Browsing by Author "Mhase, P. P."
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ThesisItem Open Access Bacteriological Studies on Mastitis In Goats (Capra falconeri)(PKV, Akola, 1997) Mhase, P. P.; Harne, S. D.ThesisItem Open Access Characterization of Pasteurella spp.From Respiratory Tract of Small Ruminants(MAFSU, Nagpur, 2017) Daphal, S. H.; Mhase, P. P.ThesisItem Open Access Detection of Brucellosis By Serological And Molecular Techniques in Bovines(MAFSU, Nagpur, 2015) Walunj, T. D.; Mhase, P. P.Brucellosis is an important infectious disease of livestock affecting a wide range of animal species including human and is characterized by abortions, fetal death, reproductive tract complications and arthritis in animals with great losses. Early precise diagnosis with more sensitive and specific test is a very important for its control and eradication. For the diagnosis of brucellosis different serological tests are used widely, but often they come with certain disadvantages like longevity and lack specificity and gold standard isolation cultural test requires special biosecurity facilities and poses a danger of infection. Hence, highly sensitive genus specific molecular techniques are preferred. Hence present studies dealt with the screening of farms having clinical brucellosis with serological tests; RBPT and iELISA followed by the PCR assay. Total 401 recently aborted and ‘In-contact’ animals were screened with RBPT belonging to the organized and unorganized farms of Pune and Ahmednagar region out of whihc 200 samples each were processed by iELISA employing two kits, one indigenous (ELISA-1) and another was commercial (ELISA-2). The serum and whole blood samples (24 each) were processed with genus specific BCSP 31 PCR for detection of brucellosis and comparative evaluation of serological tests. With the RBPT, ELISA-1 and ELISA-2 the percent seropositivity in cattle of farms in Pune region was 27.18%, 42.00% and 48.00% respectively, while from the Ahmednagar region it was 36.76%, 44.00% and 50.00% respectively, thus seropositivity observed was 30.99% , 43.00% and 49.00%, respectively. The percent seropositivity detected in Buffaloes of Pune region was 35.71%, 45.00% and 47.50% respectively, while from the Ahmednagar region it was 41.48%, 48.33 and 50.00% respectively, hence total was 40.08%, 47.00% and 49.00%, respectively. With RBPT total percent seropositivity was 36.40%, with ELISA-1 it was 45.00% and with ELISA-2 it was 49.00% in buffaloes. The percent seropositivity for brucellosis in the cattle of organized farm of Pune indicated 25.86%, 41.37% and 48.27% while on the unorganized farm it was 28.88%, 42.85% and 47.61%, respectively. Similarly in the cattle from Ahmednagar district seropositivity detected on organized farms was 35.55%, 50.00% and 59.37% while in unorganized farms it was 39.13%, 33.33% and 33.33%, respectively. Overall seropositivity in cattle from organized farms was 30.09%, 45.90% and 54.09% and in unorganized farms was 32.35%, 38.46% and 41.02% respectively, thus overall in cattle it was 30.99%, 43.00% and 49.00%, respectively. The percent seropositivity in buffaloes of Pune organized farms was 34.61%, 42.30% and 46.15% while in unorganized it was 37.50%, 50.00% and 50.00%, respectively. Percent seropositivity in organized farm buffaloes was 39.86%, 46.15% and 49.23% respectively, while unorganized farms it was 41.66%, 48.57% and 48.50 % respectively. Hence, total was 40.08%, 47.00% and 49.00%, respectively. The aborted cattle and buffaloes with of Pune serologically were 42.85% and 66.66%, 72.72% and 66.66%, 81.81% and 55.55%, while Ahmednagar region were 47.61% and 34.54, 53.33% and 66.66 %, 73.33% and 77.77%, positive respectively. The In-contact cattle and buffaloes of Pune were 23.17% and 20.00%, 33.33% and 38.07%, 38.46% and 45.16% while of Ahmednagar were 31.91% and 44.36%, 40.00% and 39.53 %, 40.00% and 38.09%, positive, respectively. It was concluded that brucellosis was more in animals belonging to Ahmednagar than Pune, was higher in buffaloes than in cattle, was more in the aborted than In-contact animals and higher in animals of organized farms than unorganized farms. The seropositivity was marginally higher in ELISA-2 kit than Indigenous