Characterization of Campylobacter jejuni An Important Zoonotic Food Borne Pathogen from Mastitis Milk and Raw Milk Samples

Campylobacteriosis is the most frequently reported zoonosis and the main bacterial foodborne disease in humans (Zendehbad et al., 2015). Campylobacter is a major medical and economic problem worldwide, with numbers of cases continuing to increase (Kaakoush et al., 2015). Campylobacter is 1 of 4 key global causes of diarrheal diseases considered to be the most common bacterial cause of human gastroenteritis in the world (WHO, 2018). Campylobacter jejuni is a leading cause of bacterial diarrheal disease worldwide and it is responsible for zoonoses (Facciola et al., 2017). The pathogenesis of C. jejuni is poorly understood as compared to other enteric pathogens (Rizal et al., 2010). Campylobacter spp. can be transferred from animals to humans by contaminated food of animal origin, especially undercooked poultry meat and unpasteurized milk/dairy products (Luangtongkum et al., 2012).

Campylobacter jejuni can cause mastitis in cow and the bovine udder is potential source of C. jejuni in raw milk (Lander and Gill, 1980). Poor pretreatment of the teats with disinfectant or contact of the milking cluster with the parlor floor result into higher levels of fecal Campylobacter contamination (Beumer et al., 1988) Campylobacter generally colonize in the lower gastrointestinal tract where there is a lower pH environment in the rumen. The stratified epithelium of the rumen lacks the necessary receptors to sustain a persistent high level of Campylobacter colonization (Grahamm et al., 2005). The common vehicles for Campylobacter foodborne disease transmission are unpasteurized bovine milk and milk products and Campylobacter spp. in raw milk derive most commonly from secondary fecal contamination during the milking process (Oliver et al., 2005).

Campylobacter is one of the most common pathogen-related causes of diarrheal illnesses globally and has been recognized as a significant factor of human disease for more than three decades (Magana et al., 2017). In humans, clinical signs of Campylobacteriosis include diarrhea, abdominal pain, fever, headache, nausea and vomiting. Most of Campylobacter are sporadic and self-limiting, but there are post-infection complications, for example, Guillain-Barres syndrome (Hansson et al., 2018).

Campylobacter jejuni is difficult to grow and identify and common milk contaminant due to fastidious growth requirement. Hence this study was attempted to isolate and identify the presence of C. jejuni using cultural, biochemical and PCR technique and compare these techniques for detection of C. jejuni in milk 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 72 samples comprising Mastitis milk sample (20) were collected from Department of Clinics, Madras Veterinary College, Chennai and raw milk samples (52) from cattle farms. 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 and Lastovica and Allos, 2008). The Campylobacter jejuni (ATCC 33291) was used as standard and reference strain.
 
Phenotypic characterization
 
Cultural examination
 
Samples were enriched in Blood free 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 (Microaerophilic gas pack CampyPack-BD oxoid). On selective agar colonies were grey/white or creamy grey in colour, smooth, glistening and convex with entire edges and moist in appearance, dew drop with the tendency to spread. Campylobacter species are gram negative rods, 0.5 - 8 μm long and 0.2 - 0.5 μm wide with characteristically curved, spiral, or S-shaped cells.
 
Biochemical test
 
The isolates were identified as C. jejuni based on their morphological and biochemical tests. Suspected colonies were sub-cultured and confirmed by biochemical tests as catalase, oxidase, nitrate and hippurate hydrolysis, Ninhydrin test, H₂S production.
 
Molecular confirmation of Campylobacter jejuni
 
The biochemically identified isolates were further employed for molecular confirmation as C. jejuni by polymerase chain reaction amplifying specific target gene using species-specific oligonucleotide primers. 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
 
Polymerase chain reaction was carried out using primers for species specific genes. The PCR was performed in a thermal cycler (Applied Biosystem). The hipO gene region is the hippuricase gene, specific for C. jejuni. Primers for specific identification were designed using the hipO gene sequences of C. jejuni based on the sequences available in the GenBank.

The isolates were confirmed by PCR using designed primers in the study as forward primer (5-TTCCATGACC ACCTCTTCC-3) and reverse primer (5-CTACTTCTTTATT GCTTGCTGC-3).

The primers used for amplification of MAP A gene were forward primer (5-CTATTTTATTTTTGAGTGCTTGTG-3) and reverse primers (5-GCTTTATTTGCCATTTGTTTTATTA-3) (Khoshbakht et al., 2015).

