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Kerala Veterinary and Animal Sciences University, Wayanad
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ThesisItem Open Access ASSESSMENT OF CELL MEDIATED AND HUMORAL IMMUNE RESPONSE TO SUBUNIT VACCINE AGAINST RIEMERELLOSIS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2019-12-30) RINSHA BALAN; Priya P. M.Riemerellosis is a bacterial disease among ducks, caused by Riemerella anatipestifer, which has been well documented as a cause of considerable economic loss to the duck production in Kerala. At least 21 serotypes of the organism have been identified globally. Since vaccination is the mainstay for the control of the disease, a research work was undertaken to prepare subunit vaccine employing recombinant OmpA of R. anatipestifer and assessment of cell mediated and humoral immune responses of the vaccine and also to evaluate the comparative efficacy with that of the developed inactivated vaccine. Broth culture of R. anatipestifer at a concentration of 2.5 OD values at 525 nm with a dose of 1 mL per bird subcutaneously was selected as LD50. L per bird subcutaneously was selected as LD50. A total of 52, day-old ducklings were divided into three treatment groups with ten birds each. They were injected with 0.5 mL of different types of vaccine subcutaneously. Group I (T1) served as control with 22 birds including six birds each for challenge control of inactivated and subunit vaccine. Group II (T2) was injected with an inactivated vaccine (7x109 cfu/mL), which was prepared as per the protocol standardised in the Department of Veterinary Microbiology and group III (T3) and group IV (T4) were administrated with different antigen concentration of subunit vaccine (equal quantity of the rompA protein (250µg and 500µg) and montanide, respectively). A booster dose was given at third week post-primary vaccination to T2, T3 and T4. It was observed that, by using both crude Omp and rOmpA based ELISA, inactivated vaccine birds (T2) produced higher antibody titre during early age while in the subunit vaccine group, the titre was higher during later stage. An early antibody response is required to lower the mortality rate in riemerellosis as the organism targets young ducklings. Thus, it could be inferred from this study that inactivated vaccine was more effective than subunit vaccine A significant CMI response was also shown by inactivated vaccine groups on 14th and 28th day post-vaccination by lymphocyte proliferation assay (LPA). Challenge studies to assess the protective response revealed 100 per cent protection for inactivated vaccine group (T2), 80 per cent protection for T4 group and 70 percent protection for T3 group. All the vaccinated birds were having significantly less gross lesion when compared to the challenge control groups. On analysing the cytokine mRNA expression levels using real-time PCR, the inactivated vaccine group showed significantly higher (p< 0.05) mRNA levels of IL-6, IL-12B and IFN-γ gene on day 28 than the two subunit vaccine groups. It was found that the inactivated vaccine was superior in terms of results obtained from the challenge study, antibody titre, CMI response and gene expression analysis than the subunit one. Hence, it is desirable to advocate the use of inactivated vaccine in field condition owing to its easiness to prepare and low cost.ThesisItem Open Access COMPARATIVE PHARMACOKINETICS OF SPARFLOXACIN IN GLIBENCLAMIDE TREATED AND UNTREATED DIABETIC RATS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD, 2019-11-07) S.V., SURAJ; N Nair, SureshDiabetes mellitus (DM) is an endocrine related metabolic diseases that result in hyperglycaemia. In diabetes, patients are prone for multiple diseases. Hence to control these complications diabetic patients use medications like antihypertensive drugs, antiarrhythmic, antibiotics, and antiplatelet aggregant drugs along with the regular antidiabetic drugs. Diabetes can cause alteration in pharmacokinetics and pharmacodynamics due to alteration in drug transporters like P-glycoprotein, along with this multidrug usage may result in drug interactions. Hence the effect of alteration in pharmacokinetics of drug and also the interaction kinetics has to be found out in diabetes condition to ensure safe and effective use for the wellbeing of animals. Sparfloxacin is a third generation fluoroquinolone antimicrobial agent which is having