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tection of Helicobacter species in the gastrointestinal tract ringtail possum and koala: Possible influence of diet, on thet microbiota

osaporn Coldham a, Karrie Rose b, Jani O’Rourke a, Brett A. Neilan a,len Dalton a, Adrian Lee a, Hazel Mitchell a,*

ool of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia

terinary & Quarantine Centre, Taronga Zoo, Australia

ntroduction

One important component of the gastrointestinalsystem is the subset of microbiota that colonise theface of the gastrointestinal mucosa of mammals. To bee to colonise the gastrointestinal tract (GIT) this

plex community of organisms must be able to thrivehe environment and utilise nutritional conditions thatst there. Often these mucosa-associated microorgan-s are actively motile, spiral-shaped organisms that

differ significantly to the microorganisms found in thelumen of GIT (Lee, 1991). In addition the composition ofthese microorganisms has been reported to not only varyat different locations within the GIT but also amongdifferent animal species. The major bacterial genera thatcompose the actively motile, spiral-shaped mucosa-associated microbiota include Borrelia, Spirillum, Helico-

bacter and Campylobacter (Solnick and Schauer, 2001). Todate members of the genus Helicobacter have been culturedfrom the GIT of humans as well as from a range of placentalmammals and birds (Solnick and Schauer, 2001; Fox,2002). In addition a recent study by our group has reportedup to three morphologically different – comma, fusiformand spiral shaped Helicobacter species colonising mucus

T I C L E I N F O

le history:

ived 7 October 2012

ived in revised form 21 May 2013

pted 19 June 2013

ords:

tail possum

la

cobacter species

logenetic study

smission electron microscopy

A B S T R A C T

The presence of Helicobacter spp. was examined in the liver and in different regions of the

gastrointestinal tract (GIT) including the stomach, 3 cm above ileum, ileum, caecum, colon

and rectum of 10 ringtail possums (RTPs) and 3 koalas using a combination of microscopy,

culture and PCR. Helicobacter was detected in the distal end of the GIT of 7 of 10 RTPs by

direct PCR and in all (10/10) RTPs by nested PCR. Five ‘S’ shaped isolates with bipolar

sheathed flagella were isolated from the lower bowel of 3 of the 10 RTPs. 16S rRNA

sequence analysis of these 5 isolates confirmed them as potentially novel Helicobacter

species. No Helicobacter species were cultured from the koalas, however Helicobacter DNA

was detected, in the majority of liver and/or stomach samples of the three koalas and in the

colonic region of one koala, using nested PCR. The 16S rRNA gene was sequenced directly

from DNA extracted from the homogenised livers and mucus scrapings of the stomach

from koala 1 and were confirmed to be Helicobacter species. Based on histopathological

examination of sections from the liver and intestine no evidence of infection could be

related to the presence of helicobacters in either the RTP or koala. Based on our results, it is

possible that diet may influence the detection of Helicobacter species; however this

required further investigation.

� 2013 Elsevier B.V. All rights reserved.

Corresponding author. Tel.: +61 02 9385 2040; fax: +61 02 9385 1483.

E-mail address: H.Mitchell@unsw.edu.au (H. Mitchell).

Contents lists available at SciVerse ScienceDirect

Veterinary Microbiology

jo u rn al ho m epag e: ww w.els evier .c o m/lo cat e/vetmic

8-1135/$ – see front matter � 2013 Elsevier B.V. All rights reserved.

://dx.doi.org/10.1016/j.vetmic.2013.06.026

T. Coldham et al. / Veterinary Microbiology 166 (2013) 429–437430

layer of the lower bowel of an Australian marsupial, thecommon brushtail possum (Trichosurus vulpecula, BTP)(Coldham et al., 2011).

Only four tree-dwelling Australian marsupial herbi-vores that feed mainly on eucalypt leaves have beendescribed, the common brushtail possum, the commonringtail possum (Pseudocheirus peregrinus, RTP), the koala(Phascolarctos cinereus) and the greater glider (Petauroides

volans) (Kerle, 2001). All of these are caecum fermentersthus the majority of plant cell wall digestion takes place ina greatly expanded caecum (Hume, 1999).

