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ThesisItem Open Access ASSESSMENT OF IMMUNOGENICITY OF LIVE ATTENUATED GOATPOX VACCINE AGAINST LUMPY SKIN DISEASE IN CATTLE(SRI VENKATESWARA VETERINARY UNIVERSITY, TIRUPATI - 517 502. (A.P.) INDIA, 2024-03) SAI SINDHU MUMMADISETTY; DEEPTHI .B (MAJOR); SRIVANI .M; SUBHASHINI .NLumpy skin disease (LSD) is a rapidly emerging, transboundary and notifiable disease listed by the World Organization for Animal Health. LSD is being reported regularly with escalated incidence in India ever since its first appearance in 2019. Severe systemic disease is reported in the affected cattle, resulting in devastating economic losses. Mass vaccination of susceptible animals is the foremost approach in tackling infectious diseases. However, several heterologous and homologous vaccines are being used as a prophylactic measure across the globe to control LSD outbreaks. Although the efficacy and immunogenicity of homologous LSD vaccines is known to be excellent, cost of vaccine production along with the Neethling responses observed in the vaccinated animals urged the need for alternative vaccine candidates. Owing to its high degree of co linearity and amino acid identity of LSDV with other Capripoxviruses (CaPVs), heterologous vaccines employing Goatpox vaccines (GPV) and Sheeppox vaccines (SPV) can be safely used to protect against LSDV. The present study deals with an objective to determine the optimum dose of Goatpox vaccine against LSD infections in cattle. A total of 31 samples were collected from clinical cases suspected for LSD, in different areas of Andhra Pradesh during September to December, 2022. Of the 31 samples, 26 were found positive for LSDV by PCR using primers targeting CaPV- specific P32 gene and LSDV- specific RPO30 gene. Clinical samples (n=10) confirmed by PCR were considered for isolation of LSDV in Madin-Darby Bovine Kidney (MDBK) cell line. LSDV (LSDV/Cattle/VJA PNGR/SVVU/2022) could be isolated from two samples (nasal swab and skin scab, both collected from a single clinical case) in the fifth passage showing characteristic cytopathic changes. MDBK amplified virus was adapted to Vero cell line and obtained infectivity titer of 10-7.595 per 100 μL is further employed for use in serum neutralization test. Phylogenetic analysis of the LSDV isolate based on full length ORF036 gene (RPO30) showed 100 % identity and formed a distinct clade with Middle-east Asia and African isolates. Amino acid specific signatures to differentiate CaPVs were noticed and estimates of evolutionary divergence over sequence pairs between clusters was determined that showed a distance of 9.52 % between vaccine strains and the field isolate in the present study. Vaccination trial was conducted in randomly selected heifers placed into four groups (A, B, C and D) of eight animals each. Group A served as control group, while groups B, C and D were vaccinated with 1mL, 2mL and 3mL of 1 X 103.0 TCID50/dose of Goatpox vaccine respectively. Group D vaccinated with 3 times the dose used in goats produced the best humoral immunity that was persistent till the end of the trial i.e., 35 days post vaccination(p<0.05). Thus, this study shows thrice the dose of Goatpox vaccine used in goats is considered as the optimum dose in cattle against LSD.ThesisItem Open Access DETECTION OF GENETIC DETERMINANTS OF ANTIBIOTIC RESISTANCE AND VIRULENCE FACTORS OF Mannheimia ISOLATES FROM BRONCHOPNEUMONIA CASES OF SHEEP AND GOAT, AND EXPLORATION OF ANTIMICROBIAL PROPERTIES OF PHYTOCHEMICALS(SRI VENKATESWARA VETERINARY UNIVERSITY, TIRUPATI - 517 502. (A.P.) INDIA, 2024-02) MARUTHI SWAROOP THATAVARTHI .N.S.R.K; SIVARAMA KRISHNA .G (MAJOR); ANAND KUMAR .P; ANNIE SUPRIYA .RThe respiratory infections of small ruminants are a burning problem for the communities who depend on sheep and goat rearing as major income source in India. The Mannheimia species is one of the major pathogens implicated in pneumonia of small ruminants and responsible for huge economic loses to livestock sector worldwide. The Mannheimia is a Gram negative, non-motile, non-spore forming, oxidase positive, catalase positive coccobacillary/bipolar