First evidence of Zika virus venereal transmission in Aedes aegypti mosquitoes

BACKGROUND Aedes aegypti is considered the main Zika virus (ZIKV) vector, and is thought to be responsible for the 2015-2016 outbreak in Brazil. Zika positive Ae. aegypti males collected in the field suggest that vertical and/or venereal transmission of ZIKV may occur. OBJECTIVES In this study, we aimed to demonstrate that venereal transmission of ZIKV by Ae. aegypti can occur under laboratory conditions. METHODS Ae. aegypti collected in the city of Manaus, confirmed as negative for Zika, Dengue and Chikungunya virus by reverse transcription real-time polymerase chain reaction (RT-qPCR) (AaM3V- strain), were reared under laboratory conditions and used for the experiments. The ZIKV used in this study was isolated from a patient presenting with symptoms; ZIKV was confirmed by RT-qPCR. Experiment 1: virgin male mosquitoes of AaM3V- strain were intrathoracically inoculated with a ZIKV suspension; four days after injection, they were transferred to a cage containing virgin females of AaM3V- strain and left to copulate for five days. Experiment 2: virgin female mosquitoes of AaM3V- strain were orally infected with a ZIKV suspension by blood feeding membrane assay; nine days after blood feeding, they were placed in cages with Ae. aegypti AaM3V- virgin males and left to copulate for four days. After copulation, all mosquitoes were individually evaluated for viral infection by RT-qPCR. FINDINGS The mean infection rate in Experiment 1 and Experiment 2 was 45% and 35%, respectively. In both experiments, cycle threshold values ranged from 13 to 35, indicating the presence of viral genomes. MAIN CONCLUSION Ae. aegypti males intrathoracically inoculated with a ZIKV suspension are infected and can transmit the virus to uninfected females by mating. Moreover, Ae. aegypti females orally infected with a ZIKV suspension can transmit the virus to uninfected males by copulation. This study shows that ZIKV infection of Ae. aegypti mosquitoes occurs not only during blood feeding, but also during copulation.

Production and characterization of monoclonal antibodies against Campylobacter fetus subsp. venerealis / Produção e caracterização de anticorpos monoespecíficos contra Campylobacter fetus subsp. venerealis

Myeloma cells Sp2/0-Ag14 and spleen cells from BALB/c mouse immunized with sonicated Campylobacter fetus subsp. venerealis NCTC 10354 were fused with polyethylene glycol (PEG) for the selection of clones producing antibodies. Clones were obtained by limiting dilution and screened for the production of specific antibodies to C. fetus subsp. venerealis NCTC 10354 by indirect ELISA and western blot against a panel of bacteria: C. fetus subsp. venerealis NCTC 10354, C. fetus subsp fetus ADRI 1812, C. sputorum biovar sputorum LMG 6647, C. lari NCTC 11352, and Arcobacter skirrowii LMG 6621 for the ELISA and C. fetus subsp. venerealis NCTC 10354 and C. sputorum biovar sputorum LMG 6647 for the western blotting. Fifteen clones producing monoclonal antibodies (MAbs) anti-C. fetus subsp. venerealis of the IgM (1) and IgG (14) classes were further screened for species-specificity. Four clones of the 15 obtained were producers of species-specific monoclonal antibodies (MAbs): two were specific for C. fetus subsp. venerealis and two were specific for C. fetus subsp. fetus. None of the clones were reactive against C. sputorum biovar sputorum LMG 6647. All clones recognized a protein with molecular mass of approximately 148 kDa from lysed C. fetus subsp. venerealis NCTC 10354.

Para a produção de anticorpos monoclonais contra Campylobacter fetus subsp. venerealis foram utilizadas as linhagens de células de mieloma Sp2/0-Ag14 e células de baço de camundongos BALB/c imunizados com sonicado de C. fetus subsp. venerealis NCTC 10354. A detecção dos anticorpos monoclonais foi realizada por ELISA indireto utilizando antígeno sonicado de C. fetus subsp. venerealis NCTC 10354. A clonagem foi realizada por diluição limitante e os clones foram caracterizados por ELISA indireto utilizando um painel de bactérias escolhidas em função da prevalência e habitats: C. fetus subsp. venerealis NCTC 10354, C. fetus subsp. fetus ADRI 1812, C. sputorum biovar sputorum LMG 6647, C. lari NCTC 11352 e Arcobacter skirrowii LMG 6621; e no "western blotting" utilizando antígenos sonicados de C. fetus subsp. venerealis NCTC 10354 e C. sputorum biovar sputorum LMG 6647. Foram obtidos 15 clones produtores de anticorpos anti- C. fetus subsp. venerealis das classes IgM (1) e IgG (14). Quatro clones dentre os 15 clones obtidos foram produtores de anticorpos monoclonais espécie-específicos: dois clones reagiram com maior especificidade contra C. fetus subsp. venerealis NCTC 10354 e dois clones reagiram com maior especificidade contra C. fetus subsp. fetus ADRI 1812. Nenhum dos clones reagiu contra C. sputorum biovar sputorum LMG 6647, comprovando a especificidade dos anticorpos monoclonais testados. Todos os clones reconheceram uma proteína de massa molecular de aproximadamente 148 kDa no sonicado de C. fetus subsp. venerealis NCTC 10354.

