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Theses

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2003 - 20067
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Subject: Veterinary Microbiology ร— End date: 2006 ร— Start date: 2000 ร— Type: Thesis ร—
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Now showing 1 - 7 of 7
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    ThesisItemOpen Access
    Polymerase chain reaction for the detection of canine parvovirus in faeces of dogs
    (Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 2004) Josemi, Mathew; KAU; Mini, M
    A study was undertaken to compare the ability of haemagglutination (HA), polymerase chain reaction (PCR) and seminested PCR for the diagnosis of canine parvovirus (CPV) infection by detecting CPV from faecal samples of clinically suspected dogs. Characterization of vaccine strain and field strains of CPV was performed by restriction enzyme analysis (REA) in this study. Attempts were made to isolate CPV from faecal samples in MDCK cell line. One hundred and twentysix faecal samples were collected from dogs suspected for CPV infection and 40 faecal samples were also collected from normal healthy non-vaccinated as well as vaccinated dogs after 15 days of vaccination, that were brought to veterinary hospitals attached to KAU. All the samples were screened by HA, PCR and seminested PCR to detect CPV. No significant difference in HA titre could be appreciated on comparing the titre using PBS, pH 7.2 and PBS โ€“ BSA as diluents. The haemagglutination reaction by CPV was found to be favoured by a slightly acidic pH in the range of 4.0 to 6.0. Chloroform treatment of faecal samples had no influence on HA titres above 5 log2. Among 126 faecal samples screened, 43.65 per cent, 61.11 per cent and 73.81 per cent were tested positive for CPV infection by HA, PCR and seminested PCR respectively. Therefore, seminested PCR was found to be a more sensitive and specific method over HA and PCR for the early diagnosis of CPV infection. All the faecal samples from healthy non-vaccinated and vaccinated dogs after 15 days of vaccination were tested negative by HA, PCR and seminested PCR. Restriction enzyme analysis using HinfI, Rsa I and Sau 961 revealed no difference in the fragment length patterns between CPV vaccine strain and field strain. Attempts to isolate CPV from faecal samples in MDCK cell line were found unsuccessful. The occurrence of CPV infection among non-vaccinated dogs was found to be higher than that of vaccinated dogs. Vaccine failure was also observed after MLV vaccination. The distribution of CPV infection was highest among dogs between two to four months of age. Breed-wise distribution of CPV infection showed highest distribution in German shepherd dogs when compared to other breeds. Most of the cases of CPV were noticed during February and July in the year 2003 and during March to June in the year 2004.
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    ThesisItemOpen Access
    Molecular methods based detection of pathogenic mycoplasmas of chicken
    (Department of Veterinery Microbiology, College of Veterinary and Animal Sciences, 2006) Dipu, M K; KAU; Jayaprakasan, V
    A study was undertaken for the detection of Mycoplasma DNA from the specimens by Polymerase Chain Reaction (PCR), differentiate the three significantly pathogenic mycoplasmas of chicken namely M. gallisepticum, M. synoviae and M. iowae from the other less pathogenic ones based on the result of polymerase chain reaction. Attempts were also carried out to isolate Mycoplasma from clinical samples testing positive by polymerase chain reaction and from a few randomly selected negative samples and to comprehend the different strains of M. gallisepticum, if any, among chicken of various age groups. Out of a total of 225 birds subjected for the study 25 were found positive for the presence of avian Mycoplasma by genus-specific PCR. Thirty samples from these twenty-five birds were positive. Tracheal swabs form all these birds were positive. Fifteen isolates were obtained from these twenty five birds when the tracheal swabs were directly streaked onto BHI agar whereas when these tracheal swabs were collected in BHI broth initially and later subcultured onto BHI agar following a positive result in PCR / colour change of the broth, only 13 of them yielded colonies. Five of the isolates were found to be M. gallisepticum by MG-PCR and these were obtained upon direct inoculation of BHI agar with tracheal swabs from birds. Those samples collected in BHI broth yielded MG colonies only in two cases. Non-MG colonies were found interspersed with MG colonies in agar plates and thus the selective isolation of MG colonies must be performed prior to subculture. The utility of PBS and buffered peptone water supplemented with glycerol as transit media for clinical samples intended for PCR was evidenced. The isolates obtained were successfully lyophilized and stored at โ€“70ยฐC throughout the span of the study. The PCR-RFLP pattern of the obtained MG isolates revealed uniformity among the isolates. None of the samples yielded positive result in MS-PCR and MI-PCR. The sensitivity and usefulness of molecular methods based detection of pathogenic mycoplasmas of chicken over isolation techniques could be appreciated.
