Thumbnail Image

Thesis

Browse

2000 - 200922010 - 20193
Reset filters
Subject: Veterinary Microbiology × Start date: 1979 × Thesis Type: Ph.D ×
Reset filters

Search Results

Now showing 1 - 5 of 5
  • Thumbnail Image
    ThesisItemOpen Access
    MOLECULAR EPIDEMIOLOGY OF CANINE PARVOVIRUS IN ANDHRA PRADESH
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-08) DEEPIKA KUMARI, G; Ramani Pushpa, R.N(MAJOR); Subramanyam, K.V.; Srinivasa Rao, T; Satheesh, K
    Canine parvovirus-2 (CPV-2) is one of the most pathogenic viral etiologic agents causing hemorrhagic enteritis in dogs. CPV is a small, non-enveloped, single-stranded DNA virus of approximately 5 kb genome and belongs to the genus Protoparvovirus, in the family Parvoviridae.The objective of the present work is to detect, isolate and characterize circulating CPV in different locations of Andhra Pradesh from fecal samples of diarrhoeic dogs and its phylogenetic characterization. A total of 342 fecal samples were obtained from diarrhoeic dogs and preliminary screening of the samples was carried out by haemagglutination assay (HA) using 0.8 % swine RBC, out of which 71 were positive with a titre ranging from 1 in 32 to 1 in 512 (20.76%). The results of HA was confirmed by haemagglutination inhibition assay (HI) using hyperimmune serum raised in rabbits. All 342 fecal samples were screened by PCR using CPV-2ab primers and 233 samples produced an amplicon size of 681 bp. Genotyping of CPV was done by employing multiplex PCR using CPV-2ab and CPV-2b primer pairs. Out of 233 positive samples, 216 (92.70%) samples produced an amplicon size of 681 bp characteristic of CPV-2a and 17 samples (7.29%) yielded two specific amplicons of 681 bp and 427 bp and thus categorized as CPV-2b. The fecal samples not reacted with CPV-2ab and CPV-2b primers were further analysed by PCR-RFLP using restriction enzyme MboII for the detection of CPV-2c strain. Only one sample reacted with 555 primers produced an amplicon size of 583 bp but remain undigested with restriction enzyme. The overall prevalence rate of CPV in Andhra Pradesh was found to be 68.42%. Virus isolation studies from the PCR positive fecal samples were carried out using CRFK and MDCK cell lines. Successful isolation was observed in both the cell lines with varied cytopathic effects. Sixteen out of thirty four processed fecal samples in CRFK cell lines could produce a mild CPE after 72h post infection (PI) at fifth passage level which include increased granularity, rounding of cells and degenerative changes. Five out of nine CPV positive samples produced marked CPE like increased granularity, rounding of cells and complete detachment of cells in MDCK cell lines after 72h PI at third passage. The presence of virus at each passage level was confirmed by PCR assay using H primers. Transmission electron microscopic studies revealed spherical virus like particles of approximately 20 nm in diameter. The Partial VP2 gene sequencing of eighteen CPV field isolates along with the vaccine strain was performed in AB13730 DNA Analyzer and with Chromas lite software. The VP2 gene sequences were compared with CPV reference strains available in the GenBank by BLASTn analysis and were found to be highly specific revealing a maximum identity of 99.84% to 100% with CPV-2a and 99.35 with CPV-2b. One sample which only reacted with 555 primer on sequence analysis, exhibited a nucleotide homology of 99.66 % with CPV-2a. Multiple sequence alignment for the 18 CPV field isolates along with the vaccine strain was done using Clustal W 1.8 program and compared with the reference strains of CPV. The Partial VP2 nucleotide sequences identity of the field isolates when compared with the reference strains was 99.99 % to 100% and a similar identity was also observed among the field isolates. The nucleotide sequences for local CPV-2a isolates exhibited 99.99 % of homology with those of CPV-2b isolates. The nucleotide homology levels between the vaccine strain (CPV-2) and the analysed CPV-2a, CPV-2b isolates was 99.98 and 99.97 %, respectively. The antigenic types CPV-2a and CPV-2b differed from the original CPV-2 in atleast four amino acid positions of the VP2 capsid protein. Amino acid sequence analysis was done using MEGA 7.0 and revealed that thirteen out of 18 found to be new CPV-2a, one as a variant of new CPV-2a and four were characterised as new CPV-2b. Based on the typing systems using key amino acid positions, 77.77% were new CPV-2a and 22.22% were new CPV-2b with no CPV-2c or original CPV-2 found. The phylogenetic analysis revealed that the 14 new CPV-2a isolates were having close ancestral relationship with the Indian and Chinese strains of CPV-2a whereas CPV-2b strains formed a separate clade with the Indian CPV-2b strains. Seroprevalence of CPV was done by Indirect ELISA and HI for a total of 542 sera samples. Indirect ELISA recorded 94.65% in vaccinated and 72.50% in unvaccinated dogs whereas HI detected 56.10% in vaccinated and 16.42% in unvaccinated dogs. The variation in the detection of CPV antibodies by Indirect ELISA and HI from vaccinated and unvaccinated dogs was statistically significant (P < 0.05).
  • Thumbnail Image
    ThesisItemOpen Access
    MOLECULAR EPIDEMIOLOGY OF FOOT AND MOUTH DISEASE VIRUS ISOLATES FROM ANDHRA PRADESH
