voordracht arcobacter 2009 ppt 97 wavfh -...

ArcobacterArcobacter

Kurt HoufKurt HoufDepartment of Veterinary Public HealthDepartment of Veterinary Public Health

Ghent University Ghent University BelgiumBelgium

Deel IDeel I

-- historiekhistoriek

-- taxonomietaxonomie

-- ArcobacterArcobacter bij de mensbij de mens

-- ArcobacterArcobacter transmissietransmissie

Deel IIDeel II

-- transmissie in pluimveetransmissie in pluimvee

-- transmissie in varkenstransmissie in varkens

-- conclusiesconclusies

Deel IIIDeel III

-- vragenvragen

HHistoriekistoriek

18861886

(1857-1911)(Publicatie herondekt in 1985, Manfred Kist) (Publicatie herondekt in 1985, Manfred Kist)

18861886 19111911

Sir McFadyean John(1853-1941)

Royal Veterinary College LondonVeterinary pathology

Sir Stockman Stewart(1869-1926)

Royal Veterinary College London

HHistoriekistoriek

Spirillen - infectieuze abortus runderen

18861886 19111911

HHistoriekistoriek

18861886 19111911

Sir McFadyean John(1853-1941)

Royal Veterinary College LondonVeterinary pathology

Sir Stockman Stewart(1869-1926)

Royal Veterinary College London

HHistoriekistoriek

18861886 19111911

Campylobacter

1991 1991 -- 19921992

HHistoriekistoriek

Prof. J.-P. ButzlerSt. Pierre Hospital - VUB

&

Dr. Paul Dekeyser, M.D.

National Institute for Veterinary Research, Brussels

1968196819381938

Elizabeth King 1966

“related Vibrio”

18861886 19111911

HHistoriekistoriek

19771977

Ellis Ellis et alet al.. (Noord(Noord--Ierland)Ierland)

bovine and porcine fetuses bovine and porcine fetuses

spirillum / spirillum / VibrioVibrio--like organismslike organisms

19781978

Neill Neill et alet al. . (Noord(Noord--Ierland)Ierland)

organs of bovine and porcine fetuses:organs of bovine and porcine fetuses:

Group 1 strains = Campylobacter fetusGroup 1 strains = Campylobacter fetus++Group 2 strainsGroup 2 strains = = aerotolerant campylobactersaerotolerant campylobacters

18861886 1978197819771977 1983198319111911

HHistoriekistoriek

Campylobacter nitrofigilisCampylobacter nitrofigilis (Mc Clung et al.)(Mc Clung et al.)

18861886 1978197819771977 198519851983198319111911

HHistoriekistoriek

Campylobacter nitrofigilisCampylobacter nitrofigilis (Mc Clung et al.)(Mc Clung et al.)

Neill Neill et alet al. .

biochemical identification of group 2 biochemical identification of group 2

aerotolerant campylobactersaerotolerant campylobacters

+ Campylobacter cryaerophila+ Campylobacter cryaerophila sp. nov. sp. nov.

18861886 1978197819771977 198519851983198319111911

HHistoriekistoriek

1991 1991 -- 19921992

Arcobacter genusArcobacter genus

A. butzleri A. butzleri (Vandamme et al.)(Vandamme et al.)A. cryaerophilus A. cryaerophilus (Vandamme et al.)(Vandamme et al.)A. skirrowii A. skirrowii (Vandamme et al.)(Vandamme et al.)

A. nitrofigilisA. nitrofigilis

20052005

A. cibarius A. cibarius (Houf et al.)(Houf et al.)

A. halophilus A. halophilus (Donachie et al.)(Donachie et al.)

20092009

A. mytili A. mytili (Colado et al.)(Colado et al.)

A. thereius A. thereius (Houf et al.)(Houf et al.)

Arcobacter sp. nov Arcobacter sp. nov (De Smet (De Smet -- Houf et al.)Houf et al.)