The aim of the current study was to determine if, giventhe high abundance of tannins and phenols obtained fromingestion of eucalypt leaves in the RTP and koalas,Helicobacter spp., are present in the GIT and the liver ofthese marsupials. To answer this question the presence ofHelicobacter spp., was determined in different regions of theGIT of RTPs and koalas using a combination of threemethods: microscopy, culture and PCR. This approach isessential as no individual method is suitable for thedetection of Helicobacter species, as firstly bacterial mor-phology (from fresh smear or silver stain section) can onlyserve as a guide for the detection of helicobacters, secondlysome Helicobacter species are likely to be non-cultivableusing current methodologies and thirdly Helicobacter genusspecific PCR based on a selected primer set might not detectall Helicobacter species/strains, including unknown Helico-

bacter spp. that might present in Australian marsupials.Furthermore PCR inhibitors may be present in the DNAextracted from the tissue samples. To reduce any effect ofinhibitors that could be present in the samples, nested PCRwas performed in parallel to direct PCR. In addition, thecolonisation location of Helicobacter spp. in the GIT wasexamined using Fluorescent in situ hybridisation (FISH) ontissue sections in which Helicobacter spp. had been detectedby all three methods. To investigate signs of any pathologyassociated with Helicobacter spp. as has been observed inother animals histopathological analysis was conducted onall specimens.

The detection and isolation rates of these bacteria in theRTP were then compared with that in koalas to determineany possible impact of diet, feeding strategies or the type ofdigestive system on the colonisation of the GIT byHelicobacter spp. Currently only three of four tree-dwellingAustralian marsupial herbivores have been studied the RTPand koala in this study and the BTP in a previous study byour group (Coldham et al., 2011). Given that the samemethodologies were used in both studies we compared theresults obtained in the RTP and koala with those previouslyreported in the BTP.

2. Materials and methods

2.1. Animal and collection of specimens

Liver and GIT specimens from 10 ringtail possums: 6female and 2 male adults, 1 unspecified sex juvenile and 1unspecified sex and age (named RTP 1–RTP 10) and 3koalas: 1 male juvenile, 1 female adult and 1 male adult,(named koala 1 – koala 3) were collected from theVeterinary & Quarantine centre, Taronga Zoo, Sydney,

Australia. All RTPs were non-caged animals while thekoalas were zoo animals. These marsupials had beeninjured or were in ill health and had subsequently died orhad been euthanised for compassionate reasons. For eachanimal, three samples of tissue were collected from each ofthe following sites: the liver, stomach, mid ileum, ileum at3 cm above the caecum (3-ileum), caecum, colon andrectum. The first sample from a particular location wasfrozen at �70 8C for DNA extraction and PCR amplification.The second sample was frozen in Brain heart Infusion–Glycerol medium (BHIG) and kept at �70 8C until cultured.The third sample was fixed in formalin for histology.

2.2. Bacterial isolation and biochemical characterisation

Homogenised liver samples and scrapings of gastro-intestinal mucus were cultured on horse blood agar plate(HBA-composition per 100 ml: sterile defibrinated horseblood 5 ml, Amphotericin 25 mg and blood agar base No. 2(Oxoid) 3.5 g) and on campylobacter selective agar plates[CSA: 100 ml of HBA plus Skirrow’s selective supplement(Polymyxin B (Sigma) 2.5 mg/ml, Vancomycin (Eli Lilly &Co., Australia) 10 mg/ml and Trimethoprim (Sigma) 5 mg/ml)]. Both the direct inoculation method and a selectivefiltration method (Robertson et al., 2001; Coldham et al.,2011) were used in this study.

The phenotypic characteristics of all isolates weredetermined using standardised methods recommended inan extensive identification scheme designed for Campylo-

bacter, Helicobacter and related bacteria (On et al., 1996;Coldham et al., 2011). Nitrate reduction and gamma-glutamyl transferase activity were examined using theAPI-Campy Identification System (BioMe’rieux, Marcy-I’Etoile, France). Indoxyl acetate hydrolysis was examinedusing the disc method (Hodge et al., 1990). Alkalinephosphatase and hippurate hydrolysis was examinedusing Rosco diagnostic tablets (UTEC diagnostics, Den-mark). Susceptibility to nalidixic acid (30 mg, NA 30,Oxoid), cephalothin (30 mg, KF 30, Oxoid) and metronida-zole (5 mg, MTZ 5, Oxoid) was determined by disc diffusionon HBA plates. Strains were determined as sensitive ifthere was a zone of inhibition and resistant if there was nozone (Karmali et al., 1980; Coldham et al., 2011).