Campilobacteriose genital bovina e tricomonose genital bovina: epidemiologia, diagnóstico e controle / Bovine genital campylobacteriosis and bovine genital trichomonosis: epidemiology, diagnosis and control

A presente atualização trata de duas das mais importantes doenças sexualmente transmitidas de bovinos, a campilobacteriose genital bovina e a tricomonose genital bovina. São abordados aspectos relacionados à epidemiologia destas doenças, principalmente em relação a sua distribuição no Brasil. Também são revisados aspectos importantes de diagnóstico, incluindo as técnicas e interpretação dos resultados, além de medidas de controle para ambas as doenças.

The present update deals with two of the most important sexually transmitted diseases of cattle: bovine genital campylobacteriosis and bovine genital trichomonosis. Epidemiological aspects, mainly their distribution in Brazil, alongside with their diagnosis in cattle are presented and commented. The main points in their diagnoses, including the description of the techniques and the interpretation of the results are also reviewed. Finally the control and prevention of both diseases are discussed.

Quantitative analysis of campylobacter fetus venerealis adhesion to bovine reproductive tract cell cultures / Análise quantitativa da adesão de campylobacter fetus venerealis em culturas de células do aparelho reprodutor bovino

Campylobacter fetus is the etiological agent of bovine genital campylobacteriosis, a sexually transmitted disease which is associated with reproductive losses in bovines. Campylobacter colonizes the vagina and the uterus and then infects the epithelial cells of the endometrium. The objective of this work was to develop an ex vivo model to quantify the adhesion of Campylobacter to its natural specific target cells; this is a key step for the establishment of infection and studies regarding the adherence and cytotoxicity on the natural host cells are not available. The assays were carried out by seeding Campylobacter fetus venerealis on bovine vaginal and uterine epithelial cell cultures. HeLa cells were used as control. Bacterial adhesion was corroborated by optical microscopy and determination of the percentage of adherent bacteria was performed on immunochemically-stained slides. Results are presented as percentage of cells with adherent Campylobacter and as number of bacteria per cell. In comparison to the control HeLa cells, the statistical analysis revealed that primary cultures show a higher percentage of infected cells and a lower variation of the evaluated parameters. This primary culture model might be useful for studies on cytopathogenicity and adhesion of different field strains of Campylobacter fetus.

Campylobacter fetus é o agente etiológico da campilobacteriose genital bovina, uma doença sexualmente transmissível que está associada com perdas reprodutivas em bovinos. Campylobacter coloniza a vagina e o útero e então infecta as células epiteliais do endométrio. O objetivo deste trabalho foi desenvolver um modelo ex vivo para quantificar a adesão de Campylobacter às células-alvo naturais específicas; este é um passo fundamental para o estabelecimento da infecção e estudos acerca da adesão e citotoxicidade sobre as células do hospedeiro natural não estão disponíveis. Os ensaios foram realizados a través da semeadura de Campylobacter fetus venerealis em culturas celulares epiteliais vaginais e uterinas.Células HeLa foram utilizadas como controle.A aderência bacteriana foi confirmada por microscopia óptica e a determinação da porcentagem de bactérias aderidas foi realizada em lâminas tingidas imunoquimicamente. Os resultados são apresentados como porcentagem de células com Campylobacter aderente e como o número de bactérias por células. Em comparação com as células HeLa controle, a análise estatística revelou que as culturas primárias mostram uma maior porcentagem de células infectadas e uma menor variação dos parâmetros avaliados. Este modelo de cultura primária pode ser útil para estudos sobre citopatogenicidade e adesão de diferentes cepas de campo de Campylobacter fetus.

Clinical utility of a life quality score in dogs with canine transmissible venereal tumor treated by vincristine chemotherapy / Utilidade clínica de escala de qualidade de vida durante o tratamento quimioterápico de cães com tumor venéreo transmissível canino (TVTC) com vincristina

The clinical utility of a life quality score during vincristine chemotherapy of dogs with canine transmissible venereal tumor (CTVT) was investigated using 93 tumor-bearing dogs in this prospective study. At diagnosis, clinical data and the performance status were evaluated according to a modified Karnofsky score (CKS) adapted for dogs. The animals were treated with vincristine sulphate (0.025mg/kg) at weekly intervals until the tumor had macroscopically disappeared. The time until the first adverse event and death were recorded. In a pilot study, CKS revealed a high inter-observer concordance (kappa=0.735; weighted kappa=0.835). CKS permitted a detailed monitoring of adverse effects during therapy. Cox regressions showed that low performance score and reduced body weight were independent predictive factors for death during chemotherapy. The modified Karnofsky performance score is a simple and reproducible clinical instrument, able to estimate patient outcome after treatment of CTVT.

Investigou-se a utilidade clínica de uma escala de qualidade de vida durante o tratamento quimioterápico de cães com tumor venéreo transmissível canino (TVTC) em 93 animais com TVTC que compuseram este estudo prospectivo. Ao diagnóstico, foram avaliados os dados clínicos e o escore de qualidade de vida adaptado para cães (CKS). Os animais foram tratados com sulfato de vincristina (0.025mg/kg) semanalmente até o desaparecimento macroscópico do tumor. Foram anotados o tempo até o aparecimento do primeiro evento adverso e a morte. Em estudo piloto do CKS, alta concordância entre os observadores foi demonstrada (kappa = 0.735; weighted kappa = 0.835). O CKS permitiu um detalhado monitoramento dos eventos adversos durante a terapia. A regressão Cox multivariada demonstrou que o baixo escore de qualidade de vida e o reduzido peso corporal foram fatores preditivos independentes para o óbito durante a quimioterapia. A escala modificada de Karnofsky é um instrumento clínico simples e reproduzível, hábil em estimar os efeitos do tratamento no TVTC.