Recombinant BoHV-5 glycoprotein (rgD5) elicits long-lasting protective immunity in cattle.

BoHV-5 is a worldwide distributed pathogen usually associated with a lethal neurological disease in dairy and beef cattle resulting in important economic losses due to the cattle industry. Using recombinant gD5, we evaluated the long-duration humoral immunity of the recombinant vaccines in a cattle model. Here we report that two doses of intramuscular immunization, particularly with the rgD5ISA vaccine, induce long-lasting antibody responses. Recombinant gD5 antigen elicited tightly mRNA transcription of the Bcl6 and the chemokine receptor CXCR5 which mediate memory B cells and long-lived plasma cells in germinal centers. In addition, using an in-house indirect ELISA we observed higher and earlier responses of rgD5-specific IgG antibody and the upregulation of mRNA transcription of IL2, IL4, IL10, IL15, and IFN-γ in rgD5 vaccinated cattle, indicating a mixed immune response. We further show that rgD5 immunization protects against both BoHV -1 and -5. Our findings indicate that the rgD5-based vaccine represents an effective vaccine strategy to induce an efficient control of herpesviruses.

Immunogenicity of an inactivated vaccine for intravaginal application against bovine alphaherpesvirus type 5 (BoHV-5).

The present study was carried out to evaluate the intravaginal vaccine potential against bovine alphaherpesvirus type 5 (BoHV-5). Sixty three cows were divided into seven groups (n: 9) and inoculated intravaginally (VA) or intramuscularly (IM) with inactivated BoHV-5, associated with the recombinant B subunit of the heat-labile enterotoxin of E. coli (rLTB), 2-hydroxyethylcellulose (Drug Delivery System A - DDS-A) or Poloxamer 407 (Drug Delivery System B - DDS-B) as follows: G1 (DDS-A + BoHV-5 + rLTB), G2 (DDS-A + BoHV-5), G3 (DDS-B + BoHV-5 + rLTB), G4 (DDS-B + BoHV-5), G5 (BoHV-5 + rLTB), G6 (Negative control) e G7 (Positive control). The local and systemic humoral responses were measured by indirect ELISA (IgA and IgG) and serum neutralization tests, and the cellular response was measured by a quantitative direct ELISA (IL-2 and IFN-Gamma). The results showed the group inoculated by the IM route, G5, demonstrated the highest levels of IgG in the vaginal mucosa among the experimental groups (p < 0.05). In the groups tested with polymers (G1 and G3) in the vaginal mucosa, even higher levels of IgG were seen in comparison to the positive control (G7; p < 0.01). Higher levels of IgA were also noted in relation to the other groups (p < 0.05) on days 30, 60 and 90 post-inoculations. The groups G1 and G3 also provided higher titers of neutralizing antibodies (Log2) in relation to other treatments (p < 0.01) 90 days after inoculation. In the nasal mucosa, there was an increase in the levels of IgA and IgG with the use of vaccines from groups G1 and G3, in relation to the positive control, G7 (p < 0.05) at 60 and 90 days after the first inoculation. Moreover, neutralizing antibodies titers were detected at 60 and 90 days by serum neutralization. The inclusion of the evaluated polymers resulted in a superior response (p < 0.05) of immunoglobulins and IL-2 and IFN-Gamma in relation to the treatment using only rLTB (G5). This data demonstrates the capabilities of a vaccine with an intravaginal application in cattle to stimulate a local and systemic immune response.

Static Magnetic Stimulation Induces Changes in the Oxidative Status and Cell Viability Parameters in a Primary Culture Model of Astrocytes.

Astrocytes play an important role in the central nervous system function and may contribute to brain plasticity response during static magnetic fields (SMF) brain therapy. However, most studies evaluate SMF stimulation in brain plasticity while few studies evaluate the consequences of SMF at the cellular level. Thus, we here evaluate the effects of SMF at 305 mT (medium-intensity) in a primary culture of healthy/normal cortical astrocytes obtained from neonatal (1 to 2-day-old) Wistar rats. After reaching confluence, cells were daily subjected to SMF stimulation for 5 min, 15 min, 30 min, and 40 min during 7 consecutive days. Oxidative stress parameters, cell cycle, cell viability, and mitochondrial function were analyzed. The antioxidant capacity was reduced in groups stimulated for 5 and 40 min. Although no difference was observed in the enzymatic activity of superoxide dismutase and catalase or the total thiol content, lipid peroxidation was increased in all stimulated groups. The cell cycle was changed after 40 min of SMF stimulation while 15, 30, and 40 min led cells to death by necrosis. Mitochondrial function was reduced after SMF stimulation, although imaging analysis did not reveal substantial changes in the mitochondrial network. Results mainly revealed that SMF compromised healthy astrocytes' oxidative status and viability. This finding reveals how important is to understand the SMF stimulation at the cellular level since this therapeutic approach has been largely used against neurological and psychiatric diseases.

Alternatives for obtaining a continuous cell line from Apis mellifera / Alternativas para a obtenção de uma linhagem celular contínua de abelhas Apis mellifera

ABSTRACT: In worldwide there are reports of a significant decrease in colonies of the species Apis mellifera, caused by several factors, including viral infections. In order to study and diagnose illnesses caused by viruses, in vitro cell culture is used as a valuable tool. Yet, there are still no immortalized cell lines of honey bee Apis mellifera. Primary cell cultures are promising for this purpose and can supply the lack of continuous strains, but their establishment is difficult and laborious, which often makes them unfeasible for many research centers. Through the use of cell immortalization techniques, it is possible to develop continuous cell lines and thus benefit, in different ways, research related to different species of bees. The choice of technique is challenging, since in addition to the ability to remain viable for countless passages, cells must keep the genotype and phenotype similar or identical to the original tissue. This review intends to present methodologies that can be used to immortalize Apis mellifera cells, aiming to establish a cell line. The genotypic and phenotypic implications of each technique are evaluated, and the purpose of the cell line to be developed.

