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ThesisItem Open Access ISOLATION AND MOLECULAR CHARACTERIZATION OF ANTIMICROBIAL RESISTANT ESCHERICHIA COLI IN PORK PRODUCTION CHAIN IN HASSAN DISTRICT OF KARNATAKA(SRI VENKATESWARA VETERINARY UNIVERSITY, TIRUPATI - 517 502. (A.P.) INDIA, 2023-06) SUDARSHAN .S; ESWARA RAO .B (MAJOR); WILFRED RUBAN .S; NAGA MALLIKA .E; SRINIVASA RAO .T; NARENDRA NATH .DThe present investigation was carried out to evaluate the occurrence of AMR E. coli in pork production chain in and around Hassan. Initial survey indicated that majority of the pig farmers were marginal farmers (65%) and kitchen / hotel waste was the major source of feed. The major antibiotics used were enrofloxacin followed by streptopenicillin, tetracycline, fortified procaine penicillin sulphonamides (Co-trimazole) and antibiotics were administered by themselves (92%). A total of 280 samples (40 each from piglets, weaners and adults, 20 feed, 30 water and 20 hand swabs from 10 pig farms and 20 each from meat samples, cutting board, hand swabs and knives swab and 10 water sample from retail pork outlets ) were screened and 227 E. coli isolates were used for further characterization. The average E. coli count ranged from 6.104 to 6.464 log10cfu/g. The overall occurrence of E. coli from various samples from pig production system was 67.89 per cent (129/190). The highest per cent of AMR E. coli counts as proportion of total E. coli was observed in human hand swabs (83.90%) followed by water trough (30.56%) and piglets (22.06%) samples as compared to other samples in this study. The overall prevalence of tetracycline, fluoroquinolone, ESBL resistant and colistin resistant E. coli in the entire pork production chain irrespective of the samples was 52.11, 37.37, 17.89 and 13.16 per cent, respectively. Of the 208 isolates, 191 isolates (91.83%) carried either one or the other gene, whereas, only 8.17 per cent of the isolates did not harbor any of the genes screened. Majority of the isolates carried tetA (68.38%), followed by blaTEM (55.77%), qnrS (44.23%), and none of the isolates carried colistin resistance gene (mcr1 to mcr5). Antimicrobial susceptibility testing revealed that majority of the isolates showed resistance to tetracycline (86.96-100%) followed by extended-spectrum b lactamase inhibitors and fluoroquinolones and complete sensitivity to carbapenem followed by aminoglycosides. It was observed that 96.92 per cent of E. coli isolates were MDR. The occurrence of ESBL and colistin resistant E. coli was 17.89 and 0.714 per cent per cent, respectively.ThesisItem Open Access IDENTIFICATION OF SINGLE NUCLEOTIDE POLYMORPHISMS AFFECTING SEMEN QUALITY TRAITS IN AI BULLS OF ANDHRA PRADESH(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2023-01) DEEPTHI, CHANDAKA; VINOO, R(MAJOR); SUDHAKAR, K; NARENDRA NATH, D; ASWANI KUMAR, KBull fertility is an important factor for improving the economic value of the herd as the single bull can be used to inseminate many cows using artificial insemination. The bull fertility is measured in terms of semen quality traits like sperm motility and plasma membrane integrity of semen. The scrotal circumference of the bulls influences the semen quality in terms of sperm concentration and percentage of normal sperms of bulls ejaculate. These traits were influenced by both genetic and non-genetic factors. The genes MAP3K1, SPEF2 and PLCZ1 influencing the semen quality traits like scrotal circumference, sperm motility and plasma membrane integrity of semen were identified based on GWAS through Chilibot and Connected paper web-based bioinformatic tools. The SNPs rs463712269 (MAP3K1) and rs722354121 (SPEF2) were identified by screening these genes for missense mutations using Ensembl database and their RFLP pattern was verified using NEBcutterV2.0 software. For PLCZ1 gene, the perusal of literature revealed the presence of rs208019489 affecting the plasma membrane integrity of semen. A total of five frozen semen straws per bull and the relevant data were collected from 239 bulls of different breeds residing in FSBS of Andhra Pradesh. A suitable DNA isolation protocol viz., modified high salt method was selected through comparison of five different DNA isolation procedures in frozen semen. PCR-RFLP polymorphisms were examined in each group by using CviQI, HpyCH4V, and AvaII restriction enzymes for digestion of PCR products in MAP3K1, SPEF2 and PLCZ1 genes respectively. Association of MAP3K1, SPEF2 and PLCZ1 genotypes on scrotal circumference, sperm motility and plasma membrane integrity respectively were worked out using General linear model of SPSS Statistics base 26. The MAP3K1/CviQ1 PCR-RFLP assay revealed that the T allele frequency was more in Ongole population while in other cattle breeds C allele frequency was more. The highest homozygosity values were observed in Jersey and a reversal of this was observed in Ongole and HF population. Negative FIS values in the studied population are indicative of heterozygous excess due to outcrossing. In Murrah, the T allele frequency was more wiaaaaaaaaath effective number of allele of 1.13 and heterozygosity excess at this locus. Fixation of T allele was observed in both the exotic pure breeds (HF and Jersey) and presence of