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Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar
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ThesisItem Restricted Detection of staphylococcus aureus in bovine sub clinical mastitis using polymerase chain reaction (PCR)(LUVAS, 2007) Sindhu, Neelesh; Jain, V.K.The present investigation was undertaken to standardize polymerase chain reaction (PCR) assay using ten different sets of primers for detection of sub clinical mastitis (SCM) directly from milk samples of apparently healthy animals. A total of 1230 milk samples including 367 samples from cow farm, 228 samples from buffalo farm and 635 samples brought in Veterinary College Central Laboratory, CCS Haryana Agricultural University, Hisar, were screened by PCR assay and other conventional tests (cultural examination, California mastitis test, Electrical conductivity test and Somatic cell count). Staphylococci isolates were characterized phenotypically and genotypically. Screening of cow farm using universal primer yielded 56.52 per cent animals and 33.24 per cent quarters to be positive for SCM whereas18.85 per cent quarters were found positive for Staphylococcus aureus by 16S-23S rRNA interspacer region primers. As many as 15.78 per cent animals and 5.70 per cent quarters were found positive by using universal primer while 15.38 per cent quarters were positive for Staphylococcus aureus by 16S-23S rRNA interspacer region primers from buffalo farm. A total of 15.90 per cent samples of College Central Laboratory were found positive for Staphylococcus aureus by 16S-23S rRNA interspacer region primers and majority of samples found negative by cultural examination and positive by PCR were from animals who received prior treatment with antibiotics. The Genus specific primers encoding gap gene and 16S-23S rRNA region could detect all Staphylococcus spp. Among species specific primers the primers encoding 16S-23S rRNA region were found to be 100 per cent sensitive followed by aroA gene (98.41 per cent), nuc gene (97.62 per cent) and coa gene (93.65 per cent). A total of 99.20 per cent of isolates were found positive by ubiquitous PCR assay. On the basis of results of different conventional diagnostic tests, CMT was found to be good animal side test for primary screening of large herd to identify subclinically infected animals within a very short period economically. Among phenotypic tests, thermostable nuclease test was most sensitive in comparison to latex and coagulase test. No false-positive reactions were detected by any of the tests applied on staphylococci other than Staphylococcus aureus. Genotypic characterization of staphylococcal isolates yielded similar results as were obtained by PCR of milk samples directly indicating PCR assay as an important tool and an alternative to phenotypic characterization. PCR was found to be most rapid, sensitive and specific assay when compared to bacteriological examination and other conventional tests and can be adapted for screening of large herd for detection of Staphylococcus aureus directly from milk.ThesisItem Restricted Clinico-biochemical and therapeutic investigations on hypocuprosis in buffaloes(LUVAS, 2008) Panwar, Manik; Rana, Yudhbir SinghA total of 108 blood samples of buffaloes from the four districts of Haryana were collected and analysed to study serum copper status in that areas. The prevalence of Cu deficiency was found to be 52.17, 51.28, 45.83 and 45.45 per cent in Fatehabad, Hisar, Rohtak and Sonipat, respectively. The overall Cu deficiency in buffaloes was 49.07 per cent, out of which 52.38 per cent heifers and 44.44 per cent adults were having Cu deficiency. Higher prevalence rate of Cu deficiency was observed in animals reared under village conditions (55.99%) than the animals kept at organized dairy farms (32.14%). Pregnant animals (55.88%) showed higher Cu deficiency than the non-pregnant (37.50%) once. Clinical signs were observed in 28 99 (52.83%) out of 53 Cu-deficient buffaloes. Achromatrichia, leucoderma and alopecia were observed in 85.71, 71.42 and 50.00 per cent of buffaloes, where as non-specific