Neospora caninum DNA in feces of crab-eating fox (Cerdocyon thous - Linnaeus, 1776) from northeastern Brazil.

Neospora caninum is an obligate intracellular tissue cyst-forming coccidian parasite and it was first described in dogs. Despite the relevance of wild canids in the transmission chain of N. caninum, there are few studies in Brazil. The aim of the present study was to detect N. caninum DNA in feces of free-range and captive crab-eating fox (Cerdocyon thous) from different states of northeastern Brazil. Fecal samples of eighteen crab-eating foxes (fifteen individually and three pools) were collected in sterile containers and were kept cool at -20 °C until further processing. All fecal samples were subjected to DNA extraction. A nested PCR targeting the ITS-1 gene was performed for N. caninum. All the positive bands were extracted from the gel and purified. Forward and reverse strands were sequenced and the nucleotide sequences obtained were compared with N. caninum sequences deposited in Genbank. The alignment was edited and phylogenetic reconstruction was based on the ITS1 gene sequences. Thirteen stool samples were PCR-positive for N. caninum DNA. Nine out of thirteen positive samples showed similarity between 99%-100% for N. caninum in relation to the sequence U25044.1 stored at GenBank. The crab-eating fox could have an important role in the sylvatic cycle of Neospora caninum in Brazil. Experimental infections studies involving these wild canids may confirm if the crab-eating foxes are definitive hosts of N. caninum.

Peripheral and placental immune responses in goats after primoinfection with Neospora caninum at early, mid and late gestation.

Neospora caninum can cause reproductive failure in goats. However, the pathogenesis of neosporosis in this domestic species remains largely unknown. We recently demonstrated that the outcome of experimental infection by N. caninum in pregnant goats is highly dependent on the time of gestation, during which infection occurs. In the present study, we examined the peripheral and placental immune responses in these groups of goats infected with 106 tachyzoites of the Nc-Spain7 isolate at early (G1, at day 40 of gestation, dg), mid (G2, 90 dg) and late (G3, 120 dg) gestation, together with a group of non-infected goats as a control group (G4). Seroconversion was observed as early as day 10 post-infection (pi) in all goats from G1 that aborted earlier (10-11 pi). The remaining infected goats had seroconverted by day 14 pi. Similar IFN-γ kinetic profiles were found in sera from goats in G1 and G2 with a significant increase in the IFN-γ levels on days 7 and 10 pi. This increase was not observed in G3. A similar pattern of placental cytokine expression was found in all infected groups. IFN-γ and IL-4 showed the highest increase, followed by a weaker up-regulation in TNF-α and IL-10. The lowest up-regulation was observed for IL-12 expression. In summary, this study provides information regarding the dynamics of immune responses and their relationship with the outcome of N. caninum infection in goats during gestation.

Detection of Mycobacterium avium subsp. paratuberculosis in bovine milk from the state of Pernambuco, Brazil

Abstract The aim of this study was to detect the IS900 region of Mycobacterium avium subsp. paratuberculosis (MAP) in bovine milk samples using real-time polymerase chain reaction (qPCR) and conventional PCR, and to study the agreement between these tests. A total of 121 bovine milk samples were collected from herds considered positive for MAP, from the State of Pernambuco, Brazil. MAP DNA was detected in 20 samples (16.5%) using conventional PCR and in 34 samples (28.1%) using qPCR. MAP DNA was detected in all of the 6 animal farms studied. Moderate agreement was found between qPCR and conventional PCR results, where the sensitivity and specificity of conventional PCR in relation to qPCR were 50% and 96.6%, respectively. Thus, the IS900 region of MAP was found in bovine milk samples from the State of Pernambuco. To the best of our knowledge, this is the first report of MAP DNA found in bovine milk in Northeast Brazil. We also demonstrated the qPCR technique is more sensitive than conventional PCR with respect to detection of MAP in milk samples.

Detection of Mycobacterium avium subsp. paratuberculosis in bovine milk from the state of Pernambuco, Brazil.