ELISA-1 followed by RBPT. A 223 bp amplification product was observed in 30/48 samples with genus specific BCSP31PCR out of which 62.50% each of cattle and buffaloes’ samples yielded positive. The proportion of positive cases in aborted animals was higher (82.14%) than ‘In-contact’ (44.44%) cases. In serum (66.66%) positivity was higher than in blood (58.33%) samples. In aborted (91.66) cases positivity was higher than In-Contact cases (33.33%). The positivity in Ahmednagar (70.83%) was higher than in Pune(54.16%). The results of PCR compared with RBPT revealed cent percent sensitivity (100%) but the specificity was 83.33% and agreement was 90.91 %. ELISA-1 revealed sensitivity of 90.00% but cent percent (100%) specificity and 90.00% agreement while ELISA-2 presented best results with cent percent specificity (100%) and 96.66% sensitivity with 96.67% agreement with PCR in detecting Brucellosis.ThesisItem Open Access Detection of Multidrug Resistance MDR Staphylococcus Aureus from Mastitis Infected Cows(MAFSU, NAGPUR, 2023-02-20) Ramteke Manali Diwakar; Mhase, P. P.Cattle are frequently affected by mastitis, an infection of the mammary gland that lowers milk production and ultimately has an effect on the economy. One of the most common bacteria that cause mastitis is Staphylococcus aureus. S. aureus occurring in mastitis frequently exhibits resistance to commonly employed antibiotics which is of great concern. Therefore, current investigation was planned for studying mastitis in dairy cows, characterization of the major pathogen S. aureus, and its present scenario, especially regarding the antibiotic sensitivity pattern in the area around Shirwal, Satara district in western Maharashtra. For this study a total of 250 Holstein Friesian (HF) crossbred cows from unorganized dairy farms were screened for mastitis. Out of these 68% of cows tested positive for mastitis. Mastitic milk samples were processed for the isolation of pathogenic microorganisms and their antibiotic sensitivity pattern. S. aureus strains were targeted for characterization with phenotypic as well as genotypic methods using 16s rRNA gene and species-specific nuc gene analysis. S. aureus strains were also presented for Antibiotic Susceptibility Test (ABST), and resistant strains showing multidrug resistance (MDR) were subcultured. S. aureus represented the highest resistance to ß lactam group of antibiotics (98.05%) followed by macrolide group (95%). Amongst the drugresistance genes studies, highest number of S. aureus carried ermA (78.33%) gene followed by tetK (61.67%) gene. An attempt was made to synthesize and employ the Green Silver Nanoparticles (GAgNPs) as an alternative therapeutic agent. Different concentrations of an aqueous leaf extract of Azadirachta indica were used successfully as a reducing agent for the biosynthesis of GAgNP from Silver Nitrate producing brown colored suspension in 48 hours. GAgNPs were characterized by Spectrophotometry where peak absorbance was produced at 405 nm. Transmission Electron Microscopy (TEM) of GAgNPs revealed spherical nanoparticles of 10 to 70 nm in size which readily attached to the bacterial cell membrane, disrupting it and demonstrating bactericidal activity.ThesisItem Open Access Efficacy of Biodegradable Lipid Glyceryl Monostearate Lipomer of Dosyclycline Against Brucella Abortus(MAFSU, Nagpur, 2012) Mhase, P. P.; Bannalikar, A. S.The efficacy of doxycycline hydrochloride (DH), doxycycline hydrochloride lipomer (DH LNP), rifampicin (RIF), rifampicin entrapped in polymer Gantrez 119 (NP RIF) and the combinations of these drug formulations against B. abortus was evaluated in vitro in U937 human macrophage cell line and in vivo in mice. All the drug formulations were subjected to cytotoxicity assays, uptake studies and estimation of minimum inhibitory concentration (MIC) before taking up efficacy studies. The assessment of cytotoxicity by MTT assay and trypan blue dye exclusion technique revealed that the maximum non-toxic concentration of DH was 5 μg/ml whereas that of DH LNP was 2 μg/ml. The maximum non-toxic concentration of RIF was 10 μg/mL while that of NP RIF 5 μg/mL. The maximum non-toxic concentration of combinations DH + RIF and DH LNP + NP RIF DH + RIF was 