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Anaplasma marginale is an important agent for animal livestock, its presence in herds of cattle, sheep and goats leads to losses for Brazilian agribusiness. The present study aimed to describe an isolation technique for A. marginale, using chicken embryo fibroblast (CEF) cell culture. For this, blood and tick samples were collected from 5 calves, between 2 and 3 months of age, which due to anemia, jaundiced mucous membranes and prostration, were considered supposedly infected with A.marginale. For the diagnosis, DNA extraction and PCR was performed from the blood and tick samples collected. All the tick and blood samples were positive in the PCR test. Additionally, ticks were crushed with the aid of a blender for inoculation in CEF cell culture. After inoculation, the cultures were kept at 37ºC and 5% CO2 for 15 days. The cell supernatant of cell cultures were again analyzed by PCR and by the technique Wrighte stain to confirm A. marginale isolation. Cell cultures were positive in PCR and the presence of the agent was demonstrated by Wrighte stain. Therefore, using CEF cell culture was possible to isolate and amplify the A. marginale in a concentration of 1.3 x 107.2 bodies per ml. The CEF cells are undemanding, easy to maintain and they are an option for isolation and production of A. marginale in the laboratory condition.

Anaplasma marginale (A. marginale) is a worldwide pathogen that infects a variety ofruminants, but mostly cattle. The present study aimed to describe an isolation technique for A. marginale, using chicken embryo Þ broblast (CEF) cell culture. Blood and tick samples were collected from 5 calves from 2 to 3 months old, which were considered to be infected with A.marginale due to anemia, jaundiced mucous membranes, and prostration. DNA extractionand PCR were performed for diagnosis using blood and tick samples. All tick and blood samples tested positive in PCR. Additionally,ticks were crushed with the aid of a blender for inoculation in CEF cell culture. After inoculation, the cultures were kept at 37ºC and 5%CO2 for 15 days. The cell supernatant of cell cultures was again analyzed using PCR and Wright stain method to conÞ rm A. marginaleisolation. Cell cultures tested positive in PCR, and the presence of the agent was demonstrated by Wright stain. Therefore, by using CEFcell culture it was possible to isolate and amplify the A. marginale in a concentration of 1.3x 107.2 bodies per mL. The CEF cells are undemanding and easy to preserve; they are anoption for isolation and production of A. marginale under laboratory conditions.

RESUMO

Anaplasma marginale (A. marginale) is a worldwide pathogen that infects a variety of ruminants, but mostly cattle. The present study aimed to describe an isolation technique for A. marginale, using chicken embryo Þ broblast (CEF) cell culture. Blood and tick samples were collected from 5 calves from 2 to 3 months old, which were considered to be infected with A.marginale due to anemia, jaundiced mucous membranes, and prostration. DNA extraction and PCR were performed for diagnosis using blood and tick samples. All tick and blood samples tested positive in PCR. Additionally, ticks were crushed with the aid of a blender for inoculation in CEF cell culture. After inoculation, the cultures were kept at 37ºC and 5% CO2 for 15 days. The cell supernatant of cell cultures was again analyzed using PCR and Wright stain method to conÞ rm A. marginale isolation. Cell cultures tested positive in PCR, and the presence of the agent was demonstrated by Wright stain. Therefore, by using CEF cell culture it was possible to isolate and amplify the A. marginale in a concentration of 1.3 x 107.2 bodies per mL. The CEF cells are undemanding and easy to preserve; they are an option for isolation and production of A. marginale under laboratory conditions.(AU)