RESUMO: Ao redor do mundo há relatos da diminuição significativa de colônias da espécie Apis mellifera, causada por diversos fatores, incluindo infecções virais. Para estudo e diagnóstico de enfermidades causadas por vírus utiliza-se, como uma ferramenta valiosa, o cultivo celular in vitro. Contudo, ainda não existem linhagens celulares imortalizadas de abelhas Apis mellifera. Os cultivos celulares primários são promissores para este fim e podem suprir a falta de linhagens contínuas, porém seu estabelecimento é difícil e laborioso o que, muitas vezes, os torna inviáveis para muitos centros de pesquisa. Através do uso de técnicas de imortalização celular é possível desenvolver linhagens contínuas de células e assim beneficiar, de diversas formas, as pesquisas relacionadas às diferentes espécies de abelhas. A escolha da técnica é desafiadora, visto que, além da capacidade de permanecer viável por inúmeras passagens, as células devem manter o genótipo e fenótipo semelhante ou idêntico ao tecido original. O objetivo deste trabalho é apresentar metodologias que podem ser utilizadas para imortalização de células de Apis mellifera, visando o estabelecimento de uma linhagem celular. São avaliadas as implicações genotípicas e fenotípicas de cada técnica, e a finalidade da linhagem celular a ser desenvolvida.

Molecular Detection of Human Adenovirus and Rotavirus in Feces of White-Eared Opossums.

The white-eared opossums (Didelphis albiventris) is the largest Brazilian marsupial and a great example of animal synanthropy. Considering the high potential as a carrier of viruses originating from environmental contamination, the presence of Human adenovirus (AdV) and rotavirus was investigated in the feces of rescued white-eared opossums, which were in the process of rehabilitation. The feces of 49 animals were initially investigated by immunochromatography, with three samples positive for AdV and one sample positive for rotavirus. When submitted to PCR and nested PCR, the samples of six animals were positive for AdV and three animals were positive for group A rotavirus. Two positive samples in the immunochromatographic assay were not confirmed by PCR. After sequencing and phylogenetic analysis of AdV samples, all were identified within the genus Mastadenovirus, one being HAdV-C, four HAdV-E, and one being similar to a Mastadenovirus found in primates. This is the first report of molecular confirmation of human adenovirus and rotavirus in white-eared opossums. These data could be important of anticipation some emerging diseases and their effects on ecosystems health.

Black queen cell virus and Nosema ceranae coinfection in Africanized honey bees from southern Brazil / Coinfecção por vírus da realeira negra (BQCV) e Nosema ceranae em abelhas africanizadas no sul do Brasil

Bees are fundamental in several aspects, especially in relation to plant biodiversity and pollination. Recently, immense losses are being faced in the number of Brazilian colonies, mainly in southern states of the country, which has a strong beekeeping activity. There are indications that, among the reasons for the losses, pathogens that affect the health of bees may be involved. Among them, the microsporidium Nosema and the black queen cell virus (BQCV) stand out for their prevalence. In this study, 92 colonies of 17 apiaries from southern Brazil were evaluated for infection by Nosema ceranae, Nosema apis and BQCV. Nucleic acid extractions and cDNA synthesis were performed from adult bee samples, followed by Reverse Transcription Polymerase Chain Reaction (RT-PCR) and multiplex PCR. Eight BQCV positive samples were subjected to sequencing. The results showed that N. ceranae and BQCV are circulating in the Southern region of the country, which may be the reason for the loss of colonies. N. apis was not found. N. ceranae was found in 57.6% (53/92) of the colonies and BQCV in 32.6% (30/92). Co-infection was found in 25% (23/92) of the colonies studied, a factor that is suggested to be reducing the hosts' longevity due to the synergistic action of the pathogens. The samples submitted to sequencing indicated similarity of 96.8 to 100% between them, in addition to strong similarity with sequences from Asia, United States, Germany and Peru. This study reports the circulation of N. ceranae and BQCV in apiaries in southern Brazil, in addition to being the first phylogenetic analysis of the Brazilian BQCV sequence.(AU)

As abelhas mostram-se fundamentais em diversos aspectos, especialmente com relação à biodiversidade de plantas e polinização. Recentemente, estão sendo enfrentadas imensas perdas no número de colônias brasileiras, principalmente nos estados do sul do país, com forte atividade apícola. Há indicativos de que, dentre as razões para as perdas, possam estar envolvidos patógenos que afetam a saúde das abelhas. Dentre eles, o microsporídio Nosema e o vírus da realeira negra (BQCV) destacam-se pela prevalência. Neste estudo, foram avaliadas 92 colônias, de 17 apiários do sul do Brasil, a respeito da infecção por Nosema ceranae, Nosema apis e BQCV. Foram realizadas extrações de ácidos nucleicos e síntese de cDNA a partir de amostras de abelhas adultas, seguidos de Reação em Cadeia da Polimerase-Transcriptase Reversa (RT-PCR). Oito amostras positivas para BQCV foram submetidas a sequenciamento. Os resultados mostraram que N. ceranae e BQCV estão circulando na região sul do país, podendo ser a razão para as perdas de colônias. N. apis não foi encontrado. N. ceranae foi encontrado em 57.6% (53/92) das colônias e BQCV em 32.6% (30/92). Foi encontrada coinfecção por ambos em 25% (23/92) das colônias estudadas, fator que sugere a diminuição da longevidade do hospedeiro por ação sinérgica dos patógenos. As amostras submetidas ao sequenciamento indicaram similaridade de 96.8 a 100% entre elas, além de forte similaridade com sequências da Ásia, Estados Unidos, Alemanha e Peru. Este estudo relata a circulação de N. ceranae e BQCV nos apiários do sul do Brasil, além de ser a primeira análise filogenética da sequência do BQCV brasileiro.(AU)

