P221

HSUT: P15HSUT: P60d

HS37: P229HS37: P248

HS8,17: P236

Matrix

Sero

type

:PF

GE

c) Comparison of Subtypes in Ducks with Other Matrices

Figure 22d and e: Dairy Faeces and Beef Faeces: Subtypes are also represented in other ruminantanimal faeces and in derived meat products, as well as chicken carcasses and pork offal, althoughobserved diversity is lower. The subtypes, when identified in water and duck faeces, occur at alow prevalence.

0%

5%

10%

15%

20%

25%

30%

Isol

atio

n Fr

eque

ncy

Dai

ry

Hum

an

Wat

er

Duc

k

Beef

Shee

p

Beef

Offa

l

Shee

p O

ffal

Chi

cken

Car

cass

Pork

Offa

l

HS2

: P20

6H

S2: P

3H

S2: P

33H

S23,

36: P

19f

HS3

5: P

31H

S4c:

P52

H

S53:

P29

H

S11:

P35

H

S4c:

P34

H

S23,

36: P

19b

Matrix

Serotype: PFGE

d) Comparison of Subtypes in Dairy with Other Matrices


0%

5%

10%

15%

20%

25%

30%

Isol

atio

n fr

eque

ncy

Beef

Hum

an

Wat

er

Duc

k

Dai

ry

Shee

p

Beef

Offa

l

Shee

p O

ffal

Chi

cken

Car

cass

Pork

Offa

l

HS1

9: P

12

HS3

5: P

44H

S11:

P35

HS1

9: P

3d

HS2

: P3

HS3

5: P

31

HS4

c: P

34a

HS2

3,36

: P19

b H

S2: P

33

HS4

c: P

34

Matrix

Serotype: PFGE

e) Comparison of Subtypes in Beef with Other Matrices

0%

5%

10%

15%

20%

25%

30%

Isol

atio

n fr

eque

ncy

Shee

p

Hum

an

Wat

er

Duc

k

Dai

ry

Beef

Beef

Offa

l

Shee

p O

ffal

Chi

cken

Car

cass

Pork

Offa

l

HS1

0: P

18

HS4

c: P

34b

HS2

3,36

: P19

b

HS4

c: P

34

HS6

: PN

C

HS2

7: P

25

HS5

: P22

2b

Matrix

Serotype: PFGE

f) Comparison of Sheep Subtypes with Other Matrices

Figure 22f: Sheep Faeces. Overall, fewer subtypes were identified at greater than 2%prevalence from this matrix when compared with the other ruminant animal matrices (Figure28f, Appendix 7). The subtypes in Figure 22f were identified at a lower prevalence in theother ruminant animal and meat product matrices, except for the subtypes HS23,36:P19b andHS4complex:P34. The sheep faecal subtypes were identified at a low prevalence in thehuman and water matrices and rarely in duck faeces.


Figure 22g: Beef Offal. All subtypes identified are presented, because only a small number ofsamples were positive for Campylobacter in this matrix. The subtypes identified are commonlyidentified in other ruminant animal faeces and derived meat products and some of the subtypesoccur at lower prevalences in human faeces. Beef offal subtypes were not frequently isolatedfrom water and duck faeces.

Figure 22h. Sheep offal. Compared with other matrices the highest diversity of subtypes at greaterthan 2% prevalence were identified in this matrix (Figure 28h, Appendix 7). The majority ofsubtypes identified in sheep offal were not identified in other matrices (Figure 22h). A cluster ofsubtypes, however, was represented in other ruminant animal, human and meat product matrices.The sheep offal subtypes were not well represented in water matrices, and if identified, wereisolated at low prevalences. These subtypes were rarely isolated from duck faeces.

0%

2%

4%

6%

8%

10%

12%

14%

Isol

atio

n Fr

eque

ncy

Beef

Offa

l

Hum

an

Wat

er

Duc

k

Dai

ry

Beef

Shee

p

Shee

p O

ffal

Chi

cken

Car

cass

Pork

Offa

l

HS1

,44:

P33

HS1

,44:

P3a

HS2

: P3

HS2

3,36

: P22

HS3

: P24

1a

HS4

c: P

34

HS5

: P22

2b

HSU

T: P

209

HSU

T: P

34b

HS1

9: P

3g

HS3

5: P

10

HS4

c: P

34a

Matrix

Serotype: PFGE

g) Comparison of Beef Offal Subtypes with Other Matrices

0%

5%

10%

15%

20%

25%

30%

Isol

atio

n Fr

eque

ncy

Shee

p O

ffal

Hum

an

Wat

er

Duc

k

Dai

ry

Beef

Shee

p

Beef

Offa

l

Chi

cken

Car

cass

Pork

Offa

l

HS2

: P16

HS2

: P52

HS2

3,36

:P19

HS2

3,36

: P22

H

S4c:

P20

4 H

S4c:

P34

b H

S5: P

222

HS5

: P26

H

S8,1

7: P

33

HSU

T: P

207

HSU

T: P

54a

HS1

9: P

3g

HS2

3,36

: P19

b H

S4c:

P34

H

S1,4

4: P

33

HS2

: P3

HS1

,44:

P3a

Matrix

Serotype; PFGE

h) Comparison of Sheep Offal Subtypes with Other Matrices


Figure 22 i.Chicken carcasses: This matrix demonstrated a high diversity of subtypes (Figure 28i,Appendix 7) and the majority of subtypes were not identified in the other matrices (Figure 22i).Only HS2:P3 was represented at high prevalences in the other matrices. This subtype wasidentified in all matrices except for water.

0%

2%

4%

6%

8%

10%

12%

14%

Isol

atio

n Fr

eque

ncy

Chi

cken

Car

cass

Hum

an

Wat

er

Duc

k

Dai

ry

Beef

Shee

p

Beef

Offa

l

Shee

p O

ffal

Pork

Offa

l

HS1

,44:

P24

6 H

S1,4

4: P

30

HS2

; P3i

H

S21:

P25

H

S21:

PN

C

HS3

1: P

29

HS4

c: P

1 H

S42:

P25

H

S8,1

7: P

244

HSU

T: P

29

HSU

T: P

4 H

SUT:

P22

3 H

S2: P

3 H

S21:

P60

a

MatrixSerotype: PFGE

i) Comparison of Chicken Subtypes with Other Matrices

Figure 22j. Pork Offal: All subtypes are represented because of the small number of samples positivefor Campylobacter. HS2:P3 was identified in all matrices except water. HS4complex:P34 wasidentified in beef and sheep faeces and beef and sheep offal at relatively high prevalences and in thewater matrix at a low prevalence.

0%

5%

10%

15%

20%

25%

Isol

atio

n Fr

eque

ncy

Pork

Offa

l

Hum

an

Wat

er

Duc

k

Dai

ry

Beef

Shee

p

Beef

Offa

l

Shee

p O

ffal

Chi

cken

Car

cass

HS2: P3

HS2: PNC

HS35: P10c

HS4c: P34

HS8,17: P3

HS23,36: P226

HS35: P44

Matrix

Serotype: PFGE

j) Comparison of Types in Pork Offal with Other Matrices


4.8 Temporal and spatial clustering of subtypes

Analysis was performed to determine whether clustering of subtypes was occurring either

temporally or spatially. If the same subtype was isolated from the meat products at the same

retailer over time this would suggest that the sampling plan was not obtaining independent

samples and that cross contamination was a complicating factor.

Table 31 (Appendix 8) shows the relationship between subtypes isolated over time from

each meat retailer. Twenty sampling events occurred where the same subtype was isolated

from meat products from one meat retailer at one time point. There were no spatial/temporal

events where the same subtype was isolated from both chicken carcasses and offal products.

Eleven of the 20 sampling events were exclusively between chicken carcasses samples

collected at the same time and place. The remaining nine sampling events were between the

same or different subtypes of offal products and the data is presented in Table 31

(Appendix 8). There were no systematic patterns of clustering of subtypes evident from this

data, beyond the isolation of the same subtype from one location and time point.

Only one subtype from the composite ruminant animal sample was isolated from each farm

at one time point. The analysis of cluster patterns of subtypes isolated from ruminant

animals on individual farms revealed that there were few occasions when the subtypes were

re-isolated in the next sampling event (data not shown). The exceptions were:

• The predominant dairy faecal subtype C. jejuni HS23,36:P19b was isolated regularly

from several farms which had been sampled on a monthly rotation, (the maximum

isolation of this subtype from one farm occurred from six out of 14 sampling events).

• C. jejuni subtype HS10:P18 was isolated on two consecutive fortnightly sampling events

from dairy faeces on one dairy farm and from two consecutive monthly sampling events

from sheep faeces on one sheep farm.

Identification of clustering of subtypes from water is complicated by the nature of this

matrix, as it may contain bacteria from many sources and any consistent patterns cannot be

directly attributed to individual spatial/temporal events.


For duck faeces there were two incidences of the same subtype being isolated from two

consecutive sampling events. This number of events producing isolates of the same subtype

was lower than expected because each sampling event involved a minimum of 3 composite

samples from the same duck pond. Therefore, there was a higher likelihood of less diversity

of subtypes in comparison to the ruminant faecal samples. In conjunction with what was

observed in the spatial and temporal patterns of subtypes isolated from meat products, it was

concluded that isolations of subtypes were independent events.

4.9 Czekanowski Index

In Table 19 through Table 21 the Czekanowski index of similarity analyses are presented.

As the serotype results include a category of “untypable”; and the PFGE subtype results

include a category of “non-cutting” special care needs to be taken in calculating similarity

indices. Therefore two numbers were calculated for each combination of matrices. The first

number assumed all isolates categorised as “untypable” or “non-cutting” were the same. The

second number assumed all isolates categorised as “untypable” or “non-cutting” were

completely different. The true situation should lie between these two assumptions and

therefore the underlying similarity index value should lie between these two numbers.

Table 19 shows the values generated by the Czekanowski index for C. jejuni serotype data.

The highest similarity is between isolates from human cases and beef cattle faeces. This is

closely followed by associations between most of the following, dairy cattle faeces, beef

cattle faeces, beef offal, sheep offal and human faeces. Another association of interest was

also observed between duck faeces and water. However, the large difference between the

first and second similarity indices (66/0.43) demonstrates the presence of many untypable

isolates and leads to the conclusion that the underlying similarity index is somewhat lower

than those relating to the ruminant and human matrices.

Table 20 as for Table 19, shows a group of matrices, which demonstrated the highest

similarity between ruminant faeces and ruminant offal. These include dairy cow, beef and

sheep faeces plus beef and sheep offal. The values are lower than those shown in Table 19

due to the high number of infrequently isolated subtypes included. For human cases the

most similar matrices are the beef and sheep faeces and the sheep offal, followed by lower


similarities with dairy cow faeces, water and chicken carcasses. An association of interest

was also observed between duck faeces and water.

Table 21 shows the similarities when both PFGE and serotyping data are considered. The

patterns are very similar to those described for Table 20.

Pote

ntia

l Tra

nsm

issi

on R

oute

s of C

ampy

loba

cter

93Au

gust

200

2Fr

om E

nvir

onm

ent T

o H

uman

s

Tab

le 1

9Si

mila

rity

Mat

rix

of C

. jej

uni P

enne

r Se

roty

pes

Hum

anfa

eces

Wat

erD

uck

faec

esD

airy

cow

faec

es

Bee

ffa

eces

Shee

pfa

eces

Bee

f off

alSh

eep

offa

lC

hick

enca

rcas

sPo

rk o

ffal

Hum

an

faec

es

1.00

/1.0

0

Wat

er0.

46/0

.39

1.00

/1.0

0

Duc

k

faec

es

0.30

/0.2

30.

66/0

.43

1.00

/1.0

0

Dai

ry

faec

es

0.55

/0.4

90.

34/0

.28

0.18

/0.1

31.

00/1

.00

Bee

f

faec

es

0.69

/0.6

20.

41/0

.33

0.28

/0.1

90.

61/0

.55

1.00

/1.0

0

Shee

p

faec

es

0.51

/0.4

90.

49/0

.46

0.42

/0.3

90.

48/0

.46

0.52

/0.5

01.

00/1

.00

Bee

f

offa

l

0.48

/0.4

10.

49/0

.36

0.34

/0.2

00.

47/0

.41

0.61

/0.5

30.

45/0

.43

1.00

/1.0

0

Shee

p

offa

l

0.63

/0.5

60.

52/0

.43

0.36

/0.2

60.

58/0

.52

0.66

/0.5

70.

54/0

.52

0.68

/0.5

91.

00/1

.00

Chi

cken

carc

ass

0.46

/0.3

90.

57/0

.33

0.50

/0.2

80.

34/0

.28

0.39

/0.3

10.

33/0

.31

0.38

/0.2

50.

48/0

.38

1.00

/1.0

0

Pork

offa

l

0.40

/0.4

00.

23/0

.23

0.20

/0.2

00.

51/0

.51

0.53

/0.5

30.

30/0

.30

0.38

/0.3

80.

43/0

.43

0.29

/0.2

91.

00/1

.00

Whi

te sq

uare

s <0.

21, l

ight

gre

y 0.

21-0

.4, m

id g

rey

0.41

-0.6

, bla

ck >

0.6.

Pote

ntia

l Tra

nsm

issi

on R

oute

s of C

ampy

loba

cter

94Au

gust

200

2Fr

om E

nvir

onm

ent T

o H

uman

s

Tab

le 2

0Si

mila

rity

Mat

rix

of C

. jej

uni P

FGE

Sub

type

s

Hum

anfa

eces

Wat

erD

uck

faec

esD

airy

cow

faec

es

Bee

ffa

eces

Shee

pfa

eces

Bee

f off

alSh

eep

offa

lC

hick

enca

rcas

sPo

rk o

ffal

Hum

an

faec

es

1.00

/1.0

0

Wat

er0.

21/0

.18

1.00

/1.0

0

Duc

k

faec

es

0.10

/0.1

00.

29/0

.29

1.00

/1.0

0

Dai

ry

faec

es

0.21

/0.2

10.

09/0

.09

0.03

/0.0

31.

00/1

.00

Bee

f

faec

es

0.28

/0.2

70.

18/0

.17

0.07

/0.0

70.

50/0

.50

1.00

/1.0

0

Shee

p

faec

es

0.29

/0.2

60.

23/0

.21

0.13

/0.1

30.

23/0

.23

0.31

/0.3

01.

00/1

.00

Bee

f

offa

l

0.14

/0.1

40.

02/0

.02

0.02

/0.0

20.

18/0

.18

0.27

/0.2

70.

21/0

.21

1.00

/1.0

0

Shee

p

offa

l

0.28

/0.2

80.

12/0

.12

0.02

/0.0

20.

33/3

30.

41/0

.41

0.32

/0.3

20.

41/0

.41

1.00

/1.0

0

Chi

cken

carc

ass

0.21

/0.1

80.

17/0

.14

0.12

/0.1

20.

15/0

.15

0.17

/0.1

60.

22/0

.16

0.08

/0.0

80.

15/0

.15

1.00

/1.0

0

Pork

offa

l

0.04

/0.0

40.

01/0

.01

0.02

/0.0

20.

16/0

.16

0.20

/0.2

00.

09/0

.09

0.13

/0.1

30.

16/0

.16

0.14

/0.1

41.

00/1

.00

Whi

te sq

uare

s <0.

11, l

ight

gre

y 0.

11-0

.2, m

id g

rey

0.21

-0.3

, dar

kest

0.3

1-0.

4, b

lack

>0.

4.

Pote

ntia

l Tra

nsm

issi

on R

oute

s of C

ampy

loba

cter

95Au

gust

200

2Fr

om E

nvir

onm

ent T

o H

uman

s

Tab

le 2

1Si

mila

rity

Mat

rix

of C

. jej

uni S

erot

ype

and

PFG

E S

ubty

pes

Hum

anfa

eces

Wat

erD

uck

faec

esD

airy

cow

faec

es

Bee

ffa

eces

Shee

pfa

eces

Bee

f off

alSh

eep

offa

lC

hick

enca

rcas

sPo

rk o

ffal

Hum

an

faec

es

1.00

/1.0

0

Wat

er0.

14/0

.06

1.00

/1.0

0

Duc

k

faec

es

0.07

/0.0

50.

16/0

.10

1.00

/1.0

0

Dai

ry

faec

es

0.16

/0.1

50.

06/0

.06

0.02

/0.0

21.

00/1

.00

Bee

f

faec

es

0.19

/0.1

60.

11/0

.10

0.03

/0.0

30.

44/0

.41

1.00

/1.0

0

Shee

p

faec

es

0.20

/0.1

60.

12/0

.09

0.07

/0.0

60.

21/0

.21

0.27

/0.2

61.

00/1

.00

Bee

f

offa

l

0.11

/0.1

10.

02/0

.02

0.02

/0.0

20.

12/0

.12

0.17

/0.1

70.

15/0

.15

1.00

/1.0

0

Shee

p

offa

l

0.20

/0.2

00.

08/0

.08

0.02

/0.0

20.

22/0

.20

0.26

/0.2

40.

23/0

.23

0.35

/0.3

41.

00/1

.00

Chi

cken

carc

ass

0.09

/0.0

70.

03/0

.03

0.07

/0.0

50.

08/0

.06

0.10

/0.0

70.

06/0

.06

0.07

/0.0

70.

10/.0

101.

00/1

.00

Pork

offa

l

0.02

/0.0

20.

01/0

.01

0.02

/0.0

20.

14/0

.14

0.17

/0.1

70.

08/0

.08

0.13

/0.1

30.

11/0

.11

0.11

/0.1

11.

00/1

.00

Whi

te sq

uare

s <0.

11, l

ight

gre

y 0.

11-0

.2, m

id g

rey

0.21

-0.3

, dar

kest

>0.

3


4.10 Association between C. jejuni “Subtypes” from Human Cases and RiskFactors identified from Questionnaire

Exposures to potential risk factor information were collected from cases after notification.

Table 22 presents the prevalence of the known risk factors for Campylobacter for the

human cases with C. jejuni and tests for any association between these risk factors and the

organism typing results for C. jejuni for those human cases. The results are considered

both at the serotype and at the serotype:PFGE subtype levels. These results identify

whether there is any evidence that the human cases with exposure to any of the potential

risk factors (either on their own or in conjunction with other risk factors) are associated

with different subtypes than the cases without exposure to these risk factors. These results

must be considered with caution, as the human cases where there were both risk factor

information and Campylobacter typing results were limited in number. The 56 human

cases with C. jejuni were spread across 44 combined serotype:PFGE subtypes, therefore

the majority of “subtypes” in this subset of the data are unique to one individual. It is also

important to note that the table provides statistics that are the results of multiple univariate

tests or comparisons, therefore the p-values should only be considered relative to each

other. Note that the total number responded varies from risk factor to risk factor, this is due

to the fact that not all questions were answered by all cases, any non-responses or don’t

know responses were excluded from the analysis. Due to the even smaller number of

C. coli human cases (six cases), the risk factor analysis was not carried out for C. coli.


Table 22 Association between C. jejuni “Subtypes” from Human Cases and Risk Factors†

p-values‡Risk Factor Number ofcases

Totalnumber

responded

Percentexposure Serotype3 Serotype x

PFGE3

Occupational exposure to animals 17 56 30% 0.4559 0.3587Occupational exposure - Dairy farmer/worker 6 56 11% 0.1566 0.2193

Animal Contact (last 10 days)Dairy cattle 15 56 27% 0.0275** 0.1137Non-dairy cattle 10 55 18% 0.8648 0.6862Calves 16 54 30% 0.3235 0.3108

Any Bovine contact - dairy, non-dairy, or calves 21 56 38% 0.0451** 0.0209**

Dogs 38 55 69% 0.1065 0.0764*Cats 38 55 69% 0.4310 0.5970Sheep 25 55 45% 0.7752 0.6225Pigs 4 55 7% 0.8188 0.4614Chickens 12 54 22% 0.0020** 0.0495**Ducks 3 55 5% 0.4151 0.8042Wild birds 2 55 4% 0.6330 0.9219Other animal 11 54 20% 0.6430 0.5099

Food/drink consumption (last 10 days)Beef 43 49 88% 0.7373 0.4676

at home 38 54 70% 0.8520 0.0871*At other home 33 48 69% 0.7858 0.1981Other 17 49 35% 0.6764 0.2967

Chicken 47 54 87% 0.9284 0.7838at home 45 53 85% 0.8596 0.7422At other home 34 48 71% 0.0656* 0.3599Other 15 50 30% 0.5306 0.5292

Duck 2 44 5% 0.2548 0.7822at home 2 54 4% 0.3571 0.9189At other home 1 47 2% 0.4681 0.7021

Eggs 45 52 87% 0.3383 0.3870at home 44 53 83% 0.3966 0.3066At other home 34 46 74% 0.4276 0.9497Other 4 47 9% 0.7945 0.4005

Fish – at home 28 54 52% 0.3792 0.3236Lamb 22 48 46% 0.6652 0.3588

at home 20 54 37% 0.5586 0.4711At other home 17 48 35% 0.5736 0.5976

† Note that the total number responded varies from risk factor to risk factor, this is due to the fact that not allquestions were answered by all cases, any non-responses or don’t know responses were excluded from theanalysis

‡ p-values are the results of univariate Fishers exact tests

3 * statistically significant at 90% level of significance** statistically significant at 95% level of significance


p-values‡Risk Factor Number ofcases

Totalnumber

responded

Percentexposure Serotype3 Serotype x

PFGE3

Other 3 48 6% 0.9214 0.8076Pork 32 51 63% 0.4219 0.4879

at home 28 54 52% 0.6165 0.8995At other home 24 48 50% 0.9188 0.8742Other 7 49 14% 0.7647 0.3192

Unpasteurised milk 9 44 20% 0.1954 0.2294at home 8 53 15% 0.2130 0.4588At other home 5 47 11% 0.6612 0.1447Other 1 47 0.5319 0.8511

Water supplyRainwater/tank 1 56 2% 0.1071 0.6250Spring 1 56 2% 0.4643 0.6250Stream/river/lake 3 56 5% 0.8872 0.5485Town 32 56 57% 0.0546* 0.3166Well/bore 21 56 38% 0.0578* 0.6117

Water elsewhere last 10 days 19 53 36% 0.4062 0.2553Water untreated last 10 days 26 52 50% 0.0763* 0.2257Recreational contact - water (last 10 days) 10 55 18% 0.7719 0.6862

Pool 6 55 11% 0.7022 0.2193River/sea 3 55 5% 0.4317 0.8105

School 9 56 16% 0.4531 0.5303Person to person contact 8 56 14% 0.8654 0.6162Faeces contact 4 53 8% 0.8184 0.9277Household animal contact 34 53 64% 0.4132 0.5685Animal dung contact 22 55 40% 0.4549 0.5752

Overseas travel 2 56 4% 1.0000 0.7091NZ travel 10 53 19% 0.3126 0.5208Farm visit / live on farm 30 52 58% 0.1663 0.6975

The two potential risk factors that showed the strongest association at both the serotype

and serotype:PFGE subtype levels are animal contact with bovine animals (dairy and/or

non-dairy cattle and/or calves) and chickens. Table 32 to Table 40 demonstrate the

breakdown of “subtype” by these key risk factors (Appendix 9). However the relationship

between the subtypes with risk factors is not particularly clear-cut. That is, there is not a

clear association between exposure and groups of subtypes, mostly due to the small

number of cases with any one subtype. From the tables in Appendix 9 there are several

serotypes that appeared to have some association with exposure to these two key risk

factors. Note that only subtypes where there were two or more individuals with the same

subtype are discussed in the following text.


At the serotype level HS 23,36; 10 and 22 appeared to be associated with exposure to

bovine animals. Comparing these serotypes to those found in other relevant matrices it was

found that HS 23,36 was in both dairy and beef cattle faeces, whereas HS 10 was found in

dairy cattle faeces and HS 22 was found in beef cattle faeces. At the serotype:PFGE

subtype level only a few subtypes had large enough numbers to make any indicative

conclusions; i.e. HS2:P16, HS23,36:P19b and HS22:P28 appeared to be associated with

bovine contact. Of these subtypes only HS23,36:P19b was found in dairy and beef cattle

faeces. Note that some serotypes are not examined at the serotype:PFGE level when the

subtypes are unique to one individual. As an alternative measure of possible associations,

the Czekanowski Index comparing the subtypes of those with bovine contact with those

without bovine contact at the serotype:PFGE level, results in a similarity index of 0.08.

For contact with live chickens HS10, 18 and 22 appeared to be associated with the risk

factor. At the serotype:PFGE subtype level there is only one subtype, which has been

identified in sufficiently high numbers to make any indicative conclusions, i.e HS22:P28

which appeared to be associated with live chicken contact. The only other potentially

relevant matrix for live chickens was that of chicken carcasses, none of the above

serotypes or subtypes were found in chicken carcasses. As an alternative measure of

possible associations, the Czekanowski Index comparing the subtypes of those with

chicken contact with those without chicken contact at the serotype:PFGE level results in a

similarity index of 0.02.

There were other potential risk factors with weaker but still important associations, which

are briefly described below. Once again it is important to note that these results are

indicative only, as numbers were small, only subtypes identified in more than 2 human

cases have been reviewed.


Contact with dairy cattle was significant at the serotype level. Serotypes HS 10, and 23,36

appeared to be associated with dairy cattle contact. In comparing these serotypes to those

found in other relevant matrices, HS 23,36 and HS 10 were both found in dairy cattle

faeces.

Contact with dogs was significant at the serotype:PFGE level. Serotype:PFGE subtypes

HS11:P35, HS23,36:P19b, HS2:P16, HS2:P18a/18c (clonally related) and HS2:P1c

appeared to be associated with contact with dogs. In comparing these subtypes to those

found in other relevant matrices, HS23,36:P19b was found in dairy cattle, beef cattle and

sheep faeces, HS11:P35 was found in dairy and beef cattle faeces and HS2:P16 was found

in sheep offal.

Access to town water supply was significant at the serotype level. Serotypes 18, 19 and 6

appeared to be associated with town water supply. Also of interest is that 7/8 of the

serotype 4 complex (all but HS 4 complex:P54a) also appeared to be associated with town

water supply. In comparing these serotypes (18, 19, 6 and 4 complex) to those found in

other relevant matrices, all of these serotypes were found in the water samples.