20 μg/mL Both the methods i.e. MTT assay and trypan blue dye exclusion technique yielded similar results and were found equally suitable. The minimum inhibitory concentration (MIC) of the drug formulations against B. abortus was estimated by resazurin microtitre assay (REMA) and broth microdilution method (BMM). The MIC of DH, DH LNP, RIF, NP RIF and DH + RIF was 0.5 μg/ml while that of combination DH LNP + NP RIF was 0.25 μg/ml. The results of MIC produced by both the methods, REMA and BMM were concordant with each other. The studies on uptake of the nanoparticulate drugs by the macrophages by fluorescence microscopy revealed that at higher doses the formulations were taken up rapidly while the uptake was poor at lower concentrations. All the formulations attained optimum intracellular concentration at 1 μg/ml between 30 to 60 min. Evaluation of in vitro efficacy DH and DH LNP against B. abortus in macrophages revealed that the reduction in log10 cfu/ml values achieved in DH treated cells was slightly greater that that of DH LNP treated cells. The DH LNP thus did not prove adequately effective in reducing the intracellular B. abortus. The RIF and NP RIF both caused significant reduction in the intracellular B. abortus counts at all the concentrations tested. Comparison of log10 reduction values by unpaired ‘t’ test revealed that the reduction in intracellular B. abortus counts achieved by NP RIF was significantly greater than that of RIF at all the doses ranging from 0.250 – 4 μg / ml. The results thus revealed a definite superiority of NP RIF over RIF in clearance of intracellular B. abortus. The combinations of DH + RIF and DH LNP + NP RIF were found effective against B. abortus and caused significant reduction in intracellular counts as compared to untreated control. The comparison of results of DH + RIF and DH LNP + NP RIF unpaired ‘t’ test revealed that the log10 reductions achieved in the cells treated DH LNP + NP RIF were significantly higher than those of DH + RIF treated cells at all the concentrations tested. Overall comparison of the values of log10 reductions achieved in response to treatment with different drug formulations revealed that the greatest log10 reduction in intracellular B. abortus counts was evident in cells treated with NP RIF followed by those treated with DH LNP + NP RIF. The drug formulations next in order in terms of efficacy were plain RIF and combination of DH + RIF. The evaluation of in vivo efficacy of DH and DH LNP in mice revealed that the reduction in splenic B. abortus counts achieved with both the treatments was almost equal. The DH LNP treatment thus did not prove superior to DH. The RIF and NP RIF both caused reduction in mean log10 cfu per spleen values as compared to untreated control mice. The comparison of log10 reduction between RIF and NP RIF treated mice revealed that the reduction achieved in NP RIF treated mice was significantly greater (over two fold) than that of RIF treated mice. Evaluation of in vivo efficacy of combination RIF + NP RIF revealed that there was a complete clearance of B. abortus from spleens of infected mice. Comparison of log10 reductions achieved in spleens of mice treated RIF and RIF + NP RIF revealed that the reduction in RIF + NP RIF treated mice was significantly higher (4.5294+ 0.001) causing complete clearance of B. abortus from spleens. The combination RIF + NP RIF thus could be of potential value in therapeutic management of brucellosis in animals and human. The future research should focus on evaluation of RIF + NP RIF combination in clinical cases of brucellosis in animals.ThesisItem Open Access Molecular Characterization of Bacterial Pathogens Associated with Bovine Mastitis(MAFSU, Nagpur, 2015) Rohokale, J. S.; Mhase, P. P.Mastitis is multi-etiological disease of dairy animals characterized by physical, chemical and usually bacteriological changes in milk and pathological changes in glandular tissues of udder. Mastitis has evolved as a disease of highest economic significance particularly to the dairy industry worldwide. Financial losses arise from the costs incurred on treatment, culling, decreased milk production, sometimes permanent loss of udder, decreased milk value and even death of