Chick heterophils release DNA extracellular traps (DETs) in vitro and in vivo upon Aspergillus fumigatus conidia exposure.

Aspergillosis is a common fungal disease in avian species, causing high mortality in young chicks in agricultural farms and yards. It is caused by fungi belonging to the genus Aspergillus. Aspergillosis occurs by inhalation of fungal conidia, and in chickens, effective infection control relies on a rapid and large influx of heterophils to the lungs. Heterophils, upon different stimuli, release to the extracellular milieu their chromatin associated with several proteins that ensnare and kill different pathogens similarly to neutrophil extracellular traps. Here, we showed that Aspergillus fumigatus conidia and the peptidogalactomannan (PGM), isolated from the fungus cell wall, induce the release of DNA extracellular traps (DETs) in chicks' blood and lung heterophils. We demonstrated that reactive oxygen species, elastase and peptidyl arginine deiminase (PAD) were involved in DETs extrusion, the occurrence of DETs in the lungs of A. fumigatus-exposed chicks in vivo, and its role in chick survival. These results may contribute to developing more efficient tools for the therapeutic and diagnosis of aspergillosis.

The in vitro antileishmanial activity of essential oil from Aloysia gratissima and guaiol, its major sesquiterpene against Leishmania amazonensis.

Leishmaniases is a tropical disease caused by protozoa of the genus Leishmania for which the current treatment is expensive, besides increasing reports of parasite resistance. This study investigated the anti-Leishmania amazonensis activity of the essential oil from Aloysia gratissima (AgEO) and guaiol, the major sesquiterpene constituent in the oil. Our results showed that AgEO killed promastigotes and intracellular amastigotes at an IC50 of 25 and 0·16 µg mL-1, respectively, while guaiol killed amastigotes at an IC50 of 0·01 µg mL-1. Both AgEO and guaiol were safe for macrophages up to 100 µg mL-1, as evaluated by the dehydrogenase activity, membrane integrity and phagocytic capacity. AgEO and guaiol did not induce nitrite oxide (NO) in resting macrophages and inhibited the production of NO in lipopolysaccharide-stimulated macrophages. The ultrastructural analysis suggested that AgEO and guaiol act directly on parasites, affecting promastigotes kinetoplast, mitochondrial matrix and plasma membrane. Together, these results pointed out that AgEO and guaiol could be promising candidates to develop anti-Leishmania drugs.

Genetic variation of a foundation rockweed species affects associated communities.

Genetic variation in a foundation species may affect the composition of associated communities as well as modify ecosystem function. While the ecological consequences of genetic diversity of foundation species have been widely reported, the ability of individual genotypes to support dissimilar communities has been documented only in forest ecosystems. Here, for the first time in a marine ecosystem, we test whether the different genotypes of the rockweed Fucus vesiculosus harbor distinct community phenotypes and whether the genetic similarity of individual genotypes or their defensive compound content can explain the variation of the associated communities. We reared replicated genotypes in a common garden in the sea and analyzed their associated communities of periphytic algae and invertebrates as well as determined their contents of defense compounds, phlorotannins, and genetic distance based on neutral molecular markers. The periphytic community was abundant in mid-summer and its biovolume, diversity and community composition varied among the rockweed genotypes. The diversity of the periphytic community decreased with its increasing biovolume. In autumn, when grazers were abundant, periphytic community biomass was lower and less variable among rockweed genotypes, indicating different relative importance of bottom-up regulation through heritable variation of the foundation species and top-down regulation through grazing intensity. Similarly, composition of the invertebrate community varied among the rockweed genotypes. Although the genotype explained about 10-18% of the variation in associated communities, the variation was explained neither by the genetic distance nor the phlorotannin content. Thus, neither neutral genetic markers nor a single phenotypic trait could provide a mechanistic understanding of the genetic basis of community specificity. Therefore, a more comprehensive mapping of quantitative trait variation is needed to understand the underlying mechanisms. The community specificity implies that genetic variation within a foundation species is crucial for the biodiversity and assembly of associated organisms and, thus, for the functioning of associated communities. The result highlights the importance of ensuring the genetic variation of foundation species as a conservation target.

Naturally acidified habitat selects for ocean acidification-tolerant mussels.

