Impact of persistent bovine viral diarrhea virus infection on indicators of innate and adaptive immune function in holstein calves and cows / Impacto da infecção persistente pelo vírus da diarreia viral bovina sobre indicadores de função imunológica inata e adaptativa em bezerras e vacas holandesas

We evaluated some indicators of innate and humoral immune response in persistently infected (PI) Holstein calves and cows from 1 to 36 months of age matched with controls from the same herd. The effects were cataloged by grouping animals into the following age groups: <12 months, 13 to 24 months, and 25 to 36 months of age. Blood samples were collected once from each animal to measure total serum protein, haptoglobin, and neutralizing antibodies titers induced by respiratory virus vaccination. Total serum protein (g/dL) was lowest in PI calves younger until 24 months old, while haptoglobin concentration was higher in PI cattle. The serum neutralizing titers against BVDV and BRSV were lower in all PI calves and cattle than in controls. PI cattle have a high serum concentration of haptoglobin, and its possible dysregulated innate immune response appears to impact the efficacy of their adaptative immune responses, resulting in poor vaccine responsiveness.

O objetivo desta pesquisa foi avaliar alguns indicadores da resposta imune inata e humoral em bezerros a vacas persistentemente infectadas, entre um a 36 meses de idade, pareados com controles oriundos de um mesmo rebanho. As variáveis respostas foram avaliadas agrupando-se os animais nos seguintes grupos etários: < 12 meses, 13 a 24 meses, 25 a 36 meses de idade. Amostras sanguíneas foram coletadas para mensurar as concentrações séricas de proteína, haptoglobina e anticorpos neutralizantes induzidos pela vacinação contra as viroses respiratórias. Os teores de proteína sérica total (g/dL) foram menores nos animais persistentemente infectados (PI) jovens até 24 meses de idade, enquanto que a concentração de haptoglobina foi maior nos animais PI mais velhos (25 a 36 meses). Os títulos de anticorpos neutralizantes contra o BVDV e BRSV foi menor nos animais PIs independentemente da idade, comparado com o grupo controle. Os valores reduzidos ou nulos de anticorpos contra as viroses respiratórias, combinado com a evidência de resposta imune inata desregulada, contribui com a susceptibilidade dos animais PIs para as infecções secundárias.

Effects of neutrophil extracellular traps during human respiratory syncytial virus infection in vitro

Abstract The human respiratory syncytial virus (hRSV) is the most common cause of severe lower respiratory tract diseases in young children worldwide, leading to a high number of hospitalizations and significant expenditures for health systems. Neutrophils are massively recruited to the lung tissue of patients with acute respiratory diseases. At the infection site, they release neutrophil extracellular traps (NETs) that can capture and/or inactivate different types of microorganisms, including viruses. Evidence has shown that the accumulation of NETs results in direct cytotoxic effects on endothelial and epithelial cells. Neutrophils stimulated by the hRSV-F protein generate NETs that are able to capture hRSV particles, thus reducing their transmission. However, the massive production of NETs obstructs the airways and increases disease severity. Therefore, further knowledge about the effects of NETs during hRSV infections is essential for the development of new specific and effective treatments. This study evaluated the effects of NETs on the previous or posterior contact with hRSV-infected Hep-2 cells. Hep-2 cells were infected with different hRSV multiplicity of infection (MOI 0.5 or 1.0), either before or after incubation with NETs (0.516 g/mL). Infected and untreated cells showed decreased cellular viability and intense staining with trypan blue, which was accompanied by the formation of many large syncytia. Previous contact between NETs and cells did not result in a protective effect. Cells in monolayers showed a reduced number and area of syncytia, but cell death was similar in infected and non-treated cells. The addition of NETs to infected tissues maintained a similar virus-induced cell death rate and an increased syncytial area, indicating cytotoxic and deleterious damages. Our results corroborate previously reported findings that NETs contribute to the immunopathology developed by patients infected with hRSV.

