Serological response to Neospora caninum infection in goats and agreement between three diagnostic techniques to detect caprine neosporosis / Resposta sorológica à infecção por Neospora caninum em cabras e concordância entre três técnicas diagnósticas para detecção de neosporose caprina

The present study aimed to measure the serological response of goats infected with Neospora caninum by assessing the diagnostic performance and agreement between three techniques (indirect immunofluorescent antibody test, IFAT; Neospora agglutitation test, NAT; enzyme-linked immunosorbent assay, ELISA). The panel of sera were comprised of 500 samples of goats, and 60 reference serum samples. These reference and field serum samples were tested by ELISA, NAT, and IFAT. In the field serum samples tested, the seroprevalences of anti-N. caninum antibodies were 3.2%, 4.6%, and 6.4% in the NAT, IFAT and ELISA, respectively. Using the IFAT as the gold standard, the NAT and the ELISA agreement was considered weak (k=0.28) and strong (k=0.75), respectively. When the IFAT performance was used for comparison purposes, the ELISA showed 91.3% sensitivity and 97.7%, specificity with a PPV of 65.2% and a NPV of 99.6%; The NAT presented sensitivity of 26.1% and specificity of 97.9% with a PPV of 37.5% and a NPV of 96.5%. Accordingly, the IFAT should remain the assay of choice for studies about N. caninum infection in goats in individual serum samples. A combination of serological assays with high sensitivity and specificity is recommended in serosurveys of caprine neosporosis.(AU)

Objetivou-se avaliar a resposta sorológica de caprinos infectados com Neospora caninum mediante o estudo da performance e concordância de três técnicas sorológicas (RIFI, NAT e ELISA). O painel de soros testes foi composto por 500 amostras de caprinos e ainda 60 soros classificados como de referência. Todos os soros de referência e de campo foram testados por ELISA, NAT e RIFI. Nos soros de campo, as soroprevalências de anticorpos anti-N. caninum foram de 3,2% no NAT, 4,6% na RIFI e 6,4% no ELISA. Utilizando a RIFI como técnica de referência, a concordância de NAT e ELISA foi considerada fraca (k=0,28) e substancial (k=0,75), respectivamente. Ainda utilizando a RIFI como comparação, foram obtidos valores de sensibilidade de 91,3% e 97,7% de especificidade no ELISA, e valores preditivos positivo de 65,2% e negativo de 99,6%; NAT apresentou resultados de sensibilidade de 26,1% e de especificidade de 97,9% com valores preditivos positivo de 37,5% e negativo de 96,5%. Com base nos resultados deste trabalho, sugerimos que a RIFI permaneça como técnica de escolha no estudo da neosporose caprina em amostras individuais, resguardando as recomendações e pontos de corte adotados neste estudo. Indicamos a associação de técnicas sorológicas de alta sensibilidade e especificidade.(AU)

Padronização de multiplex PCR para detecção de dermatófitos em pelos e crostas de cães e gatos / Standardization of a PCR multiplex for the detection of dermatophytes in dogs and cats fur and crusts

Objetivou-se padronizar uma reação do tipo multiplex PCR (mPCR) para detectar Microsporum canis, Microsporum gypseum e o complexo Trichophyton mentagrophytes em amostras de pelos e/ou crostas de cães e gatos. 250 amostras de pelos e/ou crostas de cães e gatos foram analisadas por meio de exame direto e cultura, o DNA das mesmas foi extraído para mPCR. Primers foram desenhados e como controle positivo da reação utilizou-se o DNA extraído de colônias de M. canis (URM 6273), M. gypseum (URM 6921) e T. mentagrophytes (URM 6211), provenientes da Coleção de Culturas (Micoteca URM), Departamento de Micologia, Centro de Ciências Biológicas da Universidade Federal de Pernambuco (CCB/UFPE). Como controles negativos de reação, utilizou-se água destilada esterilizada e DNA extraído de Alternaria sp. para verificar a especificidade dos primers. Do total de amostras analisadas, 15 (6%) foram identificadas, em cultura, como dermatófitos, e destas, 10 foram M. canis, três M. gypseum e dois T. mentagrophytes (complexo). Destas 15 amostras positivas, 11 (73,3%) foram detectadas por meio da mPCR. Além destas, seis outras, negativas em cultura, foram identificadas como M. gypseum. Verificou-se uma boa concordância entre os resultados da cultura e mPCR (Kappa: 0,66). O protocolo padronizado neste estudo pode ser utilizado como um método de triagem, por apresentar uma sensibilidade maior que a da cultura, usado paralelamente aos exames de rotina, permitindo um diagnóstico em menor tempo.(AU)