Ocean acidification severely affects bivalves, especially their larval stages. Consequently, the fate of this ecologically and economically important group depends on the capacity and rate of evolutionary adaptation to altered ocean carbonate chemistry. We document successful settlement of wild mussel larvae (Mytilus edulis) in a periodically CO2-enriched habitat. The larval fitness of the population originating from the CO2-enriched habitat was compared to the response of a population from a nonenriched habitat in a common garden experiment. The high CO2-adapted population showed higher fitness under elevated Pco2 (partial pressure of CO2) than the non-adapted cohort, demonstrating, for the first time, an evolutionary response of a natural mussel population to ocean acidification. To assess the rate of adaptation, we performed a selection experiment over three generations. CO2 tolerance differed substantially between the families within the F1 generation, and survival was drastically decreased in the highest, yet realistic, Pco2 treatment. Selection of CO2-tolerant F1 animals resulted in higher calcification performance of F2 larvae during early shell formation but did not improve overall survival. Our results thus reveal significant short-term selective responses of traits directly affected by ocean acidification and long-term adaptation potential in a key bivalve species. Because immediate response to selection did not directly translate into increased fitness, multigenerational studies need to take into consideration the multivariate nature of selection acting in natural habitats. Combinations of short-term selection with long-term adaptation in populations from CO2-enriched versus nonenriched natural habitats represent promising approaches for estimating adaptive potential of organisms facing global change.

Antigenotoxic Effect of Piperine in Broiler Chickens Intoxicated with Aflatoxin B1.

Piperine is an abundant amide extracted from black pepper seeds which has been shown to have protective effects against cytotoxic and genotoxic carcinogenesis induced by certain chemical carcinogens and aflatoxin B1 (AFB1) in vitro. The aim of this work was to study, in vivo, the antigenotoxic potential of feed-added piperine on broiler chickens experimentally intoxicated with AFB1, using micronucleus and comet assays. The antigenotoxicity assessment of 9-day-old chicks was performed on a total of 60 chickens divided into four groups of 15 broilers each: (C) control, (P) 60 mg·piperine kg-1 feed, (A) 0.5 mg·AFB1·kg-1 body weight, (daily by oral route), and (P + A) co-treatment with piperine and AFB1. The experiment was conducted for 26 days. Chicks intoxicated with AFB1 showed significant genotoxic effects in the first 24 h post intoxication, and the effects remained in the other periods analyzed (48, 72, and 96 h and 26 days of treatment). The DNA damage in peripheral blood cells, the number of erythrocytes with micronuclei, and polychromatic-to-normochromatic erythrocyte ratio were significantly reduced or absent in the piperine/AFB1 group. No significant differences were observed between the group piperine/AFB1 and the control and piperine-alone groups. The addition 60 mg·kg-1 of piperine to the diet of the broiler chicks was safe, promoting beneficial effects in poultry health with respect to the toxic effects 0.5 mg·AFB1·kg-1 body weight.

Prevalence of feline leukemia virus infection in domestic cats in Rio de Janeiro.

Peripheral blood smears of 1094 domestic cats were collected and tested by indirect immunofluorescence antibody assay for p27 antigen in cells to study the prevalence and risk factors for feline leukemia virus (FeLV) in the state of Rio de Janeiro. Sex, age, breed, outdoor access, neutering status, type of habitation (household, shelter, veterinary clinics and other places), number of household cats and clinical signs were registered on a form. Among the tested samples, 11.52% were positive. Risk factors for FeLV infection included outdoor access, age range between 1 and 5 years old, and cohabitation with numerous cats.

Evaluation of a recombinant p24 antigen for the detection of feline immunodeficiency virus-specific antibodies / Avaliação do antígeno recombinante p24 para a detecção de anticorpos específicos do vírus da imunodeficiência felina

Feline Immunodeficiency Virus is a worldwide infection and is considered a significant pathogen. The diagnosis of FIV infections is mainly based on commercially available rapid tests that are highly expensive in Brazil, hence it is rarely performed in the country. Furthermore, lentiviruses grow slowly and poorly in tissue cultures, making the production of viral antigen by classic means and thus the establishment of FIV immunodiagnosis impracticable. In order to deal with this, recombinant DNA techniques were adopted to produce the protein p24, a viral capsid antigen. The protein's reactivity evaluation analyzed by Western blot indicated that this recombinant antigen can be a useful tool for the immunodiagnostic of FIV infections.