A. marinus A. marinus (Hwang et al.)(Hwang et al.)

Campylobacter

1992

ProteobacteriaProteobacteriarRNA superfamily VIrRNA superfamily VI

ArcobacterArcobacter

Sulfurospirillum

Wolinella

Helicobacter

TTaxonomieaxonomie

AArcobacter rcobacter CCampylobacterampylobacter

Gram negatiefGram negatief

niet spore vormendniet spore vormend

motiel door 1 polaire flagellum zonder schede aan 1 of beide zijmotiel door 1 polaire flagellum zonder schede aan 1 of beide zijdenden

mmetabolisch inetabolisch inertert

Slanke gebogen staafjes Slanke gebogen staafjes ––pleiomorfpleiomorf

groei : 15 groei : 15 -- 37 37 °°C C -------- 42 42 °°CC

microaeroob tot aeroobmicroaeroob tot aeroob

spirilspiril

groei : 30 groei : 30 -- 42 42 °°CC

microaeroobmicroaeroob

© Butzler J.P.

© Houf K.

TTaxonomieaxonomie

TTaxonomieaxonomie

Campylobacteraceae

Campylobacter

Arcobacter

Arcobacter nitrofigilis (1992)

Arcobacter halophilus (2005)

Arcobacter mytili (2009)

Arcobacter marinus (2009)

19921992

TTaxonomieaxonomieTTaxonomieaxonomie

McClung et al. 1983.

Campylobacter nitrofigilis sp. nov., a nitrogen-fixing bacterium associated with roots of Spartina alterniflora Loisel.

Int. J. Syst. Bacteriol. 33:605-612.

TTaxonomieaxonomie

Wirsen et al. , 2002. Characterization of an autotrophic sulfide-oxidizing marine Arcobacter sp. that produces filamentous sulfur. Appl. Environ. Microbiol. 68:316-325.

Transmission electron micrographs of negatively stained cells of the filamentous-sulfur-producing microbe "Candidatus Arcobacter sulfidicus" showing four polar flagella.

Thin-section transmission electron micrographs showing the multilaminar polar membrane structure found in the filamentous-sulfur-producing microbe "Candidatus Arcobacter sulfidicus."

FISH staining "Candidatus Arcobacter sulfidicus."

TTaxonomieaxonomie

Donachie et al. 2005.

Arcobacter halophilus sp. nov., the first obligate halophile in the genus Arcobacter.

Int. J. Syst. Evol. Microbiol. 55:1271-1277.

water collected from a hypersaline lagoon on Laysan Atoll in the North-western Hawaiian Islands.

TTaxonomieaxonomie

Gevertz et al. ,2000.

Isolation and characterization of strains CVO and FWKO B, two novol nitrate-reducing, sulfide-oxidizing bacteria isolated from oil field brine.

Appl. Environ. Microbiol. 66:2491-2501.

Watanabe et al. 2000.

Molecular characterization of bacterial populations in petroleum-contaminated groundwater discharged from underground crude oil storage cavities.

Appl Environ Microbiol. 66:4803-9.

TTaxonomieaxonomie

Campylobacteraceae

Campylobacter

Arcobacter

Arcobacter nitrofigilis (1992)

Arcobacter halophilus (2005)

Arcobacter mytili (2009)

Arcobacter marinus (2009)

19921992

Arcobacter butzleri (1992)

Arcobacter cryaerophilus (1992)

Arcobacter skirrowii (1992)

Arcobacter cibarius (2005)

Arcobacter thereuis (2009)

Arcobacter sp. nov (….)

1987 1987 -- 19911991

eerste raporteringen van humane infecties met

aerotolerante Campylobacter &

Campylobacter cryaerophila

gecontamineerd drinkwater

Taylor Taylor et alet al. 1991, . 1991,

AArcobacter rcobacter -- mensmens

Bangkok

India


MatricesMatrices van humane isolatenvan humane isolaten

stoelgangstoelgang

bloedbloed

buikvochtbuikvochtbiopsie van mitralis klepbiopsie van mitralis klep

©© De Smet S.De Smet S.


Enteritis Enteritis -- septicemiesepticemie

% pati% patiëënten met symptomennten met symptomen

diarreediarree

buikpijnbuikpijn

koortskoorts

misselijkheidmisselijkheid

brakenbraken

malaisemalaise

mucus in stoelgangmucus in stoelgang

blood in stoelgangblood in stoelgang

70%70%

55%55%

34%34%

31%31%

24%24%

22%22%

4%4%

3%3%

©© De Smet S.De Smet S.