High occurrence of felid alphaherpesvirus 1 and feline calicivirus in domestic cats from southern Brazil / Alta ocorrência de alphaherpesvirus felideo e calicivirus felino em gatos domésticos no Sul do Brasil

Felid alphaherpesvirus 1 (FeHV-1) and feline calicivirus (FCV) affect cats worldwide. The aim of this study was to evaluate the frequency of occurrence of FeHV-1 and FCV in cats with clinical signs of respiratory, oral and/or ocular disease. Samples were collected from cats cared for in veterinary ambulatory and clinics and submitted to molecular detection and viral isolation. Of the 49 cats evaluated, 45 (92%) were positive for at least one of the viruses; 82% (40/49) were positive for FeHV-1 and 41% (20/49) for FCV. Of these, 31% (15/49) were coinfection cases. For FeHV-1, 45% (18/40) of the cats tested were positive from the collection of eye swab, and the same percentage (9/20) was obtained for the FCV by the oral swab. FeHV-1 and/or FCV were isolated in 35% (17/49) of the samples. The main clinical sign observed was ocular secretion in 71% (35/49) of cats, characterized as mild serous, purulent or serosanguineous, and in some cases associated with ocular injury and marked chemosis. Our findings demonstrate the high occurrence of FeHV-1 and FCV in domestic cats in southern Brazil and indicate that measures should be implemented to improve the diagnostic, prevention and management against of these important diseases.(AU)

Alphaherpesvírus felídeo 1 (FeHV-1) e calicivírus felino (FCV) afetam gatos mundialmente. O objetivo deste estudo foi identificar a frequência de ocorrência de FeHV-1 e FCV em gatos com sinais clínicos de doença respiratória, oral e/ou ocular. Amostras foram coletadas de gatos atendidos em ambulatório e clínicas veterinárias e submetidas à detecção molecular e isolamento viral. Dos 49 gatos avaliados, 45 (92%) foram positivos para ao menos um dos vírus; 82% (40/49) foram positivos para o FeHV-1 e 41% (20/49) para o FCV. Destes, 31% (15/49) foram casos de coinfecção. Para o FeHV-1, 45% (18/40) dos gatos foram positivos na coleta do swab ocular, e o mesmo percentual (9/20) foi obtido para o FCV a partir do swab oral. FeHV-1 e/ou FCV foram isolados em 35% (17/49) das amostras. O principal sinal clínico observado foi secreção ocular em 71% (35/49) dos gatos, caracterizada como serosa, purulenta ou serossanguinolenta e, em alguns casos, associada à lesão e quemose. Nossos resultados demonstram a alta ocorrência de FeHV-1 e FCV em gatos domésticos na região Sul do Brasil e indicam que devem ser implementadas medidas para melhorar o diagnóstico, a prevenção e o manejo contra essas importantes doenças.(AU)

Identification of Clade E Avipoxvirus in Brazil.

Avian poxvirus (APV) is an enveloped double-stranded DNA virus that affects many domestic and wild birds worldwide. APVs are classified into three clades (A to C), represented by fowlpox (FP) virus (clade A), canarypox virus (clade B), and psittacinepox virus (clade C), although two additional clades (D and E) have been proposed. In this study, a tumorlike skin lesion found in a domestic fowl was submitted for molecular diagnosis of Avipoxvirus by PCR and sequencing. The phylogenetic analysis revealed that the amplified segment of the corelike 4b protein and polymerase genes clustered in clade E. The APVs in clade E were previously reported from outbreaks in Hungary (flock of turkeys) and in Mozambique (layer chickens), associated with a possible vaccine failure to protect against clade E viruses. To our knowledge, this report is the first identification of clade E in this country, providing new information about host range and genetic diversity of APVs in Brazil, and may represent a potential risk of FP disease outbreaks in commercial poultry.

Reporte de caso- Identificación del Avipoxvirus clado E en Brasil. El poxvirus aviar (APV) es un virus de ADN bicatenario envuelto que afecta a muchas aves domésticas y silvestres en todo el mundo. Los poxvirus aviares se clasifican en tres clados (A, B y C), representados por el virus de la viruela aviar (FP) (clado A), el virus de la viruela del canario (clado B) y el virus de la viruela de los psitácidos (clado C), aunque dos clados adicionales (D y E) han sido propuestos. En este estudio, una lesión cutánea similar a un tumor encontrada en una gallina doméstica fue sometida a diagnóstico molecular de Avipoxvirus por PCR y secuenciación. El análisis filogenético reveló que el segmento amplificado de los genes de la proteína del centro 4b y de la polimerasa se agruparon en el clado E. Los poxvirus aviares en el clado E se reportaron previamente de brotes en Hungría (parvada de pavos) y en Mozambique (gallinas de postura), asociados con una posible falla de la vacuna para proteger contra los virus del clado E. De acuerdo con el conocimiento de los autores, este informe es la primera identificación del clado E en este país, brindando nueva información sobre el rango de hospedadores y la diversidad genética de poxvirus aviares en Brasil, y puede representar un riesgo potencial de brotes de viruela aviar en aves comerciales.

Antiviral activity of native banana lectin against bovine viral diarrhea virus and bovine alphaherpesvirus type 1.

Bovine viral diarrhea virus (BVDV) and bovine alphaherpesvirus type 1 (BoHV-1) are responsible for major economic losses of livestock worldwide, making their eradication an important objective of veterinary research. Vaccines against these infectious agents are commercially available but have some limitations due to the specific features of these viral agents. The development of new antiviral drugs is therefore essential. Native banana lectin (BanLec) is a lectin isolated from banana fruit (Musa acuminata) and has a high affinity for mannose glycans found in several viral envelopes. The inhibitory properties of this lectin against several viruses has already been demonstrated. The aim of this work was therefore to test the antiviral and virucidal activities of BanLec against BVDV-1 and BoHV-1. Its antiviral activity was assessed by measuring the viral titer and viability of susceptible Madin-Darby Bovine Kidney cells (MDBK) treated with BanLec before and after viral infection. The virucidal properties of BanLec were determined by preincubation of the lectin with the viruses, followed by measurement of the viral load in exposed cells. Treatment with 25 µg/mL BanLec resulted in high levels of inhibition against BVDV-1 (99.98%) and BoHV-1 (99.68%) without affecting cell viability, demonstrating promising potential as an antiviral agent.