activity against wide range of organisms. There are very limited studies related to pharmacokinetics of drugs in diabetes condition. The current study was undertaken to investigate the pharmacokinetic parameters of sparfloxacin in diabetic rats after oral administration and also to study alteration in pharmacokinetic parameters of sparfloxacin in glibenclamide treated diabetic rats when compared to control rats. The analysis was carried out using RP- HPLC with the mobile phase of 1% aqueous acetic acid and acetonitrile (20:80) on a phenomenex luna 5µ C18 column at the flow rate of 0.7 ml/min and column temperature of 300C. The photodiode array detector (PDA) set to 280 nm was used for detection. The method was validated with respect to accuracy, precision, specificity and linearity. The retention time obtained for sparfloxacin was 8.5 min. The recovery was found to be 87±1.547 % in spiked plasma sample. The intraday precision with the co-efficient of variation ranged from 2.474 to 8.218 %. And interday mean error ranged from 0.0035 to 0.0512. Control rats, HFD rats, diabetic rats and glibenclamide treated rats were given sparfloxacin at the dose rate of 200 mg/kg and blood was collected at different time intervals and was analysed using RP- HPLC. Pharmacokinetics of orally administered sparfloxacin in control, HFD and diabetic rats were fitted into two compartment model where as in glibenclamide treated diabetic rats 22 the pharmacokinetics of sparfloxacin were fitted to non-compartment model. In HFD, diabetic and glibenclamide treated diabetic animals, the maximum plasma concentration achieved by sparfloxacin gradually decreased in the respective order. The rate of absorption of sparfloxacin was maximum in diabetic rats than control rats which may be due to 11.02 fold and 15.9 fold change down-regulation of intestinal (colon) ABCB1a/mdr1a and ABCB1b/mdr1b genes expression. The rate absorption of sparfloxacin from the intestine was somehow decreased when glibenclamide was coadministered, even though there was 42.79 fold and 25.86 fold change down-regulation of intestinal (colon) ABCB1a/mdr1a and ABCB1b/mdr1b genes expression respectively. Probable reasons may be the pre-enterocytic interaction of glibenclamide with sparfloxacin as indicated by increased Tmax, decreased slope of absorption, decreased AUC (0-t) and AUC (0-∞). The maximum plasma concentration achieved by the drug is less in HFD rats group, diabetic rats group and glibenclamide treated diabetic rats group due to higher apparent volume of distribution. Rate of elimination in diabetic rats is faster, due to polyuria hence half-life of sparfloxacin is less in diabetic rat. However, in glibenclamide treated diabetic rats the half-life is increased due to slower rate of absorption of drug and correction of polyuria. In both diabetic rats and glibenclamide treated diabetic rats group, maximum plasma concentration achieved by sparfloxacin is decreased when compared to control rats. Sparfloxacin being an antibiotic, depend on its attained plasma concentration for its pharmacodynamic activity. From this it can be concluded that dose adjustment of sparfloxacin is required in diabetes and in glibenclamide treatment, to attain effective therapeutic concentration.ThesisItem Open Access MOLECULAR DETECTION AND CHARACTERISATION OF INFECTIOUS BURSAL DISEASE VIRUS (IBDV) IN POULTRY(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD, 2019-07-15) D., NANDHAKUMAR; R., RajasekharRecurrent infectious bursal disease (IBD) outbreaks were reported in different regions of Kerala, India. This study reports the molecular detection and comparative genetic analysis of the hypervariable region of the VP2 gene of IBD virus from the field outbreaks in Kerala. Genetic analysis of 22 isolates from 41 suspected field outbreaks of IBD were carried out in this study. In phylogenetic analysis, the obtained field isolates fall into genogroup 1 and 3. In genogroup 3, all vvIBDV isolates shared a common ancestor with other south Indian isolates but isolates 9/CVASP/IBDV, 10/CVASP/IBDV, 12/CVASP/IBDV, 14/CVASP/IBDV and 17/CVASP/IBDV are most recently evolved and is diverged from the south Indian isolates and other isolates obtained in the study. The amino acid sequence of 22 isolates were analysed, out of which 18 had serine rich heptapeptide virulence sequence ‘SWSASGS’ and conserved amino acids which are characteristic of