Campylobacter is a leading cause of human gastroenteritis worldwide . Poultry is considered to be the major reservoirs of Campylobacter, and unhygienic handling or consumption of raw or undercooked poultry products is the main cause of Campylobacter infections  . Campylobacter strains detected in feces from slaughtered poultry can contaminate poultry carcasses if hygienic practices failed during the gastrointestinal removing process  . In the last decade, Campylobacter isolated from human or animal samples is showing more and more resistance to a variety of antibiotics . However, the epidemiology of Campylobacter is still not elucidated in many countries. In Burkina Faso, there is a lack of sufficient resources to routinely employ modern methods of clinical and molecular microbiology for screening, prevention and policy-making purposes related to the gastroenteric disease. Conventional biochemical-based assays for the identification of Campylobacter species are time-consuming and laborious due to the fastidious growth requirements of these species and the paucity of informative biochemical characteristics . Moreover, Campylobacter strains identification requested selective protocol, which is relatively expensive for developing countries. It still remains a high research priority to improve the strategies for management as well as an improved method for the isolation and identification of these pathogens. Molecular methods, especially Polymerase chain reaction (PCR)-based species identification for Campylobacter spp. provide more reliable identification of thermophilic Campylobacters, as they enhance the sensitivity and specificity of the detection process . In Burkina Faso, very few studies have been undertaken to determine the prevalence of Campylobacter in humans and/or poultry  . To better understand the epidemiology of campylobacteriosis in Burkina Faso, this study aims to determine the prevalence of Campylobacter in poultry feces using an optimized identification protocol and multiplex PCR.
2. Materials and Methods
From July to August 2017, fecal samples from slaughtered chickens (n = 54) were collected from the local poultry sellers in two retail markets of Ouagadougou, Burkina Faso. There were no records available concerning poultry farms, but according to the poultry sellers, the chickens originated from many farms across the country. Immediately after the poultry was slaughtered, the whole intestine was collected after evisceration, placed in sterile plastic bags, and transported in a cool box (4˚C) to the laboratory. The cecal content of poultry was put in a sterile cryotube and kept at −20˚C for future Campylobacter isolation. The cryotubes were sent to the laboratory “Chaire de Recherche en Salubrité des Viandes (CRSV)” at the faculty of veterinary medicine /the University of Montréal for isolation and identification.
Campylobacter isolation and identification
Two methods were used for Campylobacter isolation in CRSV:
A loopful of poultry cecal content was streaked directly onto modified charcoal cefoperazone desoxycholate agar (mCCDA) (Oxoid, Nepean, Ontario) and incubated at 42˚C for 48 h in a microaerobic atmosphere using Oxoid’s microaerobic atmosphere generation system (Oxoid).
In parallel, 1 g of caecal content was inoculated into 9 ml of Bolton broth (Oxoid, Nepean, Ontario) supplemented with selective supplements (SR0155 E, Oxoid, Nepean, Ontario). Other antibiotics were tested as an additive to limit the overgrowth of cephalosporin-resistant enterobacteria. Three different antibiotics with different concentrations were used to make a serial dilution using Bolton broth (10−1 to 10−7) by inoculating the reference strain of Camylobacter (ATCC 33291) and samples. These antibiotics were: rifampicin (Oxoid, Nepean, Ontario) with 10 mg/L, colistin (Oxoid, Nepean, Ontario) with 1 mg/mL or 2 mg/mL as well as trimethoprim (Oxoid, Nepean, Ontario) with 10 mg/L. The retained antibiotic for the modified Bolton broth was colistin 2 mg/mL because this antibiotic was more selective for Campylobacter compare to the other tested antibiotics.
Then, for the samples processing, 1 g of poultry cecal content was added to 9 ml of modified Bolton broth, mixed and incubated at 42˚C for 48 h in a microaerobic atmosphere. For the control strain (C. jejuni strain ATCC 33291), one colony from the blood Agar plate was added to 9 ml of modified Bolton broth, mixed and incubated at 42˚C for 48 h in a microaerobic atmosphere. After enrichment, a loopful of the modified Bolton broth was streaked onto modified charcoal cefoperazone desoxycholate agar (mCCDA) (Oxoid) and incubated at 42˚C for 48 h in a microaerobic atmosphere.