Freshly grown bacteria from HBA plates were negativelystained with 2% uranyl acetate on a colloidal/carbon grid(483 or 400 mesh), and were examined using transmissionelectron microscopy (H7000-Hitachi, Tokyo, Japan).

2.3. DNA extraction and PCR

DNA from the liver and GIT mucus scrapings of all 10RTPs and 3 koalas was extracted using the phenolchloroform method. Briefly, DNA was extracted usingproteinase K digestion, followed by phenol, phenol–chloroform–isoamyl alcohol (25:24:1) and chloroformextraction and finally ethanol precipitation. DNA frombacterial cells was extracted using the puregene DNAisolation kit (Gentra systems, QIAGEN, Australia).

Helicobacter genus-specific PCR (direct PCR) wasperformed using primers H276f (50-CTATGACGGG-TATCCGGC-30, E. coli position 276–293) and H676r (50-

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T. Coldham et al. / Veterinary Microbiology 166 (2013) 429–437 431

CCACCTACCTCTCCCA-30, E. coli position 676–658) (Rileyal., 1996) as described previously by Coldham et al.11). The PCR reactions underwent an initial denaturationiod at 94 8C for 5 min followed by 35 cycles ofaturation at 94 8C for 5 s, annealing at 57 8C for 5 s and

ension at 72 8C for 30 s followed by a final extension at8C for 2 min. The PCR was conducted in a Sprint thermaller (Hybaid, Ashford, Middlesex, United Kingdom).Nested PCR was conducted by amplifying the prokar-e 16S RNA gene using the universal bacterial primers,

(50-AGAGTTGATCCTGGCTCAG-30, E. coli position 8–27) R1494 (50-TACGGCTACCTTGTTACGAC-30, E. coli posi-

1494–1513) (Neilan et al., 1997; Weisburg et al.,1). Reactions in the first round of the PCR underwent anial denaturation period at 94 8C for 5 min, followed bycycles of 94 8C for 10 s, 50 8C for 15 s and 72 8C for 2 min

a final extension at 72 8C for 7 min. The PCR product0 dilution) of this first round PCR was then used as aplate to amplify Helicobacter genus specific DNA (direct) in the second round PCR, using primers H276f and6r as described above.

Sequence analysis

PCR amplification of the 16S rRNA gene was conductedng the universal bacterial primers F27 and R1494ilan et al., 1997; Weisburg et al., 1991). The PCRduct obtained from this reaction was then used as DNAplate for the sequencing reaction, which employed

h of the following primers: H276f, H676r (Riley et al.,6), F27, R1494, 341R, 530F, 1115F and 1220R (Neilanl., 1997; Weisburg et al., 1991). 16S rRNA sequences

re assembled and compared with the sequences of otherteria in the GenBank. Sequences were aligned usingnment tools in the ClustalX package (Thompson et al.,7) and the phylogenetic tree was constructed by the

ghbour-joining method of Saitou and Nei (1987) ascribed in the paper by Coldham et al. (2011).

Fluorescent in situ hybridisation (FISH)

The oligonucleotide probes used were EUB338-FITCbacterial 16S rRNA probe, 50-GCTGCCTCCCGTAGGAGT-30

(Amann et al., 1992) labelled with fluorescein-isothiocya-nate (FITC)] and HRh [Helicobacter genus specific probe, 50-TCTCAGGCCGGATACCCGTCATAGCCT-30 (Fox et al., 1992),labelled with tetramethyl-rhodamine-isothiocyanate(TRITC) (GENSET Pacific Pty. Ltd., Lismore, Australia]. Thecell control suspensions were fixed in 4% paraformaldehyde.Fixed H. pylori strain SS1 (positive control) and Psychrobacter

spp. strain SW5 (negative control) were used as bacterial cellcontrols in every test. A full description of the FISHtechnique has been described previously in the paper byColdham et al. (2011).