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    ThesisItemOpen Access
    Nucleic Acid based detection of salmonellae in poultry
    (Department of Veterinary Microbiology, College of Veterinary and Animal Science, Mannuthy, 2005) Muthuramalingam, M; KAU; Mini, M
    In the present study detection of salmonellae by Polymerase Chain Reaction in avian bio-materials was carried out. Isolation of salmonellae from avian bio-materials was also done. Differentiation of salmonellae based on molecular methods was carried out. Thirteen isolates from chicken and two from quails were characterized as Salmonella gallinarum using standard bacteriological procedures. With regard to the fermentation of the sugars all isolates fermented dulcitol, maltose, arabinose, trehalose, and mannitol. Variation in fermentation pattern was observed with xylose and sorbitol. All isolates were uniformly sensitive to chloramphenicol, ciprofloxacin, enrofloxacin, and pefloxacin, while all were resistant to tetracycline, furazolidone and cloxacillin. Two isolates were serotyped as Salmonella gallinarum by the National Salmonella and Escherichia Center, Kasauli. Forty-six samples were positive by both genus specific as well as serovar specific PCR. The genus specific and serovar specific PCR were used to confirm the identity of the isolates. Performing PCR on template DNA prepared from RV broth enriched sample was found to be an extremely rapid method for detection of Salmonella. Restriction enzyme analysis of the amplicon from the rfbS gene with enzyme Tfi ะ† of all isolates revealed the expected 235 bp digestion. All the isolates carried plasmids. Two plasmid profiles were observed among the isolates examined. A multiplex PCR for virulence plasmid was carried out. The expected 571 bp level amplification, which is specific for Spv virulence region and 284 bp level amplification, which is specific for genus Salmonella, were obtained in all the isolates. An allele-specific PCR method was developed for serotype-specific detection of S. gallinarum. The expected 187 bp level amplicons were obtained in all the isolates. The sequence of the rfbS gene product has been submitted to the Genbank and has been assigned the accession No AF 442573 ATCC 9184 . The sequence showed 99 per cent identity with Salmonella gallinarum.
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    ThesisItemOpen Access
    Detection of pathogenic haemolytic bacteria in respiratory tract infections of livestock
    (Department of Veterinery Microbiology, College of Veterinary and Animal Science, Mannuthy, 2005) Aparna, S; KAU; Krishnan Nair, G
    A study was undertaken to elucidate the role of haemolytic bacteria in respiratory tract infections of livestock. This envisaged the isolation and identification of the haemolytic bacteria, determining their antibiogram patterns and testing the pathogenicity of the isolates in mice. The study also envisaged the detection of Mannheimia haemolytica by polymerase chain reaction, determining the genetic relationship of Staphylococcus aureus isolates from different animal hosts using RAPD-PCR technique and also analysis of plasmid profiles of Escherichia coli isolates. Samples were collected from clinically ill livestock and at random from apparently healthy animals from in an around Thrissur district. A total of 309 samples were taken which consisted of nasal swabs, tracheal swabs, lung samples and blood samples. Samples were cultured on blood agar and on Mannheimia haemolytica selective medium. Mannheimia haemolytica could not be isolated from any of the samples. But pooled nasal swabs when cultured on blood agar gave an isolate with characteristics almost similar to Mannheimia haemolytica, but showed variations for ornithine decarboxylase activity and utilization of sugars like trehalose and salicin. As, no reference