Resumo O vírus sincicial respiratório humano (hRSV) é a causa mais comum de doenças graves do trato respiratório inferior em crianças pequenas em todo o mundo, resultando em grande número de hospitalizações e gastos significativos para os sistemas de saúde. Neutrófilos são recrutados em massa para o tecido pulmonar de pacientes com doenças respiratórias agudas. No local da infecção, eles liberam armadilhas extracelulares de neutrófilos (NETs) que podem capturar e/ou inativar diferentes tipos de microrganismos, incluindo vírus. Evidências demonstraram que o acúmulo de NETs resulta em efeitos citotóxicos diretos nas células endoteliais e epiteliais. Os neutrófilos estimulados pela proteína F do vírus sincicial respiratório (hRSV-F) geram NETs que são capazes de capturar partículas virais, reduzindo assim sua transmissão. No entanto, a produção maciça de NETs obstrui as vias aéreas e aumenta a gravidade da doença. Assim, um maior conhecimento sobre os efeitos das NETs durante as infecções por hRSV é essencial para o desenvolvimento de novos tratamentos específicos e eficazes. Este estudo avaliou os efeitos das NETs no contato prévio ou posterior à infecção de células Hep-2 com hRSV. As células Hep-2 foram infectadas com diferentes quantidades de hRSV (multiplicidade de infecção ou MOI 0,5 ou 1,0), antes ou após a incubação com NETs (0,516 g/mL). Células infectadas e não tratadas mostraram redução da viabilidade celular e intensa coloração com azul de tripano, que foi acompanhada pela formação de sincícios numerosos e grandes. O contato prévio entre as NETs e as células não resultou em efeito protetor. As células em monocamadas mostraram um número e área de sincícios reduzidos, mas a morte celular foi semelhante àquela apresentada por células infectadas e não tratadas. A adição de NETs aos tecidos infectados manteve taxa de morte celular e formação de sincícios semelhantes àqueles induzidos pelo vírus em células não tratadas, indicando danos citotóxicos e deletérios. Nossos resultados corroboram achados relatados anteriormente de que as NETs contribuem para a imunopatologia desenvolvida por pacientes infectados com hRSV.

Effects of neutrophil extracellular traps during human respiratory syncytial virus infection in vitro / Efeitos das redes extracelulares dos neutrófilos durante a infecção in vitro pelo vírus sincicial respiratório

The human respiratory syncytial virus (hRSV) is the most common cause of severe lower respiratory tract diseases in young children worldwide, leading to a high number of hospitalizations and significant expenditures for health systems. Neutrophils are massively recruited to the lung tissue of patients with acute respiratory diseases. At the infection site, they release neutrophil extracellular traps (NETs) that can capture and/or inactivate different types of microorganisms, including viruses. Evidence has shown that the accumulation of NETs results in direct cytotoxic effects on endothelial and epithelial cells. Neutrophils stimulated by the hRSV-F protein generate NETs that are able to capture hRSV particles, thus reducing their transmission. However, the massive production of NETs obstructs the airways and increases disease severity. Therefore, further knowledge about the effects of NETs during hRSV infections is essential for the development of new specific and effective treatments. This study evaluated the effects of NETs on the previous or posterior contact with hRSV-infected Hep-2 cells. Hep-2 cells were infected with different hRSV multiplicity of infection (MOI 0.5 or 1.0), either before or after incubation with NETs (0.5–16 μg/mL). Infected and untreated cells showed decreased cellular viability and intense staining with trypan blue, which was accompanied by the formation of many large syncytia. Previous contact between NETs and cells did not result in a protective effect. Cells in monolayers showed a reduced number and area of syncytia, but cell death was similar in infected and non-treated cells. The addition of NETs to infected tissues maintained a similar virus-induced cell death rate and an increased syncytial area, indicating cytotoxic and deleterious damages. Our results corroborate previously reported findings that NETs contribute to the immunopathology developed by patients infected with hRSV.

