Proliferation and Contamination of Campylobacter Jejuni Recovered from Frozen Buffalo Meat in New Delhi, India

Genetics and Molecular Biology: Current Research

Ujjal Sen Received Date : 01 Nov, 2019 Research Article Accepted Date : 08 Nov, 2019 Volume 1 : Issue 1 Published Date : 15 Nov, 2019

Ujjal Sen* and Anil Mahadeo Garode Department of Microbiology, Kalinga University, Village Kotni, Near Mantralaya, Chattisgarh, India

*Corresponding author: Ujjal Sen, Depertment of Microbiology, Kalinga University, Village Kotni, Near Mantralaya, Naya Raipur - 492101, Chattisgarh, India.

Citation: Sen U and Garode AM. (2019) Proliferation and Contamination of Campylobacter Jejuni Recovered from Frozen Buffalo Meat in New Delhi, India. Genet Mol Biol Curr Res. 1: 001. GMBCR-001.000001

Abstract Prior to 1970 Campylobacter known to veterinary microbiologist as causative agent of spontaneous abortions in cattle and sheep along with other animals. Positively C. jejuny isolated from frozen bovine meat according to Stern et al. Campylobacter jejuny is microaerophilic, produces heat labile enterotoxin, can cause in human campylobacteriosis with symptoms of meningitis, pneumonia, miscarriage and a severe form of Guillain - Barré syndrome, non-life threatening, diarrhoea to mimicking acute appendicitis. Microscopically C. jejuny is gram negative rods with comma shaped. This research finds the result, out of 61 samples of frozen buffalo meat 49 samples found positive and 12 samples negative for C. jejuny in frozen condition. Campylobacter Agar Base with charcoal and sheep blood exhibit thick translucent white or gray or colorless growth with spreading, film-like transparent flat or moist growth at 37oC and 42oC for 2 - 5 days. Antibiotic assay test revealed C. jejuny more sensitive to 10 µg amoxyclave & little sensitive or resistant to 5 µg methicillin. Approximately other sources of contamination and risk assessment tested at Abattoir like air - out of 25 sample 100% samples are positive, out of 25 samples of water 35% samples are positive, out of 25 samples of slaughter instrument 100% are positive & Food handlers hand swab out of 25 samples 100% are positive. Therefore, the tolerances are very high in frozen buffalo meat and resulted due to unhygienic practice at abattoir, processing of buffalo meat and create food borne diseases which decreases quality and increases the level of risk of food borne disease. Keywords Campylobacter jejuni; Frozen Buffalo Meat; Campylobacteriosis

Introduction of improperly handled and unhygienically processed frozen buffalo meat at abattoir in India. C. jejuni is a zoonotic foodborne pathogen Campylobacter jejuni S-shaped, curved or spiral Gram and one of the causes of colitis in zoonotic countries. The main negative bacillus. The bacteria are highly motile with corkscrew reservoir of this bacteria in alimentary tract of different animals like motion. It grows mostly on microaerophilic condition i.e. at and at present here in buffalo alimentary tract in New Delhi, India reduced 5% O & added 10% CO . At present Campylobacteriosis 2 2 abattoir. The optimum growth temperature is 42°C and also can is a major human food borne disease noticed worldwide [1,2], grow in 37°C. Two or five days after consumption of injected which is dominantly believed to be associated with consumption or infested contaminated frozen bovine meat gastrointestinal