C allele was almost insignificant in cattle SPEF2/ HpyCH4V assay. The low selective pressure on SPEF2 locus was observed and the negative FIS values were observed in the cattle groups studied. In Murrah, the abundance of heterozygotes was observed indicating the selective advantage of C allele in this species and they deviated from the Hardy Weinberg equilibrium suggesting a high selection pressure on this locus. The GG (0.64) genotype frequency was higher than the CG (0.22) in Ongole cattle in PLCZ1/AvaII assay. Except in HF crossbred none off the other cattle genetic groups were with CC genotype which could be an inadvertent result of crossbreeding in HF crossbred. The HF crossbred and indigenous Ongole cattle (P<0.01) were not in agreement with the Hardy Weinberg equilibrium. In Murrah buffaloes, the near fixation of G allele (0.98) was observed accounting for the selection. The presence of low genetic diversity and heterozygosity excess in the Murrah population at this locus was found. Association of MAP3K1/CviQ1 and SPEF2/HpyCH4V polymorphisms had no significant influence on scrotal circumference and pre-thaw and post-thaw sperm motility respectively in both cattle and buffaloes. The PLCZ1/AvaII genotypes have significant influence on the plasma membrane integrity of semen in Holstein Freisiean, Jersey (P<0.05) and Ongole bulls (P<0.01). The plasma membrane integrity of semen was reported to be high in heterozygotes (CG) of Jersey and Ongole cattle. In Murrah, the genotypes have no significant effect on plasma membrane integrity of semen. The present study, conclude the importance of PLCZ1 gene as a marker for semen quality assessment and selection in bulls, owing to the association of PLCZ1 polymorphisms with plasma membrane integrity of bulls. Identification of few more SNPs in the vicinity of the verified SNPs suggests the importance of investigations aiming at screening for further casual mutation responsible for the variation of these traits with higher number of bulls.ThesisItem Open Access DEVELOPMENT OF PACKAGING FILMS EMBEDDED WITH DIFFERENT NANO PARTICLES TO ENHANCE THE SHELLIFE OF PORK(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2022-05) NAVEEN, ZILLABATHULA; ESWARA RAO, B(MAJOR); NAGA MALLIKA, E; SRINIVASA RAO, T; NARENDRA NATH, D; PRASAD, T.N.V.K.VIn the present study an attempt was made to develop, standardize and evaluate packaging films embedded with different nanoparticles to enhance the shelf life of pork. Nanoparticulates of Zinc Oxide and Silver were synthesized by sol-gel method. The characterization of Nanoparticles was done by Dynamic Light Scattering technique, Fourier Transform Infrared spectroscopic analysis, X-Ray Diffraction analysis, Scanning Electron Microscopy analysis and Energy Dispersive X-Ray Spectrometer analysis. Nanoparticles were embedded in LDPE base, at different levels to enhance the shelf life of pork. Blown films were prepared with ZnO, Silver and ZnO + Silver nanoparticles at three different levels of 0.5%, 1.0%, 1.5% each. The packaging films embedded with three different combinations of NPs at three different levels each, were further evaluated for physical strength and antimicrobial properties. The film characteristics, like tensile strength, tear resistance, per cent elongation at break, bursting strength, puncture strength, fragmentation tests, antimicrobial activity and water sorption kinetics were evaluated. Fourier Transform Infrared spectroscopic analysis, X-Ray Diffraction analysis, Scanning Electron Microscopy analysis and Energy Dispersive X-Ray Spectrometer analysis of packaging films embedded with three different NPs were also evaluated. The Overall Mean ± SE tensile strength, tear resistance, per cent elongation at break, bursting strength, puncture strength, fragmentation of packaging films embedded with ZnO NPs at 1.0 % level was significantly (P<0.05) higher compared to packaging films embedded with other NPs at different levels. The anti-microbial activity, measured as diameter of inhibition zones (mm) of Silver NPs embedded packaging films were significantly superior (P<0.05) compared to all other levels of each NP. Based on the evaluation of film characteristics, the packaging film embedded with ZnO nanoparticles at 1.0% level was statistically selected and was evaluated for its efficacy to enhance the shelf-life of pork patties under refrigerated (4 ± 1°C) storage. pH, 2- TBARS values, Tyrosine values and Microbial counts of pork packaged in packaging film embedded with ZnO nanoparticles at 1.0% level were significantly (P<0.05) lower than those of pork packed in control film, while the sensory quality of pork packaged in packaging film embedded with ZnO nanoparticles at 1.0% level was significantly (P<0.05) higher than those in control film. The pork packaged in control film was acceptable up to ten days and in packaging film embedded with ZnO nanoparticles at 1.0% level were acceptable up to 12 days of refrigerated storage.ThesisItem Open Access SELECTIVE INDICATORS FOR DIAGNOSIS AND ASSESSMENT OF THERAPEUTIC EFFICACY FOR ENDOMETRITIS IN GRADED MURRAH BUFFALOES (Bubalus bubalis)(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2022-06) THANGAMANI, A; SRINIVAS, M(MAJOR); MUTHA RAO, M; SREENU, MAKKENA; ASWANI