Helicobacter pullorum in broiler chickens and the farm environment : A One Health approach

Aim: This study aimed to investigate the occurrence of Helicobacter pullorum in broiler chickens and their farm environment. Materials and Methods: The ceca from 100 broiler chickens from ten farms were sampled from processing sites or markets. The cecal contents were aseptically collected from each cecum and cultured. The farms were visited, and environmental samples were collected which included water, house flies, floor swabs and soils in chicken houses. Results: H. pullorum was present in 51% of the broilers; 17.5% of the flies were found to carry H. pullorum and Campylobacter spp., 30% of house floors were positive, while all water samples were negative. Conclusion: Flies could have picked up the organisms from the chickens’ feces and/or the environment of the chicken houses or they could be one of the sources in the spread of the organisms. This study also showed that broiler chickens are potential reservoirs for H. pullorum and may serve as a source of infection for humans through the food chain.


Introduction
Helicobacter pullorum is a common resident in the ceca of healthy poultry flocks such as broilers, laying hens, turkeys, and birds [1][2][3].The organism has also been isolated from laying hens with avian hepatitis [4,5].The modes by which these broiler chickens become colonized with H. pullorum have not been fully understood [6][7][8].Several molecular epidemiologic studies showed that H. pullorum colonization may occur with a single strain that disseminates throughout the flock [9,10].
Published data are lacking on the survival of H. pullorum outside the gastrointestinal niches.Helicobacter pylori and Campylobacter spp.have been shown to survive in water as viable but non-culturable organisms [11,12], and hence, water may also play a role in the transmission of H. pullorum to chickens in the farms.House flies have been reported to carry H. pylori and other enteric bacteria such as Salmonella [13][14][15] and Campylobacter in poultry flocks [16][17][18].The transmission of the organisms is presumed to occur through small quantities of contaminated materials carried on the proboscis, legs, and body hairs or from materials regurgitated or defecated by the flies.Environmental sources, such as litter, feed, drinkers, and air, have been reported as having a potential role in the transmission of Campylobacter in poultry houses [14,[19][20][21][22][23].Ceelen et al. [24] isolated H. pullorum from farmers' boots.Therefore, environmental sources too may play an important role in the transmission of H. pullorum to chickens in the farms.Additionally, H. pullorum has also been reported to be associated with gastroenteritis, diarrhea, liver and gall bladder diseases as well as Crohn's disease in human patient [24].
In Malaysia , there is relatively very little information on the occurrence of H. pullorum in broiler chickens and the farm environment.Thus, this study aimed to determine the occurrence of H. pullorum in broiler chickens in the farm and to detect the presence of H. pullorum in the farm environment, namely water, flies, and floors and soils in the chicken houses.

Ethical approval
This study was performed as per the guidelines for the care and use of animals by Institutional Animal Care and Use Committee of Universiti Putra Malaysia and Animal Welfare Act.

Sample collection
The intestines of 100 chickens were collected from processing sites or markets.The ceca were carefully and aseptically removed from the intestine, placed in a sterile Petri dish, sealed with Parafilm, Available at www.onehealthjournal.org/Vol.5/3.pdf and kept in a cool box.Visits were made to the farms where the chickens were reared in 10 farms in different locations.Water samples were collected from various sources on the farm, which included tap water, well or groundwater, and drinkers.A total of 40 water samples (100 mL in each bottle) at four samples per farm were collected in sterile bottles.Flies in the poultry farms were collected using Glue Stick Fly Traps.Five flies were pooled in a bottle containing 1 mL of Brucella Broth (BD -Becton Dickinson and Co.).At least 10 flies were caught inside the farm, and another 10 flies were caught outside the farm within 50 m from the chicken houses.Each sample of floor swabs and soils from closed-house farms and floor swab samples from open-house farms were collected in a sterile bottle containing Brucella Broth (BD -Becton Dickinson and Co.).All samples were kept cool during transport to the Veterinary Public Health Laboratory, Faculty of Veterinary Medicine, Universiti Putra Malaysia.The samples were cultured within 4-6 h after sampling.

