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ThesisItem Open Access MITIGATION OF ENTEROHAEMORRHAGIC ESCHERICHIA COLI ON MEAT CONTACT SURFACES USING PHYTOCHEMICALS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2018) THORAT AJAY BALKRISHNA; C. SethulekshmiThe present study was undertaken to study the occurrence of Enterohaemorrhagic E. coli (EHEC) on meat contact surfaces from retail meat outlets. A detailed study was also done regarding the biofilm forming ability of EHEC isolates and the effect of phytochemicals viz., trans-cinnamaldehyde and eugenol in reducing the growth of EHEC on meat contact surfaces. The antibiofilm effect of these phytochemicals was analysed. A total of 245 swab samples comprising of meat cutting board and knife surfaces were collected from four retail meat outlets located in and around Thrissur district during the period of study from November 2017 to June 2018. All the swab samples were subjected to isolation and identification of E. coli and Enterohaemorrhagic E. coli by conventional culture technique. The characteristic colonies from Cefixime Tellurite-Sorbitol MacConkey Agar were selected and inoculated into tryptic soya broth (TSB). The broth culture was subjected to multiplex PCR for detecting the virulence genes viz., stx1, stx2, eaeA and hlyA. A total of 40 EHEC isolates comprising of 30 isolates from cutting board and 10 isolates from knife surfaces were obtained. The occurrence of EHEC recorded from four retail meat outlets was 12 per cent, 21.42 per cent, 18.88 per cent and 5.71 per cent respectively. The higher occurrence was recorded from second retail meat outlet (21.42%) in comparison with other retail outlets from Thrissur district. All the 40 positive isolates were analysed for its biofilm forming ability and of which only five per cent of isolates had biofilm forming ability. Further, minimum inhibitory concentration (MIC) of phytochemicals was determined against biofilm producing EHEC and the MIC for trans-cinnamaldehyde and eugenol was found to be 3.5 per cent and 5.5 per cent respectively. The effect of these phytochemicals in reducing the growth of EHEC on meat contact surfaces was also analysed. The two concentrations of each phytochemical was studied. A concentration of 3.5 per cent and 4.0 per cent of trans-cinnamaldehyde brought about 0.66 and 0.69 log10 cfu/ml reduction of EHEC organisms on wood in comparison with control of 5.42 log10 cfu/ml respectively. Eugenol at 5.5 per cent and 6.0 per cent showed total reduction of EHEC organisms on wood. To study the anti-biofilm effect of these phytochemicals, EHEC biofilms were artificially created on wodden and fiber surfaces and the effect of two different concentrations of each phytochemical were tested. A concentration of 3.5 per cent of trans-cinnamaldehdye and 5.5 per cent of eugenol showed reduced growth of EHEC biofilm whereas concentrations of 4.0 per cent of trans-cinnamaldehyde and 6.0 per cent of eugenol completely inhibited biofilm forming EHEC. Hence these phytochemicals can be used as a good sanitizers in order to reduce the growth of EHEC which also had a significant effect on its biofilm forming ability.ThesisItem Open Access DETECTION AND QUANTITATION OF RESIDUES OF COMMONLY USED ANTIBIOTICS IN RAW MILK(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2018) KUMARSWAMY N P; C. LATHAThe present study was undertaken to detect and quantitate the residues of commonly used antibiotics in raw milk in Thrissur district of Kerala. The cross sectional survey was conducted among the farmers to assess their knowledge and awareness level on the use of antibiotics. A total of 440 milk samples were collected from ULF and FRDS, Mannuthy, private farms in Thrissur and individual households during the period from August 2017 to July 2018. All the samples collected were subjected for screening of antibiotic residues by microbial inhibition assay (MIA). The positive samples were analysed by Charm assay to determine the group and level of antibiotic residues. The amount of residues of oxytetracycline, enrofloxacin and cloxacillin in positive samples was quantitated using High Performance Liquid Chromatography (HPLC). The survey among the farmers showed that 78 per cent of people were aware of the antibiotic use and 43 per cent of people had knowledge about the withdrawal period. The most commonly used antibiotics were oxytetracycline (54 per cent), ßlactams (44 per cent), enrofloxacin (36 per cent), sulphonamides (14 per cent) and gentamycin (12 per cent). Of 440 milk samples screened by MIA, the antibiotic residues were detected in 7.41, 18.57 and 13.33 per cent of samples from ULF and FRDS, private farms and individual households respectively. The overall occurrence