Campylobacter jejuni Detection in Proboscis Monkey (Nasalis larvatus)

Bibi Jacquelyn Julian (35597)

Bachelor of Science with Honours

(Resource Biotechnology)

2015

I

ACKNOWLEDGEMENT

I thank God that this study was able to be completed and for these people that have

been supporting me. My deepest gratefulness goes to my family especially to my parents for

their moral, financial support and above all for believing in me. I owe my sincere thanks to my

supervisor, Dr. Chong Yee Ling, for her guidance, constant reminders and specially for never

giving up on me. I wanted to say my appreciation as well to my co-supervisor, Dr. Lesley

Maurice Bilung, for allowing me to complete my laboratory work at Microbiology lab.

Not to forget, the persons that have indirectly contributed in completing this project especially

for Mr. Ng Kar Hon, whom had been really generous to share his collected samples from Bako

national Park with me and guiding me through my first ever sampling trip experience; Mdm.

Ernie Suhaiza, whom never get tired with my questions and keep guiding me during my

laboratory work; Mr. Trevor, Mr. Azis, other zoology and molecular biology departments’

postgraduates and the rangers that been helping me during my sampling trip in Samunsam

Wildlife Sanctuary as well as the whole team of Samunsam Wildlife Sanctuary fieldtrip.

Many thanks go to Abid for providing me the GPS reading and guiding me during my sampling

in Samunsam Wildlife Sanctuary, my lab-mates Hasbi, Jennifer, Claire, Hasna, Emma, Vivian,

Haziq and others in Microbiology lab. Last but not least, I would love to thank these people

for their helps and supports throughout the process of completing my laboratory work; Gabang,

Oney, Ludiah, Evie, Shanne and Bani, thank you.

II

DECLARATION

I hereby declare that the thesis is based on my original work except quotations and citation,

which have been duly acknowledgements. I also declare that it has not been previously or

concurrently submitted for any degree at Universiti Malaysia Sarawak or other institutions.

12th May 2015

Name: Bibi Jacquelyn Julian

Matric No: 35597

IC No: 930607-12-5418

III

TABLE OF CONTENTS

Acknowledgement I

Declaration II

Table of Contents III

List of Abbreviations IV

List of Tables and Figures V

Abstract 1

1.0 Introduction 2-3

2.0 Literature Review

2.1 Campylobacter jejuni

2.2 Proboscis Monkey

2.3 C. jejuni in Non-Human Primates

2.4 C. jejuni Detection

4

4

4-5

5-6

6-7

3.0 Materials and Method

3.1 Sample Collections

3.2 Samples Processing

3.3 Isolations of DNA from Bacterial Cells Using Boiling Method

3.4 Polymerase Chain Reaction (PCR)-based Detection of HipO gene

3.5 Agarose Gel Electrophoresis

8

8

9

10

10-11

11

4.0 Results

4.1 Detection of C. jejuni in Fecal Samples Collected from Bako National Park

4.2 Detection of C. jejuni in Fecal samples Collected from Samunsam Wildlife

sanctuary

4.3 Prevalence of Campylobacter spp. between Bako National Park and

Samunsam Wildlife Sanctuary

12

12-13

14-15

15-16

5.0 Discussion 17-19

6.0 Conclusion 20

7.0 References 21-24

IV

LIST OF ABBREVIATIONS

CFA Campyfood agar

mCCDA modified Charcoal Cefoperazone Deoxycholate Agar

PCR Polymerase Chain Reaction

qPCR Real-time Quantitative Polymerase Chain Reaction

NIHSJ-02 National Institute Health Science Japan-02

ISO 10272-1:2006 International for Standardization 10272-1:2006

EDTA Ethylenediaminetetraacetic acid / Edetic acid

SEM Scanning Electron Microscopy

IUCN

GBS

International Union for Conservation of Nature

Guillain Barré syndrome

V

LIST OF FIGURES

Page

Figure 1. Greyish, flat and moistened colonies morphology was

observed on modified charcoal cefoperazone deoxycholate agar in

one of the isolates from Bako National Park, indicating positive for

Campylobacter spp..

12

Figure 2. S-shaped rod morphology one of the isolates from Bako

National Park using negative Gram staining and viewed using 100X

magnification of compound microscope.

