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Efficiency of an indirect ELISA using two antigen preparations of Brucella canis for immunodiagnosis
Abstracts / Veterinary Immunology and Immunopathology 128 (2009) 211–347
makes extremely hard the diagnosis by serological techniques of triage. The law does not establish any test for Ig detection in semen of reproductive bulls. The purpose of this study was to investigate the seroprevalence of brucellosis using blood serum and seminal plasma from 71 bulls provided of 8 properties in Uberlandia County–MG State, Brazil. This work has compared the results of two tests for diagnosis of bovine brucellosis, applying the tests of RB (of triage) and 2-ME (confirmatory). The results found in these two tests present a negative correlation, considering that: when used the blood serum, it has presented a decrease in number of positive bulls for the brucellosis diagnosis (2 positive animals). On the other hand, when used the seminal plasma, the number of animals with positive diagnosis has increased (7 positive animals). This study results highlight the immediate necessity to also investigate antibodies to Brucella in seminal plasma. doi:10.1016/j.vetimm.2008.10.058 Efficiency of an indirect ELISA using two antigen preparations of Brucella canis for immunodiagnosis Maria Z.D. Oliveira 1,3,∗ , Songeli M. Freire 3 , Roberto Meyer 3 , Lara Keid 4 , Stella M. Barrouin-Melo 1,2 , Palis Paulo 1,2,3,4 1
Laboratório de Infectologia Veterinária, Escola de Medicina Veterinária, Universidade Federal da Bahia (UFBA), Av. Ademar de Barros, 500 Salvador, Bahia CEP 40170-000, Brazil 2 Departamento de Patologia e Clínicas, Escola de Medicina Veterinária, UFBA, Brazil 3 Laboratório de Imunologia e Biologia Molecular, Instituto de Ciências da Saúde, UFBA, AvReitor Miguel Calmon S/N Vale do Canela Salvador, Bahia CEP40.110-100, Brazil 4 Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo, Brazil Keywords: Brucella canis; Immunodiagnostics; ELISA; Dog E-mail address: [email protected] (M.Z.D. Oliveira). Species: Canine Introduction: Canine brucellosis is a zoonotic bacterial infection whose clinical diagnosis is difficult to perform due to frequent asymptomatic cases. The disease is important for Public Health. Human infections have been reported, being laboratory technicians and dog owners the most exposed to B. canis. Having a worldwide distribution, the infection represents a great cause of economic losses in breeding kennels, since it causes abortion and infertility in dogs. Despite molecular techniques have been recently described, blood culture is still considered the only routine definitive test for the diagnosis of canine infection. Different serological tests are routinely used, but there is a need for a fast, reliable and low cost immunological test to improve the immunodiagnosis and surveillance studies of canine brucellosis. The aim of this study was to compare two antigen preparations made from a culture of B. canis in ELISA tests using sera from dogs with distinct profiles of infection, defined as positive and negative.
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Material and methods: A B. canis strain, isolated from aborted placenta and fetuses of a bitch, was used for producing two antigen preparations: a heat soluble bacterial extract and a sonically obtained extract. After centrifugation steps, the soluble fractions of both preparations were applied in two indirect ELISA tests, performed with sera from 85 dogs, for detection of B. canis-specific IgG. Fiftyone from these 85 animals were diagnosed positive for canine brucellosis, by blood cultures and serology in agargel immunodiffusion test (AGID). Thirty-four of these dogs were healthy negative animals, with no history of breeding. The optical density readings were used to calculate specificity, sensitivity and accuracy of the ELISAs by ROC analysis. Results: The sensitivity, specificity and accuracy were of 100, 84.30 and 90.6% for the ELISA test with sonically obtained extract, and of 91.2, 100% and 94.4% for the ELISA test with heat soluble extract, respectively. Conclusions: Both ELISA tests using the different antigen preparations obtained from a wild B. canis were considered applicable for serodiagnosis of canine brucellosis, showing good specificity and sensitivity. doi:10.1016/j.vetimm.2008.10.059 Inhibition of Ehrlichia canis infection by interferon gamma in vitro Tomoko Tajima 1,∗ , Makoto Wada 1 , Misao Onuma 2 1
Laboratory of Veterinary Microbiology, Graduate School of Life and Environmental Science, Osaka Prefecture University, Osaka, Japan 2 Laboratory of Infectious Disease, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan Keywords: Ehrlichia canis; Interferon gamma E-mail address: [email protected] (T. Tajima).
