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Kerala Veterinary and Animal Sciences University, Wayanad
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ThesisItem Open Access VIRULENCE GENE PROFILING OF Escherichia coli FROM BOVINE MASTITIS AND THERAPEUTIC MANAGEMENT OF COLIFORM MASTITIS(Kerala Veterinary and Animal Sciences Mannuthy, Thrissur, 2019-12-30) M.REVATHI; S. SulficarColiforms are the major etiological agents of bovine mastitis which is an economically devastating disease causing substantial loss to the dairy farmers through reduction in the milk production. The present study was conducted to detect the virulence genes of E. coli by Polymerase Chain Reaction (PCR), to study the therapeutic efficacy of various antibiotics in E. coli mastitis and to identify the risk factors associated with coliform mastitis. Epidemiological investigations revealed that affected cows were in younger age and early stage of lactation having average milk yield. Absence of hygienic practices for clean milk production such as teat dipping resulted in poor udder hygiene which led to the occurrence of coliform mastitis. The affected animals had elevated temperature with varying degree of udder oedema and pale yellow coloured milk. In the present study, out of 168 animals affected with clinical mastitis, 123 bacterial isolates could be isolated of which 26 constituted coliforms such as Escherichia coli (14), Klebsiella spp. (10), Enterobacter spp. (1) and Citrobacter spp. (1) with the prevalence of 21.13 per cent and the remaining were Gram positive isolates. In vitro antibacterial sensitivity test of coliform isolates revealed that ceftizoxime was the most sensitive drug. Treatment of affected cases was done with sensitive antibiotics according to the antibiogram, fluid therapy, flunixin meglumine and trisodium citrate were administered based on the resolution of clinical signs. After treatment, clinical recovery with increase in milk yield was noticed in all the cases but for the resolution of udder oedema. Haematological analysis prior and after treatment revealed significant increase in total blood count and reduction in leukocyte counts. Polymerase chain reaction was performed to identify the virulence genes of E.coli viz., traT, stx 1, stx 2, eaeA and aerobactin (iucD) which revealed that among 14 isolates, two were positive for traT gene, two were positive for aerobactin gene and one was positive for stx 2 gene. Thus, the present study revealed the presence of virulence genes among E.coli isolates causing bovine mastitis.ThesisItem Open Access VIRULENCE GENE PROFILING OF Escherichia coli FROM BOVINE MASTITIS AND THERAPEUTIC MANAGEMENT OF COLIFORM MASTITIS(Kerala Veterinary and Animal Sciences Mannuthy, Thrissur, 2019-09-30) M.REVATHI; S. SulficarColiforms are the major etiological agents of bovine mastitis which is an economically devastating disease causing substantial loss to the dairy farmers through reduction in the milk production. The present study was conducted to detect the virulence genes of E. coli by Polymerase Chain Reaction (PCR), to study the therapeutic efficacy of various antibiotics in E. coli mastitis and to identify the risk factors associated with coliform mastitis. Epidemiological investigations revealed that affected cows were in younger age and early stage of lactation having average milk yield. Absence of hygienic practices for clean milk production such as teat dipping resulted in poor udder hygiene which led to the occurrence of coliform mastitis. The affected animals had elevated temperature with varying degree of udder oedema and pale yellow coloured milk. In the present study, out of 168 animals affected with clinical mastitis, 123 bacterial isolates could be isolated of which 26 constituted coliforms such as Escherichia coli (14), Klebsiella spp. (10), Enterobacter spp. (1) and Citrobacter spp. (1) with the prevalence of 21.13 per cent and the remaining were Gram positive isolates. In vitro antibacterial sensitivity test of coliform isolates revealed that ceftizoxime was the most sensitive drug. Treatment of affected cases was done with sensitive antibiotics according to the antibiogram, fluid therapy, flunixin meglumine and trisodium citrate were administered based on the resolution of clinical signs. After treatment, clinical recovery with increase in milk yield was noticed in all the cases but for the resolution of udder oedema. Haematological analysis prior and after treatment revealed significant increase in total blood count and reduction in leukocyte counts. Polymerase chain reaction was performed to identify the virulence genes of E.coli viz., traT, stx 1, stx 2, eaeA and aerobactin (iucD) which revealed that among 14 isolates, two were positive for traT gene, two were positive for aerobactin gene and one was positive for stx 2 gene. Thus, the present study revealed the presence of virulence genes among E.coli isolates causing bovine mastitis.ThesisItem Open Access Clinico-pathological and therapeutic