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Isolation and Characterization of Novel MAP Strains from Ugandan Cattle
146:1, 2012
ESVP/ECVP Proceedings 2011
69
IMMUNOHISTOCHEMICAL LOCALIZATION OF HAPTOGLOBIN IN PORCINE SALIVARY GLAND AND DIAPHRAGM es y, J. G omez-Laguna *, A. Guti errez y, F. J. Pallar I. M. Rodrıguez-G omez z, I. Barranco z, J. J. Cer on y and L. Carrasco Libradoz *CICAP, Pozoblanco, yUniversity of Murcia, Murcia and z University of Cordoba, Cordoba, Spain Introduction: Changes in the concentration of haptoglobin (Hp) have been reported in saliva and meat juice samples; however the extrahepatic localization of this protein is uncertain. In this study, immunohistochemistry (IHC) was employed to localize Hp. Materials and Methods: Five healthy and five diseased conventional pigs from a finishing unit in which animals were seropositive to PRRSv, PCV2, Mycoplasma hyopneumoniae and A. pleuropneumoniae were used in the present study. Samples from the liver, salivary gland and diaphragm were collected and fixed in 10% neutral buffered formalin. IHC was performed by the ABC method using an in-house monoclonal antibody against porcine Hp. Results: In diseased animals, the expression of Hp was confirmed by means of immunolabelling in the liver. In the salivary gland, Hp was detected in the cytoplasm of scattered glandular epithelial cells, as well as within the cytoplasm of duct epithelial cells. Multifocal labelling of myofibres of skeletal muscle was observed. In contrast, healthy pigs displayed mild to poor immunolabelling of Hp in salivary gland and diaphragmatic muscle. Conclusions: The extrahepatic localization of Hp observed would suggest a contribution of these tissues to the increment of Hp levels found in saliva and meat juice samples in inflammatory conditions.
ISOLATION AND CHARACTERIZATION OF NOVEL MAP STRAINS FROM UGANDAN CATTLE J. B. Okuni *, C. I. Dovas z, I. Bouzalas z, P. Loukopoulos z, P. D. Katete y and L. Ojok* *Department of Veterinary Pathology, School of Veterinary Medicine, y Department of Medical Micriobiology, College of Health Sciences, Makerere University, Kampala, Uganda and zFaculty of Veterinary Medicine, Aristotle University, Thessaloniki, Greece Introduction: Mycobacterium avium subspecies paratuberculosis (MAP) infection has been confirmed only recently among Ugandan cattle. Hence, no information exists on the diversity of MAP strains from the country. The aim of this study was to isolate and characterize MAP from faeces of ELISA-positive cattle and tissues with histologically-suspected lesions of Johne’s disease. Materials and Methods: Twenty-one MAP isolates, confirmed by their mycobactin dependence on Herrold’s egg yolk medium and IS900 PCR, were characterized using molecular markers comprising short sequence repeats (SSR, loci 1, 2 and 8), mycobacterial interspersed repeat units (MIRU, loci 2 and 3), variable number tandem repeat units (VNTR, locus 32) and IS1311 PCR-REA analysis. Results: The 21 isolates were differentiated into 10 different strains using a combination of all the markers. These strains were distributed throughout the country. The results show the existence of the cattle and bison type strains in the country. Two isolates showed a yet unreported IS1311 pattern designated as X type. The study has also shown the existence of new SSR genotypes of MAP previously unreported in cattle elsewhere.
FIRST REPORT OF ALEUTIAN DISEASE IN A LEAST WEASEL (MUSTELA NIVALIS) P. Loukopoulos *, C. Billinis y and E. Tsaliey *Faculty of Veterinary Medicine, Aristotle University, Thessaloniki and y Faculty of Veterinary Medicine, University of Thessaly, Karditsa, Greece Introduction: Aleutian disease (AD) is a slow infection caused by a parvovirus (ADV) and almost exclusively affects mink, although antibodies have been demonstrated in other species. Materials and Methods: Gross pathology, histopathology and PCR were used to establish the diagnosis of AD in an 8-month-old male least weasel. Results: The animal was found dead at the Thessaloniki Zoo. It had decreased appetite for 3e4 days. It had been kept as a pet and was donated to the zoo 2 months prior to its death. In both places it was housed alone. On necropsy examination, it was cachectic and showed diffuse alopecia. Multiple small white foci were scattered throughout the lungs. The liver and spleen were markedly enlarged. Histologically, severe multifocal or diffuse plasma cell infiltration of various organs was observed. Using PCR, ADV DNA was detected in various organs. Conclusions: The source of infection in the present case is unknown; given the fact that no mink or other Mustelidae were kept at the zoo. It is possible that it was another animal carrying the virus, that came in contact with the weasel, most likely while it was kept as a pet. AD is reported for the first time in this species.
