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ThesisItem Open Access SELECTION FOR EGG PRODUCTION IN NATIVE CHICKEN AND PERFORMANCE OF ITS CROSSBREDS WITH WHITE LEGHORN(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2019-12-30) HARIKRISHNAN SA study was conducted at All India Co-ordinated Research Project (AICRP) on Poultry for Eggs, Mannuthy centre, to evaluate the phenotypic and production characteristics of native chicken of Kerala and to improve its egg production through selection. From the base generation (G0) of native chicken, 775 female and 200 male native chicken (G1) were produced through a pedigreed hatch and the pullets were evaluated till 40 weeks of age for their production performance. Based on egg number 40, selection was carried out in the population using Osborne’s index and 300 dams and 50 sires were selected for producing G2 generation through a pedigreed hatch. The pullets were evaluated for their production performance till 40 weeks of age. Heritability and correlation estimates were also worked out for egg production and various correlated traits of native chicken in both G1 and G2 generations. The native chicken of Kerala had a hen housed production of 69.83 eggs, hen day egg production of 70.72 and survivor’s egg production of 70.97. Based on the selection for egg number 40 in G1 generation of native chicken, the hen day egg production (4.56 eggs) and survivor’s egg production (5.90 eggs) was significantly (p<0.05) improved but the improvement in hen housed egg production was not evident due to higher mortality occurred in G2 generation as a result of incidence of neoplastic disease of infectious origin in the flock. However, a positive phenotypic response of 2.26 eggs was obtained on hen housed basis as a result of selection for egg number 40 in G1 generation. The age at sexual maturity of native chicken was significantly (p<0.05) improved in G2 generation. Improvement was noticed in the clutch size of the birds in G2 generation while per cent broodiness was reduced as a result of selection for egg number 40. The majority of egg shell colour noticed in native chicken of Kerala was tinted followed by medium brown, white and light brown. The performance of egg quality, fertility and hatchability percentage were comparable in both generations. The sire+dam component of heritability (h²s+d ) for ASM was 0.464 in G1 generation and 0.238 in G2 generation. For the trait egg number at 40 weeks of age, the values observed were 0.364 and 0.218 in G1 and G2 generation, respectively. The realised heritability worked out was 0.19. The h²s+d estimates for egg weight in G1 and G2 generation had no much variation among generation, consequent to selection. The phenotypic correlation (rp) between body weight 16 and egg number 40 was of low magnitude while egg number and egg weights were nearing zero. The rp between ASM and egg number was negative. Genetic correlation (rg) between body weight 16 and egg number, between egg weight 28 and egg weight 40 were positive with high magnitude while ASM with egg number was strong negative. The rg between egg number and egg weights was not significant. Upon estimating genetic correlation, it was evident that correlation between most of the traits was higher in G1.The average effective selection differential for egg number 40 in the generation was 12.03 and the selection intensity was 0.45. The genetic parameters and phenotypic response for egg production and various correlated traits revealed that there is further scope for selection in native chicken of Kerala to improve its egg production. The study was also aimed at evaluating the production performances of the selected native chicken in G1 and G2 generation with improved ‘N’ strain of White Leghorn (IWN). Based on the results of crossbreds (Native x IWN), significantly (p<0.05) higher number of eggs with early sexual maturity in birds was observed for the progeny of the birds with IWN as sire and native chicken as dam (ND) than its reciprocal cross (DN). The performance of the crossbreds with respect to egg weight and egg quality traits was comparable. The feed intake was higher for ND birds compared to DN, while livability, broodiness and presence of fawn colour plumage was higher for DN birds compared to ND. Based on the study of the crossbreds, it could be observed that ND birds were better in egg production while DN birds were better in terms of livability, broodiness and plumage. However, field