clinical signs including anemia, repeat breeding, anoestrous, mastitis, diarrhoea, hoof deformity, paralysis and sudden falling of animals in 67.85, 57.00, 32.14, 10.71, 7.14, 7.14, 3.47 and 3.57 per cent of Cu deficient buffaloes showing clinical signs. Haematobiochemical investigations were carried out in clinical cases of hypo-cuperosis which were divided into two groups (A and B) for therapeutic trial and 10 normal healthy animals kept as healthy control (group C). Animals of group A were treated with copper glycinate injection given s/c along with mineral mixture orally and group B treated with oral copper sulphate with mineral mixture. Observations were recorded before treatment on day 0 and after treatment on day 15 and 30. On day 0, significantly lower levels of mean serum ceruloplasmin, Cu, Co, Zn, Mn and hair Cu concentration were observed in both the groups (A and B) as compared to group C where as no significant difference was observed in serum Fe and Mo level. Blood examination revealed significant low levels of Hb, PCV and TEC in animals of both the groups (A and B) in comparison to group C.After the treatment on day 15, non-significant changes were observed in all the above parameters of both the groups (A and B). However on day 30, serum ceruloplasmin and Cu levels were increased significantly in group A, while in group B increased non-significantly in comparison to their day 0 levels. Hair Cu concentration, serum Co, Zn and Mn were increased non-significantly in both the groups (A and B), but better increase was observed in group A than the group B. Likewise, mean Hb and PCV were increased significantly in group A, while non-significantly in group B. Then, on the basis of recovery in clinical signs and restoration in serum Cu level, Cu glycinate s/c was found more efficacious (55.44%) than the oral copper sulphate (30.00%), but incomplete recovery in clinical signs and low level of serum copper in some of the animals on day 30 indicated that more aggressive and longer duration of the treatment is required.ThesisItem Restricted Immunological and molecular techniques for diagnosis of subclinical mastitis caused by staphylococcus aureus in buffaloes(LUVAS, 2008) Tehlan, Monika; Yudhbir SinghMastitis is an inflammatory reaction of udder parenchyma caused most commonly by bacterial pathogens and their toxins. Clinical mastitis can be easily diagnosed by visible udder changes, whereas subclinical mastitis (SCM) is diagnosed by indirect tests. SCM may contribute upto 70 percent of the total mastitis cases. Staphylococcus aureus is the most frequently isolated organism from SCM milk samples in majority of the buffalo dairy herds in India and abroad, and is a leading cause of gastroenteritis in humans consuming contaminated milk or cream. Various tests based on cellular and biochemical changes, such as somatic cell count (SCC), NAGase, California mastitis test, etc. are in use for diagnosis of SCM, without giving information about the etiological agent(s). Therefore, the present study was planned in order to develop some suitable immunological and molecular diagnostic tests for diagnosis of SCM caused by of S. aureus in buffaloes and to study prevalence of SCM in buffaloes at the HAU Farm. D-SN, D-SP, PPV and NPV of these tests were also determined. One hundred sixty quarters of 40 buffaloes were screened during April 2006 to October 2007 by cultural examination. Colonies of S. aureus, other Staphylococcus spp., Streptococcus spp. and Gram −ve rods were obtained in a total of seventy eight quarters, but only 15 samples were confirmed to be having coagulase positive staphylococci on basis of coagulase tube test. LAT was developed for the detection of S. aureus exoproteins (EP) and cell membrane (CM) antigens in the milk samples from 140 quarters of 35 buffaloes, 14 (10%) and 11 (7.8%) were found positive. Similarly, sandwich ELISA for detection of S. aureus EP and CM antigens was able to detect milk 16 (11.4%) and 14 (10%) samples positive from the 140 quarters. The detection limit for ELISA-CM was 10 ng/ml and for ELISA-EP, it was 100 ng/ml. D-SN and D-SP for LAT-EP and LAT-CM were 