vvIBDV. In all the vvIBDV isolates obtained in the study had phenylalanine and Valine at the position 240 and 294 respectively similar to recently evolved Indian IBDV isolates. But we observed T269A and S299N mutations in the isolate 6/CVASP/IBDV and is the first report of such mutations at this positions in India IBDV isolates. The isolate 11/CVASP/IBDV had a unique mutation of V225A which is not yet reported in IBDV isolates. Among these 18 isolates, 9 isolates obtained from vaccinated flocks. Two isolates (15/CVASP/IBDV and 18/CVASP/IBDV) were similar to intermediate plus vaccine strain. The isolates 8/CVASP/IBDV and 19/CVASP/IBDV had amino acids unique for the intermediate vaccine with mutations observed at H253Q and V256I in 19/CVASP/IBDV, T270A and novel mutation N279Y in isolate 8/CVASP/IBDV. These two isolates had non-virulent classical heptapeptide sequence ‘SWSARGS’ nevertheless they produce field outbreaks of IBD. This is the first genetic characterisation study of field IBDV isolates in Kerala, IndiaThesisItem Open Access DETECTION AND MOLECULAR CHARACTERISATION OF ROTAVIRUS OF PIGS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD, 2019-07-15) G, LOGESHWARAN; Ravishankar, ChintuPig farming is a source of income for small and marginal farmers in Kerala. One of the important health problems in suckling and recently weaned piglets is neonatal diarrhoea. Rotaviruses belonging to Group A are one of the most frequently detected viral agents associated with diarrhoea in swine. Though the incidence of rotaviruses in Kerala has been established, a thorough study of the agent with respect to genotypes of the virus has not been carried out. Hence this study was undertaken to detect the presence of rotavirus in faecal samples of piglets by reverse transcriptase polymerase chain reaction (RT-PCR) and to genotype the virus by nucleotide sequencing. A total of 100 diarrhoeic faecal samples were collected from piglets reared in organized farms in Wayanad, Kozhikode, Palakkad, Thrissur and Ernakulam districts of Kerala. All these faecal samples were screened for the presence of porcine rotavirus (PRV) by RT-PCR. Of the 100 diarrhoeic faecal samples, 12 (12 per cent) samples was found to be positive for VP6 gene as evidenced by a 309 bp amplicon. Positive samples were obtained from Palakkad and Wayanad districts only. On analysis of the nucleic acid sequence of VP6 gene, it was observed that majority of the viruses were of inner capsid type I5 and one was of I14 type. When the positive samples were tested by RT - PCR for VP7 and VP4 gene, 10 (83.33 per cent) and 11 (91.66 per cent) samples were positive in the first round RT-PCRs yielding amplicons of 1062 and 876 bp respectively. When representative samples were tested in the semi nested PCR for VP7 gene amplicons corresponding to G2, G4, G5, G6 and G9 genotypes were obtained. In the semi nested RT-PCR for VP4 gene, P[6], P[19] and P21-5 genotypes were detected. Some of the sequences showed close similarity to rotaviruses isolated from humans and from bovines. Of the G and P types detected, G2, G9 and P[6] have been reported in humans in Kerala. Hence the results of the study indicated that the rotaviruses of pigs in Kerala are genetically diverse.ThesisItem Open Access MOLECULAR DETECTION OF INFECTIOUS BRONCHITIS VIRUS IN CHICKEN(College of Veterinary and animal Science,Mannuthy, 2019) NIRANJANA S. RAJALAKSHMI; Surya SankarAvian infectious bronchitis, an acute highly contagious disease of chickens, affects birds of all age groups. The present study envisaged the isolation, identification and molecular characterisation of IBV. For the study, samples were collected from chickens of different ages with signs of respiratory tract infection. Out of the 95 samples collected, 42 were positive for IBV using specific UP and DOWN primers targeting the highly conserved 5’UTR region. The samples were inoculated into nine to eleven day-old embryonated chicken eggs via., allantoic cavity. Virus inoculated embryos after six passages exhibited the characteristic curling and dwarfing. The harvested allantoic fluid revealed the presence of IBV by RT-PCR in 42 samples. Reverse transcriptase polymerase chain reaction identified IBV in three more allantoic fluid. A Taqman probe based real time PCR was developed targeting the highly conserved 5’UTR and N genes which detected 45 positives in suspected tissue samples and allantoic