The aim of this study was to standardize a multiplex PCR (mPCR) reaction to detect Microsporum canis, Microsporum gypseum and the Trichophyton mentagrophytes complex in dog and cat fur and/or crusts. 250 fur and/or crusts samples from dogs and cats were analyzed by direct examination and culture, DNA from them was extracted for mPCR. Primers were designed and the DNA extracted from colonies of M. canis (URM 6273), M. gypseum (URM 6921) and T. mentagrophytes (URM 6211) from the Collection of Cultures - URM Micoteca - Department of Mycology, Biological Sciences Center of the Federal University of Pernambuco (CCB / UFPE). As negative controls, sterile distilled water and DNA extracted from Alternaria sp., were used to verify the specificity of the primers. Of the total samples analyzed, 15 (6%) were identified in culture as dermatophytes, and of these, 10 were M. canis, three M. gypseum and two T. mentagrophytes (complex). Of these 15 positive samples, 11 (73.3%) were detected by mPCR. Besides these, six others, negative in culture, were identified as M. gypseum. There was good agreement between culture results and mPCR (Kappa: 0.66). The protocol standardized in this study can be used as a screening method, because it has a sensitivity greater than that of the culture, used in parallel to the routine exams, allowing a diagnosis in a shorter time.(AU)

Detecção de Aeromonas spp. e do gene de virulência aerolisina em tilápias do Nilo (Oreochromis niloticus) com a técnica de mPCR / Detection of Aeromonas spp. and virulence gene aerolysin in Nile tilapia (Oreochromis niloticus) using PCR technique

As infecções causadas por bactérias do gênero Aeromonas estão entre as doenças mais comuns em peixes cultivados em todo o mundo, com ocorrência de aeromoniose em todos os países que possuem cultivo de tilápia do Nilo (Oreochromis niloticus). O presente trabalho descreve o desenvolvimento de uma nova multiplex PCR (mPCR) para diagnóstico de Aeromonas spp. e identificação do gene aerolisina (aerA). Para padronização da mPCR foram utilizadas cepas de referência de várias espécies do gênero Aeromonas e de outros gêneros. Também foram usadas cepas de campo de A. hydrophila oriundas de cultivos de peixes pacamãs (Lophiosilurus alexandri) e Aeromonas spp. de tilápias do Nilo. Os primers foram desenhados com base na região 16S rRNA e aerA. Para verificar a melhor temperatura de anelamento foram utilizados gradientes entre 59°C a 61°C com 40ng de DNA molde. Os produtos da amplificação da região 16S rRNA e do gene aerA apresentaram 786 e 550pb, respectivamente. A mPCR apresentou melhor temperatura de anelamento a 57,6°C com limite de detecção das concentrações de DNA em ambos genes (16S rRNA and aerA) de 10-10g/μL. A mPCR padronizada é rápida, sensível e específica no diagnóstico de Aeromonas spp. e identificação do gene aerolisina. Esta metodologia apresenta vantagens quando comparada aos métodos de diagnóstico convencionais, podendo ser utilizada em cultivos comerciais de tilápias do Nilo ou outros peixes. A identificação do gene aerolisina é uma importante ferramenta na determinação do potencial patogênico dos isolados de Aeromonas spp. estudados.(AU)

