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Browsing Thesis by Author "A. K. Kataria"
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ThesisItem Open Access Genotypic Characterization of Staphylococcus aureus from Clinical Mastitis in Cattle in Relation to Some Virulence Factors(Rajasthan University of Veterinary and Animal Sciences, Bikaner - 334001, 2011) VIKAS KHICHAR; A. K. KatariaBovine mastitis caused by Staphylococcus aureus is a worldwide disease of high economical importance. Because of great heterogeneity in the phenotypic properties of natural populations of this organism it is of value to use DNA based methods for its identification with certainty. This organism acquires resistance very early towards commonly used antibiotics making the treatment of S. aureus infections difficult. Additionally, S. aureus organisms are blessed with many of the virulence factors which make them to evade host immune system. In the present study S. aureus isolates from milk obtained from H-F crossbred and Rathi cattle with clinical mastitis were characterized for their phenotypic properties, antibiogram determination and genotypic identification along with characterization of these isolates for some virulence genes viz. coa, spa, cap genes. Among the cultural and biochemical properties of all the 28 S. aureus isolates it was recorded that the colonies of organisms developed were glistening, smooth and round with variable pigmentation. On the basis of fermentation reactions using 11 different sugars the isolates were divisible into three fermentation groups in one of which all the isolates fermented mannitol, maltose, dextrose, sucrose and mannose; in another 96.4% of the isolates fermented fructose and in the third one 92.8% of the isolates fermented lactose. None of the isolates fermented dulcitol, raffinose, inositol and arabinose. All the isolates produced coagulase and the overall strongest coagulation reaction was recorded with plasma from rabbit followed by buffalo, cattle, camel, human, goat, sheep, dog, horse, chicken and pig. All the isolates were haemolytic and produced and toxins but not . The titre of -toxin from H-F isolates ranged between 1:40 and 1:1280 and for Rathi isolates it ranged from 1:160 to 1:2560. The titre of -toxin was between 1:5 and 1:30, and between 1:5 and 1:60 for isolates from H-F and Rathi cattle, respectively. The most effective antibiotic was azithromycin to which 92.86% of the isolates were susceptible, followed by 89.29% isolates to cloxacillin and methicillin, 78.5% to gentamicin and 57.14% to chlororamphinicol. Multiple resistance to four antibiotics viz. ampicillin, cefexime, metronidazole and nalidixic acid was shown by all the isolates. Overall a higher resistance was shown by the isolates. All the isolates were confirmed genotypically by 23S rRNA ribotyping in which a species specific amplicon of 1250 bp was obtained. Polymorphism was recorded in coa and spa genes. The coa gene produced one amplicon in each isolate but of variable sizes viz. 510, 600, 710 and 850 bp with more variability in Rathi isolates. The restriction endonuclease generated three and five RFLP patterns with isolates from H-F and Rathi cattle, respectively. The PCR amplification of X-region of spa gene responsible for production of protein A yielded amplicons of seven different sizes viz. 339, 306, 292, 277, 262, 243 and 206 bp with calculated number of 12, 11, 10, 10, 9, 8 and 7 repeats, respectively indicating presence of highly pathogenic strains. Among all the spa types four were common to both animal groups, one was unique to H-F cattle and two were unique to Rathi cattle. No such study on this aspect was conducted previously in this area. All the isolates possessed either cap5k or cap8k responsible for CP5 or CP8 capsular types, respectively. In this study 26 isolates (92.86%) produced amplicons of 361 bp indicating presence of cap5k gene and two isolates (7.14%) produced 173 bp amplicon indicating presence of cap8k gene. In conclusion, the S. aureus isolates in the present study showed variable phenotypic properties with increased antibiotic resistance and showed polymorphism in coa and spa genes. All the isolates were capsulated and highly pathogenic. One isolate (no.7) in the study was different from other isolates as it produced mustard yellow pigmentation and did not ferment fructose.ThesisItem Open Access Phenotypic and genotypic characterization of Staphylococcus aureus of mastitic milk origin from cattle and buffalo for some virulence properties(Rajasthan University of Veterinary and Animal Sciences, Bikaner - 334001, 2013) RAHUL YADAV; A. K. KatariaMastitis is one of the