Italy - (1983, Pugina) – Australia (1987, Tee) – Thailand (1989,Taylor - 2001, Yan) - USA – (1991, Kiehlbauch - 1999, Rice) Canada (1991 – Kiehlbauch) – Switzerland (1992, Burnes) - Germany (1994, Lerner) – United Kingdom (1995, On)South Africa (1998, Lastovica) – Denmark (2000, Engberg), Hong-Kong (2001, Woo – 2002, 2003 – Lau) Belgium (2004, Vandenberg, Wybo) – France (2006, Prouzet-Mauléon)

ArcobacterArcobacter isolaties uit patiisolaties uit patiëëntennten

-- single case reportssingle case reports

wereldwijdwereldwijdleeftijd: < 1leeftijd: < 1-- 8686= sexe distributie= sexe distributievaak met onderliggende pathologivaak met onderliggende pathologiëënn

infectiebron infectiebron ““nooitnooit”” gegeïïdentificeerd dentificeerd (drinkwater)(drinkwater)

- uitbrakenuitbraken

ItalItalïïe (bron onbekend e (bron onbekend –– horizontale transmissie)horizontale transmissie)Canada (drink water Canada (drink water –– A. butzleriA. butzleri))


-- geen routine analyse in klinisch labogeen routine analyse in klinisch labo

-- verkeerde fenotypische identificatieverkeerde fenotypische identificatie

-- data 2 surveillance networks:data 2 surveillance networks:

A. butzleriA. butzleri = 4= 4°° meest voorkomende Campylobacteraceaemeest voorkomende Campylobacteraceae

-- BelgiBelgiëë (Vandenberg et al. 2004)(Vandenberg et al. 2004)

-- Frankrijk (ProuzetFrankrijk (Prouzet--MaulMaulééon et al., 2006)on et al., 2006)

77,33

11,44

4,46

3,52

C. jejuni subsp. jejuni C. coli Campylobacter upsaliensisArcobacter butzleri Campylobacter concisus Campylobacter fetus subsp fetusCampylobacter curvus Arcobacter cryaerophilus Campylobacter hyoinstestinalisCampylobacter lari Helicobacter pullorum Campylobacter sputorum subsp sp


1. niet1. niet--selectieve mediaselectieve media

1. filter plate method 1. filter plate method (Lastovica et al,2003 (Lastovica et al,2003 -- Abdelbaqi et al. JCM Abdelbaqi et al. JCM in pressin press))

2. selectieve media2. selectieve media

1. 1. YersiniaYersinia protocol protocol ((Collins et al.Collins et al. --> Burnens et al. 1992)> Burnens et al. 1992)

2. Leptospirra 2. Leptospirra (Ellis et al.)(Ellis et al.)

3. modified 3. modified CampylobacterCampylobacter medium medium (CCDA, (CCDA, CATCAT, , ……. ). )

4. 4. ArcobacterArcobacter semisemi--solid medium solid medium ((de Boer et al.,1996de Boer et al.,1996--> Abdelbaqi et al. JCM > Abdelbaqi et al. JCM in pressin press)

5. 5. ArcobacterArcobacter selective medium selective medium ((Houf et al., 2001Houf et al., 2001 --> Houf et al. 2007)> Houf et al. 2007)

AArcobacter rcobacter -- isolatieisolatie

Method Steele and Mc Dermott (1984)

Isolation on a nonIsolation on a non--selective medium selective medium based on bacterial size and motilitybased on bacterial size and motility

-- high detection limit (clinical samples)high detection limit (clinical samples)-- limited selectivity (motile bacteria)limited selectivity (motile bacteria)


de Boer et al. (1996)de Boer et al. (1996)

-- [cefoperazone] and [piperacilin] to high (Houf et al. 2001)[cefoperazone] and [piperacilin] to high (Houf et al. 2001)-- strain selectivitystrain selectivity

Isolation on a selective medium Isolation on a selective medium based on antibiotics and bacterial motilitybased on antibiotics and bacterial motility


Modified Modified CampylobacterCampylobacter mediummedium

CATCAT

8 mg/l cefoperazone10 mg/l amphotericin4 mg /l teicoplanin

+ sensitivity- specificity

Isolation on a selective medium Isolation on a selective medium based on antibioticsbased on antibiotics


selective isolation selective isolation brothbroth and and agaragar mediummedium-- 24 g/l 24 g/l ArcobacterArcobacter brothbroth