Anaplasma marginale (A. marginale) é um patógeno mundial que infecta uma variedade de ruminantes, mas principalmente bovinos. O presente estudo teve como objetivo descrever uma técnica de isolamento para A. marginale, utilizando cultivo celular de Þ broblastos de embriões (CFE) de galinhas. Para isso, foram coletadas amostras de sangue e de carrapatos de 5 bezerros, entre 2 e 3 meses de idade, os quais, devido a anemia, icterícia de mucosas e prostração, foram considerados supostamente infectados com A. marginale. Ethics Approval This study was approved by Credenciamento Institucional para Atividades com Animais em Ensino ou Pesquisa (CIAEP: 02.0420.2021). Consent to participate Not applicable Consent to publish Not applicable Data availability Not applicable Para o diagnóstico, realizaram-se extração de DNA e posterior PCR a partir das amostras de sangue e de carrapatos coletados. Todos os carrapatos e amostras de sangue foram positivas para o teste de PCR. Além disso, os carrapatos foram triturados com o auxílio de um liquidiÞ cador para inoculação em CFE. Após a inoculação, as culturas foram mantidas a 37ºC e a 5% de CO2 durante 15 dias. O sobrenadante celular das culturas foi novamente analisado por PCR e pela técnica de coloração de Wright para conÞ rmar o isolamento de Anaplasma marginale. As culturas celulares foram po sitivas por PCR, e a presença do agente foi comprovada por meio da coloração de Wright. Portanto, utilizando CFE, foi possível isolar e ampliÞ car o A. marginale em uma concentração de 1,3x107,2 bactérias por ml. As células da CEF são pouco exigentes, de fácil manutenção e uma boa opção para isolamento e produção de A. marginale em condição laboratorial.(AU)

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Porcine circovirus 2 (PCV2) has a considerable economic impact on the pork industry worldwide for more than two decades. In 2016, a new circovirus, porcine circovirus 3 (PCV3), was described; since then, it has been reported to be associated with diseased or even in clinically healthy swine in several countries. Considering the importance of wild boars as reservoirs of swine pathogens and the extensive distribution of these animals in Rio Grande do Sul and throughout the national territory, we searched for PCV2 and PCV3 in twenty-six wild boars coupled with necropsy and histologic examination of the sampled animals. Using PCR, 182 tissue samples were analyzed, including the heart, kidneys, liver, lung, lymph nodes, spleen, and tonsils. PCV2 and PCV3 were detected in 57.7% (15/26) and 15.4% (4/26) of wild boars, respectively. Furthermore, co-infection with PCV2 and PCV3 was detected in one of these animals, with PCV2 or PCV3 DNA detection in multiple organs. Histological examination showed mild to moderate and multifocal lymphoplasmacytic interstitial nephritis distributed randomly throughout the renal cortex, apparently unrelated to PCV2 or PCV3 detection. The wild boar population in Brazil is extensive, indicating the presence of a larger number of swine pathogen hosts. In the present study, more than half of the wild boars harbored PCV2; and although less frequently, PCV3 was also detected. Therefore, free-living wild boars can serve as reservoirs of swine circoviruses in southern Brazil.

O circovírus suíno 2 (PCV2) tem causado impacto econômico na indústria suína em todo o mundo por mais de duas décadas. Em 2016, um novo circovírus foi descrito - circovírus suíno 3 (PCV3) - e desde então tem sido relatado em vários países associado a doenças ou mesmo suínos saudáveis. Diante da importância dos javalis como reservatórios de patógenos suínos, e da ampla distribuição desses animais no Rio Grande do Sul e em todo o território nacional, foi realizada pesquisa de PCV2 e PCV3 em vinte e seis javalis (10 fêmeas e 16 machos). Necropsia e exame histológico foram realizados. Utilizando PCR, foram analisadas 182 amostras de tecidos incluindo: coração, rins, fígado, pulmão, linfonodos, baço e tonsila. PCV2 e PCV3 foram detectados por PCR em 57,7% (15/26) e 15,4% (4/26) dos javalis, respectivamente. Um destes animais estava co-infectado por PCV2 e PCV3. O DNA do PCV2 ou PCV3 foi detectado em multiplos órgãos. No exame histológico foi observada nefrite intersticial linfoplasmocitária multifocal leve a moderada, distribuída aleatoriamente pelo córtex renal, aparentemente sem relação com a detecção de DNA viral. A população de javalis no Brasil é extensa, resultando em maior número de hospedeiros para patógenos de suínos. No presente estudo, mais da metade dos javalis capturados abrigavam PCV2 e, embora menos frequente, PCV3 também foi detectado. Os javalis de vida livre podem servir como reservatórios de circovírus suínos no sul do Brasil.