strain was available it was not possible to make a comparison and confirm the isolate as Mannheimia haemolytica. From the samples cultured on ordinary blood agar medium a total of 20 haemolytic bacterial isolates could be obtained. The different bacterial isolates were Staphylococcus aureus (40 per cent), Staphylococcus epidermidis (10 per cent), Escherichia coli (30 per cent), Klebsiella pneumoniae (5 per cent), Streptococcus pyogenes (5 per cent), Streptococcus agalactiae (5 per cent) and Arcanobacterium pyogenes (5 per cent). The haemolytic bacteria were identified based on morphology, cultural characteristics and biochemical tests. Antimicrobial sensitivity pattern of the isolates showed that almost all the isolates had high sensitivity to pefloxacin and ampicillin. Antimicrobial resistance was shown maximum to erythromycin. The three Staphylococcus aureus isolates, all the E. coli isolates and Klebsiella isolate caused death of mice. Rest of the five Staphylococcus aureus isolates, Streptococcus pyogenes , Streptococcus agalactiae and Arcanobacterium pyogenes did not cause death of mice but produced internal lesions and could be re-isolated. Staphylococcus epidermidis isolates could neither cause death, nor produce internal lesions and could not be re-isolated. None of the nasal and tracheal swabs, lung samples and blood samples gave a positive result for Mannheimia haemolytica specific PCR. A pooled sample of nasal swabs from cattle with respiratory infection gave a positive result for it. But PCR of the culture could not yield a positive result. Moreover, no reference strain was available to make a comparison and confirm the result. RAPD-PCR of the Staphylococcus aureus isolates showed that there was considerable genetic relationship between Staphylococcus aureus isolates of different species and also there was noticeable genetic diversity of the isolates within the host species. Plasmids could be isolated only from two of the six isolates of Escherichia coli studied. Plasmid profile analysis of the isolates could not ascertain any correlation between the virulence, antibiotic resistance and presence of plasmids.
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    ThesisItemOpen Access
    Detection and identification of pathogenic leptospires in bio-meterials
    (Department of Veterinary Microbiology, College of Veterinary and Animal Science, Mannuthy, 2005) Dhannia, A; KAU; Jayaprakashan, V
    In the present study an attempt has been made to detect and differentiate leptospires in bio-materials employing molecular techniques such as genus specific PCR, multiplex PCR, nested PCR, AP-PCR, LS-PCR and PCR-REA. Isolation trials for Leptospira were also made in the study and the isolates were tried to be differentiated employing the molecular methods mentioned above. The genus specific primers A and B were used to detect leptospires in clinical samples and samples from rodents. Out of the 147 samples only nine were positive for leptospiral DNA. Out of the nine positive samples eight were serum samples (four from cattle and four from dogs) and one was kidney of a bandicoot. Multiplex PCR, using the primers G1/G2 and B64-I/B64-II could differentiate leptospires into pathogenic and non-pathogenic ones. Among the pathogenic leptospires it could differentiate the species L. kirschneri, from other six pathogenic species viz L . interrogans, L. borgpetersenii, L. santarosai, L. noguchii, L. inadai and L. weillii. All the six isolates were found to be belonging to any of these six species. Nested PCR using the primers designed based on the sequence of L. borgpetersenii and L. interrogans amplified DNA from all the ten reference strains including the non-pathogenic serovar patoc and rachmati of L. kirschneri species. All the six isolates were amplified giving PCR products of expected sizes, 571 bp and 370 bp.
