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2012 - 20135
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Subject: Veterinary Microbiology × End date: 2013 × Start date: 2012 × Type: Thesis ×
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    ThesisItemOpen Access
    CHARACTERIZATION OF AN INDIAN ISOLATE OF BLUETONGUE VIRUS SEROTYPE 16
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2012-03) YOGANAND, BRUNDAVANAM; NARASIMHA REDDY, Y(MAJOR); DHANALAKSHMI, K; NAGENDRA HEGDE, R
    ABSTRACT: Bluetongue is an arthropod-borne disease, transmitting to ruminants through the bites of Culicoides. Bluetongue is listed as a ‘notifiable disease’ by the Office International des Epozooties (OIE), causing severe hemorrhagic disease with fever, lameness, coronitis, edematous lips and tongue and death. Export of animals and animal products from Bluetongue endemic countries like India are seriously being hampered. Bluetongue virus (BTV) belongs to the family Reoviridae, genus Orbivirus. Out of 24 serotypes identified worldwide, 12 serotypes have been reported by isolation and 11 serotypes by serology in India. VP2 gene is highly variable and responsible for neutralization and serotype specificity of BTV. Characterization of isolates which are definitively sero-typed is very much essential for developing suitable vaccine to control the disease in a given geographical region. This research work is focused on the characterization of an Indian isolate (VJW784) of bluetongue virus serotype 16, by RT-PCR and molecular techniques. This is the first time to document the complete sequencing of protein coding region of VP2 gene of an Indian isolate of bluetongue virus serotype 16. The VJW784 isolate was propagated on BHK-21 cell lines and harvested. dsRNA extracted from BHK-21 cells was used for testing by RT-PCR with serotype specific primers of BTV 1, 9, 10, 16, 21 and 23. Further serotype-specific primers for VP2 gene of BTV 16 were designed and used for specific amplification of segment 2. Cloning was carried out using pDK101 vector. The positive plasmid DNA and PCR amplicons were sequenced. The sequence information was aligned with DNAstar software and BLAST analysis was carried out. The sequence information provided herein may help to determine the geographic origin of VJW784isolate and defined the phylogenetic relationship of this isolate to other BTV strains.
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    ThesisItemOpen Access
    SAFETY AND IMMUNOGENICITY OF BRUCELLA ABORTUS STRAIN-19 REDUCED DOSE VACCINE THROUGH CONJUNCTIVAL ROUTE IN CATTLE - A TRIAL
    (SRI VENKATESWARA VETERINARY UNIVERSITY , TIRUPATI – 517502. (A.P.) INDIA, 2013-04) MANOJ KUMAR, V; NARASIMHA REDDY, Y(Major); DHANALAKSHMI, K; ANAND KUMAR, A
    ABSTRACT : The present study is taken up with a view to study the efficacy of brucella vaccine at reduced dose (5 x 109 to 8 x 109 CFU/dose) through conjunctival route and compare it with conventional standard dose (40 x 109 to 80 x 109) vaccine. Female cattle above 8 months age were selected for the study. Thirty animals were inoculated with reduced dose conjunctival vaccine and ten with standard subcutaneous vaccine using Brucella abortus S19 antigen. The efficacy of vaccine was studied in terms of both humoral and cell mediated immune responses against brucella for a period of 120 days. Humoral immunity techniques consisted of rose bengal plate test (RBPT), standard tube agglutination test (SAT) and indirect ELISA (i-ELISA). Cell mediated immune response was studied by using interferon gamma (IFN-γ) assay after in vitro stimulation of lymphocytes with both S19 and B. abortus 544 as antigens in indirect competitive ELISA (Enzyme Linked Immunosorbent Assay). Of the humoral immunity tests employed for studying the efficacy of vaccines RBPT was least sensitive. It detected 20 to 18.51% positive responders at 21 and 60 days PV in reduced dose conjunctival vaccine group. Similarly it detected 60% and 50% responders on 21 and 60 days PV in standard group. Standard tube agglutination test (SAT) detected more responders against vaccinates than the RBPT. The positive percentage ranged from 26 to 66% among reduced dose vaccine group and 40 to 80% among standard dose vaccine group. I-ELISA was the most sensitive as it detected up to 78.9% responders among reduced dose vaccination group and up to 100% in standard dose vaccinates group. Humoral immune responses declined after 90 days PV. IFN-γ production as indicator of cell mediated immune response in vaccinates was more consistent. IFN-gamma production was observed during the entire period of the trial (120 days). It is concluded that CMI responses are better indicators of immune response to brucella than the humoral immunity.
