Heminested reverse-transcriptase polymerase chain reaction (hnRT-PCR) as a tool for rabies virus detection in stored and decomposed samples

<p>Abstract</p> <p>Background</p> <p>The use of methods, both sensitive and specific, for rabies diagnosis are important tools for the control and prophylaxis of the disease. Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) has been used in rabies diagnosis with good results, even in decomposed materials. Additionally, molecular techniques have been used for epidemiological studies and to gain a better knowledge of viral epidemiology.</p> <p>Findings</p> <p>The aim of this work was to evaluate the RT-PCR and hnRT-PCR for rabies virus detection in original tissues stored at -20°C for different periods considering their use for rabies virus detection in stored and decomposed samples. RT-PCR and hnRT-PCR were evaluated in 151 brain samples from different animal species, thawed and left at room temperature for 72 hours for decomposition.</p> <p>The RT-PCR and hnRT-PCR results were compared with previous results from Direct Fluorescent Antibody Test and Mouse Inoculation Test. From the 50 positive fresh samples, 26 (52%) were positive for RT-PCR and 45 (90%) for hnRT-PCR. From the 48 positive decomposed samples, 17 (34, 3%) were positive for RT-PCR and 36 (75%) for hnRT-PCR. No false-positives results were found in the negatives samples evaluated to the molecular techniques.</p> <p>Conclusion</p> <p>These results show that the hnRT-PCR was more sensitive than RT-PCR, and both techniques presented lower sensibility in decomposed samples. The hnRT-PCR demonstrated efficacy in rabies virus detection in stored and decomposed materials suggesting it's application for rabies virus retrospective epidemiological studies.</p

Diagnóstico laboratorial de raiva em quirópteros realizado em área metropolitana na região sudeste do Brasil

No período de janeiro de 1988 adezembro de 1992, foi realizado diagnóstico de raiva em 289 morcegos através das técnicas de imunofluorescência direta e de inoculação intracerebral em camundongos. Dois morcegos insetívoros da espécie Nyctinomops macrotis se apresentaram positivos, representando 0,69% da amostra. Esses morcegos foram capturados, ainda vivos, em 1988 e 1990, na sala de um apartamento no sétimo andar e no muro de uma casa, respectivamente. Ambos em bairros residenciais. Apresentaram período de incubação de 13 e 11 dias, respectivamente, na prova biológica. A existência de morcegos insetívoros infectados com o vírus da raiva é preocupante, uma vez que essa população parece ser cada vez mais freqüente em áreas urbanas, porém isto não justifica ações predatórias indiscriminadas contra as espécies, principalmente levando-se em consideração a importância do morcego no equilíbrio ecológico da população de insetos, abundante em área urbana.Between January, 1988 and December, 1992 the S. Paulo Animal Disease Control Center subjected 289 bats to rabies examinations, utilizing the direct immunofluorescence and biological techniques. Two insectivorous bats belonging to the species Nyctinomopsmacrotis, representing 0.69% of the total sample, tested positive. Both animals had been captured alive in residential neighborhoods of the city, one in 1988 in the living room of a 7' th floor apartment and the other in 1990 on top of a wall surrounding a private house. In the biological tests, the disease showed incubation periods of 13 and 11 days respectively.Although the existence of infected insectiverous bats in urban areas is cause for concern, indiscriminate predatory action against such species can in no way be justified-particularly bearing in mind their importance in preserving the ecological balance of the insect population so prevalent in cities

Isolamento do vírus rábico de morcego insetívoro Myotis nigricans

The following is a case study of the natural infection by the rabies virus of an insectiverous bat belonging to the species Myotis nigricans in the municipality of Ribeirão Pires, Greater S. Paulo. Diagnosis was made by means of immunofluorescence and intracerebral innoculation of mice with nervous and intrascapular muscular tissues.Relata-se um caso de infecção natural de raiva em morcego insetívoro Myotis nigricans, no Município de Ribeirão Pires, Grande São Paulo (Brasil). O diagnóstico realizou-se através das técnicas de imunofluorescência e inoculação intracerebral em camundongos, do tecido nervoso e da musculatura da região interescapular do morcego

Caracterização do vírus da raiva isolado de uma colônia de morcegos Eptesicus furinalis, do Brasil

Some bat species have adapted to the expanding human population by acquiring the ability to roost in urban buildings, increasing the exposure risk for people and domestic animals, and consequently, the likelihood of transmitting rabies. Three dead bats were found in the yard of a house in an urban area of Jundiaí city in the state of São Paulo in southeast Brazil. Two of the three bats tested positive for rabies, using Fluorescent Antibody and Mouse Inoculation techniques. A large colony of Eptesicus furinalis was found in the house's attic, and of the 119 bats captured, four more tested positive for rabies. The objectives of this study were to report the rabies diagnosis, characterize the isolated virus antigenically and genetically, and study the epidemiology of the colony.Algumas espécies de morcegos têm se adaptado ao uso de abrigos em construções urbanas, aumentando a possibilidade de contato desses morcegos com pessoas e animais domésticos e conseqüentemente, o potencial risco de transmissão de raiva. Três morcegos foram encontrados no jardim de uma casa na área urbana da cidade de Jundiaí, Estado de São Paulo, Sudeste do Brasil, dois deles foram positivos para raiva pelas técnicas de imunofluorescência e inoculação em camundongos. Uma grande colônia de E. furinalis foi identificada, vivendo no sótão da casa e 119 morcegos foram encaminhados para diagnóstico de raiva, com mais quatro morcegos positivos. O objetivo desse estudo é apresentar a caracterização genética e antigênica do vírus da raiva isolado desses morcegos e o estudo epidemiológico da colônia

Reactive Oxygen Species Production and Mitochondrial Dysfunction Contribute to Quercetin Induced Death in Leishmania amazonensis

BACKGROUND: Leishmaniasis, a parasitic disease caused by protozoa of the genus Leishmania, affects more than 12 million people worldwide. Quercetin has generated considerable interest as a pharmaceutical compound with a wide range of therapeutic activities. One such activity is exhibited against the bloodstream parasite Trypanosoma brucei and amastigotes of Leishmania donovani. However, the mechanism of protozoan action of quercetin has not been studied. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we report here the mechanism for the antileishmanial activity of quercetin against Leishmania amazonensis promastigotes. Quercetin inhibited L. amazonensis promastigote growth in a dose- and time- dependent manner beginning at 48 hours of treatment and with maximum growth inhibition observed at 96 hours. The IC(50) for quercetin at 48 hours was 31.4 µM. Quercetin increased ROS generation in a dose-dependent manner after 48 hours of treatment. The antioxidant GSH and NAC each significantly reduced quercetin-induced cell death. In addition, quercetin caused mitochondrial dysfunction due to collapse of mitochondrial membrane potential. CONCLUSIONS/SIGNIFICANCE: The effects of several drugs that interfere directly with mitochondrial physiology in parasites such as Leishmania have been described. The unique mitochondrial features of Leishmania make this organelle an ideal drug target while minimizing toxicity. Quercetin has been described as a pro-oxidant, generating ROS which are responsible for cell death in some cancer cells. Mitochondrial membrane potential loss can be brought about by ROS added directly in vitro or induced by chemical agents. Taken together, our results demonstrate that quercetin eventually exerts its antileishmanial effect on L. amazonensis promastigotes due to the generation of ROS and disrupted parasite mitochondrial function