--12 g/l agar technical N12 g/l agar technical N°°33

-- 50 ml lysed horse blood50 ml lysed horse blood

-- selective supplementselective supplement* 100 mg/l 5* 100 mg/l 5--fluorouracilfluorouracil

* 10 mg/l amphotericin B* 10 mg/l amphotericin B

* 16 mg/l cefoperazone* 16 mg/l cefoperazone

* 32 mg/l novobiocin* 32 mg/l novobiocin

* 64 mg/l trimethoprim* 64 mg/l trimethoprim

Houf et al. (2001; 2007)

Quantitative method (cfu/g)Quantitative method (cfu/g)

Qualitative method (present/absence)Qualitative method (present/absence)

Validation parameters

repeatabilityreproducibilitydetection limitsensitivityspecificitymatrix effect


AArcobacterrcobacter = commensaal ?= commensaal ?

Specimen N° sex Region in Switzerland age species

28 male Central part 41 years A. cryaerophilus

36 male Northern part 44 years A. cryaerophilus

55 male Western Part 34 years A. cryaerophilus




456 male Western part 46 years A. cryaerophilus

2000 2000 -- Engberg et al. Engberg et al. –– 107 personen : 0% arcobacers 107 personen : 0% arcobacers (filter method)(filter method)

2007 2007 -- Houf and Stephan Houf and Stephan –– 500 personen 500 personen (method Houf et al., 2001)(method Houf et al., 2001)

1,4 %1,4 %


AArcobacter rcobacter genomicsgenomics **

**

*

AArcobacter rcobacter genomicsgenomics

Douidah et al. Presence of virulence-associated genes in 319 Arcobacter strains detected by gene-specific PCR assaysN°: number; -: no strains available

In vitroIn vitro studiesstudies

-- continuous cell lines continuous cell lines (Hep(Hep--2, HeLa, Vero, Caco, IPI2, HeLa, Vero, Caco, IPI--2I, CHO, 2I, CHO, …….).)

-- adhesion adhesion –– invasion invasion

Musmanno 1997 Musmanno 1997 -- Houf 2007 Houf 2007 -- Ho 2007Ho 2007

-- cytotoxic effectcytotoxic effect

Fernandez 1995 Fernandez 1995 -- Kalman 1996 Kalman 1996 –– Musmanno 1997Musmanno 1997

-- ileal loop testileal loop test

-- ratrat Fernandez 1995Fernandez 1995

-- rabbit and pigrabbit and pig Carter and Wesley, 1996Carter and Wesley, 1996

AArcobacter rcobacter genomicsgenomics

abortion abortion ––reproductive reproductive disordersdisorders

cowcowpigpig

mastitismastitis cowcow

gastric ulcersgastric ulcers pigpig

enteritisenteritisruminantsruminantspigpigprimatesprimates

AArcobacter rcobacter in landbouwhuisdierenin landbouwhuisdieren

EEpidemiologiepidemiologie

11

Outbreak of recurrent abdominal cramps associated with Arcobacter butzleri in an Italian school.Vandamme P, Pugina P, Benzi G, Van Etterijck R, Vlaes L, Kersters K, Butzler JP, Lior H, Lauwers S.

In the autumn of 1983, an outbreak of recurrent abdominal cramps occurred in a nursery and primary school in the Rovigo area in Italy. Noneof the 10 affected children had diarrhea. An atypical Campylobacter-like organism was isolated from feces in all cases. Conventionalenteropathogens were searched for but not detected. The Campylobacter-like organism was identified as Arcobacter butzleri by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins and cellular fatty acid analysis. Its identity was confirmedby DNA-DNA hybridizations versus Arcobacter reference strains. All of the preserved outbreak strains have identical protein profiles and phenotypiccharacteristics and belong to serogroup 1 of the Lior serotyping scheme on the basis of slide agglutination of crude and absorbed antisera of A. butzlerireference strains versus heat-labile antigens of live bacteria. These data point to an epidemiological relationship. The successive timing of the cases suggests person-to-person transmission.