studies on colibacillosis in neonatal calves(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD, 2019-08-19) MATHEW, XAVIER; A., JanusColibacillosis is an economically important disease, causing neonatal diarrhoea and septicaemia in calves. It has worldwide occurrence and has been reported from almost all states of India. The present study was conducted to identify different strains of Escherichia coli associated with colibacillosis in calves from Wayanad district by identification of virulence factors through PCR, assessment of clinical and haemato-biochemical changes associated with colibacillosis and to suggest a suitable treatment protocol for neonatal colibacillosis. Virulence factors of pathogenic E. coli were targeted using specific primers against K99, F41, STa, intimin, Stx1 and Stx2 genes. Seventeen samples were found positive for Intimin, Stx1 and Stx2 together or alone suggesting the presence of EHEC/ EPEC. One milk sample and one drinking water sample were found positive for intimin and Stx1 which confirmed EHEC in the sample. A multiscreen Ag ELISA kit was used for ruling out bovine Rotavirus, Coronavirus, E coli F15 and Cryptosporidium antigens but it showed negative results for all the pathogens. Haemato-biochemical values and clinical parameters of all the thirty calves when compared with control, showed significant difference and all the abnormal parameters came to normal range after treatment. Sulphamethoxazole/ trimethoprim gave sensitivity to 18 isolates which was similar to clinical response, that is usage of sulphadiazine -trimethoprim showed fast recovery in 18 cases. In six cases ceftriaxone was used five cases amoxicillin antibiotic was used as per antibiogram and both the antibiotics showed good response. Calves were treated with balanced electrolyte solutions and sodium bicarbonate to correct metabolic acidosis, dehydration and electrolyte imbalances administration was very helpful to control metabolic acidosis. Detailed study to identify the diarrhoeagenic E coli strains is necessary as E coli could affect livestock farming and even, they can enter food chain tooThesisItem Open Access CLINICO-EPIDEMIOLOGICAL STUDIES AND MOLECULAR DIAGNOSIS OF FELINE PANLEUKOPENIA IN CATS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD, 2019-10-21) BAKDE, RIYA ASHOK; R. L, RathishThe present study envisaged to undertake serosurveillance and to investigate the clinico-epidemiological features of feline panleukopenia in North Kerala. The study also aimed at comparing haemagglutination inhibition test (HI) and Loop Mediated Isothermal Amplification (LAMP) with Polymerase chain reaction (PCR) for detection of Feline Panleukopenia Virus (FPV) from faecal samples of affected cats. An indirect sandwich ELISA was done on 79 unvaccinated healthy cats to detect circulating antibodies against FPV, which revealed a prevalence of 29.11 per cent. Analysis across various epidemiological profiles revealed no statistical difference, indicating equal rate of exposure across all epidemiological parameters. Feline panleukopenia virus DNA was detected in 34 out of 40 faecal samples by PCR. Epidemiological analysis revealed higher occurrence of feline panleukopenia in cats aged less than six months, among females and non-descript cats. Stray cats, multiple cat households and cats without any deworming history were more frequently affected. Pyrexia, dysentery, vomiting, dehydration and respiratory distress were predominant clinical findings. The affected cats suffered from anaemia, thrombocytopenia, leucopoenia and elevated liver enzymes. Ultrasonography revealed gas and fluid filled intestinal loops. PCR targeting the VP2 gene of FPV resulted in 698bp amplicons which was sequenced. BLAST analysis of the sequence revealed similarity to feline panleukopenia virus. Haemagglutination was observed in 25 faecal samples, out of which haemagglutination inhibition assay confirmed the presence of FPV in ten samples and a kappa score of 0.1 indicated poor agreement to PCR. In contrast, LAMP detected 36 out of 40 samples tested by PCR. Even though all the PCR negative samples were lamp negative, two of the PCR negative samples were positive by LAMP. Statistical analysis revealed a high quotient of agreement between PCR and LAMP. LAMP had 100 per cent sensitivity and positive predictive value with 67 per cent specificity and 94 per cent negative predictive value compared to PCR. The potential of LAMP as a diagnostic tool in limited resource settings can be exploited for rapid diagnosis of infectious diseases.ThesisItem Open Access IDENTIFICATION OF INFECTIOUS ETIOLOGIES OF RESPIRATORY DISEASES IN PSITTACINES(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD, 2019-10-21) THOMAS, RANI MARIA; P.M, DeepaThe present study was conducted in the Department of Veterinary Epidemiology and Preventive Medicine, College of Veterinary and Animal Sciences, Pookode to investigate the major infectious causes of respiratory diseases in psittacines in Kerala. A total of 30 