Indian Journal of Animal Research

  • Chief EditorK.M.L. Pathak

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Indian Journal of Animal Research, volume 58 issue 3 (march 2024) : 478-483

Morphological, Biochemical and Genotypic Analysis of Zoonotic Campylobacter jejuni Isolated from Chicken Meat Samples

Sumedha Bobade1, K. Vijayarani1, K.G. Tirumurugaan1, A. Thangavelu1, S. Vairamuthu1
1Department of Animal Biotechnology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai-600 007, Tamil Nadu, India.
Cite article:- Bobade Sumedha, Vijayarani K., Tirumurugaan K.G., Thangavelu A., Vairamuthu S. (2024). Morphological, Biochemical and Genotypic Analysis of Zoonotic Campylobacter jejuni Isolated from Chicken Meat Samples . Indian Journal of Animal Research. 58(3): 478-483. doi: 10.18805/IJAR.B-4497.
Background: Campylobacter species are a leading cause of most important food-borne diarrhoeal illness worldwide while, poultry has been identified as a significant cause of Campylobacter infection in humans. C. jejuni is highly effective in colonizing chicken intestinal mucosa without causing any clinical manifestations and the consumption of poultry meat is the major source of transmission of bacteria to humans.

Methods: The total of 19 chicken meat samples collected from retail markets in Chennai were screened by cultural examination, further subjected to phenotypic characterization using biochemical test and genotypic characterization using polymerase chain reaction assay targeting hip O and map A genes.

Result: All the isolates showed growth on modified blood free charcoal cefoperazone deoxycholate agar media (mCCDA) and 18 (94.73%) samples showed typical morphological characteristics. The 12 (63.15%) isolates showed biochemical reactions positive. The results from polymerase chain reaction showed that 10 (83.33%) isolates were positive for C. jejuni. This study suggested that, it is essential to investigate the incidence of Campylobacter jejuni infection in poultry and the risk factors at all production stages of meat production to help reducing the disease in humans in terms of food safety.
Campylobacter is considered as a principal cause of most important zoonotic food-borne disease in humans for approximately 166 million diarrheal cases and 37,600 deaths per year globally (Oh et al., 2018). Campylobacter is 1 of 4 key global causes of diarrheal diseases of human gastroenteritis in the world (WHO, 2018). C. jejuni has been found to be the predominant species isolated from poultry samples and responsible for the majority of human Campylobacteriosis (Natsos et al., 2019). Poultry and poultry products are considered as the most important sources of human infections. Campylobacter jejuni is main foodborne pathogens and broiler meat is considered as the most important source of human Campylobacteriosis (Duque et al., 2021). The proliferation of C. jejuni in broilers suggests that the feed formulated to promote avian growth provides the nutrients required by these bacteria (Greene et al., 2020).

In humans, clinical signs of Campylobacteriosis include diarrhea, abdominal pain, fever, headache, nausea and vomiting. The main recognized sequelae are Guillain-Barré Syndrome (GBS), the Reactive Arthritis (REA) and irritable bowel syndrome (IBS).Thermo tolerant Campylobacter which has a clinical significance are C. jejuni and its represents more than 90% of human infections (Mikulic et al., 2016).

Campylobacter jejuni showed multiple drug resistance against some antibiotics, which is of public health concern and may pose serious problems in the treatment of animals (Shekhar et al., 2010). The control of Campylobacter in poultry seems crucial for the reduction of human Campylobacteriosis cases (Natsos et al., 2016). Hence this study was attempted to detect the presence of C. jejuni using cultural, biochemical and PCR technique and compare these techniques for detection among raw chicken meat samples.
Collection of samples
The present study was conducted at Department of Animal Biotechnology, Madras Veterinary College, Chennai, Tamil Nadu in the year 2018-19. A total of 19 samples comprising raw chicken meat were collected from retail markets around Chennai. All the samples were collected using sterile containers and transported immediately to the laboratory under cold conditions for microbiological analysis.