13

Figure 3. Greyish, flat and moistened colonies morphology was

observed on modified charcoal cefoperazone deoxycholate agar in

one of the isolates from Samunsam Wildlife Sanctuary, indicating

positive for Campylobacter spp..

14

Figure 4. S-shaped rod morphology one of the isolates from

Samunsam Wildlife Sanctuary using negative Gram staining and

viewed using 100X magnification of compound microscope.

15

1

Name: Bibi Jacquelyn Julian

Title: Campylobacter jejuni Detection in Proboscis Monkey (Nasalis Larvatus)

ABSTRACT

This study was done to detect the prevalence of Campylobacter jejuni in proboscis monkey’s fecal

samples collected from Bako National Park (n = 5) and Samunsam Wildlife Sanctuary, Sarawak (n = 37). C.

jejuni is commonly found in wildlife and domestic animals. The stool samples were enriched with Bolton broth

(ISO 10272-1: 2006), followed by cultured on selective agar, modified charcoal cefoperazone deoxycholate agar

(mCCDA) and were then sub-cultured on Bolton broth. Pure cultures of the isolated stool samples were tested

with Gram staining for morphological identification. In this study, Boiling method protocol was used for

purification of total DNA from the established pure cultures of the stool samples. All 42 established pure cultures

of the stool samples gave no reaction with the PCR assay targeting hippuricase gene for species level detection

of C. jejuni. However, high numbers of positive cultures were observed on the selective agar, mCCDA, which

indicating C. jejuni positive colonies. Furthermore, morphological test on the cultures observed S-shaped rod

characteristic. The prevalence of Campylobacter spp. detected in fecal samples collected from Samunsam

Wildlife Sanctuary (86.486%) was higher than of Bako National Park (60%). Although the specific species of

Campylobacter cannot be identified via PCR approach, the presence of colonies on mCCDA with high prevalence

indicates that proboscis monkey may be considered as a potential reservoir of Campylobacter spp.

Keywords: Campylobacters jejuni, Campylobacter spp., proboscis monkeys, hippuricase gene, modified charcoal

cefoperazone deoxycholate agar

ABSTRAK

Tujuan kajian ini adalah untuk mengesan sebaran C. jejuni melalui sampel najis yang dikutip daripada

Taman Negara Bako (n = 5) dan Taman Hidupan Liar Samunsam, Sarawak (n = 37). C. jejuni kerap dikesan pada

hidupan liar dan haiwan-haiwan domestic. Sampel najis yang diperoleh dibiakkan dengan menggunakan Bolton

broth (ISO 10272-1: 2006), kemudian dikultur pada agar terpilih, modified charcoal cefoperazone deoxycholate

agar (mCCDA) dan kemudian dikultur semula pada Bolton broth. Pewarnaan gram juga dilakukan pada kultur

untuk identifikasi karakter morfologi. Kaedah pendidihan digunakan untuk memperolehi DNA bakteria daripada

kultur tersebut. Tiada spesies bakteria yg dapat dikenal pasti melalui ujian PCR yang dijalankan pada sejumlah

42 kultur dengan mensasarkan hippuricase gene. Namun, kultur-kultur yang menunjukkan pembiakan koloni

bakteria pada agar dapat dilihat dalam jumlah yang agak tinggi. Melalui ujian morfologi yang dijalankan, karakter

morfologi yang berbentuk S dapat dilihat. Sebaran Campylobacter spp. yang dikesan pada sampel najis yang

diambil dari Taman Hidupan Liar Samunsam (86.486%) adalah tinggi berbanding yang dikesan pada sampel najis

dari Taman Negara Bako (60%). Walaupun spesies bakteria campylobacter tidak dapat disahkan melalui ujian

PCR, pembiakan koloni pada agar mCCD dalam peratusan yang tinggi menunjukkan bahawa monyet belanda

berpotensi untuk menjadi waduk kepada Campylobacter spp..