O vírus sincicial respiratório humano (hRSV) é a causa mais comum de doenças graves do trato respiratório inferior em crianças pequenas em todo o mundo, resultando em grande número de hospitalizações e gastos significativos para os sistemas de saúde. Neutrófilos são recrutados em massa para o tecido pulmonar de pacientes com doenças respiratórias agudas. No local da infecção, eles liberam armadilhas extracelulares de neutrófilos (NETs) que podem capturar e/ou inativar diferentes tipos de microrganismos, incluindo vírus. Evidências demonstraram que o acúmulo de NETs resulta em efeitos citotóxicos diretos nas células endoteliais e epiteliais. Os neutrófilos estimulados pela proteína F do vírus sincicial respiratório (hRSV-F) geram NETs que são capazes de capturar partículas virais, reduzindo assim sua transmissão. No entanto, a produção maciça de NETs obstrui as vias aéreas e aumenta a gravidade da doença. Assim, um maior conhecimento sobre os efeitos das NETs durante as infecções por hRSV é essencial para o desenvolvimento de novos tratamentos específicos e eficazes. Este estudo avaliou os efeitos das NETs no contato prévio ou posterior à infecção de células Hep-2 com hRSV. As células Hep-2 foram infectadas com diferentes quantidades de hRSV (multiplicidade de infecção ou MOI 0,5 ou 1,0), antes ou após a incubação com NETs (0,5–16 μg/mL). Células infectadas e não tratadas mostraram redução da viabilidade celular e intensa coloração com azul de tripano, que foi acompanhada pela formação de sincícios numerosos e grandes. O contato prévio entre as NETs e as células não resultou em efeito protetor. As células em monocamadas mostraram um número e área de sincícios reduzidos, mas a morte celular foi semelhante àquela apresentada por células infectadas e não tratadas. A adição de NETs aos tecidos infectados manteve taxa de morte celular e formação de sincícios [...].

Effects of neutrophil extracellular traps during human respiratory syncytial virus infection in vitro / Efeitos das redes extracelulares dos neutrófilos durante a infecção in vitro pelo vírus sincicial respiratório

The human respiratory syncytial virus (hRSV) is the most common cause of severe lower respiratory tract diseases in young children worldwide, leading to a high number of hospitalizations and significant expenditures for health systems. Neutrophils are massively recruited to the lung tissue of patients with acute respiratory diseases. At the infection site, they release neutrophil extracellular traps (NETs) that can capture and/or inactivate different types of microorganisms, including viruses. Evidence has shown that the accumulation of NETs results in direct cytotoxic effects on endothelial and epithelial cells. Neutrophils stimulated by the hRSV-F protein generate NETs that are able to capture hRSV particles, thus reducing their transmission. However, the massive production of NETs obstructs the airways and increases disease severity. Therefore, further knowledge about the effects of NETs during hRSV infections is essential for the development of new specific and effective treatments. This study evaluated the effects of NETs on the previous or posterior contact with hRSV-infected Hep-2 cells. Hep-2 cells were infected with different hRSV multiplicity of infection (MOI 0.5 or 1.0), either before or after incubation with NETs (0.5–16 μg/mL). Infected and untreated cells showed decreased cellular viability and intense staining with trypan blue, which was accompanied by the formation of many large syncytia. Previous contact between NETs and cells did not result in a protective effect. Cells in monolayers showed a reduced number and area of syncytia, but cell death was similar in infected and non-treated cells. The addition of NETs to infected tissues maintained a similar virus-induced cell death rate and an increased syncytial area, indicating cytotoxic and deleterious damages. Our results corroborate previously reported findings that NETs contribute to the immunopathology developed by patients infected with hRSV.(AU)