Rumen-protected methionine in cattle: influences on reproduction, immune response, and productive performance / Metionina protegida da degradação ruminal em bovinos: influências na reprodução, resposta imune e desempenho produtivo

Nowadays, information and knowledge generated about the main ingredients used in cattle diets have enabled greater assertiveness in their formulation. Among the ingredients, amino acids stand out as a promising tool, capable of reducing total crude protein (CP) levels and adjusting optimal metabolizable protein concentrations in diets. Nutritional programs allow diets due to amino acid requirements, providing the opportunity to increase the efficiency of the use of dietary nitrogen, increasing the scarce protein concentrates, maintaining or even boosting performance. This review aimed to present the influences of methionine, in its formulation protected from ruminal degradation, according to reproductive parameters, immune response, and productive performance in cattle. Reproduction-related benefits are linked to the early days of embryonic implantation in the uterine environment, which initially secretes through the histotroph produced by endometrial glands, the nutrients needed to develop the conceptus until implantation, and vascular communication with the mother. Given the immune response, the main results are related to the benefits of innate immunity, with marked increase in phagocytic capacity of neutrophils and monocytes, as well as an important antioxidant effect mediated by methionine products, such as glutathione. When evaluating the influences on productivity, the most evident correlation is the increase in protein in the milk of cows supplemented with methionine. Over the past decade, studies investigating the potential benefits of this strategic supplementation in beef cattle were intensified, expanding the opportunities for the development of new experimental projects.(AU)

Atualmente, as informações e o conhecimento gerado sobre os principais ingredientes utilizados em dietas para bovinos possibilitaram maior assertividade em suas formulações. Dentre esses ingredientes, os aminoácidos se destacam como uma ferramenta promissora, capaz de levar à redução nos níveis totais de proteína bruta e ajustar as concentrações ideais de proteína metabolizável nas dietas. Programas nutricionais permitem formular dietas por exigências de aminoácidos, oportunizando o aumento na eficiência de utilização do nitrogênio dietético, reduzindo dispêndios com concentrados ricos em proteína, com a manutenção, ou ainda, incremento de desempenho. Esta revisão buscou apresentar as influências da metionina, em sua formulação protegida da degradação ruminal, frente a parâmetros reprodutivos, resposta imune e desempenho produtivo em bovinos. Os benefícios relacionados à reprodução se mostram ligados aos primeiros dias de implantação embrionária no ambiente uterino, que inicialmente secreta, através do histotrofo produzido por glândulas endometriais, os nutrientes necessários para o desenvolvimento do concepto até a implantação e comunicação vascular com a mãe. Dada a resposta imune, os principais resultados estão relacionados aos benefícios da imunidade inata, com aumento acentuado da capacidade fagocitária de neutrófilos e monócitos, assim como um importante efeito antioxidante mediado por produtos originários da metionina, como a glutationa. Por fim, quando avaliadas as influências em produtividade, a correlação mais evidente é o incremento em proteína no leite de vacas suplementadas com metionina. Na última década, os estudos que investigam os potenciais benefícios dessa suplementação estratégica em bovinos de corte foram intensificados, abrindo um caminho de oportunidades para o desenvolvimento de novos projetos experimentais.(AU)

Viruses that affect Apis mellifera and their occurrence in Brazil / Vírus que acometem Apis mellifera e sua ocorrência no Brasil

ABSTRACT: Bees are very important insects for agriculture, fulfilling an important role in pollination and renewal of the ecosystem. However, in several countries significant losses of colonies and population decline of honeybees and native bees have been reported in recent years. Most researchers reported that premature losses are linked to several factors, including viruses that have a great impact on the colonies. This article reports the identification of new viruses, some transmission routes, the association of these parasites with the symptoms of the diseases that affect the health of honeybees, as well as viruses that have been described in Brazil.

RESUMO: As abelhas são insetos de grande importância na agricultura, cumprindo um papel muito importante na polinização e na renovação do ecossistema. No entanto, em diversos países têm sido relatadas perdas significativas de colônias e declínio da população de abelhas melíferas e nativas, nos últimos anos. Com isso, surgiram novos estudos sobre este assunto. A maioria dos pesquisadores relata que as perdas prematuras estão ligadas a diversos fatores, dentre eles os vírus, que causam grande impacto nas colônias. Este artigo visa expor as novas identificações de vírus, algumas rotas de transmissão, a associação dos mesmos a parasitas e a sintomatologia das enfermidades que afetam a saúde de abelhas melíferas, bem como os vírus que já foram descritos no Brasil.

Immune responses in bovines to recombinant glycoprotein D of bovine herpesvirus type 5 as vaccine antigen.

Bovine herpesvirus 5 (BoHV-5) is responsible for outbreaks of meningoencephalitis that cause important economic losses in young cattle. BoHV-5 glycoprotein D (gD5) is essential for attachment and penetration into permissive cells and targeting of host immune systems, inducing strong humoral and cellular immune responses. The aim of this study was to evaluate the vaccinal immune response of vaccines formulated with the recombinant BoHV-5 gD (rgD5) in bovines. For the experiment, 72 heifers were randomly allotted into 6 different groups with 12 animals each. Group 1: vaccine formulated using inactivated BoHV-5 (iBoHV-5) adjuvanted with ISA50V2; Group 2: iBoHV-5 associated with 100 µg of rgD5 adjuvanted with ISA50V2; Group 3: 100 µg of rgD5 adjuvanted with ISA50V2; Group 4: 100 µg of rgD5 adjuvanted with Al(OH)3; Group 5: commercial vaccine; and Group 6: control group. Two doses were administered in a 26-day interval and the third after 357 days from primo vaccination. Cattle vaccinated with the vaccines formulated with iBoHV-5 plus rgD5 showed a significant (p < 0.01) five-fold increase in total immunoglobulin G (IgG) for BoHV-5, BoHV-1, and rgD5 as compared with the commercial and control groups. Also, a significant (p < 0.05) increase in IgG1 and IgG2a levels was induced in serum for rgD5. In addition, these same vaccines showed significant (p < 0.01) four-fold higher titers of BoHV-1 and -5 neutralizing antibodies. The results demonstrated that the rgD5 conserved important epitopes that were able to stimulate bovine humoral immunity response capable of viral neutralization of BoHV-1 and -5, suggesting it as a promising vaccine antigen to be used in vaccine for BoHV-1 and -5 endemic areas.

