Comparison of six methods of DNA extraction for the diagnosis of bovine brucellosis by real-time PCR.

Brucellosis is an infectious disease caused by bacteria of the genus Brucella, which affects domestic animals and is transmissible to humans. The objective of this study was to evaluate six methods of DNA extraction directly from bovine tissue to detect Brucella spp. The Cq values for all samples were above 30 and varied according to the extraction kit used, but four kits showed no statistical difference in sensitivity. This work demonstrates the importance of choosing the best extraction kit before validation of a molecular diagnostic technique.

Molecular characterization of infectious laryngotracheitis virus in naturally infected egg layer chickens in a multi-age flock in Brazil.

The virus responsible for an outbreak of infectious laryngotracheitis (ILT) in a multi-age flock of egg layer chickens under quarantine in Brazil was characterized. Layer chickens from this area with circulating gallid herpesvirus 1 (GaHV 1) were evaluated using histopathology and molecular characterization techniques based on sequences of infected-cell polypeptide 4 (ICP4) and thymidine kinase (TK) genes. The infected chickens that were analyzed were PCR-positive for GaHV-1 in the trachea and negative in most trigeminal ganglia. The lack of ILT lesions in the conjunctiva and respiratory tissues, combined with detection of viral DNA in the trachea, was found to be associated with latent infection. The sequences from five farms obtained in the present study were identical, and there were no deletions within the 272- to 283-bp region of the ICP4 gene, as observed in the sequences of vaccine strains (CEO and TCO). The lack of a deletion in the ICP4 fragment analyzed in this study indicates that the chickens were infected with a field virus. The absence of the T252M mutation in a fragment of the TK gene, in addition to the low mortality rate observed, suggests that the outbreak in the state of Minas Gerais was not caused by a highly virulent strain but rather by a field virus of lower virulence. In addition, using phylogenetic reconstructions, it was found that this field strain was grouped together in a separate branch, apart from the previously characterized Brazilian strains. The introduction of vectored vaccines apparently has been effective in reducing clinical disease and lesions, and preventing new outbreaks of disease.

Fatal toxoplasmosis in a vinaceous Amazon parrot (Amazona vinacea).

Toxoplasmosis was diagnosed in a vinaceous Amazon parrot based on histopathology and immunohistochemistry. The bird was prostrate on the bottom of the cage and died. Necropsy revealed edema and congestion of the lungs, cloudy air sacs, and mild hepatomegaly. Histopathology revealed severe pulmonary congestion and edema and interstitial mononuclear cell inflammation associated with many cysts containing bradyzoites of Toxoplasma gondii scattered throughout. The heart had mild multifocal lymphocytic myocarditis and free tachyzoites in the muscle fibers, and the kidneys had mild interstitial nephritis and a few cysts containing bradyzoites of T. gondii. Immunohistochemistry was negative for Sarcocystis falcatula and Neospora caninum and confirmed the protozoa as T. gondii. This is the first description of T. gondii in an endangered species ofa Brazilian psittacine.

Recovery of Brucella in raw milk Minas artisanal cheese approved for consumption by official inspection agency in Brazil: assessment of prevalence and risk factors through One Health integrated approaches.

BACKGROUND: Minas artisanal cheese (MAC) from the Serro region is a Brazilian intangible cultural heritage. Produced from raw milk, it may carry zoonotic pathogens such as Brucella. This study included a randomized survey for the prevalence of Brucella-positive MAC and its associated factors. METHODS: MAC samples (n=55), each one from a different rural family-based cheese-processing agroindustry, were analysed for Brucella by direct polymerase chain reaction (PCR) species-specific DNA detection and cultivation-based approaches. RESULTS: Among 55 MACs that were analysed, we found 17 Brucella DNA-positive samples (30.9% [95% confidence interval {CI} 18.7 to 43.1]) by PCR and, for the first time, from one MAC (1.8% [95% CI 0.5 to 9.7]), viable Brucella abortus was recovered by cultivation. Higher values for two variables, the number of lactating cows per herd (p=0.043) and daily milk production per herd (p=0.043), were each associated with Brucella-positive MAC, which concentrated in three high-risk and one low-risk spatial clusters. CONCLUSIONS: MAC may be a source of Brucella for humans, since the positive samples were from batches that were sold by cheesemakers. This should be of concern and encourage cooperation between the health and agriculture sectors in order to mitigate this public health risk through One Health integrated approaches.