66.6%, 98.2 % and 26.6%, 98.4% whereas for ELISA-EP and ELISA-CM were 55.5%, 93% and 33.3, 92.78%, respectively. PPV of these tests viz LAT-EP, LAT-CM, ELISA-EP, ELISA-CM were 71.3 %, 66.6%, 50.0%, 35.1% respectively whereas their NPV were 96.1%, 91.4%, 94.0%, 92.01% respectively. The immunological tests developed were highly specific so that they were suitable as confirmatory tests. The sensitivity of all the tests was however less than desired and needs to be further improved. PCR was used to detect coa gene of S. aureus in a few samples. This is the first report to use PCR for coagulase gene (coa) of S. aureus strains associated with SCM in buffaloes or any other animal species in India. No coa gene polymorphism was detected in S. aureus strains circulating in buffalo farm at HAU. Overall prevalence of S. aureus associated SCM in buffalo farm was found to be 14%, 11%, 16% and 14% by LAT-EP, LAT-CM, ELISA-EP and ELISA-CM respectively. The prevalence of S. aureus associated SCM in buffaloes in Haryana in the present study indicated the need for improvement of managemental practices in the organized farm.ThesisItem Restricted Some aspects of diagnosis, pathogenesis and therapy of sub-clinical mastitis in buffaloes(LUVAS, 2008) Basiri, Mohammad Shapoor; SridharThe present investigation was conducted on lactating buffaloes from Animal Farm CCS HAU Hisar and also from Hisar city and surrounding areas on some aspects of pathogenesis and therapy of sub-clinical mastitis. Indirect laboratory diagnostic tests like California mastitis test (CMT), somatic cell count (SCC) and milk conductivity test (MCT) were employed to diagnose these cases by which animal wise prevalence rate was found to be 9.62 %(18 out of 187 animals) whereas quarter wise prevalence rate was 2.44 % (18 out of 736 quarters).These rates were quite lower than those reported in literature by workers from same areas It was also found that CMT and SCC were comparatively superior tests for such diagnosis compared to MCT since former two tests were able to diagnose all the 18 cases which were considered positive while by MCT, 4 out of these 18 cases gave negative results. Hematological examination of diseased animals revealed leukocytosis and neutrophilia along with relative lymphopenia/monocytopenia and eosinophilia which was in accordance with the reports in literature. Absolute counts of individual erythrocytes also followed more or less similar pattern except in case of lymphocytes. Assessment of oxidative stress related changes showed a significantly lower level of serum inorganic phosphorus in animals with sub-clinical mastitis along with a significantly higher level of blood/milk malondialdehyde which is an indicator of lipid peroxidation in the body. These changes clearly indicated that oxidative stress is a component of pathogenesis of sub-clinical mastitis. Therapeutic studies conducted on cases of sub-clinical mastitis in buffaloes indicated that a commonly used antibiotic enrofloxacin when combined with an antioxidant (ascorbic acid) as adjunct was highly effective (100.00%) as compared to enrofloxacin alone (83.33%) or a homeopathic drug combination (Phytolacca 200, Bryonia 200, Silicea 200, Aconite 200 and Belladonna 200), efficacy of which was found to be 66.66%. Results of therapeutic trial also revealed that oxidative stress has a considerable role in the pathogenesis of sub-clinical mastitis and ascorbic acid has a potential to be an ideal adjunct to antibiotic therapy of this disease.ThesisItem Restricted Therapeutic studies on ceftiofur sodium and antioxidants in sub-clinical mastitis in cows(LUVAS, 2008) Mahesh Kumar; Goel, ParveenThe present investigation was carried out to study the prevalence of SCM in cows, to find out the oxidative stress and selenium status in sub-clinical mastitis and to study the efficacy of ceftiofur sodium and antioxidants for the treatment of SCM. As per IDF Criteria 20.39, 1.49 and 3.98 per cent quarters (out of 201 quarters and 51 animals screened) were positive for sub-clinical, latent and non specific mastitis, respectively. While considering the cultural examination and somatic