O vírus sincicial respiratório humano (hRSV) é a causa mais comum de doenças graves do trato respiratório inferior em crianças pequenas em todo o mundo, resultando em grande número de hospitalizações e gastos significativos para os sistemas de saúde. Neutrófilos são recrutados em massa para o tecido pulmonar de pacientes com doenças respiratórias agudas. No local da infecção, eles liberam armadilhas extracelulares de neutrófilos (NETs) que podem capturar e/ou inativar diferentes tipos de microrganismos, incluindo vírus. Evidências demonstraram que o acúmulo de NETs resulta em efeitos citotóxicos diretos nas células endoteliais e epiteliais. Os neutrófilos estimulados pela proteína F do vírus sincicial respiratório (hRSV-F) geram NETs que são capazes de capturar partículas virais, reduzindo assim sua transmissão. No entanto, a produção maciça de NETs obstrui as vias aéreas e aumenta a gravidade da doença. Assim, um maior conhecimento sobre os efeitos das NETs durante as infecções por hRSV é essencial para o desenvolvimento de novos tratamentos específicos e eficazes. Este estudo avaliou os efeitos das NETs no contato prévio ou posterior à infecção de células Hep-2 com hRSV. As células Hep-2 foram infectadas com diferentes quantidades de hRSV (multiplicidade de infecção ou MOI 0,5 ou 1,0), antes ou após a incubação com NETs (0,5–16 μg/mL). Células infectadas e não tratadas mostraram redução da viabilidade celular e intensa coloração com azul de tripano, que foi acompanhada pela formação de sincícios numerosos e grandes. O contato prévio entre as NETs e as células não resultou em efeito protetor. As células em monocamadas mostraram um número e área de sincícios reduzidos, mas a morte celular foi semelhante àquela apresentada por células infectadas e não tratadas. A adição de NETs aos tecidos infectados manteve taxa de morte celular e formação de sincícios [...].(AU)

Mansoa alliacea extract improves the growth performance and innate immune response of Arapaima gigas challenged with Aeromonas hydrophila and handling stress / Extrato de Mansoa alliacea melhora o desempenho de crescimento e resposta imunológica de Arapaima gigas desafiado com Aeromonas hydrophila e estresse de manejo

This study investigated the effects of dietary supplementation with Mansoa alliacea hydroalcoholic extracts on growth, blood and immune parameters of Arapaima gigas. Fish were fed for 30 days with diets enriched with 0, 4, 8, and 12 g kg-1 of M. alliacea hydroalcoholic extract and subjected to infection with Aeromonas hydrophila and handling stress. Fish fed with 8 g kg-1 of extract showed significant increase in final weight, specific growth rate and feed efficiency when compared to the other groups. Glucose, triglycerides, total proteins, and globulins increased significantly in fish fed with 8 g kg-1 of extract, whereas albumin decreased. The number of thrombocytes increased significantly with the dietary supplementation of 8 and 12 g kg-1 of extract. After the challenge with A. hydrophila and handling stress, fish fed with 8 g kg-1 of extract had significantly higher levels of glucose, globulins, and albumins, and fish fed with 8 and 12 g kg-1 of extract showed an increment of respiratory burst. Triglyceride levels dropped significantly in fish fed with 4, 8, and 12 g kg-1 of extract, whereas the number of neutrophils increased, and total thrombocytes, leukocytes and lymphocytes were higher in fish fed with 12 g kg-1 of extract. Dietary supplementation with M. alliacea extract at 8 g kg-1 was efficient in improving the growth and innate immunity of A. gigas, being potentially useful in fish farming to control the development of A. hydrophila infections.(AU)