most important diseases of lactating animals leading to severe financial loss. Cattle and buffalo are maximum milk producing animals in India, so the occurrence of mastitis in these species is an issue of serious concern. Of the 277 many causes Staphylococcus aureus is the most important infection. This organism is blessed with many virulence factors such as, toxins, enzymes and a variety of extracellular or cell wall associated factors and the ability to develop antibiotic resistance after short period of exposure. In the present investigation the overall prevalence of S. aureus in both cattle and buffalo mastitic milk was 35.95%. These isolates were studied for their phenotypic properties including colony characteristics, fermentation of various sugars, haemolysis on blood agar, production of coagulase in tube test, slime production, DNase activity, thermostable nuclease activity, beta-lactamase activity, typing as well as titration of haemolysins and antibiogram against antibiotics of different generations and production. The genotypic confirmation of S. aureus was done by polymerase chain reaction using species-specific 23S rRNA based primers. Further the isolates were characterized by PCR for some of the genes such as coa, spa X-region, clfA, hla and hlb responsible for different virulent properties. On aerobic cultivation on nutrient agar colonies obtained were round, smooth and glistening but with variable pigment colors. Of the 12 sugars eight were fermented by variable number of isolates whereas dulcitol, inositol, arabinose and raffinose were not fermented by any of the isolates. On the basis of sugar fermentation reactions the cattle isolates were divisible into six groups and buffalo isolates into seven. All the isolates produced coagulase and human plasma showed the best coagulation reaction. THE OVERALL HAEMOLYTIC REACTIONS ON SHEEP BLOOD AGAR SHOWED THAT 15.62, 62.50, 12.50 AND 9.37% ISOLATES SHOWED COMPLETE, PARTIAL, BOTH AND NO HAEMOLYSIS REACTIONS, RESPECTIVELY. DELTA (δ) HAEMOLYSIN ON HORSE BLOOD AGAR WAS SHOWN BY NINE ISOLATES (28.12%) ONLY. On toxin titration all the isolates were found to produce alpha-toxin (maximum titre being1:5120) whereas beta-toxin (maximum titre being1:1280) was produced by 68.75% cattle isolates and by 43.75% buffalo isolates. Of the total, 15.62% isolates (two from cattle and three from buffalo) detected as delta-toxin (maximum titre being 1:80) producer. In present investigation 93.7% isolates showed DNase activity and all isolates exhibited thermonuclease activity. Slime production was recorded in very high percentage (96.87%) of isolates. We developed antibiograms against 33 antibiotics belonging to different categories and generations. Seven antibiotics namely chloramphenicol, doxycycline, gentamicin, methicillin, netiline, rifampisin and tobramycin were 100% effective against all the cattle isolates and eight antibiotics namely cefdinir, doxycycline, gentamicin, linezolid, methicillin, netiline, rifampisin and 278 tobramycin showed 100% efficacy against buffalo isolates. Both cattle and buffalo isolates were resistant to cephalexin, polymixin B, cefixime, cephalexin and amoxicillin. Beta-lactams production was detected only in 12.5% isolates from cattle and in 56.20% buffalo isolates. The ribotyping produced an amplicon of 1250 bp in all the isolates confirming them to be S. aureus. The cattle isolates were grouped into four coagulase types based on the size of coa gene amplicons (650, 600, 510 and 400 bp) and buffalo isolates were divisible into five groups (680, 650, 600, 510 and 400 bp). In the present investigation, six RFLP patterns of coa gene amplicon were generated using AluI restriction enzymes from five different coagulase types. The cattle isolates produced spa amplicons of 120, 150, 200, 250, 280, 300 and 330 bp with calculated number of repeats of 3, 4, 6, 8, 10, 11 and 12, respectively while buffalo isolates produced five different types and the sizes of spa amplicons were 150, 200, 250, 330 and 380 with 5, 7, 9, 12, and 14, respectively. The present study suggested a great variability in cattle and buffalo mastitic S. aureus isolates in regards to spa typing. Of the total, 75% isolates were considered pathogenic based on spa typing. The clfA gene was amplified in 84.30% isolates from both cattle and buffalo, producing two different amplicons of 900 (71.80%) and 1000 bp (12.50%) sizes showing polymorphism. In the present study 15 cattle and all the buffalo isolates (96.80%) amplified hla gene producing amplicons of 534 bp and the hlb gene was amplified by 13 cattle and 14 buffalo isolates (84.30%) producing single amplicon of 833 bp in each.