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2007-03) POORNIMA, M; SATYANARAYANA CHETTY, M(MAJOR); SREENIVASULU, D; NARASA REDDY, G.V; HAFEEZ, Md.; ESWARA PRASAD, P
    ABSTRACT: Hither to the prophylactic vaccinations were being employed to protect the livestock from devastating FMD. Even then the occurrence of outbreaks were recorded. Hence, it has triggered the idea in the minds of the scientists to study the divergency of antigenicity of FMDV isolates which could be incorporated in the vaccination programmes. Hence, a modest attempt has been made on exposition of various dimensions of molecular epidemiology its applications and implications in disease management to evolve a suitable vaccine strain to cater the needs of farmers lest the occurrence will continue. The disease was recorded round the year from different agro-climatic zones of Andhra Pradesh, both among vaccinates and unvaccinated animals with maximum incidences in cattle species (1 7.45%) with type 0. The complementation of results of antigenic detection in clinical samples showed virus isolation as golden standard while RT-PCR as sensitive and specific where as ELISA as dependable diagnostic tool. The FMDV field isolates of diffment ago-climatic zones of Andhra Pradesh were catalogued and master banks were prepared. In FMDV detection of OP fluids from carrier animals more percentage of positives were recorded by RT-PCR technique (47.8%). The phylogenetic analysis of type 0 and A isolates, revealed circulation of genetically heterologus genotypes that might have antigenically drifted away from current vaccine strains in vogue. It has been extrapolated the genetic variancy of deduced amino acid sequences to understand the heterogenicity in antigenecity. The isolates recovered corroborated serological relatedness to vaccine strains OIND R2/75 and AIND 17/82
  • Thumbnail Image
    ThesisItemOpen Access
    Molecular Characterization Of Egg Drop Syndrome-76(EDS-76 ) Virus
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2000-05) Vijaya Krishna, S; Subba Rao, V(MAJOR); Venkata Reddy, T; Choudhuri, P.C; Alaha Singari, N; Sreenivasulu, P
  • Thumbnail Image
    ThesisItemOpen Access
    Untitled
    (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.
  • Thumbnail Image
    ThesisItemOpen Access
    SEROEPIDEMIOLOGY AND MOLECULAR CHARACTERIZATION OF LEPTOSPIROSIS IN ANDHRA PRADESH
    (Sri Venkateswara Veterinary University, TIRUPATI – 517 502,A.P, 2010-11) RANI PRAMEELA, D; SREENIVASULU, D (Major); UMAMAHESWARA RAO, S; ESWAR PRASAD, P; SRILATHA, Ch
    ABSTRACT: Leptospirosis the world wide zoonosis is considered as reemerging disease. Besides economic losses caused by leptospira to animal production, its zoonotic character makes it an important public health problem. Due to the endemicity of the disease in Tamil Nadu, Kerala and Karnataka the adjoining states of Andhra Pradesh and absence of detailed information on leptospirosis in the state, the present work was planed to study seroepidemiology, isolation, characterization of leptospira and development of inactivated adjuvanted vaccine and to asses immune response in rabbits. The seroepidemiological study conducted using MAT on 2320 serum samples collected from apparently healthy cattle, sheep, goat, dogs and pigs revealed 20.9 percent positivity. Similarly 33.37 percent positivity was recorded from clinically suspected cases of cattle, sheep, pigs, dogs and humans. High seroprevalence in coastal region (23.09 percent) followed by Rayalaseema (17.49 percent) and Telangana (16.30 percent) was observed.. High seropositivity was recorded during north east monsoon (28.29 percent) followed by south west monsoon (21.45 percent) and lowest in summer (7.26 percent). Biochemical analysis of serum samples from cattle positive for MAT showed elevated levels of total bilirubin, SGPT and SGOT. Clinical samples collected from cattle (26) sheep (42) dogs (13), Pigs (15), Humans (53), and stagnated water in rice fields (10) were inoculated in EMJH Liquid medium with tween 80, antibiotics and 5- Flurouracil. A total of 17 isolates were recovered from sheep (5), rat (5), pigs (4), Humans (2) and rice field (1) were purified and maintained in EMJH liquid medium and semi solid medium. Physiochemical characterization of isolates at 130C, growth in the presence of 8-Azaguanine and lipase activity revealed the pathogencity of the isolates. PCR detected 12 isolates, of 17 isolates tested. RAPD DNA analysis was found to be simple and rapid test for identifying serovars of leptospira. The test identified the leptopsiral isolates as L. hardjo, L. autumnalis and L.pomona. 16S rRNA PCR sequence analysis of leptospiral isolates recovered from sheep were identified as Leptonema illini, L. hardjo and L.inadai, from rats were identified as Leptonema illini, L. noghuchi and from pigs identified as L. Pomona. A trivalent inactivated vaccine was prepared with three commonly circulating serovars namely L. grippotyposa, L. hardjo and L. autumnalis. The vaccine was adjuvanted with alumminium hydroxide (Vaccine-I) and Montanide (Vaccine-II) and the immune response in rabbits was studied. There is no significant difference between the vaccines. Both the adjuvanted vaccines yielded satisfactory immune response up to 150 days post vaccination.