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Oxidative stress is known to be involved in development of numerous diseases including cardiovascular, respiratory, renal, kidney and cancer. Thus, investigations that mimic oxidative stress in vitro may play an important role to find new strategies to control oxidative stress and subsequent consequences are important. Rotenone, widely used as a pesticide has been used as a model to simulate oxidative stress. However, this chemical was found to produce several diseases. Therefore, the aim of this study was to investigate the antioxidant and cytoprotective effect of avocado (Persea americana Mill) extract and oil in monkey kidney epithelial cells (VERO) exposed to rotenone. VERO cells were exposed to IC50 of rotenone in conjunction with different concentrations of avocado extract and oil (ranging from 1 to 1000 µg/ml), for 24 hr. Subsequently, cell viability and oxidative metabolism were assessed. Data demonstrated that avocado extract and oil in the presence of rotenone increased cellular viability at all tested concentrations compared to cells exposed only to rotenone. In addition, extract and avocado oil exhibited antioxidant action as evidenced by decreased levels of reactive oxygen species (ROS), superoxide ion, and lipid peroxidation, generated by rotenone. Further, avocado extract and oil appeared to be safe, since these compounds did not affect cell viability and or generate oxidative stress. Therefore, avocado appears to display a promising antioxidant potential by decreasing oxidative stress.

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BACKGROUND: Prostate cancer is the most common visceral neoplasia in men and frequently presents chemotherapy resistance. In this context, lemongrass (Cymbopogon citratus (D.C.) Stapf) has been studied since it presents many important biological activities, such as anticancer. OBJECTIVE: We investigated the antitumor effect of lemongrass and in chemotherapy activity using prostate cancer cells line (DU-145). METHODS: DU-145 cells were exposed to different concentrations of aqueous extract of lemongrass (30; 100; 300; 500 and 1000 µg/mL), isolated and in combination with docetaxel, during 24 and 72 hours. After, cell viability and proliferation, oxidative metabolism, colony formation and cell cycle analyses were performed. Also, we exposed the African green monkey kidney cell line (VERO) to the same lemongrass concentrations to investigate a possible toxicity of this extract. RESULTS: Our findings suggested that lemongrass presented an antitumor effect and improved docetaxel chemotherapy activity by decreasing cell viability and proliferation as well as colony formation. Moreover, we found an oxidative stress increased and cell cycle arresting in G0 /G1 phase. In addition, this extract presented selectivity action for cancer cells, since it did not cause cytotoxicity in normal cells, ensuring non-toxic therapeutic concentrations. CONCLUSION: Lemongrass is a promising medicinal plant that could be used during chemotherapeutic treatment, in order to potentiate the antitumor response and decrease the resistance of prostate cancer.

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Senecavirus A (SVA) has been a problem in Brazil since the end of 2014. The infections caused by SVA have disrupted the productive chain in Brazil, as it can be confused with foot-and-mouth disease. Although, the virus has remained endemic in the country, an increase in the number of cases of the disease was observed in 2018. The aim of the present study was to conduct the differential diagnosis of foot-and-mouth disease in an outbreak of vesicular disease in finishing swine. Animals (160-170 days old) were kept on a farm with 6000 pigs in Minas Gerais State, Brazil. The morbidity and mortality rates were 20% and 2.2%, respectively. The diagnosis was performed by RT-PCR, using primers that determine the amplification of an internal region of the 3D gene. Furthermore, samples were inoculated into BHK-21 cell culture for viral isolation. In the first passage under cultivation, a cytopathogenic effect compatible with SVA replication (rounding and detachment of the cell monolayer) was observed. The viral identity was confirmed using two additional assays: indirect immunofluorescence assay (IFA) and nucleotide sequencing. Both tests confirmed that the infection was caused by SVA. In summary, we described a method for the diagnosis and viral isolation of SVA, a virus that arrived in Brazil in 2014 and has become endemic in the country.(AU)