Citation: Sen U and Garode AM. (2019) Proliferation and Contamination of Campylobacter Jejuni Recovered from Frozen Buffalo Meat in New 1 Delhi, India. Genet Mol Biol Curr Res. 1: 001. GMBCR-001.000001 Volume 1 : Issue 1 Ujjal Sen© symptoms including abdominal cramps, fever and diarrhea will frozen poultry meat in Brazil. [13] It is also reported in USA and appear, vomiting usually not present but diarrhea may sometime New Zealand during storage of beef trimming in freezing and become horrific, turns to bloody diarrhea or dysentery [7,18,19]. frozen condition C. jejuni causes a wide range of debilitating, but Campylobacter is highly susceptible to environmental stresses, non – life - threatening, symptoms, from diarrhoea to mimicking like variations in temperature, humidity, osmolarity, presence acute appendicitis. In addition, the rare immune - mediated of sunlight and atmospheric oxygen. [2] This study conducted on neuropathic Guillain – Barré syndrome that is characterized by frozen bovine meat after processing at very low temperature but, ascending paralysis and occasional death. [14] In a study of raw sampling resulted and busted out with tolerance and survivability meat of beef and chicken it is reported that samples contaminated of the organisms O2 at bovine abattoir in India. Studies elsewhere with C. jejuni [15]. have demonstrated that Campylobacter survive in raw and cooked poultry meat during refrigerated or frozen storage. [3,4,5,6] The Morphology and Cultural Characteristics objective of this study were to determine potential sources of Campylobacter are microaerophilic (reduced 5% O2 & added human disease along with prevalence and survivability of the 10% CO2, Oxygen quenching agents such as Hemin and Charcoal organism connection in zoonotic foodborne illness at a point in used in media), gram negative rods with comma, S or “gull - wing” slaughter house after thorough processing samples collected and shapes, 0.5–8 µm in length and 0.2 – 0.5 µm in width, cells are examine the influence of organism and recovery of C. jejuni when pleomorphic, non – spore forming, very small, slender, curved or gaining familiarity with C. jejuni isolation procedures. spiral shape and one or more polar or amphitrichous flagella shows corkscrew movement of bacilli, as old cultures spiral or curved History and Disease Report bacilli are replaced by round forms. Campylobacter jejuni are

Veterinary Microbiologist prior to 1970 known Campylobater cultured in microaerophilic condition at reduced 5% O2 & added as an organism that cause spontaneous abortions in cattle and 10% CO2 in media, charcoal used as effective substitute in media. sheep and cause of other animal pathogens. It appears sensitive On blood agar it is non - haemolytic. Here Campylobacter Blood to freeze. C. jejuni reported isolated from frozen bovine muscle Charcoal Agar Base supplemented with 5-7% sterile lysed horse meat. [9] C. jejuni is occasionally invasive and infections are blood or 10% sterile defibrinated sheep blood and Campylobacter manifested as meningitis, pneumonia, miscarriage and a severe supplement I (Blaser - Wang) or Campylobacter Supplement III form of Guillain - Barré syndrome. [10.11] C. jejuni produces heat (Skirrow) and Charcoal is used [16]. labile enterotoxins. It is reported that in 2007 in Kuala Lumpur, Malaysia different samples of chilled and frozen chicken carcasses Materials and Methods have been tested and C. jejuni confirmed present in the samples. Sample Collection: - Collect 100 gm of frozen buffalo meat [12] Human Campylobacteriosis also reported in chilled and sample, transport them in an insulated shipping container enough gel - type to maintain refrigerant at 6°C or below. Upon receipt in the laboratory, store the samples at 4°C and analyze immediately. If analysis cannot be started within 4 days after collection, freeze samples promptly and store at -20°C until examined. Thaw at room temperature and proceed with analysis as usual. Maintain frozen samples at -20°C until examined. Method: -Within 100gm sample 25gm frozen buffalo meat samples are weighed aseptically inside laminar air flow and add 225mL of 0.1% peptone water homogenized the mixture in a blender and used as pre-enrichment solution according [16] sampling method as mentioned above. Incubate the pre-enrichment at 37oC for 24hrs. After incubation pour plate on the Campylobacter Blood Figure 1: After 5 days of incubation colorless or grey, flat, moist, Charcoal Agar Base supplemented with 5-7% sterile lysed horse translucent and young colonies look like water droplets on Char- blood or 10% sterile defibrinated sheep blood and Campylobacter coal – Blood agar base. Hence Campylobacter present.

Citation: Sen U and Garode AM. (2019) Proliferation and Contamination of Campylobacter Jejuni Recovered from Frozen Buffalo Meat in New 2 Delhi, India. Genet Mol Biol Curr Res. 1: 001. GMBCR-001.000001 Volume 1 : Issue 1 Ujjal Sen©

Figure 2: A, C Pictures exhibit Creamish-White colonies and Yellow colonies of different strains of Campylobacter jejuni, but with Char- coal - blood agar exhibit different haemolytic colonies. supplement I (Blaser-Wang) or Campylobacter Supplement III Biochemical Confirmation (Skirrow) and Charcoal. Incubate at 37oC and 42oC for 2 - 5 days Campylobacter jejuni is microscopically gram negative rods undergo moist, microaerophilic condition in a anaerobic jar or and biochemically confirmed as motile, oxidase positive, catalase wrapped it with aluminium foil. Observe the plates for presence or positive, ferment carbohydrates, H S on TSI negative. absence of colonies. 2

Observation Antibiotic Sensitivity of C. jejuni Antibiogram test on recovered positive C. jejuni isolates Campylobacter jejuni exhibit thick translucent white or gray conducted to look for acquired whether antibiotic resistant C. or colourless growth to spreading, film-like transparent growth or jejuni organisms takes part in creating food borne diseases during watery, convex, pinpointed, round, irregular and smooth edges human consumption. These assessment measure actual potential colonies and also show water droplets on charcoal agar.