organism. In the present study a total of 92 nasal swab samples were collected form clinical cases of pneumonic sheep and goats, out of which the Mannheimia was isolated from 30 samples (25 from sheep and 5 from goats). Morphologically, all the isolates characterized as bipolar & cocco-bacillary in Gram’s staining. Culturally, the Mannheimia isolates developed small pin-point pink coloured colonies on MacConkey agar, and small greyish haemolytic colonies on Blood agar. The phenotypically confirmed Mannheimia isolates were further genotyped by using genus specific primers targeting the gene coding for 16S rRNA. The genotypic primers specifically reacted and typed the isolates as belongs to the genera Mannheimia with an amplified product size of 304 bp. Overall the prevalence of Mannheimia was found to be 32.6%. The phylogenetic analysis revealed that the Mannheimia isolates from Andhra Pradesh formed a separate clade with close relation to M. hemolytica, Canada and M. caviae, Denmark strains. The biofilm forming ability of the Mannheimia isolates was evaluated by three methods such as Standard Tube method (ST), Congo red agar method (CRA) and Microtiter plate assay (MTP). Among, MTP assay was considered as best method which detected and quantified the biofilm production by 29 (96.6%) isolates. The antibiogram pattern of Mannheimia isolates revealed that high resistance to Ampicillin (80%) followed by Gentamicin (50%), Co-trimoxazole (43%), Tetracycline (40%), Amoxicillin-Clavulanic acid (40%), Ceftriaxone (36.6%) and Enrofloxacin (26.6%). On analyzing the multiple antibiotic resistance index (MAR), none of the samples revealed a MAR index of less than 0.2, which indicates a potential threat of rising multi-drug resistant (MDR) bacteria. The genes coding for antibiotic resistance of streptomycin and extended spectrum β-lactamases (ESBL) were identified using gene specific primers in multiplex PCR with a prevalence rate of 70% for strA gene, followed by 16% for blaOXA, 13.3% for blaTEM and 10% for blaSHV genes. The genes coding for virulence factor leukotoxin (Lkt) was found in only two isolates. The antimicrobial properties of selected phytochemicals were evaluated which can be tested as alternative approach to combat the threat of rising MDR strains of Mannheimia. The Eugenol, an extract of Syzygium aeromaticum, was found to effective with MIC ranging between 32-2052 µg/ml against the Mannheimia isolates whereas the MIC of Cinnamic acid was found to be ranging between 1.25 to 2.50 mg/ml. In conclusion, our study provides the detailed information on prevalence of Mannheimia in clinical cases of Pneumonia of sheep and Goats, its phylogenetic relations, pathogenic potential, danger of rising MDR strains and phytochemicals, Eugenol and Cinnamic acid, as alternative therapies to combat the MDR strains.ThesisItem Open Access MOLECULAR CHARACTERIZATION OF LUMPY SKIN DISEASE VIRUS FROM CATTLE OF ANDHRA PRADESH(SRI VENKATESWARA VETERINARY UNIVERSITY, TIRUPATI - 517 502. (A.P.) INDIA, 2024-02) SAIKUMAR GANGITLA; RAMANI PUSHPA .R.N (MAJOR); ANAND KUMAR .P; ANNIE SUPRIYAEver since 2019, there have been reports of outbreaks of Lumpy skin disease (LSD) in cattle from several regions of India, with Andhra Pradesh as one of the main impacted states where animals were extremely suffered resulting in severe economic loss to farmers. Hence, a modest attempt has been made to study the LSD with regard to detection, isolation and characterization of LSDV isolates from various districts of Andhra Pradesh and its phylogenetic analysis. The clinical signs noticed in the ailing animals include papular and nodular lesions across the body ranging from 0.5 to 5 cm in diameter, enlargement of superficial lymph nodes, a rise in body temperature, oculo-nasal discharges, lameness owing to edematous swelling in their limbs. These signs were more severe in the crossbred cattle. In the current investigation, a total of 81 clinical samples (38 skin scabs, 21 nasal swabs, 18 blood samples and 4 faecal samples) from 54 clinically infected animals from different regions of Andhra Pradesh were collected. Preliminary screening of the samples was done by