O vírus da imunodeficiência felina tem distribuição mundial e é considerado um patógeno significativo. No Brasil, a prática diagnóstica é baseada principalmente em teste rápidos, importados e de custo elevado, disponíveis comercialmente. Devido ao seu custo proibitivo em nosso país, o diagnóstico da infecção pelo FIV é raramente realizado. Ademais, os lentivírus se multiplicam lenta e pobremente em cultura de células, o que torna a produção de antígeno por meios clássicos e o estabelecimento do imunodiagnóstico impraticável. Com o objetivo de lidar com esta questão, técnicas de DNA recombinante foram utilizadas para produção de um antígeno do capsídeo viral, a proteína p24. A avaliação da reatividade realizada por Western blot indicou que este antígeno recombinante pode ser útil para o imunodiagnóstico de infecções pelo FIV.

Oral administration of piperine for the control of aflatoxin intoxication in rats

Aflatoxins are mycotoxins that have important toxic effects on human and animal health, even if consumed at low doses. The oral administration of piperine (1.12 mg/kg) during 23 days in rats seemingly interfered with the toxicity of aflatoxins, decreasing hepatic injuries and the leukocyte depletion in experimentally intoxicated animals.

Characterization of neutrophil extracellular traps in cats naturally infected with feline leukemia virus.

Feline leukemia virus (FeLV), a common, naturally occurring gammaretrovirus in domestic cats, is associated with degenerative diseases of the haematopoietic system, immunodeficiency and neoplasia. FeLV infection causes an important suppression of neutrophil function, leading to opportunistic infections. Recently, a new microbicidal mechanism named NETosis was described in human, bovine and fish neutrophils, as well as in chicken heterophils. The purpose of the present study was to characterize NETosis in feline neutrophils, as well as to evaluate neutrophil function in FeLV naturally infected symptomatic and asymptomatic cats through the phagocytosis process, release of neutrophil extracellular traps (NETs) and myeloperoxidase (MPO) activity. The results showed that feline neutrophils stimulated with protozoa parasites released structures comprising DNA and histones, which were characterized as NETs by immunofluorescence. Quantification of NETs after neutrophil stimulation showed a significant increase in NET release by neutrophils from FeLV(-) and FeLV(+) asymptomatic cats compared with FeLV(+) symptomatic cats. Moreover, the number of released NETs and MPO activity in unstimulated neutrophils of FeLV(+) symptomatic cats were higher than those in unstimulated neutrophils from FeLV(-) and FeLV(+) asymptomatic cats. This study reports, for the first time, NET release by feline neutrophils, along with the fact that NET induction may be modulated by a viral infection. The results indicate that the NET mechanism appears to be overactivated in FeLV(+) cats and that this feature could be considered a marker of disease progression in FeLV infection.

Oral administration of piperine for the control of aflatoxin intoxication in rats.

Aflatoxins are mycotoxins that have important toxic effects on human and animal health, even if consumed at low doses. The oral administration of piperine (1.12 mg/kg) during 23 days in rats seemingly interfered with the toxicity of aflatoxins, decreasing hepatic injuries and the leukocyte depletion in experimentally intoxicated animals.

Administração oral de piperina em frangos de corte / Piperine oral administration on broiler' chikens

O efeito tóxico da piperina, principal amida presente em diversas espécies de pimenta, foi avaliada em frangos de corte por meio da administração oral (0,00, 1,12, 2,25 e 4,5mg kg-1 peso vivo) por 14 dias consecutivos. Foram empregados 60 pintos machos (Cobb Avian 48) com sete dias de idade, distribuídos aleatoriamente em quatro grupos experimentais (n=15). Foram avaliados parâmetros como: ganho de peso, peso relativo do fígado e alterações hematológicas e anatomopatológicas. A administração oral de piperina não interferiu no ganho de peso ou no peso relativo do fígado, além de não promover alteração no tamanho e na coloração dos órgãos ou no aparecimento de lesões de parênquima e nas mucosas do órgão. Todavia, alterações histopatológicas dose-dependentes foram observadas. A piperina não foi capaz de alterar significativamente os parâmetros hematológicos analisados, com exceção do leucograma, em que as doses de 1,12mg e 2,25mg kg-1 promoveram aumento do número de heterófilos e do número total de leucócitos, respectivamente. Os resultados sugerem que a dose oral de 1,12mg de piperina por quilo não é tóxica para frangos de corte, sendo semelhante aos resultados obtidos para ratos e camundongos. Além disso, constatou-se a capacidade da piperina em aumentar o número total de leucócitos circulantes a partir da dose de 2,25mg kg-1 nessa espécie animal.