Access to well/bore water supply was significant at the serotype level. Serotypes HS 10,

and 23,36 appeared to be associated with well/bore water supply. (N.B. Most human cases

not on town water supply were on well/bore water supply). In comparing these subtypes to

those found in other relevant matrices, only HS23,36 was found in water samples.

Consumption of untreated water in the last ten days was significant at the serotype level.

Serotypes HS 10, and 23,36 appeared to be associated with the consumption of untreated

water. (N.B. Most well/bore water supplies were classified as untreated water). In

comparing these subtypes to those found in other relevant matrices, only HS23,36 was

found in water samples.

Consumption of chicken at another house was significant at the serotype level. Serotypes

HS 1,44; 10, 11, 18, 19, and 6, appeared to be associated with chicken consumption at

another house. In comparing these subtypes to those found in other relevant matrices

serotypes HS 1,44 and 6 were found in chicken carcasses.


Consumption of beef at home was significant at the serotype:PFGE level. Serotype:PFGE

subtypes HS11:P35, HS1,44:P33, HS2:P1c, HS22:P28 and HS23,36:P19b appeared to be

associated with beef consumption. In comparing these subtypes to those found in other

relevant matrices HS1,44:P33 was found in beef offal, however HS11:P35 and

HS23,36:P19b were both found in dairy and beef cattle faeces.

4.10.1 Analysis of water supplies

A breakdown of the types of water supply as recorded by the respondents to the

questionnaire is provided below. A definition of town water supply is presented in the

glossary. However, it is important to note that a town water supply is not necessarily

treated. The addresses of the human cases were also matched with their water supply zones

and showed some misclassifications by the respondents as to the source of their water

supply.

Twenty-four C. jejuni cases responded that they were not on town water supply. Two cases

were on stream/river/lake, one on rainwater/tank, and one on spring water supplies, all of

which were categorised as untreated water supplies. The remaining 20 cases were on

well/bore water supplies. However, of these 24 cases, four were identified from their

addresses to have been on town supply (one on stream/river/lake, and three on well/bore

water), and one was unable to be matched to a water supply. To make up for these

misclassified cases, four cases that responded that they were on town water supply were

identified as having private water supplies. Of the 20 cases that responded that they were

on well/bore water, 17 specified that their water supply was untreated, two specified that

their water was treated and one did not answer the question. The two cases that specified

that their water was treated were matched to private water supplies.

Of the 32 C. jejuni cases that responded that they were on town water supply, 23 reside in

Ashburton itself and their addresses matched to the Ashburton water supply. There were,

also, two cases that responded that they were on stream/river/lake or well/bore water

supplies that were matched to the Ashburton water supply. The Ashburton water supply


had three groundwater sources that were not disinfected and one source from the

Ashburton River, which was disinfected by chlorine.

The nine remaining C. jejuni cases that responded that they were on town supply were

located outside of the Ashburton Township. Of these nine cases, four were identified as

having private water supplies, and two cases that had responded that they were not on town

water supply were matched to Highbank (non-secure groundwater, no disinfection) and the

Methven/Springfield rural water supply (surface water source, no disinfection)). The

remaining cases had the following water supplies: three were matched to Methven

township (infiltration gallery from river, UV treated), one was matched to Fairton (non-

secure groundwater, no disinfection) and one was matched to Winchmore (non-secure

groundwater, no disinfection).

To conclude, a previous study has shown the presence of C. jejuni in reticulated water

from the Ashburton supply (and others) albeit at low levels (Savill et al, 2001a). It is

therefore possible that some of the C. jejuni cases noted in the previous paragraphs arise

from the drinking water consumed. Table 22 shows significant associations between the

subtypes of campylobacteriosis and exposure to some types of drinking water source. It

cannot, however, be concluded from these results that drinking water is a significant risk

factor for C. jejuni infection as there are other confounding factors. In particular it can be

noted that of the human cases that consumed untreated water in the last ten days that 92%

(23/25) also either lived on or visited a farm. This is in comparison with the cases that did

not consume untreated water where only 25% (6/24) either lived or visited a farm. Please

note that only 49 cases responded to both questions on farm visit and consumption of

untreated water. A similar pattern can be seen with either town or well/bore water supply

as the majority of farms are more likely to be on well/bore supply as opposed to town

supply. Also note that the majority of cases that identified themselves as being on

well/bore water supply also identified themselves as having consumed untreated water.

4.11 Potential Linkages Identified for Campylobacter

Potential linkages between indistinguishable and clonally related subtypes isolated from

environmental matrices and human cases are presented in Table 23 for C. coli subtypes and


Table 24 for C. jejuni subtypes. Finding indistinguishable isolates in animals, water and

human samples does not imply a definite linkage between them. Please refer to the

discussion section of the report. For a more detailed description of each case at the level of

individual analysis of questionnaire responses refer to Appendix 10. Bold lettering

highlights those potential linkages based on the collation of spatial, temporal and

epidemiological data. If one or more of these factors is missing the potential linkage is less

certain and therefore does not appear as bold print in the table.

Pote

ntia

l Tra

nsm

issi

on R

oute

s of C

ampy

loba

cter

104

Augu

st 2

002

From

Env

iron

men

t To

Hum

ans

Tab

le 2

3Po

tent

ial L

inka

ges I

dent

ified

for

C. c

oli a

s iso

late

d in

Ash

burt

on D

istr

ict d

urin

g th

e Sa

mpl

ing

Peri

od o

f 200

1

Find

ing

indi

stin

guis

habl

e is

olat

es in

ani

mal

s, w

ater

and

hum

an sa

mpl

es d

oes n

ot im

ply

a de

finite

link

age

betw

een

them

. Ple

ase

refe

r to

the

disc

ussi

on se

ctio

n of

the

repo

rt.

C. c

oli P

FGE

Subt

ype

(sin

gle

or r

elat

edPF

GE

) iso

late

dfr

om m

atri

ces

and

case

s

Ani

mal

rese

rvoi

r –

Rum

inan

t/D

uck

faec

es

Wat

er(a

ndte

mpo

ral

dist

ance

from

anim

al)

Food

- M

eat

or c

hick

en(a

ndte

mpo

ral

dist

ance

from

anim

al)

Hum

an C

ase

(and

tem

pora

ldi

stan

ce fr

omot

her

mat

rice

s)

Rel

evan

t ri

skfa

ctor

info

rmat

ion

Com

men

ts

P 2

and

33Sh

eep/

beef

cattl

e fa

eces

Nil

Shee

p of

fal

(No

tem

pora

llin

k to

anim

als)

1 C

ase

(no

tem

pora

llin

k to

ani

mal

s,1

day

afte

r off

al)

Cas

e liv

es

on

ada

iry

farm

.C

onta

ct

with

shee

p an

d do

gs.

A o

ne-d

ay in

cuba

tion

perio

d fo

r she

ep o

ffal

to h

uman

cas

e m

akes

it u

nlik

ely

that

thes

eev

ents

ar

e re

late

d.

This

ca

se

may

dem

onst

rate

di

rect

an

imal

co

ntac

t is

sign

ifica

nt.

P3Sh

eep

faec

esN

ilN

il2

Cas

es(1

1 an

d 38

day

s(w

inte

r-tim

e)af

ter s

heep

faec

es)

Bot

h ca

ses

dairy

farm

wor

kers

.Pl

ausi

ble

incu

batio

n pe

riods

ass

umin

g so

me

surv

ival

in

faec

es.

No

cont

act

with

she

epre

porte

d so

exa

mpl

e la

rgel

y ne

gate

d.

Pote

ntia

l Tra

nsm

issi

on R

oute

s of C

ampy

loba

cter

105

Augu

st 2

002

From

Env

iron

men

t To

Hum

ans

C. c

oli P

FGE

Subt

ype

(sin

gle

or r

elat

edPF

GE

) iso

late

dfr

om m

atri

ces

and

case

s

Ani

mal

rese

rvoi

r –

Rum

inan

t/D

uck

faec

es

Wat

er(a

ndte

mpo

ral

dist

ance

from

anim

al)

Food

- M

eat

or c

hick

en(a

ndte

mpo

ral

dist

ance

from

anim

al)

Hum

an C

ase

(and

tem

pora

ldi

stan

ce fr

omot

her

mat

rice

s)

Rel

evan

t ri

skfa

ctor

info

rmat

ion

Com

men

ts

P10

and

29

Dai

ry

cow

faec

esW

ater

(23

days

afte

r da

iryco

w)

Nil

1 C

ase

(63

days

afte

rco

w, 4

0 da

ysaf

ter w

ater

)

Cas

e liv

es

on

afa

rm,

had

cons

umed

w

ater

from

un

safe

sour

ces.

Thes

e ev

ents

occ

urre

d in

the

win

ter

and

soth

e po

tent

ial

surv

ival

tim

es

ofC

ampy

loba

cter

in

w

ater

an

d fa

eces

ar

epr

olon

ged.

How

ever

, un

less

the

sam

e C

.co

li su

btyp

e w

as b

eing

she

d an

d w

ashe

din

to t

he r

iver

ove

r so

me

time,

the

per

iod

betw

een

dete

ctio

n in

wat

er a

nd d

ate

ofon

set i

s too

long

to su

gges

t a p

ossi

ble

link.

AP

11/1

1aSh

eep

faec

esN

ote

mpo

ral

link

Nil

Shee

p of

fal

(fro

m lo

cal

sour

ce)

1 C

ase

(23

days

aft

erof

fal)

Tra

velle

dou

tsid

e ar

ea (b

utw

ithin

N

ewZe

alan

d).

The

tim

e be

twee

n de

tect

ion

in o

ffal

and

the

date

of o

nset

of i

llnes

s is

cre

dibl

e, b

utat

the

upp

er e

nd o

f th

e po

ssib

le t

ime

cour

se. T

he d

ata

may

onl

y de

mon

stra

te a

pote

ntia

l lin

k.P

11/1

1aC

attle

faec

esW

ater

(14

days

afte

r ca

ttle

faec

es)

Nil

Nil

N/A

Indi

cate

s po

ssib

le w

ashi

ng o

f C

. col

i fr

omca

ttle

padd

ock

into

the

river

P17

Nil

Wat

erN

il1

Cas

e9

and

23 d

ayaf

ter w

ater

Ove

rsea

s tra

vel.

The

two

incu

batio

n pe

riods

gi

ven

asis

olat

ions

wer

e m

ade

on tw

o di

ffer

ent d

ays.

Trav

el la

rgel

y ne

gate

s thi

s exa

mpl

e.

A R

ows

mar

ked

by b

old

lette

ring

dem

onst

rate

a p

oten

tial

trans

mis

sion

rou

te f

rom

env

ironm

enta

l m

atric

es t

o hu

man

cas

es b

ased

on

a co

llatio

n of

spa

tial,

tem

pora

l an

dep

idem

iolo

gcal

dat

a.

Pote

ntia

l Tra

nsm

issi

on R

oute

s of C

ampy

loba

cter

106

Augu

st 2

002

From

Env

iron

men

t To

Hum

ans

Tab

le 2

4Po

tent

ial L

inka

ges I

dent

ified

for

C. j

ejun

i as i

sola

ted

in A

shbu

rton

Dis

tric

t dur

ing

the

Sam

plin

g Pe

riod

of 2

001.

Find

ing

indi

stin

guis

habl

e is

olat

es in

ani

mal

s, w

ater

and

hum

an sa

mpl

es d

oes n

ot im

ply

a de

finite

link

age

betw

een

them

. Ple

ase

refe

r to

the

disc

ussi

on se

ctio

n of

the

repo

rt.

C. j

ejun

i Pen

ner

Sero

type

:PF

GE

Sub

type

(sin

gle

orcl

onal

ly r

elat

edgr

oup)

Rum

inan

t /B

ird

rese

rvoi

rW

ater

Food

res

ervo

ir(m

eat p

rodu

cts)

Hum

an c

ases

Rel

evan

t ri

skfa

ctor

info

rmat

ion

Com

men

ts

AH

S1:P

3aSh

eep

faec

esSh

eep

offa

l(N

o te

mpo

ral

link

to sh

eep

faec

es)

1 C

ase

11 a

nd 2

5 da

ysaf

ter

offa

l

Subt

ype

isol

ated

from

8%

sh

eep

offa

l an

d 7%

beef

of

fal.

Cas

ew

as

a fr

eezi

ngw

orke

r.

Evi

denc

e fo

r lin

kpr

ovid

ed

as

case

at

em

eat

prod

ucts

, bu

toc

cupa

tiona

l ex

posu

rem

ay b

e re

spon

sibl

e fo

rth

e ca

se.

HS1

:P33

Rum

inan

t fae

ces

Nil

Shee

p of

fal (

note

mpo

ral

link

to sh

eep

faec

es)

2 C

ases

31 d

ays (

Cas

e 1)

and

7 w

eeks

(Cas

e2)

afte

r off

al

Subt

ype

isol

ated

from

6%

of

shee

pof

fal

sam

ples

.C

ase

2 liv

ed o

n a

farm

an

dco

nsum

edun

treat

ed w

ater

Perio

ds

betw

een

food

and

case

s to

o lo

ng

solin

k no

t de

mon

stra

ted.

Pote

ntia

l lin

k on

ly.

A R

ows

mar

ked

by b

old

lette

ring

repr

esen

t cas

es, w

hich

dem

onst

rate

a p

oten

tial t

rans

mis

sion

rout

e fr

om th

e en

viro

nmen

tal m

atric

es to

hum

an c

ases

of c

ampy

loba

cter

iosi

s,ba

sed

on th

e co

llatio

n of

spat

ial,

tem

pora

l and

epi

dem

iolo

gica

l dat

a.

Pote

ntia

l Tra

nsm

issi

on R

oute

s of C

ampy

loba

cter

107

Augu

st 2

002

From

Env

iron

men

t To

Hum

ans

C. j

ejun

i Pen

ner

Sero

type

:PF

GE

Sub

type

(rel

ated

PFG

E)

Rum

inan

t /B

ird

rese

rvoi

rW

ater

Food

res

ervo

ir(m

eat p

rodu

cts)

Hum

an c

ases

Rel

evan

t ri

skfa

ctor

info

rmat

ion

Com

men

ts

HS2

:P3/

P33

and

rela

ted

subt

ypes

Shee

p, d

airy

and

beef

cat

tle fa

eces

Wat

erSu

btyp

e H

S:P3

don

ly

Pork

, she

ep a

ndbe

ef o

ffal

(35

days

from

beef

cat

tle fa

eces

)

4 C

ases

Cas

e 1:

27

days

from

cat

tle fa

eces

Cas

e 3:

(sum

mer

)32

day

s fro

mca

ttle

faec

esA

nd 3

3 da

ys fr

omsh

eep

faec

es

HS2

:P33

is

olat

edfr

om

dairy

(3

%)

and

cattl

e fa

eces

(10%

) an

d sh

eep

faec

es (1

%)

HS2

:P3

isol

ated

from

eve

ry m

atrix

at

abov

e 2%

prev

alen

ce, e

xcep

tfo

r wat

er

Cas

e 1

labo

urer

in

gu

tho

use

(she

ep c

onta

ct)

Cas

e 2

lived

on

fa

rm,

cont

act:

dairy

and

pig

s.C

ase

3:

Dai

ry

farm

er(d

airy

co

w

and

shee

pco

ntac

t)C

ase

4 C

hild

liv

ing

onlif

esty

le b

lock

Con

tinue

d ex

posu

re t

o a

pote

ntia

l so

urce

of

infe

ctio

n.Ti

me

inte

rval

too

lon

g to

dem

onst

rate

lin

k. C

ase

3an

d an

imal

s. C

ase

1 di

dno

t re

port

expo

sure

to

beef

ca

ttle.

G

iven

th

eub

iqui

ty o

f th

e su

btyp

e,lin

ks

wou

ld

be

hard

to

draw

.

Pote

ntia

l Tra

nsm

issi

on R

oute

s of C

ampy

loba

cter

108

Augu

st 2

002

From

Env

iron

men

t To

Hum

ans

C. j

ejun

i Pen

ner

Sero

type

:PF

GE

Sub

type

(rel

ated

PFG

E)

Rum

inan

t /B

ird

rese

rvoi

rW

ater

Food

res

ervo

ir(m

eat p

rodu

cts)

Hum

an c

ases

Rel

evan

t ri

skfa

ctor

info

rmat

ion

Com

men

ts

HS2

:P16

Wat

er(N

o te

mpo

ral

links

)

Shee

p of

fal

2 C

ases

:0

and

28 d

ays

afte

r off

al

Bot

h ca

ses

visi

ted

or

wor

ked

onfa

rms

and

dran

kun

treat

ed w

ater

.

Pote

ntia

l lin

k on

ly a

s tim

ebe

twee

n of

fal

and

case

sto

o sh

ort o

r at t

he li

mit

ofan

ac

cept

able

tim

e.M

ultip

le

othe

rop

portu

nitie

s to

be

com

ein

fect

ed.

HS2

:P18

a/1

8cD

uck

(the

only

sam

ple,

besi

des h

uman

case

s)

Nil

Nil

5 C

ases

No

tem

pora

l lin

kto

duc

k

No

othe

r m

atric

esfr

om

whi

ch

this

subt

ype

was

isol

ated

, ex

cept

the

one

duck

sam

ple

No

obvi

ous

tem

pora

l or

pers

on-to

-per

son

cont

act

betw

een

case

s, w

hich

all

occu

rred

with

in:

Dec

26,

2000

-Mar

19,

200

1.

HS2

:P54

Shee

p an

dC

attle

faec

esN

ilSh

eep

offa

l(n

o te

mpo

ral l

ink

to a

nim

als)

1 C

ase

1 da

y fr

om sh

eep

faec

es

Isol

ated

onc

e fr

omca

ttle

and

shee

pfa

eces

an

d sh

eep

liver

Tim

e in

terv

al to

o sh

ort t

ode

mon

stra

te

link.

C

ase

had

expo

sure

to

be

efca

ttle

and

calv

esTo

wn

wat

er

supp

ly

and

untre

ated

riv

er

wat

er.

Oth

er

expo

sure

s m

ayth

eref

ore

expl

ain

case

.

HS2

:P20

6D

airy

, cat

tleFa

eces

Nil

Nil

1 C

ase

No

tem

pora

l lin

kses

tabl

ishe

d

Subt

ype

isol

ated

from

da

iry

(5%

)an

d ca

ttle

faec

es(1

%)

Cas

e is

a c

hild

who

liv

eson

fa

rm

and

in

cont

act

with

shee

p an

d ch

icke

ns

Pote

ntia

l Tra

nsm

issi

on R

oute

s of C

ampy

loba

cter

109

Augu

st 2

002

From

Env

iron

men

t To

Hum

ans

C. j

ejun

i Pen

ner

Sero

type

:PF

GE

Sub

type

(rel

ated

PFG

E)

Rum

inan

t /B

ird

rese

rvoi

rW

ater

Food

res

ervo

ir(m

eat

prod

ucts

)

Hum

an c

ases

Rel

evan

t ri

skfa

ctor

info

rmat

ion

Com

men

ts

Dai

ry fa

eces

Cat

tle fa

eces

Nil

Shee

p of

fal

Chi

cken

car

cass

Cas

e 1

14 d

ays a

fter d

airy

faec

esC

ase

21

day

afte

r off

al;

Subt

ype

isol

ated

from

raw

chi

cken

(4%

)C

ase

1: N

o co

ntac

tw

ith fa

rm a

nim

als

Cas

e 2:

N

ot

inco

ntac

t w

ithan

imal

s.

Cas

e 1:

C

ase

had

eate

nch

icke

n,

but

the

subt

ype

was

no

t is

olat

ed

from

chic

ken

prio

r to

case

.C

ase

2: D

urat

ion

too

shor

tto

dem

onst

rate

link

to o

ffal

and

too

long

for c

hick

en.

HS4

com

plex

:P1

Nil

Nil

Chi

cken

Cas

e 2

30 d

ays a

fter

chic

ken

Chi

cken

ca

rcas

sis

olat

ion

(4%

)C

ase

1and

2: n

o ex

posu

re to

farm

an

imal

s, bu

t ha

dco

nsum

ed

chic

ken.

How

ever

dur

atio

n be

twee

nde

tect

ion

in

chic

ken

and

dise

ase

is to

o lo

ng fo

r a li

nkto

be

es

tabl

ishe

d,

and

chic

ken

cons

umpt

ion

isco

mm

on.

HS4

co

mpl

ex:

P52

Dai

ry a

nd sh

eep

faec

esN

ilN

il1

case

No

tem

pora

l lin

kto

ani

mal

s

Subt

ype

isol

ated

from

da

iry

(3%

)an

d sh

eep

faec

es(2

%)

Occ

upat

iona

l ex

posu

re

toho

rses

, cal

ves,

pigs

. Con

tact

with

dai

ry c

ows.

No

dire

ctlin

k m

ade.

Pote

ntia

l Tra

nsm

issi

on R

oute

s of C

ampy

loba

cter

110

Augu

st 2

002

From

Env

iron

men

t To

Hum

ans

C. j

ejun

i Pen

ner

Sero

type

:PF

GE

Sub

type

(sin

gle

or r

elat

edPF

GE

)

Rum

inan

t /B

ird

rese

rvoi

rW

ater

Food

res

ervo

ir(m

eat

prod

ucts

)

Hum

an c

ases

Rel

evan

t ri

skfa

ctor

info

rmat

ion

Com

men

ts

HS1

0:P1

8/18

b/3h

Dai

ry, b

eef c

attle

and

shee

p fa

eces

Nil

Shee

p of

fal

(loca

l sou

rce)

No

tem

pora

llin

k to

ani

mal

s

2 C

ases

8 da

ys fr

om o

ffal

to C

ase

1(ca

se a

ndof

fal b

oth

subt

ype

HS1

0:P1

8)

Wat

er

from

an

unsa

fe s

ourc

e w

asco

nsum

ed.

Expo

sure

to

rum

inan

t fae

ces.

(HS1

0:P1

8 =4

% in

shee

p fa

eces

)

Alth

ough

th

e tim

e fr

ame

betw

een

the

food

and

cas

eis

pl

ausi

ble,

th

e m

ultip

leex

posu

res

to

othe

r ris

kfa

ctor

s pr

even

t a li

nk b

eing

mad

e.

HS1

1:P3

5C

attle

faec

esD

airy

faec

esW

ater

(14

days

from

dai

ryfa

eces

)

Nil

4 C

ases

:16

, 23,

43

days

from

cat

tle fa

ecal

sam

ple

to 3

hum

an c

ases

Subt

ype

isol

ated

from

da

iry

(5%

)an

d ca

ttle

faec

es(4

%).

All

3 ca

ses

had

cont

act

with

farm

ani

mal

s.

The

timin

g is

pla

usib

le f

ortw

o of

the

thr

ee c

ases

, and

whe

re 4

3 da

ys w

as re

cord

edth

e an

imal

m

ay

have

cont

inue

d to

she

d af

ter

the

sam

ple

was

take

n.

HS1

5:P6

0bN

ilN

ilW

ater

1 C

ase

No

tem

pora

l lin

kto

wat

er

Foun

d on

ly

inw

ater

and

one

cas

eC

ase:

w

ater

su

pply

is

untre

ated

wel

l/bor

e w

ater

Pote

ntia

l Tra

nsm

issi

on R

oute

s of C

ampy

loba

cter

111

Augu

st 2

002

From

Env

iron

men

t To

Hum

ans

C. j

ejun

i Pen

ner

Sero

type

:PF

GE

Sub

type

(rel

ated

PFG

E)

Rum

inan

t /B

ird

rese

rvoi

rW

ater

Food

res

ervo

ir(m

eat p

rodu

cts)

Hum

an c

ases

Rel

evan

t ri

skfa

ctor

info

rmat

ion

Com

men

ts

Dai

ry, b

eef c

attle

and

shee

p fa

eces

Wat

er(7

day

s fro

msh

eep,

dai

ry a

ndca

ttle

faec

es)

Shee

p of

fal

Cas

e 1

24 d

ays f

rom

shee

p of

fal;

17 a

nd 3

1 da

ysfr

om d

airy

faec

es;

3 da

ys fr

om sh

eep

faec

esN

o te

mpo

ral l

ink

to w

ater

Cas

e 1:

(w

inte

rtim

e) A

farm

w

orke

r(s)

an

ddr

ank

wat

er

from

an

unsa

fe

sour

ce.

Link

poss

ible

gi

ven

the

dive

rse

mat

rices

cont

aini

ng

the

subt

ype

and

the

expo

sure

s.ΑH

S23,

36:

P19b

Dai

ry, s

heep

and

beef

cat

tle fa

eces

Wat

er0,

7,16

day

s fro

mda

iry

faec

es;

7 da

ys fr

omsh

eep

and

beef

catt

le fa

eces

Shee

p of

fal

Cas

e 2

7 da

ys fr

om o

ffal

,14

day

s fro

mbe

ef c

attle

faec

es

Com

mon

subt

ype

in

rum

inan

tfa

eces

and

she

epof

fal

(Dai

ryfa

eces

29%

, be

effa

eces

7%

, sh

eep

faec

es

6%

and

shee

p of

fal 5

%)

Cas

e 2:

D

rank

unpa

steu

rise

d m

ilk,

dair

y fa

rm

wor

ker.

Lin

k po

ssib

le g

iven

the

dive

rse

mat

rice

sco

ntai

ning

the

sub

type

and

the

expo

sure

s.

Α R

ows

mar

ked

by b

old

lette

ring

dem

onst

rate

a p

oten

tial t

rans

mis

sion

rou

te f

rom

env

ironm

enta

l mat

rices

to h

uman

cas

es b

ased

on

the

colla

tion

of s

patia

l, te

mpo

ral a

ndep

idem

iolo

gcal

dat

a.