DIAGNOSIS OF CAPRINE TUBERCULOSIS USING ESAT-6/ CFP-10 PEPTIDES IN PERSISTENLY INFECTED HERDS A. J. Buendia *, J. A. Navarro *, J. Salinas *, H. M. Vordermeier y, A. Aranaz z, J. Bezos z, C. Penafiel-Verdu *, N. Ortega *, R.G. Hewinson y and J. Sanchez* *Facultad de Veterinaria, Univesidad de Murcia, Spain, yAnimal Health and Veterinary Laboratories Agency, Addlestone, UK and zVISAVET, Facultad de Veterinaria, Universidad Complutense de Madrid, Spain Introduction: A caprine tuberculosis eradication programme based on the comparative tuberculin skin test is being implemented in the southeast of Spain. Although initial progress was promising, the programme subsequently stalled. Two observations made were that the presence of paratuberculosis in the flocks and the desensitization caused by repeat tuberculin skin test could lead to a decrease in the skin test sensitivity. Materials and Methods: To evaluate the efficacy of the alternative blood-based IFN-g assay in conjunction with peptides derived from the specific antigens ESAT-6/CFP-10 for the diagnosis of caprine tuberculosis, two goat herds with persistent tuberculosis co-infected with paratuberculosis were selected for study. The results obtained using these antigens were compared with skin test and the IFN-g assay using avian and bovine tuberculin. Several rounds of testing with the three techniques were carried out in each herd and test-positive goats were killed after each test round to establish the presence of tuberculosis infection by macroscopical and microscopical analysis of lesions and M. caprae isolation. Results: The IFN-g assay using ESAT-6/CFP-10 performed better (72% sensitivity and 90% specificity) than the other skin test and IFN-g assay using PPD (sensitivities of 40% and 68%, specificities of 45% and 84%, respectively). The false-negative animals detected in the study had small single lesions, generally fibrocalcified, that could correspond to non-active/latent infections.
ESVP/ECVP Proceedings 2011
69
IMMUNOHISTOCHEMICAL LOCALIZATION OF HAPTOGLOBIN IN PORCINE SALIVARY GLAND AND DIAPHRAGM es y, J. G omez-Laguna *, A. Guti errez y, F. J. Pallar I. M. Rodrıguez-G omez z, I. Barranco z, J. J. Cer on y and L. Carrasco Libradoz *CICAP, Pozoblanco, yUniversity of Murcia, Murcia and z University of Cordoba, Cordoba, Spain Introduction: Changes in the concentration of haptoglobin (Hp) have been reported in saliva and meat juice samples; however the extrahepatic localization of this protein is uncertain. In this study, immunohistochemistry (IHC) was employed to localize Hp. Materials and Methods: Five healthy and five diseased conventional pigs from a finishing unit in which animals were seropositive to PRRSv, PCV2, Mycoplasma hyopneumoniae and A. pleuropneumoniae were used in the present study. Samples from the liver, salivary gland and diaphragm were collected and fixed in 10% neutral buffered formalin. IHC was performed by the ABC method using an in-house monoclonal antibody against porcine Hp. Results: In diseased animals, the expression of Hp was confirmed by means of immunolabelling in the liver. In the salivary gland, Hp was detected in the cytoplasm of scattered glandular epithelial cells, as well as within the cytoplasm of duct epithelial cells. Multifocal labelling of myofibres of skeletal muscle was observed. In contrast, healthy pigs displayed mild to poor immunolabelling of Hp in salivary gland and diaphragmatic muscle. Conclusions: The extrahepatic localization of Hp observed would suggest a contribution of these tissues to the increment of Hp levels found in saliva and meat juice samples in inflammatory conditions.