Processing of samples
The isolation was performed according to Man (2011) and the isolates were identified by biochemical tests as described by (Fitzgerald and Nachamkin, 2007; Lastovica and Allos, 2008).
Phenotypic characterization
Cultural examination
Sample was enriched in modified Charcoal Cefoperazone Deoxycholate (mCCDA) broth (Hutchinson and Bolton, 1984) with CCDA supplement under microaerophillic conditions (candle jar method) by using internal gas generation system using (Microaerophilic gas pack CampyPack-BD oxoid).
Biochemical test
The isolates were identified as C. jejuni based on their morphological and biochemical tests. Suspected colonies were sub-cultured and confirmed by catalase, oxidase, nitrate and hippurate hydrolysis, Ninhydrin test, H2S production.
Molecular confirmation of Campylobacter jejuni
The biochemically confirmed isolates employed for molecular confirmation as C. jejuni by polymerase chain reaction amplifying specific target gene. DNA were extracted by Phenol-Chloroform extraction method and the DNA concentration was quantified by nanodrop and stored at -20°C until further processing.
Genotypic confirmation of isolates by polymerase chain reaction for hip O gene and map A gene
The isolates were confirmed by PCR using designed primers in the study as forward primer (5-TTCCATGACCACCTC TTCC-3) and reverse primer (5-CTACTTCTTTATTGCTT GCTGC-3) for hip O gene. The primers used for amplification of map A gene were forward primer (5-CTATTTTATTTTT
GAGTGCTTGTG-3) and reverse primers (5-GCTTT ATTTGCCA TTTGTTTTATTA-3) (Khoshbakht et al., 2015).

The PCR reactions were performed in 25 μl reaction mixture. The amplification conditions consisted of initial denaturation at 94°C for 3 min, 35 cycles with denaturation at 94°C for 1 min, annealing at 53°C for hip O 1 min, at 72°C for 1 min, followed by a final extension at 72°C for 5 min respectively (Al-Amri et al., 2007). The annealing temperature for Map A gene was optimized as 52°C for 1 min (Khoshbakht et al., 2015). The DNA from C. jejuni (ATCC 3329) was included as positive control for PCR identification of the isolates. The amplified products were observed and photographed using gel documentation System (Applied Biosystems).
Campylobacteriosis is one of the most common bacterial infections worldwide causing economic costs (Soro et al., 2020). Modified blood free Charcoal cefoperazone deoxycholate agar is commonly used worldwide (Hutchinson and Bolton, 1984). In current study a total of 19 samples were processed for isolation of Campylobacter, all samples showed growth on mCCDA agar plates. The isolates showed typical grey/white or creamy grey in colour and moist spreading type colonies with sticky nature were confirmed phenotypically as Campylobacter. The suspected colonies were examined for morphological characteristics, motility, Gram’s staining. Campylobacter species are Gram negative rods with characteristically curved, spiral, or S-shaped cells. The overall incidence of Campylobacter was found to be 18 (94.73%) by cultural examination.
Biochemical characterization
The isolates were processed for phenotypic characterization and identified by biochemical tests. The test for hippurate hydrolysis is critical for separation of Campylobacter jejuni and C. coli strains. Glycine and benzoic acid are formed when hippurate is hydrolysed by C. jejuni (Morris et al., 1985). Twelve (63.15%) isolates were positive for catalase, oxidase, nitrate and hippurate hydrolysis, Ninhydrin test (Fig 1). The samples from raw chicken meat (9) were positive for H2S production. C. jejuni biotype 2 strains are H2S positive, whereas C. jejuni biotype 1 strains are H2S negative (Penner, 1988). In this study nine isolates belong to biotyope 2 while three belong to biotype 1 of C. jejuni. All the samples found to be positive for ninhydrin test confirmed as C. jejuni. Malik et al., (2014) studied hundre sis test positive for two isolates, categorized as C. jejuni and negative for 30 isolates while twelve (63.15%) isolates were positive in this study.

Fig 1: Characterization of C. jejuni isolates by biochemical tests.

Genotypic characterization
The size of PCR product for hip O gene was 270 bp and the size of the PCR product for map A gene was 589 bp (Fig 2). The incidence of C. jejuni from raw chicken meat using three screening tests was given in Table 1, Fig 3. The total of 10 (83.33%) isolates were confirmed as C. jejuni.

Table 1: Result of cultural, phenotypic and genotypic characterization of C. jejuni isolates.

Fig 2: Agarose gel showing the amplification product of hip O and map A gene of Campylobacter jejuni (270 bp and 589 bp) (L=100 bp DNA ladder).

Fig 3: Comparative result of phenotypic and genotypic study of C. jejuni from raw chicken meat samples.