Pote

ntia

l Tra

nsm

issi

on R

oute

s of C

ampy

loba

cter

112

Augu

st 2

002

From

Env

iron

men

t To

Hum

ans

C. j

ejun

i Pen

ner

Sero

type

:PF

GE

Sub

type

(sin

gle

or r

elat

edPF

GE

)

Rum

inan

t /B

ird

rese

rvoi

rW

ater

Food

res

ervo

ir(m

eat p

rodu

cts)

Hum

an c

ases

Rel

evan

t ri

skfa

ctor

info

rmat

ion

Com

men

ts

HS2

3,36

:P2

2B

eef f

aece

sN

ilO

ffal

1 C

ase

No

tem

pora

l lin

ksto

faec

es o

r off

al

Subt

ype

iden

tifie

din

bee

f off

al (7

%),

shee

p of

fal

(7%

)an

d be

ef

faec

es(1

%)

Cas

e liv

ed

on

farm

.C

onta

ct w

ith d

airy

cow

s,ca

lves

, sh

eep

and

pigs

.D

irect

lin

k no

t sh

own,

but

pres

ence

of

Subt

ype

in

anim

al

faec

es

and

mea

t pro

duct

s in

dica

tes

apo

tent

ial l

ink.

HS3

5:P1

0, 3

1D

airy

faec

esW

ater

(28

days

from

dairy

faec

es b

utno

t rel

ated

tohu

man

cas

e)

Pork

, she

ep a

ndbe

ef o

ffal

1 C

ase

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5. DISCUSSION

This study was unable to sample matrices from throughout New Zealand, therefore the

results are dependent, in part, upon the sampling scheme and that the study concentrated on

the Ashburton region. Further as environmental (animal, food and water) samples are only

random samples of what might be present in the environment at a particular time and

location, the fact there are not many direct spatial and temporal linkages between

environmental and human samples does not infer that they do not exist. The finding that a

particular Campylobacter subtype exists in a particular matrix means that there is the

potential for those bacteria to be passed onto another matrix, in particular to cause infection

in human cases.

5.1 Isolation of Campylobacter from the Matrices Tested

The prevalence of Campylobacter in the matrices examined was largely what would have

been expected from previous work in New Zealand and information from overseas. In

contrast to previous studies (Hudson et al., 1999) only 27.5% of chicken samples tested

were positive. This observed lower prevalence may be due to several reasons. All the

chicken was supplied by one company, which may produce chicken with a lower prevalence

than the average prevalence of around 55% samples positive for Campylobacter. In the CTR

study only whole chickens were sampled and it may be that whole chickens are less

contaminated. It is unlikely that differences in methodology led to a low prevalence in

chickens in this CTR study as the same enrichment method was used in both the CTR and

Hudson et al. (1999) studies.

Theoretically all human faecal samples should have yielded an isolate on subsequent sub-

culture as they had all originated from laboratory-confirmed cases of campylobacteriosis,

but not all did. It is possible that there were a low number of organisms present in some of

the original samples that declined to undetectable levels during storage and transport. Faecal

specimens could have been stored inappropriately or for too long. The screening

laboratories may have identified species other than C. jejuni and C. coli, but these would not

have been detected by the methods used in the CTR project. The proportion of C. coli to C.

jejuni (10 : 90) in human faeces was as expected from previous experience and reports

(Lawson et al., 1999).


The CTR samples were composite samples from five ducks, therefore isolations from ducks,

at 65.2%, were somewhat higher than would have been expected from overseas data where

prevalences of 30-40% have been reported (Luechtefeld et al., 1980; Fallacara et al., 2001).

Ducks in the CTR study were sampled from two locations in Ashburton, both were domains

with significant human ingress and activity. This limited sampling and the “unnatural”

locations of the sampling sites might explain the observed higher percentage of positive

samples.

Feral rabbits (n=72) and possums (n=197) were sampled for the detection of Campylobacter

in a Ministry of Agriculture and Forestry funded project on the same Ashburton farms

sampled for the CTR study (Savill et al., 2001b). C. jejuni was not identified in rabbit or

possum faecal samples. One rabbit faecal sample was positive for C. coli. Campylobacter

Rates of isolation of around 50% are expected from river water samples, and the CTR study

results concurred (see literature review). Seasonal variation is usually observed in water

samples due to differences in sunlight and water temperature, with isolations of

Campylobacter spp. being more frequent in winter. The data in Figure 14 indicate that the

temperature of the Ashburton river water peaked at approximately 14 - 18oC in the summer,

while in the winter temperatures in the range of 2 - 4oC were recorded. Figure 10 and Figure

11 showed that C. jejuni and C. coli had slightly higher prevalences in the Ashburton River

in the winter months

High prevalences were observed for ruminant faeces. However, since these data are from

composite samples the prevalences are higher than would be detected if individual animals

had been sampled. The data produced by the CTR study do not represent isolation rates for

individual animals. Highly variable carriage rates in ruminants have been reported, from a

few percent in adult animals to 54% in calves. In New Zealand, Meanger and Marshall

(1989) reported isolation rates between 12 and 31% for dairy cows depending on the season.

It was not the intention of the CTR work to determine prevalence on an individual animal

basis and so the prevalence in New Zealand ruminants remains largely undefined.


C. coli was isolated most frequently from ruminant faeces, and almost half of the composite

sheep faeces samples contained this species. This was unexpected as the main association

noted in the literature between C. coli and animal is with pigs (Christensen and Sorenson,

1999).

The contamination of offal by C. jejuni was dependent on the animal species from which the

offal was derived, with sheep offal being the most often contaminated (38.9%), followed by

beef (9.0%) and pork (4.8%). Pork offal was contaminated to the same level as C. jejuni by

C. coli, but the higher prevalence of C. coli in sheep faeces was not reflected in a higher

number of isolations of this species from the derived offal. The higher prevalence of

Campylobacter in offal has been discussed in the literature review, and has been noted

previously for New Zealand offal (Hudson, 1997). The prevalence of C. jejuni in sheep offal

was expected, but the lower prevalences in offal from the other two sources were not.

5.2 Temporal and spatial clustering of subtypes

There was a need to identify if clustering of subtypes was occurring in a temporal or spatial

distribution. If the same subtype was isolated from the same retailer over time this would

suggest that the sampling plan was not achieving an analysis of independent samples and

that cross contamination was a complicating factor. In the analysis of subtype data isolated

from ruminant animals and ducks, it was important to establish if there was clustering

occurring in consecutive sampling events rather than during the same sampling event. This

was because only one subtype was isolated from one farm or duck pond at any one time

point.

No evidence of clustering of subtypes was obtained for either meat products or animal and

bird isolates. There were a few occasions when the ruminant faecal subtypes were re-

isolated from the same farm in the next sampling event. One of these occasions was dairy

faecal subtype C. jejuni HS23,36:19b, which was isolated regularly from several farms on a

monthly rotation. This subtype was the most prevalent subtype identified in dairy faeces

(29%) and had lower prevalence in sheep faeces (6%) and beef faeces (7%). Therefore the

identification of this subtype from the same spatial locations does not exclude its isolation

being an independent event because it was a dominant subtype. To ascertain if this subtype


was present as a commensal micro-organism or a transient subtype would require a closer

investigation of the ecosystem.

Table 31 (Appendix 8) presents the data for cluster analysis of meat products collected from

the same meat retailer at the same sampling event (spatial/temporal events). For meat

products, a larger number of sampling events (20) resulted in the same subtype being

isolated from meat products from a single retailer at a single time point. There were no

spatial/temporal events where the same subtype was isolated from chicken carcasses and

offal products. This was not unexpected as the chicken carcasses were processed separately

to the offal and arrived pre-packaged at the meat retailers. Eleven of the 20 sampling events,

where the same subtype was isolated, were exclusively between chicken carcasses

purchased at the same time and place. It was known that all chicken carcasses, in the CTR

study, originated from the same supplier and therefore it would be reasonable to assume that

chickens from these sampling events were from one flock and slaughtered at the same time.

A flock of chickens is often infected with only one or two C. jejuni subtypes (Payne et al.,

1999). Isolation of the same subtype at the same time, therefore, does not necessarily imply

cross contamination, but rather the subtype could be present as part of the normal flora of

the chickens from which the samples were derived.

The observation of sampling events where the same subtype was isolated (on the same day

from the same retailer) from different offal products could be due to cross contamination.

However, the possibility that the same subtypes were isolated by coincidence from different

animals at the same time cannot be ignored, as the CTR results demonstrated that some

subtypes were prevalent in many matrices (Figure 22). This again raises the question of

whether the isolate was a commensal organism or a transient subtype.

The CTR study did not observe the consistent identification of the same subtype in

consecutive sampling times. The analysis of the spatial and temporal patterns of subtypes

led to the conclusion that the isolations of subtypes were independent events. This was not

unexpected as the sampling plan was based on fortnightly/monthly sampling intervals. The

high turnover of meat products and the standard cleaning and sanitising procedures

implemented by retailers would further preclude isolation of the same subtype.


5.3 Penner Serotypes of C. jejuni

5.3.1 Subtypes Identified in the CTR Study

Eighty-nine (14.4%) of the 616 isolates serotyped were untypable. This proportion is similar

to that reported in other studies (e.g. Nicol and Wright, 1998). Figures as low as 1.5% have

been reported (Nielsen and Nielsen, 1999) but only after repeated subculture and testing.

The most frequently encountered serotypes in the CTR study were HS 2 (13.6%); 4 complex

(11.7%); 23,26 (10.7%); 1,44 (7.8%); 5 (6.2%); 6 (3.7%); 19 (3.7%); 8,17 (3.7%); 35

(3.2%); 21 (2.9%); 11 (2.7%); 37 (2.3%) and 10 (1.8%). For human cases HS 2 (29.8%) and

4 complex (14.0%) again dominated. HS 23,36 was isolated from only 5.3% of clinical

cases compared to 10.7% of the overall sample set, and this reflects the high prevalence in

ruminant animals in this study (isolated from 39.0% of dairy cattle faeces).

Among the water isolates HS 8,17; 6; 5 and 1,44 are common, while HS 23,36; 4 complex

and 2 were rarely isolated. The latter two serotypes account for 43.8% of the isolates from

human cases.

Among the serotypes isolated from human cases, HS 2 was common. This might imply that

this serotype is particularly associated with human disease (HS2 is one of the most common

serotypes isolated worldwide from humans and other sources). The only other matrix that

yielded this serotype frequently was beef faeces, but at the level of analysis of serotype only

it is not possible to link this reservoir/transmission route to disease in humans.

Duck faeces contained HS 37 and 5 most frequently, the latter also being commonly

obtained from river water. HS 23,36 predominated in dairy cattle (34/87, 39%, isolates). It is

possible therefore that this serotype is associated with this animal reservoir. Interestingly the

same observations could not be made for beef cattle, where HS 2 was the most frequently

isolated serotype. Chicken carcasses were contaminated most often with HS 21 and HS 2.

For some matrices, e.g. pork offal, the number of isolates is too small to draw any

conclusions.

5.3.2 Comparison with Prior New Zealand Data


Nicol and Wright (1998) reported on the results of three New Zealand studies. HS 2 was

isolated from bovine and ovine sources and composed 26% of the human isolates. HS 4 was

also isolated from bovine sources and human cases. In the second study of human cases

frequent serotypes were HS 2 (16%); 4 (8%) and 8 (8%). In the third study (Hudson et al.,

1999) it was found that the most frequently isolated serotypes from human and veterinary

cases, raw chicken and water were HS 4 complex (14.8%); 2 (14.2%); 33 (9.9%); 6 (8.6%)

and 12 (7.4%). In the winter most human cases were caused by HS 4 complex (52.6%) and

2 (21.7%), but in the summer the most prevalent serotypes were HS 2 (26.1%); 33 (21.7%)

and 6 (15.2%). HS 6 was also associated with raw chicken meat in the summer. These data

are largely in agreement in that HS 2 and 4 complex are consistently associated with human

disease, both in New Zealand and worldwide. Serotypes involved with three New Zealand

outbreaks of campylobacteriosis were HS4; 2 and 23,36 (Nicol and Wright, 1998).

Results from the CTR work also showed that HS 2 and 4 complex occurred frequently in

human isolates. HS 2 and 4 complex were also frequently isolated from bovine faeces.

Contrary to previous New Zealand data no HS 8 or 33 isolates were obtained from human

cases, but they were isolated from other matrices studied. Two human cases caused by HS 6

isolates were identified in the CTR study. Overall therefore, the major serotypes associated

with human disease (HS 2 and HS 4 complex) were the same for the CTR project as has

been recorded in previous studies of New Zealand isolates. Variations among the less

frequently occurring serotypes were observed. At the level of serotype, therefore, the range

of isolates obtained for the CTR study is very much in accordance with that observed

previously in New Zealand.

HS 21 was also the serotype most frequently isolated from chickens in the CTR study, with

HS 2 being the next most frequent. HS 21 was also the most frequently isolated serotype

identified in chicken portions in a Christchurch study (Hudson et al., 1999), but was not

detected in humans in either the Christchurch or CTR study. However 14% of

Wellington/Hutt Valley case isolates subtyped in 1997 were identified as HS 21. These

apparently contradictory observations may reflect differences in transmission of the disease,

possibly in terms of sporadic case versus outbreak epidemiology (no outbreaks were

identified in the CTR study), or may be the result of limited sampling data.


The results discussed above have not included less frequently occurring serotypes isolated at

less than 2% of the total. There were 19 of these serotypes in the CTR study.

5.3.3 Comparison with Overseas Data

Serotypes identified for human isolates in the CTR study differ in terms of frequency of

isolation from those found in the USA (Patton et al., 1993). A significant percentage of New

Zealand CTR clinical isolates were HS 2, but this serotype was only the sixth most

commonly isolated in the American study. The USA data showed quite marked differences

in HS subtypes between states, and the data for California showed greater similarity with

CTR data, HS 2 and 4 complex being the most common. HS 2 and 23,36 were identified in

12/15 (80%) outbreaks in the USA from 1978 to 1989. An outbreak in Vermont caused by

consumption of contaminated raw milk was caused by C. jejuni HS 2 (Vogt et al., 1984). An

outbreak in the UK implicating cattle as the source of infection was caused by cross-

reacting serotypes 13, 16, 43 and 50 (Bradbury et al., 1984). This group is now regarded as

part of HS 4 complex.

Serotypes 1, 2 and 4 were the most frequently identified in isolates from cases of

campylobacteriosis in British Columbia (McMyne et al., 1982) and in South Western

England (Jones et al., 1984). A similar pattern of distribution was found for isolates from

patients with gastroenteritis in Toronto between 1978 and 1980 where HS 2 was always the

most frequently isolated. HS 4, 3, 8 and 1 were also commonly isolated (Karmali et al.,

1983). These results concur with CTR data.

HS 53; 15 and 22 predominated in isolates from clinical cases in Bangladesh (Neogi and

Shahid, 1987). While these serotypes were identified in the CTR study, they occurred at 1%

or less of total isolates typed.


There is a broad agreement among similar countries that HS 2 and 4 are the serotypes most

often associated with human disease. Other serotypes tend to contribute a small proportion

of cases individually, but represent a large component when combined. Developing

countries may show different patterns of C. jejuni serotypes, presumably because the routes

of infection, health status of the population and environments are quite different, and there

may also be differences in the testing methods used.

Isolates from Danish poultry products demonstrated serotypes similar to those reported in

the CTR project. The five most frequently isolated Danish serotypes were HS 1,44; 2; 3; 4

complex and 5 (Nielsen and Nielsen, 1999). Some differences in serotype distribution were

observed between different subtypes of poultry: 16.3% of chicken isolates and 81.3% of

poussin isolates were HS 2.

More recent Danish work compared serotype distribution in wildlife, human cases and

broilers. Human cases were most often associated with HS 2 and 4 complex, a similar

pattern to the CTR results. Wildlife (e.g. hedgehogs, squirrels ducks) isolates were

predominantly HS 4 complex, HS 12 and 4. HS 1,44 was confined to human cases and

broiler flocks. It was concluded that wildlife did not contribute to serotypes causing human

disease or infecting broilers, but that there was significant overlap of broiler and human case

serotypes (Petersen et al., 2001).

Three ducks were tested in the Danish study (Petersen et al., 2001). Two of the three

isolates were of serotypes also identified in the CTR study. HS 37, found in one of the

Danish ducks, is common in CTR duck isolates but was not isolated from them exclusively.

HS 37 was not identified in human cases in the CTR project. It is possible that this serotype

might have an association with ducks, although not exclusively.

Chickens yielded mostly HS 1; 4 complex; 6,7 and 2 in a British study (Jones et al., 1984).

HS 1; 2 and 4 complex were also isolated from chicken intestines by Munroe et al. (1983),

along with HS 3; 5 and 31. HS 4 complex was the most frequent chicken isolate serotype in

a British study conducted in the mid 1980s, but no single serotype or group of serotypes

predominated (Hood et al., 1988). Fricker and Park (1989), again in a British survey,


detected mostly HS 8; 1 and 2 in chicken isolates. HS 21 was most frequently isolated from

chickens in the CTR study with serotype 2 the next most frequent.

In a British study pond and river water yielded mostly HS 1; 4 complex; 2 and 19 (Jones et

al., 1984). Fricker and Park (1989) detailed the ten most common serotypes in human cases,

two of which were not found in river water. The eight serotypes reported were: HS 13/16

(10.4%); 4 (9.5%); 2 (8.3%); 8 (5.7%); 1 (4.8%) with 3, 23 and 10 each at 1.9%. Ashburton

river water yielded a diverse range of serotypes in the CTR study, as might be expected

given the numerous inputs into a river system. Unlike other studies HS 2 and 4 were only

the fifth equal most frequently isolated serotypes from Ashburton waterways.

Cattle yielded mostly HS 4 complex; 2; 1; 11 and 27,31 (Jones et al., 1984). HS 1; 2 and 4

complex were isolated by Munroe et al. (1983), with HS 2 apparently present more

frequently in cows with diarrhoea. Of the beef isolates tested in a British study, 40% were

HS 2 (Fricker and Park, 1989). Sheep yielded mostly HS 1, 2, 4 complex, 6,7 and 23 (Jones

et al., 1984). Only a few isolates from lamb meat were serotyped by Fricker and Park

(1989), these were mostly HS 2; 4; 9 and 48. The same authors reported a predominance of

HS 2 and 4 in offal, although the animal of origin was not recorded. Serotypes 2 and 4

complex were isolated quite frequently from ruminants and offal in the CTR project.

5.4 Pulsed Field Gel Electrophoresis Subtypes of C. jejuni

It is not possible to compare PFGE data from this study directly with those from overseas as

subtyping systems and nomenclature vary between researchers, despite the fact that most

PFGE subtyping carried out uses the same general methodology. This underscores the

desirability of New Zealand’s participation in international subtyping databases, which use

uniform protocols and nomenclature. In this and subsequent sections comment is made on

the CTR data, and comparisons drawn with a previous New Zealand study where the same

methodology was used.


5.4.1 CTR Data

Because multiple PFGE groups were identified, and no direct international comparisons can

be made, comment on PFGE data alone will be brief. Almost 7% (n=42) of isolates typed as

PFGE macrorestriction profile 19b, P19b. More than half of these were isolated from dairy

cow faeces, with a few isolates occurring in other matrices tested with the exception of duck

faeces, pork offal and beef offal). Seventeen isolates of P21 were identified, 15/17 were

from water, 1/17 from beef faeces and 1/17 from sheep faeces.

Isolates of P25 constituted another large group (n=34). These were found in all matrices

except for offal, perhaps indicating that survival in foods is poor. The 28 isolates of P28

came from all matrices except for water, and approximately 30% of them were from raw

chickens. Most of the 22 P33 isolates came from ruminant animals, and three human cases

were caused by this PFGE subtype. In contrast, there were 31 isolations of P34, but nearly

all were confined to ruminant faeces or offal, no human cases were caused by this subtype.

The isolation pattern of these two subtypes, P33 and P34 might indicate that there are

subtypes present in ruminant animals. However, these observations need to be verified.

5.4.2 Comparisons with Previous New Zealand Data

The most relevant comparison is with isolates described by Hudson et al. (1999) from the

Christchurch area. Most of the PFGE subtypes reported in the Christchurch study were also

identified in the isolates from Ashburton. The most predominant subtype isolated in

Christchurch, P25, came from human cases, raw chicken and water samples. This was also

the case with the P25 isolates identified in the CTR study, but the same PFGE group was

also isolated from duck and ruminant animal faeces. PFGE subtypes P25a, 25b, 25c, 25d

and 73, clonally related to P25 (for criteria for determining clonal relatedness see Materials

and Methods Section and Table 30, Appendix 6 for a table of related C. jejuni PFGE

subtypes), were only isolated from water, duck faeces and chicken. P4 is also related to the

P25 group and the pattern of isolate sources is the same. More than 50 isolates from this

study were assigned to P subtype 25 and its clonal relatives. The absence of P subtypes

clonally related to P25, but not P25 itself, from humans and ruminant animals could be


significant, but further work analysing more isolates would need to be undertaken to

confirm a possible variation in human pathogenic potential between these PFGE subtypes.

PFGE subtype 18, 18a and 18b, which are clonally related, and P3, were isolated quite

frequently in the Christchurch study. Because of the interpretative criteria applied in

designation of clonal relatedness in this study, it is difficult to determine the contribution of

other, related, PFGE subtypes in the CTR dataset, but these groups comprise only a few

isolates. The P18/P3 group was isolated by Hudson et al. (1999) from human cases (in the

summer only), chicken (in the winter only) and from the faeces of two sick ruminant

animals, but not from water. In the present study P3 is a large group (n=28) isolated from all

matrices except water. P18, 18a, 18b and 18c were isolated from most matrices with the

exception of water, dairy cow faeces, beef cattle faeces and foods. The observed lack of

isolation of these PFGE subtypes from water samples is notable given that, over the course

of both studies, more than 60 isolates of this subtype have been obtained. It is possible that

isolates of this PFGE group are poor survivors in water. Further experimentation would be

needed to confirm this. Representatives of all these clonal groups have been isolated from

human cases.

Among the smaller groups, P1 and 1a were only isolated from human cases in the

Christchurch study. In the current study, isolates of this subtype were not isolated from

human cases exclusively, although they were absent from water, duck faeces, sheep faeces,

beef offal and pork offal. The number of isolates in this group is too small however, to make

further comment. Six isolates of P19 recovered from human, chicken and water samples

were reported in the Christchurch study, and 42 isolates of subtype P19b (clonally related to

P19) were identified in the CTR dataset. The Ashburton isolates were predominantly from

bovine faeces, a matrix not tested in the Christchurch work. Other PFGE subtypes were only

infrequently isolated by Hudson et al. (1999).

5.5 C. jejuni PFGE and Penner Subtypes

Isolates belonging to these discrete Penner serotype:PFGE groups could not be

distinguished from each other by either subtyping method, and therefore are considered as


subtypes. Complex inter-relationships exist between and among isolates of differing

relatedness.

5.5.1 CTR Data

The panels in Figure 22 compare the most frequently isolated subtype in each matrix with

the isolates of those subtypes in each other matrix. For example Figure 22a compares the

nine most common subtypes from human cases to isolates of those subtypes in all other

matrices. From this graph it might be inferred that there is no overlap with isolates from

pork offal. Caution must used in interpretation of these graphs. For example, the greatest

numerical overlap of subtypes from human cases is with isolates from water samples, where

five subtypes are common to both sources. This does not indicate a direction of flow

however, in that humans may contaminate water, and contaminated water infect humans

(presumably both directions of flow operate). Both matrices may have been contaminated

by a third unknown transmission route. Similarly, one of the most common subtypes in

human cases was isolated from only one of the other matrices – ducks. Again, direction of

pathogen flow cannot be assumed. The common subtype HS23,36:P19b was found in

human cases and constituted around 25% of isolates from dairy cow faeces. The observation

that a few human cases caused by this subtype also occurred does not necessarily imply a

direct dairy cow to human transmission route.

It is of interest that none of the most frequently isolated subtypes from water were isolated

from dairy cow faeces, beef offal, chicken carcasses or pork offal. It might be expected that

Campylobacter subtypes in faeces deposited onto paddocks would be washed into

waterways, but those frequently isolated subtypes were not. Certain subtypes identified in

beef cattle and sheep faeces were also isolated from water. Possibly the subtypes found in

cow faeces are poor survivors in water. The overlap between water and duck subtypes

would be expected from direct and indirect deposition of duck faeces into waterways.

Isolates from ducks largely appear to be a discrete group as there is little overlap with

subtypes isolated from other matrices with the exception of water. No link with human cases

can be discerned from these data.


Several subtypes are common to both dairy faeces and human cases, but the common dairy

faecal isolate subtypes are also common among the other ruminant animal and derived

products. Similar observations can also be made for beef cattle faeces and sheep faeces.

The data shown in Figure 22g and Figure 22h demonstrate considerable overlap between the

subtypes isolated from beef offal and sheep offal, and, to a lesser extent, between beef offal

and ruminant faecal isolates. Interestingly certain subtypes, present in both offal and human

isolates, were not detected in the faeces of ruminant animals. It might be that that the offal

tested were obtained from animals raised outside the region of study or might indicate that

the contamination came from an unidentified source. Subtypes HS1,44:33 and HS1,44:P3a

were only isolated from human faeces and ruminant offal.

The dominant subtypes isolated from chicken carcasses showed very little overlap with

those from human cases (Figure 22i). Only two of 14 subtypes were common to both

matrices, one of these was HS2:P3, which was isolated from all matrices tested with the

exception of water. Overall, there was little similarity in subtypes between the chicken

isolates and those from any other source. This observation is unexpected as the consumption

of, or cross contamination from, raw chicken has been identified as a major cause of human

campylobacteriosis in numerous studies. Duck faecal isolates and chicken carcass isolates

might also have been expected to show overlap, given the similarity in the physiology of the

source animals. However, chickens tested were probably not raised in the same area as the

duck faeces tested.

Pork offal isolates are discussed separately from bovine offal isolates, as pigs are mono

gastric, not ruminant. They may also be raised intensively, and so the spread between

animals is different from animals raised in paddocks. Isolates from pork offal included the

common subtype HSS2:P3. There was also overlap in subtype of one pork offal isolate

(HS4c:P34) with isolates from ruminant animal faeces and meat products.