fluid. The hypervariable regions 1, 2 and 3 of spike gene of all the positive samples were amplified by RT-PCR using three different primers and the respective amplicons were sequenced to study the genetic diversity of the isolates. The sequences of the isolates were compared with those of commercial vaccine strain (H120) and other Indian isolates available in NCBI GenBank database. All the sequences showed more than 95 per cent similarity with the commercial vaccine strain as well as with the Indian isolates from various regions. Phylogenetic tree was constructed based on the obtained sequences. On phylogenetic analysis of hypervariable regions 1 and 2, all the four Thrissur isolates clustered with each other and also with the IBV isolates from different regions. Whereas, on phylogenetic analysis of hypervariable region 3, all the four Thrissur isolates were branched separately among themselves and also with the IBV isolates from different regions of the country. Thus, a comprehensive analysis of these variations could aid in the selection of a suitable candidate vaccine strain.ThesisItem Open Access COMPARISON OF DIFFERENT MOLECULAR TYPING METHODS FOR CHARACTERISATION OF Riemerella anatipestifer ISOLATES(College of Veterinary and animal Science,Mannuthy, 2019) C. DEVIGASRI; Priya P. MThe pathogen causing septicaemia and infectious serositis in ducks and wide variety of wild birds, Riemerella anatipestifer has been categorised as a member of Flavobacteriaceae by 16S rRNA sequence analysis. At present, 21 serotypes have been characterised using serum agglutination test. So far, the molecular determinants for R. anatipestifer serotyping are not clear; moreover, serotype analysis is time-consuming and labour-intensive. To find a better molecular typing method for R. anatipestifer, serotype-associated genetic variations has to be determined. Hence, the 20 field isolates (maintained as lyophilised culture and also collected from the suspected cases reported to the Department of Veterinary Microbiology) were subjected to different molecular methods like Polymerase chain reaction- Restriction fragment length polymorphism (PCR-RFLP), Repetitive-sequence based PCR (REP- PCR), Internal transcribed spacer (ITS) nucleotide sequencing, Omp A gene sequencing and pulsed field gel electrophoresis (PFGE) to determine the genetic variations. The phylogenetic trees constructed based on the variations of the ITS region grouped the organism into four clusters and the amino acid sequence variation of OmpA region categorised it into two clusters. The Omp A sequence correlated much better with serotyping than ITS. By comparing the pattern based methods, REP-PCR and PCR-RFLP showed similarity with PFGE which distinguished the isolates into three pulsotypes, Although all methods displaying broadly similar discriminatory powers, REP-PCR subtyping proved to be a much easier, cheaper and more rapid method. To conclude many techniques are available but each of them has advantages and limitations which making them useful in some studies and restrictive in others. Hence to group the local isolates with universally categorised isolates, one or more of the different typing methods need to be applied in conjunction with conventional methods.ThesisItem Open Access DETECTION OF LEPTOSPIRA IN SEROREACTIVE GOATS BY ISOLATION AND POLYMERASE CHAIN REACTION(College of Veterinary and animal Science,Mannuthy, 2019) DHIVAHAR M; Ambily R.Leptospirosis is a worldwide zoonotic disease that in pigs primarily causes reproductive disturbances. Samples collected from Teaching Veterinary Clinical Complex (TVCC) and University Sheep and Goat Farm, CVAS, Mannuthy, Livestock Research Station (LRS), Thiruvizhamkunnu, District Veterinary Centres (DVC), Kannur and Malappuram as well as from selected private farms and households in five districts of Kerala viz., Kozhikode, Kannur, Malappuram, Palakkad and Thrissur. This included 250 serum samples collected from both healthy as well as goats with history of abortion belonging to different age groups were used for the present study. Serum samples (n=250) were tested using microscopic agglutination test (MAT) and an overall seropositivity of 10.80 per cent could be detected. The serogroup Pomona (24.32 per cent) was the most prevalent among the 27 positive samples followed by serogroups Australis (16.22 per cent), Autumnalis, Hebdomadis and Sejroe (10.81 per