Journal of Clinical Microbiology, 1992, 30: 2335-2337

11

22


77°°C C –– drinking waterdrinking water

Van Driessche E . Van Driessche E . –– Houf K.Houf K. JJournal of Applied Microbiology, 2008ournal of Applied Microbiology, 2008

Survival capacity of Survival capacity of ArcobacterArcobacter species in waterspecies in water

0%

20%

40%

60%

80%

100%

120%

0 21 42 63 84 105 126 147 168 189

incubation time (d)

% in

itial

Log

cfu

/

A.butzleri A.cryaerophilus A.skirrowii

77°°C C –– drinking water + organic materialdrinking water + organic material

0%

20%

40%

60%

80%

100%

120%

0 21 42 63 84 105 126 147 168 189

incubation time (d)

% in

itial

Log

cfu

/






52°C – drinking water

0%

20%

40%

60%

80%

100%

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

incubation time (min)

% in

itial

Log

cfu

/




0%

20%

40%

60%

80%

100%

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

incubation time

% in

itial

Log

cfu

/


52°C – drinking water + organic material

11

22

33


PREVALENCE OF PREVALENCE OF ARCOBACTERARCOBACTER IN HEALTHY DOGS AND CATSIN HEALTHY DOGS AND CATS

Houf K. et al. Houf K. et al. -- Veterinary Microbiology, 2008Veterinary Microbiology, 2008

Arcobacter was not isolated from the feces and swab samples from cats.

5 dogs shed arcobacters in the feces and 2 other dogs carried arcobacters in the oral cavity.

1 dog A. butzleri > 3 weeks

Rest of the isolates were identified as A. cryaerophilus strains

3 dogs came from the same household where the other 10 dogs present were negative.

Arcobacter species and prevalence in healthy dogs = healthy humans (Houf and Stephan, 2007).

stool buccal swab

dogs 5/252 2/252

cats 0/52 0/52

11

22

33

44


11

22

33

44


AArcobacter rcobacter in eetwaren van dierlijke oorsprongin eetwaren van dierlijke oorsprong pluimveeproductenpluimveeproducten

Product N n % Country Reference

Skin

Meat

Carcass rinseMeat/skin

Eggs

4012520117050374510524480756192241108022032142552151009425309657

401211628942293998204564919151620105253071634323582530510

10010097978181

52,352,38484

78,378,38787959583839595

65,365,331.131.16060737348481001006565

24,124,10050506464

65,465,4202023236262100100100100535300

CanadaCanadaFrance

GermanyU.S.A.

BelgiumBelgiumBelgiumBelgiumBelgiumTurkeyCzechU.S.A.

AustraliaJapan

ThailandFrance

The NetherlandsItaly

BelgiumBelgiumBelgiumU.S.A.Japan

IrelandU.K.

TurkeySpainItaly

Lammerding, 1994Lammerding, 1996Marinescu et al., 1996Harraß et al., 1998Johnson en Murano, 1999bHouf et al., 2000Houf et al., 2001aHouf et al., 2001aHouf et al., 2002aHouf et al., 2002bAtabay et al., 2002aVytřasová et al., 2003Villarruel-López et al., 2003Rivas et al., 2004Morita et al., 2004Morita et al., 2004Festy et al., 1993de Boer et al., 1996Zanetti et al., 1996bHouf et al., 2000Houf et al., 2001aHouf et al., 2001aVillarruel-López et al., 2003Kabeya et al., 2004Scullion et al., 2006Atabay en Corry, 1997/1998Atabay et al., 2006Gonzalez et al., 2000Zanetti et al., 1996b


Turkey Meat 3039517

03034

07724

ItalyU.S.A.

Denmark

Zanetti et al., 1996bManke et al., 1998Atabay et al., 2001

Turkey/Duck

MeatMeat/skin

950

046

092

CzechIreland

Vytřasová et al., 2003Scullion et al., 2004

Duck Carcass 1010

87

8070

U.K.Denmark

Ridsdale et al., 1998Atabay et al., 2001

Rabbit Meat 8 0 0 Czech Vytřasová et al., 2003

AArcobacter rcobacter in eetwaren van dierlijke oorsprongin eetwaren van dierlijke oorsprong pluimvee en konijnpluimvee en konijn


Beef Ground meat

Minced meat

453290

108689

97

1372

37155

28,9222,2341,5

55,65,1

U.S.A.Australia

JapanIreland

The NetherlandsCzechTurkey

Villarruel-López et al., 2003Rivas et al., 2004Kabeya et al., 2004Scullion et al., 2006de Boer et al., 1996Vytřasová et al., 2003Ongör et al., 2004

Pork Ground meat

Minced meat

299272004521100101194416

1671

642367

35100

55,83,732

51.1297

350.500

U.S.A.Italy

U.S.A.U.S.A.