KUMAR, K; CHANDRASHEKAR REDDY, KThe present research was conducted to identify the selective indicators for diagnosis of endometritis and to evaluate the efficacy of intrauterine immunomodulators (E.coli LPS, Oyster glycogen and autologous Platelet-rich plasma) for the treatment of endometritis (subclinical and clinical) in graded Murrah buffaloes. The characterization of repeat breeding based on affections of reproductive tract in buffaloes (n=130), revealed that most predominant cause of uterine affections was endometritis with an occurrence of 89.80 per cent (88/98). Occurrence of subclinical endometritis based on per rectal examination, appearance of uterine discharge (score), pH, white side test, leucocyte esterase strip test, optical density and uterine discharge cytology (flushing) was 82.95, 63.34, 72.73, 65.91, 78.41, 75.00 and 67.04 per cent, respectively. While, occurrence of clinical endometritis was 17.05, 36.36, 27.27, 34.09, 21.59, 25.00 and 32.95 per cent, respectively. Uterine discharge cytology (flushing technique) by Leishman’s stain and Field’s stain could be effectively used under field conditions for staining of uterine discharge smears. The mean levels of physical characteristics and analytical parameters were significantly (P≤0.05) higher in CE as compared to SCE affected and normal healthy buffaloes. However, the mean levels of physical characteristics and analytical parameters were non-significantly (P>0.05) higher in the uterine discharge of SCE than normal healthy buffaloes. The haematological changes in whole blood sample revealed no significant (P>0.05) variations in the mean Hb (gm%) and PCV (%) concentrations of normal, endometric buffaloes. While, the mean neutrophil and lymphocyte counts (%) were significantly (P≤0.01) higher in endometritis affected buffaloes than normal healthy buffaloes. However, there was no significant difference between the SCE and CE affected buffaloes. Biochemical parameters like, Urea, cholesterol, alkaline phosphatase, creatine kinase and zinc had a highly significant (P≤0.01) difference between normal and endometritis affected buffaloes. Uterine discharge of buffaloes with greater than 39.78 mg/dl, 144.79 IU/L and 98.12 IU/L of threshold concentrations in urea, ALP and CK, respectively were at a higher risk to be affected with SCE. While, uterine discharges in buffaloes with less than 50.90 mg/dl and 141.27 μg/dl of threshold concentrations in cholesterol and Zn, respectively were at a greater risk to be affected with SCE. Differentially expressed protein profiles of uterine discharge that the mean concentrations of Heat shock protein (HSP-70) and C-reactive protein (CRP) were significantly (P≤0.01) higher in endometritis affected buffaloes than normal healthy buffaloes. However, there was a non-significant (P≥0.05) variation in HSP-70 (ng/ml) between CE and SCE affected buffaloes. On the other hand CRP (mg/L) concentration were significantly (P≤0.05) higher in CE than SCE and it was showed CRP levels an increasing trend with the increase in severity of uterine infections. The differentially expressed protein profile by SDS-PAGE analysis showed a total of 2 different protein bands were recorded with molecular weights ranging between 60 to 70 kilodaltons (kDa) in uterine discharge of normal buffaloes at the time of estrus, whereas 4 different protein bands with molecular weights ranging between 50 to 120 kilodaltons (kDa) in uterine discharge of SCE affected buffaloes. Relative quantification of pro-inflammatory cytokines gene expression (Toll like receptor-4) revealed that buffaloes with SCE showed 1.14 folds up-regulation of TLR-4 mRNA compared to normal buffaloes without SCE. In the present study, intrauterine administration of E.coli LPS (100μg) and Platelet rich plasma (autologous) yielded promising recovery (90.00% and 80.00%) and conception rates (77.77% and 68.75%) in endometritis affected buffaloes as compared to Oyster glycogen treatment (65.00% and 46.15%). In conclusion, therapy with E.coli LPS and Platelet rich plasma (autologous) could be effectively used as an alternative to the traditional use of antibiotics and other antimicrobial agents with better efficacy to resolve the uterine infection (both SCE and CE) by promoting uterine defense mechanism. While, PRP enriched the endometrial layers with the growth factors required for implantation and development of embryo and cured endometritis by its anti-inflammatory properties.ThesisItem Open Access HISTOPATHOLOGICAL EVALUATION AND IMMUNOHISTOCHEMICAL DETECTION OF CERTAIN TUMOR MARKERS OF CANINE MAMMARY TUMORS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2020-10) SUDHA RANI CHOWDARY, CH.; RAMA DEVI, V (MAJOR); SATHEESH, K; RAVI KUMAR, P; SUDHAKAR, KThe present study was undertaken to know the occurrence of canine mammary tumors (CMTs), to classify and study their gross and microscopic lesions, evaluate different tumor markers by IHC and to determine the variations of p53 gene in CMTs. In the present study, the animals with mammary tumors ranged from 1.5 to 14 years of age with highest occurrence in 9-10 years age group and noticed only in female dogs. Highest occurrence of canine mammary tumors was recorded in non-descript dogs followed by Spitz and German