Senecavírus A (SVA) é um problema no Brasil desde o final de 2014. As infecções causadas pelo SVA têm causado problemas para a cadeia produtiva no Brasil, pois podem ser confundidas com febre aftosa. Embora o vírus permaneça endêmico no país, foi observado um aumento no número de casos em 2018. O objetivo do presente estudo foi realizar o diagnostico diferencial de febre aftosa em um surto de doença vesicular em suínos de terminação. Os animais (160 a 170 dias de idade) eram mantidos em uma granja com 6.000 suínos no estado de Minas Gerais, Brasil. As taxas de morbidade e mortalidade foram de 20% e 2,2%, respectivamente. O diagnostico foi realizado por RT-PCR, utilizando primers que determinam a amplificação de uma região interna do gene 3D. Além disso, as amostras foram inoculadas na cultura de células BHK-21 para isolamento viral. Na primeira passagem em cultivo, foi observado efeito citopatogênico compatível com a replicação do SVA (arredondamento e descolamento da monocamada celular). A identidade viral foi confirmada usando duas técnicas adicionais: ensaio de imunofluorescência indireta (IFA) e sequenciamento de nucleotídeos. Ambos os testes confirmaram que a infecção foi causada por SVA. Em resumo, descrevemos um método para o diagnóstico e isolamento viral do SVA, um vírus que chegou ao Brasil em 2014 e se tornou endêmico no país.(AU)

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Bovine alphaherpesvirus 2 (BoHV-2) is the agent of herpetic mammilitis (BHM), a cutaneous and self-limiting disease affecting the udder and teats of cows. The pathogenesis of BoHV-2 is pourly understood, hampering the development of therapeutic drugs, vaccines and other control measures. This study investigated the pathogenesis of BoHV-2 in calves after inoculation through different routes. Three- to four-months seronegative calves were inoculated with BoHV-2 (107TCID50.mL-1) intramuscular (IM, n=4), intravenous (IV, n=4) or transdermal (TD) after mild scarification (n=4) and submitted to virological, clinical and serological monitoring. Calves inoculated by the IV route presented as light increase in body temperature between days 6 to 9 post-inoculation (pi). Virus inoculation by the TD route resulted in mild inflammatory lesions at the sites of inoculation, characterized by hyperemia, small vesicles, mild exudation and scab formation, between days 2 and 8pi. Virus or viral DNA was detected by PCR in the crusts/swabs collected from lesions of 3 out of 4 animals inoculated TD from day 2 to 8pi. Viremia was detected in 3/4 animals of the IM group (from day 4 to 8pi); in 2/4 animals of the IV group (days 6 and 8pi) but not in the TD group. Calves from all inoculated groups seroconverted to BoHV-2 in titers from 4 to 64, as indicated by virus-neutralizing (VN) assays performed in sera collected at day 15pi. Administration of dexamethasone (Dex) to the inoculated calves at day 48pi, did not result in virus reactivation as indicated by lack of virus detection in the blood and/or in inoculation sites and no increase in VN antibody titers. These results demonstrated that BoHV-2 was able to replicate efficiently in calves following different routes of exposure, produced viremia after IM and IV inoculation and was not reactivated by Dex treatment.(AU)

O alfaherpesvírus bovino 2 (BoHV-2) é um agente etiológico da mamilite herpética (BHM), uma doença cutânea e autolimitante do úbere e tetos de vacas. Pouco se sabe sobre a patogênese do BoHV-2, dificultando o desenvolvimento de medicamentos terapêuticos e vacinas. Este estudo investigou a patogênese do BoHV-2 em bezerros após a inoculação por diferentes vias. Bezerros soronegativos de três a quatro meses foram inoculados com BoHV-2 (107TCID50.mL-1) por via intramuscular (IM, n=4), por via intravenosa (IV, n=4) ou transdérmica (TD, n=4) após escarificação leve e submetidos a monitoramento virológico, clínico e sorológico. Os bezerros inoculados pela via IV apresentaram aumento leve da temperatura corporal entre os dias 6 a 9 pós-inoculação (pi). A inoculação do vírus pela via TD resultou em lesões inflamatórias leves nos locais de inoculação, caracterizadas por hiperemia, pequenas vesículas, exsudação leve e formação de crostas, entre os dias 2 e 8pi. O vírus ou DNA viral foi detectado por PCR nas crostas/swabs coletados de lesões de 3 de 4 animais inoculados TD do dia 2 ao 8pi. Viremia foi detectada em 3/4 dos animais do grupo IM (do dia 4 ao 8pi); em 2/4 animais do grupo IV (dias 6 e 8pi), mas não no grupo TD. Bezerros de todos os grupos inoculados soroconverteram o BoHV-2 em títulos de 4 a 64, conforme indicado por ensaios de vírus-neutralização (VN) realizados em soro coletado no dia 15pi. Administração de dexametasona (Dex) nos bezerros inoculados no dia 48pi, não resultou em reativação do vírus, como indicado pela falta de detecção de vírus no sangue e/ou nos locais de inoculação e pela ausência de aumento nos títulos de anticorpos. Estes resultados demonstraram que o BoHV-2 foi capaz de replicar eficientemente em bezerros seguindo diferentes vias de inoculação, produziu viremia após a inoculação IM e IV e não foi reativado pelo tratamento com Dex.(AU)