Antigenic and immunogenic properties of the canine distemper virus nucleocapsid protein expressed in Escherichia coli employing codon optimized synthetic gene / Propriedades antigênicas e imunogênicas da proteína do nucleocapsídeo do virus da cinomose canina expressa em Escherichia coli empregando gene sintético com codons otimizados

Despite common occurrence and importance of canine distemper disease the majority of tests currently available for diagnosis are hampered by either low sensitivity or specificity. In this study it was evaluated antigenic and immunogenic characteristics of a conserved region of nucleocapsid protein of canine distemper virus (rCDV NP) expressed in Escherichia coli employing a codon optimized synthetic gene. The expression of rCDVNP in Star strain (mean 300μg/mL, purified) was confirmed by SDS-PAGE and Western blot analysis by using His-Tag monoclonal antibodies. Western blot and ELISA, employing positive and negative control dog sera, demonstrated the rCDVNP antigenicity. The rCDVNP was inoculated in hens and immunoglobulin Y (IgY) was purified from the egg yolk. The mean yield of IgY was 28.55mg/mL. IgY reacted with the recombinant protein as demonstrated by Western blot and ELISA assays. In summary, our findings demonstrated that rCDVNP is antigenic since CDV positive dog sera recognized the protein in vitro. Additionally, the rCDVNP proved to be immunogenic in hens being possible to isolate a high concentration of specific IgY antibodies from the egg yolk. Taken together, these results indicate that the rCDVNP along with the specific IgY could be useful tools for development of the canine distemper immunodiagnostic assays.(AU)

Apesar da ocorrência comum e importância da cinomose canina, a maioria dos testes atualmente disponíveis para diagnóstico são prejudicados pela baixa sensibilidade ou especificidade. Neste estudo foram avaliadas características antigênicas e imunogênicas de uma região conservada da proteína do nucleocapsídeo do virus da cinomose canina (rCDV NP) expressa em Escherichia coli empregando um gene sintético e codons otimizados. A expressão na cepa Star (média de 300μg/mL, purificada) foi confirmada por SDS-PAGE e Western blot utilizando anticorpos monoclonais anti-His-Tag. A antigenicidade da rCDVNP foi demonstrada por western blot e ELISA empregando soros de cães positivos e negativos. A rCDVNP foi inoculada em galinhas e imunoglobulina Y (gY) foi obtida e purificada a partir da gema. A produção média de IgY foi 28.55mg/mL. Anticorpos IgY reagiram com a proteína recombinante, quando analisados por Western blot e ELISA. Em resumo, nossos achados demonstram que a rCDVNP produzida é antigênica, uma vez que os anticorpos de soro de cães positivos para CDV reconheceram a proteína in vitro. Além disso, a rCDVNP foi imunogênica em galinhas, sendo possível isolar anticorpos IgY específicos a partir da gema do ovo em altas concentrações. Tomados em conjunto, estes resultados indicam que a rCDVNP juntamente com a IgY específica podem ser ferramentas úteis para elaborar ensaios de imunodiagnóstico de cinomose canina.(AU)

Potencial antiviral e virucida da melitina e apamina contra herpesvírus bovino tipo 1 e vírus da diarreia viral bovina / Antiviral and virucidal potential of melittin and apamin against bovine herpesvirus type 1 and bovine viral diarrhea virus

A busca por alternativa aos fármacos sintéticos têm revelado descobertas no campo da farmacologia e, nesse sentido, melitina e apamina, dois constituintes do veneno de abelhas, foram descritas com várias ações farmacológicas. Este estudo objetivou avaliar in vitro as capacidades antiviral e virucida destes componentes. Para tanto, células MDBK (Madin Darby Bovine Kidney), após verificação das respectivas doses tóxicas por ensaio MTT ((3-(4,5 dimetiltiazol-2yl)-2-5-difenil-2H tetrazolato de bromo), foram cultivadas em microplacas e tratadas com diferentes concentrações de apamina, melitina e sua associação. Esse tratamento ocorreu antes e após a infecção com 0,1 MOI (multiplicidade de infecção) de cepas citopatogênicas de herpesvírus bovino tipo 1 (BoHV-1) cepa Los Angeles e vírus da diarreia viral bovina (BVDV) cepa NADL. Após incubação por 72 horas, 37oC, as células foram submetidas ao ensaio MTT para estimativa da viabilidade celular. Em experimento paralelo, placas que foram submetidas ao mesmo procedimento sofreram ciclo de congelamento e descongelamento das células, para rompimento das mesmas e mensuração dos títulos virais. O ensaio virucida foi realizado incubando-se suspensões de BoHV-1 e BVDV com as soluções de apamina, melitina e associação por 24 horas a 37oC e 22oC. O título viral foi avaliado às 0 horas, 1, 2, 4, 8 e 24 horas de incubação. A concentração citotóxica para 50% das células (CC50) de melitina foi 2,32 μg/ml e apamina não demonstrou toxicidade à maior concentração testada (100μg/ml). Houve efeito antiviral da melitina sobre BoHV-1, especialmente na concentração de 2μg/ml, onde observou-se 85,96% de viabilidade celular quando o tratamento foi realizado antes da infecção e 86,78% de viabilidade quando o tratamento foi realizado após a infecção. Houve ainda redução de 90% das partículas virais de BoHV-1. Em menores concentrações (1 e 1,5μg/ml) de melitina não houve atividade antiviral, pois a viabilidade celular foi baixa, demonstrando efeito citopático do vírus. Na associação das duas substâncias houve queda no título de BVDV e observou-se maior viabilidade celular quando comparados à ação isolada dos composto sobre este vírus. Isso se confirma na atividade virucida, uma vez que houve decréscimo de 90% das partículas virais de BVDV com a associação dos dois compostos do veneno de abelhas. Atuando individualmente, melitina apresentou efeito antiviral e virucida frente ao BoHV-1, zerando seu título em apenas 2 horas a 37oC. Conclui-se que melitina tem ação antiviral e virucida frente ao BoHV-1 e sua associação com apamina potencializou seus efeitos frente ao BVDV.(AU)