The aim of this study was to detect the IS900 region of Mycobacterium avium subsp. paratuberculosis (MAP) in bovine milk samples using real-time polymerase chain reaction (qPCR) and conventional PCR, and to study the agreement between these tests. A total of 121 bovine milk samples were collected from herds considered positive for MAP, from the State of Pernambuco, Brazil. MAP DNA was detected in 20 samples (16.5%) using conventional PCR and in 34 samples (28.1%) using qPCR. MAP DNA was detected in all of the 6 animal farms studied. Moderate agreement was found between qPCR and conventional PCR results, where the sensitivity and specificity of conventional PCR in relation to qPCR were 50% and 96.6%, respectively. Thus, the IS900 region of MAP was found in bovine milk samples from the State of Pernambuco. To the best of our knowledge, this is the first report of MAP DNA found in bovine milk in Northeast Brazil. We also demonstrated the qPCR technique is more sensitive than conventional PCR with respect to detection of MAP in milk samples.

Experimental caprine neosporosis: the influence of gestational stage on the outcome of infection.

Here, we assessed outcome of experimental infection by Neospora caninum in goats intravenously inoculated with 10(6) tachyzoites of the Nc-Spain7 isolate at 40 (G1), 90 (G2) and 120 (G3) days of gestation. Infected goats had fever between 5 and 9 days post inoculation (dpi); all were seropositive at the time of abortion/birth. Foetal death occurred in G1 from 10 to 21 dpi (n = 7) and in G2 from 27 to 35 dpi (n = 4). Goats in G2 also had seropositive stillbirth (n = 1) and healthy kids (n = 2). G3 goats (n = 7) had 3 seropositive and 3 seronegative weak kids, and 2 seronegative healthy kids. Parasite DNA detection in placentomes was 100% in G2, 85.7% in G3 and in G1 was detected only in placentomes from the goats with foetal losses from 17 dpi (100%). Parasites were detected in foetal/kid brain (>85.7%) and liver (≥ 50%) of G2 and G3, and in G1 after 17 dpi (100%). The highest parasite loads were detected in the placentomes of G1 from 17 dpi and G2, and in foetal tissues of G1 from 17 dpi and G3. Multifocal necrotic lesions were observed in the placentas of the three groups, but they were larger and more frequent in G1 and G2. Similar lesions were observed in foetal tissues, but they were more frequent in G3. These findings suggest that, as observed in cattle and sheep, the clinical consequences of N. caninum in pregnant goats are dependent in part on the time of gestation when animals were infected.

Application of different techniques to detect Toxoplasma gondii in slaughtered sheep for human consumption.

The aim of this study was to investigate occurrence of Toxoplasma gondii in sheep slaughtered in the state of Alagoas, Brazil, by means of different diagnosis techniques. Serum samples and tissues from 100 slaughtered sheep were used. To detect antibodies, the indirect immunofluorescence antibody test (IFAT) was used, and tissues from seropositive animals (cut-off ≥1:64) were submitted to Polymerase Chain Reaction (PCR) and immunohistochemistry (IHC). To assess the concordance between the direct techniques, the kappa test was used. In the IFAT, it was observed that 14% (14/100) of the ovine samples were serum-positive. In the PCR, 21.43% (3/14) of the animals were positive and in IHC, it was observed that 7.14% (1/14) were positively stained for T. gondii in cerebral tissue. Histopathologically, the predominant finding was the presence of mononuclear cell infiltrate in the heart and a perivascular cuff in the cerebrum and cerebellum. The concordance between the direct diagnosis techniques was moderate (k=0.44). Thus, it is important to use different direct techniques in diagnosing toxoplasmosis in naturally infected sheep.