rate of antibiotic residues observed was 13.18 per cent. On analysing 58 positive samples in MIA by Charm Rapid One Step Assay (ROSA), it was found that 2.95 per cent of samples were positive for tetracyclines residues, 3.64 per cent of samples for enrofloxacin residues and 2.27 per cent of samples for ß-lactams residues. The HPLC conditions, sample extraction and analytical methods for detection and quantitation of oxytetracycline, enrofloxacin and cloxacillin residues were optimized and validated. The analysis of oxytetracycline was carried out on C18 column using a mobile phase of 0.03 M Oxalic acid : Acetonitrile : Methanol (70: 15 : 15) delivered at a flow rate of 1 ml/min. Photodiode array detector was set at 354 nm for oxytertracycline detection. The mobile phase consisting of 0.1 M Orthophosphoric acid (pH adjusted to 2.5 with triethylamine): Acetonitrile: Methanol (80:17:3) was used for enrofloxacin detection on C18 column. Flow rate of 0.8 ml/min and detection wavelength of 278 nm were found to be optimal. Chromatography conditions of cloxacillin consisted mobile phase of 0.1 per cent Trifluroacetic acid: Acetonitrile (50:50), flow rate of 1 ml/min and detection wavelength of 210 nm. The retention time noticed for oxytetracycline, enrofloxacin and cloxacillin was 5.6, 7.7 and 7.48 min respectively. A good linearity with coefficient of determination greater than 0.99 was obtained for all antibiotics. Of the 13 samples tested by HPLC for oxytetracycline, 12 samples showed oxytetracycline residues with a mean concentration of 1450.20 ± 182.09 ng/ml. Enrofloxacin was detected in all the 16 samples tested by HPLC with highest concentration of 709.28 ng/ml. Cloxacillin could be dectected only in two samples out of ten samples tested by HPLC with mean concentration of 118.77 ± 29.76 ng/ml. The presence of antibiotic residues in edible tissues of animals can cause adverse effects in human health and also lead to development of antimicrobial resistance which is one of the most serious global public health threats in this century. Thus, judicious and proper use of antibiotics, strict adherence to withdrawal period and maintenance of treatment records are the necessary steps to prevent the occurrence of antibiotic residues in milk. Control of antibiotic residues in foods of animal origin needs strict monitoring and surveillance studies so as to discard the food products that are not safe for human consumption.ThesisItem Open Access OCCURRENCE OF CAMPYLOBACTER SPP. IN SWINE PRODUCTION FACILITIES AND PORK PROCESSING LINES(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2018) MURALIKRISHNA P; B. SUNILThe present investigation was undertaken to determine the occurrence of Campylobacter spp. in swine production facilities, pork processing line, molecular confirmation and antibiotic resistance profile of the positive isolates. Comparative analysis regarding occurrence, weekly prevalence and antibiogram of Campylobacter spp. in piglets from both farms. The study also assessed the multiple drug resistant (MDR) and multiple antimicrobial resistance index (MAR) of the isolates. A total of 505 samples comprising of 340 rectal swabs from pigs and piglets, 40 faecal samples of pigs, 20 wild bird faecal droppings, 20 samples each feed, drinking water, wallowing tank water, soil and worker’s hand swabs and foot swabs were collected from two farms (F1 and F2) in Thrissur. In addition, five samples which includes three dog rectal swabs and two human faecal samples were collected from F1. To establish critical control points and to identify contamination points in pork processing line, a total of 484 samples was screened from various points of pork processing line from processing units, M1 and M2. All the samples were subjected to isolation and identification by conventional culture technique. Confirmation and species level identification was done by multiplex polymerase chain reaction. The antibiotic resistance profiling and multiple antimicrobial resistance index (MAR) analysis was carried out for all the positive isolates. Higher occurrence of Campylobacter spp. was recorded from F1 (33.3 per cent) when compared to F2 (21.70 per cent). From both the farms, piglets started excreting Campylobacter from fourth week after birth. Higher occurrence of Campylobacter spp. was recorded from F1 where 33.33 per cent of piglets selected for study excreted organisms from fourth week, while only 20 per cent of piglets from F2 were excreting Campylobacter spp. By the end of tenth week, occurrence of Campylobacter coli from both the farms were almost similar with 66.67 per cent of piglets positive from F1 and 60 per cent from F2. Shedding pattern of Campylobacter spp. in piglets was regular from F1, while