Kata kunci: Campylobacter jejuni, Campylobacter spp. monyet belanda, hippuricase gene, modified charcoal

cefoperazone deoxycholate agar

2

1.0 INTRODUCTION

Campylobacter jejuni is a curved Gram negative bacterium that possesses several unique

morphological characteristics such as cytochrome oxidase positive, microaerophilic and

corkscrew motility (Levin, 2007). It is also stated by Levin (2007) that this bacterium colonizes

the intestinal tracts of many wildlife and domestic animals, especially in poultry, in which

some of the healthy animals may show asymptomatic infection. In most countries, C. jejuni

had been known as the main causes of disease termed campylobacteriosis (Ugarte-Ruiz et al.,

2012). Other than that, Young et al. (2007) stated that this bacterium exist in water in large

amount. Non-human primates are one of the animals that are susceptible to the infection of C.

jejuni and the transmission is commonly via fecal or oral route. C. jejuni infection in non-

human primates will cause diarrhea, which resulting in mild to severe enteritis (Vilardo et al.,

2005). For example, a study done by Vilardo et al. (2005) on the prevalence of Campylobacters

in non-human primates such as cynomolgus monkeys (Macaca fascicularis), rhesus macaque

(Macaca mulatta) and squirrel monkeys (Saimiri sp.) showed that C. jejuni is one of the

Campylobacter spp. that most frequently isolated from stool samples. Furthermore, C. jejuni

is accounted for most of the infections (80% - 90% of infections were caused by C. jejuni) and

some serotypes are related with Guillain Barré syndrome (Vilardo et al., 2005). Guillain Barré

syndrome (GBS) is a common cause of neuromuscular paralysis and C. jejuni infection is one

of the factors that play an important role in the pathogenesis of GBS (Doorn et al., 2008). Thus,

C. jejuni may become a threat to human health. However, the study on C. jejuni detection in

proboscis monkeys (Nasalis larvatus) has yet been done. Proboscis monkeys may be infected

by this organism through environmental contaminants in water and food. As this bacterium

can be found in other non-human primates, hence, proboscis monkey are likely to become the

3

sources of infection from this disease-causing bacterium. Thus, this study is important to

understand the possible circulation of this bacterium in proboscis monkeys. Research

objectives of this study are:

1. To isolate C. jejuni from the proboscis monkey’s faeces and further confirm via

Polymerase Chain Reaction targeting hippuricase gene.

2. To compare the prevalence of C. jejuni between different sampling locations, Bako

National Park and Samunsam Wildlife Sanctuary.

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2.0 LITERATURE REVIEW

2.1 Campylobacter jejuni

C. jejuni is a thermophilic and highly motile bacterium, curved Gram negative, non-

spore forming and is positive for hippurate hydrolysis, oxidase and catalase (Snelling et al.,

2005). In countries across the world, C. jejuni is considered to effectuate enteric illness, which

causing severe symptoms in human but some may cause serious infections as well, such as

infection of the extremities and permanent neurological symptoms (Levin, 2007). C. jejuni was

observed and isolated most frequently from faeces of both asymptomatic and clinically

affected wildlife, including non-human primates (Levin, 2007). Furthermore, certain animals

infected by C.jejuni may exhibit similar symptom of disease that are seen on human such as

diarrhea and inflammation (Young et al., 2007). Vilardo et al. (2005) stated that C. jejuni is

closely associated with Guillain Barré syndrome, which caused neuromuscular paralysis in

human.

2.2 Proboscis Monkeys

Proboscis monkey (Nasalis larvatus) or also known as Long-nosed monkey is

considered as an endangered species and was considered to be facing a high risk of extinction

in the wild as it is threaten by activities such as ongoing hunting and habitat destruction

(Meijaard et al., 2008). During the year 2000, it was classified as an endangered species by

International Union for Conservation of Nature (IUCN). Meijaard et al. (2008) stated that there

are less than 1000 proboscis monkeys exist in unevenly divided populations in Sarawak. This

reddish-brown arboreal old world monkey is recognized as one of the largest monkey species

5

native to Asia, sexually dimorphic and can be found in the island of Borneo (Boonratana,

2000).

Furthermore, it inhabits mangroves, swamps and riverine forests that are linked with

waterways but always stay 1 km away from the river (Muhammad et al., 2009). However, it

often congregates near river banks to sleep and would return to its sleeping sites every evening

(Muhammad et al., 2009). Matsuda et al. (2011) stated that proboscis monkeys will move to

new sleeping sites every day to minimize competition for food resources and sleeping trees.

Other than that, they will choose tall trees as their sleeping trees in order to avoid predators

attack (Matsuda et al., 2011). Occasionally, they will venture onto land to search for food and

sometimes swim across the river (Boonratana, 2000). It was stated by Matsuda et al. (2011)

that the proboscis monkeys’ diet is mainly on leaves, seeds and unripe fruits.