trials have to be conducted to confirm the present results under backyard conditions.ThesisItem Open Access DEVELOPMENT AND EVALUATION OF FUNCTIONAL CHICKEN NOODLES(COLLEGE OF VETERINARY & ANIMAL SCIENCES MANNUTHY, THRISSUR, 2019-12-30) PAVAN. M; Sathu TThe rapid expansion of knowledge among the people about the influence of food on well-being and health, increased the demand for functional foods. Now the people in the developed and developing countries are demanding the food items which have beneficial and positive psychological effects and that are convenient to use. Chicken meat products have wider acceptability by the consumer because of good nutritional and flavour profiles. A functional meat product can be made by means of technological approaches like incorporating functional food ingredients like, addition of natural plant extracts, natural colours, natural antioxidants, bioactive compounds during product development etc. The dietary fibers, natural antioxidants and natural colours used as functional food ingredients are shown to have anticarcinogenic, antimutagenic, antidiabetic and antistress activity (Arhiara, 2006). The current study was conducted in Department of Livestock Products Technology to develop Functional Chicken Noodles by incorporating natural antioxidant and natural colour and its physico-chemical, nutritional, sensory attributes, microbiological qualities and upto 60th day of storage under ambient temprature. The standardized functional noodles and control stored in laminated pouches under ambient temperature were analyzed for physico-chemical, water hydration properties, colour parameters, Hunter L*, a*, b* values, Thio-barbituric Acid Reactive Substances (TBARS), Tyrosine Value (TV), DPPH (2,2- diphenylpicrylhydrazyl) assay, Total Phenolics (TP), microbiological qualities and sensory attributes on 0, 15th, 30th ,40th, 50th and 60th days of shelf life. pH was significantly (p<0.001) higher for control noodles than SFCN on all the storage days and there was no significant difference across the storage days for both. There was no significant difference in the TBARS and TV across the storage days, but the values were relatively higher for SFCN than control. However, the physicochemical parameters were same as it was in the preliminary experiments. There was no significant increase in the total viable counts of the noodles and the yeast and mold growth was not detected upto 60th day of storage study. The sensory evaluation revealed no significant changes in most of the sensory attributes along the storage days proving that the product was shelf stable even on 60th day of storage. The sensory scores did not show any significant difference when it was compared to the commercially available chicken noodles. The cost of production per kilogram of chicken noodles was Rs 280.89 and that of control noodles was Rs 184.39. From the above studies it can be inferred that the instant chicken noodles with natural antioxidant aloe vera and paprika oleoresin can be prepared and marketed at ambient temperature in laminated pouches for minimum 60 days with good nutritional and sensory properties. This nutrient rich noodle will be a good source of instant food for children, teenagers, sport persons, pregnant and lactating women.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 DEVELOPMENT AND EVALUATION OF RECOMBINANT PROTEIN BASED ELISA FOR THE DIAGNOSIS OF INTESTINAL SCHISTOSOMOSIS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2019-12-30) PRIYA M. N.; Bindu LakshmananAnimal schistosomosis caused by Schistosoma spindale is an economically considerable blood fluke infection that adversely affects the livestock sector in India. The aim of the present study was to clone, express, purify and analyse 22.6 kDa tegument protein of S. spindale (rSs22.6 kDa) in prokaryotic system and to assess its usefulness as a diagnostic candidate for seroprevalence studies. An abattoir survey of intestinal schistosomosis in bovines conducted in Thrissur corporation slaughter house, Kuriachira from August, 2017 to July, 2018 revealed an overall prevalence of 25 per cent of intestinal schistosomosis in cattle with 24.34 per cent of S. spindale and 0.66 per cent of S. indicum infection. The highest prevalence of infection (26.32 per cent) was observed during monsoon season. Majority of the samples showed low intensity infections (81.57 per cent). Occurrence of the disease or the intensity of the infection