Infections caused by bacteria of the genus Aeromonas are among the most common diseases in fish farming systems worldwide, and this disease occurs in all countries which have Nile tilapia (Oreochromis niloticus) farmed. The present work describes the development of a new multiplex PCR (mPCR) technique that diagnosis the genus Aeromonas and detects aerolysin gene (aerA). Reference strains of several Aeromonas species and other genera were used for standardization of mPCR. Strains of A. hydrophila from "pacaman" fish (Lophiosilurus alexandri) and Aeromonas spp. from Nile tilapia from farming systems were used too. Primers were designed based on the 16S rRNA region and aerA (aerolysin toxin). To verify a better annealing temperature were used gradients between 59°C and 61°C with 40ng of the DNA template. The 16S rRNA gene and the aerA gene amplification products showed 786 and 550 bp, respectively. The mPCR showed better annealing temperature at 57.6°C, and the detection limit for both genes (16S rRNA and aerA) was 10-10g/μL of the DNA. The standardized mPCR is quick, sensitive, and specific for Aeromonas spp. diagnosis and to detect aerolysin gene. This method showed advantages when compared to the conventional diagnostic methods and can be used in Nile tilapia or other fish farming systems. The detection of aerolysin gene is an important tool to determine the potential pathogenicity of Aeromonas spp. isolates.(AU)

Molecular and serological investigation of infectious diseases in captive and free-range crab-eating fox (Cerdocyon thous - Linnaeus, 1776) from northeastern Brazil.

The aim of this study was to detect DNA and antibodies anti-Leishmania spp., Neospora caninum and Toxoplasma gondii in captive and free-range crab-eating fox (Cerdocyon thous) from northeastern Brazil. Twenty-five crab-eating foxes from different states of northeastern Brazil were sampled by this study. Blood samples were collected by cephalic or jugular vein punctures. The whole blood was submitted to PCR, and the sera samples to the serological analysis as follows: MAT for T. gondii, NAT for N. caninum, and ELISA for L. chagasi. The frequence of antibodies anti-T. gondii was 50% and 29.41% for free-range and captive wild canids, respectively. The frequence of antibodies anti-N. caninum observed by this study was 62.50% and 23.52% for free-range and captive wild canids, respectively. The frequence of antibodies anti-L. chagasi was 4.0% for captive wild canids. Co-infections cases were identified as follows: one captive wild canid seropositive for T. gondii and L. chagasi and two free-range animals seropositive for T. gondii and N. caninum. All PCR assays performed were negative for the pathogens analyzed. This study describes the presence of antibodies anti-T. gondii, N. caninum e L. chagasi in wild canids from northeastern Brazil and highlights the necessity of further studies on infectious diseases in free-range and captive wild canids.

Transplacental transmission of Neospora caninum in naturally infected small ruminants from northeastern Brazil / Transmissão transplacentária de Neospora caninum em pequenos ruminantes infectados naturalmente do Nordeste do Brasil

Toxoplasma gondii and Neospora caninum are causative agents of abortion in sheep and goats. Thus, the present study aimed to describe the transplacental transmission of these protozoans in small ruminants of northeastern Brazil. Seventeen fetuses (6 goats and 11 sheep) from farms with history of abortion were necropsied and samples were collected from different tissues (brain, liver, lung, kidney and heart). The samples were analyzed by PCR, histopathology (HP) and immunohistochemistry (IHC) to evaluate whether T. gondii and/or N. caninum infection were the cause of abortion. None of the samples was positive for T. gondii according to PCR and IHC results. Some brain, liver, lung, kidney and heart samples of goat fetuses were positive for N. caninum by PCR. In the histopathology, mild mononuclear infiltration and necrosis with calcification were observed in the liver and brain of one goat fetus, respectively, that also was positive for N. caninum by PCR and IHC. The results confirmed vertical transmission of N. caninum in naturally infected goats of northeastern, Brazil.(AU)