capripox generic PCR. Thirty four out of 38 skin scabs (89.48%), 7 out of 21 nasal samples (33.33%) and 1 out of 4 faecal samples (25%) were found positive and produced the amplification with product size of 192bp. The blood samples did not react with any primers. All the 81 samples were further screened using A33R gene primer pair. The positive percentage obtained for skin scabs, nasal samples and faecal samples by A33R gene-based PCR assay was same as that of p32 gene-based PCR assay and produced the amplification product of 588bp. Hence, the new technique of amplifying LSDV A33R genome for the detection of LSDV in clinical samples is equivalent to the OIE-recommended PCR technique that amplifies the p32 gene. The LSDV specific fusion (F) gene was detected in 29 skin scabs(76.3%), 3 nasal swabs (14.29%) and 1 faecal sample (25%) and produced the amplification with product size of 472bp. None of the blood samples reacted with the primers. The coding region of A33R gene from three field isolates was sequenced and the blast analysis showed 100% similarity with isolates from Turkey, Russia, and Kazakhstan. A phylogenetic tree was constructed with the sequences of field isolates and circulating strains available in GenBank. It was revealed that the field isolates were clustered with LSDV isolates from Turkey, Serbia, Russia, and Kazakhstan. However, vaccine strains clustered separately. Similarly, the genomic region of partial F gene from six field isolates was sequenced and blast analysis showed 100% genomic similarity with strains from Kenya, China, Russia, Bangladesh, and India. The similarity with other LSDV vaccine strains was 98% to 99%. The phylogenetic tree generated showed that the isolates from Vizianagaram region were clustered with Kenya isolate and KSGP 0240 strain. The isolate from Srikakulam was in cluster with isolates from the field outbreaks in Tamil Nadu and Odisha during 2019. Isolates from Krishna and Machilipatnam regions were in one node and clustered separately as well as Palamaner isolate which also clustered separately. All the vaccine strains were grouped together in one node The skin scab from clinically infected cattle which tested positive in PCR assay was propagated in primary lamb testicular cells (PLT). No CPE was observed until 5 days post infection (PI). The supernatant from the infected cells was used to infect fresh PLT cells (first blind passage). The PLT cells began to exhibit CPE in the form of cell rounding, cell aggregation, shrinking of cells after 72 hrs PI and prominent CPE was evident after 96 hrs PI. The virus was also propagated through chorio allantoic membrane (CAM) route in 10-day-old embryonated chicken eggs (ECE) up to 5th passage using skin scabs which were tested positive by PCR. The mortality of chicken embryos was observed after 96 h at each passage level. The isolates produced the characteristic pock lesions on CAM after 4th passage and became clear after 5th passage. The virus in both PLT cells and CAM was further confirmed by PCR using LSDV specific primers (Fusion gene). The CAM of LSDV infected chicken embryo was subjected to histopathological examination. The Haematoxylin and Eosin-stained tissue sections revealed thickening of the membrane over inoculation site with congested blood vessels and haemorrhages over the membrane. The epithelial cells showed vacuolar degeneration and often containing eosinophilic, intra-cytoplasmic inclusions which is the characteristic of pox virus. The histopathological findings of infected skin scabs revealed hyperkeratosis in the epidermis, infiltration of mononuclear cells, macrophages, lymphocytes and fibroblasts in the dermis. Eosinophilic intracytoplasmic inclusion bodies in the epithelial cells were also noticed.ThesisItem Open Access DETECTION AND MOLECULAR TYPING OF TOXINS OF Clostridium perfringens ISOLATES FROM SMALL RUMINANTS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2023-02) SAI MANI DEEPIKA, CHEDE; SRIVANI, M(MAJOR); ANAND KUMAR, P; ASWANI KUMAR, KA study was carried out on the isolation, biochemical, molecular characterization and toxin typing of C. perfringens isolated from healthy and diarrhoeic sheep