2.6. Histopathology

Four-micron sections of paraffin embedded sampleswere cut and stained using a modified Steiner silver stainand a haematoxylin & eosin (H&E) stain. The silver stainedsections were examined for the presences of spiral/helicalshaped bacteria and the H&E stained sections forhistopathological changes.

3. Results

3.1. Bacterial cultivation

Following bacterial culture, phase contrast microscopywas conducted on suspect colonies, and any bacteria with athin spiral or fusiform shape that were actively motilewere selected for confirmation using a Helicobacter genusspecific PCR. In general these bacteria appeared as a thinsmear or very small and clear colonies on both HBA andCSA (Fig. 1). In cases where no typical colonies showingactively motile thin spiral or fusiform shape bacteria wereobserved, at least one to two isolates of curved to spiralshaped bacteria were collected for confirmation from eachanimal. Of the 10 RTPs examined, 5 of 13 individualcultures of spiral to curved shaped bacteria were identifiedas Helicobacter species using the Helicobacter genus specificPCR. These isolates cultured from 3 different RTPs (RTP1,RTP5 and RTP 9) were further examined using transmis-sion electron microscopy (TEM). The negative stainsshowed that they were all ‘S’ shaped; measuring 0.3 by2.5 mm in size; with bipolar sheathed flagella and no

Fig. 1. The appearance of tiny pinpoint colonies (A) and a thin water-like film (B) on the HBA or CSA plates.

T. Coldham et al. / Veterinary Microbiology 166 (2013) 429–437432

periplasmic fibres (Fig. 2). These were designated asisolates RTP1S to RTP5S.

From the 3 koalas, no bacteria having a typical thinspiral or fusiform shape were observed on microscopy andno thin smears or any typical small and clear colonies werefound on HBA or CSA. Although six individual isolateshaving a curved rod shape appearance were investigated,none were identified as Helicobacter species using theHelicobacter genus specific PCR. Based on their non-typicalcolony appearance, bacterial morphology and the results ofPCR we concluded that these isolates were not Helicobacter

species. As this study aimed to determine the presence ofHelicobacter spp., we did not undertake any furtheridentification of these isolates.

The results of culture, the location from which the isolateswere cultivated and the direct and nested PCR resultsobtained from the 10 RTP’s and 3 koalas are shown in Table 1.

3.2. 16S rRNA gene sequencing analysis and biochemical

characteristics

Sequencing of the near complete 16S rRNA gene(�1500 bp) of the 5 isolates from the RTPs was undertaken

and compared to other sequences in the GenBankdatabase. The 16S rRNA sequences have been depositedin GenBank and the accession numbers for RTP 1 to RTP 5are AY554130–AY554134 respectively. The sequencesobtained from these 5 isolates showed 95% similarity toHelicobacter sp. MIT 99-5507 isolated from rhesus monkey2 (accession number AF33334) (Fox et al., 2001) and 94%identical to H. canis and H. hepaticus. Given this low level ofhomology, it is likely that the RTP isolates constitute a newspecies. The similarity among the 16S rRNA genesequences of the 5 isolates was 99.6–99.9%, indicatingthat the five isolates potentially are the same species.

Helicobacter species DNA was detected by nested PCR inthe homogenised livers of all 3 koalas as well as from thestomach of koala 1 and koala 3. To confirm that these weretruly Helicobacter species we amplified and sequenced the16S rRNA gene directly from DNA extracted from thehomogenised liver of koala 1 (1318 bp) and mucusscrapings of the stomach of koala 1 (1071 bp). Comparisonof these sequences to other sequences in the GenBankdatabase showed the DNA from the liver of koala 1 to have99% similarity to Helicobacter bilis and the DNA from thestomach of koala 1 to have 99% similarity to Helicobacter

felis. The 16S rRNA accession numbers in GenBank for koala1 liver is AY583532 and for koala 1 stomach is AY583533.