Investigou-se os efeitos da suplementação com extrato hidroalcólico de Mansoa alliacea sobre o crescimento e parâmetros sanguíneos e imunológicos de Arapaima gigas. Os peixes foram alimentados por 30 dias com dietas enriquecidas com 0, 4, 8 e 12 g kg-1 de extrato hidroalcoólico de M. alliacea e submetidos à infecção por Aeromonas hydrophila e estresse de manejo. Os peixes alimentados com 8 g kg-1 de extrato apresentaram aumento significativo no peso final, taxa de crescimento específico e eficiência alimentar quando comparados aos demais grupos. Glicose, triglicerídeos, proteínas totais e globulinas aumentaram significativamente nos peixes alimentados com 8 g kg-1 de extrato, enquanto a albumina diminuiu. O número de trombócitos aumentou significativamente com a suplementação dietética de 8 e 12 g kg-1 de extrato. Após infecção com A. hydrophila e estresse de manejo, os peixes alimentados com 8 g kg-1 de extrato apresentaram níveis significativamente mais altos de glicose, globulinas e albuminas, e os peixes alimentados com 8 e 12 g kg-1 de extrato apresentaram incremento de explosão respiratória. Os níveis de triglicerídeos decresceram nos peixes alimentados com 4, 8 e 12 g kg-1 de extrato, enquanto o número de neutrófilos aumentou, e o número total de trombócitos, leucócitos e linfócitos foi maior nos peixes alimentados com 12 g kg-1 de extrato. A suplementação com a 8 g kg-1 de extrato de M. alliacea foi eficiente em melhorar o crescimento e a imunidade inata de A. gigas, sendo potencialmente útil na piscicultura para controlar o desenvolvimento de infecções por A. hydrophila.(AU)

Effects of neutrophil extracellular traps during human respiratory syncytial virus infection in vitro / Efeitos das redes extracelulares dos neutrófilos durante a infecção in vitro pelo vírus sincicial respiratório

Abstract The human respiratory syncytial virus (hRSV) is the most common cause of severe lower respiratory tract diseases in young children worldwide, leading to a high number of hospitalizations and significant expenditures for health systems. Neutrophils are massively recruited to the lung tissue of patients with acute respiratory diseases. At the infection site, they release neutrophil extracellular traps (NETs) that can capture and/or inactivate different types of microorganisms, including viruses. Evidence has shown that the accumulation of NETs results in direct cytotoxic effects on endothelial and epithelial cells. Neutrophils stimulated by the hRSV-F protein generate NETs that are able to capture hRSV particles, thus reducing their transmission. However, the massive production of NETs obstructs the airways and increases disease severity. Therefore, further knowledge about the effects of NETs during hRSV infections is essential for the development of new specific and effective treatments. This study evaluated the effects of NETs on the previous or posterior contact with hRSV-infected Hep-2 cells. Hep-2 cells were infected with different hRSV multiplicity of infection (MOI 0.5 or 1.0), either before or after incubation with NETs (0.5-16 μg/mL). Infected and untreated cells showed decreased cellular viability and intense staining with trypan blue, which was accompanied by the formation of many large syncytia. Previous contact between NETs and cells did not result in a protective effect. Cells in monolayers showed a reduced number and area of syncytia, but cell death was similar in infected and non-treated cells. The addition of NETs to infected tissues maintained a similar virus-induced cell death rate and an increased syncytial area, indicating cytotoxic and deleterious damages. Our results corroborate previously reported findings that NETs contribute to the immunopathology developed by patients infected with hRSV.

Resumo O vírus sincicial respiratório humano (hRSV) é a causa mais comum de doenças graves do trato respiratório inferior em crianças pequenas em todo o mundo, resultando em grande número de hospitalizações e gastos significativos para os sistemas de saúde. Neutrófilos são recrutados em massa para o tecido pulmonar de pacientes com doenças respiratórias agudas. No local da infecção, eles liberam armadilhas extracelulares de neutrófilos (NETs) que podem capturar e/ou inativar diferentes tipos de microrganismos, incluindo vírus. Evidências demonstraram que o acúmulo de NETs resulta em efeitos citotóxicos diretos nas células endoteliais e epiteliais. Os neutrófilos estimulados pela proteína F do vírus sincicial respiratório (hRSV-F) geram NETs que são capazes de capturar partículas virais, reduzindo assim sua transmissão. No entanto, a produção maciça de NETs obstrui as vias aéreas e aumenta a gravidade da doença. Assim, um maior conhecimento sobre os efeitos das NETs durante as infecções por hRSV é essencial para o desenvolvimento de novos tratamentos específicos e eficazes. Este estudo avaliou os efeitos das NETs no contato prévio ou posterior à infecção de células Hep-2 com hRSV. As células Hep-2 foram infectadas com diferentes quantidades de hRSV (multiplicidade de infecção ou MOI 0,5 ou 1,0), antes ou após a incubação com NETs (0,5-16 μg/mL). Células infectadas e não tratadas mostraram redução da viabilidade celular e intensa coloração com azul de tripano, que foi acompanhada pela formação de sincícios numerosos e grandes. O contato prévio entre as NETs e as células não resultou em efeito protetor. As células em monocamadas mostraram um número e área de sincícios reduzidos, mas a morte celular foi semelhante àquela apresentada por células infectadas e não tratadas. A adição de NETs aos tecidos infectados manteve taxa de morte celular e formação de sincícios semelhantes àqueles induzidos pelo vírus em células não tratadas, indicando danos citotóxicos e deletérios. Nossos resultados corroboram achados relatados anteriormente de que as NETs contribuem para a imunopatologia desenvolvida por pacientes infectados com hRSV.