The toxic effect of piperine, the main amide compound of different pepper species, was evaluated on broiler chickens by oral administration at 0.00, 1.12, 2.25 and 4.50mg kg-1 concentrations for 14 days. Sixty seven days old male chicks (Cobb Avian 48) randomly allocated to four experimental groups (n=15) were used in this work. Parameters such as: body weight gain, liver relative weight, hematological and anatomopathological alterations were analyzed. The oral route administration did not interfere on weight gain or liver relative weight, as well as, any modification on size and organs' color and/or parenchyma/mucous membranes injuries were observed; however, liver histopathological changes were noticed in a dose-dependent manner. In addition, piperine did not alter hematological parameters, except for leukocytes counting, which at 1.12 and from 2.25mg kg-1 caused an increase of heterophils and in the total number of leukocytes, respectively. The results suggest that 1.12mg kg-1 of piperine orally administrated is not toxic for broiler chickens, as previously observed for rats and mice. Moreover, 2.25mg kg-1 of piperine seems to increase the total number of leukocytes for this animal specie.

Performance of a latex agglutination test in the diagnosis of acute gastroenteritis by rotavirus

The aim of this work was to determine the performance of latex agglutination test (LAT) for evaluating children acute gastroenteritis by rotavirus. The LAT showed good sensitivity, as well as specificity and predictive positive value and due to its simplicity and speed, it has been suitable for rotavirus diagnosis in hospital laboratories.

O objetivo deste trabalho foi determinar o desempenho do teste de aglutinação em látex (TAL), no diagnóstico das gastroenterites agudas em crianças, causadas por rotavírus. O TAL mostrou boa sensibilidade, especificidade e valor preditivo positivo e devido à sua simplicidade e rapidez, o teste é apropriado para uso em hospitais para o diagnóstico de rotaviroses.

Ineficiência da Thuja occidentalis no tratamento dos poxvirus aviários / Thuja occidentalis ineficiency in avian poxviruses treatment

Medicações à base de Tuia (Thuja occidentalis) são comuns em tratamentos homeopáticos e fitoterápicos, na medicina humana e veterinária. Suas propriedades imunoestimulantes e antivirais são descritas e sua utilização empírica no tratamento das poxviroses aviárias é recomendada por criadores e veterinários. Para avaliar o potencial terapêutico da Tuia sobre estas infecções, dois grupos (controle e teste) de dez aves jovens foram inoculados por escarificação, com uma amostra de campo de poxvírus aviário. As aves inoculadas foram examinadas diariamente, quanto ao início e evolução das lesões, registrando-se o número e tamanho destas, até a cicatrização. Após o surgimento das lesões, o grupo teste recebeu a tintura alcoólica diluída na água de consumo. A análise experimental indicou que, nas condições empregadas, a utilização da Tuia não favoreceu a regressão ou restrição no desenvolvimento das lesões de pele promovidas pela infecção experimental.

Importância da resistência osmótica na estabilidade do antígeno celular de Mycoplasma mycoides subesp. mycoides tipo LC em ensaio imunoenzimático (ELISA) / Osmotic resistance importance on Mycoplasma mycoides subsp. mycoides l type cellular antigen LC stabilization on immunoenzymatic assay (ELISA)

O trabalho avalia a importância das condições de armazenamento do antígeno celular de Mycoplasma Mycoides subesp. Mycoides Tipo LC no teste de ELISA. Os resultados mostram a importância da preservação da integridade da célula micoplásmica na estabilidade do antígeno empregado no teste.