Isolation of H. pullorum
Fresh cecal contents were obtained aseptically from the ceca of each chicken and subjected to the method of Miller et al. [25] which was slightly modified as described in Wai et al. [26].Briefly, approximately 2 g of cecal contents were squeezed into 4 mL (1:2) of 0.85% sterile saline.Then, 100 μL of the suspension was further diluted in 400 μL of a sterile enrichment broth mixture (EBM) containing 25 mL of Brucella Broth (BD -Becton Dickinson and Co.), 5 mL of inactivated horse serum (Oxoid, UK), and 7.5 g of glucose (Sigma Chemical Co).Six drops of the aliquot in EMB were placed on 0.45-μm pore size, sterile cellulose acetate membrane filter (Sartorius) which was earlier placed on 10% sheep blood agar (SBA) (Oxoid, UK) plate and incubated upright in a hydrogen-enriched microaerophilic atmosphere, generated using a gas generating pack BR0038B (Oxoid, UK) without palladium catalyst also recommended by le Roux and Lastovica [27], at 42°C for 1 h to allow passive filtration.Using sterile forceps, the filters were carefully removed, and the plates were incubated at 42°C for 48 h under the same atmospheric conditions as described above.The suspected colonies were selected and subcultured on SBA.Campylobacter jejuni ATCC 29428 and H. pullorum CCUG 33837 and ATCC 51863 were used as reference strains.All field isolates and reference strains were stored at −20°C and −80°C, respectively.
Isolation of H. pullorum from water samples was carried out as per the method described by Diergaardt et al. [28].Briefly, 100 mL of each water sample was filtered; then, the filter was rolled, placed in a sterile bottle containing 9 mL of EBM, and incubated at 42°C for 24 h under similar condition described previously.After incubation, 250 µL of the culture in EBM was dropped onto the surface of a membrane filter placed earlier on SBA surface.Similar procedure as described above was used for the isolation of H. pullorum from water samples.The pooled samples of flies in each bottle were crushed, and 2 mL of EBM was added.The procedure for the isolation of H. pullorum from flies and floor swab samples was similarly carried for cecal contents.

Phenotypic identification of H. pullorum
All suspected H. pullorum colonies were examined for phenotypic characteristics (Gram-negative, slightly curved, slender rod) and subjected to oxidase, catalase, and indoxyl acetate hydrolysis tests for presumptive identification.The motility of the isolates was determined by phase-contrast microscopy of a wet mount prepared from cultures.Other phenotypic identification was hippurate hydrolysis test, sensitivity to polymyxin B and nalidixic acid, resistance to cephalothin, and growth at 25°C, 37°C and 42°C in aerobic, anaerobic, and microaerobic conditions.