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    ThesisItemOpen Access
    Role of newcastle disease, infectious bronchitis and egg drop syndrome-76 viruses in low egg production of chicken in Kerala
    (Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 2004) Arun, A; KAU; Mini, M
    The present study has been undertaken to assess the prevalence of Newcastle disease (ND), infectious bronchitis (IB) and egg drop syndrome-76 (EDS-76) viruses in apparently healthy layer chicken in Kerala by seroprevalence studies and virus isolation trials and to associate these viruses with low egg production. Sera, cloacal and tracheal swab samples were collected from two to five per cent of total population of a farm to carry out the study. Samples were collected from Regional Poultry Farms (RPF) of Kerala, University Poultry Farm, Mannuthy and also from birds brought to the department of microbiology for disease investigation. To assess the production performance of the layers data pertaining to the egg production were collected, for the past one to five years depending upon the availability. The haemagglutination inhibition (HI) test was used to study the seroprevalence of ND. Out of 615 samples tested, 548 samples were found to be positive. Newcastle disease vaccination was carried out regularly in all the organized farms and the titre is to be considered as an effect of vaccination. Seven virus isolates were recovered from cloacal swab samples and was identified as NDV by HI test. The isolates were subjected to conventional characterization by mean death time, intracerebral pathogenicity index, intravenous pathogenicity index, and stability of haemagglutinins at 56ยบ C, agglutination of mammalian erythrocytes, and adsorption of haemagglutinins by chick embryo brain cells. Results of these tests identified all the seven isolates as lentogenic. The isolates produced mild cytopathic effects (CPE) like syncytia, pyknosis of nuclei and vacuolation of nuclei in chick embryo fibroblast (CEF) monolayer culture. Immunofluorescence test (IFT) was carried out in CEF monolayer using NDV antiserum and specific fluorescence was noticed in the cytoplasm. All the isolates were subjected to reverse transcriptase polymerase chain reaction (RT-PCR) for amplifying 254 bp fusion protein cleavage site (FPCS) sequence gene. Sera samples were tested by ELISA for detecting IB antibodies due to the erratic results of HI test. Out of 418 sera samples screened, 311 (74.40 per cent) samples were found to be positive for IB. Since IB vaccination was not followed in any of the farms in Kerala, the antibodies might be due to past/subclinical infection. One IBV isolate was recovered from tissue samples of ailing broiler bird and was identified by AGID test. The ciliostatic effect of IBV was studied in tracheal organ culture (TOC) and the virus was identified in TOC by IFT and immunoperoxidase test (IPT). The IB virus was confirmed by RT-PCR using primers for a 464 bp nucleotide sequence of S1gene. The antibodies against EDS-76 virus were identified by HI test. Out of 615 chicken sera samples tested, 58 (9.43 per cent) were found to be positive. None of the swab and tissue samples yielded EDS-76 virus. By correlating the production performance of chicken with seroprevalence and isolation trials ND, IB and EDS-76 viruses, this study revealed the possible involvement of IB and EDS-76 viruses in low egg production of chicken in Kerala, because the birds are well protected against ND by vaccination.
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    ThesisItemOpen Access
    Assessment of different experimental vaccines against Chlamydophila abortus (Chlamydia psittaci)in rabbits
    (Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 2003) Sanjeetha, L; KAU; Mini, M
    Two isolates of Chlamydia psittaci viz., M-430 and M-28, maintained in the Department of Microbiology, were used in the study. M-430 was used for the preparation of inactivated yolk sac and elementary body vaccines and M-28 was used for challenge experiment. These isolates were revived by inoculating to six to eight day-old chicken embryo through yolk sac route. Both isolates produced characteristic lesions in the embryo and yolk sac membrane. M-430 was also propagated in Mc Coy cell line for high yield of elementary bodies for vaccine preparation. Homogenous suspensions of the yolk sac and elementary body vaccines were inactivated with formalin to a final concentration of 0.4 per cent. Pure and safe preparations were used for vaccination trial. Immunogenic potential of the vaccines were tested in rabbits by giving two doses of each of the vaccines. The first dose was given at three months of age and second dose was given 14 days after the first. Three vaccinated and control rabbits were challenged with M-28 isolate on 28th day post vaccination and rest three on 70th day post vaccination. The clearance of elementary bodies from the tissues (lung, liver lymphnode and spleen) of vaccinated rabbits was an indication of the protection conferred by the vaccines. Better response was noticed with EB vaccine than yolk sac vaccine. The sera were collected from rabbits at regular intervals of 0, 7, 14, 21, 28, 42, 56 and 70 for passive haemagglutination and serum neutralization test. Both vaccines elicited good immune response. The greater humoral immune response of the rabbits that received EB vaccine suggests its slight superiority over the yolk sac vaccine. More evaluation and elaborated field trials on target species are required before advocating the vaccine for field use