AustraliaJapan

IrelandThe Netherlands

ItalyCzech

Collins et al., 1996aZanetti et al., 1996bOhlendorf en Murano, 2002Villarruel-López et al., 2003Rivas et al., 2004Kabeya et al., 2004Scullion et al., 2006de Boer et al., 1996Zanetti et al., 1996bVytřasová et al., 2003

Pork/Beef Minced meat

613

30

4.90

The NetherlandsCzech

de Boer et al., 1996Vytřasová et al., 2003

Sheep Ground meat

13 2 15 Australia Rivas et al., 2004

AArcobacter rcobacter in eetwaren van dierlijke oorsprongin eetwaren van dierlijke oorsprong rood vleesrood vlees

AArcobacter rcobacter in landbouwhuisdierenin landbouwhuisdieren

Rectaal (of droppings) Rectaal (of droppings) genomen faeces stalen genomen faeces stalen van van nietniet--gerelateerdegerelateerdedierendieren

51 runderen51 runderen82 varkens82 varkens62 schapen62 schapen13 paarden13 paarden84 braadkippen84 braadkippen

rund 39.2% varken 43.9% schaap 16.1% paard 15.4%kip 0.0 %

Ante-mortem

Arcobacter contamination on Belgian bovine carcassesDe Smet S. – Houf et al. JAM

AArcobacter rcobacter in Belgisch rundsvleesin Belgisch rundsvlees

post-mortem

rund 39.2%


post-mortem



post-mortem



post-mortem

- 9 % Arcobacter contaminatie-vnl. A. butzleri- aantallen < 100 kve / g


directly p.e.(n=18)

6 h p.e. (n=80)

24 h p.e. (n=71)

foreleg 18 74 52

chest 17 75 56

pelvis 17 47 13

ham 15 57 22

Characterization of the Arcobacter contaminationon Belgian pork carcasses

Van Driessche E and Houf et al. International Journal of Food microbiology,

23,8%

19,2%

1-1000 kve / gram

31 detailhandels

AArcobacter rcobacter in Belgisch varkenvleesin Belgisch varkenvlees

AArcobacter rcobacter in Belgische pluimveeproductenin Belgische pluimveeproducten

Onderzoek van 10 g monster

Arcobacter Campylobacter

Braadkip (huid) 90 % 55 %

Braadkip filet 65 % 42 %

Soepkip (huid) 100 % 88 %

contaminatie aantal : 101 – 103 cfu / g

species : A. butzleriA. cryaerophilus

A. skirrowii Houf et al.,2002; 2003

AArcobacter rcobacter transmissietransmissie

AArcobacter rcobacter transmissie transmissie -- varkenvarken

12 14 16 18 20 22 24 26 28 30Leeftijd (weken)

1 2 3 4 5 6 7 8 9 10Nr.

staalname

Aankomst vleesvarkensstal

Slachtrijp

Opfokperiode

Hok 1

10 varkens

Hok 2

10 varkens

middengang

Hok 3

10 varkens


Identificatie- Species niveau: multiplex-PCR (Houf et al., 2000)- Genus niveau: genus-PCR (Harmon en Wesley, 1996)

Karakterisatie- ERIC PCR (Houf et al., 2002)- AFLP (Debruyne L, Houf et al., J. Syst. Bact.)


B

AC

Doxycycline


Bedrijf Species identificatiex

A. butzleri A. cryaerophilus A. skirrowii A. thereius

A 11 (1/10) 3 (0/3) 1 (0/1) 50 (47/3)

B 10 (0/10) 0 (0/0) 37 (37/0) 78 (68/10)

C 44 (10/34) 2 (1/1) 1 (1/0) 1 (0/1)

D 215 (78/137) 7 (5/2) 0 (0/0) 10 (7/3)

( )x: aantal isolaten na telling / aantal isolaten na aanrijking

+ 16 isolaten niet identificeerbaar tot op species niveau


Heterogeniteit van Arcobacter

100

90807060

96.4

95.6

94.6

92.9

88.7

76.1

95.6

94.7

57.4

98.7

96.5

95.8

94.7

52.1

G 127G 128

G 113

G 55G 129

G 206

G 92G 31

G 194

G 195G 166

G 190G 54

G 188

G 32

G206G113G128G92G129G55G127G54G166G190G32G188G31G195G194

AFLP ERIC15 A. butzleri isolaten in Varken 92

Bedrijf D (staalname 10)