Toxoplasma gondii e Neospora caninum são reconhecidos como protozoário causadores de aborto em ovinos e caprinos. Desta forma, objetivou-se descrever a transmissão transplacentária desses agentes em pequenos ruminantes na região Nordeste do Brasil. Foram examinados seis fetos caprinos e onze fetos ovinos, totalizando 78 amostras de diferentes tecidos (cérebro, fígado, pulmão, rim e coração) provenientes de propriedades rurais com histórico de aborto. As amostras foram analisadas por Reação em Cadeia da Polimerase (PCR), Histopatologia (HP) e Imunohistoquímica (IHQ), com a finalidade de associar o abortamento a T. gondii e/ou N. caninum. Nenhuma amostra foi positiva na PCR e IHQ para T. gondii. Algumas amostras de cérebro, fígado, pulmão, rim e coração de fetos de caprinos e ovinos foram positivas na PCR para N. caninum. Na histopatologia foi observado leve infiltrado mononuclear no fígado e necrose com calcificação no SNC de um caprino, associada à imunomarcação positiva para N. caninum na IHQ e PCR positiva. Os resultados confirmam a transmissão vertical de N. caninum em caprinos naturalmente infectados na região nordeste do Brasil.(AU)

Incidence and vertical transmission rate of Neospora caninum in sheep.

The infection by Neospora caninum in sheep can lead to abortion and the birth of weak and debilitated lambs. The aim of this study was to assess the incidence of natural infection by Neospora caninum and the vertical transmission rate among sheep. A flock of 50 sheep was monitored for serum antibody titres against N. caninum and seroconversion over a period of six months using an indirect ELISA technique. The offspring of the herd was also investigated regarding anti-N. caninum antibodies to determine the vertical transmission rate through the indirect immunofluorescence technique. The initial and final prevalences of infection by N. caninum were 26.0% (13/50) and 72.0% (36/50), respectively, and the incidence of infection by N. caninum in the present study was 62.2% (23/37). The vertical transmission rate found was 15.4% (2/13). A high incidence of infection by N. caninum in sheep was observed, and this is the first report assessing the incidence of N. caninum among naturally infected sheep.

Achados microbiológicos, moleculares e histopatológicos em pequenos ruminantes experimentalmente infectados com Actinobacillus seminis / Microbiological, molecular and histopathological findings in small ruminants experimentally infected with Actinobacillus seminis

O objetivo deste trabalho foi avaliar a patogenicidade, em ovinos, de uma cepa de Actinobacillus seminis isolada de caprino no Brasil. Foram utilizadas amostras de sêmen, punção e fragmentos de epidídimo, ducto deferente, testículos e glândulas seminíferas de dois caprinos (animais 1 e 2) e dois ovinos (animais 3 e 4), e foram realizados exame histopatológico, cultivo microbiológico e diagnóstico molecular. O inóculo foi preparado com solução salina na diluição de 10-2 correspondendo ao padrão 1,0 da escala de McFarland, com colônias previamente cultivadas de A. seminis e administrado no volume de 2 mL pelas vias intra-prepucial (animais 1 e 3) e na cauda do epidídimo (animais 2 e 4). Na avaliação clínica observou-se aumento unilateral de consistência firme após 30 dias no epidídimo e testículo do animal 4 que continuou até o dia da eutanásia, bem como o animal 1 apresentou discreto aumento unilateral dos testículos. As lesões macroscópicas e microscópicas observadas nos animais 3 e 4 foram compatíveis com aquelas causadas pela infecção por A. seminis. A. seminis foi isolado de material de punção e sêmen de um ovino (animal 4). Conclui-se que o modelo de infecção experimental utilizando caprinos e ovinos comprovou a patogenicidade da amostra de A. seminis, isolada de um caprino no semiárido brasileiro e reproduzida em um ovino, comprovando a predileção do agente pelo epidídimo, com quadro clinico, achados histopatológicos, isolamento bacteriano e diagnóstico molecular positivo.(AU)