Figure 3: Campylobacter jejuni colonies denotes non-haemolytic and few are haemolytic may be β-haemolytic, or gamma - haemolytic colonies.

Citation: Sen U and Garode AM. (2019) Proliferation and Contamination of Campylobacter Jejuni Recovered from Frozen Buffalo Meat in New 3 Delhi, India. Genet Mol Biol Curr Res. 1: 001. GMBCR-001.000001 Volume 1 : Issue 1 Ujjal Sen©

Figure 4: Biochemical Tests: Motility-Non-motile, TSI - H2S Positive, Urease- Positive, Citrate - Positive, Indole - Negative, VP - Nega- tive, MR - Positive, Catalase positive, Oxidase - Positive. Hence, after biochemical confirmation it is observed that Campylobacter is confirmed appeared on Campylobacter agar base media which has from it’s pure culture. source may come from cattle feed and may come from insertion disc, 10µg amoxyclave discs which incubated at 37oC for 24 hours. of antibiotic to control diseases in buffalo. These conveyed as 1.7 cm zone of inhibition appears on around methicillin disc and natural phenomena and transferred through genetically acquired 7mm zone of inhibition appears around methicillin disc. Hence antibiotic resistant plasmid DNA to human after the meat Campylobacter more sensitive to 10µg amoxyclave disc but less consumption. As a result, when disease acquired higher dose or sensitive to 5µg methicillin disc. higher generation of antibiotic used to cure the disease. Lower dose of antibiotic become ineffective. In this research few samples Risk Assessment from The Potential Sources of isolated positive C. jejuni colonies analyzed. Campylobacter of Abattoir Environment jejuni positive colonies picked from few samples of frozen buffalo The assessment of potential risk is important to signify meat and tested with three antibiotic discs like 5µg methicillin that potential risk depend on different contaminant present in abattoir environment which contaminate frozen buffalo meat while processing and resultant in spoilage. To assess and analyze these potential risk sources are summarize and significant in all respect. Most importantly, air present inside abattoir where aerosols are major potent source which need to analyze frequently with conventional way i.e. by sedimentation technique of exposing the plates in plant environment. Food handler’s hand, equipment, machineries, cracks and crevices with wall and floor swab analysis can provide potential source to identify contaminating areas. On the other hand, potential and significant source can identify through most probable number (MPN) test of water which used in washing floors and other used utensils. During assessing the risk of Campylobacter jejuni from air, it is observed that out of 25 samples almost 100% of the samples are positive for C. jejuni.

Figure 5: Whitish Campylobacter colonies are enriched in pep- While screening the water samples used in plant by pour plate tone broth and incubate at 37oC for 24hours. Then, incubated technique, it is evident out of 25 samples, 35% of the samples found liquid culture pour plate in Nutrient Agar and gently mix. After positive. During investigation of other pathogenic organisms solidification, place 5µg methicillin disc and 10 µg amoxyclave disc and incubate at 37oC for 24 hours. 1.7cm zone of inhibition through swabbing slaughter house instrument after wash out of 25 appears around amoxyclave disc and 7mm zone of inhibition ap- swab samples analysis yield 100% of the samples are positive. Even pears on around methicillin disc. Hence, it is observed recovered when check and analyze 25 swabbing samples of food handlers and Campylobacter jejuni sensitive to both antibiotics. butchers where 100% of the samples observed as positive. It means

