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20213
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Subject: Veterinary Microbiology × End date: 2021 × Start date: 2021 ×
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    VIRULENCE GENE PROFILE AND ANTIMICROBIAL RESISTANCE OF STREPTOCOCCUS ISOLATES ASSOCIATED WITH BOVINE MASTITIS.
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2021-05) TEJASWI, PENUGONDA; RAMANI PUSHPA, R.N (MAJOR); ANAND KUMAR, P; SRINIVASA RAO, T
    Bovine mastitis is recognised as the most economically important disease affecting Dairy industry in India and all over the world. Most prevalent bacterial etiological agents identified in bovine mastitis are Staphylococcus aureus, Streptococcus species, E. coli and other Gram-negative organisms. The preferred treatment regime during the past decades for mastitis is antibiotic therapy. Streptococcus species are one of the most important causative agents of mastitis. Usually mastitis caused by Streptococci is of the subclinical type, so early detection is important. Among Streptococcus species S. agalactiae, S. dysgalactiae and S. uberis are predominant next to Staphylococcus species. The ability of Streptococcus to initiate growth in vivo and stably infect a host requires acquisition of virulence factors capable of neutralizing the mechanisms of host defence. Hence the present study is on antimicrobial susceptibility and detection of virulence factors of Streptococcus species associated with bovine mastitis. A total of 108 Bovine mastitis milk samples were examined and out of this 60 (55.5 %) samples were positive for Streptococcus species. The most prevalent isolate obtained was S. uberis 31 (51.7 %) followed by S. agalactiae 13 (21.7 %) and S. dysgalactiae 11 (18.3 %). All the isolates of Streptococcus spp. were identified based on morphology, cultural examination and further subjected to various biochemical tests viz., catalase test, oxidase test, ninhydrin test and sugar fermentation tests. Streptococcus selection broth was used for primary isolation followed by inoculation on to Edward’s medium with 5% sheep blood. The samples with Gram positive cocci in chains on Gram’s staining and cultures showing greyish, pinpointed colonies and/or esculin hydrolysis on edward’s medium were tentatively identified as Streptococcus species. All Streptococcus species isolates were further confirmed by PCR using genus and species specific primers. All the isolates of S. agalactiae were also subjected to CAMP test. Five Streptococcus isolates did not react to any of the specific primers used for S. uberis, S. agalactiae and S. dysgalactiae, hence categorised under Streptococcus other than these three species. Virulence factors of Streptococcus species were detected by using PCR. pauA gene was detected in eighteen (58.1 %) isolates and skc was detected in six (19.3 %) isolates of S. uberis. cfb gene (CAMP factor) was detected in twelve (92.3 %) isolates of S. agalactiae but phenotypically only five isolates were positive for CAMP test. In S. dysgalactiae mig gene was detected in 6 (54.5 %) isolates and skc gene was detected in one (9.1 %) isolate. The luxS gene responsible for biofilm formation was detected in eleven (35.4 %) isolates of S. uberis. xv On antibiotic sensitivity test the S. uberis isolates were resistant to ampicillin/ sulbactum followed by kanamycin, penicillin, ceftriaxone, tetracyclin, gentamicin and cotrimoxazole. Least resistance was observed for chloramphenicol, enrofloxacin and amoxicillin/ clavulanic acid. S. agalactiae isolates showed high resistance towards ampicillin/ sulbactum followed by kanamycin, penicillin G, enrofloxacin, chloramphenicol and gentamicin. Least resistance was found to ceftriaxone, amoxicillin/ clavulanic acid, tetracyclin and cotrimoxazole. Antibiogram of S. dysgalactiae revealed that the isolates were resistant to ampicillin/ sulbactum followed by kanamycin, enrofloxacin, penicillin G, chloramphenicol, gentamicin, ceftriaxone, tetracyclin and cotrimoxazole. These isolates showed least resistance towards Amoxicillin/ Clavulanic acid. Minimum inhibitory concentrations (MICs) of selected panel of antibiotics against S. uberis, S. agalactiae and S. dysgalactiae were studied by conducting modified resazurin dye microdilution assy. The MIC of S. uberis for penicillin-G is 15.62 μg/ml followed by 3.90 μg/ml for amoxicillin, 7.80 μg/ml for enrofloxacin, 78 μg/ml for gentamicin, 312.50 μg/ml for sulphamethoxazole + trimethoprim and 1250 μg/ml for sulphamethoxazole. For S. agalactiae the MICs for penicillin G, amoxicillin, enrofloxacin, gentamicin, sulphamethoxazole + trimethoprim and sulphamethoxazole were 15.62 μg/ml, 3.90 μg/ml, 31.25 μg/ml, 156 μg/ml, 625 μg/ml and 2500 μg/ml respectively. For S. dysgalactiae the MICs for penicillin G, amoxicillin, enrofloxacin, gentamicin, sulphamethoxazole + trimethoprim and sulphamethoxazole were 7.80 μg/ml, 0.98 μg/ml, 3.90 μg/ml, 39 μg/ml, 1250 μg/ml and 2500 μg/ml respectively.