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    ThesisItemOpen Access
    DETECTION OF VIRULENCE FACTORS GENES AND INVESTIGATION OF ANTIBIOTIC RESISTANCE PATTERN IN Staphylococcus aureus ISOLATED FROM MASTITIC MILK SAMPLES OF BUFFALOES
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI – 517 502. (A.P) INDIA, 2013-04) RAM BABU, G; ANAND KUMAR, P (Major); SUBRAMANYAM, K V; RAMADEVI, V; SRINIVASA RAO, G
    ABSTRACT: Mastitis in buffaloes is responsible for heavy economic losses to Indian dairy industry. Staphylococcus species are the most common pathogens responsible for mastitis in cows and buffaloes. Staphylococcus aureus is implicated in the sub-clinical mastitis. Rapid and accurate detection of S. aureus is important for controlling mastitis in a herd. Many virulence factors responsible for pathogenicity are reported in S. aureus. Detection of the virulence factors helps to identify the strains of S. aureus circulating in the area. Among the important virulence factors coagulase and enterotoxins assume significance. Genes coding for these virulence factors in S. aureus can be detected by polymerase chain reaction (PCR) test. Reports on antibiotic resistance phenomenon also prompt to test the S. aureus isolates from bubaline mastitis cases for antibiotic sensitivity. Based on isolation on selective media, haemolysis on blood agar, catalase test, oxidase test and coagulase test 57.33% of the mastitic milk samples collected in the present study were provisionally confirmed for Staphylococcus species, which were later subjected to confirmation by PCR test. On screening by California Mastitis Test (CMT), 69.76% of these mastitic milk samples were found to be from sub-clinical mastitis cases of buffaloes. For PCR test, bacterial DNA from the enriched bacterial cultures was extracted by inexpensive high salt method, which yielded required quality and concentration of DNA. All the 43 mastitic milk samples that were provisionally confirmed for Staphylococcus species were tested with Staphylococcus genus specific oligonucleotide primers (Sta I and Sta II) and S. aureus specific oligonucleotide primers (Staur 4 and Staur 6) in PCR test. All the 43 samples were confirmed as Staphylococcus species (PCR products of 100 to 200 bp), whereas 69.76% (n=30) of them were confirmed as S. aureus (PCR product of 1250 bp). Bacterial cultures that were confirmed as S. aureus by PCR test were further tested for coagulase gene (coa) in PCR test using coa gene specific primers. Out of the 30 S. aureus isolates from mastitic milk samples 8 samples (26.66%) did not yield any PCR products on reactivity with coa gene specific primers. The remaining 22 S. aureus isolates (73.33%) yielded PCR products of different sizes ranging from 610 bp to 960 bp. The S. aureus isolates TVCC 18 and GV 6 yielded a PCR product of 610 bp. Six S. aureus isolates viz. TVCC 23, GDV 1, KSP 3, KSP 4, KSP 6 and KSP 7 yielded PCR product of 740 bp. Twelve isolates of S. aureus viz. TVCC 2, TVCC 4, TVCC 6, TVCC 7, TVCC 8, TVCC 9, TVCC 10, GDV 2, GDV 3, GV 1, GV2 and KSP 21 yielded a PCR product of 870 bp. Two S. aureus isolates KSP 17 and KSP 25 yielded PCR product of 960 bp. The present study confirms coa gene polymorphism in S. aureus isolates from bubaline mastitis. It is interesting to note that the S. aureus isolates GDV 5, GV 7, KSP 14, KSP 15 and KSP 16 were negative for coagulase activity in tube coagulase test and also didn’t yield any PCR product with coa gene specific primers in PCR test. These S. aureus isolates are surmised to be to be coagulase negative variants. All the 30 S. aureus isolates were tested for