2.3 C. jejuni in Non-human Primates

C. jejuni is one of the bacteria that frequently isolated from faeces of healthy non-

human primates (Okwori et al., 2014). C. jejuni colonizes the intestinal tracts of many wildlife

and domestic animals, especially in poultry, in which some of the healthy animals may show

asymptomatic infection (Levin, 2007). Non-human primates are one of the animals that are

susceptible to the infection of C. jejuni from contaminated water and food sources. In addition,

the transmission is commonly via fecal or oral route. C. jejuni infection in non-human primates

will cause watery diarrhea that affected jejunum, ileum and colon, which resulting in mild to

severe enteritis (Vilardo et al., 2006). A study done by Ngotho et al. (2006) on C. jejuni in

vervet monkeys showed that wild non-human primates may act as a reservoir of this bacterium.

C. jejuni pathology in infected monkeys is similar to C. jejuni pathology in the human and in

piglets except for the lesions in the stomach is more prominent in monkeys (Ngotho et al.,

6

2006). There is no study on C. jejuni detection in proboscis monkeys (Nasalis larvatus) has

been done.

2.4 C. jejuni Detection

There are several methods in C. jejuni detection such as conventional methods and

molecular methods. A study done by Ugarte-Ruiz et al. (2012) compared four types of methods

(i.e. direct plating, Bolton enrichment, Preston enrichment and real-time quantitative

polymerase chain reaction, qPCR) for the detection of thermophilic campylobacter at different

stages in the food chain (i.e. poultry faeces, neck skin and packed fresh meat). The study

identified that the fast, simple and cheap method was through direct plating, which yield the

best isolation efficiency for campylobacter in highly contaminated sample such as faeces and

neck skin samples. Other than that, Linton et al. (1997) stated that C. jejuni is consider closely

related to C. coli, both are differentiated through biochemical test such as hippurate hydrolysis

test in which only C. jejuni is able to hydrolyze hippurate, but sometimes certain C. jejuni

strains (e.g. C. jejuni subsp. jejuni D 603) failed to do so.

Several enrichment methods (e.g. Bolton enrichment, Preston enrichment) were shown

to be effective in samples that were less contaminated with C. jejuni such as retail chilled

minced chicken and packed fresh meat. For example, in NIHSJ-02 method as described by

Momose et al. (2013), Preston broth was used for the enrichment step instead of Bolton broth

as recommended in ISO 10272-1:2006 in which the growth of background bacteria was able

to be reduced. Momose et al. (2013) managed to observe a lower frequency of occurrence of

background bacteria in NIHSJ-02 method compared to ISO 10272-1:2006 method which are

43.1% and 92.6%, respectively. Other than that, the selectivity of standard Bolton broth can be

restored by adding 2 mg/l potassium clavulanate as described by Moran et al. (2011).

7

While for qPCR detection, Ugarte-Ruiz et al. (2012) described this method as the most

sensitive method for detection for all types of samples, as it yielded the highest positive

samples. A study done by Lin et al. (2008) shown that clinical samples can be directly detected

by using qPCR after DNA extraction without pre-enrichment step. The number of positive

samples detected by qPCR was consistent with the number of positive samples detected using

culture-based method. It also provides faster performance with lower detection limit and the

genes that commonly used in molecular-dependent technique are 16S r RNA gene, hipO gene

and ceuE gene (Ertas et al., 2003). However, without culture-based method, some phenotypic

properties cannot be tested and information regarding the subtyping or antimicrobial resistance

testing cannot be obtained (Ugarte-Ruiz et al., 2012).

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3.0 MATERIALS AND METTHODS

3.1 Sample Collections

A sampling trip to Samunsam Wildlife Sanctuary was conducted for a week from 26th

February 2015 until 4th March 2015. The collection of samples was done as early as 6 am in

the morning. During the first day of sampling, fecal samples were collected according to the

places which groups of proboscis monkeys were found along the riverside since tagging of the

proboscis monkeys’ sleeping sites was unable to be done on the day before. The samples were

collected using a plastic forceps and placed into sterile zip lock bags, which then kept on ice

in a polystyrene box. Fecal samples that were collected within two meters area were considered

from one individual. The fecal samples collected were differentiated by groups based on the

areas it was collected.