aviaries from seven districts namely Thiruvananthapuram, Alappuzha, Ernakulam, Thrissur, Kozhikode, Wayanad and Kannur was visited and 190 birds with respiratory signs like tail bobbing, dyspnoea, nasal discharge, conjunctivitis, etc was sampled. Faecal, choanal, conjunctival , nasal swabs and tissues of dead birds like trachea, liver, lungs and air sacs were taken. DNA and RNA was isolated from all the samples. Polymerase chain reaction was conducted for Mycoplasma gallisepticum targeting lipoprotein gene, 16SrRNA for Chlamydia spps and Mycobacterium spp and fusion protein gene for Paramyxoviral infection. An epidemiological questionnaire was distributed among the breeders of these 30 aviaries. Out of 190 birds, 71 (37.37 per cent) was positive for Chlamydia and sequencing results showed 100 per cent identity with C.psittaci. M.gallisepticum was positive in 60 birds with a prevalence of 31.58 per cent and sequencing revealed 99.48 per cent identity with M.gallisepticum isolate from Neerkunnam, Israel and USA. Least occurrence was for Paramyxoviral infection with 5.79 per cent and sequence analysis revealed 100 per cent identity with Newcastle disease virus. Mycobacterium spp. was not identified. Major clinical signs seen in Chlamydia infection were conjunctivitis and greenish diarrhoea. Birds positive for Mycoplasmal infection had tail-bobbing and conjunctivitis and in Paramyxoviral infection dyspnoea and neurological signs were seen. Budgerigars were most commonly affected with Chlamydial infection whereas small psittacines and finches were susceptible for Mycoplasmal infection. Cockatiels and alexandrine parakeets were positive for Paramyxoviral infection. The probability of getting positive results of Mycoplasma was highest in nasal swabs followed by conjunctival and tissues. Chlamydia and Paramyxovirus was mostly identified in conjunctival swabs and faecal swabs respectively. Source of the bird and poor biosecurity practices was found to play a major role in spread of infections among psittacine birds in Kerala.ThesisItem Open Access CLINICO-THERAPEUTIC STUDIES ON BOVINE THEILERIOSIS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2018) JACKSON BENNY; P.V. TresamolBovine theileriosis is an economically important tick-borne protozoan disease that affects cattle all over the world. The present study titled “Clinico-therapeutic studies on bovine theileriosis” envisages diagnosis and treatment of oriental theileriosis in dairy cattle. Microscopical examination of stained blood smears from 187 animals revealed Theileria spp. piroplasms in 61 cases, inclusions suggestive of A. marginale in 18 cases, concurrent infection of theileria and anaplasma in 32 cases and piroplasms of Babesia bigemina in one animal. Blood smears from positive cases were later confirmed by Acridine orange staining. Genus specific PCR for Theileria spp. yielded positive results in 58 clinical cases and 37 in-contact animals. Out of 58 positive cases, 50 were confirmed as T. orientalis using species specific PCR. Among the in-contact animals, all 37 positive cases were positive for T. orientalis. None of the samples from 58 clinical cases and 37 in-contact animals yielded positive results of T. annulata. Major clinical signs observed were anaemia, pyrexia, lymph node enlargement, lethargy and drop in milk production. Haemato-biochemical analysis revealed anaemia with low Hb, PCV and TEC and hyperproteinaemia with hyperglobulinaemia in the affected animals. Treatment with single dose of buparvaquone @ 2.5 mg/kg BW intramuscularly was found to be more effective than two doses of long acting oxytetracycline @ 20 mg/kg BW intramuscularly 48 hours apart.ThesisItem Open Access VIRULENCE GENE PROFILING OF Escherichia coli FROM BOVINE MASTITIS AND THERAPEUTIC MANAGEMENT OF COLIFORM MASTITIS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2018) M.REVATHI; S. SulficarColiforms are the major etiological agents of bovine mastitis which is an economically devastating disease causing substantial loss to the dairy farmers through reduction in the milk production. The present study was conducted to detect the virulence genes of E. coli by Polymerase Chain Reaction (PCR), to study the therapeutic efficacy of various antibiotics in E. coli mastitis and to identify the risk factors associated with coliform mastitis. Epidemiological investigations revealed that affected cows were in younger age and early stage of lactation having average milk yield. Absence of hygienic practices for clean milk production such as teat dipping resulted in poor udder hygiene which led to the occurrence of coliform mastitis. The affected animals had elevated temperature with varying degree of udder oedema and pale yellow coloured milk. In the present study, out of 168 animals affected with clinical mastitis, 123 bacterial isolates could be isolated of which 26 constituted coliforms such as Escherichia coli (14), Klebsiella spp. (10), Enterobacter spp. (1) and Citrobacter spp. (1) with the prevalence of 21.13 