Hok 1

B11

B1

B2

Hok 2

B4B11

C1

B8B10

B12

Hok 3

B4

B1B7

B1’


Hok 1 Hok 2

Hok 3

Genotype B4staalname 2, 3 en 4 (14, 16 en 18 wkn) Hok 2

staalname 5 (20 wkn) Hok 2 en 3Staalname 9 (28 wkn) Hok 1, 2 en 3

Hok 1 Hok 2

Hok 3

Genotype B4Staalname 10 (28 wkn) Hok 2 en 3

Hok 1

B11

B1

B2

Hok 2

B4B11

C1

B8B10

B12

Hok 3

B4

B1B7

B1’

Genotype C1 Bedrijf D (staalname 10)

Hok 1 Hok 2

Hok 3

Staalname 5 (20 wkn) Hok 1Staalname 9 (28 wkn) Hok 1 en 2 Staalname 10 (30 wkn) Hok 2

hoge graad van genetische diversiteit op dier-, hok- en bedrijfsniveau (ERIC-PCR > AFLP)

Arcobacter stammen kunnen persisteren en spreiden in de vleesvarkenstal gedurende de opfok (12-30 weken)

naast faeco-orale transmissie, mogelijke rol van andere transmissie routes niet uitsluiten


Arcobacter thereius sp. nov. (n=140) werd voor het eerst geïsoleerd uit 43 faecale stalen

een potentieel nieuw Arcobacter species (n=16) werd geïsoleerd uit de faeces van varkens (12, 14 en 16 wkn )

aanwezigheid van arcobacters in faeces van varkens op slachtleeftijd → contaminatie varkenskarkassen →infectierisico mens


AArcobacter rcobacter transmissie transmissie -- kipkip

Broiler flock A (n = 3000) Broiler flock A (n = 3000) –– first flock slaughtered on mondayfirst flock slaughtered on monday

-- just before slaughterjust before slaughter

* 20 chickens randomly taken from transport crates* 20 chickens randomly taken from transport crates

euthanazationeuthanazation

* samples:* samples: -- feathersfeathers

-- neck skinneck skin

-- breast & thigh skinbreast & thigh skin

-- skin around cloacaskin around cloaca

-- bile / spleen / liver / stomachs / cropbile / spleen / liver / stomachs / crop

-- content of cecum, cloaca and small content of cecum, cloaca and small intestineintestine

* 120 cloacal swabs* 120 cloacal swabs

* 10 swabs from transport crates* 10 swabs from transport crates

-- At slaughter : 45 neck skin samplesAt slaughter : 45 neck skin samplesdetectable prevalence > 2.25 % (= 68 birds)detectable prevalence > 2.25 % (= 68 birds)


SampleSample 20 birds20 birds

FeathersFeathers 00

Neck skinNeck skin 00

Breast skinBreast skin 00

Thigh skinThigh skin 00

Skin around cloacaSkin around cloaca 11*1*1

BileBile 00

SpleenSpleen 00

LiverLiver 00

StomachsStomachs 00

CropCrop 00

Cloacal swabCloacal swab 11*2*2

Cecal contentsCecal contents 00

Content of small Content of small intestineintestine

00

11 Ac+As, Ac+As, 22 AcAc


* No arcobacters isolated from the cloacal swab (n=120)

* Transport crates

2

18

A. butzleri A. cryaerophilus

7/10 transport crates positive for Arcobacter


27%

40%

33%

Ab Ac Ab+Ac

Neck skin samples (n=45 Neck skin samples (n=45 -- first 10 birds first 10 birds –– then every 20then every 20thth birdbird))

-- all samples positiveall samples positive

-- contaminated with > 10contaminated with > 1033 cfu/gcfu/g


Method validation in terms ofMethod validation in terms of-- rrepeatabilityepeatability-- rreproducibilityeproducibility-- ssensitivityensitivity-- sspecificitypecificity-- detection limitdetection limit