Tracking of proboscis monkeys was done on the evening at 5 pm along Sungai

Samunsam from Samunsam Jetty to Samunsam Buta. GPS readings were taken in each area,

which the groups of proboscis monkeys were found (Appendix A). Other than that, each place

was tagged using warning tapes to indicate the sleeping sites of the proboscis monkeys. Fecal

samples were collected from some of the sleeping sites in the next morning. These sampling

protocols were done for four days of sampling, which were on 27th February 2015, 28th

February 2015, 1st March 2015 and 3rd March 2015. Sampling was unable to be done on 2nd

March 2015 due to adverse weather condition (heavy rain and high tide). Additional five fecal

samples of proboscis monkeys were given by Mr. Ng Kar Hon. These fecal samples were taken

based on sampling at Bako National Park on October 2014.

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3.2 Sample Processing

Five samples from Bako National Park were processed on February 2015 and 37

samples from Samunsam Wildlife Sanctuary were processed on March 2015. All samples were

processed in Microbiology Lab of Faculty Resource Science and Technology. Processing of

the fecal samples was done based on the standard ISO 10272-1:2006 protocols with minor

modifications.

First, 0.50 g of fecal samples was weighed and dissolved in 0.45 ml Bolton selective

enrichment broth (Merck, USA) in small sterilized air-tight bag. All samples were kept inside

an anaerobic jar (Oxoid, UK) with a gas-pack kit (Anaerocult; Merck, USA) inside to generate

microaerobic condition. For pre-enrichment incubation, the samples were incubated at 37 °C

for six hours, which were transferred to an incubator shaker (Infors HT, Switzerland) and

incubated at 42 °C for additional 48 hours.

After 48 hours, 50 µl of the enriched samples were cultured on modified charcoal

cefoperazone deoxycholate agar (mCCDA; Merck, USA) by spread plate method using hockey

stick. Only a plate was made for each sample. The plates were kept inside an anaerobic jar with

a gas-pack kit to generate microaeroic condition. The plates were then incubated for 48 hours

at 42 °C in an incubator shaker. Greyish, flat and moistened colonies growth was expected to

be observed on the plates after 48 hours of incubation. mCCDA is a selective media for

Campylobacter species, hence, other species of campylobacter such as Campylobacter coli,

Campylobacter lari and Campylobacter fetus were suspected to grow on the media especially

at the less selective temperature of 37 °C. Next, two single colonies from each plate were

picked and sub-cultured onto Bolton broth in two different 2 ml Eppendorf tubes.

10

The Eppendorf tubes were kept in anaerobic jar with a gas pack kit to generate microaerobic

condition for 48 hours incubation at 42 °C. Gram staining was performed to determine the

morphological characteristics.

3.2 Isolation of DNA from Bacterial Cells Using Boiling Method

100 µl of samples that were sub-cultured onto a Bolton broth were transferred into 1.5

ml Eppendorf tubes. The samples were centrifuged at 15 000 rpm for 15 minutes to obtain

bacterial pellets. The supernatants were discarded before the pellets were suspended in 40 µl

sterile distilled water. Next, the suspended pellets were washed by centrifugation at 15 000

rpm for 10 minutes and the supernatants were discarded afterwards. The pellets obtained were

re-suspended with 40 µl sterile distilled water and were boiled in a water bath at 100 °C for 10

minutes. After boiling for 10 minutes, the samples were cooled on ice for 15 minutes and later

centrifuged at 15 000 rpm for 10 seconds. The templates DNA were stored at -20 °C and only

2 µl of template DNA were used for Polymerase Chain Reaction.

3.3 Polymerasse Chain Reaction (PCR)-Based Detection of HipO Gene

The HipO gene-PCR provides detection of Campylobacter jejuni, which targeting

hippuricase gene. Species-specific primers, HIP400F and HIP1134R (adopted from Aldraghi,

2014), which give PCR product of about 735 bp were used. The oligonucleotide sequences of

forward primer and reverse primer used for PCR analysis are 5’-GAA GAG GGT TTG GGT

GGT G-3’ (Amresco, USA) and 5’-AGC TAG CTT CGC ATA ATA ACT-3’ (Amresco,

USA), respectively.

