A polymerase chain reaction for the detection of Brucella canis in semen of naturally infected dogs

The objective was to evaluate a PCR assay for the detection of Brucella canis in canine semen, comparing its performance with that of bacterial isolation, serological tests and PCR assay of blood. Fifty-two male dogs were examined clinically to detect reproductive abnormalities and their serum was tested by the rapid slide agglutination test, with and without 2-mercaptoethanol (2ME-RSAT and RSAT, respectively). In addition, microbiological culture and PCR assays were performed on blood and semen samples. The findings of the semen PCR were compared (Kappa coefficient and McNemar test) to those of blood PCR, culture of blood and semen, RSAT, and 2ME-RSAT. Nucleic acid extracts from semen collected from dogs not infected with B. canis were spiked with decreasing amounts of B. canis RM6/66 DNA and the resulting samples subjected to PCR. In addition, semen samples of non-infected dogs were spiked with decreasing amounts of B. canis CFU and the resulting suspensions were used for DNA extraction and amplification. of the 52 dogs that were examined, the following tests were positive: RSAT, 16 (30.7%); 2ME-RSAT, 5 (9.6%); blood culture, 14 (26.9%); semen culture, 11 (21.1%); blood PCR, 18 (34.6%); semen PCR, 18 (34.6%). The PCR assay detected as few as 3.8 fg of B. canis DNA experimentally diluted in 444.9 ng of canine DNA (extracted from semen samples of noninfected dogs). In addition, the PCR assay amplified B. canis genetic sequences from semen samples containing as little as 1.0 x 10(0) cfu/mL. We concluded that PCR assay of semen was a good candidate as a confirmatory test for the diagnosis of brucellosis in dogs; its diagnostic performance was similar to blood culture or blood PCR. Furthermore, the PCR assay of semen was more sensitive than the 2ME-RSAT or semen culture. Examination of semen by PCR should be included for diagnosis of brucellosis prior to natural mating or AI; in that regard, some dogs that were negative on serological and microbiological examinations as well as blood PCR were positive on PCR of semen. (c) 2007 Elsevier B.V. All rights reserved.

A polymerase chain reaction for detection of Brucella canis in vaginal swabs of naturally infected bitches

A PCR assay for the detection of Brucella canis in canine vaginal swab samples was evaluated, comparing its performance with that of bacterial isolation, serological tests, and a blood PCR assay. One hundred and forty-four female dogs were clinically examined to detect reproductive problems and they were tested by the rapid slide agglutination test, with and without 2-mercaptoethanol (2ME-RSAT and RSAT, respectively). In addition, microbiological culture and PCR were performed on blood and vaginal swab samples. The results of the vaginal swab PCR were compared to those of the other tests using the Kappa coefficient and McNemar test. of the 144 females that were examined, 66 (45.8%) were RSAT positive, 23 (15.9%) were 2ME-RSAT positive, 49 (34.02%) were blood culture positive, 6 (4.1%) were vaginal swab culture positive, 54 (37.5%) were blood PCR positive, 52 (36.2%) were vaginal swab PCR positive, and 50.69% (73/144) were positive by the combined PCR. The PCR was able to detect as few as 3.8 fg of B. canis DNA experimentally diluted in 54 ng of canine DNA, extracted from vaginal swab samples of non-infected bitches. In addition, the PCR assay amplified B. canis genetic sequences from vaginal swab samples containing 1.0 x 10(0) cfu/mL. In conclusion, vaginal swab PCR was a good candidate as a confirmatory test for brucellosis diagnosis in bitches suspected to be infected, especially those negative on blood culture or blood PCR; these animals may be important reservoirs of infection and could complicate attempts to eradicate the disease in confined populations. (C) 2007 Elsevier B.V. All rights reserved.