Shepherd breeds. The occurrence was highest in intact bitches when compared to spayed dogs. Out of 72 cases, 47 cases had a single mammary gland affected whereas 25 cases had tumors in multiple glands. The highest occurrence was recorded in the inguinal (50.49%), followed by caudal abdominal (33.98 %) glands. Majority of the cases showed TNM stage III tumors (59.42%), followed by stage II (21.74%), stage IV (8.7%), stage V (7.25%) and stage I tumors (2.90%). Overall, the cytological diagnosis had 75% correlation to histopathological diagnosis. The cytologic diagnosis correlated with the histologic diagnosis for benign and malignant tumors in 60 and 79.59% of cases respectively. Grossly, the size of the tumors ranged from 2 to 19 cm in greatest diameter with different shapes and consistencies. Sixty three cases (87.5%) had malignant tumors and 9 cases (12.5%) showed benign tumors. Histologically, they were classified into 20 major subtypes and among them, solid carcinoma had the highest frequency (20.6%), followed by complex carcinoma (15.9%), intraductal papillary carcinoma (14.3%). In the present study, metastasis of CMTs to lymphatic vessels, lymph nodes and visceral organs was recorded in 13 cases (18.1%). Maximum number of carcinomas were of grade II (46.7%) followed by grade III (40%) and grade I category (13.3%). The mean AgNOR number per cell ranged from 1.1 to 6.7 and was higher (4.58) in malignant tumors than in benign tumors (1.4). Of the 72 CMTs subjected to immunostaining of different tumor markers, 38.9% of tumors showed Ki-67 expression, 37.5% and 25% of CMTs showed ERα and PR expression respectively, whereas 89.1%, 25% and 16.7 % of the cases showed VEGF, survivin and p53 expression respectively. Of the 20 CMTs subjected to molecular studies, three mutations in exon 4 were detected in two tumor tissues which included two missense mutations in a complex carcinoma and a silent mutation in a carcinosarcoma. However, concordance of p53 phenotype and p53 genotype was found only in one case which had a silent mutation. On Kaplan - Meier analysis, early TNM stage, positive expression of ERα and PR, negative expression of p53 and survivin were found to be associated with longer overall survival. However, only TNM stage and PR expression were found to be independent prognostic factors on multivariate analysis. In the present study, various factors such as tumor stage, histological grade and features, expression of different tumor markers and p53 gene variations along with epidemiological data were evaluated in CMTs. It was revealed that the factors like TNM stage and PR expression have significant prognostic value and can influence the clinical outcome of canine mammary tumors.ThesisItem Open Access IDENTIFICATION OF POLYMORPHISM AND EPIGENETIC MODIFICATION OF PARTIAL PROMOTER OF ACACB GENE VIS-A-VIS EFFECT OF POLYMORPHISM ON EGG PRODUCTION AND QUALITY TRAITS IN LAYERS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2021-03) SHIVAPRASAD, C.H; Vinoo, R (MAJOR); Chatterjee, R.N; Muralidhar, M; Narendranath, D; Aswani kumar, KPoultry eggs are rich source of inexpensive, high-quality protein, vitamins namely A, B2, B6, B12, D, E and K, minerals like zinc, iron selenium, and copper etc. Egg yolk contains more calories and fat. In the recent past, there is demand for consumption of food sources of poly unsaturated fatty acids like dodecanoic acid, nonadecanoic acid, myristoleic acid, alpha linoleic acid and omega-3 fatty acids. The fatty acid composition depends on type of diet and genetic composition. Among the candidate genes, the acetyl-coenzyme A carboxylase beta (ACACB) is a candidate gene that can influence fatty acid composition. Perusal of literature revealed that the identification of partial promoter of ACACB gene was not done in chicken. Hence the present study is aimed at identification of partial promoter region, genetic polymorphism, gene expression and epigenetic regulation of ACACB gene in chicken. Further, the study also aimed at unraveling the association of the genetic polymorphism with body weight, egg production and egg quality traits in the two-layer lines, IWI and IWK. The study could successfully predict the minimal promoter region which is in the upstream 0.5 kb region. The region included certain transcription factors like CEBP, COUP, HNF4, PPARA, SREBP1, and T3R which are known to be involved in lipid metabolism. The minimal promoter region could potentially translate the gene in chicken embryo fibroblasts. Further, the minimal promoter region is polymorphic with 12 haplotypes and 18 haplogroups based on SSCP analysis. The haplotype h5 is least frequent in both IWI and IWK lines while h11 haplotype is most frequent in IWI line and h4 haplotype is the most frequent one in IWK line. Sequencing of the haplotypes revealed indels and substitutes in these haplotypes. The h8h8 haplogroup is associated with higher body weight at 8th, 16th and 20th week in IWI line. The haplogroup h12h12 is strongly associated with higher egg production in IWI line up to 52 weeks of age. The egg weight didn’t vary significantly within the line in any of the age except h1h2 haplogroup in IWI line at 72nd week, which is associated with higher egg weight. The shell weight is not particularly