5.6 Comparisons with Prior New Zealand Data

Eight of the ten PFGE/Penner subtypes reported by Hudson et al. (1999) were represented

among the isolates in the CTR study. The only group that the Christchurch study identified


as occurring in both summer and winter was HS2:P18/18a. This group was isolated from all

matrices tested except water. The inter-relationships within this group is, however, not

simple (Figure 23). Discussion will focus on the three core PFGE subtypes 18, 18a and 18c.

Figure 23 Genetic Relationships among the Clonal Group P18

18C3 18A

183h 18B

211

(Arrows indicate clonal relationships, the numbers a the designated PFGE pattern numbers)

In the CTR project this group comprises HS2:P18a and HS2:P18c as no HS2:P18 isolates

were identified. Only a few isolates of this subtype were obtained from Ashburton, five

from water and one from duck faeces. Although the number of isolates was small, an

association with human cases holds as, of 19 isolates of this subtype from both surveys, 14

originated from human cases. The other sources were veterinary cases (2), duck faeces (1)

and chicken (2).

HS2:P3 isolates were all from human cases in the Christchurch study. PFGE subtype 3 is

clonally related to two other PFGE groups; (18, 33 and 47) and (3a, 3b, 3c, 3d, 3e, 3g and

47a). Isolates of the subtypes HS2:P3, HS2:P33 and HS2:P47 (n=35) were however, not

confined to human cases in the CTR study. Isolates of those subtypes were also derived

from ruminant faeces (19/35 isolates), chicken carcasses (7/35), all offal (6/35), and duck

faeces (1/35), in addition to human cases (2/35). The Christchurch study did not examine

isolates from the faeces of healthy ruminant animals. There were too few isolates identified

in the HS2:P3b, 3c and 3d group to make comment.

The four PFGE subtypes 1, 1a, 1b and 1d are a largely cohesive group, except that 1a and 1b

are not directly related, although they are both related to P1. All the Christchurch S4:P1

isolates were from human cases. While two HS4:P1/P1a isolates were from human cases, all

HS4:P1b isolates were from ruminants and the HS4:P1 isolates were from chicken and


sheep offal. However, there are too few isolates from the current study in this group to draw

conclusions.

Subtype HS12:P4 was relatively common (nine isolates) among isolates in the Christchurch

study but only one isolate of this subtype was identified among the CTR isolates. Too few

CTR isolates designated S6:P25 and S21:P25 were available to allow comparison with the

data of Hudson et al. (1999).

HS33:P25 was the most frequently isolated single subtype of C. jejuni isolated in the

Christchurch study. It was absent from winter isolates but predominant among those

obtained in the summer. It was only isolated from human cases and chicken portions. No

isolates of this subtype were identified from the CTR study. This might be due to

geographic or demographic reasons, or to fluctuations in different extant subtypes.

5.7 Pulsed Field Gel Electrophoresis Subtypes of C. coli

There is little published information with which C. coli subtyping data can be compared

except in Hudson et al. (1999). Caution must be applied in the comparison, as the animal

isolates obtained for that study were faeces from sick animals, and the isolation methods

used were different. Only one PFGE subtype was common to human faecal isolates in both

studies, P 14, represented by only a single isolate in each study. This subtype was also

isolated from a CTR water sample. One of the veterinary isolates from the Christchurch

study and isolates from sheep offal, dairy cow faeces, beef cattle faeces and sheep faeces

from the CTR study were designated P1. However, given the small number of isolates tested

by Hudson et al. (1999) little else can be said in comparison of the two sets of data.

5.8 Czekanowski Similarity Indices

Discussion will be largely confined to the combined PFGE and serotyping data as these

represent the most rigorous assessment of similarity between isolates from various matrices.

The lack of similarity in the subtypes isolated from two matrices does not exclude the

possibility of links between the two, but it can be considered that these links are the

exception rather than the rule.


Human case isolates were similar to those from ruminant faeces and offal, the greatest

similarity being with isolates from sheep faeces and offal. This provides some indication

that these animals are a source of human pathogenic subtypes, either by direct contact with

animals or through food derived from them. It should be borne in mind that offal was used

as a surrogate for more frequently consumed meat products derived from the same animals.

The finding of Campylobacter in the ruminant faeces provides an indication of the effect of

human contact with live animals.

Some of the highest similarity values were recorded between ruminant faeces (cattle and

sheep) themselves and offal. This information can be interpreted as reflecting the generally

held view that meat products are contaminated during slaughter and processing either

directly or indirectly from animal faeces. The observation that isolates from ruminant

animals have similar subtype distributions to one another (within and between animal

species) is presumably a reflection of the similar physiology of the animals from which they

came, indicating possible selection for particular subtypes or common subtypes.

In general, isolates from water were not highly similar to those from other matrices. The

greatest similarities between water were to humans, ducks and ruminant animals (although

not dairy cows). This might be expected due to faeces being directly deposited into water

from ducks, or via run off from farms. The similarity between subtypes of water isolates and

those identified in human cases was affected by the number of untypable isolates making the

interpretation of relationships less certain. Water, may however need to be considered

differently, because it is the recipient of Campylobacter from, a large number of disparate

inputs. These will presumably include pigs, deer and geese among others, in addition to the

inputs described here. This potential large diversity of subtypes may mean that water

isolates are likely to be more dissimilar to subtypes from any one source. Therefore, water

may require much more rigorous sampling to reflect the expected diversity of subtypes.

Four matrices yielded isolates that were dissimilar to those isolated from human cases, and

often, from any other matrix. Pork offal isolates showed little similarity with most other sets

of isolates, although there was some similarity between pork offal isolates and those from

bovine faeces. Since pigs are monogastric animals, this lack of similarity might be expected,


and the observed similarity with the bovine isolates might reflect cross contamination in

butchers’ shops. Isolates from chicken carcasses most closely resembled those from offal,

and interestingly, there was only a very low similarity with isolates from human cases. Duck

faecal isolates were most similar to water isolates, this particular association would be

expected for the reasons described above.

5.9 Potential Linkages of Campylobacter between matrices

This was the only analysis that considered clonally related PFGE subtypes. It is not possible

to take this approach with the other main statistical analyses used, as the relationships are

complex.

Due to the nature of the sampling time frame it was impossible to sample every matrix every

week. In addition, not all isolates were amenable to subtyping. This has led to some

limitations in the data for isolates from various matrices. For example, the isolation of a

particular subtype from offal one week but not the following week does not exclude the

possibility that the same subtype was actually present in offal sampled in the second week.

This situation might arise because the subtype may have been present in a different sample

or portion of sample not selected for subsequent isolation. Similarly there are no data on

whether subtypes can be isolated on a continuing basis from ruminant faeces or whether

subtypes turn over rapidly.

Given the data in Table 23 the information provided for these potential C. coli linkages is

far from compelling. However, it should be noted that three of the five cases lived or

worked on farms and in one case, consumed unsafe water. A further case had holidayed on a

Pacific Island immediately prior to onset of symptoms. This person could not have

consumed the water from which the same subtype was isolated nine days prior to the onset

of disease.

Few, if any, of the potential C. jejuni linkages shown in Table 24 are supported by sufficient

data to confirm them as transmission routes. As with the results for C. coli, most of the cases

had multiple potential exposures to C. jejuni, even if subtyping data did not support these

other exposures. However, the data presented here and for C. jejuni are highly suggestive of


a link between a rural lifestyle and infection by these organisms. Of the cases recorded, 58%

lived on a farm, or had visited a farm, in the 10 days prior to disease onset. Direct contact

with animals was also common, for example, 45% of cases had been in contact with sheep.

Other direct “rural” exposures included cases who had consumed water and unpasteurised

milk (Table 22). Other exposures, such as contact with cats and dogs and consumption of

animal derived foods, were also common.

From Table 24 the best evidence for a link between a matrix and a human case (dairy

farmer) comes from one of the two cases of infection by C. jejuni subtype HS23,36:P19b

(Case 2). Here isolates of the same subtype were obtained from sheep offal and cattle

faeces. The cattle faeces were obtained from samples in area A, the area where the dairy

farmer worked. The offal sample could also have originated from an animal grown in the

local area as the retailer of the offal obtains meat from a local processing plant. The times

between isolation from these matrices and the onset of disease are plausible. The case also

drank raw milk, but if this was the source of this infection, animal faeces would have been

the most likely source of contamination of the raw milk. However, a large degree of

uncertainty surrounding this potential link remains.

Attempts were made to subdivide the cases into “rural” and “urban” with an urban case

taken as not having visited or worked on a farm, and not consuming untreated milk or water.

However when this was performed the number of true “urban” cases (a maximum of ten

with appropriate data) was too small to be analysed.

Two pairs of cases could be considered as occurring in “urban” people. One pair comprised

two isolates of HS4 complex:P1 and the other pair two isolates of HS6:PNC (non-cutting).

Unfortunately, since the second pair was untypable by PFGE they cannot be considered as

indistinguishable isolates. The other pair of cases caused by HS4 complex:P1 were similar

in that the cases had no contact with animals of any species. This subtype had been isolated

twice from chickens and once from sheep offal during the course of this study, but not

immediately before the cases occurred. This subtype had also been isolated in a previous

study conducted in Christchurch by Hudson et al., (1999), but only from human cases and

not from chicken. This means that a total of 7/10 isolates of this subtype have been from

human cases. This subtype may therefore be associated with humans, and occasionally with


poultry products. The history of neither case in this study indicates person-to-person

transmission. Alternatively, an as yet unrecognised transmission route may be the cause of

these cases.

The greatest number of cases caused by a particular subtype was four. These involved C.

jejuni subtype HS11:P35. One of the cases was not recorded on EpiSurv and therefore

relevant risk factors are unknown. Two of the cases lived in the same town, and one at the

boundary of the study area. The dates of onset of illness for the two cases from the same

town were two weeks apart. One of these two cases had fallen headlong into a cow pat,

suggesting a likely source of infection, the same subtype having been isolated from dairy

and beef cattle faeces. The other case occurred prior to the commencement of sample

collection, and may have occurred as a result of person-to-person transmission. There does

not seem to have been a common cause to link these four cases.

Three cases have visited local swimming pools, but there was no subtyping evidence to link

them.

C. jejuni subtype HS2:P18a/18c was isolated from five human cases in the Ashburton

District and one duck faecal sample. The cases occurred between 26th December and 19th

March, 2001. There was no known temporal linkage or person-to-person contact between

any of the cases. However, as the subtype was isolated within a defined time period, it is

suggestive that further investigation of the epidemiology of cases may have increased the

likelihood of determining its source. Early recognition of the link by subtype may have

allowed the Health Protection Officer to reassess potential common exposures between

these cases.

5.10 Characteristics of human cases

The demographic characteristics of the cases was fairly typical of that seen for

campylobacteriosis in New Zealand with the highest incidence in children under 5 years,

and a secondary peak among young adults. The absence of Maori and Pacific Island cases

reflects the demographic make-up of the Ashburton population (4.6% Maori and 0.5%

Pacific Islander), the small study size, and the relatively low rates of enteric infections


reporting in Maori and Pacific people relative to Europeans. Cases differed from the New

Zealand population in having a more rural distribution.

5.11 Exposure histories of human cases

Most of the cases in this study would have been infected from exposure to a source of

Campylobacter infection in the Ashburton district. Only two cases (4%) had been overseas

in the previous 10 days, though ten cases (19%) had travelled outside Ashburton during that

period. All cases also appear to have been sporadic infections. There was no evidence of

common source outbreaks in this population. Person-to-person contact with another case

was only reported by eight cases (14%). Based on the limited information recorded on the

timing and nature of this contact, plus the fact that very few of the related cases had

provided a faecal sample for testing, none were able to be definitively identified as

secondary cases.

A relatively high proportion of cases in this study had contact with farm animals and rural

environments. Living on a farm or visiting a farm during the previous 10 days was reported

by 58% of cases. Direct contact with farm animals was also relatively common in this

sample, and included contact with sheep (45%), cattle (38%), and chickens (22%) Of the

cases, 30% had occupational exposure to animals, 69% contact with dogs and 69% with

cats.

The majority of cases reported eating foods of animal origin in the preceding ten days,

including beef (88%), chicken (87%), eggs (87%), and pork (63%). Other exposures

included consumption of water (urban and rural, Table 22) and unpasteurised milk.

5.12 Characteristics of Campylobacter infecting humans

Human infection was predominantly by C. jejuni (83% of cases), with 9% infected with C.

coli, and one case with dual infection included in both proportions. There were 7 (10%) of

samples that did not yield any Campylobacter.


More than half the identifiable C. jejuni serotypes (53%, 17/32) were found in humans.

Using PFGE, these serotypes were divided into 250 Serotype-PFGE subtypes of which 19%

(44/250) were found in humans. Only 9% (5/34) of the distinguishable PFGE C. jejuni

subtypes were found in humans.

The range of subtypes infecting humans was diverse. There were 44 subtypes of C. jejuni

found in 56 isolates (a diversity of 78.5%) and 5 subtypes of C. coli for 6 isolates (a

diversity of 83%). (Note: for the purposes of further discussions of subtypes, that human

isolates with untypable serotypes with the same PFGE subtype, and those with non- cutting

PFGE subtypes with the same serotype are not assumed to have the same infection. This

impacts on two subtypes: HSUT:P25 and HS6:P non cutting, which both involved two

cases). Most subtypes of C. jejuni infected a single person (39) or two people (5). Only 1

subtype infected three people, and 1 subtype infected four people.

There were four subtypes incorporating untypable serotypes or non-cutting PFGE subtypes,

encompassing 6 cases. Nineteen (48 %) of the remaining subtypes founds in humans were

also found in other matrices. Twenty-one subtypes were unique to humans in this study, and

these subtypes only accounted for 46 % of cases. This leaves 27 human C. jejuni cases

where subtyping data can be used to explore relationships with subtyping information

obtained from samples collected from these other matrices. In the case of C. coli all of the

PFGE subtypes found in humans were also found in other matrices.

At a population level, most of isolate subtypes from humans were also found in all matrices.

Based on the 56 human cases infected with C. jejuni, the Czekanowski index suggests that

the serotypes found in humans were most similar to the serotypes isolated from cattle.

However, at the PFGE level the same level of association did not apply.

Further analysis is possible to test the hypothesis that humans who report contact with a

particular potential source are infected with subtypes that are more similar to those isolated

from that source than other cases who do not report exposure to that source. However due to

the small sample size, level of diversity and result of performing multiple univariate tests or

comparisons the statistical results can not be seen as definitive but only as indicative results

that need to be considered along with other results to direct further focused research. This


analysis suggested a significant association between subtypes of C. jejuni (combined

serotype and PFGE subtypes) infecting humans who reported direct contact with dairy

cows, cattle, and live chickens and the subtypes isolated from these animals.

For human cases who reported eating chicken at someone else’s home, there was a weak

association, at 90% significance level, with the serotype of C. jejuni with which they were

infected and with that isolated from chicken. Similarly, there was a weak association for

human cases who reported eating beef at home and the serotype of C. jejuni with which they

were infected and those isolated from beef products. The serotype isolated from cases who

reported drinking untreated water in the last 10 days were also associated, at 90%

significance level, with the same serotypes of C. jejuni as those isolated from water.

5.13 Conclusions about Linkages

The animal and water components of this study have shown that New Zealanders living in

rural South Canterbury live in an environmental sea of Campylobacter. These organisms are

ubiquitous in animal and bird reservoirs, which in turn contaminate surface water and the

terrestrial environment through their infected faeces. Any person living and working in this

environment is likely to be heavily exposed to this micro-organism.

This study has also shown, at least among people with campylobacteriosis, that risk

behaviour is common, including consumption of unpasteurised milk and drinking water

(rural and urban). Given that many of the supplies are likely to be located on rural properties

the most probable source of any Campylobacter contamination would be from livestock. An

exception might be roof water where contamination from birds may also be significant,

however only one human case indicated that their water supply was sourced from rainwater.

A large river water study was conducted (The Freshwater Microbiological Programme,

MfE) to investigate the presence of both pathogens and indicators in water. This study

identified both the presence and numbers of Campylobacter in a high percentage of rivers.

Campylobacter was detected in 60% of water samples from the 25 recreational fresh water

sites tested throughout NZ (Till et al., 2002).


A total of 20% of cases consumed raw milk (Table 22). The most likely source of

Campylobacter in raw milk is from bovine faecal contamination during milking and hence

the subtypes isolated from raw milk could be predicted to be broadly similar to those found

in the dairy herd being milked. However, raw milk was not sampled in the CTR study.

These findings are consistent with what is already known about the sources of human

Campylobacter infection in New Zealand and other developed countries. They support, in

part, the results of the MAGIC study (Eberhardt-Phillips et al., 1997), which found

significantly elevated risks of disease among those who reported consuming chicken (an

aspect of the epidemiology not reflected to any large degree in the CTR results) and

handling calf faeces.

A similar approach as that used in the CTR study could be applied to a more predominantly

urban population. It is interesting to note that, from the most recent data available (March

2002), campylobacteriosis rates higher than the national average were recorded in

Wellington, Hutt, Taupo, South Canterbury, Waikato, North West Auckland, Central

Auckland, Taranaki and Hawkes Bay Health Districts. No rural/urban divide is discernible

among the areas affected by higher than average rates of campylobacteriosis. The two most

urban health districts in New Zealand, Wellington and Central Auckland, are included with

others that are predominantly rural, for example, South Canterbury. It could be that

distinctly different sources of infection exist in rural and urban areas, this supposition has

previously not been considered in depth.

5.14 Limitations of this analysis

The potential for this study to identify transmission pathways/routes/linkages was limited by

the following:

Small size of the Pilot Study

This limitation was particularly important for human cases, where both epidemiological

information and typable isolates were only obtained for 61 people. Approximately twice as


many human cases had been expected in this population based on previously observed rates

of infection.


Lack of dominant micro-organism subtypes

A striking feature of these results, at least with the subtyping systems being used here, is the

absence of dominant Campylobacter subtypes in the matrices examined. It is only at the

species level that any dominant infection is apparent, as is seen for C. jejuni which appears

to be dominant in humans, water and all animals except sheep (where C. coli was isolated

just as frequently). This feature of the biological system inevitably limits the power of the

study to propose definitive transmission pathways and is also exacerbated by the resultant

small sample size. In particular the analysis of human exposures was hampered by a

combination of a relatively small sample size and a large diversity in the number of

subtypes; therefore only very simple analyses were undertaken which were only able to

provide indicative results.

Sampling issues in food, water, animal and environmental

This study suggests that food, water, animals and the environment are being contaminated

with a wide range of Campylobacter subtypes. It will therefore be difficult for such a study

design to sample from these matrices in a way that conclusively establishes infection

sources for human cases, or transmission pathways/routes/linkages within the environment.

This is in part because it is impossible to be continuously sampling every matrix and every

unit within each matrix. An isolation of one subtype from, say, offal one week and then not

the following week, does not exclude the possibility that the same subtype was actually

present in offal in that second week. The subtype may have been present in the following

week but on a different piece of offal or a portion of sample not selected for isolation.

However, it is also likely that if a subtype was isolated from a unit in a matrix then it is

highly likely that there are other units in that matrix that are likely to also to have that

subtype.

Exeter medium, the enrichment medium used to culture Campylobacter, is a selective

medium, which excludes most non-Campylobacter species and non-Thermophilic

campylobacters. It is recognised that not all C. jejuni or C. coli strains are equally enriched


by Exeter, but by maintaining the same procedure for all matrices, the bias of different

enrichment methods was minimised.

Similarly there are no data on whether subtypes can be isolated on a continuing basis from

faeces from the same animal or herd or whether subtypes turn over rapidly. Arguably,

human isolates have far fewer associated sampling issues as the study aimed to include all

detected human cases.

It is likely that a proportion of the samples tested contained a number of subtypes, only one

of which was isolated and identified. Since very little information is available concerning

how many subtypes might be expected per sample type, it was considered that single

isolations from many samples in a matrix would be more representative than numerous

isolations from a much smaller number of samples. A small survey of multiple isolates from

water samples (Ministry of Health Report, 2001, FW0149) demonstrated that the majority of

isolates (12 of 13 isolates) from each water sample were of one PFGE subtype.

In addition, some potential animal reservoirs were not sampled at all, including domestic

animals (cats, dogs) and wild animals and birds, other than ducks.

Genomic Stability

The genome of Campylobacter undergoes recombinational events quite readily, therefore

genotypic subtyping results need to be interpreted with caution when proposing definitive

transmission pathways/routes/linkages. In the example of PFGE subtyping, one band shift in

the PFGE pattern may indicate that the two isolates are clonally related subtypes. This is in

contrast to E. coli O157, which is regarded as a highly clonal organism with infrequent

genotypic changes. Therefore a one band shift between two E. coli 0157 isolates typed by

PFGE may indicate that the two isolates are not clonally related (Ribot, 2002).

General Application of CTR study conclusions to other regions

A further limitation of this study is the general application of the conclusions from the CTR

study to other regions. For good reasons it has focused on a single geographical area.


Inevitably this area is not representative of New Zealand as a whole. Obvious differences

include the low proportion of Maori and Pacific People, and the relatively high proportion

of people living in rural areas. Some of the foods available in this area, such as chicken,

came from a single supplier, which again is not a typical situation. Findings from this study

therefore need to be interpreted with caution when applying them to the New Zealand

population as a whole.

5.15 Implications for public health

Findings from this study support public health advice in the following areas:

• Farmers and their families should take precautions to avoid becoming infected following

contact with farm animals and birds. Such precautions include careful handwashing

following contact with animals and the farm environment, and especially prior to eating

or smoking where ingestion of the organism might occur.

General points to be reiterated include:

• The public should avoid drinking untreated water and unpasteurised milk.

• The public should thoroughly cook chicken and offal derived from cattle, sheep and

pigs, and avoid cross-contamination of other foods through contact with raw chicken

and red meat products.


RECOMMENDATIONS

1. Conduct an enteric disease (campylobacteriosis) intervention study in a rural area,

based on the findings of the CTR study. This study could be carried out in the Ashburton

area to build on data from this present research project.

2. Include other potential reservoirs in additional future studies, notably companion

animals and asymptomatic household members.

3. Further investigate potential transmission routes to humans on farms, particularly the

role of direct animal contact, consumption of unpasteurised milk and untreated water

and the effects of farming practices.

4. Carry out an investigation of potential Campylobacter linkages in an urban population

by focusing on a larger number of samples from a smaller number of reservoirs and/or

transmission routes.


6. CONCLUSIONS

It is important to note that the results of this study are relevant to the Ashburton area

specifically, and to our particular sampling scheme, and may not be completely applicable

to New Zealand as a whole.

In terms of the prevalence of Campylobacter in the samples tested the results concurred

with previous studies, with the exception that isolations from chicken were less frequent. In

addition the ratio of C. jejuni to C. coli isolated from human cases was congruent with the

widely recognised pattern of disease. C. coli was isolated at higher prevalence than was

expected from sheep faeces, although this did not extend to isolates from sheep offal.

Generally, subtypes isolated were similar to those previously found in New Zealand.

Comparison of existing C. jejuni Penner serotyping data with those for CTR isolates

indicate that the serotypes isolated from this study generally concur. Therefore the pattern of

Campylobacter species and subtypes in the Ashburton area is unlikely to be either unusual

or different from the overall New Zealand situation.

Analysis of the CTR data employed three major approaches 1) use of the Czekanowski

Index to estimate the similarity in the spectrum of isolates obtained from each of the

matrices in a pairwise analysis; 2) analysis of the subtypes in human cases exposed to a

potential risk factor compared to those human cases who were not and 3) descriptive

analysis of potential linkages based on the collation of data derived from subtyping

(Penner/PFGE), spatial, temporal and epidemiological analyses. The results produced by

these three approaches were largely consistent, however , the three analyses, in particular

human risk factor analysis can only be considered to be indicative due to the small sample

size, level of diversity and multiple univariate tests or comparisons undertaken.

Briefly, analysis by use of the Czekanowski index showed that subtypes of C. jejuni isolated

from ruminant animal sources, whether faeces or meat, were the most similar to one another.

They were also the most similar to those isolated from human cases.


The data was too sparse in that there were too many Campylobacter subtypes distributed

among the small number of human cases for firm conclusions to be made from risk factor

analysis. However, indicative results are that contact with bovine animals and live chickens

are the more important risk factors for this study population.

Analysis on a case-by-case basis largely failed to provide compelling evidence to identify

definitive transmission routes/linkages (third approach) by use of bacterial subtyping,

temporal and geographical data. Any analyses of this nature were necessarily complicated

by the numerous potential exposures reported by the cases. The linkages identified

indistinguishable Campylobacter subtypes common to ruminant animals (faeces and meat)

and humans. This linkage data supported the findings of the Czekanowski analysis.

The main conclusion that can be drawn from the three analyses is that, for the population

sampled, bovine animal contact, direct or indirect, was the highest risk factor identified in

the CTR study.

While, for the purposes of the current study, there was good reason to focus on the

Ashburton area, the question of how typical this area is of New Zealand as a whole, must be

addressed. It could be speculated that this largely rural community is actually quite typical

of New Zealand rural communities in general and that the epidemiology prevailing here

would also apply in those other rural areas. It is not possible however, to generalise these

findings to the New Zealand population as a whole. The sources of infection in large urban

areas such as Auckland and Wellington are likely to be different since exposures to farm

animals, untreated water and unpasteurised milk would be fewer.

A proposed follow-up study should confirm the rural connection and identify the sites of

transmission. These linkages could be investigated for other zoonotic organisms. Finally,

the truly urban lifestyle of New Zealand could be investigated.

The results of the current study are extremely useful in identifying risk management options

for rural communities. Intervention messages, such as, educational messages counselling

against the consumption of raw milk and avoiding the consumption of untreated water could

be conveyed to the general public. Farmers, farm workers, people living on farms, people


visiting farms and others with occupational exposure to animals may not be aware that

ruminant faeces and faeces from other animals, may contain pathogenic bacteria. If this fact

were common knowledge then contact of this nature might be avoided. People in direct

contact with animals need to wash their hands thoroughly prior to activities such as eating

and smoking, where cross contamination and inadvertent consumption of Campylobacter

could occur.