Freqüência de anticorpos anti-Neospora caninum em soros de caprinos do estado de São Paulo e sua relação com o manejo dos animais

Com o objetivo de se avaliar a freqüência de caprinos leiteiros soropositivos para Neospora caninum, no estado de São Paulo, e se verificarem possíveis associações com idade, sexo e problemas reprodutivos, nos capris, e, também, presença de cães, nas propriedades, foram obtidos soros de 923 caprinos de ambos os sexos e idade acima de 3 meses. Os animais eram provenientes de 17 propriedades de diferentes municípios. Para o diagnóstico, foi utilizado o teste de aglutinação para Neospora (NATe25), e, em todos os capris, aplicou-se um inquérito a partir do qual se obtiveram informações epidemiológicas e de esfera reprodutiva. Todos os resultados estatísticos foram discutidos no nível de 5% de significância. Assim, chegou-se à conclusão de que a freqüência percentual de positividade para N. caninum foi de 19,77%, e, em apenas uma propriedade, não houve registro de animal soropositivo, o que revela difusão do agente, no Estado. Não foram verificadas diferenças significativas entre freqüências de positividade quanto ao sexo, idade ou problemas reprodutivos. Porém, ressalta-se que a presença de cães, nos capris, foi associada a uma maior freqüência de caprinos soropositivos a N. caninum. A representação geográfica da distribuição de caprinos soropositivos para o protozoário, em mapa coroplético em hachuras, pode implicar em um ganho considerável para estudos da epidemiologia geográfica, na elaboração de um planejamento de controle da enfermidade.

Soropositividade para brucelose em suínos em abatedouros

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação do teste do anel em leite na vigilância epidemiológica da brucelose bovina em rebanhos e em laticínios

Analisaram-se 464 amostras individuais de leite e 54 amostras de leite de latões, oriundos de leite dos mesmos animais, por meio do teste do anel do leite (TAL) visando à sua aplicação no diagnóstico individual e de rebanhos da brucelose bovina. Foram também avaliadas 464 amostras de soro sangüíneo por meio de provas do antígeno tamponado acidificado (ATA), soroaglutinação lenta em tubos (SAL) e 2-mercaptoetanol (2-ME), todas para brucelose. Cento e vinte e três amostras (26,5%) testadas pelo TAL apresentaram resultados positivos. Dessas, 30 resultaram positivas ao ATA, 28 ao ATA, à SAL e ao 2-ME e 18 à SAL. Das amostras positivas ao TAL, 95 pertenciam a animais sorologicamente negativos ao 2-ME, caracterizando 77,2% (95/123) das reações falso-positivas; dos resultados negativos ao TAL, 4 pertenciam a animais sorologicamente positivos, caracterizando 1,2% (4/341) de reações falso-negativas no TAL individual. Das 54 amostras de leite de latões analisadas pelo TAL, 17 foram consideradas positivas, das quais uma foi caracterizada como falso-positivo, pois todos os animais que a compunham foram negativos ao 2-ME. de 37 latões considerados negativos ao TAL, três continham leite de animais positivos ao 2-ME, caracterizando 8,1% de falso-negativos. O TAL individual demonstrou elevado percentual de resultados falso-positivos, enquanto o TAL em amostras de leite obtidas em latões detectou 84,2% de latões contaminados e 75% de rebanhos infectados.

Comparison of infection by Brucella spp. in free-ranging and captive wild animals from São Paulo State, Brazil

The aim of the current study was to evaluate the infection rate by Brucella spp. in wild and in captive animals. Serum samples from 121 animals (94 free-ranging and 27 captive) of different mammal species were evaluated. Sera were submitted to rose Bengal test (RBT) for screening and serum agglutination tests (SAT) and 2-mercaptoethanol test (2-ME) for confirmatory results. Nine animals (five free-ranging and four captive) tested positive in RBT, but negative in the confirmatory tests. Several domestic animal diseases that have control programs are not focused on wild reservoirs, such as brucellosis in Brazil. The study of new reservoirs in wildlife is essential to prevent emerging diseases.