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Bovine alphaherpesvirus 2 (BoHV-2) is the agent of herpetic mammilitis (BHM), a cutaneous and self-limiting disease affecting the udder and teats of cows. The pathogenesis of BoHV-2 is pourly understood, hampering the development of therapeutic drugs, vaccines and other control measures. This study investigated the pathogenesis of BoHV-2 in calves after inoculation through different routes. Three- to four-months seronegative calves were inoculated with BoHV-2 (107TCID50.mL-1) intramuscular (IM, n=4), intravenous (IV, n=4) or transdermal (TD) after mild scarification (n=4) and submitted to virological, clinical and serological monitoring. Calves inoculated by the IV route presented as light increase in body temperature between days 6 to 9 post-inoculation (pi). Virus inoculation by the TD route resulted in mild inflammatory lesions at the sites of inoculation, characterized by hyperemia, small vesicles, mild exudation and scab formation, between days 2 and 8pi. Virus or viral DNA was detected by PCR in the crusts/swabs collected from lesions of 3 out of 4 animals inoculated TD from day 2 to 8pi. Viremia was detected in 3/4 animals of the IM group (from day 4 to 8pi); in 2/4 animals of the IV group (days 6 and 8pi) but not in the TD group. Calves from all inoculated groups seroconverted to BoHV-2 in titers from 4 to 64, as indicated by virus-neutralizing (VN) assays performed in sera collected at day 15pi. Administration of dexamethasone (Dex) to the inoculated calves at day 48pi, did not result in virus reactivation as indicated by lack of virus detection in the blood and/or in inoculation sites and no increase in VN antibody titers. These results demonstrated that BoHV-2 was able to replicate efficiently in calves following different routes of exposure, produced viremia after IM and IV inoculation and was not reactivated by Dex treatment.(AU)

O alfaherpesvírus bovino 2 (BoHV-2) é um agente etiológico da mamilite herpética (BHM), uma doença cutânea e autolimitante do úbere e tetos de vacas. Pouco se sabe sobre a patogênese do BoHV-2, dificultando o desenvolvimento de medicamentos terapêuticos e vacinas. Este estudo investigou a patogênese do BoHV-2 em bezerros após a inoculação por diferentes vias. Bezerros soronegativos de três a quatro meses foram inoculados com BoHV-2 (107TCID50.mL-1) por via intramuscular (IM, n=4), por via intravenosa (IV, n=4) ou transdérmica (TD, n=4) após escarificação leve e submetidos a monitoramento virológico, clínico e sorológico. Os bezerros inoculados pela via IV apresentaram aumento leve da temperatura corporal entre os dias 6 a 9 pós-inoculação (pi). A inoculação do vírus pela via TD resultou em lesões inflamatórias leves nos locais de inoculação, caracterizadas por hiperemia, pequenas vesículas, exsudação leve e formação de crostas, entre os dias 2 e 8pi. O vírus ou DNA viral foi detectado por PCR nas crostas/swabs coletados de lesões de 3 de 4 animais inoculados TD do dia 2 ao 8pi. Viremia foi detectada em 3/4 dos animais do grupo IM (do dia 4 ao 8pi); em 2/4 animais do grupo IV (dias 6 e 8pi), mas não no grupo TD. Bezerros de todos os grupos inoculados soroconverteram o BoHV-2 em títulos de 4 a 64, conforme indicado por ensaios de vírus-neutralização (VN) realizados em soro coletado no dia 15pi. Administração de dexametasona (Dex) nos bezerros inoculados no dia 48pi, não resultou em reativação do vírus, como indicado pela falta de detecção de vírus no sangue e/ou nos locais de inoculação e pela ausência de aumento nos títulos de anticorpos. Estes resultados demonstraram que o BoHV-2 foi capaz de replicar eficientemente em bezerros seguindo diferentes vias de inoculação, produziu viremia após a inoculação IM e IV e não foi reativado pelo tratamento com Dex.(AU)