irregular shedding pattern was recorded from F2. Higher occurrence of Campylobacter spp. was noticed in pigs (37.5 per cent), house crows and egrets (30 per cent), workers hand swabs and foot swabs after operations (40 per cent) and food waste mixed with raw chicken waste (20 per cent) from F1. Lower occurrence was noticed in F2, with pigs and crows having an occurrence of 20 per cent. Higher occurrence of Campylobacter spp. was noticed from environmental samples obtained from F2 with an occurrence of 20 per cent from wallowing tank water and soil samples while F1 had an occurrence of 16 per cent. Migratory pathway of infected wild reservoirs resulted in cross-transmission among birds and pigs. On analysis of contamination points from both the processing units, pigs brought to slaughter was the prime source of contamination for Campylobacter coli. From both the units, pigs carried the organism after stunning till scalding. No organisms could be detected after scalding. In M1 Campylobacter coli could be detected from carcass swabs obtained from jowl and belly after evisceration. Organisms could be also detected from carcass swabs after splitting of carcass. From M2, Campylobacter coli could not be detected after scalding till packing. In M1, meat samples which were positive before freezing were negative after freezing at -20°C for 24 hours. So freezing temperature could be considered as a CCP2 in M1 while scalding can be considered as a CCP2 in M2. All the C. coli isolates showed higher resistance against multiple antibiotics with cent per cent resistance against Ceftazidime, Co-trimoxazole and Ofloxacin. Cent per cent of the C. jejuni isolates showed resistance against Ceftazidime. Difference in resistance pattern was noticed in swine production system based on management practices followed in production facilities. In antibiotic free systems (F1) there was less prevalence of resistant strains compared to farms using antibiotics (F2). The multiple antimicrobial resistance (MAR) index of isolates was in the range of 0.21-0.87, with maximum number of isolates showing resistance for more than three groups of antimicrobials and pigs acting as a major source of multiple drug resistant strain of Campylobacter spp. (86.95 per cent). Thus proper implementation of biosecurity measures and bio containment in swine production systems will reduce the risk of Campylobacter infections and promote food safety through farm to fork concept. Control of foodborne diseases and emergence of multidrug resistance Campylobacters requires a multifaceted one health approach and surveillance programmes.ThesisItem Open Access IDENTIFICATION OF CAMPYLOBACTER SPP. CRITICAL CONTROL POINTS IN BEEF PRODUCTION CHAIN(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2018) VANI R. PILLAI; K. Vrinda MenonThe present study was undertaken to identify the critical control points of Campylobacter spp. in beef production chain. The study also assessed the antibiotic resistance profile of the isolates. A total of 500 samples were collected from two meat processing plants and University Livestock Farm and Fodder Research Development Scheme, Mannuthy (ULF and FRDS) during November 2017 to May 2018. The critical control points in the beef production chain to confine the occurrence of organism in beef were also investigated. All the samples were subjected to isolation of Campylobacter spp. by conventional cultural technique. The samples were also subjected to direct PCR using the genus specific 16S rRNA gene, C. jejuni specific mapA gene, C. coli specific ceuE gene and virulence gene cadF. The antibiotic sensitivity test of the positive isolates was performed using standard disc diffusion method to find out antibiotic of choice in treatment. Out of 236 samples examined from ULF & FRDS, Mannuthy, four per cent dung samples were found to have Campylobacter spp. by culture technique. The overall occurrence of Campylobacter spp. in ULF & FRDS was found to be 5.94 per cent by PCR. The samples (264) from different stages in the beef processing line of two meat processing plants were also assessed for detection of Campylobacter spp. The occurrence rate of Campylobacter spp from meat processing plant I and II was found to be 9.16 and 15.9 per cent respectively. As, process of dehiding and evisceration helps to reduce the microbial load, they were identified as critical control points (CCP2). Animal, floor surface, operator hands, water, knife, table surface are the critical points of contamination noticed in the present study. The confirmed isolates were sensitive to amikacin, amoxycillin, chloramphenicol, ciprofloxacin, clindamycin, dorepenem, doxycycline, gentamicin, imipenem and meropenem while they were resistant to