Based on our results we constructed a phylogenetic treeusing the 5 RTP isolates, the 18 BTP isolates obtained fromour previous study on brushtail possums (Coldham et al.,2011), 35 members of the genera Helicobacter (including32 validly published species) and three closely relatedspecies; Wolinella succinogenes, Arcobacter butzleri andCampylobacter jejuni (Fig. 3). The sequences obtained from‘BTP1C to BTP9C’ (comma shaped), ‘BTP1F to BTP6F’(fusiform shaped), ‘BTP 1S to BTP3S’ (S shaped) and ‘RTP1Sto RTP5S’ (S shaped) isolates were shown to cluster into 4groups, supporting the view that these 4 groups constitute4 different species.

As 16S rRNA analysis alone does not always provideconclusive evidence for species level identification andmay prove highly misleading (Vandamme et al., 2000), all 5RTP isolates were further tested for biochemical char-acteristics and susceptibly to nalidixic acid, cephalothinand metronidazole. The results of these tests and a

Fig. 2. A transmission electron micrograph of an ‘S’ shaped Helicobacter

isolate, RTP5S. The bacterium measured 0.3 mm � 2.5 mm and had

bipolar-sheathed flagella.

Table 1

The results of Helicobacter culture, direct and nested PCR obtained from the 10 RTPs and 3 koalas.

Animals Regions

Liver Stomach Mid ileum 3-Ileum Caecum Colon Rectum

RTP 1 _ N N C, N N C, dP, N dP, N

RTP 2 _ _ _ _ _ _ N

RTP 3 _ _ _ _ _ N N

RTP 4 _ _ _ _ _ dP, N dP, N

RTP 5 _ _ _ N N C, N dP, N

RTP 6 _ _ _ _ _ N N

RTP 7 _ _ _ _ _ N dP, N

RTP 8 _ _ _ _ _ N dP, N

RTP 9 _ _ _ _ N C, dP, N C, dP, N

RTP 10 _ _ _ _ _ _ dP, N

Koala 1 N N N _ _ N _

Koala 2 N _ _ _ _ _ _

Koala 3 N N _ _ _ _ _

C = Helicobacter culture positive; dP = direct PCR positive; N = nested PCR positive; – = Helicobacter culture, direct and nested PCR, all negative.

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T. Coldham et al. / Veterinary Microbiology 166 (2013) 429–437 433

parison with other validly published Helicobacter

cies is shown in Table 2. All 5 RTP isolates were foundossess characteristics common to Helicobacter species,

luding catalase positivity, hippurate hydrolysis nega-, urease activity negative, sensitive to cephalothin and

istance to nalidixic acid.

The spatial distribution of mucus-associated

roorganisms in fixed sections of the liver and different

ions of the GIT of RTPs and koalas

No microorganisms were observed in any of the silverined sections of the liver of RTPs or koalas using lightroscopy. Due to a large number of clumps ofteria-like particles present in the luminal contents,ection of mucus-associated microorganisms in theer stained sections from different regions of the

gtail possum GIT was problematic. However smallbers of long, curved to spiral shaped organisms were

erved in the mucus layer overlying the epithelium in the crypts of the caecum, colon and rectum of RTPTP 5 and RTP 9. Examples of the distribution of thesecus-associated bacteria in the colonic region of RTP 9

shown in Fig. 4. Microscopic examination of silverined sections obtained from the 3 koalas showed 2dominant morphological types of microorganisms,ci and filamentous bacillary forms. Small numbers ofg, curved to spiral shaped organisms observed in the

caecum and colonic regions of koala 1 are shown inFig. 4.

As isolation and detection of helicobacters using cultureand direct PCR was unsuccessful in the koala the spatialdistribution of bacteria belonging to the genus Helicobac-

ter, using FISH, was not performed in any of the koalas aswe considered that the numbers of helicobacters if present,would be too low for microscopic detection. The FISHtechnique was however performed to determine colonisa-tion location in a formalin-fixed colonic section of RTP 9 inwhich Helicobacter species had been isolated as well asdetected by PCR. Bacteria belonging to the Helicobacter

genus were observed predominantly colonising the mucuslayer lining the epithelium and in the crypts of the colon(Fig. 5).