ISOLATION AND CHARACTERIZATION OF NOVEL MAP STRAINS FROM UGANDAN CATTLE J. B. Okuni *, C. I. Dovas z, I. Bouzalas z, P. Loukopoulos z, P. D. Katete y and L. Ojok* *Department of Veterinary Pathology, School of Veterinary Medicine, y Department of Medical Micriobiology, College of Health Sciences, Makerere University, Kampala, Uganda and zFaculty of Veterinary Medicine, Aristotle University, Thessaloniki, Greece Introduction: Mycobacterium avium subspecies paratuberculosis (MAP) infection has been confirmed only recently among Ugandan cattle. Hence, no information exists on the diversity of MAP strains from the country. The aim of this study was to isolate and characterize MAP from faeces of ELISA-positive cattle and tissues with histologically-suspected lesions of Johne’s disease. Materials and Methods: Twenty-one MAP isolates, confirmed by their mycobactin dependence on Herrold’s egg yolk medium and IS900 PCR, were characterized using molecular markers comprising short sequence repeats (SSR, loci 1, 2 and 8), mycobacterial interspersed repeat units (MIRU, loci 2 and 3), variable number tandem repeat units (VNTR, locus 32) and IS1311 PCR-REA analysis. Results: The 21 isolates were differentiated into 10 different strains using a combination of all the markers. These strains were distributed throughout the country. The results show the existence of the cattle and bison type strains in the country. Two isolates showed a yet unreported IS1311 pattern designated as X type. The study has also shown the existence of new SSR genotypes of MAP previously unreported in cattle elsewhere.
FIRST REPORT OF ALEUTIAN DISEASE IN A LEAST WEASEL (MUSTELA NIVALIS) P. Loukopoulos *, C. Billinis y and E. Tsaliey *Faculty of Veterinary Medicine, Aristotle University, Thessaloniki and y Faculty of Veterinary Medicine, University of Thessaly, Karditsa, Greece Introduction: Aleutian disease (AD) is a slow infection caused by a parvovirus (ADV) and almost exclusively affects mink, although antibodies have been demonstrated in other species. Materials and Methods: Gross pathology, histopathology and PCR were used to establish the diagnosis of AD in an 8-month-old male least weasel. Results: The animal was found dead at the Thessaloniki Zoo. It had decreased appetite for 3e4 days. It had been kept as a pet and was donated to the zoo 2 months prior to its death. In both places it was housed alone. On necropsy examination, it was cachectic and showed diffuse alopecia. Multiple small white foci were scattered throughout the lungs. The liver and spleen were markedly enlarged. Histologically, severe multifocal or diffuse plasma cell infiltration of various organs was observed. Using PCR, ADV DNA was detected in various organs. Conclusions: The source of infection in the present case is unknown; given the fact that no mink or other Mustelidae were kept at the zoo. It is possible that it was another animal carrying the virus, that came in contact with the weasel, most likely while it was kept as a pet. AD is reported for the first time in this species.
DIAGNOSIS OF CAPRINE TUBERCULOSIS USING ESAT-6/ CFP-10 PEPTIDES IN PERSISTENLY INFECTED HERDS A. J. Buendia *, J. A. Navarro *, J. Salinas *, H. M. Vordermeier y, A. Aranaz z, J. Bezos z, C. Penafiel-Verdu *, N. Ortega *, R.G. Hewinson y and J. Sanchez* *Facultad de Veterinaria, Univesidad de Murcia, Spain, yAnimal Health and Veterinary Laboratories Agency, Addlestone, UK and zVISAVET, Facultad de Veterinaria, Universidad Complutense de Madrid, Spain Introduction: A caprine tuberculosis eradication programme based on the comparative tuberculin skin test is being implemented in the southeast of Spain. Although initial progress was promising, the programme subsequently stalled. Two observations made were that the presence of paratuberculosis in the flocks and the desensitization caused by repeat tuberculin skin test could lead to a decrease in the skin test sensitivity. Materials and Methods: To evaluate the efficacy of the alternative blood-based IFN-g assay in conjunction with peptides derived from the specific antigens ESAT-6/CFP-10 for the diagnosis of caprine tuberculosis, two goat herds with persistent tuberculosis co-infected with paratuberculosis were selected for study. The results obtained using these antigens were compared with skin test and the IFN-g assay using avian and bovine tuberculin. Several rounds of testing with the three techniques were carried out in each herd and test-positive goats were killed after each test round to establish the presence of tuberculosis infection by macroscopical and microscopical analysis of lesions and M. caprae isolation. Results: The IFN-g assay using ESAT-6/CFP-10 performed better (72% sensitivity and 90% specificity) than the other skin test and IFN-g assay using PPD (sensitivities of 40% and 68%, specificities of 45% and 84%, respectively). The false-negative animals detected in the study had small single lesions, generally fibrocalcified, that could correspond to non-active/latent infections.