Campylobacter is the most commonly notified disease in New Zealand accounting for

almost 50% of notifications in 2001. Results of the CTR study suggest that bovine animals

may be an important reservoir and source of infection for rural New Zealanders. Although

this link has been observed in international studies it could have greater significance for the

New Zealand setting. A high proportion of New Zealanders live in or have contact with

rural environments. The role of bovine animals as a source of human Campylobacter

infection needs to be confirmed and quantified. It would also be useful to investigate the

role of this animal source for other important enteric diseases, notable salmonellosis,

giardiasis, cryptosporidiosis, and STEC. Such work would support the development of

effective interventions.


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GLOSSARY

Clonally related

Genetically related isolates are said to be clones. Tenover et al., (1995) describes clones as

“isolates that are indistinguishable from each other by a variety of genetic tests (e.g., PFGE

and ribotyping) or that are so similar that they are presumed to be derived from a common

parent [Given the potential for cryptic genetic changes detectable only by DNA sequencing

or other specific analyses, evidence for clonality is best considered relative rather than

absolute (Eisentein, 1989)].”

Commensal

An organism, which is regarded as part of the normal flora of a host. It resides in its host

without causing disease.

CTR

Campylobacter Transmission Routes. The name given to the study discussed in this report.

Decimal reduction time

Time for 90% of organisms to die off, i.e. 1 log10 removal

Episurv

Surveillance network database of notifications of infectious disease within New Zealand. It

contains the results of questionnaires completed by cases reporting a notifiable disease.

Genome

The genetic constitution of a microorganism.

Genotype

The recognised “subtype” of a micro-organism’s genome.


Log

Abbreviation for logarithm, which is the index of the power to which a fixed number or base

must be raised to produce the number".

Natural competence

The ability of a bacterial species to take up foreign DNA from the environment and

incorporate it into its own genome.

Notified Disease

The decision to make a disease notifiable is based on the disease’s public health importance,

as measured by such criteria as incidence, impact and preventability. Notification confers

special status. It provides a legal requirement for reporting, enables cases of disease to be

notified without breaching the Privacy Act, and should ensure more complete identification

of cases.

Odds Ratio

The ratio of two odds (e.g. the odds of disease in individuals exposed and unexposed to a

factor). Often taken as an estimate of the relative risk in a case-control study.

Penner Serotyping

Penner Serotyping is a phenotypic subtyping method, which relies on the detection of

antigens present on the surface of microorganisms. It was developed by Penner and

Hennessy (1980). It uses the technique of passive haemagglutination to differentiate

Campylobacter species isolates on the basis of their soluble heat-stable (HS) antigens.

Phenotype

The measurable, expressed, physical and biochemical characteristics of an organism, which

are a result of the interaction between its genotype and environment.


Polymerase Chain Reaction (PCR)

Two oligonucleotide primers, complementary to two regions of the target DNA to be

amplified, are added to the target DNA, in the presence of excess deoxynucleotides and a

heat-stable DNA polymerase. In a series of temperature cycles (typically 30), the target

DNA is repeatedly denatured at 95°C, annealed to the primers at 50-60°C and a daughter

strand extended from the primers, at 72°C. As the daughter strands, themselves, act as

templates for subsequent cycles, DNA fragments matching both primers are amplified

exponentially, rather than linearly.

Prevalence

Is the proportion of a group of samples that are positive by PCR or culture.

Pulsed Field Gel Electrophoresis (PFGE)

Pulsed field gel electrophoresis (PFGE) is a genotypic method, which cleaves the entire

bacterial genome with rare-cutting restriction endonucleases. The resulting DNA fragments

are separated by size difference in an agarose gel. The gel is run under special

electrophoretic conditions that switch the orientation of the electric field in a pulsed manner

to separate the large (20 to 500kb) DNA fragments.

Reservoir

The habitat in which an infectious agent normally lives, grows and multiplies. Reservoirs

include human reservoirs, animal reservoirs, and environmental reservoirs.

Transmission Route

Any mode or mechanism by which an infectious agent is spread through the environment or

to another person.

Restriction Endonuclease

Enzymes produced by micro-organisms which each recognise specific short palindromic

base sequences in DNA. They cut the DNA helix at a particular point within the recognised

sequence.


Strain

A strain is an isolate that can be distinguished from other isolates of the same species by

phenotypic and/or genotypic characteristics. A strain is a descriptive subdivision of a

species (Tenover et al., 1995).

Subtype

Subtyping to differentiate isolates to the strain level of a bacterial species by employing a

combination of subtyping methods. In the CTR study Penner serotyping and PFGE

subtyping were the combination used to derive a strain of C. jejuni.

Town water supply

This term is the equivalent of community drinking water supply as defined in the Drinking

Water Standards for New Zealand 2000: “A publicly or privately owned drinking water

supply which serves more than 25 people for at least 60 days per year.”

Well/Bore water

Well/ Bore water is derived from ground water, which is water extracted from an

underground aquifer.


APPENDIX 1: DESCRIPTIONS OF SUBTYPING SYSTEMS

Table 25 Description of Phenotypic Subtyping Systems

Subtyping Method Target Brief DescriptionPenner Serotyping Heat stable antigens

on bacterial surfacePassive haemagglutination to differentiateCampylobacter strains on the basis of solubleheat-stable (HS) antigens

Laboratory of EntericPathogens (LEP)

Heat stable antigenson bacterial surface

Modification of Penner serotyping system,which uses absorbed antisera in an effort toovercome cross reactivity associated with thePenner scheme

Lior serotyping Heat labile antigenson bacterial surface

Slide agglutination procedure using livebacteria together with unabsorbed andabsorbed antisera

Multi Locus EnzymeElectrophoresis (MLEE)

Isoenzymes i.e.alleles of the sameenzyme

Protein extracts are electrophoresed throughstarch gels and screened for various enzymes.The different mobilities of each enzyme aredependent on allelic differences.

Plasmid Profile Analysis Plasmids The presence /absence of plasmids isascertained for each bacterial isolate (basedon a plasmid size library for each bacterialspecies)

Phage subtyping Bacteriophage The susceptibility to lysis by a panel of phageis ascertained for each bacterial isolate

Table 26 Description of Genotypic Subtyping Systems

Subtyping Method Target Brief DescriptionPulsed Field GelElectrophoresis (PFGE)

Entire Genome Restriction enzyme (RE)cleavage followed by DNAseparation on agarose gel.Different DNA cleavagepatterns are indicative of strainvariation.

Denaturing Gradient GelElectrophoresis (DGGE)

Entire genome or specificgene e.g. flagellin

RE Cleavage of DNA isfollowed by denaturinggradient gel electrophoresiswhich detects differences inthe melting behaviour of smallDNA fragments (200-700 bp)that differ by as little as asingle base substitution


Subtyping Method Target Brief DescriptionRestriction Fragment LengthPolymorphism (RFLP)

Gene(s) specific PCR amplification of aspecific gene(s) followed byRE cleavage and separation byelectrophoresis. DifferentDNA patterns are indicative ofstrain variation.

Random AmplifiedPolymorphic DNA (RAPD)

Entire genome PCR amplification using shortrandom (non-specific) primerswhich amplify regions of thegenome. The number andlocation of these sites variesfor different strains of abacterial species. Separationof the PCR products byelectrophoresis generatesdifferent patterns, which areindicative of strain variation.

Amplified Fragment LengthPolymorphism (AFLP)

Entire genome Restriction digestion ofgenomic DNA by two RE.PCR of the fragments by twoprimers based on the two REsequences amplifies only thosefragments flanked by both REsites. One of the primerscontains a fluorescent orradioactive label and PCRproducts are analysed ondenaturing polyacrylamidegels. 80 – 100 bands aregenerated by this technique.

Multi Locus Sequence Typing(MLST)

Entire genome Double stranded DNAsequencing of at least 7conserved genes in anorganism. Comparison of theallelic differences within eachgene is indicative of strainvariation.

Ribotyping/riboprinting Multiple copies of ribosomalRNA gene(rRNA)

Cleaved genomic DNA iselectrophoresed followed bySouthern blot hybridisationwith a probe specific forrRNA genes.NB Campylobacter containsonly three copies of rRNAgenes.


APPENDIX 2: MODIFIED CROWN PUBLIC HEALTH QUESTIONNAIRE

CAMPYLOBACTER TRANSMISSION ROUTES PROJECT

Campylobacter is the most commonly reported notifiable disease in New Zealand with an

annual rate of over 300 cases/100,000. It is more than three times higher than the rate of

Campylobacter reported by other developed countries.

The purpose of this project is to determine the likely sources of Campylobacter in the

environment. This knowledge is essential if the identified sources of contamination are to

be managed in a way that leads to a reduction in the risk of illness from Campylobacter.

The aims of this project will be achieved by analysis of the information contained in

questionnaires that will be completed by each person in the South Canterbury that contracts

Campylobacter during 2001, and analysis of animal faeces, food and water over the same

period. The Campylobacter strains isolated from these environmental reservoirs will be

compared with those from humans suffering from campylobacteriosis. Information about

species and strains present in the environmental samples and human faeces will allow a

better understanding of the movement (i.e. transmission routes) of Campylobacter through

the environment and food chain to the human population.

The Campylobacter strain that caused your infection will be obtained from the pathology

laboratory, which diagnosed the disease. There will be no need for you to provide a further

stool sample.

The information obtained from this questionnaire will be confidential and your anonymity is

assured. Only the case identification number will identify the details supplied by the

questionnaire. The cover sheet, which is the only place where your personal details are

recorded, will be separated from the body of the questionnaire and stored in a secure cabinet

separate from the questionnaire. Only the project leader of the Transmission Routes of

Campylobacter Project will have access to your name. Nobody else will be able to link the

information you give in the questionnaire with your name. This information will be

destroyed at the completion of the project.


PARTICIPANT CONSENT

I, ………………………………… consent to ESR to type the Campylobacter strains that

have already been isolated by the pathology laboratory and to use the information provided

in the questionnaire for the Campylobacter Transmission Routes Project. I do so on the

assurance that my / my child’s personal information is not given to any third party and that

the information will be destroyed at the completion of the project.

Signed ………………………………………… Date ………………………

Witnessed …………………………………….. Date ………………………

I wish / do not wish to receive a summary of the report’s findings upon completion of theproject. Please strike out that which is not applicable.

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ere

you

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Yes

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163

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ase

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ased

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lam

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es

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al (l

iver

, kid

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ils.

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In th

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beco

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165

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9

If y

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type

s use

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ell/b

ore

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ode

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……

……

……

10In

the

10 d

ays

befo

re th

e st

art o

f yo

ur il

lnes

s, di

d yo

u dr

ink

wat

er f

rom

any

whe

re o

ther

than

you

r ho

me,

wor

k, sc

hool

or p

resc

hool

?

Yes

No

Unk

now

n

Pote

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167

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If y

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Num

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/Nam

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Pre

mis

es/T

own/

City

Zone

C

ode

……

……

……

……

11D

id y

ou d

rink

any

untre

ated

wat

er (e

.g. f

rom

a ri

ver,

stre

am, l

ake,

wel

l, et

c), w

ell/b

ore

wat

er o

r rai

nwat

er in

the

10 d

ays b

efor

e th

e st

art o

f you

r illn

ess?

Yes

No

Unk

now

n

If y

es, s

peci

fy d

etai

ls:

Rec

reat

iona

l Wat

er C

onta

ct

12D

id y

ou h

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recr

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nal c

onta

ct w

ith w

ater

dur

ing

the

10 d

ays b

efor

e th

e st

art o

f you

r illn

ess?

Yes

No

Unk

now

n

Pub

lic sw

imm

ing

pool

, spa

poo

l, or

any

oth

er ty

pe o

f poo

l (e.

g. sc

hool

, hos

pita

l, m

otel

, priv

ate

pool

)

Nam

e of

poo

l(s)

Dat

e of

exp

osur

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omm

ents

(in

clud

ing

whi

ch p

ool(s

) yo

u sw

am i

n if

a la

rge

com

plex

)

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…./…

……

./……

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……

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……

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….

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./……

….

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ntia

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nsm

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on R

oute

s of C

ampy

loba

cter

168

Augu

st 2

002

From

Env

iron

men

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ans

Stre

ams,

river

s, be

ache

s, et

c

Nam

e of

stre

am/ri

ver/b

each

Dat

e of

exp

osur

eC

omm

ents

……

…./…

……

./……

….

……

…./…

……

./……

….

Oth

er re

crea

tiona

l con

tact

with

wat

er (p

leas

e sp

ecify

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(e.g

. an

y ac

tivity

in

whi

ch w

ater

can

be

swal

low

ed,

e.g.

sur

fing,

kaya

king

, win

d su

rfin

g, d

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g, e

tc)

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……

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13If

the

case

is a

chi

ld, d

o th

ey a

ttend

scho

ol, p

resc

hool

or c

hild

care

?

Yes

No

Unk

now

n

If y

es, s

peci

fy d

etai

ls:

Num

ber/S

treet

/Nam

e of

Pre

mis

esSu

burb

Tow

n/C

ityO

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se O

nly

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Cod

e…

……

…

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ntia

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nsm

issi

on R

oute

s of C

ampy

loba

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169

Augu

st 2

002

From

Env

iron

men

t To

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ans

14A

re y

ou a

war

e of

any

oth

er p

eopl

e w

ho h

ave

rece

ntly

:

(a)

been

told

they

hav

e C

ampy

loba

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?Y

es

N

o

U

nkno

wn

(b)

expe

rienc

ed si

mila

r illn

ess?

Yes

No

Unk

now

n

Nam

e of

per

son

with

illn

ess

Dat

e ill

ness

star

ted

Rel

atio

nshi

p to

th

e ca

se

(e.g

. pa

rent

,ch

ild)

……

…./…

……

./…

……

.

……

…./…

……

./…

……

.

……

…./…

……

./…

……

.

15D

id y

ou c

hang

e an

y ba

bies

’ nap

pies

, or

have

oth

er c

onta

ct w

ith s

ewag

e or

oth

er s

ubty

pes

ofhu

man

faec

al m

atte

r du

ring

the

10 d

ays b

efor

e th

e st

art o

f the

illn

ess?

Yes

N

o

U

nkno

wn

If

yes,

wha

t di

d yo

u ha

veco

ntac

t with

?

Ani

mal

Con

tact

16a

Did

you

hav

e co

ntac

t with

(liv

e or

dea

d) a

nim

als d

urin

g th

e 10

day

s bef

ore

the

star

t of y

our i

llnes

s?

No

U

nkno

wn

Pote

ntia

l Tra

nsm

issi

on R

oute

s of C

ampy

loba

cter

170

Augu

st 2

002

From

Env

iron

men

t To

Hum

ans

If y

es, s

peci

fy s

ubty

pes

of a

nim

als

you

have

had

con

tact

with

and

not

e if

they

wer

e de

ad o

r had

diar

rhoe

a (“

scou

rs”)

.(T

ick

all a

ppro

pria

te b

oxes

).

Dog

s/pu

ppie

sY

es

No

Don

’t kn

ow

Dea

d A

live

Had

dia

rrho

ea

Cat

s/ki

ttens

Yes

N

o D

on’t

know

D

ead

Aliv

e H

ad d

iarr

hoea

Dai

ry c

ows

Yes

N

o D

on’t

know

D

ead

Aliv

e H

ad d

iarr

hoea

Cal

ves

Yes

N

o D

on’t

know

D

ead

Aliv

e H

ad d

iarr

hoea

Non

-dai

ry c

attle

Yes

N

o D

on’t

know

D

ead

Aliv

e H

ad d

iarr

hoea

Shee

p/la

mbs

Yes

N

o D

on’t

know

D

ead

Aliv

e H

ad d

iarr

hoea

Pigs

Yes

N

o D

on’t

know

D

ead

Aliv

e H

ad d

iarr

hoea

Chi

cken

sY

es

No

Don

’t kn

ow

Dea

d A

live

Had

dia

rrho

ea

Duc

ksY

es

No

Don

’t kn

ow

Dea

d A

live

Had

dia

rrho

ea

Wild

bird

sY

es

No

Don

’t kn

ow

Dea

d A

live

Had

dia

rrho

ea

Oth

er a

nim

als

Yes

N

o D

on’t

know

(s

peci

fy)

Dea

d A

live

Had

dia

rrho

ea

Pote

ntia

l Tra

nsm

issi

on R

oute

s of C

ampy

loba

cter

171

Augu

st 2

002

From

Env

iron

men

t To

Hum

ans

16b

Did

any

oth

er m

embe

r of t

he h

ouse

hold

hav

e co

ntac

t with

ani

mal

s dur

ing

this

per

iod?

Yes

No

Unk

now

n

If y

es, w

hat a

nim

als d

id th

ey h

ave

cont

act w

ith?

16c

Did

you

han

dle

anim

al m

anur

e or

dun

g as

you

mig

ht w

hen

gard

enin

g or

cle

anin

g up

afte

r a

pet

durin

g th

e 10

day

s be

fore

sym

ptom

s beg

an?

Yes

No

Unk

now

n

If so

, wha

t typ

e(s)

of a

nim

al d

ung?

16d

Do

you

smok

e ci

gare

ttes?

Yes

No

Unk

now

n

Ove

rsea

s Tra

vel

17W

ere

you

over

seas

in th

e 10

day

s bef

ore

the

onse

t of i

llnes

s?Y

es

N

o

U

nkno

wn

If y

es, d

ate

of a

rriv

al in

New

Zea

land

……

…./…

……

./……

….

Sequ

ence

Cou

ntrie

s Vis

ited

Dat

e of

arr

ival

in th

atco

untry

Last

……

…./…

……

./……

….

Seco

nd la

st…

……

./……

…./…

……

.

Third

last

……

…./…

……

./……

….

Oth

er T

rave

l

18D

id y

ou tr

avel

with

in N

Z du

ring

the

10 d

ays b

efor

e yo

ur il

lnes

s sta

rted?

Yes

No

Unk

now

n

If y

es, w

hich

pla

ces d

id y

ou v

isit?

Dat

es…

……

./……

…./…

……

.

Pote

ntia

l Tra

nsm

issi

on R

oute

s of C

ampy

loba

cter

172

Augu

st 2

002

From

Env

iron

men

t To

Hum

ans

……

…./…

……

./……

….

……

…./…

……

./……

.

19In

the

10 d

ays b

efor

e yo

ur il

lnes

s beg

an, w

ere

you

invo

lved

in a

ny o

f the

follo

win

g ac

tiviti

es?

If so

, whe

re?

Cam

ping

(incl

udin

g sc

hool

or s

imila

r typ

e ca

mps

)

Tram

ping

Farm

vis

it

Oth

er

Oth

er C

omm

ents

20A

re th

ere

any

othe

r com

men

ts y

ou w

ish

to a

dd (i

.e. o

ther

pos

sibl

e so

urce

s you

con

side

r may

hav

e ca

used

you

r illn

ess?

).

Sign

atur

e of

per

son

com

plet

ing

the

ques

tionn

aire

Dat

e…

……

./……

…./

……

….

Tha

nk y

ou fo

r co

mpl

etin

g th

is q

uest

ionn

aire

.

Plea

se r

etur

n it

in th

e st

ampe

d se

lf-ad

dres

sed

enve

lope

pro

vide

d.

Pote

ntia

l Tra

nsm

issi

on R

oute

s of C

ampy

loba

cter

173

Augu

st 2

002

From

Env

iron

men

t To

Hum

ans

Off

ice

Use

Onl

y

SOU

RC

EW

as a

sour

ce id

entif

ied?

Def

inite

Sus

pect

No

Unk

now

n

If d

efin

ite o

r su

spec

t, sp

ecify

sour

ce:

Pers

on to

per

son

cont

act w

ith a

noth

er c

ase,

spec

ify re

latio

nshi

p to

cas

e

From

con

sum

ptio

n of

con

tam

inat

ed fo

od o

r drin

k, sp

ecify

food

or d

rink

From

con

tact

with

infe

cted

ani

mal

, spe

cify

type

of a

nim

al

If d

efin

ite o

r su

spec

t, ho

w so

urce

was

impl

icat

ed:

Part

of a

n id

entif

ied

com

mon

sour

ce o

utbr

eak

Org

anis

m o

r tox

in o

f sam

e ty

pe id

entif

ied

in fo

od o

r drin

k co

nsum

ed b

y ca

se

Oth

er m

etho

d fo

r ide

ntify

ing

sour

ce, s

peci

fy m

etho

d

Tra

vel H

isto

ry

Prio

r his

tory

of t

rave

l – re

cord

as n

o in

all

inst

ance

s

CA

SE M

AN

AG

EM

EN

TC

ase

excl

uded

from

wor

k or

scho

ol/p

resc

hool

/chi

ldca

re u

ntil

wel

l?Y

es

N

o

N

A

U

nkno

wn

If th

e ca

se w

orks

as

a fo

od h

andl

er, o

r is

em

ploy

ed t

o ca

re f

or p

atie

nts,

elde

rly, o

rch

ildre

n le

ss

than

5

year

s of

ag

e,

was

th

e ca

se

excl

uded

fr

om

wor

k un

tilm

icro

biol

ogic

al c

lear

ance

ach

ieve

d?Y

es

N

o

N

A

U

nkno

wn


APPENDIX 3: TABLE OF MEAT PRODUCT SALES IN ASHBURTON

Table 27 A Comparison of Meat Volumes sold by Retailers in Ashburton and TinwaldTownships

Retailer FreshChickenNumbers perweek(% of totalvol.)

SheepVolume(kg)

(% of totalvol.)

Beef volume(kg)

(% of totalvol.)

Pigvolume(kg)

(% of totalvol.)

A 178 whole +90 rotisserie(57%)

180(7.6%)

1200(18.5%)

260(12.5%)

B 48(10%)

225(9.5%)

250(4%)

300(14.5%)

C zero 90-120(5%)

250(4 %)

60(3%)

D zero 160-220(9.3%)

350(5.4%)

180-240(11.5%)

E zero 120(5%)

1500(23%)

360(17.4%)

F 60(13%)

800(34%)

1440(22%)

450(21.7%)

G 93(20%)

700(30%)

1500(23%)

400(19%)


APPENDIX 4: LABORATORY PROTOCOLS FOR DETECTION OFCAMPYLOBACTER FROM ENVIRONMENTAL MATRICES

SECTION A: LABORATORY PROTOCOLS FOR ENRICHMENT OFCAMPYLOBACTER FROM ENVIRONMENTAL MATRICES

A.1 Procedure For Campylobacter Isolation From Samples Of Offal, Faeces, WaterAnd Chicken Carcass

Lab coats, gloves and eye protection must be worn at all times when carrying out thisprocedure. Wherever it is practical sample manipulations involving open tubes must becarried out in an approved biohazard cabinet.

A.2 Procedure for Samples that can be Homogenised: Meat (offal) and FaecalSamples (Figure 25)

A2.1 Materials and Equipment

35 ml Universal bottles (# LBS 3722W)Whirl-Pak Bags (# BO1020 WA, Nasco, Life Technologies)Gas jar (Oxoid, Basingstoke, Hampshire, England)CampyGen™ system (Oxoid, Basingstoke, Hampshire, England) or 10% CO2 incubatorIncubators: 42 ± 1ºC, 37 ± 0.5ºC“Exeter” agar plates (Section F)“Exeter” enrichment brothColumbia Blood Agar plates with 5% defibrinated sheep blood

A2.2 Procedure for offal and animal or human faecal samples

Please do not dispose of any of the original samples until two weeks after initialarrival. Samples should be stored at 4ºC.

A2.2.a Meat: Using sterile utensils, prepare a homogenate by weighing 10 g of diced meat(e.g. offal) into a sterile “Whirl Pak” bag and adding 90ml of “Exeter” enrichmentbroth (Section F). Stomach for 1 minute to mix in a Colworth Stomacher 400.

A2.2.b Faeces: Each animal faecal sample received will be a composite of 5 differentanimals in one sampling pottle to increase the likelihood of detectingCampylobacter. Therefore the animal samples will need to be mixed well with asterile spatula, before removing a representative 2.5 gram faecal sample.

Using a sterile spatula, prepare a homogenate by weighing 2.5 grams of faeces intoa sterile “Whirl Pak” bag and adding 50ml of “Exeter” enrichment broth (SectionF). Stomach for 15 seconds to mix. For chicken faeces the weight of faecal materialadded to the “Whirl Pak” bag is 1.0 gram (NB. chicken faeces are not routinelyanalysed in the PH3 project).


[Please note that if there is less than 2.5 grams of faecal material supplied, then thebest option is to add about 20 ml of enrichment broth to the sampling pottle andmix it with the faeces before transferring, aseptically, to the “Whirl Pak” bag.Another volume of Exeter broth can be used to rinse out the pottle to ensurecomplete transfer of all faecal material to the “Whirl Pak” bag.]

A2.2.c Primary Enrichment Broth: Incubate enrichment broth at 37 ± 0.5ºC for aminimum of 4 hours in a microaerophilic atmosphere. Transfer the jar containingenrichments to an incubator operating at 42 ± 0.5oC as soon as possible after the 4hours and continue incubation up to a total of 48 h.

A2.2.d Secondary Enrichment Broth: Using a sterile pipette, transfer 0.1 ml of theenrichment broth into a 10 ml “Exeter” enrichment broth and incubate in amicroaerophilic atmosphere at 42 ± 0.5ºC for 24 h.

A2.2.e Performance of controls is checked before any results of samples are recorded(refer Section B).

A.3 Procedure for Poultry Carcasses


35 ml Universal bottles (# LBS 3722W)Sterile plastic bag, Model 3500 from Seward Stomacher Lab SystemWhirl-Pak Bags (# BO1020 WA, Nasco, Life Technologies)Gas jar (Oxoid, Basingstoke, Hampshire, England)CampyGen™ system (Oxoid, Basingstoke, Hampshire, England) or 10% CO2 incubatorIncubators: 42 ± 1ºC, 37 ± 0.5ºC“Exeter” agar plates“Exeter” enrichment brothColumbia Blood agar plates with 5% defibrinated sheep blood

A3.2 Procedure for Poultry Carcasses


A3.2.a Using sterile utensils, place the packaged chicken carcass into a sterile plastic bagwith 250 ml of buffered peptone water, BPW (Section F). Ensure that the bag issecurely closed. Rinse the surface by massaging the carcass with the BPW diluent.