3.4. Histopathology

Histopathological examination of the liver sectionsfrom the RTP showed all to be within the normal range. Nosignificant lesions were observed in the stomachs. Thelower bowel of all RTPs include RTP 1, RTP 5 and RTP 9 fromwhich Helicobacter species were isolated, was normalshowing only a few infiltrations of mononuclear cells andeosinophils in the intestinal mucosa.

No significant change in histopathology was observedin the liver, stomachs or the lower bowel of any of thekoalas.

3. Phylogenetic tree for RTP1S-RTP5S isolates, 18 BTP isolates (Coldham et al., 2011), 34 members of the genera Helicobacter (include 32 validly

lished species), and three closely related species: Wolinella succinogenes, Arcobacter butzleri and Campylobacter jejuni. The scale bar represents 0.01

cted substitutes per site.

T. Coldham et al. / Veterinary Microbiology 166 (2013) 429–437434

4. Discussion

In the current study we used a combination ofmicroscopic examination, culture and PCR to detectHelicobacter species in the RTP’s and koalas as weconsidered that none of these methods alone was sufficientfor the detection of new or unknown Helicobacter species.As spiral or fusiform shape is not limited to Helicobacter

species, microscopic examination can only be used as aguideline for the detection of Helicobacter species. Amongthe three methods used in this study, culture is the goldstandard and PCR, and in particular nested PCR, is the mostsensitive detection method. In the current study aHelicobacter genus specific PCR using H276f and H676rprimers was employed for the detection of Helicobacter

spp., in DNA extracted from the liver and tissue mucusscrapings, as well as for confirmation of the identity ofbacterial isolates. As this primer pair had previously beenshown to amplify some Wolinella species, a genus closelyrelated to Helicobacter spp. (Oxley et al., 2004), weconfirmed the results of the Helicobacter genus specificPCR positive samples by sequencing the 16S rRNA gene andcomparing the sequence with those sequences in GenBank.

In any study where primers based on current knowl-edge of a particular genus are used to detect unknownspecies/strains it is impossible to predict the possibility ofmismatches. This proved to be so in the current study, as asingle internal mismatch between the Helicobacter genusspecific primers we employed and the Helicobacter speciesisolated from the RTP. (C:T (H676r primer 50-ATTCCACC-TACCTCTCCCA-30: 5 RTP isolates 50-ATTCCACT-TACCTCTCCCA-30) was observed. However, given theinternal location of this mismatch it is unlikely to havehad any significant effect on the PCR product yield (Kwoket al., 1990), a view supported by the fact that H276f andH676r successfully amplified all 5 RTP isolates.

As shown in Table 1, Helicobacter species were foundcolonising the distal end (colon and rectum) of the GIT ofall RTP’s, except RTP1 in which one Helicobacter spp. wasisolated from the ileum at 3 cm above the caecum. In thisanimal, Helicobacter DNA was detected in all regions of theGIT except for the liver. According to this animals’ history,it was found dead, and thus it is impossible to rule out thepossibility that bacteria from the distal end of the GIT mayhave translocated to other sites (the stomach and smallintestine) following death, prior to sample collection. InRTP 2, 3 and 6, Helicobacter DNA was detected only at thedistal end of the GIT using nested PCR, suggesting thatHelicobacters were present in very low numbers in theseanimals. The colonisation of Helicobacter species appearedto have no impact on the histopathology of the liver or GITof the RTPs.

The cultivation rate of Helicobacter species, as comparedwith detection using PCR, was low. In the rectal region ofthe RTP, only one Helicobacter isolate was cultured (RTP 9)while Helicobacter DNA was detected in the rectal region of7 RTPs using direct PCR and in 10 using nested PCR. Thislow cultivation rate of Helicobacter species in the RTP’smight relate to the fact that specific nutritional require-ments were absent in the media used for their culture. Forexample, it is possible that they may require specific plantT