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    ThesisItemOpen Access
    MOLECULAR SEROTYPING OF Avibacterium paragallinarum AND DETECTION OF THE GENETIC DETERMINANTS OF ITS VIRULENCE FACTORS
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2021-04) MOHAMMED UMAR, SHAIK; ANAND KUMAR, P (MAJOR); LAKSHMI KAVITHA, K; ASWANI KUMAR, K
    Infectious coryza caused by Avibacterium paragallinarum is a highly contagious respiratory infection in poultry affecting the upper respiratory tract with the involvement of nasal passages, infra orbital and paranasal sinuses of chickens. In the present investigation samples were collected from suspected cases of infectious coryza in commercial poultry farms and backyard poultry in East and West Godavari districts of Andhra Pradesh, for detection of A. paragallinarum. To detect A. paragallinarum, the samples were suwith the samples. Out of total 150 samples collected, 35 samples did not yield any result specific to A. paragallinarum as they were processed after six months of their collection due to COVID 19 national lockdown. The remaining 115 samples were processed immediately after collection to detect A. paragallinarum. A total of twelve samples (B1, B2, S1, S2, J1, J2, BA1, BA2, BA3, BA4, BA5 and BA6) were found to be positive for A. paragallinarum in cultural (with satellitism) and biochemical tests. In both the culture PCR test and direct swab PCR test with oligonucleotide primers specific to A. paragallinarum, all the 12 isolates yielded a specific PCR product of 500 bp. The direct swab PCR test is found to be sensitive to detect A. paragallinarum in samples collected from infectious coryza cases, without the need of culture step, which is very helpful in rapid diagnosis. In PCR test for detecting genetic determinant of putative virulence factor haemagglutinin (hagA) only 4 isolates of A. paragallinarum viz. B1, B2, J1 and J2 yielded specific PCR product of 900 bp. However, the genetic determinant of iron acquisition protein (fur) was not detected in all the 12 isolates of A. paragallinarum. In molecular serotyping by multiplex PCR (mPCR) test using oligonucleotide primers specific to Page serotypes A, B and C, 8 isolates of A. paragallinarum viz. B1, B2, S1, S2, J1, J2, BA3 and BA4 were identified as serotype A with a PCR product of 372 bp, though the A. paragallinarum vaccine used as positive control in this study yielded PCR products of 372 bp and 800 bp. The remaining 4 isolates viz. BA1, BA2, BA5 and BA6 were identified as serotype B with a PCR product of 1100 bp. None of the A. paragallinarum isolates in this study were identified as serotype C. The results of the present investigation with the molecular serotyping indicates that serotype A and B of A. paragallinarum are circulating in East Godavari and West Godavari Districts of A.P. However, further research is required with large number of samples to identify the circulating serotypes of A. paragallinarum in A.P.
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    ThesisItemOpen Access
    INVESTIGATION OF INTERCONNECTION BETWEEN ANTIMICROBIAL RESISTANCE TO BIOCIDES AND ANTIBIOTICS IN Staphylococcus SPECIES ISOLATED FROM BUFFALOES
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2021-04) NAVYA, M; ANAND KUMAR, P (MAJOR); RAMANI PUSHPA, N; BINDU KIRANMAY, CH
    The present study is undertaken to investigate the interconnection between antimicrobial resistance to biocides and antibiotics in Staphylococcus species isolated from buffaloes and to know the distribution of these genetic determinants in the isolates. Based on culture on mannitol salt agar and conventional biochemical tests (catalase test, oxidase test and coagulase test) 73 samples (60.84%) out of 120 collected in the present study were provisionally confirmed for Staphylococcus species. In PCR test with Staphylococcus genus specific primers 16S rRNA, all the 73 samples yielded PCR product of 228 bp. In PCR test with S. aureus specific oligonucleotide primers Staur 4 and Staur 6, 19 out of 73 Staphylococcus species isolates yielded specific PCR product of 1250 bp. Certain Staphylococcus species (other than S. aureus) are found to be coagulase negative staphylococci (CoNS). The S. aureus isolate P1 is a mannitol non-fermenter, but positive for coagulase production. With regard to antibiotic sensitivity of 73 Staphylococcus species isolates in in vitro antibiotic sensitivity test (AST) with antibiotic discs, majority isolates were sensitive to Gentamicin (n=71, 97.26%) followed by Amoxicillin/Clavulanic acid (n=70, 95.89%), Chloramphenicol (n=68, 93.15%) and Enrofloxacin (n=63, 86.30%), but highly resistant to Oxacillin (n=64, 87.67%). The Multiple Antibiotic Resistance index results with all the Staphylococcus species isolates, including S. aureus showed that a total of 31 samples (42.46%) showed the MAR index below 0.2 and remaining 42 samples (57.53%) showed the MAR index ≥ 0.2. In PCR test for detecting the genetic determinants of antibiotic and biocide resistance, out of 73 isolates of Staphylococcus species (including S. aureus) a total of 44 (60.27%) were blaZ positive, 42 (57.53%) were mecA positive, 32 (43.83%) were tetK positive, 6 (8.22%) were qacA/B positive and 5 (6.85%) were smr positive. In Staphylococcus species other than S. aureus, the coexistence of genetic determinants of biocide and antibiotic resistance were detected only in 3 isolates viz. M5, M7 and S11. In S. aureus isolates the coexistence of genetic determinants of biocide and antibiotic resistance were detected only in 5 isolates viz. M9, M10, N4, N5 and N6. In conclusion, perhaps for the first time the genetic determinants of biocides and antibiotic resistance in Staphylococcus species isolated from buffaloes is investigated in Andhra Pradesh. However, further detailed investigation is required with a greater number of samples from different settings.