and goat. The antibiotic resistance pattern was studied in representative samples. A total of 158 samples (rectal swabs and intestinal tissues) were collected from healthy and diarrhoeic sheep and goat, from which 34 (21.51%) C. perfringens isolates were obtained. A higher isolation rate was obtained from intestinal content samples (67.65%) than faecal samples (32.35%). The samples that exhibited characteristic cultural and biochemical characters were subjected to PCR, which revealed 34 samples were positive for C. perfringens. The sugars like galactose, cellobiose and raffinose were used to differentiate toxin type A, D and F from other toxin types, respectively. Out of 34 positive samples, a high prevalence rate was observed among diarrhoeic lambs (50.0%) followed by diarrhoeic sheep (44.12%) and healthy sheep (5.88%). Multiplex PCR for six toxin genes (alpha, beta, epsilon, iota, entero and beta2) of 34 isolates revealed the production of four toxins namely alpha, epsilon, entero and beta2 toxins belonging to three toxin types i.e., type A, D and F. Out of these isolates, 30 (88.23%) isolates were characterized as C. perfringens type A, one (2.94%) isolate was characterized as C. perfringens type D and three (8.82%) isolates were characterized as C. perfringens type F. In the present study, C. perfringens type A was found to be predominant among all the toxin types. Among diarrhoeic sheep, 86.67% and 13.33% of C. perfringens type A and type F were detected, respectively. The isolates obtained from healthy sheep were detected as type A and among diarrhoeic lambs, 88.24%, 5.88% and 5.88% of type A, D and F were detected, respectively. Antibiogram of randomly selected isolates showed resistance to sulphamethizole (66.67%) followed by co-trimoxazole (58.33%), enrofloxacin (41.67%) and tetracycline (33.33%) and high sensitivity observed against gentamicin (91.67%) followed by cefotaxime/clavulanic acid (91.67%), norfloxacin (91.67%) followed by chloramphenicol (83.33%), cefotaxime (75.0%) and streptomycin (66.67%). Sequencing results of selected isolates showed 100% homology with corresponding published toxin genes confirming the amplicons as C. perfringens (481 bp), alpha (324 bp) and epsilon (376 bp) toxin genes of C. perfringens. The present study concluded that the most of the isolates were characterized as C. perfringens type A. Therefore, it may be recommended to include C. perfringens type A anaculture in the existing vaccine, to prevent C. perfringens infections in this geographic region (Krishna and West Godavari districts of Andhra Pradesh).ThesisItem Open Access DETECTION OF ANTIBIOTIC RESISTANCE PROFILES AND ANTIBIOTIC RESISTANCE GENES (ARGs) IN Escherichia coli ISOLATES FROM MASTITIS MILK SAMPLES OF BUFFALOES AND THE ENVIRONMENT(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2023-02) RATNAMRUTHA, NUNNA; ANAND KUMAR, P(MAJOR); SRIVANI, M; SUBHASHINI, NThe present study is undertaken to investigate the antibiotic resistance patterns in E. coli isolates from mastitis milk samples of buffaloes and environment (water) samples in the vicinity of the same dairy animals and to know the distribution of the selected genetic determinants of antibiotics in these isolates. Based on cultural, biochemical and molecular (PCR test) tests, 52 samples out of 79 mastitis milk samples and 31 samples out of 54 water samples were confirmed as E. coli. For isolation of E. coli presumptively producing ESBL/AmpC, the mastitis milk samples and water samples were cultured on 1 mg/L of cefotaxime (CTX) supplemented MacConkey agar (MCA) and Tryptone Bile Glucuronic (TBX) agar, respectively. A total of 27 mastitis milk samples and 14 water samples were identified as ESBL/AmpC producing E. coli by cultural, biochemical and molecular tests. With regard to antibiotic resistance pattern of E. coli isolates (including ESBL/AmpC producing E. coli) from mastitis milk samples in in vitro disc diffusion based antibiotic sensitivity test (AST) with primary panel of nine antibiotics, majority of isolates were resistant to cefotaxime followed by ampicillin, cotrimoxazole, imipenem, enrofloxacin, amoxyclav, tetracycline, chloramphenicol