cent), Canicola (8.11 per cent), Bataviae, Javanica and Pyrogenes (5.41 per cent) and Grippotyphosa (2.70 per cent). The titre ranged from 1:100 to 1:400. Samples of whole blood (n=52), urine (n=26) and aborted foetus (n=2) when tested using PCR for the presence of lipl32 gene of Leptospira, and none found positive. Among these areas, Thrissur district showed the greater seroprevalence rate (16.67 per cent). The highest seroprevalence was recorded in aborted animals (25.81 percent) compared to healthy ones (5.85 per cent). Serovar Pomona found to be the most prevalent serovar among the aborted goats. Age wise seroprevalence was recorded and found goats with age group of > three years showed greater seroprevalence (12.41 per cent). On statistical analysis by Chi-square test, significant difference noticed between the district wise seroprevalence and between aborted and healthy goats and no significant difference noticed between the seroprevalence rate of age group of animals.ThesisItem Open Access COMPARATIVE EFFICACY OF LOOP-MEDIATED ISOTHERMAL AMPLIFICATION, REAL TIME POLYMERASE CHAIN REACTION AND IMMUNOCHROMATOGRAPHY FOR THE DETECTION OF Brucella IN BOVINES(College of Veterinary and animal Science,Mannuthy, 2019) RESHMA S.; Binu K. ManiThe present research was undertaken to compare the efficacy of LAMP and real time PCR with immunochromatography for diagnosis of brucellosis in bovines. One hundred and one samples were collected from University Livestock farms as well as from private cattle farms in and around Thrissur. The samples collected were aborted foetal internal organs and stomach contents, uterine discharges from aborted dams within two weeks after abortion and vaginal discharges from repeat breeder animals. All the samples were subjected to immunochromatography, LAMP and real time PCR. Among 101 samples tested, 8 (7.92 per cent) were found to be positive by immunochromatography; 19 (18.80 per cent) were found to be positive by real time PCR and 21 (20.79 per cent) were found to be positive by LAMP. However, LAMP showed a higher sensitivity (100 per cent) and specificity (86 per cent) in comparison to real time PCR (sensitivity 50 per cent and specificity 83.9 per cent) by taking immunochromatography as the standard test. More number of positive cases was reported from private farms compared to government farms when analysed by LAMP and real time PCR. Uterine discharges collected from aborted dams and the specimens from aborted foetus showed higher proportion of positive cases compared to vaginal discharges from repeat breeder animals.ThesisItem Open Access ISOLATION AND IDENTIFICATION OF BACTERIAL AGENTS ASSOCIATED WITH POLYARTHRITIS IN CALVES(College of Veterinary and animal Science,Mannuthy, 2019) NAIR ASWATHY; Priya P. MBlood and synovial fluid/wound swabs were collected from thirty one calves with polyarthritis presented to University Veterinary Hospitals, Mannuthy and Kokkalai, organised and unorganised farms of Thrissur district to identify the bacterial etiology. Out of 18 bacterial isolates obtained, 12 were Gram- positive and six were Gram-negative bacteria. Based on cultural, morphological and biochemical tests, they were identified as Staphylococcus aureus (38.9 per cent), S. epidermidis (11.1per cent), Streptococcus spp. (11.1 per cent), Corynebacterium jeikeium (5.6 per cent), Escherichia coli (5.6 per cent), Klebsiella spp. (5.6 per cent), Pseudomonas aeruginosa (5.6 per cent) and Salmonella spp. (5.6 per cent). Polymerase chain reaction (PCR) was standardised using designed primers for Corynebacterium spp. and Staphylococcus spp. for their confirmation. One and nine E. coli isolates were obtained from the wound and rectal swabs respectively. The identity of the E. coli isolated were confirmed by pho A based PCR. On screening against virulence genes like lt, fim H, k99, f41, stx1, stx2, eaeA, cnf1, cnf2, ehlyA, cdt III and f17. E. coli isolated from rectal swabs were positive for stx1, eae A, cnf1 and cdt III. Whereas all genes were negative for the E.coli isolated from wound swab. On genus specific PCR, none of the DNA extracted from the synovial fluids were positive for mycoplasmal infection. Gentamicin was found to be the most effective drug against the isolates obtained. The blood parameters revealed leukocytosis, thrombocytosis and granulocytosis. Increase in total protein and leucocytosis were observed on synovial fluid analysis.