For samples and control strains, after incubation, the typical looking Campylobacter colonies were purified on mCCDA (Oxoid) agar plates and incubated at 42˚C for 48 h in a microaerobic atmosphere. Then, the pure colonies were streaked onto blood agar (Oxoid) and incubated at 42˚C for 48 h in a microaerobic atmosphere. The colonies with typical Campylobacter morphology on blood agar were kept and further identified down to the species level by polymerase chain reaction (PCR).
The presence and identification of Campylobacter strains were detected by multiplex PCR. The primers and PCR conditions were as previously described , with slight modification. Briefly, for DNA extraction, a single loopful of culture was inoculated in 50 µl NaOH (25 mM) in a 1.5 ml microcentrifuge tube using a disposable loop (1 mm diameter), and the cell mixture was heated at 100˚C for 10 min. After neutralization with 50 ml tris/HCl buffer (80 mM, pH 7.5), cell debris was pelleted by centrifugation at 20,000 g, 4˚C, for 5 min, and the supernatant was used as template DNA. The final multiplex PCR comprised: 1 ml template DNA; 0.2 mM primers C412F, C1228R, C-1, C-3, CC18F, CC519R, CU61F, CU146R, MG3F, CF359R, CLF, CLR, HYO1F and HYOFET23SR; and 0.56Q solution (Qiagen, Montréal, Canada) in 16 multiplex PCR master mix (Qiagen, Montréal,Canada). The final volume per well was adjusted to 25 µl with sterile water. Primers sequences were previously described by Yamazaki-Matsune et al. .
The cycling conditions used were 95˚C for 15 min, 25 cycles each of 95˚C for 0.5 min, 58˚C for 1.5 min and 72˚C for 1 min and a final extension at 72˚C for 7 min. Samples were held at 4˚C prior to analysis. PCR amplicons were visualized on a 1% agarose (Thermo, Fisher Scientific, Ottawa, Canada) gel stained with Sybrsafe (Thermo, Fisher Scientific, Ottawa, Canada). The positive control consisted of DNA extracted from C. jejuni strain ATCC 33291 while the negative control contained no DNA.
Optimization of the isolation method
The results show that direct plating of poultry feces on mCCDA plates was not selective like enrichment in Bolton broth supplemented with additional antibiotics. The enrichment method using Bolton broth supplemented with additional colistin (2 mg/mL) has given good results in selecting the campylobacter strains and inhibiting the other flora. This selective broth yielded significantly better isolation rate of Campylobacter than those achieved on using normal Bolton broth supplemented without additional antibiotic or direct plating. Enrichment in selective broth allowed the detection of 18 (34.61%) Campylobacter strains from 52 feces samples and direct plating on mCCDA 11 (21.15%) Campylobacter strains (p < 0.05).
Prevalence of Campylobacter
A multiplex PCR assay has been used for the identification of six common Campylobacter species, namely Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalis subsp. hyointestinalis, Campylobacter jejuni, Campylobacter lari and Campylobacter upsaliensis. Figure 1 shows picture of a typical electrophoretogram from PCR. Out of the 52 feces samples, 18 (34.61%) were positive for Campylobacter genus.
The PCR results have shown that 17 (94.45%) of the Campylobacter strains detected belonged to Campylobacter coli and 1 (5.55%) strains belonged to Campylobacter jejuni.
Poultry is an important reservoir of Campylobacter and chicken and poultry product has been involved in campylobacteriosis worldwide. In this study, we investigated using optimization protocol and multiplex PCR to detect Campylobacter presence in poultry feces. The result shows that the enrichment method using Bolton broth supplemented with additional colistin at 2 mg/mL yielded significantly better isolation rate and selectivity than those achieved on using normal Bolton broth supplemented without additional antibiotic or direct plating on mCCDA. From the direct plating, the inconvenience is the growth of
Legend: Well 1 and 20 = Ladder; Well 2 (−) Reference strain (C. jejuni); Well 3 (+, Campylobacter sp); Well 4 (+, Campylobacter sp); Well 5 (−); Well 6 (+, Campylobacter sp); Well 7 (+, Campylobacter sp); Well 8 (+, Campylobacter sp); Well 9 (+, Campylobacter sp); Well 10 (−); Well 11 (+, Campylobacter sp); Well 12 (+, Campylobacter sp); Well 13 (+, Campylobacter sp); Well 14 (+, Campylobacter sp); Well 15 (+, Campylobacter sp); Well 16 (−); Well 17 (+, Campylobacter sp); Well 18 (+, Campylobacter sp); Well 19 (−).