cefixime, cefotaxime, cefuroxime, ceftazidime, enrofloxacin and tetracycline. Hygienic practices throughout the rearing, transportation and slaughtering of cattle should be adopted to eliminate contamination of beef with Campylobacter spp.ThesisItem Open Access EVALUATION OF NOVEL RECOMBINANT OmpF PROTEIN AS A VACCINE CANDIDATE AGAINST SALMONELLA TYPHIMURIUM IN POULTRY(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD, 2018) ANNIE NAVOMI PHILIP; PrejitThe present investigation was carried out to evaluate the suitability of the use of recombinant outer membrane protein F (OmpF) of S. Typhimurium expressed in prokaryotic expression system in inducing protective immune response against S. Tyhphimurium in layer birds. The ompF gene of S. Typhimurium was amplified, cloned in pRham expression vector and expressed in E. coli as a fusion protein. The rOmpF protein was expressed in E. coli at an optimal temperature of 25°C with 0.2 per cent rhamnose for 16 h auto induction. The expression was analysed by SDS-PAGE and confirmed by western blot using anti-His-tag conjugate. This confirmed that rOmpF is a fusion protein with a size of ~57kDa. The expressed rOmpF was in insoluble form and was purified under denaturation condition by Ni- NTA purification and further concentrated by dialysis for obtaining single protein band. Similarly, rOmpC clone that was previously developed in the lab was induced and expressed. Both rOmpF and rOmpC was purified in largescale. SDS-PAGE analysis revealed the presence of 57 kDa (rOmpF) and 58 kDa (rOmpC) proteins. A total of 80 birds were divided into four groups (rOmpF+adjuvant, rOmpC+adjuvant, commercial vaccine and PBS control) and immunized at the dose of rate of 100 µg/bird i/m. Humoral immune response by ELISA revealed that the antibody level in vaccinated groups was more and highly significant (P<0.001), when compared with control group in all the six weeks. Afterwards, the birds were challenged orally with virulent S. Typhimurium culture. The protective index was calculated in the 12th, 13th, 14th week. Statistical analysis showed highly significant (p<0.01) variation among overall Salmonella positive samples between vaccinated and control groups. There was no significant variation in between the vaccinated groups. The protective index of the three vaccinated groups after 3 weeks post challenge was found to be as follows, commercial vaccine group= 87.5 per cent, rOmpC=81.3 per cent and rOmpF group= 75 per cent. This study concluded that rOmpF protein proved to show protection against S.Typhimurium infection in poultry.ThesisItem Open Access DEVELOPMENT OF RECOMBINANT OmpF PROTEIN BASED INDIRECT ELISA FOR THE DETECTION OF SALMONELLA ANTIBODIES IN POULTRY(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD, 2018) NIMISHA SOMAN; PrejitSalmonellosis is one of the most frequently reported poultry disease worldwide. Screening and identification of salmonellosis among poultry flock have assumed principal role in preventing transmission of this pathogen to humans from chickens. The objective of this study was to develop a highly promising and reliable diagnostic tool for detection of Salmonella antibodies using rOmpF protein. The ompF gene of S. Typhimurium was amplified, cloned in pRham expression vector and expressed in E. coli as a fusion protein. The rOmpF protein was expressed in E. coli at an optimal temperature of 25°C with 0.2 per cent rhamnose for 16 h auto induction. The expression was analysed by SDS-PAGE and confirmed by western blot using anti-His-tag conjugate. This confirmed that rOmpF is a fusion protein with a size of ~57kDa. The expressed rOmpF was in insoluble form and was purified under denaturation condition by Ni-NTA purification and further concentrated by dialysis for obtaining single protein band. The rOmpF was assessed for its immunoreactivity as a diagnostic antigen by immunoblotting. The immunogenic reactivity of expressed rOmpF was optimized in indirect ELISA using known true positive and negative sera of poultry with respect to OmpF antibodies. The relative diagnostic sensitivity and specificity of the assay (95.24 per cent and 100 per cent respectively) was observed at a cut off value of 0.897. The diagnostic efficiency was comparable with the reference test (rOmpC based I-ELISA). Standardized rOmpF based IELISA was used for analysing 210 poultry serum samples from Wayanad distirct, of that 42 sera were tested positive for Salmonella antibodies. In this study, the overall occurrence of Salmonella in poultry stock of Wayanad was found to be 20 per cent. This study revealed that indirect rOmpF based I-ELISA is a promising diagnostic tool for diagnosis of salmonellosis in poultry and also a safe antigen to be used for sero-diagnosis.