Application of different techniques to detect Toxoplasma gondii in slaughtered sheep for human consumption / Utilização de diferentes técnicas para detecção de Toxoplasma gondii em ovinos abatidos para consumo humano

Abstract The aim of this study was to investigate occurrence of Toxoplasma gondii in sheep slaughtered in the state of Alagoas, Brazil, by means of different diagnosis techniques. Serum samples and tissues from 100 slaughtered sheep were used. To detect antibodies, the indirect immunofluorescence antibody test (IFAT) was used, and tissues from seropositive animals (cut-off ≥1:64) were submitted to Polymerase Chain Reaction (PCR) and immunohistochemistry (IHC). To assess the concordance between the direct techniques, the kappa test was used. In the IFAT, it was observed that 14% (14/100) of the ovine samples were serum-positive. In the PCR, 21.43% (3/14) of the animals were positive and in IHC, it was observed that 7.14% (1/14) were positively stained for T. gondii in cerebral tissue. Histopathologically, the predominant finding was the presence of mononuclear cell infiltrate in the heart and a perivascular cuff in the cerebrum and cerebellum. The concordance between the direct diagnosis techniques was moderate (k=0.44). Thus, it is important to use different direct techniques in diagnosing toxoplasmosis in naturally infected sheep.

Resumo O objetivo deste estudo foi pesquisar a ocorrência de Toxoplasma gondii em ovinos abatidos no Estado de Alagoas, Brasil por meio de diferentes técnicas de diagnóstico. Foram utilizadas amostras de soros e tecidos de 100 ovinos abatidos. Para a pesquisa de anticorpos foi utilizada a Reação de Imunofluorescência Indireta (RIFI), e os tecidos dos animais soropositivos (ponto de corte ≥1:64) foram submetidos às técnicas de Reação de Cadeia da Polimerase (PCR) e Imunohistoquímica (IHQ). Para o estudo da concordância entre as técnicas diretas foi empregado o teste Kappa. Na RIFI, 14% (14/100) das amostras foram soro-positivas. Na PCR, 21,43% (3/14) dos animais foram positivos e, na IHC, 7,14% (1/14) apresentaram marcação positiva para T. gondii no tecido cerebral. Na histopatologia, o achado predominante foi o infiltrado celular mononuclear no coração e manguito perivascular no cérebro e cerebelo. A concordância entre as técnicas diretas de diagnóstico foi moderada (K= 0,44). Desse modo, é importante utilizar diferentes técnicas diretas no diagnóstico da toxoplasmose em ovinos naturalmente infectados.

Isolation of Actinobacillus seminis from a goat with clinical epididymo-orchitis in Brazil

The present study reports the first isolation of Actinobacillus seminis from a goat in Brazil. A four-year-old Moxotó breeding goat in a flock of 70 goats and 65 sheep reared together in the county of Patos, semiarid region of Northeastern Brazil, showed clinical signs of unilateral orchitis and epididymitis. Diagnosis of A. seminis infection was confirmed by association of clinical findings, bacterial isolation and 16S rRNA gene sequencing. This result suggests that A. seminis may be an additional cause of infertility in goats, and that sheep may be the source of infection because the mixed farming system allows the contact between sheep and goats in the semiarid region of Northeastern Brazil.

Detection of Ureaplasma spp. in semen samples from sheep in Brazil

A study was conducted to verify the presence of mycoplasmas and ureaplasmas DNA in sheep semen samples from the State of Pernambuco. The PCR assay was conducted of according with standard protocols with generic primers. Mollicutes DNA was detected in 26.0% and Ureaplasma spp. in 12.0% of semen samples.

Inquérito sorológico e molecular da brucelose canina no município de Natal, Estado do Rio Grande do Norte / Serological and molecular survey of canine brucellosis in the county of Natal, Rio Grande do Norte state