The aim of this study was to evaluate, in sheep, the pathogenicity of an Actinobacillus seminis strain isolated from a goat in Brazil. Samples of semen, puncture and fragments of epididymis, deferent duct, testicles and seminal vesicles from two goats (animals 1 and 2) and two sheep (animals 3 and 4) were used, and histopathological, microbiological culture and molecular diagnoses were performed. The inoculum was prepared with saline solution at 10-2 dilution corresponding to 1.0 McFarland standard, with A. seminis colonies previously cultured and administered on 2mL volume by intra-preputial (animals 1 and 3) and epididymis tail (animals 2 and 4) routes. At clinical evaluation it were found unilateral swelling of firm consistency after 30 days in epididymis and testicle from animal 4 that continued until the day of euthanasia, as well as animal 1 shown discrete unilateral swelling of testicles. Gross and microscopic lesions in animals 3 and 4 were compatibles with that caused by A. seminis infection. A. seminis was isolated from material of puncture and semen of one sheep (animal 4). It is concluded that the experimental infection model using goats and sheep has proved the pathogenicity of the A. seminis strain isolated from a goat in the Brazilian semiarid and reproduced in a sheep, which confirm the prediletion of the agent for epididymis, with clinical signs, histopathological findings, bacterial isolation and positive molecular diagnosis.(AU)

Identificação de Avibacterium paragallinarum em frangos de corte e poedeiras comerciais no Estado de Pernambuco / Avibacterium paragallinarum identification in broiler and commercial laying hens in the state of Pernambuco, Brazil

As aves da indústria avícola brasileira são suscetíveis a surtos de doenças infecciosas relacionadas a perdas econômicas, principalmente por enfermidades infecciosas causadas por Mycoplasma, Avibacterium paragallinarum e vírus que acometem o trato respiratório. Objetivou-se neste estudo pesquisar o envolvimento de A. paragallinarum em surto de doença respiratória aviária. Foram coletados 18 swabs da região infraorbitária dos seios nasais de aves, sendo seis amostras de frangos de corte com sinais clínicos e 12 de poedeiras com sinais clínicos. As amostras foram cultivadas em meios específicos para o agente e também testadas com a técnica molecular Reação em Cadeia de Polimerase. Das amostras analisadas no isolamento, 100% foram negativas. Na PCR, duas (16,66%) foram positivas. A detecção de A. paragallinarum na PCR em galinhas poedeiras com sinais clínicos respiratótios indica que esta bactéria está associada à etiologia da síndrome respiratória das aves nas granjas no estado de Pernambuco...

Pullets of the Brazilian poultry industry are susceptible to outbreaks of infectious diseases with economic losses, mainly caused by Mycoplasma, Avibacterium paragallinarum and viruses affecting the respiratory tract. The objective of this study was to verify the involvement of A. paragallinarum in an outbreak of avian respiratory disease. Eighteen swabs from the infra-orbital sinuses were collected, six samples from broilers and twelve from hens with clinical signs. The samples were cultured in specific media for the agent and also tested with the molecular technique of Polymerase Chain Reaction (PCR). All samples cultured in specific media were negative; PCR revealed two samples (16.66%) as positive. The detection of A. paragallinarum by PCR in laying hens with clinical respiratory signs indicates that this bacterium is associated with the etiology of respiratory syndrome in poultry farms in the state of Pernambuco...

Prevalence of Toxoplasma gondii in slaughtered pigs in the state of Pernambuco, Brazil.

The object of this study was to investigate Toxoplasma gondii antibodies and parasite DNA in pigs in the state of Pernambuco, Brazil. Blood samples were collected from 305 slaughtered pigs in 11 municipalities, and their sera were tested for T. gondii antibodies using the indirect fluorescent antibody test (IFAT, cutoff 1:64); 38 (12.5%) samples were positive. Attempts were made to detect T. gondii DNA in the heart tissue of seropositive pigs using the B 1 gene and PCR; 21 (55.2%) of the 38 hearts were positive. This is the first detection of T. gondii DNA in tissues of serologically positive swine in the State of Pernambuco, Brazil.