A3.2.b Using a sterile pipette, transfer 10 ml of the rinsings into a sterile “Whirl Pak” bag.Add 90 ml of “Exeter” enrichment broth. Stomach for 15 seconds.

A3.2.c Primary Enrichment Broth: Incubate enrichment broth at 37 ± 0.5ºC for aminimum of 4 hours in a microaerophilic atmosphere. Transfer the jar containingenrichments to an incubator operating at 42 ± 0.5oC as soon as possible after the 4hours and continue incubation up to a total of 48 h.


A3.2.d Secondary Enrichment Broth: Using a sterile pipette, transfer 0.1 ml of theenrichment broth (mix well) into a 10 ml “Exeter” enrichment broth and incubatein a microaerophilic atmosphere at 42 ± 0.5ºC for 24 h.

A3.2.e Set up controls for all enrichment batches as outlined in Section B.

A3.3 References for food analysis

Compendium of Methods for the Microbiological Examination of Foods, 3rd ed. APHA(1992) 29.4.

Adapted from Humphrey et al (1995) International Journal of Food Microbiology,26 295-303.

A.4 Procedure for Water Analysis


Vacuum/pressure pumpFilter apparatus, sterileMembrane filters, 0.45µm, sterileForceps: smooth-tipped100 µL pipettesIncubators: 42 ± 1ºC, 37 ± 0.5ºCAnaerobic jarGas jar (Oxoid, Basingstoke, Hampshire, England)CampyGen™ system (Oxoid, Basingstoke, Hampshire, England) or 10% CO2 incubatorIncubators: 42 ± 1ºC, 37 ± 0.5ºC

A4.2 Procedure for water analysis (Figure 25)


A sterile funnel must be used for each sample. Alternatively, funnels can bedisinfected between samples by immersing in boiling distilled water for 5 minutes andallowing to cool or by immersing in alcohol and flaming.

A4.2.a Using sterile forceps, place a sterile membrane filter on a filter-support base andattach the funnel.

A4.2.b Shake the sample bottle at least 25 times.

A4.2.c Filter 500ml of water. Apply vacuum to draw sample through. Rinse the sides ofthe funnel twice with 20-30 ml of sterile rinsing buffer (BPW) and turn the vacuumoff once the rinsing buffer has passed through the filter. Due to the large volume ofwater use as many filters as necessary if the first filter becomes clogged withdebris. Maintain aseptic techniques during the changing of any filters.


A4.2.d Carefully remove the filter with sterile forceps and place into a 100 ml “Exeter”enrichment broth in a “Whirl-Pak” Bag.

A4.2.e Primary Enrichment Broth: Incubate enrichment broth at 37 ± 0.5ºC for aminimum of 4 hours in a microaerophilic atmosphere. Transfer the jar containingenrichment broths to an incubator operating at 42 ± 0.5oC as soon as possible afterthe four hours and continue incubation up to a total of 48 h in a microaerophilicatmosphere.

A4.2.f Secondary Enrichment Broth: After the 48 hour incubation, mix each of theenrichment broths by inversion or gentle swirling then. Using a sterile pipette,transfer 0.1 ml of the enrichment broth into a 10 ml “Exeter” enrichment broth andincubate in a microaerophilic atmosphere at 42 ± 0.5ºC for 24 h.

A4.2.g Set up controls for all enrichment batches as outlined in Section B.


SECTION B: CONTROLS

B.1 All media is to be validated as required for the MfE project.

B.2 Test each batch of Exeter broth (Section F) against our Campylobacter multiplexprimer set as for the MfE project:

B.3 Set up controls for enrichment batches (Figure 24)For each batch of samples processed, positive, negative and sterility controls are tobe included at all stages in the procedure as listed below:

For primary enrichment:Positive controlsA) “Exeter” enrichment broth spiked with 100 µL aliquot Campylobacter

jejuni (NCTC 11351) grown 48 hours in “Exeter” brothB) 100 µL aliquot of Campylobacter coli (NCTC 11366) grown 48 hours in

“Exeter” broth.Negative control “Exeter” enrichment broth spiked with Escherichia coli

(ATCC 25922) grown 24 hours in Nutrient Broth.Sterility control Uninoculated “Exeter” enrichment brothRefrigerated Uninoculated “Exeter” enrichment broth stored at 4ºC

Please plate out all controls to check their culturability on Exeter plates.

Performance of controls is checked before the results of any samples are recorded.


Figure 24 Controls for Campylobacter Enrichment Process

Controls for Campylobacter enrichment in Exeter brothControls for enrichment : C. jejuni / C. coli / E. coli / uninoculated broth

On Tuesday Set up Primary enrichment controls = P 1

Transfer primary controls into secondary enrichment broths

=P 2

Plate out P 1 controls

On Thursday

On Friday

Plate out P 2 controls Secondary enrichment broths are sent to Molecular Bio. Lab for the harvesting and washing of cells from P 2 enrichment broths

Read plated controls from secondary enrichment Sunday

SaturdayRead plated controls of primary enrichment


Figure 25 Procedure for Enrichment of Campylobacter cells

Water sample

filtered

Cells in broth washed

Cells collected

Cells lysedto get DNA

DNA used for PCR

Enrichment 137ºC - 4 hrs42ºC - 44 hrs

Enrichment 1Filter placed

in tubein broth

Enrichment 242ºC - 24 hrs

Food/faecal sample

Stomach≤ 1min

Enrichment 1Whirl Pak

Bag


SECTION C: PREPARATION OF ENRICHMENT BROTH CELLS FORTESTING BY PCR

Secondary “Exeter” enrichment broths are received from the Public Health Lab (PHL) onthe Friday morning and the cells present in the enrichment broths are washed as outlinedbelow.

C.1 Cell Harvest and Washing (for further details refer to Figure 26).

Lab coats, gloves and eye protection must be worn at all times when carrying out thisprocedure. Wherever it is practical sample manipulations involving open tubes must becarried out in an approved biohazard cabinet.

C1.1 Label 1.5ml microcentrifuge tubes (2 for each sample).

C1.2 Add 1ml of the secondary “Exeter” enrichment broth sample to each of the twotubes and repeat for all samples. Note: The two sample sets are processedseparately from here on.

C1.3 Centrifuge one of the 1 ml sample sets at 7000 rpm for 20 minutes at 4ºC. C1.4 Remove supernatant from each tube and add 1ml of Phosphate Buffered Saline

(PBS), vortex to resuspend cells. Centrifuge 7000 rpm for 10 minutes. C1.5 Repeat 1.4. C1.6 Remove supernatant and add 400µl of PBS. Resuspend cells in the PBS and store

at -20ºC until required.

C.2 Long Term Sample Storage

C2.1 Prior to commencing cell washing, six glass balls are added to sufficientmicrocentrifuge tubes for the number of samples to be processed. The tubes arethen autoclaved, and dried before adding 500µl BHI broth containing 20% glycerolto each tube.

C2.2 Centrifuge the second set of samples at 3000 rpm for 20 minutes. C2.3 Remove supernatant from each tube and add 500µl Brain Heart Infusion (BHI)

broth to which 20% glycerol has been added. Resuspend cells. C2.4 Transfer the resuspended cells to the microcentrifuge tube containing the glass

balls and broth. C2.5 Label with sample number, gently mix and store at -80ºC until required.


Figure 26 Bacterial Cell Harvest and Washing

PH3 MoH Campylobacter Project - Cell harvest and washing

“Exeter” broth culture 1ml

1.5mlseppendorf

1.5mlseppendorf

1.5mlseppendorf

Centrifuge 7000 rpm20 minutes 4ºC

Remove supernatantAdd 1 ml of PBSResuspend by vortexing


Centrifuge 3000 rpm20 minutes

Remove supernatantAdd 0.5ml BHI

+20% glycerol

Resuspend cells by manual pipettingand transfer to eppendorf containing glass beads

Mix gently, label andStore at -80ºC until

required

Add 10 glass beads

Autoclave and dry

Add 0.5mls BHI+20% glycerol


Remove supernatantAdd 400 uL PBSResuspend cells by manual pipetting

Remove supernatantAdd 1 ml of PBSResuspend by vortexing

Heat blast the washed cells (12 minutes at 100o C)

Centrifuge 12,000 rpm for 10 min.

Store at 4o C for same day PCRor store at -20o C long term


C.3 PCR Detection of Viable Campylobacter Cells

The secondary enrichment step was introduced to ensure that only viable cells weredetected. For example, in a water sample, the number of Campylobacter cells required to bepresent in the original sample to give a positive result has been calculated as follows:

Volume of water sample Number of Campylobactercells/100ml

10 ml 2.9 x 106

100 ml 2.9 x 105

500 ml 5.8 x 104

C3.1 It was calculated from these figures that at least 5.8 x 104 non-viable cells wouldhave to be present in the original sample to produce a false positive result, MOHreport FW9948, 1999.

SECTION D: STANDARD PROTOCOL FOR THE DETECTION OFCAMPYLOBACTER JEJUNI AND CAMPYLOBACTER COLI BYTHE POLYMERASE CHAIN REACTION

D.1 Sample Preparation

This step is to be performed immediately before PCR. Heat-treated samples are not verystable and must be amplified as soon as possible or stored at –20ºC.

Wear safety glasses during this procedure as heated tubes can explode spilling theircontents.

D1.1 Turn on 0.5 ml tube heating block and set at 100ºC.D1.2 Defrost samples to be tested.D1.3 Heat-treat samples at 100ºC for 12 minutes (check temperature on thermometer).D1.4 Centrifuge samples at 12,000 rpm for 10 minutes at 4ºC while preparing the

premixes.

CONTROLS for the PCR reaction.

D.1 Preparation of a positive PCR control. Measure 80 µl of ddH20 into a 0.5 ml tube.Add 10 µl of each working solution (100 µg/ml) of DNA from C. jejuni and C. colito give a final concentration of 10 µg/ml DNA per bacterial species. Mix andaliquot 33 µl of PCR positive control into 2 further tubes. Store in -20ºC freezer.Add 10 µl of this solution to the premix for positive control.

Negative PCR control. Add 10 µl of autoclaved deionised water to PCR negativecontrol premix.

D.2 Preparation of Premix

Prepare sufficient premix for all samples, and positive and negative PCR controls plus aspare tube (Table 28).


D2.1 Primer preparation

All stock solutions of primers prepared (in manufacturer's tubes) are at 100 nmoles/ml(100 picomoles/µl). To prepare a working solution for PCR, dilute all stock primers 1:10(10 µl primer + 90 µl ddH2O), to produce a working concentration of 10 pmoles/µl.

D2.2 Nucleotides (dNTPs)

These are purchased individually from Life Technologies as dATP, dCTP, dGTP and dTTPeach at a concentration of 100mM. To prepare a 25 mM solution of dNTP’s add equalvolumes of each (eg 25 µl) to a 0.5 ml tube.

D2.3 PCR Buffer (supplied by PE Biosystems with the Taq Polymerase enzyme)

10 X Buffer containing 50 mM KCl, 10 mM Tris, pH 8.4, with no MgC12 is used.

D2.4 Polymerase Enzyme

Taq Polymerase enzyme was purchased from PE Biosystems: Amplitaq, 5.0 units /µl.

D2.5 Magnesium Chloride

MgC12 is purchased from PE Biosystems as a stock concentration of 25 mM. It is added tothe premix to give a final concentration of 4.0 mM. BSA is also added to help prevent anyinhibition, (refer Section F for preparation).

D2.6 Distilled Water

Dnase / Rnase- Free Water purchased from Life Technologies, Gibco #10977-015.


Table 28 Template of the Premix for C. jejuni and C. coli specific PCR

Reagents Concentration perreaction tube (µl)

Volume per reactiontube (µl)

Add H2O to make final volumeof 50µl

e.g. 17.25

25 mM MgCl2 4 mM 8

BSA2 mg/ml

0.2 mg/ml 5

10 x PCR buffer 1 x 5

DNTPs(25 mM

each)

250 µM 0.5

Taq PolymeraseAmplitaq

(5 Units/ µl)

1.25 Units 0.25

Primer stock solutions10 picomoles/µl

Primer Set 1 forwardC. coli

10 picomoles 1.0

Primer Set 1 reverseC. coli 10 picomoles 1.0

Primer Set 2 forwardC. jejuni

5 picomoles 0.5

Primer Set 2 reverseC. jejuni

5 picomoles 0.5

Primer Set 3 ForwardThermophilic Campylobacter

5 picomoles 0.5

ReverseThermophilic Campylobacter

5 picomoles 0.5

Volume of master mix Per reaction

40 µl

Amount ofDNA template

e.g. 10 µl(for heat blasted cells)

Total volume 50 µl


D.3 Amplification of DNA

Forty µl of premix was aliquoted into each 0.2ml PCR tube without oil. The Perkin Elmer9700 thermal cycler has a hot top, which negates the need for an oil overlay. Ten µl ofsample or control DNA was added to the premix and tubes gently mixed. Tubes were brieflycentrifuged to ensure the entire sample was in the bottom of the tube and run on a PerkinElmer 9700 thermal cycler under the following conditions:

94ºC for 3 minute cycle 1 (denaturing)

94ºC for 1 minute (denaturing)60ºC for 1 minute cycles 2-41 (annealing)74ºC for 1 minute (extension)

74ºC for 8 minutes cycle 42 (extension)

D.4 Detection of PCR Product

Agarose gel:

D4.1 Gel Casting

D4.1.1 Prepare individual sterile 100ml Schott bottles each containing 1 gram ofagarose. When ready to pour gel add 50 ml of 1 x Tris Borate EDTA (TBE)running buffer (Section F). Loosen cap and heat in microwave on high power for1 minute, swirl gently and repeat heating for another 20 seconds or until the gelis homogeneously melted. Wear gloves and avoid contact with steam as theagarose mixture contains ethidium bromide. Allow the gel to cool for 15 - 20minutes.

D4.1.2 While gel is cooling set up the gel casting tray, ensuring it is level and using the22 lane comb for the Midicell system.

D4.1.3 Pour the gel into the casting chamber and allow to set - approximately 15minutes.

D4.1.4 Prepare running buffer (1 X TBE) by diluting 200 ml of 10X TBE with 1800 mlof deionised water. Add 100 µl of ethidium bromide and mix thoroughly. Addenough of the running buffer to the gel chamber to just cover the gel.

D4.2 Gel Loading

D4.2.1 Dot 3µl of loading buffer onto a piece of parafilm according to the number ofsamples to be run.

D4.2.2 Add 10 µl of the PCR product to the dot of loading buffer.D4.2.3 Mix each dot with pipette tip and load carefully into well, minimising DNA

spillage. Load 10 µl of the stock solution of the ‘1kb plus’ DNA ladder (SectionF) at the ends of the gel, either side of the samples.

D4.2.4 Once all samples are loaded, close the cover of the tank, plug in the electrodes,set the voltage at 100 Volts and run the gel for one hour and forty minutes oruntil the blue dye is at the front edge of the gel.


NOTE: The steps in B4.2 should be performed without delay and interruption.D4.2.5 When the time is up, unplug gel tank, and with gloves remove the gel from the

tank.D4.2.6 Transfer to the UV transilluminator and examine gel for DNA bands. Wear UV

protective visor for eye and skin protection.D4.2.7 Take a polaroid photo. Setting red filter, exposure f 5.6 for ½ sec. using black

and white film. Double expose the film.D4.2.8 Remove photograph from the film cassette by pulling the white tab and then the

black tab in one sweeping movement.D4.2.9 Leave the photo on the bench to develop for 45 seconds. Peel away the backing

to view the photograph.D4.2.10 Examine the bands and identify the Campylobacter species present.

Positive Controls: The following bands must be present in the positive control.

Primer Set 1 = 695 bp C. coliPrimer Set 2 = 99 bp C. jejuniPrimer Set 3 = 246 bp Thermophilic Campylobacter

NB To confirm the presence of C. jejuni in a sample, 2 bands must be visualised on theagarose gel: Primer Set 2 band at 99 bp and Primer Set 3 band at 246 bp.

To confirm the presence of C. coli in a sample 2 bands must be visualised on the agarosegel: Primer Set 1 band at 695 bp and Primer Set 3 band at 246 bp.

Negative Controls: No bands should be present in the negative controls except for theprimer –dimer band. This band confirms that the PCR reaction was not inhibited.

The detection limits of this enrichment PCR for each of the matrices sampled in the CTRstudy are shown in Table 29. The detection levels of Campylobacter by the conventionalplating method are also presented to enable comparison of the two methods.


Table 29 Comparison of Detection limits of Campylobacter for the Enrichment PCR Methodand the Conventional Plating Method

PCRConventional Method:

Plating from thePrimary enrichment

Matrix Sampled

Lowestnumber of

viable C. jejuni cells

detected

Lowestnumber of

viable C. coli

cellsdetected

Lowestnumber of

viable C. jejuni cells

Detected

Lowestnumber of

viable C. coli

cellsdetected

Beef Liver(per 10 grams)

one one one one

Pig liver(per 10 grams)

one one one one

Sheep Liver(per 10 grams)

one one one one

Chicken Carcass(per 10 ml

carcass washing)

one one one seven

River Water(per 100 ml)

one one one one

Duck Faeces(per 2.5 grams)

one one Fifteen one

Chicken Faeces(per 1.0 grams)

one two one two

Human Faeces(per 2.5 grams)

two one two one

Cattle Faeces(per 2.5 grams)

two eight two eight

Dairy Faeces(per 2.5 grams)

one one one one

Sheep Faeces(per 2.5 grams)

two one two one

In addition Savill et al. ( 2001a) using a different enrichment PCR found that 47% of watersamples were positive for Campylobacter, whereas 34% of the same water samples werepositive using the conventional method of plating.


SECTION E: PROCEDURE FOR ISOLATION AND RESUSCITATION OFC. JEJUNI AND/OR C. COLI

E.1 Procedure for Isolation and Resuscitation of C. jejuni and/or C. coli

E1.1 Secondary Exeter broths that have tested positive for C. jejuni and/or C. coli by theCampylobacter multiplex PCR method, are plated onto Exeter agar and incubatedmicroaerophically for 48 hours at 42°C.

E1.2 Isolated colonies from these initial Exeter plates are streak isolated onto ColumbiaBlood Agar (CBA) for 2 consecutive times, for 48 hours each, to ensure the purityof the isolate (Figure 27).

E1.3 The identity of the selected purified isolate is confirmed by the CampylobacterMultiplex PCR of a single isolated colony. The methodology for the PCR reactionis the same as presented in Table 28, with the exception that the distilled waterincreases to 27.25 µl. This is because whole cells are being added as the DNAtemplate instead of a suspension of washed cells.

E1.4 The double distilled water (DDW) is added to the PCR tube and a small portion ofa single isolated colony is added to the distilled water.

E1.5 The bacterial cells are lysed by heating in a thermal cycler (Perkin Elmer 9700) at94°C for 3 minutes and held at 4°C until the PCR premix is added. Thereafter thePCR cycle conditions and the running of the gel and visualisation of the DNA arethe same as the methods outlined in .

E2 Mixed C. jejuni and C. coli Samples

E2.1 A few samples contain both C. jejuni and C. coli bacteria. These samples areinitially isolated onto Exeter agar from the secondary Exeter broth or from thesecondary Exeter broth stored in the -80°C freezer. The plates are incubatedmicroaerophically for 48 hours at 42°C. Faecal samples can also be used to isolateC. jejuni and C. coli from mixed samples by direct plating of faeces onto Exeteragar. Recovery of Campylobacter is enhanced by suspending approximately onegram of faeces in 10 ml of Exeter broth prior to plating out loopfuls onto Exeteragar and incubating under microaerophilic conditions at 42° C for 48 hours.

E2.2 The Campylobacter Multiplex PCR then putatively identifies 8 isolated colonies as.At the same time as a portion of the isolated colony is being added to the DDW forthe PCR reaction another portion of the same colony is plated out onto a CBAplate. The plates are incubated microaerophically for 48 hours at 42°C. One ofeach C. jejuni and C. coli identified by PCR, is further purified by streak isolatingtwo consecutive times onto CBA plates. At this stage the Campylobacter MultiplexPCR confirms the identification of the bacteria.

E.3 When the isolates have been identified as either C. jejuni or C. coli they areprepared for transportation to Kenepuru Science Centre (KSC) for serotyping andPFGE analysis and/or to PaMS Microbiology Laboratory at the University ofCanterbury for PFGE analysis. Campylobacter strains sent to KSC are transportedas Charcoal swabs, whereas, the PaMS Microbiology lab receives Campylobacterstrains on CBA plates.

Figure 27 Campylobacter Isolation and Resuscitation


Secondaryerichmentbroth plated out

Resuscitation on Exeter= initial isolation

Isolation A on Exeterfrom a single isolated colonyon the initial isolation plate

Isolation B on Bloodfrom a single isolated colonyon the initial isolation plate

After PCR confirmation of a single isolated colony from Isolation plate B:Proceed to:1) -80ºC pureculture stock2) prepare charcioal swab for C. Nicol at KSC3) send remainder of plate to J.Klena’s lab

Water samples require a further isolation from a single colony

Isolation C :isolation streak of part of the same colonyidentified by PCR from isolation plate C. After PCR confirmation proceed with steps 1-3 as outlined above for isolation B plate

For water samples only


SECTION F: MEDIA AND REAGENTS

dd H2O = double distilled water

2% Agarose gel

1 g Agarose 2%5 ml 10 x TBE 1 x TBE45 ml dd H202.5 µl Ethidium Bromide 0.5 µg/ml

(10 mg/ml stock)

Heat in microwave until all agarose has dissolved. (for details, refer to section on gelcasting).

Brain Heart Infusion (BHI) Broth containing 20% Glycerol

Brain Heart Infusion Broth (Merck 1.10493) 3.7 gGlycerol (BDH # 10118 4K) 20 mlDeionised water 80 ml

Weigh the required amount of broth into a Schott bottle and add the water. Mix thoroughlyto dissolve broth, microwaving if necessary. Autoclave at 121°C for 15 minutes. Allow tocool and check the pH is 7.4 + 0.2. Store at 2-8°C for up to 3 months.

Bovine serum albumin (BSA) preparation (2 mg/ml)

Weigh out 100 mg BSA (Albumin, Sigma A-4503 from Global Science) into a Falcon tubeAdd 50 ml sterile dd H20 (2 mg/ml)Dissolve by shaking. Filter sterilise through a 0.2 µm filterDispense aseptically in 1ml aliquots into sterile 1.5 ml tubesStore in freezer at -20ºC

Buffered Peptone Water 1% (BPW)

Peptone Water (Merck # 1.07228) 25.5 gramDistilled Water 1 litrepH 7.2Autoclave at 121oC for 21 minutes

DNA Ladder (1kb plus supplied by Life Technologies

Make stock solution:1kb plus DNA (1.0 µg/µl) 30 µl

(Gibco # 10787-018, Life Technologies)1 x TBE Buffer 150 µlLoading Dye 40 µl


Aliquot into Eppendorf tubes and store at –20ºC

Ethidium bromide (10 mg/ml)

Weigh 10 mg Ethidium bromide (Sigma E-8751) into an Eppendorf tube. Add 1 ml ofdd H20. Store at 2-8ºC.

“Exeter” medium

Nutrient broth No. 2 (Oxoid) made according to instructions per one litre volumes. Afterautoclaving, add the following per litre:50 ml lysed horse blood,5 ml filter-sterilised solution containing 4% sodium metabisulphite, 4% sodium pyruvate

and 10% iron sulphate solution, (aseptically dispense solution in 5 ml amounts andstore in the freezer) 15 mg cefaperazone, (add 2ml/litre of filter sterilised stocksolution, 7.5 mg/ml) 2 vials Oxoid supplement SR117E.

Each vial of supplements supplies 2500 i.u. polymixin B, 5 mg rifampicin, 5 mgtrimethoprim and 50mg actidione. These components vary from the “Exeter” formulation bythe inclusion of actidione, but it provides the convenience of the commercial availability ofthe antibiotic supplement.

“Exeter” agar

Add 15 g of agar to a litre of nutrient broth No. 2 and boil to dissolve before autoclaving.Proceed as above for “Exeter” medium.

Gel-loading buffer –for agarose gels

0.25% bromophenol blue (Sigma #B0128)30% glycerol (BDH #10118 4K) in waterStore at 4ºC

Phosphate buffered saline (PBS)

Dissolve one PBS tablet (Oxoid) in 100ml of dd H20. Autoclave and store at 4ºC.

10 x TBE Buffer

108 g Trizma Base (Sigma T-8524) 0.9M55 g Boric acid (Sigma B-6768) 0.9M40 ml 0.5 M EDTA pH 8.0 0.02M


Dissolve the above in 900 ml dd H20 and make up to 1L;or purchase 10 x TBE powder from USB # 70454 which comes makes 200ml aliquots whenreconstituted.

1 x TBE (working TBE)

200 ml 10 x TBE 0.09M1800 ml dd H20 100 µl Ethidium Bromide 0.5 µg/ml

(10 mg/ml stock)or dilute 10 x TBE stock solution 1:10 in distilled water


APPENDIX 5: PULSED FIELD GEL ELECTROPHORESIS

STAGE 1 - PLUG PREPARATION

DAY 1

1.1 Plate cells onto Mueller-Hinton agar plates containing 5% sheep’s blood. Incubateat 37-43C in an atmosphere of 10% CO2 for 36 to 48 hours.

DAY 3

1.2.1 Scrape cells from the plates using a sterile cotton swab and place into a test tubecontaining 2ml of PETT IV buffer until the equivalent of McFarland’s Standardnumber 1 is achieved. This equates to about 4-6 colonies from a plate.

1.2.2 Aseptically transfer 1.5 ml of the cellular growth to a sterile 1.5 ml Eppendorf tube.

1.2.3 Pellet the cells by centrifugation (5K, 5 min). Discard the supernatant andresuspend the cell pellet using 150 µl PETT IV buffer.

1.2.4 Melt 1.6% agarose in water (0.16g agarose, Pulsed Field Certified Agarose, Bio-Rad Laboratories, 162-0137). Maintain molten at 56°C.