staphylococcal enterotoxin genes seg, seh, sei in PCR test using oligonucleotide primers specific to these three enterotoxin genes. In PCR test 30% of S. aureus isolates (n=9) viz. TVCC 10, TVCC 14, TVCC 18, TVCC 23, GDV 1, GDV 3, GV 6, KSP 15 and KSP 21 were positive for seg gene; 20% of S. aureus isolates (n=6) viz. TVCC 10, TVCC 18, TVCC 23, GDV 1, KSP 3 and KSP 4 were positive for seh gene; 40% of S. aureus isolates (n=12) viz. TVCC 10, TVCC 14, TVCC 18, TVCC 23, GDV 1, GDV 3, GV 2, GV 6, GV 7, KSP 3, KSP 15 and KSP 21 were positive for sei gene. Four isolates TVCC 10, TVCC 18, TVCC 23 and GDV 1 were positive for all the three enterotoxin genes seg, seh and sei. Five isolates TVCC 14, GDV 3, GV 6, KSP 15 and KSP 21 were positive for two enterotoxin genes seg and sei. One isolate KSP 3 was positive for seh and sei genes, whereas only one isolate KSP 4 is positive for one enterotoxin gene seh. Therefore in the present study just 13.33% of S. aureus isolates were
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    ThesisItemOpen Access
    DEVELOPMENT OF INACTIVATED WHOLE CELL VACCINE AGAINST OVINE FOOT-ROT
    (Sri Venkateswara Veterinary University, TIRUPATI – 517 502,A.P, 2012-11) SANDHYA RANI, K; RANI PRAMEELA, D (Major); SREENIVASULU, D; VAIKUNTA RAO, V
    ABSTRACT : Foot rot is an infectious and contagious disease of sheep and goat caused by an anaerobic bacterium Dichelobacter nodosus. The disease results in lameness of affected sheep and goat leading to production losses. The disease is being reported in Andhra Pradesh regularly during the rainy season. Treatment of affected sheep appears to be costly and not economical. Serogroup specific vaccination found to have excellent therauptic efficacy. Hence the study was taken up to develop an inactivated whole cell vaccine against ovine Foot rot using local isolates belonging to serogroups B and I. The serogroups B and I of D. nodosus were selected as vaccine strains based on earlier isolation and serogrouping studies in Chitoor district of Andhra Pradesh. The available serogroups B and I of D. nodosus were revived in TASH agar media with 2% hoof powder. Purity of antigens were checked and the concentration of antigens were adjusted to 1x108 org/ml and 2x109 org/ml. Later the antigens were inactivated by adding formalin at a concentration of 0.5% for overnight and adjuvanted separately using Montanide and Alum. The whole cell inactivated adjuvanted vaccines thus prepared were subjected for safety and sterility tests according to British pharmacopoeia (1985). Later trials were conducted in laboratory animals and also in the field to study the efficacy of vaccine. Experimental trials were conducted in sheep and goat under laboratory conditions with two different adjuvants, namely Montanide, Alum and with two different antigenic concentrations i.e. 1x108 org/ml and 2x109 org/ml. The immune responses were evaluated in sheep and goat by measuring antibody response using microtiter plate agglutination test and ELISA. The immune response was high in vaccines prepared using antigenic concentration of 2x109 org/ml compared with antigenic concentration of 1x108 org/ml. Similarly among two adjuvants Montanide and Alum, Montanide adjuvanted vaccines were found to be better in eliciting immune response. Further field evaluation of vaccine prepared using serogroup B of D. nodosus adjuvanted with montanide was also conducted in sheep in one of the village of Chitoor district of Andhra Pradesh, where the Foot rot was reported previously. The booster injection was given on 30th day of primary dose. The vaccine induced satisfactory protective titers.