Phenotypic and antigenic variation of Mycoplasma Gallisepticum vaccine strains

Cepas vacinais de Mycoplasma gallisepticum, F e TS-11, foram examinadas quanto às suas variacões fenotípicas e antigênicas, por SDS-PAGE e através de dois métodos sorológicos (inibicão da hemaglutinacão e imunoeletroforese). A análise densitométrica das bandas obtidas nos géis de poliacrilamida mostrou pequena variabilidade fenotípica entre as amostras, sendo a banda peptidica de 75 kDa detectada apenas na amostra vacinal F. Anticorpos policlonais produzidos em galinha foram utilizados nos ensaios sorológicos para estudar a variabilidade antigênica das amostras. Houve elevada reatividade cruzada entre as amostras e os anticorpos homólogos e heterólogos. A característica mais evidente foi a resposta específica da banda peptídica de 75 kDa da vacina F ao anticorpo homólogo.

Protein profile analysis by SDS-Page of "Mycoplasma gallisepticum" strains S6(208) and F-K810 grown in Hayflick's and Frey's media

The "Mycoplasma gallisepticum" strains [wild-type S6(208) and vaccine-type F-K810] grown in Frey's and hayflick's media were analysed by SDS-PAGE. No visual changes in the protein profiles of these strains were observed regardless of the media composition used, although the polyacrylamide gel electrophoretograms showed minor differences do exist when densitometer traces of the gel are compared. Both strains were easily differentiated on SDS-PAGE analysis by a peptide band p75, that is specific for MG F-K810 strain, used as vaccine.

Diferenciaçäo de tipos de Mycoplasma mycoides na etiopatogenia da micoplasmose caprina / Differentiation of Mycoplasma mycoides types on the etiopathogeny of goat mycoplasmosis

Dentre as várias espécies e subespécies de micoplasma, isoladas de caprinos, Mycoplasma mycoides subesp. mycoides e M. mycoides subesp. capri, säo considerados importantes patógenos, por produzirem doenças com diversas manifestaçöes clínicas (infecçöes sistêmicas, pneumonia, artrite, agalaxia etc.). O propósito desse estudo foi pesquisar a presença dessas subespécies em caprinos com diagnóstico de micoplasmose, oriundos de uma propriedade localizada em Nova Friburgo, município do estado do Rio de Janeiro. Na coleta de espécimes para o diagnóstico, usou-se o método de lavagem do conduto auditivo externo de 20 animais, utilizando-se o meio líquido de Hayflick. Após a obtençäo dos lavados de ouvido, as amostras foram cultivadas segundo os procedimentos padröes. Os cultivos foram examinados em microscópio estereoscópico, visando a observaçäo de colônias típicas de micoplasma. Essas colônias foram coradas pelo método de Dienes, submetidas aos testes de sensibilidade à Digitonina e Imunoperoxidase Indireta (IPI), para posterior identificaçäo. Todos os caprinos examinados apresentaram cultivo positivo para Mycoplasma spp., i.é, 100 por cento (20/20) e desses, 80 por cento (16/20) foram positivos para M. mycoides. Das amostras positivas para M. mycoides, 70 por cento (14/16) o foram para M. mycoides subesp. mycoides tipo Colônia Grande (®Large Colony-LC¼), 45 por cento (9/16) para M. mycoides subesp. mycoides tipo Colônia Pequena (®Small Colony-SC¼) e 70 por cento (14/16) para M. mycoides subesp. capri, onde 12 dos 16 caprinos examinados portavam mais de uma subespécie e/ou tipo no conduto auditivo. No presente estudo foram encontrados micoplasmas com características de ambas as subespécies e tipos (LC e SC), no conduto auditivo externo de caprinos, sugerindo a necessidade de estudos adicionais para elucidaçäo da etiopatogenia das micoplasmoses caprinas.

N-terminal amino acid sequences of the major outer membrane proteins from a Neisseria meningitidis group B strain isolated in Brazil

The four dominant outer membrane proteins (46, 38 33 and 28 kDa) were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a semi-purified preparation of vesicle membranes of a Neisseria meningitidis (N44/89, B:4:P1.15:P5.5,7) strain isolated in Brazil. The N-terminal amino acid sequence for the 46 kDa and 28 kDa proteins matched that reported by others for class 1 and 5 proteins respectively, whereas the sequence (25 amino acids) for the 38 kDa (class 3) protein was similar to class 1 meningococcal proteins. The sequence for the 33 kDa (class 4) was unique and not homologous to any known protein.