animal. For monitoring udder health, it is necessary to implicate the reliable and affordable diagnostics method and constant need to improvise them. In the present study easy, cheap and rapid MALDI-TOF MS technique was applied for detection of pathogens associated with mastitis keeping PCR results as reference particularly for major pathogens. During present investigation a total of 235 dairy animals from unorganized and organized farms located in Pune and Satara region were examined using CMT to study prevalence of mastitis. All 136 CMT positive samples were further processed for bacterial culture in enrichment medium which generated 127 culturally positive samples from which 203 pure bacterial colonies were isolated culturally as single or as mixed infections. The major pathogens were identified upon growing them on selective media like EMB and MacConkey’s agar for E. coli and on MSA agar for S. aureus, respectively. They were confirmed by biochemical catalase, oxidase and IMViC tests. Accordingly, total 80 (62.9 %) isolates were detected as S. aureus and 53 ( 41.7 %) as E. coli. All the isolates were processed for antibiotic sensitivity testing and overall rate of sensitivity indicated, highest rate of sensitivity in 159 isolates (78.3 %) for gentamicin followed by enrofloxacin 155 (76.3 %), ceftriaxone 122 (60 %), ciprofloxacin 97 (47.7 %), chloramphenicol 73 (35.9 %), streptomycin 44 (21.6 %), oxytetracycline 25 (12.3 %) and penicillin 9 (4.4 %) and overall rate of resistance pattern of all isolates indicated highest rate of isolates i.e. 192 (94.5 %) being resistant to Penicillin followed by oxytetracycline 149 (73.4 %), streptomycin 132 (65 %), chloramphenicol 88 (43.4 %), ciprofloxacin 78 (38.4 %), ceftriaxone 52 (25.6 %), gentamycin 36(17.7 %) and enrofloxacin 34(16.7 %). Culturally detected E. coli and S. aureus were subjected to molecular detection with PCR technique so as to use the results as reference for evaluating MALDI TOF MS. Using femA gene PCR, 80 isolates identified culturally were specifically (100 %) confirmed as S. aureus which generated 132bp size product upon electrophoresis. Total 53 E. coli isolates when treated with 16SrRNA PCR were also confirmed specifically (100 %) which yielded 232 bp size bands on electrophoresis of their PCR end product. Randomly selected isolates of E. coli (n = 25) and S. aureus (n = 25) were further processed with MALDI-TOF MS technique; keeping PCR result as reference. Along with these the other gram positive (n = 32) and gram negative (n=38) bacterial isolates were also processed which were generated during bacterial culture of milk sample for their identification. MALDI-TOF MS technique was found highly accurate and rapid in detection of major bacterial pathogens like E. coli and S. aureus with highest specificity (100 %) other unidentified bacterial isolates associated with mastitis from freshly cultured media were identified confirmed on genus and probable species level with MALDI TOF MS as Salmonella anatum 4 (5.7%), Streptococcus dysgalactiae, Enterobacter cloacae, S. xylosus, Proteus mirabilis, Clostridium difficile, Enterococcus faecium, all were 3 in no. ( 4.2 %), S. hominis, Klebsiella oxytoca, Stenotrophomonas spp., S. schleiferi, Klebsiella oxytoca, Pseudomonas flurescens, S. hominis, S. epidermidis, S. cohnii, Proteus vulgaris, Streptococcus faecalis, Clostridium beijerinckii all were found 2.85 % ( 2 in no.) Lactobacillus agilis, Aeromonas encheleia, Lactobacillus aviarius, Camphylobacter jejuni (all were 1.42 %). Some of the different species were also detected during present studies which were Cuprividus hector, Azoarcus communis, Arthrobacter polychromogen. While some organisms remained as unidentified as peak score observed was not optimum. From the present findings it may be concluded that, MALDI-TOF MS is a robust assay in case of detection of disorders presenting polymicrobial etiologies like in case of mastitis with high confidence and shortest possible time. MALDITOF MS is a fast and truthful technique which has capability to replace conventional identification of multiple bacterial strains usually associated with udder environment and milk of bovine mastitis.ThesisItem Open Access Molecular Characterization of Mycobacteria Recovered from Animal and Human Specimens.