per cent and the remaining were Gram positive isolates. In vitro antibacterial sensitivity test of coliform isolates revealed that ceftizoxime was the most sensitive drug. Treatment of affected cases was done with sensitive antibiotics according to the antibiogram, fluid therapy, flunixin meglumine and trisodium citrate were administered based on the resolution of clinical signs. After treatment, clinical recovery with increase in milk yield was noticed in all the cases but for the resolution of udder oedema. Haematological analysis prior and after treatment revealed significant increase in total blood count and reduction in leukocyte counts. Polymerase chain reaction was performed to identify the virulence genes of E.coli viz., traT, stx 1, stx 2, eaeA and aerobactin (iucD) which revealed that among 14 isolates, two were positive for traT gene, two were positive for aerobactin gene and one was positive for stx 2 gene. Thus, the present study revealed the presence of virulence genes among E.coli isolates causing bovine mastitis.ThesisItem Open Access OCCURRENCE AND CHARACTERIZATION OF METHICILLIN RESISTANT Staphylococcus aureus IN PIGS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2018) SILPA RAJ. S; Justin Davis. KMethicillin resistant Staphylococcus aureus (MRSA) has been detected from pigs in many parts of the world and is having great potential to act as a human pathogen. Exposure to livestock is considered as a major risk factor for human colonization with MRSA. This study was conducted to evaluate the occurrence and molecular characteristics of MRSA among healthy pigs. Nasal swabs (n=119) were derived from four pig farms in Thrissur district and Staphylococci were enumerated from the swabs by conventional culture methods. The prevalence of S. aureus was 42.86 per cent by Polymerase Chain Reaction (PCR). S. aureus isolates were characterized by Staphylococcal Protein A (spa) typing and all the isolates belonged to six spa types with t091 (20%) being the predominant spa type. Eleven new spa types were also identified in the study. Methicillin resistance in S. aureus isolates were identified genotypically by molecular characterization of mecA gene and the prevalence was 5.89 per cent. The beta-lactam resistance gene, blaZ was detected in 62.75 per cent isolates. Antibiogram studies of all the S. aureus isolates revealed that maximum resistance was shown to gentamicin (76.47%) followed by azithromycin (74.77%), and ciprofloxacin (70.59%). Phenotypic methicillin resistance, as indicated by the resistance to oxacillin, was identified in 29.41 per cent of the isolates. The high Multiple Antibiotic Resistance (MAR) indices obtained in the study indicated a remarkable spread of resistance among the isolates due to frequent exposure to the antibiotics. This study confirmed the occurrence of nasal colonization of MRSA among healthy pigs which is a major threat to public health and should be viewed seriously.ThesisItem Open Access ETIOPATHOLOGICAL PROFILING AND THERAPEUTIC STUDIES OF NEONATAL CALF DIARRHOEA(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2018) AMRUTHA V S; K. VinodkumarNeonatal calf diarrhoea (NCD) is an economically important disease in dairy sector all over the world. Both infectious and non-infectious etiologies are reported for the disease. Major infectious etiological agents include rotavirus, coronavirus, E. coli and Cryptosporidium parvum. Present study was conducted to identify the associated risk factors, etiological agents responsible and the haemato-biochemical changes in NCD among calves reared in and around Thrissur district. Fifty diarrhoeic and twenty normal neonatal calves formed the study group. Among 50 diarrhoeic faecal samples, five were positive for Cryptosporidium spp. under acid fast staining and 19 were positive under genus specific nested PCR. Positive samples were identified as C. bovis by molecular methods. Virulence factors of pathogenic E. coli were targeted using specific primers against K99, F41, STa, intimin, Stx1 and Stx2 genes. Fifteen samples were found to possess genes for intimin, Stx1 and Stx2 together or alone as virulence factors and suggestive of the presence of EHEC/ STEC. A multi-purpose commercial sandwich antigen ELISA gave positive result for rota virus in a single sample and negative results for corona, Cryptosporidium parvum and E. coli K99 in all. Microscopic examination of faecal sample by concentration method detected the presence of ova of Strongyloid, Toxocara and unidentified nematode larvae in one sample each. Haemato-biochemical values of diarrhoeic animals when compared with normal, showed significant difference in WBC, lymphocyte count, monocyte count, serum albumin level and serum globulin levels. Since commonest pathogens found in this study are C. bovis, EAEC and EHEC in NCD, therapeutic management should aim at removal of these pathogens and correction of associated haemato- biochemical changes. Feeding of more quantities of colostrum should also be recommended.