1.2.5 Mix 240 µl of 1.6 % agarose with cells and apply cell mixture to plug mold. Letwells harden and then transfer to a refrigerator for 10-20 minutes to solidify.

1.2 6 Remove plugs and place each one into a separate Universal tube containing 2 mlEC lysis buffer containing RnaseA.

1.2.7 Add 40 µl of 20 mg/ml proteinase K solution. Incubate plugs at 50-56°C for 24 to48h.

STAGE 2 - PLUG WASHES

2.1 Label a sufficient number of orange-capped Falcon tubes (50 ml conical centrifugetubes) with a description of the plugs to be washed (eg., strain name).

2.2 Carefully transfer a plug from a Universal tube to the appropriately labelled Falcontube using an alcohol-rinsed spatula. Note: the plugs should be very clear at thistime.

2.3 Add 25-50 ml sterile distilled water to each tube.

2.4 Incubate at ambient temperature for 20-30 minutes with infrequent mixing (eg. 10minutes).


2.5 Place a green plug stopper (Bio-Rad) over the end of a Falcon tube and pour thewater out into a 1 L Tripour beaker. If the plug has been caught in the plugstopper, use a spatula to carefully place it back into the Falcon tube.

2.6 Repeat steps 2.3 to 2.5 two more times.

2.7 Add 20-50 ml 1X TE buffer to each Falcon tube and gently mix.

2.8 Incubate at ambient temperature for 20-30 minutes.

2.9 Place a green plug stopper (Bio-Rad) over the end of a Falcon tube and pour thewater out into a 1 L Tripour beaker. If the plug has been caught in the plugstopper, use a spatula to carefully place it back into the Falcon tube.

2.10 Repeat steps 2.7 – 2.9 one more time.

2.11 Add 20-50 ml 1X TE to each Falcon tube and gently mix.

2.12 Refrigerate the plugs overnight in TE. Note: plugs can remain stored in thiscondition for years, although it is recommended that they be transferred to 1.5 mlEppendorf tubes for long term storage (saves space).

STAGE 3 - PLUG DIGESTION

3.1 Label two 1.5 ml Eppendorf tubes for each plug to be digested. Place 1 ml of 1X TEin one of the two Eppendorf tubes. Note: this is only necessary if plugs are beingremoved from the Falcon tube for the first time. If plugs are already in a 1.5 mlEppendorf tube, label one 1.5 ml Eppendorf tube.

3.2 Carefully clean a glass slide with 70% alcohol. Clean with alcohol a spatula and aPasteur pipette “hook” and a razor blade.

3.3 Transfer a plug to the clean, dry glass slide. Try to leave behind as much TE aspossible.

3.4 Section the plug into 6 roughly equal-sized strips. Tease apart one strip.

3.5 Working with one strip, cut the strip into two roughly equal parts.

3.6 Place one of the two half-strip sections into one Eppendorf tube. This section willbe digested by a restriction endonuclease.

3.7 Place the remaining strips and the remaining half-strip into the second Eppendorftube containing 1 ml TE. Place these tubes into a refrigerator for long-termstorage.

3.8 Clean the slide and the spatula with alcohol.


3.9 Repeat steps 3.3 – 3.9 until all plugs are finished.

3.10 Make a Master mix of the 10X restriction enzyme buffer, the restriction enzyme andwater for the plugs to be digested. For example, if you are digesting 10 sections,the Master mix should contain:

100 ul 10X Buffer A880 ul ddH2O20 ul SmaI (20 units per reaction)

3.11 Transfer 100 µl of Master mix to the half-section in an Eppendorf tube. Make sureto completely cover the slice with the reaction mixture. Incubate the reactionmixtures containing plugs at 25C for a minimum of 4 hours, preferably overnight.

STAGE 4 - ELECTROPHORESIS

4.1 Preparation of the agarose gel

4.1.1 Clean the agarose gel casting system (this includes the two clamping ends, the comb,and the black plate, which will support the gel).

4.1.2 Assemble the system, making sure that the black plate is neatly fitted into thegrooves of the white ends and the ends are sealed. Make sure that the comb doesnot touch the black plate.

4.1.3 Prepare 2 L of 0.5X TBE buffer from 10X stock. It is important that this stock isfresh (less than 2 weeks old), otherwise the buffering capacity of the system will bealtered. Remove 100 ml of the buffer to a 250 ml Erlenmeyer flask.

4.1.4 Weigh 1 g of pulsed field gel electrophoresis Grade agarose and place it into theErlenmeyer flask containing 100 ml 0.5X TBE buffer. Weigh the flask and recordthe weight.

4.1.5 Place a cotton stopper into the mouth of the Erlenmeyer flask and microwave theflask on high until the agarose has dissolved; this will generally be for 80-90seconds.

4.1.6 Reweigh the flask. Add water to make up for any loss in weight.

4.1.7 Let the flask cool for several minutes and then pour the agarose into the tray. Allowsufficient time for the agarose to harden prior to removing the comb.

4.2 DRIII set-up

4.2.1 Please seek out someone who has been trained in the use of the DRIII prior to youruse (if you are an initiate!).

4.2.2 Make sure that the electrophoresis chamber is clean and agarose free prior to set-up.


4.2.3 Fit the guide plate into the centre of the electrophoresis chamber.

4.2.4 Add the 1.9 L of 0.5X TBE to the electrophoresis unit prior to turning the unit on.Please make sure that this buffer is near ambient temperature so that the coolingcoils do not snap-freeze.

4.2.5 Turn the unit on (main switch) and turn on the pump. Set the pump at 70.

4.2.6 Make sure that any bubbles that might interfere with buffer flow are removed fromthe hoses PRIOR to turning the chiller unit on.

4.2.7 Turn the chiller unit on and set the temperature to 14C.

4.2.8 Allow the buffer to equilibrate to 14C before fitting gel.

4.2.9 Set the parameters of the run, if necessary. These parameters will include: the initialpulse time, the final pulse time, the angle of the pulse and the duration of the run.Typical parameters for Campylobacter are TI=10 sec, Tf=35 sec; 120° for 22h. DONOT PRESS RUN UNTIL YOUR GEL HAS BEEN LOADED.

4.3 Loading the gel

4.3.1 Carefully remove the comb from the agarose gel.

4.3.2 Remove samples from 25°C water bath. Carefully remove the digestion solutionfrom the Eppendorf tube and gently work the plug up from the bottom of the tubeusing the pipette tip.

4.3.3 Using a sterile spatula, place the plug into a well. Note: aim for consistency, that is,try moving the plugs so that they are always next to the left-hand side of the well.Load the lambda marker in lanes 1, 11 and 20 (for a 20 lane sample) and 1, 11, 21and 30 for a 30 lane gel. If possible, include a known Campylobacter control ontwo spots on the gel.

4.3.4 Secure the plugs in each well using PFGE-grade agarose in water.

4.3.5 Once set, place the gel into the guide plate in the electrophoresis chamber, makingsure that the gel is completely immersed in 0.5X TBE.

4.3.6 Push start and monitor the electrophoresis periodically during the run.


APPENDIX 6: RELATIONSHIPS BETWEEN C. JEJUNI PFGE SUBTYPES

Table 30 Related PFGE Subtypes of C. jejuni

C. jejuni PFGE Subtypes Related PFGE Subtypes

1 1a/1b/1c1a 11b 11c 11d None10 10b/10c/3110b 1010c 1012 None15 None16 None18 18a/18b/3/3h18a 18/18b/18c18b 18/18a/21118c 18a19 19b19b 19/19c/d/e/f19c 19b19d 19b19e 19b19f 19b200 200a/b

200a 200200b 200201 231202 none203 none204 none205 3d/3i206 206a

206a 206207 209208 none209 none210 none211 18b and 3h214 none215 none216 28217 none218 none219 none21 21a/c21a 21/21b21b 21a21c 2122 none


C. jejuni PFGE Subtypes Related PFGE Subtypes220 none221 221a

221a 221222 222b

222a 3b222b 222222c none223 228225 none226 none227 none228 223229 229a/248

229a 229230 none231 201232 none233 none234 234a

234a 234235 none236 none237 none238 none239 239a

239a 239241 241a

241a 241242 none243 none244 none245 none246 none248 229249 none250 33251 none252 none25 4/25a/25b25a 4/25/25b/7325b 4/25/25a/7325c none25d none26 none28 21629 none30 none31 1033 33a/47/3/3d/3e/25033a 3334 34a/b34a 3434b 3435 none


C. jejuni PFGE Subtypes Related PFGE Subtypes39 none3 3a/b/c/d/e/g/h/i and 18/33/47

3a 3/3d/3e/3g/47a3b 3/222a3c 3/3i3d 3/3a/3e/3g/33/47/47a/2053e 3/3a/3d/3g/33/473g 3a/3d/3e/47a3h 3 and 18 and 2113i 3/3c/2054 25/25a/25b

41 none40 none43 none44 none45 none47 47a/33/3/3d/3e47a 47/3a/3d/3g48 none52 none53 none54 54a54a 5457a 57b57b 57a58 none58b none59 none60 60a/b/d60a 60/60b60b 60/60a/60d60d 60/60b62 none64 none71 none72 none73 25a/25b9 9a

9a 9


APPENDIX 7: DISTRIBUTION OF C. JEJUNI SUBTYPES IN INDIVIDUALMATRICES

Figure 28 Distribution of C. jejuni Subtypes (combined serotype and PFGE) in individualmatrices

a) Humann=56

0%

10%

20%

30%

40%

50%

60%

70%

HS1

,44:

P33

HS2

: P16

HS2

: P1c

HS2

: P28

HS2

3,36

: P19

b

HS6

: PN

C

HSU

: P25

HS1

1: P

35

HS2

: P18

a

subt

ype

<2%

b) Water n=152

0%

10%

20%

30%

40%

50%

60%

70%

HS1

,44:

P16

HS1

5: P

60b

HS6

: PN

C

HS5

: P21

HS5

: P25

b

HSU

T: P

25

HSU

T: P

25b

HS8

,17:

P23

6

HSU

T: P

221

subt

ype

<2%

subt

ype

<1%

c) Duck n=58

0%

10%

20%

30%

40%

50%

60%

HS1

9: P

208

HS4

c: P

221

HS5

: P24

5

HS5

2: P

221

HSU

T: P

15

HSU

T: P

60d

HS3

7: P

229

HS3

7: P

248

HS8

,17:

P23

6

subt

ype

<1%

subt

ype

<2%


Distribution of C. jejuni Subtypes (combined serotype and PFGE) in individual matrices

d) Dairyn=87

0%

5%

10%

15%

20%

25%

30%

35%

HS2

: P20

6

HS2

: P3

HS2

: P33

HS2

3,36

: P19

f

HS3

5: P

31

HS4

c: P

52

HS5

3: P

29

HS1

1: P

35

HS4

c: P

34

HS2

3,36

: P19

b

subt

ype

<2%

subt

ype

<1%

e) Beefn=71

0%5%

10%15%20%25%30%35%40%45%50%

HS1

9: P

12

HS3

5: P

44

HS1

1: P

35

HS1

9: P

3d

HS2

: P3

HS3

5: P

31

HS4

c: P

34a

HS2

3,36

: P19

b

HS2

: P33

HS4

c: P

34

subt

ype

<1%

f) Sheepn=48

0%

10%

20%

30%

40%

50%

60%

HS1

0: P

18

HS4

c:P3

4b

HS2

3,36

:P1

9b

HS4

c: P

34

HS6

: PN

C

HS2

7: P

25

HS5

:P2

22b

subt

ype

<2%



g) Beef offaln=15

0%

2%

4%

6%

8%

10%

12%

14%H

S1,4

4: P

33

HS1

,44:

P3a

HS2

: P3

HS2

3,36

: P22

HS3

: P24

1a

HS4

c: P

34

HS5

: P22

2b

HSU

T: P

209

HSU

T: P

34b

HS1

9: P

3g

HS3

5: P

10

HS4

c: P

34a

h) Sheep offaln=63

0%

5%

10%

15%

20%

25%

30%

35%

HS2

: P16

HS2

: P52

HS2

3,36

:P19

HS2

3,36

: P22

HS4

c: P

204

HS4

c: P

34b

HS5

: P22

2

HS5

: P26

HS8

,17:

P33

HSU

T: P

207

HSU

T: P

54a

HS1

9: P

3g

HS2

3,36

: P19

b

HS4

c: P

34

HS1

,44:

P33

HS2

: P3

HS1

,44:

P3a

subt

ype

<2%



i) Chickenn=56

0%

5%

10%

15%

20%

25%

HS1

,44:

P24

6

HS1

,44:

P30

HS2

; P3i

HS2

1: P

25

HS2

1: P

NC

HS3

1: P

29

HS4

c: P

1

HS4

2: P

25

HS8

,17:

P24

4

HSU

T: P

29

HSU

T: P

4

HSU

T: P

223

HS2

: P3

HS2

1: P

60a

subt

ype

<2%

j) Pork offaln=9

0%

5%

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APPENDIX 9: POTENTIAL RISK FACTOR ASSOCIATIONSTable 32 Humans who had animal

contact – Cattle (dairycows, calves or non-dairycattle) in the last 10 days

Serotype PFGE No Yes Total1,44 222a 1 1

33 2 23a 1 141 1 1

10 18 1 118b 1 1

11 35 2 1 312 4 1 115 60b 1 118 211 1 1

251 1 119 12 1 1

19c 1 12 16 2 2

18a 4 418c 1 11c 1 1 21d 1 1206a 1 1220 1 1250 1 13 1 133 1 13b 1 154 1 1

22 28 2 223,36 19b 2 2

22 1 131 25 1 135 10 1 14 complex 1 1 1

1a 1 1200a 1 1243 1 1249 1 1252 1 152 1 154a 1 1

52 25 1 157 216 1 16 Non-cutting 2 2Untypable 25 2 2

3 1 172 1 1

Total 35 21 56

Table 33 Humans who had animalcontact – chickens (last 10days)


33 1 13a 1 141 1 1

10 18 1 118b 1 1

11 35 1 2 312 4 1 115 60b 1 118 211 1 1

251 1 119 12 1 1

19c 1 12 16 2 2

18a 4 418c 1 11c 2 21d 1 1206a 1 1220 1 1250 1 13 1 133 1 13b 1 154 1 1

22 28 2 223,36 19b 2 2

22 1 131 25 1 135 10 1 14 complex 1 1 1

1a 1 1200a 1 1243 1 1249 1 152 1 154a 1 1

52 25 1 157 216 1 16 Non-cutting 2 2untypable 25 2 2

3 1 172 1 1

Total 42 12 54


Table 34 Humans who consumedchicken at other home (last10 days)

Serotype PFGE No Yes Total1,44 33 2 2

3a 1 141 1 1

10 18 1 118b 1 1

11 35 3 312 4 1 118 211 1 1

251 1 119 12 1 1

19c 1 12 16 1 1

18a 2 2 418c 1 11c 1 1 2206a 1 1220 1 1250 1 13 1 133 1 13b 1 154 1 1

22 28 2 223,36 19b 1 1

22 1 131 25 1 135 10 1 14 complex 1 1 1

1a 1 1200a 1 1243 1 152 1 154a 1 1

52 25 1 157 216 1 16 Non-

cutting2 2

untypable 25 1 13 1 172 1 1

Total 14 34 48

Table 35 Humans who consumedbeef at home (last 10 days)


33 2 23a 1 141 1 1

10 18 1 118b 1 1

11 35 3 312 4 1 115 60b 1 118 211 1 1

251 1 119 12 1 1

19c 1 12 16 2 2

18a 1 3 418c 1 11c 2 21d 1 1206a 1 1220 1 1250 1 13 1 133 1 13b 1 154 1 1

22 28 2 223,36 19b 2 2

22 1 131 25 1 135 10 1 14 complex 1 1 1

1a 1 1200a 1 1243 1 152 1 154a 1 1

52 25 1 157 216 1 16 Non-cutting 2 2untypable 25 1 1 2

3 1 172 1 1

Total 16 38 54


Table 36 Humans who consumeduntreated water (last 10days)


33 1 1 23a 1 141 1 1

10 18 1 118b 1 1

11 35 2 1 312 4 1 115 60b 1 118 211 1 1

251 1 119 12 1 1

19c 1 12 16 2 2

18a 1 2 318c 1 11c 2 21d 1 1206a 1 1220 1 1250 1 13 1 133 1 13b 1 154 1 1

23,36 19b 2 222 1 1

31 25 1 135 10 1 14 complex 1 1 1

1a 1 1200a 1 1243 1 1249 1 152 1 154a 1 1


3 1 172 1 1

Total 26 26 52

Table 37 Humans who consumedWell/Bore Water Supply(within last 10 days)


33 1 1 23a 1 141 1 1

10 18 1 118b 1 1

11 35 1 2 312 4 1 115 60b 1 118 211 1 1

251 1 119 12 1 1

19c 1 12 16 1 1 2

18a 3 1 418c 1 11c 2 21d 1 1206a 1 1220 1 1250 1 13 1 133 1 13b 1 154 1 1

22 28 1 1 223,36 19b 2 2

22 1 131 25 1 135 10 1 14 complex 1 1 1

1a 1 1200a 1 1243 1 1249 1 1252 1 152 1 154a 1 1


3 1 172 1 1

Total 35 21 56


Table 38 Humans who consumedTown Water Supply (last10 days)


33 1 1 23a 1 141 1 1

10 18 1 118b 1 1

11 35 2 1 312 4 1 115 60b 1 118 211 1 1

251 1 119 12 1 1

19c 1 12 16 2 2

18a 1 3 418c 1 11c 2 21d 1 1206a 1 1220 1 1250 1 13 1 133 1 13b 1 154 1 1

22 28 1 1 223,36 19b 2 2

22 1 131 25 1 135 10 1 14 complex 1 1 1

1a 1 1200a 1 1243 1 1249 1 1252 1 152 1 154a 1 1


3 1 172 1 1

Total 24 32 56

Table 39 Humans who had contactwith dogs (last 10 days)


33 1 1 23a 1 141 1 1

10 18 1 118b 1 1

11 35 3 312 4 1 115 60b 1 118 211 1 1

251 1 119 12 1 1

19c 1 12 16 2 2

18a 4 418c 1 11c 2 21d 1 1206a 1 1220 1 1250 1 13 1 133 1 13b 1 154 1 1

22 28 1 1 223,36 19b 2 2

22 1 131 25 1 135 10 1 14 complex 1 1 1

1a 1 1200a 1 1243 1 1249 1 152 1 154a 1 1

52 25 1 157 216 1 16 Non-cutting 1 1 2Untypable 25 2 2

3 1 172 1 1

Total 17 38 55


Table 40 Humans who had contact with dairy cattle (last 10 days)


33 2 23a 1 141 1 1

10 18 1 118b 1 1

11 35 3 312 4 1♣ 115 60b 1 118 211 1 1

251 1 119 12 1 1

19c 1 12 16 1 1 2

18a 4 418c 1 11c 1 1 21d 1 1206a 1 1220 1 1250 1 13 1 133 1 13b 1 154 1 1

22 28 1 1 223,36 19b 2 2

22 1 131 25 1 135 10 1 14 complex 1 1 1

1a 1 1200a 1 1243 1 1249 1 152 1 154a 1 1


3 1 172 1 1

Total 40 15 55


APPENDIX 10: INDIVIDUAL LEVEL ANALYSIS FOR C. COLI and c. jejuniISOLATED FROM HUMAN CASES

The data below represent results from the integration of subtyping data and questionnairesfrom human cases of campylobacteriosis. It provides descriptive information on eachspecific subtype of C. coli and C. jejuni identified during the CTR study. It describes thehuman cases, their exposure histories, and the other matrices from which these isolateswere obtained. In addition, this information provides a synopsis of the behavioural habitsof cases associated with campylobacteriosis by describing lifestyles in a rural community.

The information presented is a summary of questionnaire data. The information in thequestionnaires was not always complete and there has been no attempt to extrapolate thedata beyond the responses provided by each case. A summary of the cases with potentiallinkages to other matrices can be viewed in Table 23 and Table 24. The location of thehuman case, if not stated in the text, is provided in parentheses with the informationconcerning onset of illness.

Subtyping of C. coli was by PFGE only.

C. coli subtype P2/33Onset date 28/8/01

A three-year-old child living on a dairy farm (Rakaia) was infected with the clonallyrelated subtype C. coli P2/33. The child had contact with stock and stock-fouled areas.This clonally related subtype was present in sheep and cattle faeces and in sheep offal, butnot in dairy faeces. The case was reported to have had contact with sheep and dogs. In theten days prior to the onset of illness the case consumed chicken, eggs and lamb at homeand another home and fish at home.

C. coli subtype P3Case 1(Ashburton township): Onset date 30/8/01, Report date 15/11/01 (hospitalised 31/8)Case 2(Ashburton township): Onset date 12/10/01, Report date 9/11/01

C. coli P3 was only found in sheep faeces and three human cases of campylobacteriosis.One of the cases did not return a questionnaire. All three cases were dairy farm workersand were on town water supply. The two cases who returned questionnaires had animalcontact with dairy cows, calves, cats and dogs. One of the cases had a family member whocontracted campylobacteriosis in the same month but in this case a C. jejuni isolate wasidentified as the causative organism.

In the ten days prior to the onset of illness both cases reported home consumption of beef,chicken and eggs and unpasteurised milk. The first case had also consumed pork, lamb andfish at home prior to the onset of symptoms.


C. coli subtype P10/29Onset date 9/9/01,

C. coli P10/29 was a common subtype isolated from farm animals from Regions B and C,one sample of duck faeces and one water sample from the Ashburton Intake (Region A).The case lived on a farm (Tinwald) and had contact with cats, calves and sheep, howeverthe case did not have occupational exposure to animals. The stock water was piped from acreek and the case had frequent contact with this water. The household water supply wasfrom untreated well/bore water. The case had recreational contact with a creek and haddrunk untreated water from the same creek. In the ten days prior to the onset of illness, thecase consumed beef, chicken, eggs, pork and lamb at home and another home.

C. coli subtype P11/11aOnset date: 11/12/01

The clonally related subtypes P11/11a appear to be a common C. coli subtype in sheep. Apossible temporal link from sheep offal to a human case of campylobacteriosis wasdeduced. This was in the only human case with a co-infection of C. coli and C. jejuni. TheC. jejuni isolate was a unique subtype, which was not isolated from any other matrix in thisstudy. The case lived in Methven and reported campylobacteriosis 11 December, 2001.Prior to this date the case had travelled to Christchurch, Wellington and Auckland between5–9 December, eating at restaurants while away from home. In the ten days prior to theonset of illness there was home consumption of chicken, pork and eggs, however noinformation was provided on food consumption outside the home. No occupational riskfactors in regard to farm animals or household animal contact were associated with thiscase. The household water supply was town water.

C. coli subtype P17Onset date 26/8/01

Four water samples from the Ashburton Intake sampling site tested positive for C. coli P17and one of these was isolated nine days prior to a human case of campylobacteriosis. Thiscase resided in Ashburton Township. No water samples tested positive for this subtypeafter the human case. This C. coli subtype was only detected in the human and watermatrices, suggesting the possibility of an unidentified environmental reservoir. The casereported campylobacteriosis on 29 August, 2001 and had been holidaying in Fiji from 15-26 August. During this holiday period all meals were eaten at restaurants and the case hadrecreational contact with river and seawater. No other information was available on foodconsumption.


Individual level analysis for C. jejuni subtypes isolated from human cases

Subtyping of C. jejuni was by a combination of serotyping and PFGE subtyping schemes.

C. jejuni subtype HS1,44:P33Case 1: Onset date 6/5/01,Case 2: Onset date 17/1/02,

The subtype HS1:P33 was isolated from the faeces of two human cases ofcampylobacteriosis. One case lived in Winslow and the other, in Rakaia. This subtype wasisolated from sheep offal (6%) and found in one cattle offal sample. Retailers obtainedsome of these sheep offal from local farm sources. Clonally related isolates were alsorecovered from cattle faeces, dairy faeces and sheep faeces from all three samplingregions.

Case 1 had no occupational contact with farm animals, but contact with ducks and cats.This case was on town water supply and had been on holiday in Akaroa for three days,returning home four days prior to the onset of the campylobacteriosis. No other familymembers or friends had become ill during this time period. This case had consumed beefand pork (at home, another home and elsewhere), chicken and eggs (at home, and anotherhome), within 10 days of the onset of illness.

Case 2 was a sheep farmer who also had contact with cats and dogs. The case lived on afarm where the water supply came from untreated well/bore water. This second case hadconsumed beef (at home, and another home), chicken (at home, another home andelsewhere), eggs and pork (elsewhere) within 10 days of the onset of illness. The casecommented that they thought that take-away chicken nuggets were the cause of theirillness.

C. jejuni subtype HS1, 44:P3aOnset date 6/2/01,

Subtype HS1, P3a was isolated from sheep offal (8%) and cattle offal (7%) and one sampleof sheep faeces. Retailers obtained some of the sheep offal from local farms. The case wasa freezing-plant worker who lived in Ashburton Township and had contact with animalfaeces, live sheep, cats and dogs. The case was on town water supply and had consumedbeef and lamb (at another home), chicken (at home and another home and elsewhere), eggsand pork (at home, and another home), and fish at home, within 10 days of the onset ofillness. The case had recreational contact with river and seawater and was at the RakaiaGorge a week prior to the onset of illness.

C. jejuni subtype HS2:P16Case 1: (Rakaia Barrhill). Onset date 9/3/01,Case 2: (Winchmore, Region A) Onset date 8/12/01,

Two cases of human campylobacteriosis, which occurred at different times of the yearwere attributed to subtype HS2:P16. This subtype was isolated from 2 sheep offal samplesand the Ashburton Intake (Region A) water sampling site, but no other environmental


matrices. Retailers obtained both sheep offal samples from local farm sources. The firstcase was a Japanese visitor who stayed on a farm property within the 8 days prior to onsetof illness. The property had a private water supply, which was a water race treated byfiltration. The case had contact with calves, sheep, cats and dogs and had consumedchicken, eggs and pork at home, and another home, ten days prior to the onset of illness.