All PCR amplifications were performed in a solution containing 10.0 µl 5X Green

Buffer (Promega, USA), 2.0 µl 25 mM MgCl2 (Promega, USA), 1.0 µl dNTPs (Promega,

11

USA), 1.0 µl of each primer, 0.25 µl 5U Taq DNA Polymerase (Promega, USA), 2.0 µl of

genomic DNA and 32.75 µl ddH2O (Table 1 of Appendix B). One reaction was mixed with

sterilized distilled water to substitute the genomic DNA to be used as the negative control. The

protocol of PCR amplification was as follows (Table 2 of Appendix B): initial denaturation at

95 °C for 10 min (1 cycle); 30 cycles of denaturation at 94 °C for 30 sec, annealing at 59 °C

for 90 sec, and extension at 72 °C for 10 min. The protocol described above was based on

Mateo et al. (2005) with minor modifications. Other than that, positive control strain of

Campylobacter jejuni that was used in this study is ATCC 29428.

3.4 Agarose Gel Electrophoresis

In this study, 735 bp of molecule size is expected to be observed through gel

electrophoresis. 1.5 % agarose (Bio-Rad, USA) in 1X TBE Buffer (100 ml of 10X TBE Buffer

in 900 ml dH2O) was made. The agarose was dissolved by boiling the solution in microwave

oven for two minutes. The solution was cooled to room temperature before it was poured into

the gel tray of electrophoresis apparatus with well combs in place. It was allowed to set for

approximately 15 minutes. Next, 6 µl of 100 bp DNA ladder (Promega, USA) as molecular

weight markers was loaded into the first lane of the gel. Then, 6 µl of each amplified PCR

product was loaded to the additional wells of the gel and the last well was loaded with the

negative control. The electrophoresis was run at 100 V for 45 minutes and then was stained

with ethidium bromide solution for 25 minutes. The gel was visualized using gel

documentation system (Bio-Rad, USA) and photographed.

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4.0 Results

4.1 Detection of Campylobacter jejuni in Fecal Samples Collected from Bako National

Park

After all five samples were cultured on mCCDA, colonies growth was observed in three

(BPM 065, BPM 066, BPM 067) out of five samples from Bako National Park. Greyish, flat

and moistened colonies were observed on the three mCCDA plates after 48 hours incubation

at 42 °C as shown in figure 1. Other than that, figure 2 shows the S-shaped rod morphology

characteristic that was able to be observed in these three samples by negative Gram staining.

However, no product obtained from PCR assay specific for C. jejuni of the three samples.

Figure 1. Greyish, flat and moistened colonies morphology was observed on

modified charcoal cefoperazone deoxycholate agar in one of the isolates from Bako

National Park, indicating positive for Campylobacter spp..

13

Figure 2. S-shaped rod morphology one of the isolates from Bako National Park

using negative Gram staining and viewed using 100X magnification of compound

microscope.

4.2 Detection of C. jejuni from Fecal Samples Collected in Samunsam Wildlife Sanctuary

A total of 37 proboscis monkey’s fecal samples were collected within four days out of

seven days of the sampling. All 37 samples from Samunsam Wildlife Sanctuary were cultured

onto mCCDA. 35 plates showed growth of bacteria colonies while only two plates (SPM 002

and SPM 007) did not have any colony growing. Greyish, flat and moistened colonies were

observed on 35 mCCDA plates as shown in figure 3.

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However, only 32 samples showed S-shaped rod morphological characteristic when tested

using negative Gram staining. Figure 4 showed the image of S-shaped rod morphological

characteristic that was viewed in 32 cultured samples. The S-shaped rod morphological

characteristic was not observable in three isolates (SPM 024, SPM 029 and SPM 032) using

negative Gram staining. Other than that, no product obtained from PCR assay specific for C.

jejuni of the 32 isolates as well.

Figure 3. Greyish, flat and moistened colonies morphology was observed on

modified charcoal cefoperazone deoxycholate agar in one of the isolates from

Samunsam Wildlife Sanctuary, indicating positive for Campylobacter spp..

15

Figure 4. S-shaped rod morphology one of the isolates from Samunsam Wildlife

Sanctuary using negative Gram staining and viewed using 100X magnification of

compound microscope.