Genotypic identification of H. pullorum
A single presumptive Helicobacter colony was streaked onto SBA and incubated at 42°C for 48 h under similar atmosphere mentioned above.A few colonies were taken and washed in 1 mL Brucella Broth (BD -Becton Dickinson and Co.), in a 1.5 mL sterile microcentrifuge tube.The extraction of genomic DNA was done per DNeasy Blood and Tissue DNA Purification Kit (QIAGEN).The quality of the extracted DNA was accessed using spectrophotometer and gel electrophoresis.The resulting DNA pellet with desirable quality was stored at −20°C until used.Confirmation of the presumptive isolates was done using a modified species-specific polymerase chain reaction (PCR) assay as described by Miller et al. [25].The primers used were forward primer 5'-ATGAATGCTAGTTGTTGTGAG-3' and reverse primer 5'-GATTGGCTCCACTTCACA-3'.A total of 1 μL of DNA preparation was added to a 20 μL (final volume) reaction mixture containing 0.25 μM of each primer and Maxime PCR Premix Kit (iStar-Taq) (iNtRON Biotechnology, Korea).The PCR product was then incubated with initial denaturation at 94°C for 4 min, followed by 30 cycles of 93°C for 1 min, annealing at 65°C for 1 min, and extension at 72°C for 1 min with a final incubation at 72°C for 5 min.A 5 μL of the PCR product was electrophoresed through a 2% agarose gel containing Tris-Borate-EDTA (TBE) buffer (40 mmol/1 tris-borate, 2 mmol/1 EDTA, pH 7.5) and gel red (3 μL/mL) in TBE buffer at 75 V, for 80 min.The gel was viewed by ultraviolet (UV) transillumination.
Multiplex PCR (m-PCR) was performed to further identify the presumptive mixed isolates of H. pullorum and Campylobacter species.The primers used to detect Campylobacter were for Campylobacter genus specific: Forward C99-GCGTGGAGGATGACACCT and reverse C98-GATTTTACCCCTACACCA and for H. pullorum species-specific mentioned as above.
PCR amplifications were performed as described earlier; however, the annealing temperature used was 55°C as mentioned in Shen et al. [29].

