Dr. R.N. RAMANI PUSHPAJ. SHIVA JYOTHI2016-12-312016-12-3142324http://krishikosh.egranth.ac.in/handle/1/93811Dry period is a crucial phase in the lactation cycle of a dairy cow. An important factor that influences the manifestation of clinical mastitis in the next lactation is intramammary infection (IMI), which develops during or persists throughout the dry period. The present study was carried out to study the efficacy of ceftiofur as a dry-cow therapy in crossbred cows to cure infections that were presented during drying off and in preventing new intarmammary infections postpartum. The efficacy was calculated in comparison with the control group that was left untreated.The resistance pattern of various isolates to antibiotics was tested in vitro. The intramammary infections on the day of dry off, day 5 and day 28 postpartum were determined by isolation of bacterial pathogens from milk samples. Isolates were initially identified based on Gram’s staining and then by colony morphology. The tentatively identified organisms were subjected to biochemical characterisation for final confirmation by standard bacteriological tests. A total of 351 isolates of staphylococcus sp. 18 of Streptococcus sp. 154 of E.coli and 18 of Pseudomonas sp.were isolated from milk animals of control and treatment groups collected on day dry off, day 5 and day 28 post partum. The efficacy of Ceftiofur was studied in terms of cure rates and new intramammary infections postpartum. Results showed that the intramammary cure proportions rate in treatment group on day 5 and day 28 postpartum was 29.1% and 55.9% respectively, while in control group a spontaneous cure rate of 4% was observed. The proportion of new intramammary infections was 8.14% and 16.2% in treatment group which was comparatively less than the control group that had 40% and 48% of new intramammary infections on day 5 and day 28 post partum. Significant difference (P≤0.05) was found for cure rates and new intramammary infections between the groups. Antibiogram studies for isolates showed that ceftiofur was 90% sensitive for E.coli, 60% for Staphylococcus sp. and Pseudomonas sp. and 58% for Streptococcus species. Estimation of Somatic Cell Count (SCC) was carried out to identify cases of subclinical mastitis. The mean of SCC for staphylococcus infections was 12.4x106cells/ml, the mean of SCC with cows more than two pathogenic infections was 134x106cells/ml and the milk samples having mean of SCC less than 2x105cells/ml were culture negative. Antibiogram of the Staphylococcus isolates treated with ursolic acid showed significantly (P<0.01).higher sensitivity to antibiotics when compared to untreated isolates. Staphylococcus isolates (200) were examined for biofilm production on Congo Red Agar (77.5%), Coagulase activity (6%), Methicillin resistance on ABST (17.5%) and haemolytic activity (21.5%). Correlation studies showed that the methicillin resistance pattern and biofilm production were positively correlated (P<0.05). Similarly haemolysin production and coagulase activity were also positively correlated at (P<0.01) level. The in vitro studies of ursolic acid in inhibiting biofilm formation by Staphylococcus sp by Microtiter Plate assay (MTP) showed 71.5% and 48.6% inhibition at concentrations of 60μg/ml and 30μg/ml respectively. Statistical analysis showed a significant difference at (P<0.01) between the treated and untreated isolates. The Scanning electron microscopic images further qualitatively showed inhibition of biofilm formation when isolates were treated with ursolic acid. Molecular detection of icaD, mecA and pvl gene for biofilm production, methicillin résistance and leukocidin toxin of Staphylococcus isolates (93) showed that 73 isolates were positive for icaD, 15 isolates for mecA while all the isolates were negative for pvl gene. Comparison of diagnostic sensitivity and specificity of the tests was done to detect biofilm formation which showed that the microtiter plate method was the best method when compared to Congo red agar method. The present study showed high prevalence of infections on the day of dry off and subsequent treatment of ceftiofur was effective in eliminating subclinical intramammary infections in dairy cattle. Intramammary infections due to biofilm producing Staphylococcus sp. were difficult to treat even with intramammary antibiotics. Detailed studies on infections produced by biofilm forming pathogens are warranted. Development of therapeautic regimes with combination of antibiotic and effective antibiofilm agent may provide successful control of mastitisbacteria, mastitis, livestock, biological phenomena, drying, antibiotics, animal husbandry, economic systems, acidity, productivityThesis