cell count (SCC) alone, 21.89 per cent and 24.37 per cent quarters were found positive for SCM, respectively. Out of 44 isolates, Streptococcus dysgalactiae (50.00%) was the predominant organism isolated from SCM cases followed by Staphylococcus aureus (27.27%), Staphylococcus epidermidis (15.91%), Streptococcus agalactiae (4.55%) and Corynebacterium pyogenes (2.27%). Assessment of oxidative stress related changes showed a significantly higher level of blood/milk malondialdehyde and a significantly lower level of serum selenium in diseased cows. These results clearly indicated involvement of selenium and oxidative stress in relation to pathogenesis of sub-clinical mastitis. However, there was no significant difference in serum inorganic phosphorus levels in healthy and diseased animals. Therapeutic studies conducted on cases of sub-clinical mastitis in cows indicated that therapy with ceftiofur sodium (1.0 g intramuscularly, once a day for five days) along with vitamin E plus selenium (10ml intramuscularly, two injections at an interval of seven days) was more efficacious (83.33 %) compared to ceftiofur sodium (same dose and schedule) alone (55.55 %) or vitamin E plus selenium (same dose and schedule) alone (33.33 %). Results of therapeutic trial also revealed that oxidative stress has a considerable role in the pathogenesis of sub-clinical mastitis and vitamin E and selenium has a potential to reduce the oxidative stress of this disease.ThesisItem Restricted Detection of staphylococcus spp. by double antibody sandwich enzyme linked immuno-sorbent assay (DAS-ELISA) in bovine sub-clinical mastitis(LUVAS, 2005) Jain, Arun Kumar; Goel, ParveenIn the present investigation, milk samples from 279 quarters of 72 apparently healthy lactating crossbred cows reared at an organized farm were screened with a purpose to determine the prevalence and diagnosis of sub-clinical mastitis by DAS-ELISA and to compare DAS-ELISA with other conventional tests viz. CMT, EC, NAGase activity, SCC and cultural examination. On the basis of International Dairy Federation criteria 29.39, 12.90 and 7.17 per cent quarters were positive for sub-clinical, latent and non-specific mastitis, respectively. However, on basis of culturally examination and SCC alone were having SCM, respectively. S. epidermidis (66.94%) were found most prevalent mastitogen, followed by S. aureus (19.83%), Str. agalactiae (6.61%), Str. uberis (3.31%), Corynebacterium spp. (2.48%) and Pseudomonas aeruginosa (0.83%). Three cases of mixed infections of staphylococci and streptococci were observed. Majority of major pathogens of mastitis were associated with SCC above 5,00,000. DAS-ELISA systems specific for S. aureus and S. epidermidis were standardized separately. DAS-ELISA system developed for S. aureus can be used for screening the herd for both S. aureus and S. epidermidis mastitis. However, DAS-ELISA (S. epidermidis) is useful assay for detection of S. epidermidis mastitis only. For SCM, 31.90 per cent, 22.58 per cent, 35.13 per cent, 36.56 per cent, 42.29 per cent, 41.22 per cent and 12.90 per cent were tested positive by CMT, EC, NAGase activity, SCC, cultural examination, DAS-ELISA (S. aureus) and DAS-ELISA (S. epidermidis), respectively. While comparing conventional tests, SCC revealed highest sensitivity (69.49%) and predictive value of negative test (79.66%). On the basis of results, it can be concluded that CMT can be used as cow byre-side test and SCC as a preference laboratory method for detection of SCM. For staphylococcal mastitis using cultural examination as standard, DAS-ELISA (S. aureus) was found to be the most sensitive (74.29%) showing highest predictive value of negative test (83.54%) whereas DAS-ELISA system for S. epidermidis was found highly specific (100%) and showed highest predictive value of positive test (100%) as compared to other tests. DAS-ELISA can be used in conjunction with California mastitis test for the screening of herd for staphylococcal mastitis.ThesisItem Restricted Therapeutic efficacy of Mastacure and its effect on cellular immune response in buffalo mastitis(LUVAS, 2005) Khemani, Taruna; Yudhbir