The second case was on holiday and employed as a farm worker in Region A. At the timeof infection the case was employed in the tailing of lambs and had visited a dairy farm. Thecase also had contact with cattle, sheep, cats and dogs. The property on which the case wasstaying had an untreated water supply derived from well/bore water. This case consumedchicken, eggs and fish, at home ten days prior to the onset of illness.

C. jejuni subtype HS2:P3/P33 and related PFGE (P33/P47/P3b/P3d/P3i)Case 1: (Ashburton) Onset date 26/4/01,Case 2: (Rakaia) Onset date 22/10/01, Report date 26/10/01 (hospitalised 22/10)Case 3: (Hinds) Onset date 12/12/01,Case 4: (outskirts Ashburton township) Onset date 28/1/02,

This is a large subtype group containing several clonally related subtypes(P33/P47/P3/P3b/P3d/P3i). The subtype HS2:P3 was isolated from every matrix exceptwater at prevalences of 2% and above. It was isolated from meat and chicken products atthe highest frequencies. The clonally related subtype HS2:P3b was isolated from onehuman and the clonally related HS2:P3i subtype was isolated from chicken at a prevalenceof 4%. Subtype HS2:P33 was isolated from two human cases of campylobacteriosis, andfrom dairy faeces and cattle faeces at frequencies of 3% and 10% respectively. It wasevenly distributed throughout the three sampling regions.

The first human case of C. jejuni subtype HS2:P3 was employed as a labourer in a guthouse, where they had contact with sheep. The case lived in Ashburton and the dwelling ofthe case was on the town water supply. In the ten days prior to the onset of illness, the casehad consumed beef (at home and elsewhere), eggs and lamb at home, another home andelsewhere.

The second case was infected by the clonally related C. jejuni subtype HS2:P3b and hadcontact with dairy cows, calves, dogs and pigs. The water supply to the farm was fromuntreated well/bore water. In the ten days prior to the onset of illness, the case hadconsumed beef, chicken and eggs at home and another home, and pork (at home, anotherhome and elsewhere) and fish at home.

C. jejuni subtype HS2:P33 was isolated from two human cases of campylobacteriosis. Thissubtype was isolated from dairy faeces and cattle faeces at frequencies of 3% and 10%respectively and was evenly distributed throughout the three sampling regions. There wasno temporal link apparent between the animal faeces sampled and the human cases.

The first case, a dairy farmer, lived on a farm and was in contact with dairy cows, calves,cattle, sheep, cats, dogs and chickens. In the ten days prior to the onset of illness the caseconsumed beef, eggs, lamb and unpasteurised milk (at home and another home), pork (athome, another home and elsewhere).


The second case was caused by the clonally related subtype C. jejuni HS2:P250. The casewas a young child attending pre-school and living in a rural area on a lifestyle block. Thecase had contact with sheep and a pet bird and recreational contact with sheep, cats, dogs,chickens, and fish in addition to water at a public swimming pool.

C. jejuni subtype HS2:P18a

C. jejuni subtype HS2:P18a caused five human cases of campylobacteriosis (10.5% of totalC. jejuni cases) and was isolated from one duck faecal sample within the same time period.All cases occurred between December 26th, 2000 and 19th of March, 2001. The first casewas a freezing worker who had contact with sheep, dogs, wild birds and chicken faeces.The second case had contact with untreated water at Lake Opuha. The third case hadvisited a farm and had contact with untreated water. The fourth case was the child of aparent who worked at a rendering plant, but no other family member contractedcampylobacteriosis. The fifth case, a teacher on holiday who had been traveling in theNorth Island on 26 December, 2000 and 6 January, 2001, was infected with a clonallyrelated subtype of C. jejuni (HS2:P18c). This case had recreational contact with water in apublic swimming pool while traveling in the North Island.

Because this subtype did not occur in any other matrices, except for one duck faecalsample, a summary of the risk factors associated with these cases is presented in Table 41.

Table 41 Risk factors associated with Cases of Subtype HS2:P18

Age(years)

Animalcontact

Meat consumptionHuman casesand location

Home Outside of home

Water Supply

Case 1(Fairton)

26 Sheep, dogs,wild birds,chickens

Beef, chicken, eggs,Lamb

Beef, chicken, eggs,lamb

Well/bore water

Case 2(Ashburton)

3 dogs Beef, chicken, eggs Town water

Case 3(Ashburton)

65 Sheep, cats,dogs

Eggs, pork, fish Eggs, pork Town water

Case 4(Ashburton)

2attends

pre-school

Cats, dogs Beef, chicken, eggs,Lamb

Beef, chicken, eggs,lamb

Town water

Case 5(Methven)

37 Cats, dogs Beef, chicken, pork Town water


C. jejuni subtype HS2:P54(Mayfield township) Onset date 20/1/01

Subtype HS2:P54 caused one human case of campylobacteriosis and was also isolatedfrom beef cattle faeces, sheep faeces and sheep liver, but only at a prevalence of 2% orless. There were no temporal linkages between the animal isolates and the human case. Thecase had no occupational exposure to animals, but did have contact with non-dairy cattle,calves, cats, dogs and horses. The case is listed as being on water supply from town butalso untreated stream/river/lake water. The case consumed beef and chicken (at home,another home and elsewhere), pork (home and another home), and eggs and fish at homeand unpasteurised milk at another home in the ten days prior to the onset of illness.

C. jejuni subtype HS2:P206(Rakaia) Onset date 6/7/01

The subtype HS2:P206 was isolated from dairy faeces and beef cattle faeces at aprevalence of 3% and 1%, respectively. The subtype was not isolated from sheep. Aclonally related subtype was associated with one case of human campylobacteriosis, achild who attended pre-school in Methven. Family members and other children in thecreche did not report Campylobacter infections. The case lived on a farm and had contactwith sheep, cats, dogs, chickens and fish. The farm water supply is from well/bore water,treatment status not known. In the ten days prior to the onset of illness the case consumedbeef (elsewhere), chicken, eggs and lamb (at home and another home) and pork (at home,another home and elsewhere).

C. jejuni subtype HS 4 complex:P1 and related PFGE (P1a/P1b)Case 1: (Ashburton township) Onset date 24/5/01,Case 2: (Methven) Onset date 13/12/01,

C. jejuni subtype HS 4 complex:P1 caused two human cases of campylobacteriosis. Thisclonal group is poorly represented in most of the matrices. It was most frequently isolatedfrom the chicken matrix (4% of total positive samples). This case had no occupationalexposure to animals, but had household contact with cats. The dwelling in which the caselived was on town water supply. The case reported the consumption of chicken at homeand elsewhere in the ten days prior to the onset of illness.

C. jejuni subtype HS4 complex:P1 was isolated from chicken carcasses and sheep offalsamples prior to the second case but outside the limit which might indicate a temporal linkbetween chicken and humans. A possible temporal link might be established, however,between sheep offal and the human case. The second case reported no occupationalexposure to animals and contact with cats and dogs. The case has listed sheep shearerunder the category household contact with animals. The dwelling in which the case livedwas on town water supply. The case reported the consumption of beef and chicken (athome, another home and elsewhere), eggs, lamb and pork at home and another home in theten days prior to the onset of illness.


C. jejuni subtype HS4 complex:P52(Lismore) Onset date 27/1/01

The subtype HS4 complex:P52 was isolated from one human case of campylobacteriosisand from dairy faeces and sheep faeces at a prevalence of 3 % and 2%, respectively. Thecase had occupational exposure to horses, horse dung, calves and pigs and had contact withdairy cows. The case lived on a farm and recorded that the water supply was from townwater and from untreated well/bore water. The case had consumed beef (elsewhere) andchicken at home and elsewhere, in the ten days prior to the onset of illness.

C. jejuni subtype HS 4 complex:P243(Ashburton) Onset date 10/5/01

The subtype HS4 complex:P243 was isolated from one case of human campylobacteriosisand from one sheep faecal sample from Region A. The case had occupational exposure toanimals including cats, dogs and ducks. The case lived in a dwelling on town water supply.The case had consumed beef (at home, another home and elsewhere), chicken and lamb (athome and another home), eggs and fish at home, in the last ten days prior to the onset ofillness. The case suspected food as the source of infection.

C. jejuni subtype HS10:P18/18bCase 1: (Mayfield) Onset date 13/10/01, Report date 19/10/01Case 2: (Rakaia) Onset date 16/11/01, Report date 24/12/01 Sample taken on 19/12/01.

In two human cases of campylobacteriosis the clonally related subtype C. jejuniHS10:P18/18b was isolated from faeces. PFGE subtype 18 is clonally related to PFGEsubtype 18b. The same subtype (HS10:P18) was isolated from sheep offal 8 days prior tothe first human case and therefore is within the time frame to indicate a possible temporallink between the two matrices. There were no isolations of this subtype fromenvironmental matrices prior to the second human case. Clonally related subtypes werepresent in sheep (HS10:P18, 4% prevalence and HS10:P18b, 2% prevalence) and alsoisolated from dairy and beef cattle. All animal faecal samples were isolated from RegionsB and C.

The first human case was a one-year-old child who lived on a farm where the water supplywas untreated well/bore water. Person-to-person contact with another campylobacteriosiscase was reported. Travel to Southland was indicated for this case, but no dates wererecorded. The case had animal and dung contact with dairy cows and calves, sheep, cats,chicken and dogs. This case had consumed beef, chicken, eggs and lamb at home andanother home and fish at home within 10 days of the onset of illness.

The second case, a school-age child who lived on a farm, occurred two months after thefirst case. This case had animal contact with dairy cows and calves, sheep, cats, chickenand dogs. The farm was on an untreated well/bore water supply. This case had consumedchicken, eggs, lamb, pork and unpasteurised milk at home and another home and fish athome, within 10 days of the onset of illness.


C. jejuni subtype HS11:P35Case 1: (Rakaia) Onset date 5/1/01,Case 2: not included in Episurv database. Faecal sample received on 5/9/01,Case3: (Ashburton township) Onset date 7/9/01,Case 4: (Ashburton town ship) Onset date 28/9/01,

C. jejuni subtype HS11:P35 was isolated from 4 human cases of campylobacteriosis. Thissubtype was isolated most frequently from human (7%), dairy (5%) and beef cattle (4%)faecal matrices. Three of the human cases (Cases 2-4) were within a similar time periodand there was a temporal connection among all three cases and one cattle faecal isolate ofthe same subtype. Case two did not return a questionnaire.

Case three was a teenager who attended school, had access to town water supply and hadcontact with dogs. However, the case also stayed frequently on a friend’s dairy farm. Thecase had consumed beef (at home and another home), chicken (at home, another home andelsewhere) and fish at home in the ten days prior to illness.

Case four was a pre-school child who had contact with calves, cattle, sheep, chickens,dogs, pigs and a turkey. The water supply to their dwelling was well/bore water. The casehad fallen face-first into calf manure within the ten days prior to onset of symptoms. Thecase had consumed beef, chicken, eggs and lamb at home and another home, and fish athome, within ten days prior to the onset of illness.

Case one showed no temporal linkages with any environmental matrices as their reportedillness occurred prior to the start of the first sampling week. This case was of pre-schoolage and had person-to-person contact with both parents who had campylobacteriosis. Itwas noted that the case had faecal contact with a younger sister. The case had animalcontact with sheep including sheep dung, chickens, dogs and other animals. The watersupply to their dwelling was untreated well/bore water and the case had recreationalcontact with private swimming pool water 10 days prior to the onset of illness. The casehad consumed beef, chicken, eggs, lamb and pork (at home and another home), and fish athome, within ten days prior to the onset of illness.

C. jejuni subtype HS15:P60bOnset date 1/12/01

C. jejuni subtype HS15:P60b was isolated from human and water matrices only, andoccurred above 2% prevalence only in water. All water samples were isolated from theAshburton Intake (Region A). No temporal link was found between water samples and thehuman case. The case lived in a semi-rural area on the outskirts of Ashburton Township.There was no occupational risk factor associated with farm animals, but there was contactwith a cat. The case lived on a property where the water supply was derived from untreatedwell/bore water. The case had recreational contact with water in a public swimming pool.In the ten days prior to the onset of illness the case had consumed chicken, pork and fish athome. The case, however, did not answer all the food consumption questions.


C. jejuni subtype HS23,36:P19b and related subtypes (P19/P19d/P19f)Case 1: (Rakaia) Onset date 4/9/01Case 2: (Winchmore) Onset date 13/11/01, Report date 14/11/01 (hospitalised 14/11)

Two temporally distinct cases of human campylobacteriosis were attributed to C. jejunisubtype HS23,36:P19b. This was a large subtype group containing several clonally relatedsubtypes (P19/P19d/P19f). However both human cases were of the predominant subtypeHS23,36:P19b. The matrix with the highest prevalence of isolation of this subtype wasdairy faeces (29%) with clonally related subtypes HS23,36:P19 and P19f occurring at 1%and 3%, respectively, in dairy faecal samples. The matrices from which this predominantsubtype (P19b) was isolated with lower prevalences were beef (7%) and sheep faeces (6%)and sheep offal (5%). The animal faecal samples were evenly distributed through the threeregions of sampling. This subtype was found in two water samples (1% prevalence) fromthe Ashburton Intake (Region A).

The first human case was a farm worker who had occupational contact with dairy cows,calves, fouled equipment, cats and dogs. The case lived on a farm which had watersupplied from untreated well/bore water. The same subtype was isolated from dairy faeces,sheep faeces and sheep liver all within the maximum time period allowed to indicate apossible temporal link with a human case. The case had consumed beef and chicken (athome and elsewhere) and eggs at home in the ten days prior to the onset of illness.

In the second human case, the same subtype was isolated from cattle faeces and from sheepliver within the maximum time period assumed to indicate a possible temporal link with ahuman case. The case lived on a farm and had contact with dairy cow, cattle, calves, cats,dogs and a horse. The water supply to the farm was untreated well/bore water. In the tendays prior to the onset of illness, the case had consumed beef and chicken (at home andelsewhere), duck, eggs, lamb, pork and unpasteurised milk, at home.

The same subtype was isolated from two water samples from Region A sampling site andfrom sheep faeces, dairy faeces and beef cattle faeces from farms in all three samplingregions. The ruminant faeces were isolated within the maximum allowable time period fora possible temporal and spatial link to the two water samples.

C. jejuni subtype HS23,36:P22(Hinds) Onset date 23/11/01

C. jeuni subtype HS23,36:P22 was isolated from one human case of campylobacteriosisand from 7% of beef offal samples, 3% of sheep offal samples and 1% of beef faecalsamples. There were no temporal links between the case and the other matrices. The caselived on a farm where the water supply was untreated well/bore water. The case hadcontact with dairy cows, calves, sheep, cats and dogs and faecal contact with pigs,chickens and sheep. The case had recreational contact with water at a public swimmingpool. The case had consumed beef and eggs (at home and another home), pork (at home,another home and elsewhere), fish at home and unpasteurised milk (at home, another homeand elsewhere).


C. jejuni subtype HS31:P25(Ashburton) Onset date 7/1/01

C. jejuni subtype HS31:P25 was isolated from one human case, one duck faecal sampleand one chicken carcass. There were no temporal links between the case and the othermatrices. The case had no occupational exposure to animals, but had contact with dogs andanimal dung used as fertiliser. The case lived in a dwelling on town water supply and hadconsumed beef, chicken, and eggs at home. The case had travelled to Lake Opuha the dayprior to the onset of illness and had consumed untreated water there as well as havingrecreational contact with lake, river and seawater. Another young family membercontracted campylobacteriosis on the same date. C. jejuni isolated from this second casewas subtype HS2:18a, which is unrelated to subtype HS31:P25 and was the subtypeassociated with four other human cases and one duck faecal sample.

C. jejuni subtype HS35:P10/31(Winslow) Onset date 17/8/01

C. jejuni subtype HS35:P10/31 is a diverse group of clonally related subtypes, whichcaused one human case of campylobacteriosis. This clonally related subtype was isolatedfrom a variety of matrices, including dairy faeces and beef cattle faeces at frequencies of3% and 4%, respectively. The highest prevalences observed for this subtype were frombeef offal (13%) and pork offal (11%). The majority of animal faecal samples were fromRegion B. Matrices from which it was not isolated were duck faeces, sheep faeces andchicken carcasses. There was a possible temporal link between a clonally related dairyfaecal sample (14 days before the human case occurred) and the wintertime human case.The case was a farmer who had contact with dairy cows, calves, sheep, cats, dogs andchickens. Previously the farm had been a pig farm. No other people known to the case hadsymptoms of campylobacteriosis. The supply of water to the farm was untreated well/borewater. In the ten days prior to the onset of illness, the case had consumed beef, chicken,eggs and pork at home and another home, and fish at home.

C. jejuni subtype HS6:P non-cuttingCase 1: Onset date 12/2/01Case 2: Onset date 30/12/01

C. jejuni HS6 are recognised internationally as difficult to subtype by PFGE, thereforethese isolates cannot be designated as a definitive subtype (C. Nicol, personalcommunication). Any conclusions regarding these isolates are therefore, speculative andalthough this grouping was found in other matrices, no assessment of relationship to thosematrices or between human cases caused by these isolates can be made. The followingcomments are provided only on the basis of epidemiological observations from the cases.

This group contains two temporally distinct human cases both resident in AshburtonTownship and on town water supply. These cases have no occupational exposure toanimals, and only one case lists contact with cats and dogs.


C. jejuni subtype SUT (serotype untypable):P3.

This group cannot be regarded as a subtype, due to the inability of the isolates to beserotyped. Therefore, although this grouping was found in other matrices, no assessment ofrelationship to those matrices or between human cases can be made. The followingcomments are provided only on the basis of epidemiological observations from the cases.

One case was SUT: P3. The case lived on a dairy farm, on a non-registered, non-securedrinking water supply without monitoring or treatment (Dennis Burridge, AshburtonDistrict Council, personal communication). The case had contact with many farm animals,including calves and pet lambs. The case consumed beef (elsewhere), chicken (at homeand another home), pork (at home and another home), fish at home and unpasteurised milk(at home and another home) within the ten days prior to the onset of illness.

C. jejuni subtype SUT (serotype untypable):P25Case 1: (Ashburton township) Onset date 2/12/01Case 2: (Ashburton township) Onset date 15/1/02

SUT:P25 is a diverse group containing clonally related PFGE subtypes. This group cannotbe regarded as a subtype, due to the inability of the isolates to be serotyped. Therefore,although this grouping was found in other matrices, no assessment of a relationship tothose matrices or between human cases can be made. The following comments areprovided only on the basis of epidemiological observations from the cases.

The first case had contact with the faeces of cats and dogs and their dwelling was on townwater supply. The case had consumed chicken, eggs, lamb, pork and fish at home in the tendays prior to the onset of illness. The food consumption questions were not completed.

The second case had contact with cats and dogs and their dwelling was on town watersupply. The second case reported visiting a farm as part of their occupation, but did not listany farm animal contact. The case had recreational contact with water in a publicswimming pool. In the ten days prior to the onset of illness the case had consumed beefand chicken (at home, another home and elsewhere), duck, eggs, lamb, pork (at home andanother home), fish at home and unpasteurised milk (at home and elsewhere).

C. jejuni subtype SUT (serotype untypable):P72Case 1: Onset date 9/7/01 (returned home from school 29 /6/01),Case 2: included in Episurv, but no sample received. Onset date 14/7/01,(Both cases lived in Hinds).

SUT:P72 cannot be regarded as a subtype, due to the inability of the isolates to beserotyped. Therefore, although this grouping was found in the water matrix from Region A(2% prevalence), no assessment of relationship to this matrix or between human cases canbe made. The following comments are provided only on the basis of epidemiologicalobservations from the cases.

The first case was a school student on vacation. They helped with duties in the pigpen andhad contact with beef cattle, cats, dogs and chickens. The case consumed beef, chicken,


eggs (at home and another home) and pork (at home, another home and elsewhere) in theten days prior to the onset of illness. The water supply to the property was from a stockwater race as the well was dry. Case two was a farm worker, who lived in the same houseas Case one, also contracted Campylobacter infection. ESR however, did not receive afaecal sample from this second case. Both cases commented that they believed the sourceof infection was due to a contaminated water supply.

Human cases with no subtype relationship to other matrices.

C. jejuni subtype HS1:P41

A freezing–plant worker who lived in Tinwald, on town water supply, had contact withsheep, cats, dogs and a pet bird and faecal contact with a baby. In the ten days prior to theonset of illness, the case had consumed beef and lamb (at home, another home andelsewhere), chicken, eggs and pork (at home and another home).

C. jejuni subtype HS2:P1cCase 1: Onset date 26/9/01,Case 2: Onset date 26/10/01,

The case lived in Lismore on an untreated water supply from well/bore water. The casewas a dairy farm worker who had contact with dairy cows, calves, cats and dogs. The casehad person-to-person contact with a co-worker who had contracted campylobacteriosis.The case had consumed beef and eggs at home and elsewhere and fish at home in the tendays prior to the onset of illness.

The second case, a housewife lived in a rural area in Rakaia. The case had contact withsheep, cats and dogs. The dwelling was on an untreated water supply from well/bore water.The case had consumed beef, chicken, eggs at home and another home and fish at home inthe ten days prior to the onset of illness.

C. jejuni subtype HS2:P1d

The case lived in Tinwald, their dwelling was on town water supply. The case had nocontact with live animals, but contact with sheep manure, which was spread onto theirgarden. The case had person-to-person contact with a friend who had contractedcampylobacteriosis. The case had consumed chicken (at home), pork (elsewhere), howeverthe other food consumption questions were not completed. The case had travelled toChristchurch one day prior to the onset of illness, where they had consumed a takeaway ofsweet and sour pork.


The case was a young child who attended pre-school. The case lived on a farm inWinchmore (Region A) and had contact with sheep, sheep dung, cats and dogs. Because aparent was a veterinarian the child had contact with many animals. The dwelling was ontown water supply. The case had person-to-person contact with a brother who hadcontracted campylobacteriosis. The case had consumed beef and eggs (at home, another


home and elsewhere), chicken (at home and another home), lamb and fish at home, in theten days prior to the onset of illness.

C. jejuni subtype HS4 complex:P54a

The case was a farm worker who had contact with dairy cows, calves, cattle, sheep, dogsand deer. The farm at Lismore on which the case lived was supplied by untreated well/borewater. The case had consumed beef, eggs and pork at home and another home, andunpasteurised milk at home.

C. jejuni subtype HS4 complex:P200a

The case lived in Ashburton on town water supply. The case had contact with dogs andpuppies, faecal contact with a sibling and person-to-person contact with a parent who hadbeen ill. The case had recreational contact with water in a pool. The case had consumedchicken (at home, another home and elsewhere), and eggs and pork at home and anotherhome.

C. jejuni subtype HS4 complex:P249

The case worked as a meatworker in a fellmongery and had contact with sheep. The caselived in Ashburton and was on town water supply. The case did not record any details oftheir food consumption.

C. jejuni subtype HS4 complex:P252

The case had stopped over in Australia and Bali on the way to New Zealand. Thesymptoms began two days before arrival in NZ.


The case was retired and had contact with cats. The case lived in Ashburton and theirdwelling was on town water supply. In the ten days prior to the onset of illness the casehad consumed beef, eggs and lamb (at home and another home) chicken and fish at home.The case had consumed fish and chips the day of the onset of illness.


The case lived in Ashburton and the dwelling was on the town water supply. The case wasa young child who attended preschool and who had contact with cats. In the ten days priorto the onset of illness the case had consumed chicken and eggs at home and another home.


The case was a dairy farm worker who lived in Rakaia and their dwelling was on townwater supply. The case had contact with dairy cows, non-dairy cows, sheep, cats, chickens,dogs and a horse. The case had consumed beef, chicken, eggs and pork at home andanother home.



The case, a housewife, lived in Fairton, near Ashburton, their dwelling was on town watersupply. The case had contact with cats and wild birds. The case had consumed beef,chicken and eggs at home, another home and elsewhere, and fish at home, within the tendays prior to the onset of illness.

C. jejuni subtype HS19:P19c

The case was a fellmonger who had contact with sheep, sheep dung, sheep pelts andhousehold contact with cats. The case lived in Tinwald and the dwelling was on townwater supply. The case had consumed beef, chicken, eggs and pork at home and anotherhome in the ten days prior to the onset of illness.

C. jejuni subtype HS22:P28Case 1: Onset date 21/11/01Case 2: Onset date 28/12/00

Two human cases of campylobacteriosis were caused by C. jejuni subtype HS22:P28. Thefirst case was a homemaker who lived in Ashburton, their dwelling was on town watersupply. The case was recorded as consuming water from Methven. The case visited thefarm of a family member and had contact with dairy cows and chickens. The case hadconsumed chicken and eggs at home, and beef, pork and unpasteurised milk at home andelsewhere, in the ten days prior to the onset of illness. The case had person-to-personcontact with a family member who worked on a farm and who had contractedcampylobacteriosis caused by C. coli. However, in the case of the other family member theCampylobacter identified was a strain of C. coli.

The second case of campylobacteriosis occurred three weeks after the first case. The casewas a homemaker who lived in Hinds and whose water supply was well/bore water. Thecase had contact with non-dairy cows, calves, sheep, lambs, cats, dogs, chickens, ducksand sparrows. They had been travelling to Fisherman’s Bend a day before the onset ofsymptoms and to the Waitaki River five days prior to the onset of illness. The case hadconsumed water at both of these places. The case had consumed beef, chicken, eggs, lamband fish at home, in the ten days prior to the onset of illness.


The case had no occupational exposure to animals but had other contact with sheep, catsand dogs. The case was on holiday in New Zealand staying with family members on theirfarm. The farm water supply was untreated rainwater/tank water. The case had beenvisiting the North Island up to, and including, the date of onset of symptoms. The case hadconsumed beef, chicken, eggs and lamb at home and another home, in the ten days prior tothe onset of illness.



The case, a dairy farm worker, had contact with dairy cows, calves, and cattle dung. Thecase lived on a farm that had an untreated water supply derived from well/bore water. Inthe ten days prior to illness the case had consumed beef, chicken and pork at home andanother home.

potential transmission routes of campylobacter …

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