Species: CanineEhrlichia canis infects to macrophagesmonocytes of dog and cause canine monocytic ehrlichiosis, a persistent infection that continues for a long period even after treatment with antibiotics. We previously reported that expression of mRNA for interferon-␥ (IFN-␥) was detected in peripheral blood mononuclear cells (PBMC) from E. canis-infected dogs within three days after infection and continued for more than 50 days. To determine the role of IFN-␥ in E. canis infection, E. canis was cultured with white blood cells (WBC) or PBMC from E. canis-infected dogs. Two SPF female beagle dogs were inoculated intravenously with E. canis Oklahoma strain and the infection was confirmed by PCR using peripheral blood DNA of these dogs 14 days post-inoculation (DPI). Blood was drawn from each dog on 16 DPI, and isolated WBC or PBMC were added to E. canis-inoculated DH82 cells. WBC and PBMC from an uninfected beagle dog were used as negative controls. The cultured cells were collected daily, stained, and incidence of inclusion-positive cells was calculated by counting cells under microscope. Incidence of inclusion-positive cells was significantly reduced in the culture with WBC or PBMC from E. canis-infected dogs but not with those from uninfected
makes extremely hard the diagnosis by serological techniques of triage. The law does not establish any test for Ig detection in semen of reproductive bulls. The purpose of this study was to investigate the seroprevalence of brucellosis using blood serum and seminal plasma from 71 bulls provided of 8 properties in Uberlandia County–MG State, Brazil. This work has compared the results of two tests for diagnosis of bovine brucellosis, applying the tests of RB (of triage) and 2-ME (confirmatory). The results found in these two tests present a negative correlation, considering that: when used the blood serum, it has presented a decrease in number of positive bulls for the brucellosis diagnosis (2 positive animals). On the other hand, when used the seminal plasma, the number of animals with positive diagnosis has increased (7 positive animals). This study results highlight the immediate necessity to also investigate antibodies to Brucella in seminal plasma. doi:10.1016/j.vetimm.2008.10.058 Efficiency of an indirect ELISA using two antigen preparations of Brucella canis for immunodiagnosis Maria Z.D. Oliveira 1,3,∗ , Songeli M. Freire 3 , Roberto Meyer 3 , Lara Keid 4 , Stella M. Barrouin-Melo 1,2 , Palis Paulo 1,2,3,4 1
Laboratório de Infectologia Veterinária, Escola de Medicina Veterinária, Universidade Federal da Bahia (UFBA), Av. Ademar de Barros, 500 Salvador, Bahia CEP 40170-000, Brazil 2 Departamento de Patologia e Clínicas, Escola de Medicina Veterinária, UFBA, Brazil 3 Laboratório de Imunologia e Biologia Molecular, Instituto de Ciências da Saúde, UFBA, AvReitor Miguel Calmon S/N Vale do Canela Salvador, Bahia CEP40.110-100, Brazil 4 Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo, Brazil Keywords: Brucella canis; Immunodiagnostics; ELISA; Dog E-mail address: [email protected] (M.Z.D. Oliveira). Species: Canine Introduction: Canine brucellosis is a zoonotic bacterial infection whose clinical diagnosis is difficult to perform due to frequent asymptomatic cases. The disease is important for Public Health. Human infections have been reported, being laboratory technicians and dog owners the most exposed to B. canis. Having a worldwide distribution, the infection represents a great cause of economic losses in breeding kennels, since it causes abortion and infertility in dogs. Despite molecular techniques have been recently described, blood culture is still considered the only routine definitive test for the diagnosis of canine infection. Different serological tests are routinely used, but there is a need for a fast, reliable and low cost immunological test to improve the immunodiagnosis and surveillance studies of canine brucellosis. The aim of this study was to compare two antigen preparations made from a culture of B. canis in ELISA tests using sera from dogs with distinct profiles of infection, defined as positive and negative.
237
Material and methods: A B. canis strain, isolated from aborted placenta and fetuses of a bitch, was used for producing two antigen preparations: a heat soluble bacterial extract and a sonically obtained extract. After centrifugation steps, the soluble fractions of both preparations were applied in two indirect ELISA tests, performed with sera from 85 dogs, for detection of B. canis-specific IgG. Fiftyone from these 85 animals were diagnosed positive for canine brucellosis, by blood cultures and serology in agargel immunodiffusion test (AGID). Thirty-four of these dogs were healthy negative animals, with no history of breeding. The optical density readings were used to calculate specificity, sensitivity and accuracy of the ELISAs by ROC analysis. Results: The sensitivity, specificity and accuracy were of 100, 84.30 and 90.6% for the ELISA test with sonically obtained extract, and of 91.2, 100% and 94.4% for the ELISA test with heat soluble extract, respectively. Conclusions: Both ELISA tests using the different antigen preparations obtained from a wild B. canis were considered applicable for serodiagnosis of canine brucellosis, showing good specificity and sensitivity. doi:10.1016/j.vetimm.2008.10.059 Inhibition of Ehrlichia canis infection by interferon gamma in vitro Tomoko Tajima 1,∗ , Makoto Wada 1 , Misao Onuma 2 1
Laboratory of Veterinary Microbiology, Graduate School of Life and Environmental Science, Osaka Prefecture University, Osaka, Japan 2 Laboratory of Infectious Disease, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan Keywords: Ehrlichia canis; Interferon gamma E-mail address: [email protected] (T. Tajima).
Species: CanineEhrlichia canis infects to macrophagesmonocytes of dog and cause canine monocytic ehrlichiosis, a persistent infection that continues for a long period even after treatment with antibiotics. We previously reported that expression of mRNA for interferon-␥ (IFN-␥) was detected in peripheral blood mononuclear cells (PBMC) from E. canis-infected dogs within three days after infection and continued for more than 50 days. To determine the role of IFN-␥ in E. canis infection, E. canis was cultured with white blood cells (WBC) or PBMC from E. canis-infected dogs. Two SPF female beagle dogs were inoculated intravenously with E. canis Oklahoma strain and the infection was confirmed by PCR using peripheral blood DNA of these dogs 14 days post-inoculation (DPI). Blood was drawn from each dog on 16 DPI, and isolated WBC or PBMC were added to E. canis-inoculated DH82 cells. WBC and PBMC from an uninfected beagle dog were used as negative controls. The cultured cells were collected daily, stained, and incidence of inclusion-positive cells was calculated by counting cells under microscope. Incidence of inclusion-positive cells was significantly reduced in the culture with WBC or PBMC from E. canis-infected dogs but not with those from uninfected