SinghThe study was conducted with respect to prevalence of sub clinical mastitis (SCM) by cultural examination and somatic cell count (SCC), assessment of therapeutic efficacy of Mastacure, a homeopathic medicine in buffalo mastitis, and to compare the cellular immune response of normal and mastitic animals along with the effect of Mastacure in modulation of immune response. Prevalence rate on the basis of cultural examination alone was 13.48 per cent of the buffaloes and 7.90 per cent of the quarters examined. Streptococcus agalactiae was the most predominant organism isolated. Five buffaloes having sub clinical mastitis were subjected to mastacure treatment for 20 days. Before treatment out of 20 quarters, 12 culturally positive quarters revealed isolation of Str. agalactiae (9) and S. aureus (3). On day 5 post treatment 83.33 per cent of the quarters revealed bacteriological cure which on day 10 and 20 post treatment decreased to 75.00 and 66.60 per cent, respectively. Again on day 30 post treatment bacteriological cure rate reached to 83.33 per cent. However, there was persistence of Str. agalactiae and S. aureus in one quarter each. The difference of mean SCC from five sub clinical mastitic buffaloes (175.20+38.30x104/ml) and five healthy buffaloes (20.4+3.76x104/ml) before the start of treatment with mastacure and on day 5 post treatment was found to be statistically significant, whereas on day 10, 20 and 30 post treatment this difference in mean SCC+SE per ml was not found statistically significant. On flow cytometry, milk from mastitic buffaloes revealed significant increase in BoCD4+ and BoWC1+ T cells as compared to normal buffaloes (P<0.05). An increase was also recorded in BoCD8+ cells of mastitic buffaloes as compared to normal buffaloes but this elevation was not significant (P>0.05). In mastitic milk BoCD8+ were present in higher proportions than BoCD4+ T lymphocytes. After administration of homeopathic medicine, down regulation of BoCD8+ T cells was observed in milk from mastitic buffaloes on day 5 and then there was gradual increase which was found significant (P<0.05) on day 20. Significantly (P<0.05) higher percentage of BoCD4+ lymphocytes on day 5, 10 and 20 were found in the milk of mastitic buffaloes. This up gradation was highly significant (P<0.01) on day 5 post treatment. BoWC1+ T lymphocytes in milk of mastitic buffaloes showed marked increase from day 5 to day 30. However, significant up gradation was encountered only on day 20 (P<0.01).ThesisItem Restricted Detection of streptococcal mastitis in bovine using polymerase chain reaction assay(LUVAS, 2009) Yadav, Rajendra; Ashok KumarA total of 449 quarter milk samples of 114 apparently healthy lactating animals from an organized cow farm were screened by PCR assay and other conventional tests viz. cultural examination (CE), California mastitis test (CMT), Electrical conductivity test (ECT) and Somatic cell count (SCC). Screening of cow farm by PCR assay using universal primers yielded 55.26 per cent animals and 42.32 per cent quarters positive for sub clinical mastitis. The prevalence rate on the basis of CE was 49.12 per cent animal wise and 22.04 per cent quarter wise. Screening of cow farm by CMT yielded prevalence rate to be 71.05 per cent animal wise and 41.65 per cent quarter wise. Results of ECT indicated 53.51 per cent cows and 20.94 per cent quarters as positive. While considering SCC (≥ 5, 00,000 cells per ml of milk) alone, 32.46 per cent of the cows and 12.69 per cent of quarters were found positive for sub clinical mastitis. In addition to samples from organized cow farm, as many as 3918 quarter milk samples of 1278 lactating cows and buffaloes brought to Veterinary College Central Laboratory, CCS Haryana Agricultural University, Hisar, by individual farmers from different parts of Haryana and neighbouring parts of Rajasthan were screened by PCR assay, CE and SCC. Of these, 76.92 per cent animals and 53.52 per cent quarters were found positive by PCR assay using universal primers. CE showed 75.74 per cent animals and 51.74 per cent quarters positive for mastitis. Milk samples of 46.64 per cent animals