Figure 1. Typical electrophoretogram.
competitive Gram-negative bacteria, which influence the presence of Campylobacter. Previous studies have reported that increased selective pressure in selective agar and/or enrichment broth enhances Campylobacter isolation from various samples. Kim et al.,  reported that the addition of polymyxin B, rifampicin, or both to the Bolton selective supplements enhanced the selective isolation of Campylobacter isolation from wastewater. Yoo et al.  reported that the addition of rifampicin (10 µg/ml) or polymyxin B (5 IU/ml) to Bolton agar (Bolton agar with Bolton supplement) restrained the growth of non-Campylobacter without any inhibition of C. jejuni and C. coli in fresh produce foods. Chon et al.  demonstrated that the addition of high concentrations of polymyxin B to the Bolton supplement in enrichment procedure improved the efficiency of C. jejuni and C. coli recovery and suppressed background competing bacteria. Consistently, our results showed that the supplementation with additional colistin (2 mg/mL) improved the efficacy of Campylobacter isolation from poultry feces.
In this study, the result revealed a low prevalence (34.61%) of Campylobacter species in poultry feces, with a high predominance of C. coli. This finding could be explained by the fact that samples were frozen at −20˚C before to send to the laboratory “Chaire de Recherche en Salubrité des Viande (CRSV)”, department of veterinary medicine, University of Montréal. The result highlights a good hypothesis that C. coli could be more resistant to the environmental condition than C. jejuni, which could be tested in future studies. However, many authors demonstrated that if the birds are older, there is more C. coli because C. jejuni would be gradually replaced by C. coli  . Contrast results were reported in our previous study where, highest prevalence (67.96%) of Campylobacter in poultry, with a high predominance of C. jejuni . The lowest prevalence of campylobacter from poultry (15.52%) feces compare to our present result has been reported by Rawat et al.  in India. In contrast, Osbjer et al.,  reported 56% prevalence of Campylobacter in chicken feces in rural Cambodia. The difference in Campylobacter prevalence in poultry feces could be explained by the difference in isolation methods, the sampling conditions, geographical areas, and farming conditions.
The Campylobacter species found in this study were C. coli and C. jejuni, which have been reported worldwide as the most frequently associated with human infection  . Moreover, Whiley et al.  reported in their study that the consumption of contaminated poultry is the primary cause of developing human campylobacteriosis.
These findings show that more attention is needed during poultry carcasses processing plan to avoid carcasses contamination, and hygienic practices should be respected during carcasses preparation to avoid the risk of cross-contamination. This study highlights also the need for developed countries to invest more resources into Campylobacter research to identify possible sources of campylobacteriosis and therefore put in place adequate measures to lower the impact of the foodborne pathogens on public health.
The authors would like to thank Institute of Sciences/Ministry of Higher education and Research of Burkina Faso for the financial support to the trip of A.K. at the University of Montréal to carry out this study. We thank technicians in the laboratory “Chaire de Recherche en Salubrité des Viandes (CRSV)” for their help and collaboration.
 Steinhauserova, I., Ceskova, J., Fojtikova, K., Obrovska, I. (2001) Identification of Thermophilic Campylobacter spp. by Phenotypic and Molecular Methods. Journal of Applied Microbiology, 90, 470-475.
 Schonberg-Norio, D., Mattila, L., Lauhio, A., Katila, M.L., Kaukoranta, S.S., Koskela, M., Pajarre, S., Uksila, J., Eerola, E., Sarna, S. and Rautelin, H. (2010) Patient-Reported Complications Associated with Campylobacter jejuni Infection. Epidemiology & Infection, 138, 1004-1011.
 Taremi, M., Mehdi Soltan Dallal, M., Gachkar, L., MoezArdalan, S., Zolfagharian, K. and Reza Zali, M. (2006) Prevalence and Antimicrobial Resistance of Campylobacter Isolated from Retail Raw Chicken and Beef Meat, Tehran, Iran. International Journal of Food Microbiology, 108, 401-403.