Fatal coccidiosis by Isospora icterus (Upton & Whitaker, 2000) in captive Campo Troupial (Icterus jamacaii) in Brazil.

An outbreak of coccidiosis by Isospora icterus (I. icterus, Upton & Whitaker, 2000) in captive Campo Troupial (Icterus jamacaii) (Gmelin, 1788) at the Wild Animals Triage Center (IBAMA, Belo Horizonte, Brazil) is described. Clinical history and the necropsy findings documented diarrhea with diffuse necrotic enteritis. Sporulated oocysts (n = 100) had a bilayered wall, were subspherical, and measured 30.1 (27.5-32.5) microm in length and 28.5 (26.2-30.0) microm in width. A polar body but no micropyle was present and the length/width ratio was 1.1 (1.00-1.2). Each oocyst contained two ellipsoidal sporocysts measuring 17.6 (15.0-20.0) microm in length and 12.9 (12.5-15.0) microm in width, with a length/width ratio of 1.4 (1.2-1.5), and with Stieda and sub-Stieda bodies. Each sporocyst contained four sporozoites with granular sporocyst residuum. Oocysts were compatible with those from I. icterus, previously described in Campo Troupial.

Identification and isolation of psittacid herpesvirus from psittacids in Brazil.

Psittacid herpesvirus (PsHV) was isolated from 41 birds kept in captivity in Belo Horizonte, Minas Gerais/Brazil using chicken embryo fibroblasts (CEF) cell cultures. For this study, leukocytes or cloacal swabs of live birds were used. Also, portions of liver, spleen or kidney from birds collected at necropsy were utilized for these tests. PCR tests confirmed the presence of PsHV in 100% of samples. Thirty-three of the PCR products were sequenced and the results disclosed a 99% and 100% identity when compared with other sequences PsHV-1 (AY372243.1 and AF261756.1), previously deposited in GenBank. In addition, histopathology was performed and 19 of the 29 birds contained random multifocal lymphoplasmacytic hepatitis with necrotic foci, suggestive of viral infection. Three samples were examined by electron microscopy to visualize the viral particles obtained from cell culture. The viral structures measured 269 nm in average, had envelopes with an icosahedral capsid and tegument, consistent with herpesvirus. Thus, a total of 41 isolates were obtained from PsHV cell cultivation in CEF, confirming the circulation of the virus between parrots kept in captivity in Belo Horizonte, and affirming the importance of further studies in this area.

Validation of the multiplex PCR for identification of Brucella spp. / Validação da técnica de PCR multiplex para identificação de Brucella spp.

ABSTRACT: A multiplex PCR technique for detection of Brucella spp. in samples of bacterial suspension was validated as a complementary tool in the diagnosis of the disease. This technique allows the characterization of the agent without performing biochemical tests, which greatly reduces the time for a final diagnosis, and provides more security for the analyst by reducing the time of exposure to microorganisms. The validation was performed in accordance with the Manual of Diagnostic Tests from OIE (2008) and following the requirements present in the ABNT NBR ISO/IEC 17025:2005. The mPCR validated in this study identified the different species of Brucella (Brucella abortus, B. suis, B. ovis e B. melitensis) of bacterial suspension obtained from the slaughterhouse samples, as well as distinguished the biovars (1, 2 e 4; 3b, 5, 6 e 9) of B. abortus in grouped form and differentiated the field strains from vaccine strains, as a quick, useful and less expensive technique in diagnosis of brucellosis in Brazil.