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T. Coldham et al. / Veterinary Microbiology 166 (2013) 429–437 435

ondary metabolites that are present in their GIT forwth, or it may be that some Helicobacter species in the

are sensitive to the aerobic conditions created duringple collection. While no Helicobacter species were

ated from the 3 koalas investigated, Helicobacter DNAs detected using nested PCR in the liver of all 3 koalas,

stomach of 2 koalas (koalas 1 and 3) and the ileum andn of koala 1. Sequencing analysis of DNA extracted

the liver and stomach mucus scrapings of koala 1firmed that Helicobacter species were present in thisla.The higher cultivation and detection rates of Helico-

ter spp. in the RTP’s as compared with the koalas raises

the possibility that the koalas diet, which consists ofmainly eucalypt leaves could limit colonisation byHelicobacter in their lower bowel. Although the koalas inthis study were zoo animals while the RTPs were wild, interms of their diet, this is unlikely to have impacted on thestudy, as the koalas spent the majority of their life in treeswithin the zoo and feeding mainly on fresh eucalypt leaves.The cultivation rate of Helicobacter spp. in the wild RTPs (3of 10 animals) and in the koalas (0 of 3 animals) wasconsiderably lower than that previously reported in wildBTP’s (11 of 11 animals) (Coldham et al., 2011).

Given that RTP and BTP are very closely related species,believed to have diverged approximately 55 million years

4. Photomicrographs of silver stained GIT sections obtained from (A) the caecum of koala 1, (B and C) the colon of koala 1 and (D–F) the colon of RTP 9.

arrow indicated curve to spiral shaped bacteria. Magnification = 1000�.

T. Coldham et al. / Veterinary Microbiology 166 (2013) 429–437436

ago (Flannery, 1994), the low cultivation and detectionrates of Helicobacter spp. in the RTP’s was somewhatsurprising, given the relatively high detection rates weobserved in BTP’s. It is possible however that the differencein the detection rates of Helicobacter species in the GIT ofRTPs and BTPs may relate to differences in the ability ofHelicobacter species to utilise available nutrients and/or totolerate toxins produced within the GIT of these twomarsupials, or to differences in nutrients available in eachindividual host. For example, while RTP feed almost

entirely on leaves, shoots or flowers, from only one ortwo Eucalyptus species and are coprophagic, the diet ofBTP’s consists not only of eucalypt leaves but also of fruits,grasses, herbs, insects and meat (Kerle, 2001; Hume, 1999;Strahan, 1983). Further, unlike BTP who have four pointedcusps molars, RTP have crescent shaped molars whichincrease their ability to grind leaves into very fine particles,resulting in the release of high amounts of anti-nutrients orplant secondary metabolites present in eucalypt leaves(Kerle, 2001; Hume, 1999). Furthermore, RTP possesses aspecific mechanism for controlling the flow of differentsized particles between the caecum and the colon.Separation of different sized food particles occurs in theproximal colon, with small particles (including plantsecondary metabolites) being pushed back into thecaecum, whilst large particles continue their passagethrough the colon (Kerle, 2001; Hume, 1999). The lowdetection levels of Helicobacter in the caecum of the RTPsuggests that Helicobacter species may not have the abilityto utilise the solutes and fine particles, or tolerate the plantsecondary metabolites, implying that they may not beinvolved with fermentation. In contrast there is noselective retention of digesta in the GIT of the BTP, thusdifferent sized particles do not move through the gut atdifferent rates.

Interestingly the caecum and proximal colon of koalascontained a higher amount of small particles than eitherthe stomach or the distal colon, which may relate to theselective retention of solutes and fine particles in thecaecum and the proximal colon, which are believed to beimportant for microbial fermentation (Hume, 1999). Thus,in the caecum and proximal colon of the koala conditionsmay be unsuitable for Helicobacter spp.

In conclusion the presence of Helicobacter spp. in koalasand RTPs provides further evidence that Helicobacter

species can colonise the GIT of Australian Marsupials.The three marsupials studied to date belong to the orderDiprotodontia and are ‘hindgut fermenting’ herbivorousmarsupials. Australian Marsupials can however, based ontheir dietary requirements and the structure of their GIT,be classified into three feeding types, carnivores, omni-vores and herbivores. Future studies, which investigate thepresence of Helicobacter species in carnivores and omni-vores, as well as herbivores not covered in this study, willnot only provide important insights into the specificnatural niche of Helicobacter species, but will also increaseour understanding of the ecology of Helicobacter species.

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