related with seasons did not show any statistical significance. For the production of recombinant 22.6 kDa protein, RNA was isolated from adult live schistosome worms, cDNA synthesized and the specified 22.6 kDa tegument protein coding gene was amplified and cloned in pJET cloning vector and transformed in E. coli Top 10 cells. The confirmed product (573bp) was amplified with primers having restriction site for BbS1 and digested with BbS1 restriction enzymes. Then pET28b expression vector was digested with Xho1 and NCo1 restriction enzymes and transformed to BL21 E. coli cells. Induction of protein was done 0.6mM IPTG for four hours. Purification of the soluble fraction of newly expressed polyhistidine (6X-His) tagged fusion protein was carried out by Nickel chelating affinity chromatography using Ni-NTA agarose column. The SDS PAGE analysis of rSs22.6 protein and the following Coomassie Brilliant Blue staining of the gel revealed a bright single band of size approximately 22.6 kDa. Moreover, immunoblotting of rSs22.6 protein with schistosome positive bovine serum revealed a single immunodominant protein corresponding to the approximate molecular weight of 22.6 kDa without any cross reaction with amphistome positive bovine serum. Standardisation of IgM and IgG based Dot and indirect ELISA was carried with rSs22.6 protein. Excretory secretory antigens (ESA) were also prepared using the mesentery recovered adult schistosomes to conduct ESA based indirect IgG ELISA. Copro PCR was carried out with mitochondrial gene specific primers of Schistosoma spp. and an amplicon of approximately 454 bp size was amplified indicating the presence of S. spindale. Comparison of results of rSs22.6 IgM ELISA with that of copro PCR revealed a sensitivity of 16.67 per cent while that of copro PCR was 30 per cent with a cent percent specificity. The results showed that the sensitivity of rSs22.6 IgM ELISA was lower than that of copro PCR, suggesting unsuitability of rSs22.6 IgM indirect ELISA for seroprevalence studies. The diagnostic performance of rSs22.6 IgG ELISA was compared with ESA IgG ELISA and copro PCR using 38 known positive samples and 13 known negative samples. A sensitivity of 92.11 per cent for rSs22.6 IgG ELISA, 89.47 per cent for ESA IgG ELISA and 31.58 per cent for copro PCR was observed whereas specificity was cent per cent for all the three assays. Sensitivity of rSs22.6 IgG ELISA higher than ESA IgG ELISA. Sensitivity of copro PCR was considerably low. However, there was no statistically significant difference between the sensitivities of rSs22.6 IgG ELISA and ESA IgG ELISA while both these assays showed statistically significant difference from copro PCR. Seroprevalence study with rSs22.6 IgG ELISA of 506 sera samples of cattle from southern, central and northern zones of Kerala revealed a prevalence status of 26.88 per cent of intestinal schistosomosis. Highest prevalence of the infection was observed in Alappuzha district (42.86 per cent) and coastal sandy zone (41.18 per cent) whereas the lowest prevalence was in Ernakulam (17.19 per cent) district and Red loam (15.63 per cent) zone of Kerala without any statistically significant difference between these findings. In silico analysis of the expressed protein revealed that it is a protein with 190 amino acids. Predicted secondary structure of protein revealed the presence of alpha helices (47.89 per cent), extended strands (17.37 per cent), beta turns (8.95 per cent) and random coils (25.79 per cent). The tertiary structure of the protein revealed that it contained α helices and β sheets with EF-hand as a helix-loop- helix domain. Analysis of rSs22.6 protein showed that signal peptides were absent. Presence of transmembrane helices in the predicted protein sequence indicated the absence of transmembrane helices. The NCBI conserved domain prediction revealed the presence of two conserved domains, one EF-hand domain located in its N-terminus (residues 12–71) and a dynein light-chain domain located in its C-terminus (residues 99–186). The possible number and composition of epitopes were predicted by linear epitope prediction in Immune Epitope Database and Analysis Resource tool revealed the presence of seven epitopes with aminoacid sequences ranging from 3 to 20. Phylogenetic analysis using the Maximum Likelihood method in MEGA 5.2. revealed that it was a sister clade of S. haematobium and S. bovis while it is distinct from the clade containing S. japonicum.