and 43.98 per cent quarters were found to be positive for mastitis as they were having SCC ≥500,000/ml. Out of 2097 milk samples found positive by universal primers from samples received in Veterinary College Central Laboratory, a total of 920 and 847 samples were found positive for Streptococcus spp. using genus specific primers STRicF and STRicR, and STRI and STRII, respectively. Among species specific primers for Streptococcus agalactiae, StV1 and StV2 were found to be most sensitive followed by Sag40 and Sag445, and STRA-AgI and STRA-AgII. For detection of Streptococcus dysgalactiae, primers Sdy105-Sdy386 were more sensitive than STRD-DyI and STRD-DyII. On the similar lines, Primers Sub 1546 and Sub 2170 detected presence of Streptococcus uberis in mastitic milk with higher sensitivity than STRU-UbI and STRU-UbII. All the culturally positive milk samples revealed detection of streptococci by PCR assay also. In addition to this, some of the culturally negative milk samples from antibiotic treated animals also showed positive results by PCR Therefore, PCR was found to be most specific and sensitive assay for rapid detection of Streptococcus spp. directly from milk samples as compared to conventional methods and can be used as a routine diagnostic test not only to make specific diagnosis but also for screening of large number of milk samples from large herd for selection of infected quarters for subsequent cultural examination and antimicrobial sensitivity.ThesisItem Restricted Investigations on prevalence and therapy of ear infections with special reference to malassezia pachydermatis in dogs(LUVAS, 2009) Miakhil, Jahangir; Goel, ParveenA study on the topic of “Investigations on prevalence and therapy of ear infections with special reference to Malassezia pachydermatis in dogs” was carried out during October 2008 to April 2009. The prevalence of M. pachydermatis in normal ears was found 37.94 per cent and 62.07 per cent in roll smear cytology and cultural examination, respectively and 46.88 per cent and 68.75 per cent in roll smear cytology and cultural examination, respectively was found in otitic ears. The microorganisms obtained from 64 otitic ears revealed that M. pachydermatis was the most common organism (68.75 %) followed by Staphylococcus spp., Pseudomonas aeruginosa, Proteus spp., E-coli, Streptococcus spp., Candida spp. and Aspergillus spp. Staphylococci were the main organisms isolated in association with M pachydermatis. It was observed that the highest (77.78%) of the cases of otitis externa associated with M. pachydermatis were reported during the months of October. The highest percentage of Malassezia otitis externa was recorded in dogs with age four to six years old where German shepherds was the most susceptible breed followed by Labrador, Spitz and Pug. In clinical cases of dogs with Malassezia pachydermatis ear infections, presence of brownish-black or yellowish colour and foul smelling purulent ceruminous exudate in external ear canal with pruritus was conspicuous. Microscopically, the cells of Malassezia pachydermatis were seen as oval to pea-nut Gram-positive unicellular yeast cells which reproduced by mono-polar budding on a broad base. SDA with and without chloramphenicol has been used for routine M. pachydermatis culture work. However, blood and nutrient agar failed to support the growth of M. pachydermatis. The Malassezia pachydermatis colonies were seen as convex, smooth, opaque, cohesive and crenated having pasty texture with entire edges. Biochemically, all the strains of M. pachydermatis were urease positive and weak catalase positive while no indole and coagulase production was noticed. All the strains were found negative for Methyl red, Voges-Proskauer reactions and nitrate reduction tests. Among the drugs used for treatment of otitis externa associated with M. pachydermatis the combination of chlorhexidine with fluconazole was recorded as superior compare to others. In in-vitro sensitivity tests, M. pachydermatis showed the most sensitivity in decreasing order of efficacy to ketoconazole, Nystatin, Fluconazole, Itraconazole and Clotrimazole antimycotics.