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The clinical implications of recently discovered porcine circovirus 3 (PCV3) infections are still unknown. The potential role of this emerging virus in reproductive loss in swine has been described. Herein, we report a high prevalence of PCV3 in mummified fetuses from sows maintained in modern farms in Rio Grande do Sul, Santa Catarina, Paraná, Goiás, and Mato Grosso do Sul states, Brazil. For this analysis, 276 mummified fetuses from 11 commercial swine farms were included. The presence of PCV3 DNA was confirmed using PCR, and the complete sequence of five different viral strains was obtained. Sequences of PCV3 genomes available on GenBank were then used for phylogenetic tree construction. Of the 276 mummified fetuses examined, 270 (nearly 97%) were positive for PCV3. In 93.1% of the fetuses, co-infections with at least one of the following agents were identified: porcine parvovirus (PPV), porcine circovirus 2 (PCV2) and Leptospira spp. Twelve fetuses were positive for PCV3 alone. The amino acid sequence of the capsid gene for the five viral strains shared 98-100% homology among them. Analysis of the DNA sequence indicates that the viruses identified in this study belong to the PCV3a1 subgroup. In summary, PCV3 DNA was detected in mummified fetuses at a surprisingly high rate. The role of PCV3 in porcine circovirus-associated disease (PCVAD) is still uncertain. However, considering that PCV3 has been detected in a variety of conditions, even in healthy animals, the present results confirm the need to investigate PCV3 as a causative agent of fetal mummification in swine.

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Caprine alphaherpesvirus 1 (CpHV-1) is a worldwide pathogen of goats and is closely related to Bovine alphaherpevirus 1 (BoHV-1). We herein studied the antigenic relationships of CpHV-1 with BoHV-1 and investigated the pathogenesis of CpHV-1 in kids and calves. Monoclonal antibody reactivity revealed that CpHV-1 and BoHV-1 share immunogenic epitopes in the major envelope glycoproteins gB, gC and gD. The antigenic relationship was further demonstrated by virus-neutralizing assays, in which CpHV-1 and BoHV-1 antisera presented varied degrees of cross-neutralization against the respective heterologous viruses. Although cross-neutralization was observed between both viruses and the heterologous antisera, BoHV-1 antisera neutralized CpHV-1 with higher efficiency than CpHV-1 antisera neutralized BoHV-1. Hence, the antigenic cross-reactivity between CpHV-1 and BoHV-1 should be considered upon serologic testing of goats and cattle in regions where the two viruses co-circulate. Intranasal (IN) inoculation of CpHV-1 (WI13-46 isolate) in seven seronegative kids resulted in efficient viral replication in the respiratory tract. Additionally, mild to moderate systemic and respiratory signs were observed, including apathy, hyperthermia, nasal discharge and respiratory distress. Dexamethasone administration to the inoculated kids between days 36 and 40 pi did not result in virus shedding in nasal secretions. However, latent infection had been established, as evidenced by the detection of CpHV-1 DNA in trigeminal ganglia and olfactory bulbs of kids euthanized at day 67 pi. Contrasting with the outcome of infection in kids, IN inoculation of CpHV-1 in calves did not result in productive infection as no virus replication or shedding were detected, and the animals did not develop clinical signs nor seroconverted. The animal experiments demonstrated that CpHV-1 was able to produce respiratory disease in kids, but did not replicate to detectable levels in calves.

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