and gentamicin. The E. coli isolates (including ESBL/AmpC producing E. coli) from water samples also showed maximum resistance to cefotaxime followed by ampicillin, imipenem, cotrimoxazole, tetracycline, amoxyclav, enrofloxacin, chloramphenicol and gentamicin. Further, AST with secondary panel of five antibiotic discs for presumptive ESBL/AmpC producing E. coli isolates from mastitis milk samples and water samples showed that all the isolates were ESBL/AmpC producers and showed highest resistance to ceftazidime followed by cefoxitin and meropenem. In PCR test for detecting the genetic determinants of antibiotic resistance genes, 91.9% of the E. coli isolates (including ESBL/AmpC producers) from mastitis milk samples and water samples possessed AmpC gene followed by SHV, NDM-1, TEM and OXA genes. In the present study, AmpC gene was detected in all E. coli isolates that were showing resistance to third generation cephalosporins in AST except three E. coli isolates from water samples (W41, W48 and W52). In some (A6, A9, A16, A25, A34, A49 and A64) of the E. coli isolates, although AmpC gene was detected in PCR test, phenotypically it was not correlated in ASTs as these isolates have shown only sensitivity/intermediate resistance to third generation of cephalosporins. Further, some of the E. coli isolates that were detected with AmpC gene also didn’t show resistance to clavulanic acid. This might be due to the lack of expression of AmpC gene in those isolates.ThesisItem Open Access ASSESSMENT OF ANTIBIOGRAM AND DETECTION OF GENETIC DETERMINANTS OF SELECTED VIRULENCE FACTORS IN AVIAN PATHOGENIC ESCHERICHIA COLI ISOLATES OF CHICKEN(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2022-05) LOKESWARI, R; SIVARAMA KRISHNA, G(MAJOR); ANAND KUMAR, P; GANGU NAIDU, SThe present study was taken up for understanding the pathogenic potential and prevalence of antibiotic resistance pattern of avian E. coli strains, isolated from colibacillosis affected chickens by both traditional and molecular methods. A total of 105 samples were collected from the internal organs of colibacillosis affected chickens. Based on morphological, cultural and biochemical characterization viz., Gram staining, growth pattern on EMB agar, lactose fermentation on MacConkey agar, IMViC tests (Indole, Methyl Red, Voges-Proskauer, Citrate), Catalase, Nitrate, Oxidase and Urease tests, E. coli was isolated and characterized from 81 samples (77.14%) out of 105 samples collected. The gene coding for 16S rRNA is highly conserved among species/strains of Escherichia genus was detected in all the 81 E. coli isolates by PCR. In congo red dye uptake assay, all the isolates produced orange red to red color colonies, indicating their invasiveness and pathogenic potential. Out of 81 isolates, the production of hemolysin was phenotypically characterized in 74 (91.4%) isolates, of which 39 (48.2%) isolates are ɑ-hemolytic and 35 (43.2%) are Entero-hemolytic on 10 per cent sheep blood agar. The biofilm production was assessed by qualitative tests like Christensen borosilicate glass tube method and polypropylene tube method, and quantitatively by tissue culture plate method (TCP). Among the qualitative methods the polypropylene tube method (89.47% Sensitivity and 91.66% Specificity in comparison to TCP method) was found to be superior to the borosilicate tube method (77.1% sensitivity, 83.3% specificity in comparison to TCP method). In TCP method, 61 isolates (75.3%) were quantitatively characterized as biofilm producers, which is the most reliable method. In Embryo lethality test, based on the % embryo death rate, 52 isolates were characterized as highly pathogenic strains, 24 isolates were characterized as moderately pathogenic and five isolates were characterized as low pathogenic strains. A total of eight genes were targeted for the identification of genetic determinants of virulence factors for all the 81 avian pathogenic E. coli isolates. The hlyF gene was detected in four isolates viz., F6, H1, H2 and H3, iroN gene was detected in five isolates viz., B3, B4, B5, D1 and D3, whereas eaeA gene was detected only in H3 isolate. The genes iss, ompT, stx1, stx2 and