Arcobacter Arcobacter reference strains with a known reference strains with a known bacterial concentration for bacterial concentration for in vitro in vitro contamination of fecal samplescontamination of fecal samples

-- 101033 cfu/mlcfu/ml-- 101044 cfu/mlcfu/ml

3

3,5

4

4,5ho

rse

shee

p

cow

pig

poul

try

1 2 3 4 5 6

2

2,5

3

3,5

hors

e

shee

p

cow pig

poul

try

1 2 3 4 5 6


Flock B (n = 5000)Flock B (n = 5000)

* 250 intestinal tracts* 250 intestinal tracts

1 g of content from1 g of content from

-- small intestinesmall intestine

-- cecumcecum

sampling sampling detectable prevalence > 1.2 %detectable prevalence > 1.2 %

* 45 neck skin samples* 45 neck skin samples



1/ surface swab + 1 g content of 11/ surface swab + 1 g content of 1stst cecumcecum

2/ superficial ethanol treatment of other cecum2/ superficial ethanol treatment of other cecum

3/ surface swab + 1 g content of 23/ surface swab + 1 g content of 2ndnd cecumcecum

4/ samples small intestines4/ samples small intestines


1/ storage for 24 h at 41/ storage for 24 h at 4°°CC

2/ 2/ surface swab + 1 g content of 1 cecumsurface swab + 1 g content of 1 cecum

90%

26%

cecal contentsurface



1/ 1/ surface swab + 1 g content of 1surface swab + 1 g content of 1stst cecumcecum


3/ 3/ surface swab + 1 g content of 2surface swab + 1 g content of 2ndnd cecumcecum




2/ 2/ surface swab + 1 g content of 1 cecumsurface swab + 1 g content of 1 cecum

90%

26%


7%

0 %



1/ 1/ surface swab + 1 g content of 1surface swab + 1 g content of 1stst cecumcecum


3/ 3/ surface swab + 1 g content of 2surface swab + 1 g content of 2ndnd cecumcecum




2/ surface swab + 1 g content of 1 cecum2/ surface swab + 1 g content of 1 cecum

90%

26%


7%

0 %

100% 100%

100 % Campylobacter +


- at slaughterhouse

- presence of a continuous contamination source

- first bird slaughtered +- carcass contamination within a flock = almost 100%- large heterogeneity- contamination 10 – 10.000 cfu g skin

- in all poultry slaughterhouses ( 16 examined – capacity > 3000 birds / h)

- transport crates

- contaminated before and after loading -> cleaning / disinfection ?

molecular epidemiology : no identification of the transmission routes

living birds - Arcobacter no part of intestinal / skin floraor - Arcobacter prevalence < 1 % and at least 100 x contamination rate

Van Driessche E. and K. Houf. 2007. Discrepancy between the occurrence of Arcobacter in chickens and broiler carcass contamination. Poultry Science. 86:744-751.


CConclusiesonclusies

Arcobacter = humane pathogeen ?

-> Indicaties

- focus op A. butzleri- validatie en implementatie van methoden- studie van virulentie factoren

- genome mapping- in vitro testing - dier model ? -> Su - Ca

CConclusiesonclusies

Arcobacter = foodborne pathogen?

- aanwezig op vlees (pluimvee > varken > rund)- epidemiologie Arcobacter ≠ Campylobacter

- standardizatie / validatie van methoden- studie van de transmissie routes

- heterogeniteit - genomic variation- multiple reservoirs

- characterization methods

-> virulentie merker

Bijzonder dank aan:Bijzonder dank aan:

Dr. Ellen Van DriesscheDr. Ellen Van DriesscheDrs. Sarah De smetDrs. Sarah De smetDrs. Laid DouidahDrs. Laid DouidahDrs. Julie BarDrs. Julie Baréé

Dr. Johan VanhendeDr. Johan Vanhende

Prof. Dr. Peter VandammeProf. Dr. Peter VandammeProf. Dr. Tom CoenyeProf. Dr. Tom CoenyeDr. Lies DebruyneDr. Lies Debruyne

Dr. Stephen On (Nieuw-Zeeland)Dr. Bill Miller (USA)Prof. Dr. Hanne Igmer (Denemarken)Prof. Dr. Maria Figueras (Spanje)Prof. Dr. Roger Stephan (Zwitserland)

voordracht arcobacter 2009 ppt 97 wavfh -...

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