Comparison of a PCR assay in whole blood and serum specimens for canine brucellosis diagnosis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Detection of Toxoplasma gondii in the milk of experimentally infected Wistar female rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Genotypic characterization of Toxoplasma gondii in sheep from Brazilian slaughterhouses: New atypical genotypes and the clonal type II strain identified

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Chlamydophila psittaci and Toxoplasma gondii infection in pigeons (Columba livia) from São Paulo State, Brazil

Pigeons (Columba livia) cohabit with humans in urban and rural areas, representing a public health problem since microorganisms are transmitted through the inhalation of dust from their dry feces (chlamydiosis) and through ingestion of their undercooked or poorly refrigerated meat (toxoplasmosis). This study aimed to evaluate the presence of Chlamydophila psittaci and Toxoplasma gondii in pigeons from four cities in São Paulo State, Brazil. C psittaci was evaluated through hemi-nested polymerase chain reaction (hnPCR) using cloacal and tracheal swabs, whereas T. gondii specific antibodies were assessed by means of modified agglutination test (MAT), mouse brain and muscle bioassay, and polymerase chain reaction (PCR). To confirm the infection in mice, T. gondii antibodies were assayed by using indirect fluorescent antibody test (IFAT). Considering C. psittaci, 40/238 (16.8%; 95%CI 12.6-22.1%) samples were positive according to hnPCR, especially for the cities of São Paulo (42.5%) and Bauru (35%). As regards T. gondii, 12/238 (5%; 95%CI 2.9-8.6%) serum samples were positive according to MAT. of these, five samples had titer equal to 1:8; six samples, 1:16; and one sample, 1:32. Bioassay, IFAT and PCR were negative for mouse toxoplasmosis. The absence of T. gondii antibodies suggests that pigeons may be infected with a low concentration of the agent, not detected by the antigen test. Thus, C. psittaci represents an actual problem concerning bird health. (C) 2010 Elsevier B.V. All rights reserved.

Serological profile of Toxoplasma gondii and Neospora caninum infection in commercial sheep from São Paulo State, Brazil

Neosporosis and toxoplasmosis are two important infections in young and adult sheep, leading to low production and abortion. This study aimed to determine the frequency of antibodies to Toxoplasma gondii and Neospora caninum in sheep from the eastern region of São Paulo State, Brazil. Serum samples (382) were collected from the sheep and assayed for T. gondii through modified agglutination test (MAT) and indirect fluorescent antibody test (IFAT), and for N. caninum antibodies, through IFAT, with cut-off titers equal to 16 (T. gondii) and 25 (N. caninum). All frozen samples were sent to the Center for Zoonoses Research (NUPEZO), Department of Veterinary Hygiene and Public Health (DHSVP), FMVZ, UNESP, for serological tests. A total of 71/382(18.6%) samples reacted to T. gondii, especially at titers 16 (28; 39.4%), 64(15; 21.1%), 256 (21; 29.6%) and 1024 (6; 8.5%) by MAT, and 16(34: 47.9%), 64 (18; 25.4%), 256 (14; 19.7%) and 1024 (5; 7%) by IFAT. As regards N. caninum, 49/382 (12.8%) samples reacted at titers 25 (17; 34.7%), 50 (11; 22.5%), 100 (11; 22.5%), and >= 200 (10; 20.4%). These animals presented infection but no clinical signs. Six and ten animals had high titers for toxoplasmosis and neosporosis. No significant association was observed between antibodies for both parasites (P = 0.535) according to Fisher's exact test, and no correlation was found between T. gondii (MAT) and N. caninum antibody titers (r = -0.0068; P=0.895), T. gondii (IFAT) and N. caninum antibody titers (r = -0.0025; P = 0.961). Thus, T. gondii and N. caninum infections were observed in farms located in São Paulo State, where sheep play an important economical role for the national and regional business. (C) 2010 Elsevier By. All rights reserved.