hlyA were detected in none of the isolates tested. All the isolates were found to be multidrug resistant with a Multiple antibiotic resistance index (MAR) of more than 0.2 as assessed by disc diffusion method. The occurrence of genes coding for extended spectrum β-lactamase (ESBL) were assessed in multiplex PCR and a prevalence of 86.27 per cent of blaTEM, 3.7 per cent of blaOXA and 0 per cent of blaSHV was reported in the present study. In conclusion, our study might be the first to assess the pathogenic potential of E. coli from colibacillosis affected chickens by both traditional and molecular methods in Andhra Pradesh. The present data on characterization of pathogenic E. coli strains and existence of multidrug resistant strains of E. coli in colibacillosis affected chickens may be useful for developing novel control strategies and vaccines.ThesisItem Open Access ASSESSMENT OF ANTIBIOTIC RESISTANCE PATTERN AND BIOFILM FORMATION IN Pseudomonas aeruginosa ISOLATES FROM BUFFALOES AND DOGS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2022-03) RAMA NARAYANA, P; DEEPIKA KUMARI, G (MAJOR); ANAND KUMAR, P; BINDU KIRANMAYI, ChThe current research is conducted to assess the antibiotic resistance pattern and biofilm formation ability in Pseudomonas aeruginosa isolates from the clinical samples of buffaloes and dogs. Samples were inoculated in Brain Heart Infusion (BHI) broth and cultured on Pseudomonas isolation agar. Based on pigment production and biochemical tests (catalase, oxidase, IMViC), 52 samples (34.8%) out of 149 collected in the present study were provisionally confirmed for Pseudomonas species. The isolates were further confirmed by Polymerase Chain Reaction (PCR) assay with Pseudomonas aeruginosa species-specific primers (16S rRNA) and all the 52 isolates yielded specific PCR product of 956 bp. One of the main objectives of the present study, antibiotic resistance pattern was determined by subjecting the PCR confirmed 52 P. aeruginosa isolates to 12 different antibiotic discs in vitro by Kirby Bauer method. The results of the test revealed that co-trimoxazole was highly resistant (98.07%), followed by ampicillin (96.15%), clindamycin (92.30%), oxytetracycline (88.46%) and amoxycillin (84.61%). Streptomycin (75%), ceftriaxone (50%) and norfloxacin (36.53%) were moderately resistant. Ciprofloxacin (5.76%), enrofloxacin (9.61%), amikacin (11.53%) and gentamicin (21.15%) were least resistant than others. The Multiple Antibiotic resistance (MAR) indices of P. aeruginosa isolates ranged from 0.33 to 0.91 in the current study, 51 isolates were confirmed as Multi Drug Resistance (MDR) and 1 isolate was found to be Extensively Drug Resistance (XDR). Phenotypic screening for extended spectrum β-lactamases production was carried by employing phenotypic screening test (PST). Isolates were tested with four cephalosporin antibiotics (cefotaxime, ceftazidime, ceftriaxone, and aztreonam). Forty seven out of the 52 PCR confirmed Pseudomonas aeruginosa isolates were found to be resistant against one or more cephalosporin discs, 44 isolates were resistant against cefotaxime, 26 were resistant against ceftriaxone, 9 were resistant to ceftazidime and 5 against aztreonam. Hence they were considered as ‘suspect ESBL producers’ as defined by the CLSI (2018) guidelines. Thereafter, the suspect ESBL producers were confirmed for Extended Spectrum β Lactamases (ESBL) presence by phenotypic confirmatory tests (PCTs). ESBL production was confirmed in 42 isolates by combination disc method (CDM) and 37 isolates exhibited double disc synergy in double disk synergy test (DDST). Further, all the 52 isolates were tested for genetic determinants of antibiotic resistance with oligonucleotide primers specific for extended spectrum genes (blaOXA, blaSHV, blaTEM). A PCR product of 564 bp, 713 bp and 800 bp size was yielded for blaSHV, blaOXA and blaTEM genes, respectively. Out of 52 isolates of Pseudomonas aeruginosa, 10 isolates were positive only for blaOXA, 9 isolates were positive only for blaSHV, 3 isolates were positive only for blaTEM. Both blaSHV and bla OXA were observed in 5 isolates (9.61 %), both blaSHV and blaTEM were observed