(MAFSU, Nagpur., 2012) Dalvi, S. A.; Mhase, P. P.The present investigation dealt with isolation of mycobacteria from human and animal specimens and their characterization by conventional and molecular techniques. Processing of 165 human and animal specimens resulted in recovery of 23 mycobacteria of which 2 (8.7%) were rapidly growing while 21 (91.3 %) were slow growing. On characterization by conventional biochemical tests 13 (56.5 %) isolates were found to be Mycobacterium tuberculosis whereas remaining 10 (43.5 %) were categorized as nontuberculous mycobacteria (NTM). A total of 12 isolates were recovered from 132 animal specimens with isolation rate of 9.09 % whereas, 11 isolates were recovered from 33 human sputa with an isolation rate of 33.33 %. Out of the 33 sputum samples 13 (39.39 %) were found positive for presence of acid-fast bacilli by direct microscopy. The cultural isolation was found have lower sensitivity as compared to direct microscopy. Characterization of 12 isolates from animal specimens by conventional tests revealed their species-wise distribution as; M. avium (8), M. fortuitum (2) and M. tuberculosis (2) whereas that of 11 isolates from human samples confirmed their identity as M. tuberculosis. All the 23 isolates and reference strains M. tuberculosis H37Rv, M. bovis 3/86 and M. avium were analyzed by PRA of hsp65 gene. In PCR using primers Tb11 and Tb12 amplification of 439 bp products was found in all the isolates. Restriction digestion of the amplicons with BstEII and HaeIII in separate reactions and analysis of RE digests by 3% agarose gel electrophoresis revealed distinct RFLP patterns characteristic for each species. Based on the results of PRA, 12 isolates recovered from animal specimens were identified as M. avium I (8) and M. fortuitum II and MTBC (2 each). The PRA of hsp65 could identify M. avium and M. fortuitum to subspecies level whereas it could not distinguish between M. tuberculosis and M. bovis. IS6110 PCR assay was applied on 13 isolates identified as MTBC on the basis of results of PRA of hsp65 and reference strains M. tuberculosis H37Rv and M. bovis 3/86. All the 13 isolates and reference strains yielded specific amplification product of 445 bp confirming their identity as the members of MTBC. In order to confirm species level identity of 13 MTBC isolates a single tube multiplex PCR targeting 12.7 Kb fragment was performed. All the 13 clinical isolates and Mycobacterium tuberculosis H37Rv yielded specific amplification product of 389 bp confirming their identity as M. tuberculosis. M. bovis 3/86 on the contrary revealed amplification of 823 bp product.ThesisItem Open Access Molecular Detection of Mycoplasma gallisepticum in Chicken(MAFSU, Nagpur, 2021) Vanjari, A. D.; Mhase, P. P.The present investigation was undertaken in periphery of hundred kilometers around Shirwal with aim of detection of M. gallisepticum infection in poultry. Out of 106 tissue samples tested by PCR, 7 (6.60 %) were positive for M. gallisepticum, whereas all 24 choanal swab samples were negative. Four flocks out 11 were positive for M. gallisepticum infection. Flocks from Bhor and Wai area were positive for M. gallisepticum by specific PCR on the pooled tissues. PCR positive samples were used for isolation of M. gallisepticum in view of their confirmation with gold standard. However, M. gallisepticum was culturally isolated from 05/99 (5.05 %) of tissue samples. Sequence analysis of PCR amplicons from standard control (M. gallisepticum vaccine strain DNA) and field sample was carried out using SeqMan DNASTAR and NCBI BLAST. Blast analysis of field sample showed highest alignment with PB1/06/Ind. The novel PSR technique with different gradient combinations using 16SrRNA and lipoprotein gene primer sets were attempted with different gradient combinations of temperature (60 ºC, 61 ºC , 62 ºC , 63 ºC, 64 ºC, 65 ºC); extension time (20 min, 30min, 40min, 50 min, 60 min, 120 min for time) and MgSO4 (2,3,4,5,6 mM) concentrations. But, none of the tested protocols yielded positive results, hence, primer sets and reaction conditions used in this study were not found optimum for PSR detection of M. gallisepticum.