Campylobacter was detected in 76% and Campylobacter jejuni was the most prevalent species (64.7%) being contaminated from broiler meat products collected in retail outlets (Nicodeme et al., 2015). The prevalence of Campylobacter spp. in chicken meat was 17.33% and twenty three Campylobacter jejuni were identified by biochemical examination and confirmed by polymerase chain reaction collected from retail meat markets, slaughter houses (Moudgil and Kumar, 2014). In the present study higher incidence rate of 12 (63.15%) was confirmed by biochemical tests and 10 (83.33%) by polymerase chain reaction.

Sixty eight C. jejuni isolates were obtained with an overall prevalence rate of 34% using the uniplex PCR targeting map A from different farms at Dakahlia Governorate in Egypt. (Younis et al., 2018). The isolates were confirmed as C. jejuni biochemically targeting the map A gene of C. jejuni. A total of 17% and 11.87% of broiler chicken and layer chicken samples respectively, were positive for C. jejuni collected in Egypt (Ghoneim et al., 2020). While in the present study 12 (63.15%) chicken meat samples were positive on the basis of biochemical testes and 10 (83.33%) by PCR targeting map A gene and hip O genes. Chicken samples were bacteriologically positive  for  Campylobacter isolates and 285 isolates (94.37%) were identified as C. jejuni by polymerase chain reaction targeting the hip O gene specific for C. jejuni (Barakat et al., 2020).

Campylobacter jejuni (88.1%) was most prevalent species isolated from broiler meat samples by cultural method and polymerase chain reaction assay (Zendehbad et al., 2015). Four C. jejuni (3.3%) isolated from poultry meat in Northern Poland (Szczepanska et al., 2017), while higher incidence (63.15%) was found in our study. Campylobacter jejuni (58.82%) was isolated from raw meat samples in Kolkata, India suggested that the consumption of undercooked meat possess a possible health risk for consumers (Sharma et al., 2016).

The most predominantly isolated species among Campylobacter was C. jejuni (69.5%) from chickens (Sinulingga et al., 2019). A total of 17% and 11.87% of broiler chicken and layer chicken samples respectively were positive for C. jejuni as the most commonly isolated species (Frosth et al., 2020). The risk of Campylobacteriosis in chicken meat consumers in southern Benin was studies and reported prevalence of C. jejuni (23.4%) from chicken meat by molecular methods (Kouglenou et al., 2020), while higher incidence (83.33%) was reported in our study.

Campylobacter jejuni was recognized as a main species accounting for 37 (68.5%) from chicken and turkey meat samples (Kanaan and Abdulwahid, 2019). The most frequently reported isolates were Campylobacter jejuni (22%) in poultry raw meat samples sold in retails. This suggested that “One Health” approach represent a strategy to control Campylobacter spp. and further programmes, policies, legislations and research will contribute to control contamination at primary production level and retail outlet (Gigliotti et al., 2019). Campylobacter isolates (35.4%) were recovered and analysed by multiplex PCR and four (13.8%) were identified as C. jejuni from broiler meat samples (Nafarrate et al., 2020) and their results concur with the results of our study.

C. jejuni was isolated from 17.0% of samples obtained in butcher shops and formal slaughter houses (Alaboudi et al., 2020). C. jejuni was isolated in 5 (41.7%) raw chicken samples collected from local market (Chen et al., 2020). Baali et al., (2020) reported that 65%, 55% and 70% of the cloacal swab, neck skin and caecal content were contaminated with thermotolerant Campylobacter strain, Campylobacter jejuni was the predominant species (73.5%) from broiler chickens in Batna, East Algeria and this is in accordance with the results of our study.
Poultry is an important reservoir and source of human Campylobacteriosis, although the contribution of other sources, reservoirs and transmission in reducing contamination of other meat samples. Campylobacter colonization in poultry and focus at the farm level has been successful in intervention strategies reducing the number of Campylobacter cases in several countries. Increasing farm biosecurity and education of consumers are likely to limit the risk of infection. Active on-farm biosecurity measures on chicken farms and more hygienic efforts in slaughter houses, in local chicken slaughter shops should be made for the effective control of these foodborne pathogens.
The authors wish to sincerely thank the Tamil Nadu Veterinary and Animal Sciences University, Department of Animal Biotechnology, Madras Veterinary College, Chennai. All authors contributed to the study.
All authors declare that they have no conflict of interest.

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