in 3 isolates (5.76 %), whereas blaOXA and blaTEM together were observed in 6 isolates (11.50 %). All the three genes blaSHV, blaOXA, blaTEM were observed in 3 isolates (5.67 %). No ESBL genes were observed in 13 isolates. The biofilm formation ability was assessed by processing the P. aeruginosa isolates through congo red agar method (CRA) and tube method. CRA method detected that 7 isolates (13.46%) were weak, 26 (50%) as moderate and 19 (36.53%) isolates to be strong biofilm producers, whereas, in tube method 11 (21.15%) isolates were detected weak, 18 (34.61%) moderate, 23 (44.23%) as strong biofilm producers. In conclusion, the current study investigated the presence of genetic determinants of multidrug resistant P. aeruginosa isolates from clinical samples of buffaloes and dogs in Andhra Pradesh. There is a great necessity to pursue further research on multi-drug and extended drug resistance in Pseudomonas aeruginosa isolates from dairy and companion animalsThesisItem Open Access STUDY ON ANTIMICROBIAL EFFICACY OF Lactobacillus SPECIES FROM CHICKEN AGAINST Clostridium perfringens AND E. coli(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2022-03) CHANDANA SIVA POOJA, M; SRIVANI, M (MAJOR); RAMANI PUSHPA, R.N.; ASWANI KUMAR, KA study was carried out on the isolation, molecular characterization and probiotic potency which includes antibacterial activity of Lactobacillus spp. isolated from healthy chicken against Clostridium perfringens and E. coli. A total of 73 samples from desi and commercial chicken were collected of which 50 were cloacal swabs and 23 were tissues, from which 56 (76.71%) isolates were obtained. Tissues (87.5%) have an increased incidence of Lactobacillus spp. than cloacal swabs (70%). The percentage of autoaggregation of 56 isolates increased significantly over time from 5.03 % - 77.33 % at 4 hours to 10.37 % - 82.29 % after 24 hours of incubation. The cell surface hydrophobicity of the 56 isolates ranged from 6.3 % to 90.68 % for xylene and 18.13 % to 95.84 % for n-hexadecane. Isolates exhibited high percentage of hydrophobicity to n-hexadecane when compared to xylene. Based on autoaggregation and hydrophobicity results 22 isolates were chosen. All the 22 isolates demonstrated viable colonies at all ox-bile concentrations (0.1 %, 0.3 % and 0.5 %) after 2 and 4 hours of incubation, however after 6 hours of incubation, viability varied. In the acid tolerance assay, majority of the isolates showed viability at pH 3 and all the isolates demonstrated viability at pH 6.5. Based on bile and acid viability results 16 isolates were selected for further tests. All these 16 isolates inhibited Clostridium perfringens with zone of inhibition ranging from 11mm-22mm and 10mm-20mm anaerobically and aerobically, respectively. Whereas, the zone of inhibition of E. coli ranged from 10mm-18mm in agar well diffusion assay. In the antibiofilm assay, all 16 isolates developed biofilm and reduced E. coli biofilm formation. The antibiotic resistance pattern of 16 isolates revealed 100% resistance to Nalidixic acid and Vancomycin, 93.75 % resistance to Tetracycline and Streptomycin, 75 % resistance to Ciprofloxacin and Gentamicin, 66.66 % resistance to Cotrimoxazole, 25 % resistance to Nitrofurantoin, 6.25 % resistance to Clindamycin and Cefaclor and 0 % resistance to Chloramphenicol, Ampicillin and Erythromycin. The hemolysis test for the 16 isolates indicated a complete hemolysis for 14, partial hemolysis for one and no hemolysis for one isolate, whereas all the isolates showed negative reaction for gelatin hydrolysis test. Based on the results of probiotic potency and safety assays, one isolate was finally chosen from the 56 isolates. The isolate sequence revealed 96.82% of its similarity to Lactobacillus fermentum. The present study concluded that selected Lactobacillus fermentum has potential probiotic properties with good antimicrobial activity against Clostridium perfringens and E. coli hence, may be tested in vivo in poultry as an alternative to antibiotics.ThesisItem Open Access 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, TBovine 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.