associated with any of the haplotypes in IWI line, but within IWK line, the h6h6, h9h9 haplogroups showed higher shell weight. The h9h9 and h9h10 haplogroups are associated with higher shell weight in IWK line compared to IWI line. The total egg fat% is significantly higher within IWI line, when they have h10h10 haplogroup. The same haplogroup also showed higher total fat% in IWI line compared to IWK line. There is no significant association is observed between haplogroups and HDL concentration in IWI line. But, in IWK line, the h8h8 haplogroup is associated with high serum HDL cholesterol concentration. Temporal expression pattern of the ACACB gene indicated that the gene expresses in all stages in the skeletal muscle of chicken during pre and post-hatch period. Epigenetic profile of the minimal promoter indicated that there are seven CpG islands in this region. There is negative relationship between highest and lowest expressed birds with the percent methylation in both the genetic groups. The present study suggests that the ACACB gene plays a crucial role in pre and post- hatch period in IWI and IWK lines. Further studies with spatial expression pattern of the gene and experiments aiming at precise functional analysis would provide additional insights into the role of ACACB gene in chicken in providing healthy eggs and meat.ThesisItem Open Access PATHOLOGICAL STUDIES AND MOLECULAR DETECTION OF RESPIRATORY PATHOGENS ASSOCIATED WITH RESPIRATORY DISEASE COMPLEX IN COMMERCIAL CHICKEN(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2021-03) CHANDRAVATHI, T; RAMA DEVI, V (MAJOR); SATHEESH, K; RAVI KUMAR, P; SUDHAKAR, KThe present study was planned to study the incidence and pathology of common respiratory diseases of commercial chicken and to carry out the molecular detection of pathogens associated with respiratory disease complex. A total of 639 birds with a history of suspected respiratory diseases were procured from various parts of Andhra Pradesh and representative samples were collected and subjected to pathological and molecular studies. Based on these studies, respiratory diseases were identified in 488 birds, accounting to an overall incidence of 76.36%. The major pathogens involved in respiratory disease complex in the present study were Newcastle disease virus (NDV), Infectious bronchitis virus (IBV), Fowl adenovirus (FAdV), M.gallisepticum (MG), M.synoviae (MS), Avibacterium paragallinarum, Pasteurella multocida and avian pathogenic E.coli (APEC). LPAIV and ILTV were not detected. Of these, respiratory disease caused by a single pathogen was identified in 136 (27.87%) cases and respiratory disease complex associated with multiple pathogens was identified in 352 (72.13%) of cases. xxxii Out of 488 cases, a total of 8 pathogens were detected either as a single infectious agent or as concurrent infections that resulted in respiratory disease complex. Among the single pathogen identified, NDV was detected in 159 cases (32.58%), followed by IBV in 72 cases (14.75%), FAdV infection in 31 cases (6.35%), M.gallisepticum in 190 cases (38.93%), M.synoviae in 108 cases (22.13%), A.paragallinarum in 14 cases (2.87%), P.multocida in 65 cases (13.33%) and avian pathogenic E.coli (APEC) in 308 cases (63.11%) occurring either as a single infectious agent or among concurrent infections. Among the 136 cases caused by a single infectious agent, NDV was detected in 32 cases (6.56%), followed by IBV in 20 cases (4.10%), FAdV in 8 cases (1.64%), M.gallisepticum in 18 cases (3.69%), M.synoviae in 2 cases (0.41%), A.paragallinarum in 12 cases (2.46%), P.multocida in 25 cases (5.12%) and APEC in 19 cases (3.89%). In case of concurrent infections, two to four pathogens were commonly involved in a single case and recorded in 72.13% of cases. Concurrent infection of two pathogens was recorded in 258 cases (52.87%) and three pathogens were recorded in 81 cases (16.60%). Isolation of quadruple agents involving NDV, MG, MS and APEC were identified in 13 cases (2.66%) of respiratory disease complex. Avian pathogenic E.coli was the most common organism involved in the concurrent infection in 289 (59.22%) cases. In the present study, the clinical signs, gross and histopathological findings were almost similar in the respiratory diseases and it was difficult to diagnose the diseases based on the pathological studies alone especially when multiple agents were involved in a single case. Hence, molecular techniques were employed for confirmation of various respiratory pathogens. Birds affected with different respiratory diseases showed signs of dullness, nasal discharge, gasping, coughing, open-mouth breathing, respiratory rales, dyspnoea, oedema of the face and eyelids, conjunctivitis, cyanotic combs and wattles, soiled vent and diarrhoea. Some birds also manifested paralysis and torticollis. A drop in egg production and egg quality in the flocks were recorded. Grossly, in cases of respiratory disease complex caused by multiple etiological agents, the lesions recorded included congestion, haemorrhages and catarrhal to caseous exudates in trachea, thickened and cloudy air sacs with fibrinous to caseous exudates and edema, congestion and consolidation of lungs. Further, proventricular haemorrhages, haemorrhagic