The search for an alternative to synthetic drugs have revealed discoveries in the field of pharmacology and, according to melittin and apamin, two components of bee venom which have been described were with various pharmacological actions.This study aimed to evaluate the in vitro antiviral and virucidal capabilities of these components. Therefore, after verification of their toxic doses by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, MDBK cells (Madin Darby Bovine Kidney) have been cultivated in microplates and treated with different concentrations of apamin, melittin and its association. This treatment occurred before and after infection with MOI (multiplicity of infection) 0.1 of cytopathogenic strains of bovine herpesvirus type 1 (BoHV-1) strain Los Angeles and bovine viral diarrhea virus (BVDV) strain NADL. After incubation for 72 hours, 37°C, the cells were submitted to MTT assay to estimate cell viability. In parallel experiments, plates were subjected to the same procedure suffered freezing and thawing cycle the cells to rupture the same and measurement of viral titers. The virucidal assay was performed by incubating suspension of bovine herpesvirus type-1 and BVDV with apamin solutions, melittin and association for 24 hours at 37°C and 22°C. The viral titer was evaluated at 0 hours, 1, 2, 4, 8 and 24 hours of incubation. The cytotoxic concentration to 50% of the cells (CC50) of melittin was 2.32g/mL and apamin did not show toxicity at the greater concentration tested (100μg/mL). There was antiviral effect of melittin on bovine herpesvirus type-1, especially at a concentration of 2μg/mL, where was observed 85.96% cell viability when treatment was performed before the infection and 86.78% viability when the treatment was carried out after infection. There was also a 90% reduction of viral particles of bovine herpesvirus type-1. In lower concentrations (1 and 1.5μg/mL) melittin no antiviral activity because cell viability was low, showing cytopathic effect of the virus. At the association two substances there were a decrease in the title of BVDV and there was higher cell viability when compared to the isolated action of the compounds of this virus. This is confirmed in the virucidal activity, since there was a decrease of 90% of the viral particles of BVDV with the combination of the two compounds of bee venom. Acting individually, melittin showed antiviral effect and virucidal against for BoHV-1, zeroing its title in just 2 hours at 37°C. It is concluded that melittin has antiviral and virucidal action against the BoHV-1 and its association with apamin potentiate its effects against BVDV.(AU)

Probiotics Bacillus toyonensis and Saccharomyces boulardii improve the vaccine immune response to Bovine herpesvirus type 5 in sheep.

There have been significant efforts toward the development of more efficient vaccines for animal health. A strategy that may be used to improve vaccine efficacy is the use of probiotics. Bovine herpesvirus 5 (BoHV-5) is an example of an important animal pathogen for which vaccines have provided only limited protection. In this study, we examined the use of the probiotics Bacillus toyonensis and Saccharomyces boulardii as a potential immune modulator to improve vaccine efficiency. Thirty, 5-month-old lambs were randomly grouped in three lots of 10 each and vaccinated at days 0, 21 and 42 of the experiment. They grazed on the same pasture and were fed ad libitum twice a day with commercial sheep feed supplemented with either B. toyonensis (1×106CFU/g of feed) or S. boulardii (1×107CFU/g of feed), or non-supplemented feed. The probiotic supplementation was suspended day 28; thereafter, the next 35days, they were fed with the same commercial feed as control group. Animals supplemented with probiotics showed a significant (p>0.001) increased seroconversions against BoHV-5, and higher neutralizing antibodies titres (p>0.05) to BoHV-5 than non-supplemented animals. At 63days of experiment, splenocytes from the supplemented sheep had higher mRNA transcription levels of cytokines IL-10 and IL-17A. These results suggest that these probiotics could provide a promising means of improving vaccine efficacy.

Melittin and its potential in the destruction and inhibition of the biofilm formation by Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa isolated from bovine milk.

Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa stand out in veterinary and human medicine for their role in opportunistic infections and their pathogenic mechanisms, including the biofilms formation. It was investigated the antibacterial activity of melittin and antibiofilm of such bacteria. Twelve strains of these microorganisms isolated from bovine milk were used, as well as the strains S. aureus ATCC 12600, E. coli ATCC 8739 and Pseudomonas aeruginosa ATCC 15442. The minimum inhibitory concentrations (MIC) and minimum bactericidal concentration (MBC) were determined by broth microdilution technique. The biofilms were formed in 96-well plates and melittin on these colonies was added at different concentrations and times. Bacteria previously exposed to melittin were evaluated for inhibition of biofilm production. The MIC and MBC were respectively in µg/mL: S. aureus (6-7 and 32-64), E. coli (40-42.5 and 64-128) and P. aeruginosa (65-70 and 64-128). S. aureus biofilms were more sensitive to the action of melittin, since upon exposure to a concentration 10 times lower than the MIC for 4 h, was completely destroyed. In Gram negative bacteria, the pre-formed biofilm was destroyed only when exposed for 4 h under the MIC. With respect to inhibition of biofilm production, S. aureus was the most sensitive again because produced only 37.2% of the biofilm formed by the control (without previous exposure to melittin), when exposed to the MIC, and at a concentration hundred times smaller than MIC, this microorganism produced 75.2% of the biofilm. E. coli was the most resistant bacteria and produced 56.3% of the biofilm, even if previously exposed to melittin MIC. Melittin presents desirable effects in combating microorganisms studied both at your disposal, biofilm destruction and inhibition of the formation, and maybe used in future studies of new strategies to combat infections caused by these pathogens.