 Prachantasena, S., Charununtakorn, P., Muangnoicharoen, S., Hankla, L., Techawal, N., Chaveerach, P., Tuitemwong, P., Chokesajjawatee, N., Williams, N., Humphrey, T. and Luangtongkum, T. (2016) Distribution and Genetic Profiles of Campylobacter in Commercial Broiler Production from Breeder to Slaughter in Thailand. PLoS ONE, 11, e0149585.
 Reddy, S. and Zishiri, O.T. (2017) Detection and Prevalence of Antimicrobial Resistance Genes in Campylobacter spp. Isolated from Chickens and Humans. Onderstepoort Journal of Veterinary Research, 84, Article No. a1411.
 Yamazaki-Matsune, W., Taguchi, M., Seto, K., Kawahara, R., Kawatsu, K., Kumeda, Y., Kitazato, M., Nukina, M., Misawa, N. and Tsukamoto, T. (2007) Development of a Multiplex PCR Assay for Identification of Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalis subsp hyointestinalis, Campylobacter jejuni, Campylobacter lari and Campylobacter upsaliensis. Journal of Medical Microbiology, 56, 1467-1473.
 Rawat, N., Maansi, Kumar, D. and Upadhyay, A.K. (2018) Virulence Typing and Antibiotic Susceptibility Profiling of Thermophilic Campylobacters Isolated from Poultry, Animal, and Human Species. Veterinary World, 11, 1698-1705.
 Bonkoungou, I.J.O., Haukka, K., Österblad, M., Hakanen, A.J., Traoré, A.S., Barro, N. and Siitonen, A. (2013) Bacterial and Viral Aetiology of Childhood Diarrhea in Ouagadougou, Burkina Faso. BMC Pediatrics, 13, Article No. 36.
 Kagambèga, A., Thibodeau, A., Trinetta, V., Soro, K.D., Sama, N.F., Bako, E., Bouda, S.C., Wereme, A., Fravalo, P. and Barro, N. (2018) Salmonella spp. and Campylobacter spp. in Poultry Feces and Carcasses in Ouagadougou, Burkina Faso. Food Science & Nutrition, 6, 1601-1606.
 Kim, J., Oh, E., Banting, G.S., Braithwaite, S., Chui, L., Ashbolt, N.J., Neumann, N.F. and Jeon, B. (2016) An Improved Culture Method for Selective Isolation of Campylobacter jejuni from Wastewater. Frontiers in Microbiology, 7, 1345.
 Yoo, J.H., Choi, N.Y., Bae, Y.M., Lee, J.S. and Lee, S.Y. (2014) Development of a Selective Agar Plate for the Detection of Campylobacter spp. in Fresh Produce. International Journal of Food Microbiology, 189, 67-74.
 Chon, J.W., Kim, H., Yim, J.H., Park, J.H., Kim, M.S. and Seo, K.H. (2013) Development of a Selective Enrichment Broth Supplemented with Bacteriological Charcoal and a High Concentration of Polymyxin B for the Detection of Campylobacter jejuni and Campylobacter coli in Chicken Carcass Rinses. International Journal of Food Microbiology, 162, 308-310.
 Colles, F.M., McCarthy, N.D., Bliss, C.M., Layton, R. and Maiden, M.C. (2015) The Long-Term Dynamics of Campylobacter Colonizing a Free-Range Broiler Breeder Flock: An Observational Study. Environmental Microbiology, 17, 938-946.
 Osbjer, K., Boqvist, S., Sokerya, S., Cheng, K., San, S., Davun, H., Rautelin, H. and Magnusson, U. (2016) Risk Factors Associated with Campylobacter Detected by PCR in Humans and Animals in Rural Cambodia. Epidemiology & Infection, 144, 2979-2988.
 Kaakoush, N.O., Castano-Rodriguez, N., Mitchell, H.M. and Man, S.I.M. (2015) Global Epidemiology of Campylobacter Infection. Clinical Microbiology Reviews, 28, 687-720.
 Whiley, H., Van Den Akker, B., Giglio, S., and Bentham, R. (2013) The Role of Environmental Reservoirs in Human Campylobacteriosis. International Journal of Environmental Research and Public Health, 10, 5886-5907.