The PCR reactions were performed in 25 μl reaction mixture, containing 12.5 μl PCR master mix (2X-Ampliqon), 1 μl of each primer of a 10 μM primer concentration, 1 μl MgCl₂ (25 mM), 3 μl template DNA and 6.5 μl nuclease-free water making a total volume of 25 μl. The amplification conditions were initial denaturation at 94°C for 3 min, 35 cycles with denaturation at 94°C for 1 min, annealing at 53°C for HipO gene for 1 min and extension 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 33291) was included as positive control for PCR identification of the isolates and the master mix without sample DNA used as negative control. The amplified products were observed and photographed using gel documentation System (Applied Biosystems).  

Campylobacter spp. is a major cause of gastroenteritis, there is an urgent need to control these pathogens with zoonotic and public health point of view. The current study total 52 samples studied for presence of Campylobacter from raw milk and milk from mastitis cases. The Campylobacter species are difficult to isolate but the results from inoculation studies showed that plates with either blood or charcoal had a better recovery rate than other media used for isolation. Modified blood free Charcoal Cefoperazone Deoxycholate agar is commonly used worldwide (Bolton et al., 1984; Hutchinson and Bolton, 1984). In current study a total of 72 samples were processed for isolation of Campylobacter and 52 isolates 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. The overall incidence of Campylobacter was found to be 18 (34.61%) by cultural examination.
 
Biochemical characterization
 
The isolates were processed for phenotypic characterization and identified by biochemical tests, viz. oxidase, catalase, indoxyl acetate hydrolysis tests and H₂S production in triple sugar iron test. The test for hippurate hydrolysis is critical for separation of Campylobacter jejuni strains. Glycine and benzoic acid are formed when hippurate is hydrolyzed by C. jejuni (Morris et al., 1985). In present study 10 (19.23%) isolates, 4 from mastitis milk samples and 6 from raw milk samples were positive for catalase, oxidase, nitrate and hippurate hydrolysis, Ninhydrin test. C. jejuni biotype 2 strains are H₂S positive, whereas C. jejuni biotype 1 strains are H₂S negative (Penner, 1988). Two samples from mastitis milk were positive for H₂S production belongs to biotyope 2 (Table 1). The most of the samples were negative for H₂S production categorized as biotype 1 of C. jejuni. The hippuricase enzyme hydrolyzes hippurate and this test is used routinely in the clinical laboratory. (Malik et al., 2014) studied hundred chicken caecal samples and reported   hippurate hydrolysis test positive for two isolates, categorized as C. jejuni and negative for 30 isolates. In current study all the samples found to be positive for ninhydrin test confirmed as C. jejuni.


 
Genotypic characterization
 
The isolates were confirmed by polymerase chain with species specific primers for Hip O and MAP A gene. 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 1).  The incidence of C. jejuni using three different screening tests was given in Table (2) Fig (2).

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Raw milk samples are among the potentially important sources of Campylobacter spp. A total of 100 raw milk samples were screened and revealed a prevalence of 3% (Kumar et al., 2015).

A lower prevalence rate of 1.41% was observed by (Elango et al., 2010) in Chennai, India. A total of 60 milk samples collected from different milk vendors of different areas of the Faizabad district out of which Campylobacter jejuni (4) were recovered from 60 milk samples in accordance with this study  (Shekhar et al., 2010). C. jejuni was detected in 34 (12%) bulk tank milk by specific PCR (Bianchini et al., 2014).

A total of 42 C. jejuni isolates were obtained with a prevalence rate of 1.36% (Elango et al., 2012). Campylobacter  species were detected in 7 (2.91%) raw milk samples and all the isolate identified were Campylobacter jejuni whereas none of the milk product was positive. (Modi et al., 2015), while in this study 3 isolates from mastitis milk and 4 isolates from raw milk samples found to be positive for C. jejuni Campylobacter spp. was isolated from 16 (5.3%) samples by conventional method the isolates were identified by biochemical analyses and the polymerase chain reaction which revealed that 12 (75%) samples were Campylobacter jejuni (Kilicaltun et al., 2014) in accordance with our study.

The result from our study showed that milk samples are the main source transmission of Campylobacter and related Food infections. The presence of C. jejuni in milk can also be due to contaminated sources in the dairy farm environment and direct contact as a possible reservoir for Campylobacter transmission to livestock. Possibly as a result of contact with bovine feces, contaminated water or direct contamination in dairy farm environment can be responsible for mastitis in cattle. Consumption of raw milk can be as a potential source of transmission of Campylobacter to human. The human consumption of unpasteurized or improperly pasturized milk can be result into Campylobacteriosis and gastroenteritis.