Atividade antiviral e virucida de extratos hidroalcoólicos de própolis marrom, verde e de abelhas Jataí (Tetragonisca angustula) frente ao herpersvírus bovino tipo 1 (BoHV-1) e ao vírus da diarreia viral bovina (BVDV) / Antiviral and virucidal activity of hydroalcoholic extracts of propolis brown, green and jataí bees (Tetragonisca angustula) against Bovine Herpesvirus Type-1 (BoHV-1) and Bovine Viral Diarrhea Virus (BVDV)

Dentre as propriedades biológicas da própolis, a atividade antimicrobiana tem merecido destacada atenção. No presente trabalho, descreve-se a ação antiviral e virucida de três extratos hidroalcoólicos de própolis (marrom, verde e de abelhas jataí (Tetragonisca angustula), frente ao Herpesvírus Bovino tipo (BoHV-1) e ao Vírus da Diarreia Viral Bovina (BVDV). Os três extratos hidroalcoólicos foram obtidos de extração etanólica e são oriundos do sul do Brasil. A composição química dos extratos de própolis foi determinada pela cromatografia líquida de alta eficiência acoplada a espectrômetro de massas (UFLC-PDA-ESI-TOF/MS) que identificou e quantificou compostos como: ácido cafeico e ácido p-cumárico, ácido clorogênico, ácido ferúlico, além de flavonoides como a rutina. A toxicidade celular bem como a atividade antiviral dos extratos de própolis em monocamadas de células MDBK (Madin-Darby Bovine Kidney) foi avaliada através de observação microscópica e quantificada pelo teste de MTT (3-(4,5 dimetiltiazol-2yl)-2-5-difenil-2H tetrazolato de bromo). O extrato de própolis de abelhas jataí demonstrou ser menos citotóxico (1,57µg/mL), quando comparado aos extratos verde (0,78µg/mL) e marrom (0,39µg/mL). Quanto a atividade antiviral, a própolis verde demostrou maior eficácia em ambos os tratamentos celulares (pós e pré-exposição) frente ao BoHV-1 em relação aos outros extratos, ou seja, houve maior viabilidade celular quando comparada aos controles de células e vírus. Já a de jataí apresentou atividade frente aos dois vírus (BoHV-1 e BVDV) no método pré-infecção, enquanto a própolis marrom demonstrou ação apenas frente ao BoHV-1 também no método pré-infecção. Para determinação da atividade virucida foram utilizadas diferentes diluições dos vírus, bem como temperaturas e tempos distintos de incubação. A própolis verde a 37°C propiciou a maior redução no título viral (4,33log) em relação a marrom (log = 3,5log) e de jataí (log = 3,24log). No entanto, frente ao BVDV a própolis jataí apresentou os melhores resultados em ambas as temperaturas (22oC e 37oC). Portanto, os extratos avaliados apresentaram atividade antiviral e virucida frente ao BoHV-1 e BVDV, o que os torna alvo para o desenvolvimento de novos biofármacos como alternativa ao uso de antivirais comerciais em Medicina Veterinária.(AU)

Among the biological properties of propolis, the antimicrobial activity has received prominent attention. In this paper, we describe the antiviral and virucidal effect of three hydroalcoholic extracts of propolis (brown, green and jataí bees (Tetragonisca angustula), against bovine herpesvirus type-1 (BoHV-1) and bovine viral diarrhea Virus (BVDV). All hydroalcoholic extracts were obtained from ethanol extraction. The chemical composition of propolis extracts was determined by high-performance liquid chromatography coupled to mass spectrometer (UFLC-PDA-ESI-TOF/MS) to identify and quantify compounds such as caffeic acid and p-coumaric acid, chlorogenic acid, ferulic, and flavonoids such as rutin. Cell toxicity and antiviral activity of propolis extracts in monolayers of MDBK cells (Madin-Darby Bovine Kidney) were assessed by microscopic observation and quantified by the MTT assay (3- (4.5 dimethylthiazol-2yl) -2- 5-diphenyl-2H-tetrazolato bromine). Propolis extract from Jataí bees proved to be less cytotoxic (1.57mg / ml) when compared to green extracts (0.78mg / ml) and brown (0.39mg/mL). Regarding antiviral activity, propolis has shown greater efficacy in both cellular treatments (post and pre-exposure) against BoHV-1 when compared to other extracts, ie, there was increased cell viability compared to cell and virus controls. Extracts from Jataí showed activity against both viruses (BoHV-1 and BVDV) infection in the pre-test, whereas brown propolis demonstrated action only against the BoHV-1 in the pre-infection method. To determine the virucidal activity, it were used different dilutions of virus, as well as different temperatures and incubation times. The green propolis at 37°C led to a greater reduction in viral titer (4.33log) compared to brown (3.5log) and jataí (3.24log). Jataí propolis showed the best results in both temperatures (22oC and 37oC) when tested against BVDV. In summary, the evaluated extracts showed antiviral and virucidal activity against BoHV-1 and BVDV, and may be important targets for the development of new compounds as an alternative to commercial antivirals.(AU)

Activity of the antimicrobial peptide P34 against bovine alphaherpesvirus type 1 / Atividade do peptídeo antimicrobiano P34 contra o alfaherpesvírus bovino tipo 1

ABSTRACT: Previous studies have demonstrated the antimicrobial activity of the peptide P34. In this study, the antiviral potential of P34 and the in vitro mechanism of action were investigated against bovine alphaherpesvirus type 1 (BoHV1). P34 exhibited low toxicity, a high selectivity index (22.9) and a percentage of inhibition of up to 100% in MDBK cells. Results from antiviral assays indicated that P34 did not interact with cell receptors, but it was able to inhibit the viral penetration immediately after pre-adsorption. In addition, BoHV1 growth curve in MDBK cells in the presence of P34 revealed a significant reduction in virus titer only 8h post-infection, also suggesting an important role at late stages of the replicative cycle. Virucidal effect was observed only in cytotoxic concentrations of the peptide. These findings showed that the antimicrobial peptide P34 may be considered as a potential novel inhibitor of in vitro herpesviruses and must encourage further investigation of its antiherpetic activity in animal models as well as against a wide spectrum of viruses.