Results and Discussion
The colonies of suspected H. pullorum were very small, pinpoint, translucent, hemolytic, and some were a watery film like.They were Gram-negative, motile (spiral, slow-jerky-tumbling motility under wet mount), and oxidase and mostly catalase positive (isolates from one farm were catalase negative) and all were negative to indoxyl acetate hydrolysis and hippurate hydrolysis tests.Several suspected isolates were observed to show a weak reaction to indoxyl acetate hydrolysis test.All isolates were resistant to cephalothin and sensitive to polymyxin B, and 84.3% were resistant to nalidixic acid.The suspected H. pullorum isolates and the reference strain H. pullorum ATCC 51863 yielded a single band at 447 bp (Figure -1), whereas the reference strain C. jejuni ATCC 24928 did not show any band in the H. pullorum species-specific PCR assay.The phenotypic and PCR results provided evidence that the suspected isolates were H. pullorum.Of the 100 broiler chickens sampled, 51 (51%) were positive for H. pullorum.Pure isolates of H. pullorum were obtained from 45 of 51 positive samples from seven farms.One farm (farm 18) had six of ten positive samples which were co-colonized with Campylobacter species.Two farms were negative for H. pullorum; these two farms (farms 16 and 17) were under closed-housing system (Table -1).In the m-PCR assay, all the suspected isolates showed bands at 447 bp and 296 bp, exactly to that of the reference strain H. pullorum ATCC 51863 and C. jejuni ATCC 24928, respectively (Figure -2).It was noted that each isolate with weak indoxyl hydrolysis test results showed that it consisted of H. pullorum and Campylobacter spp.which could not be separated on repeated subcultures.
All the 40 water samples were negative for H. pullorum (Table -2 The study also showed that H. pullorum was prevalent in broiler chickens in the farms.Previous studies showed high occurrences of H. pullorum in chickens ranged from 60% in the UK [30] to 78.3% in the Czech Republic [31], 100% in Italy [2], and France [32].However, some studies showed low-to-moderate isolation rates which ranged from 4% in Switzerland [5] to 13.5% in Australia [25], 33.6% in Belgium [10], and 39.3% in Egypt [8].Manfreda et al. [33] found that chickens reared in free-range farms had lower occurrence (57%) compared to those reared in conventional (84%) and organic (97%) farms, warranted further investigation by the authors.In this study, the presence of the organisms in chickens was probably   due to the unhygienic condition of housing facilities, high fly populations, presence of wild birds and rats around farm environment and in chicken houses, and lack of biosecurity measures such as absence of footbath and presence of water puddles under some of the chicken houses which were observed during the study.The presence of Campylobacter spp.together with H. pullorum was found on one farm.Shen et al. [29] had reported the simultaneous presence of Helicobacter spp.and Campylobacter spp. in cats.
It is interesting to note that H. pullorum was not isolated from chickens in two farms which practiced closed-housing system.It is most likely due to good hygiene and husbandry practices, and birds, as well as insects, had no access to the chicken houses.Furthermore, the environmental samples collected from these farms were also negative for H. pullorum.A similar finding was reported by Tang et al. [34] who did not isolate any Campylobacter in chickens in closed-housing system compared to 95% in chickens reared under open-housing system.Bull et al. [23] showed that the transmission of Campylobacter spp.can occur from puddles outside the facility to broiler flocks, feed, water, litter, and air within the house despite the implementation of standard biosecurity measures in modern broiler house.
In this study, H. pullorum was not present in the water samples.Azevedo et al. [12] reported that all Helicobacter spp.were sensitive to light and temperature (37°C) and all tested strains lost their culturability within 24 h of exposure.UV rays and higher temperatures (>42°C) have been shown to destroy and even kill Campylobacter cells [28].The absence of H. pullorum in water as well as in the environment may not only be due to a small number of H. pullorum in the sampled water but may also be due to presence of contaminating robust microorganisms or the organisms were in nonculturable but viable form could be inactivated due to warm temperature.In most of the open-house farms, the water tanks were not covered and readily exposed to warm temperature (37°C).The uncovered water tanks were exposed to high temperature and UV which may have contributed to the absence of viable H. pullorum in water and farm environments.
Flies from three farms that practiced open-housing system were found co-colonized with H. pullorum and Campylobacter spp.Rosef and Kapperub [16] isolated Campylobacter from 50.7% of flies in broiler farms, while Hald et al. [18] found an 8.2% carriage rate of C. jejuni in flies captured in ventilation inlets of broiler house.H. pylori in flies was reported to serve as a reservoir and a vector to chickens and man as well as the environment [13].From this study, it may be possible that flies may have had a role as reservoirs in the spread H. pullorum and Campylobacter to chickens in the farms or on having in contact with the organisms from the chickens and/or the contaminated environment may had disseminated the organisms.Apart from flies, wild birds have also been reported to carry Campylobacter and H. canadensis, a probable zoonotic pathogen and closely related to H. pullorum [35].Wild birds were observed around and in the chicken houses in the open-housing system.The floor samples in three open-house farms too were contaminated with H. pullorum and Campylobacter spp.It could be that the organisms shed in the feces had contaminated the floors and soils.The potential role of litter in the transmission of Campylobacter in poultry house from infected chicks was described by Montrose et al. [19].Further study is recommended to investigate the role of environment in the spread of H. pullorum.The study also showed a high occurrence of H. pullorum, indicating that they may be a frequent intestinal colonizer of broiler chickens and thus may represent a health risk to humans.

Conclusion
This study provided further information on the occurrence and spread of H. pullorum in poultry.The closed-housing system showed that good biosecurity measures and good management and husbandry practices could minimize or control the presence of H. pullorum and Campylobacter spp. in broiler chickens and the farm environment; thus, the establishment of more of such farms is highly recommended.
).Of the 40 bottles containing pooled fly samples, seven (17.5%) from three farms were positive and found to carry H. pullorum and Campylobacter spp.(Figure-2 and Table-3).Six (30%) of floor and soil swab samples from three farms were positive and consisted of H. pullorum and Campylobacter spp.(Figure-2 and Table-3).The flies and floor samples from three farms (9, 11, and 12) were positive for H. pullorum and Campylobacter spp.Farms 14 and 15 had low occurrences probably due to better environmental conditions.
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Table - 2
: Occurrence of H. pullorum isolates in broiler chickens and environmental samples in 10 farms.Positive for H. pullorum, − negative for H. pullorum.H. pullorum=Helicobacter pullorum Table-3: Occurrence of H. pullorum in water, flies, and floor and soil.