and necrotic intestines and caecal tonsils, pericarditis, perihepatitis, necrotic foci on liver, swollen and congested kidneys, congestion of ovaries, and egg peritonitis were noticed. The prominent histopathological lesions in cases of respiratory disease complex observed were haemorrhagic tracheitis, oedema, pulmonary congestion and pneumonia, fibrinous airsacculitis, fibrinous pericarditis and perihepatitis, haemorrhagic proventriculitis, catarrhal enteritis, lymphoid depletion in caecal tonsils and spleen, tubular epithelial cell degeneration, congestion, interstitial nephritis and urate tophi in kidneys, perivascular cuffing and glial cell proliferation of brain. In NDV and concurrent infections (IBV, P.multocida, MG, MS and APEC) grossly congestion, haemorrhages and catarrhal exudates in the trachea, congestion of lungs, proventricular haemorrhages, haemorrhagic and necrotic intestines and caecal tonsils were noticed. Further, necrotic foci on the liver when co-infected with P.multocida, fibrinous to caseous exudates in the air sacs, pericarditis, perihepatitis and egg peritonitis in concurrent infections of MG, MS and E.coli were found. Microscopically, tracheitis, airsacculitis, pneumonic changes, exudates in the parabronchi and lymphoid depletion in the spleen and caecal tonsils were noticed. Ultrastructurally, trachea showed total loss of cilia and epithelium, large vacuoles in the cytosol and pyknotic nuclei with margination of chromatin and in lungs erythrophagocytosis, loss of cell membrane of Type II pneumocytes, few lamellar bodies and electron dense bodies in the cytosol were noticed. In IBV and concurrent infections (NDV, MG and APEC), birds showed grossly congestion, edema and caseous exudates on the tracheal mucosa, thickened air sacs with fibrinous to caseous exudates and edema, congestion, consolidation and pneumonia of lungs. Kidneys were swollen, congested or haemorrhagic and ureters were distended with urates. Cystic oviduct, egg peritonitis and mild fibrinous pericarditis and perihepatitis were also found. White chalky urate deposits were found on the pericardium, liver and kidneys. Microscopically, tracheitis, airsacculitis, exudates in the parabronchi and haemorrhages and degenerative changes in the kidneys and interstitial nephritis were noticed. Ultrastructurally, tracheal tissue homogenates showed round electron dense virions of approximately 100 nm in diameter. In FAdV and concurrent infections (MG and APEC), congestion and thickening of the tracheal mucosa, thickened air sacs, edema in lungs, ascites, hydropericardium, swollen liver with necrotic foci, swollen haemorrhagic kidneys, fibrinous pericarditis and perihepatitis were noticed. Microscopically, tracheitis, airsacculitis, congestion and edema in lungs and intranuclear basophilic inclusion bodies in liver and kidneys were found. Ultra structurally, virus particles exhibiting oval or hexagonal configurations arranged in crystalline arrays were found in the hepatic cells. In M.gallisepticum and concurrent infections (MS and APEC), catarrhal to caseous exudates in the nasal passages, trachea, bronchi and air sacs and congested and consolidated lungs were noticed. In severe infections, fibrinous exudate was deposited on the pericardium and liver. Microscopically tracheitis, airsacculitis, pneumonia and lymphoid aggregates in the trachea, lungs, liver and kidneys, pericarditis and perihepatitis were noticed. In M.synoviae and concurrent infections (MG and APEC), congestion of trachea and lungs, accumulation of catarrhal exudates in the air sacs, mild fibrinous exudate on the pericardium and liver were seen. Microscopically, mild tracheitis, airsacculitis and congestion and edema of lungs were noticed. In birds infected by P.multocida and concurrent infections (APEC), congestion throughout the carcass, petechial haemorrhages on coronary fat and epicardium, congestion of liver, intestines, kidneys and ovaries and necrotic foci on the liver, fibrinous pneumonia, pericarditis and perihepatitis were noticed. Microscopically, congestion, fibrinous exudates in the parabronchi, thickening of parabronchial septa, degenerative changes in the heart and necrotic changes in liver were evident. In A.paragallinarum and APEC concurrent infections, the birds revealed swelling of eyes and face, conjunctivitis and a serous to mucoid discharges from nostrils and sinuses, congestion of trachea, edema and congestion of lungs, thickened air sacs and fibrinous exudates on the pericardium and liver. Microscopically, severe sinusitis, mild tracheitis and airsacculitis were noticed. In avian pathogenic E.coli infected birds trachea and lungs were severely congested and edematous and polyserositis with fibrinopurulent pericarditis, perihepatitis, airsacculitis and egg peritonitis were noticed. Out of 60 E. coli isolates, virulent genes viz. iss, vat, tsh and papC were found in 48(80%), 43(71.6 %), 31 (51.6%) and 18(30%) of isolates respectively. In the present study molecular detection of the respiratory pathogens viz. NDV, IBV, FAdV, LPAI, ILTV, M. gallisepticum, M. synoviae, A. paragallinarum, P.multocida and avian pathogenic E.coli (APEC) was carried out by PCR/RT-PCR. Of these, except LPAI and ILTV other pathogens were detected on