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    ThesisItemOpen Access
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    (Sri Venkateswara Veterinary University, TIRUPATI – 517 502,A.P, 2012-06) VINOD KUMAR, N; SREENIVASULU, D (Major); NARASIMHA REDDY, Y; SHOBHAMANI, P; ESWARA PRASAD, P
    ABSTRACT : Multiplex PCR was standardized using specific primers for alpha, beta and epsilon toxin genes of reference strains of C. perfringens. The average detection limit of the test was found to be 1- 1.5 x 104 cfu ml -1 with purified DNA extracted from the reference strains. A total of 208 clinical samples which includes 140 from ET suspected lambs and 68 from healthy slaughtered animals were subjected to multiplex PCR screening. The bacterial lysate of 24h broth culture of clinical samples were found suitable for effective screening for C. perfringens types in clinical samples. Out of 208 samples tested by multiplex PCR from ET suspected and healthy lambs, 181 were positive for C. perfringens with overall prevalence of 87.01 per cent. Out of 140 samples from ET suspected animals, 129 (92.14%) were positive, whereas 68 samples from healthy animals revealed 52 (76.47%) positives. The toxin genotyping by multiplex PCR screening showed Clostridium perfringens type A as the predominant type with prevalence of 69.61 per cent. The prevalence of 92.30 per cent of C. perfringens type A healthy animals was found to be highly significant compared with ET suspected group .C. perfringens type C is found to be the least prevalent (11.04%) among all the C. perfringens types recovered in the present study. A prevalence of C. perfringens type D was found to be 19.33 per cent with 24.82 per cent prevalence of in ET suspected animals which is significantly higher than healthy animals (5.76%). Out of 208 samples attempted for isolation, 97 (69.28%) isolates were obtained from enterotoxaemia suspected lambs and 27 (39.70%) were from healthy lambs. Among the total 124 isolates, 110 (88.71%), 4 (3.23%) and 10 ( 8.06%) belongs to C. perfringens type A type C and D, respectively. All the isolates were obtained from initial multiplex PCR positive samples. The sequence analysis and phylogenetic analysis of the amplified toxin genes of alpha, beta and epsilon reveled cent per cent homology between respective toxin genes of isolates and when compared with published sequences of respective toxin genes homology of 96 to 100 per cent was observed, all segregated in to same phylogenetic group. All were found to be showing 82.6 to 100 per cent homology with the published sequences of respective toxin genes. When all the samples collected were subjected for multiplex PCR and isolation of C. perfringens, 181sample were found to be positive for PCR reaction and 124 isolates of C. perfringens were recovered. All the isolates recovered were from PCR positive samples. Prevalence of C. perfringens types in different districts of Andhra Pradesh revealed the presence of C. perfringens type C and type D in all the districts of AP except in Viziyanagaram where only type D was noticed. Presence of type C was found to be more with the increase in the prevalence of enteritis , mortalities and case fatality rates in the enterotoxaemia suspected flocks in all the districts. Attempts for identification of most toxigenic strain from all the 10 isolates of C. perfringens type D were made. The isolate KurCpD1 from Kurnool district was found to be producing higher concentration of epsilon prototoxin both in culture supernatant (2.11± 0.03 mg/ml) and purified epsilon prototoxin (1.75± 0.04 ) among all the isolates. Plasmid profiles of six out of 10 Clostridium perfringens type D field isolates revealed similar patterns with molecular weight ranging from 1.2 MDa to 30 MDa. All C. perfringens type C isolates obtained from the outbreak of Kurnool district showed identical plasmid profile with sizes ranging from 5 MDa to 30 MDa. The plasmid profiles of type C isolates in this study could be clearly differentiated from plasmid profiles of type D isolates. Amplification of etx gene was observed from the plasmid band of size 45 kb in type D isolates . Amplification of alpha toxin genes were observed from the plasmid of 23.5 kb size in type D siolates indicating the mixing of linear chromosomal DNA with the plasmid DNA. Clostridium perfringens type C plasmids at 37 kb showed amplification of beta toxin gene. The plasmids of C. perfringens type C and D stored at - 4 0 C and -20 0 C for 24 h and 48 h revealed diffused banding patterns indicating the rapid degradation of plasmids. In conclusion, the multiplex PCR can be employed for toxin genotyping of C. perfringens. Further, the test can be used for screening of the toxin genes in the region which helps in understanding the epidemiology of C. perfringens. Unique plasmid profiles of each toxin type of C. perfringens observed in the study can be used for identification of C. perfringens toxin types based on plasmid profiles. Prevalence of C. perfringens type C was observed in the State and is found to be increasing with the severity of enterotoxaemia along with C. perfringens type D. There is a need for development of a vaccine incorporating C. perfringens type C to control clostridial infections in sheep in the region.