O objetivo do presente trabalho foi determinar a ocorrência de anticorpos anti-Brucella rugosa e anti-Brucella lisa em cães do município de Natal, Estado do Rio Grande do Norte, Brasil, bem como identificar fatores de risco associados à positividade e realizar a detecção molecular em animais soropositivos. Foram utilizados soros sanguíneos de 416 cães atendidos em clínicas veterinárias durante o período de março a novembro de 2011. Para o diagnóstico sorológico da infecção por Brucella rugosa, foi empregada a prova de imunodifusão em gel de ágar (IDGA), utilizando antígeno de lipopolissacarídeos e proteínas de Brucella ovis, amostra Reo 198 e, para o diagnóstico da infecção por Brucella lisa, foi utilizado o teste do antígeno acidificado tamponado (AAT). De animais soropositivos, foram coletadas amostras de sangue com citrato de sódio para o diagnóstico pela reação em cadeia pela polimerase (PCR). A frequência de anticorpos anti-Brucella rugosa foi de 28,9% (120/416). Todos os animais foram negativos para anticorpos anti-Brucella lisa. Dentre 80 animais soropositivos, o DNA de Brucella spp. foi amplificado em três animais (3,8%). Não foram identificados fatores de risco associados à soropositividade. Conclui-se que a infecção por Brucella rugosa está presente no município de Natal, bem como se sugere o monitoramento sorológico de animais atendidos em clínicas visando à identificação de fontes de infecção.

The aim of this study was to determine the occurrence of anti-rough Brucella and anti-smooth Brucella antibodies in dogs from the county of Natal, Rio Grande do Norte state, Brazil, as well as to identify risk factors associated with positivity and to perform molecular detection of the agent in seropositive animals. Sera from 416 dogs attended in veterinary clinics during the period from March to November 2011 were used. For the serological diagnosis of rough Brucella the agar gel immunodiffusion (AGID) test, using antigen of lipopolysaccharides and proteins from Brucella ovis, strain Reo 198, was carried, and for smooth Brucella the buffered plate agglutination test (BPAT) was used. From seropositive animals, blood samples with sodium citrate were collected for the diagnosis by polymerase chain reaction (PCR). Frequency of anti-rough Brucella antibodies was 28.9% (120/416). All animals were negative for anti-smooth Brucella antibodies. Of the 80 seropositive animals Brucella spp. DNA was amplified in three (3.8%). Risk factors associated with the seropositivity were not identified. It was concluded that rough Brucella infection is present in the county of Natal, as well as it is suggested the serological monitoring of animals attended at clinics aiming the identification of sources of infection.

Detecção de DNA de Brucella spp. em amostras de sangue e de suabe vaginal ou prepucial de cães do município de Natal, Rio Grande do Norte, Brasil / Detection of Brucella spp. DNA in samples of blood and vaginal or preputial swab from dogs in the county of Natal, Rio Grande do Norte, Brazil

Objetivou-se com este trabalho detectar o DNA de Brucella spp. em amostras de sangue e de suabe vaginal ou prepucial de 80 cães sorologicamente positivos para brucelose pela prova de imunodifusão em gel de ágar (IDGA), no município de Natal, estado do Rio Grande do Norte, Brasil. Amostras de sangue total foram colhidas com anticoagulante (citrato de sódio) juntamente com amostras de suabe vaginal e prepucial , para extração de DNA e posterior realização da rea- ção em cadeia da polimerase (PCR) empregando-se os primers ITS66 e ITS279. O DNA de Brucella spp. foi amplificado em seis animais, sendo um animal em ambas as amostras, dois cães em amostras de sangue e três em amostras de suabe do trato reprodutivo. Concluiu-se que a infecção por Brucella spp. está presente em cães no município de Natal, e que a detecção de DNA do agente em amostras de suabe do trato reprodutivo podem ser utilizadas como ferramenta suple- mentar no diagnóstico de brucelose canina.

The aim of this work was to detect Brucella spp. DNA in samples of blood and vaginal or preputial swabs in 80 sero-positive dogs for brucellosis by agar gel immunodiffusion test (AGID) from the county of Natal, Rio Grande do Norte State, Brazil. Whole blood samples were collected with anticoagulant (sodium citrate) and vaginal and preputial swab samples for DNA extraction and polymerase chain reaction (PCR) employing ITS66 and ITS279 primers. Six animals showed amplification of Brucella spp., being one animal in both samples, two dogs only in blood samples, and three only in reproductive tract swabs. It is concluded that infection due to Brucella spp. occurs in dogs from the county of Natal, and the detection of DNA of the agent in reproductive tract swabs may be used as complementary tool in the diagnosis of canine brucellosis.