4.3 Prevalence of Campylobacter spp. Between Bako National Park and Samunsam

Wildlife Sanctuary

In this study, the prevalence of C. jejuni between Bako National Park and Samunsam

Wildlife Sanctuary cannot be studied. However, the prevalence of Campylobacter spp.

Between two different geographical locations, namely Bako National Park and Samunsam

Wildlife Sanctuary was compared based on the morphological testing. The prevalence of

Campylobacter spp. in Bako National Park is 60% based on the colony morphology isolated

16

and morphological test of five samples as summarize in Table 1 (Appendix C). Whilst, the

prevalence of Campylobacter spp. in Samunsam Wildlife Sanctuary is 94.595% based on the

colony morphology that were observed in 35 isolates out of 37 isolates, refer to Table 1

(Appendix D).

17

5.0 Discussion

Campylobacter is hard to isolate due to its fastidious growth requirements and have

lack of distinguishable biochemical characteristics, which make it harder for species level

identification (Ertas et al., 2003). In this study, selective agar, modified charcoal cefoperazone

deoxycholate agar (mCCDA) was used in isolating Campylobacter spp.. mCCDA is specific

for selective isolation and differentiation of Campylobacter spp.. In a study done by Enberg et

al. (2000), mCCDA showed more significant effectiveness compared to other media used in

recovering thermophilic Campylobacter spp.. Campylobacter jejuni colony morphology on

mCCDA will appear greyish, flat, moistened and often spreading around the agar (Hald &

Madsen, 1997). These colony morphologies were observed in 38 isolates out of 42 isolates

from fecal samples that were collected in Bako National Park and Samunsam Wildlife

Sanctuary. Three isolates were from Bako national Park and 35 isolates were from Samunsam

Wildlife Sanctuary. However, the species identification via PCR assay did not showed any C.

jejuni detection. It was possible that the colony growths on those plates were by other

Campylobacter spp. as they showed almost similar colony morphology. As an example,

Campylobacter coli colony morphology on mCCDA will appears creamy-grey in colour, moist

but slightly raised and often discrete as stated by Hald and Madsen (1997).

In the morphological testing for three isolates from fecal samples collected in Bako

National Park and 35 isolates from fecal samples collected in Samunsam Wildlife Sanctuary,

a total of 35 isolates showed S-shaped rod when microscopically viewed after negative Gram

staining. All three isolates from Bako National Park showed the S-shaped rod while out of 35

isolates from Samunsam Wildlife Sanctuary, 32 isolates showed the S-shaped rod morphology.

In a study by Ng et al. (1985), different characteristics morphological were shown depending

18

the location of the cells within a colony in which spiral form was observed when the cell was

from the edge of a colony and coccoid form was observed when the cell was from center of

the colony. The observation was done by using scanning electron microscopy (SEM). The

study suggested that C. jejuni is a bacterium with an intermediate form between spiral and

coccus shape. Campylobacter coli showed almost the same characteristics morphology as C.

jejuni. However, the cell C. coli single colony was described as pleomorphic due to its ability

to alter its shape into S-shaped, gull-shaped, ribbon-shaped spiral, including dimpled and round

coccus forms (Ng et al., 1985). Hence, it is possible that the microscopic Gram stain images

obtained are either C. coli or C. jejuni. Furthermore, among Campylobacter species, C. jejuni

and C. coli were the only species that could grow at high temperature of incubation at 42 °C.

Negative result that was obtained for species level identification may due to lack optimization

of the PCR assay protocol for C. jejuni detection.

Although, the prevalence of specific species Campylobacter could not be identified but

the prevalence of Campylobacter spp. between two different sampling locations, named, Bako

National Park and Samunsam Wildlife Sanctuary were able to be generally estimated. The

percentage of Campylobacter spp. prevalence between Bako National Park and Samunsam

Wildlife Sanctuary are 60% and 86.486%, respectively. Hence, it was estimated that

Campylobacter spp. is more prevalent in proboscis monkeys at Samunsam Wildlife Sanctuary.

It may due to the habitat of the proboscis monkeys at Samunsam Wildlife Sanctuary, which is

nearer to the riverside. The proboscis monkeys may be infected by the organism through

environmental contaminants in the river. A study carried out by Okwori et al. (2014) that

determined the bacteria associated with captivated non-human primates, the percentage of the

prevalence of Campylobacter spp. in captivated non-human primate was 18.2%. The organism