Recrudescence of Toxoplasma gondii infection in chronically infected rats (Rattus norvegicus)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Seroprevalence of anti-Leptospira spp. antibodies in dogs in Bahia, Brazil

The aim of this study was to determine the prevalence of anti-Leptospira spp. antibodies in dogs living in the urban area of the city of Ilheus, Bahia, Brazil using the microscopic agglutination test (MAT) to investigate 24 serovars. A semi-structured questionnaire was administered to dog owners to collect data about demography, husbandry and environmental factors. The prevalence of anti-Leptospira spp. antibodies in the population of 282 dogs was 7.1% (95% confidence interval: 4.4-10.7%). Serovar Copenhageni was the most prevalent, followed by serovars Bratislava, Canicola and Gryppotyphosa. No risk factor was detected with regard to demography (age, gender and breed), husbandry (Leptospira vaccinations, food and water exposure through their environment, hunting habits, contact with other animals and contact with rats) and environmental factors (sewage network, garbage collection, history of flooding, river proximity and wastelands). Despite the low prevalence found in this study, the seroprevalence of Leptospira spp. in healthy dogs in Ilheus indicates the presence of this agent in the environment, which may be a source of human infection. Knowledge of the serovars present in this environment is important for understanding the epidemiology of leptospirosis and establishing public health policies aimed at its control. (C) 2012 Elsevier B.V. All rights reserved.

Toxoplasma gondii and Leishmania spp. infection in captive crab-eating foxes, Cerdocyon thous (Carnivora, Canidae) from Brazil

The zoonoses toxoplasmosis and leishmaniasis are important worldwide and also affect wild animals. Thus, the present study aimed to assess the prevalence of Leishmania spp. and Toxoplasma gondii antibodies in 52 serum samples from captive crab-eating foxes (Cerdocyon thous) kept in 17 zoos in São Paulo State, Brazil. Modified agglutination test (MAT, for toxoplasmosis) and indirect fluorescent antibody test (IFAT, for toxoplasmosis and leishmaniasis) were employed with heterologous anti-dog immunoglobulin. Antibodies to T. gondii were found in 19.2% animals, with an almost perfect concordance (kappa = 0.86; standard error = 9.31%; CI95% = 68.25-104.76%; P < 0.0001) and a strong correlation coefficient (rs = 0.87; P < 0.0001), which allows the use of heterologous anti-dog immunoglobulin to perform IFAT for toxoplasmosis in crab-eating foxes. No sample was positive for Leishmania spp. Toxoplasmosis infection occurs in wild animals from the studied Brazilian zoos, which indicates a probable environmental contamination, highlighting the importance of appropriate zoo management and the action of the parasite as a sentinel to human infection. (C) 2009 Elsevier B.V. All rights reserved.

Epidemiological Aspects in the Leptospira spp. and Toxoplasma gondii Infection in Horses from Botucatu, São Paulo, Brazil

Leptospirosis and toxoplasmosis are zoonoses with high importance because of the economic and public health impact. This study was aimed to determine the seroprevafence of leptospirosis and toxoplamosis in 714 serum samples of horses from different farms from Botucatu, São Paulo, Brazil. The samples were researched for Toxoplasma gondii antibodies by indirect immunofluorescence antibody test (IFAT) and for Leptospira spp. antibodies by microscopic agglutination test. of 714 serum samples, 128 (17.9%; 95% Cl: 15.3%-20.9%) were positive for one or more serovars of Leptospira spp., with icterohaemorrhagiae, canicola, and castellonis as the most prevalent serovars, whereas 42 (5.9%; 95% CI: 4.4%-7.9%) were positive for T gondii, of which 33 samples (78.57%; 95% CI: 64.0%-88.2%) presented a titer of 16, 7 (16.7%; 95% CI: 8.4%-30.7%) a titer of 64, and 1 (2.38%; 95% CI: 0.6%-12.3%) a titer of 256. No significant difference was found among the results obtained and the associated variables such as age and sex. (C) 2012 Elsevier B.V. All rights reserved.