RESUMO: A atividade antimicrobiana do peptídeo P34 já foi previamente demonstrada. Neste estudo, o potencial antiviral do P34 e o mecanismo de ação in vitro contra o alfaherpesvírus bovino tipo 1 (BoHV1) foram investigados. O P34 exibiu baixa toxicidade, alto índice de seletividade (22.9) e percentagem de inibição viral de até 100% em células MDBK. Os resultados dos ensaios antivirais indicaram que não interage com receptores celulares, mas é capaz de inibir a penetração viral, imediatamente após a pré-adsorção. Além disso, a curva de crescimento do BoHV1 em células MDBK na presença do P34 revelou uma significativa redução no título somente após 8h de infecção, sugerindo também uma importante atividade do peptídeo nas fases finais do ciclo replicativo. Efeito virucida frente / BoHV1 foi observado apenas em concentrações citotóxicas do peptídeo. Os dados obtidos indicam que o peptídeo antimicrobiano P34 pode ser considerado um potencial composto inibidor de herpesvírus, in vitro, e estimulam posteriores investigações sobre sua atividade anti-herpética em modelos animais, bem como contra outros vírus.

Antibodies against Neospora caninum, Sarcocystis spp. and Toxoplasma gondii detected in buffaloes from Rio Grande do Sul, Brazil / Anticorpos contra Neospora caninum, Sarcocystis spp. e Toxoplasma gondii detectados em búfalos no Rio Grande do Sul

The presence of antibodies against Neospora caninum, Sarcocystis spp. and Toxoplasma gondii was evaluated in buffaloes (Bubalus bubalis) from Rio Grande do Sul state (RS), southern Brazil. Serum samples (n=220) were analyzed for antibodies by indirect fluorescent antibody test (IFAT). Antibody presence was considered when the titers were equal or higher than 100 for these protozoa. A total of 60.5% (133/220) buffalo serum samples were positive for at least one of the protozoa evaluated in this study. Antibodies for N. caninum, Sarcocystis spp. and T. gondii were found in 36.4% (80/220), 25.5% (56/220) and 16.8% (37/220) of the buffaloes respectively, indicating a higher frequency of N. caninum infection (p=0.0133). The IFAT is a suitable method to diagnose N. caninum, Sarcocystis spp. and T. gondii infection in buffaloes for detecting IgG antibodies. This study demonstrates the presence of these three protozoa in buffalo herds in RS, Brazil, which may be source of infection to other animals. The high frequency of animals positive for N. caninum is important and could be related to reproductive problems. Additionally, the presence of Sarcocystis spp. and T. gondii in buffaloes can be a possible public health issue.(AU)

A presença de anticorpos contra Neospora caninum, Sarcocystis spp. e Toxoplasma gondii foi avaliada em búfalos (Bubalus bubalis) no estado do Rio Grande do Sul (RS), Região Sul do Brasil. Amostras de soro de 220 bubalinos foi analisada para presença de anticorpos, através de reação de imunofluorescência indireta (RIFI). Foram consideradas positivas as amostras que apresentaram títulos de anticorpos maiores ou iguais a 100, para os protozoários estudados. Um total de 60,5% (133/220) das amostras sorológicas dos búfalos foram positivas para pelo menos um dos parasitas pesquisados. Anticorpos para N. caninum, Sarcocystis spp. e T. gondii foram encontrados em 36,4% (80/220); 25,5% (56/220) e 16,8% (37/220) dos búfalos respectivamente, indicando que houve uma maior frequência de infecção de N. caninum em relação aos demais protozoários (p=0.0133). A RIFI é um método adequado para o diagnóstico sorológico da infecção por N. caninum, Sarcocystis spp. e T. gondii em búfalos. Este estudo demonstrou a presença destes três protozoários em bubalinos no RS, Brasil, que pode ser fonte de infecção para outros animais. A elevada ocorrência de animais positivos para N. caninum é importante e pode estar relacionada a problemas reprodutivos. Adicionalmente, a presença de Sarcocystis spp. e T. gondii em búfalos, pode significar um possível problema de saúde pública.(AU)

Development of an Indirect ELISA for Serological Diagnosis of Bovine herpesvirus 5.

Bovine herpesviruses 1 and 5 (BoHV-1 and BoHV-5) are economically important pathogens, associated with a variety of clinical syndromes, including respiratory and genital disease, reproductive failure and meningoencephalitis. The standard serological assay to diagnose BoHV-1 and BoHV-5 infections is the virus neutralization test (VNT), a time consuming procedure that requires manipulation of infectious virus. In the present study a highly sensitive and specific single dilution indirect ELISA was developed using recombinant glycoprotein D from BoHV-5 as antigen (rgD5ELISA). Bovine serum samples (n = 450) were screened by VNT against BoHV-5a and by rgD5ELISA. Compared with the VNT, the rgD5ELISA demonstrated accuracy of 99.8%, with 100% sensitivity, 96.7% specificity and coefficient of agreement between the tests of 0.954. The rgD5ELISA described here shows excellent agreement with the VNT and is shown to be a simple, convenient, specific and highly sensitive virus-free assay for detection of serum antibodies to BoHV-5.