RESUMO: Validou-se neste trabalho uma técnica de PCR Multiplex (mPCR) para detecção de Brucella spp. em amostras de suspensão bacteriana, como ferramenta complementar no diagnóstico da doença. Esta técnica possibilita a caracterização do agente sem que seja necessária a realização de testes bioquímicos, o que diminui consideravelmente o tempo para o diagnóstico final, além de oferecer mais segurança ao analista ao diminuir o tempo de exposição ao agente infecioso. A validação foi realizada de acordo com o Manual de Testes de Diagnósticos da OIE (2008), seguindo as exigências presentes na norma de qualidade da ABNT NBR ISO/IEC 17025:2005. A mPCR validada neste trabalho identificou as diferentes espécies de Brucella (Brucella abortus, B. suis, B. ovis e B. melitensis) em suspensão bacteriana, obtidas a partir de amostras de frigorífico. Além disso, discriminou os biovares (1, 2 e 4; 3b, 5, 6 e 9) de B. abortus, de forma agrupada, e diferenciou cepa vacinal de cepa de campo, sendo esta uma técnica rápida, útil e de menor custo para o auxílio no diagnóstico de brucelose no Brasil.

Histomorphological and immunohistochemical characterization of 172 cutaneous round cell tumours in dogs / Caracterização histomorfológica e imuno-histoquímica de 172 neoplasias cutâneas de células redondas em cães

This paper describes the use of a panel of antibodies (CD117, CD3, CD79a, CD45, cytokeratin, vimentin and E-cadherin) on formalin-fixed, paraffin-embedded sections of canine cutaneous round cell tumours. Neoplastic tumours were diagnosed by histology and histochemical stains and included 107 mast cell tumours, 31 cutaneous histiocytomas, two localized histiocytic sarcomas, 21 cutaneous lymphomas, three plasma cell tumours, one transmissible venereal tumour and seven unclassified round cell tumours. The histologic diagnosis was modified in 39.5% of the total 172 neoplasms. The staining for CD45 and Ecadherin were variable, and therefore, the final diagnoses of cutaneous histiocytoma and localized histiocytic sarcoma were made based on histology in association with negative results for CD3, CD79a, CD117 and cytokeratin. The cellular origin of unclassified round cell tumours was defined in all cases. Cutaneous B-cell lymphoma and plasma cell tumours were CD79a-positive and could be distinguished from each other by the morphological characteristics. Mast cell tumours and T cell lymphoma were CD117 and CD3 positive, respectively. The positive staining for vimentin and the negative staining for CD3, CD79a, CD117 and cytokeratin favoured the diagnosis of transmissible venereal tumours. Thus, the final diagnosis of cutaneous round cell tumours should be based on the interpretation of immunohistochemical results together with the cellular morphology observed by histology. Therefore, more studies to optimize the specific markers in formalin-fixed, paraffinembedded tissues (especially for histiocytes) are required for definitive diagnosis of round cell tumours in dogs.

Este trabalho descreve o uso de um painel de anticorpos (CD117, CD3, CD79a, CD45, citoqueratina, vimentina e e-caderina em tecidos formalizados e parafinizados para o diagnóstico de neoplasias de células redondas em cães. Os tumores foram diagnosticados usando-se a histopatologia e a marcação imuno-histoquímica. Foram incluídos 107 mastocitomas, 31 histiocitomas cutâneos, 2 sarcomas histiocíticos localizados, 21 linfomas cutâneos, 3 plasmocitomas, 1 tumor venéreo transmissível e 7 tumores de células redondas não classificados. O diagnóstico histológico foi modificado em 39,5% do total de 172 neoplasias. A marcação do anticorpo CD45 e E-caderina foi variável e, nesse sentido, o diagnóstico final de histiocitoma cutâneo e sarcoma histiocítico localizado foi baseado na histologia em associação com os resultados negativos para CD3, CD79a, CD117 e citoqueratina. A origem celular dos tumores de células redondas não classificados foi definida em todos os casos. Linfoma cutâneo de célula B e plasmocitoma foram positivos para CD79a e foram distinguidos entre si pelas características morfológicas. Marcação positiva para vimentina e negativa para CD3, CD79a, CD117 e citoqueratina favoreceram o diagnóstico dos tumores venereos transmissíveis. Assim, o diagnóstico final dos tumores de células redondas foram baseados na interpretação dos resultados da imuno-histoquímica em conjunto com a avaliação das características morfológicas observadas na histologia. Finalmente, mais estudos em relação à padronização de marcadores específicos para tecidos parafinizados (especialmente para histiócitos) são necessários para o diagnóstico definitivo das neoplasias de células redondas em cães.