PCR in the affected birds. Based on the pathogen under study, the amplicons of appropriate base pair sizes were detected by agar gel electrophoresis. Further, nucleotide sequencing and phylogenetic analysis of F gene of NDV, S1 gene of IBV and hexon gene of FAdV was also carried out. On sequencing and phylogenetic analyses, the 6 NDV samples (NDV/GVM/AP1- 6) were grouped with the isolates of Tamil Nadu, Nagpur, Assam, West Bengal and Bareilly belonging to genotype XIII. On amino acid analysis, fusion protein cleavage site amino acids at positions 112 and 116 (RRRKR) and a phenylalanine (F) at position 117, indicated that the NDV strains are of virulent type of strains. The IBV samples IBV/GVM/AP-1, 2 and 3 were grouped with vaccine strains M41, H120 and other respiratory IBV strains and IBV/GVM/AP- 4, 5 and 6 were grouped with nephropathogenic strain of IBV-470 and other nephropathogenic IBV strains. The two FAdV samples (FAdV/GVM/AP-1 and 2) isolated from the respiratory disease complex cases were grouped along with fowl adenovirus strain E. The present study indicated that respiratory disease complex is a major problem in commercial chicken and it has a potential to cause severe economic losses in poultry.ThesisItem Open Access CLINICO-DIAGNOSTIC STUDIES AND EVALUATION OF CERTAIN BIOMARKERS IN CANINE PYOMETRA(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2021-01) ANUSHA, KONJETI; SADASIVA RAO, K (MAJOR); SRINIVAS, M; SREENU, M; ASWANI KUMAR, KThe aim of the present study was to identify the frequency of SIRS in pyometra affected bitches and to study the role of certain biomarkers of diagnostic and prognostic importance, along with evaluation of blood flow velocities and hemodynamic indices in uterine artery and to study the hemato-biochemical, histopatholgical and immunohistochemical changes in canine pyometra. Out of 716 bitches presented for various gynaecological ailments, 122 bitches were diagnosed with pyometra with an overall occurrence of 17.04 per cent. Bitches in the age group of 6 to 9 years (29.51%) had high occurrence of pyometra followed by 3 to 6 years (28.69%), 9 to12 years (27.05%), 12 to 15 years (9.83%) and least in bitches upto 3 years of age (4.92%). The mean age of occurrence of pyometra was 8.03±0.26 years with a range of 2 to 15 years. The present study revealed high occurrence of pyometra in Spitz (43.44%) followed by Labrador (22.95%), Mongrel (9.83%), German Shepard (7.38%), Pug (5.74%), Pomeranian (4.10%), Great Dane (2.46%), Doberman (1.64%), Dachshund, Bull Mastiff and Saint Bernard (0.82% each). Occurrence of pyometra was highest in nulliparous (62.30%) bitches followed by primiparous (22.13%) and pluriparous (15.57%) bitches. The occurrence of open cervix (85.25%) pyometra was higher than closed cervix (14.75%) pyometra. The most predominant symptom reported by the owners was presence of vaginal discharge (80.00%) followed by inappetence (72.00%), polydipsia (74.60%), vomiting (68.00%), polyuria (57.30%) and diarrhoea (12.00%). The altered physiological findings on clinical examination were increased (28.00%) and decreased (1.33%) temperature, pale (28.00%) and hyperemic (6.67%) mucous membranes, mild (32.00%) to moderate (6.67%) dehydration, moderate (50.67%) to severe depression (34.67%) in pyometra affected bitches. On palpation of abdomen 24.00 per cent bitches expressed pain and enlarged uterus was clearly felt in 32.00 per cent bitches and unclear in 68.00 per cent bitches. The temperature and respiratory rate were significantly elevated in pyometra affected bitches compared to healthy controls. Hematological studies revealed decreased hemoglobin, packed cell volume and total erythrocyte count, while leukogram showed significant neutrophilia and leukocytosis with left shift. None of the hematological parameters differed significantly between open and closed pyometra except for TLC and percentage of band neutrophils being higher in closed cervix pyometra. Serum biochemical profile showed elevated levels of total protein, globulin, ALT, AST, ALP, BUN and creatinine and reduced albumin and A/G in pyometra affected bitches compared to healthy controls. None of the biochemical parameters differed significantly between open and closed cervix pyometra except for elevated globulin and reduced A/G in closed cervix pyometra. Estimation of CRP and SAA revealed significant elevation of these acute phase proteins in pyometra affected bitches (71.12±6.19 mg/L and 60.68±2.27 mg/L) compared to healthy controls and also, the concentrations were significantly higher in closed cervix pyometra than open cervix pyometra. In the present study 70.67 per cent bitches affected with pyometra had positive SIRS status. Positive SIRS status was noticed in 80.00 per cent of bitches with closed cervix pyometra and 68.33 per cent of bitches with open pyometra. Among the clinical criteria to assess SIRS status temperature, heart rate, TLC, percentage of band neutrophils and SAA were significantly higher in SIRS positive than SIRS negative bitches. No significant difference was noticed in respiratory rate, albumin and CRP between SIRS positive and SIRS negative bitches. Radiographic examination revealed a space occupying radiographic opacity between colon and urinary bladder in