Citation: Sen U and Garode AM. (2019) Proliferation and Contamination of Campylobacter Jejuni Recovered from Frozen Buffalo Meat in New 4 Delhi, India. Genet Mol Biol Curr Res. 1: 001. GMBCR-001.000001 Volume 1 : Issue 1 Ujjal Sen© during processing and post-processing an unhygienic practice of storage temperature, storage period and the interruption of the prevail which can contaminate and help in spoilage of the food at cooling chain on its survival have been evaluated. [27] According to very low temperature. This is significantly dangerous for human result it suggests that freezing offers a short- to long-term solution consumption and requires standard international and national to the safe storage of buffalo meat with respect to the survival of level regulatory authority to set microbiological and other signicant Campylobacter. While it does not entirely eliminate potentially parameter before export from New Delhi, India to maintain it infectious Campylobacter, the initial reduction achieved in its strictly. On the other hand, it’s a not only economic loss of the population using this method may have a substantial impact on manufacturer and buyer, but, at the same time economic loss and reducing the incidence of campylobacteriosis [29] These results honor for the country. At the end, safe food production and safe also demonstrate the existence of injured cells in the frozen consumption will open the way to new era of food production. bacterial population. To prevent campylobacteriosis, it is suggested to spray chlorinated water up to 50ppm in concentration during Results and Discussions slaughtering, skinning and evisceration under aseptic conditions Indeed, present research experiments of frozen buffalo on buffalo carcasses. Before the consumption of frozen buffalo meat where 61 samples have been pursued for analysis to identify meat food, the processed raw frozen buffalo meat must cook Campylobacter jejuni, but 49 samples found positive and 12 samples undergo temperature reach at least 77°C and 82°C respectively. found negative. Hence, as a result this finding expressed processing To model the survival kinetics of Campylobacter jejuni on frozen of buffalo meat yield the results of tolerance ofCampylobacter jejuni chicken meat studied elaborately by. [28] in Netherland. at very low temperature can withstand the temperature and survive Conclusion in very low temperature too can cause spoilage of the food. The research and investigation reported organism’s tolerances are very Campylobacter jejuni can recovered from mostly all cuts of high in frozen buffalo meat and resulted unhygienic processing of frozen buffalo meat. This research concentrated only on frozen buffalo meat, compromising with quality and increases the risk of buffalo meat recovery of C. jejuni and its survival capacity in very low freezing, but not concentrated on chilled meat recovery. The food borne diseases. The most common species of Campylobacter detection from frozen buffalo meat is pretty high and denotesC. jejuni in animals and foods are Campylobacter jejuni. C. jejuni involved survivability capacity and adaptation during freezing environment. in human gastroenteritis. C. jejuni in raw meat of animals detected Overall, as a reason of survival lead to food deterioration and in Iran [17]. Hippurate hydrolysis is the only biochemical test spoilage. Diseases caused by Campylobacter jejuni are increasing which can differentiate between C. jejuni and other Campylobacter in significance worldwide. Infact, cross contamination through species. [18,19,20,21] Present investigation of frozen buffalo meat mishandling of frozen buffalo meat during processing at abattoir reported that C. jejuni can survive frozen storage or shipment. and the failure to maintain the storage condition increasing. The Freezing is harmful to the organism and can lower isolation result show that raw frozen buffalo meat must be considered to rate. The deleterious effect of freezing has been reported. [22,23] increased C. jejuni contamination rate. Therefore, there will be a Fresh and frozen poultry collected at slaughter and recovered C. considerable risk of C. jejuni infection in consumers either through jejuni have been reported in a study. [24] In a complete study on direct contamination or cross-contamination during handling Bovine Genital Campylobacteriosis is a bacterial disease caused or processing of raw frozen meat. However, in order to prevent by Campylobacter fetus subspecies veneralis isolated from cows, Campylobacter jejuni contamination and foodborne diseases, buffalos and cattles in Australia. The disease is characterized by consistently practice good hygiene at buffalo meat processing and temporary infertility of female cattle as a result of a sub-acute diffuse handling as well as consumer education mandatorily implement. mucopurulent cervicitis, endometritis and salpingitis. Abortions occurred in small percentage of cows and buffalos months after Acknowledgement initial infection [25]. Prevalence and survivability of C. jejuni as I am grateful to the Chancellor of Kalinga University, Ph. initial level of aerobic bacteria and failure to decrease in number of D. Supervisor of Kalinga University, Principle of Shivaji Science beef trimmings during frozen storage has been reported. [26] One College, Chickli, Maharastra, India for contributing help in respect of the study investigated C. jejuni in Turky meat during storage the to provide suggestions to accomplish the vital goal of research impact of the storage factors on its survivability based on effects work on Campylobacter jejuni.

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Citation: Sen U and Garode AM. (2019) Proliferation and Contamination of Campylobacter Jejuni Recovered from Frozen Buffalo Meat in New 6 Delhi, India. Genet Mol Biol Curr Res. 1: 001. GMBCR-001.000001