moderately affected cases, while in severely affected bitches distended uterus was easily identified beyond caudo-ventral abdomen displacing the intestines cranially. On ultrasonographic examination the distended uterine horns appeared as round or oval anechoic to hypoechoic fluid filled structures dorsal to anechoic urinary bladder. The pyometra affected bitches were categorized basing on the sonographic appearance of uterus and uterine contents into groups B, C, DH and DA. The diameter of uterine horns was highest in group DA and thickness of uterine wall was highest in group DH. In pyometra, the blood flow was continuous with high velocity, low resistance and low pulsatility. In pyometra affected bitches, higher blood flow velocities (PSV and EDV) and lower hemodynamic indices (RI, PI and S/D) were recorded in group DA. The histopathological findings showed that Type II CEH-pyometra complex (51.52%) was the common pathology observed in pyometra affected bitches followed by Type III (36.36%) and Type IV (12.12%). Histopathological picture of Type II uterus was characterized by cystic endometrial hyperplasia with diffuse infiltration of the stroma by plasma cells. Type III pyometra was characterized by cystic endometrial hyperplasia with acute inflammatory reaction. Type IV pyometra was characterized by marked atrophy of endometrium with very few glands and stroma was infiltrated with plasma cells, macrophages and few neutrophils. All the histopathological types of pyometra showed cytoplasmic staining for VEGF. A variation in quick sore was noticed in luminal and glandular epithelium between different histological types of pyometra. Type II and Type IV had higher quick score in luminal epithelium whereas, the quick score in glandular epithelium was higher in Type III. The nuclear staining for Ki-67 did not yield consistent results in the present study. However cytoplasmic staining of Ki-67 was observed in stroma cells of few cases of Type II pyometra. In the present study, the mortality rate in pyometra affected bitches was 18.67 per cent of which 6.67 per cent succumbed to death after surgery and 12.00 per cent before surgery. The mean temperature, heart rate, percentage of band neutrophils, total protein, globulin, ALP, BUN, creatinine and SAA were significantly higher while hemoglobin and TEC were significantly lower in non-survivors than survivors. A significant association with mortality was noticed with the presence of SIRS (P<0.0072), closed pyometra (P<0.0279), elevated ALP (P<0.0109), elevated creatinine (P<0.0002) and elevated serum amyloid A (P<0.0001) in the pyometra affected bitches and these parameters could be used as prognostic indicators in canine pyometra.ThesisItem Open Access CLINICO-DIAGNOSTIC AND THERAPEUTIC STUDIES ON RENAL DISEASE IN DOGS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2020-12) CHAITANYA, YALAVARTHI; LAKSHMI RANI, N (MAJOR); VAIKUNTA RAO, V; RAVI KUMAR, P; SREENU, MAKKENADuring the study period of two years, the occurrence of renal diseases in dogs accounted up to 3.40 per cent in. Among the affected dogs, 18.24 per cent suffered with acute kidney injury, whereas 81.76 per cent were of chronic kidney disease. Kidney diseases were more prevalent in male dogs, aged above 8 years and the occurrence was more in Pomeranian (30.82%). In the affected dogs, the clinical presentation varied from asymptomatic to typical clinical signs. Detailed haemato- biochemical examination and imaging studies performed in 128 dogs revealed that, 25 dogs were affected with AKI and 103 dogs had CKD. The dogs with CKD were further categorized into four stages based on SDMA values as stage I, II, III and IV. The SDMA values in the affected dogs ranged from 14 to 152 μg/dL. Sub staging of CKD dogs was done based on UPC and Blood pressure. Significant difference (P0.05) was noticed in mean values of PI and RI in acute kidney injury and chronic kidney disease stage I and II with the healthy control while the same in chronic kidney disease stage III and IV varied significantly (P0.05) was noticed in mean values of LA/Ao and M mode echocardiographic findings in dogs with acute kidney injury which varied significantly (P<0.05) in chronic kidney disease. Therapy was initiated in dogs with kidney diseases by using herbal antioxidant, diuretic, phosphate binders, antibiotics, diuretic, ACE inhibitors, nutritional therapy, haematinics, fluid therapy along with specific treatment and were monitored for a period of 3 months. The survival percentage of dogs with acute kidney injury (Group I) was 84.00% per cent (21/25). The survival percentage was 100.00 per cent in Group IIa (17/17) and IIb (15/15) i.e., chronic kidney disease stage I and stage II, where as it was 80.00 per cent (20/25) in CKD stage III (Group IIc) and 56.00 per cent (26/46) in chronic kidney disease stage IV (Group IId). Significant difference (p<0.05) was recorded in the SDMA/creatinine ratio(9.20±0.18 in survivors vs 12.07 ± 0.42 in non survivors, BUN ( 82.53±1.84 mg/dL vs 148.62±12.82mg/dL), product of calcium and phosphorus(58.22±2.02 vs 89.32±1.92), sodium to potassium ratio (39.42±1.02 vs 48.93±1.32) , UPC (1.92±0.22 vs 3.86±0.52 ) and resistive index (0.62±0.56 vs 0.74±0.24) between survivors and non survivors