Epidemiological aspects of cattle tick fever in Brazil / Aspectos epidemiológicos da tristeza parasitária bovina no Brasil

The present work reviews the epidemiologic situation of Anaplasma marginale and Babesia spp. infections and the occurrence of cattle tick fever outbreaks in Brazil. In areas of tick fever enzootic instability, environmental conditions interfere with the development of Rhipicephalus (Boophilus) microplus: chilly winter in the southern region, floods in the Pantanal, and low humidity in the Caatinga. In contrast, the climatic conditions of stable zones (Cerrado, Amazon and Atlantic Forest biomes) favor tick development. In enzootic areas, tick fever is uncommon because the animals are in frequent contact with the parasite, acquiring immunity naturally during the period of innate resistance; however, outbreaks may occur when calves become infested by considerable numbers of infected ticks during this period or in adults raised in tick-free environments that become infested for the first time when transporting to stable areas. It is necessary to better understand the disease's risk factors under stable conditions and the implications of the mechanical and other vector transmission of A. marginale. To prevent tick fever outbreaks in Brazil, it is important to develop and use anaplasmosis and babesiosis vaccines in cattle from enzootic unstable regions, especially when animals are moved to stable areas.(AU)

O presente trabalho revisa a situação epidemiológica das infecções por Anaplasma marginale e Babesia spp. e a ocorrência de surtos de Tristeza parasitária bovina (TPB) no Brasil. Em áreas de instabilidade enzoótica, as condições ambientais interferem no desenvolvimento do Rhipicephalus (Boophilus) microplus: o frio do inverno na região Sul; as inundações no Pantanal; e a baixa umidade na Caatinga. Por outra parte, as condições climáticas das zonas de estabilidade (os biomas Cerrado, Amazônia e Mata Atlântica) favorecem o desenvolvimento do carrapato. A TPB não é comum nas áreas estáveis, porque os animais estão em contato frequente com os parasitas, adquirindo imunidade naturalmente. Podem, porém, ocorrer surtos quando um número considerável de carrapatos infectados infesta bezerros, durante o período de resistência inata, ou quando adultos que foram criados em ambientes livres de carrapatos infestam-se, pela primeira vez, ao serem transportados para áreas estáveis. É necessário entender melhor os fatores de risco da doença em condições de estabilidade e a implicação da transmissão de A. marginale de forma mecânica, ou por meio de vetores diferentes do carrapato. Para prevenir surtos de TPB, no Brasil, é necessário desenvolver e usar vacinas em bovinos de regiões de instabilidade, especialmente antes de transportá-los para áreas estáveis.(AU)

Innate immunity gene expression profiles in conjunctival membrane biopsies from Amazonian buffalo / Perfis de expressão de genes da imunidade inata em biopsias de membrana conjuntival de búfalos da Amazônia

Domestic buffalo production plays an economically important role in the Brazilian Amazon, but they are susceptible to many diseases favored by the tropical climate and annually flooded habitats, including ocular diseases. In this context, it is important to select genotypes that maximize innate ocular immunity in Amazonian herds. We aimed to characterise, for the first time, gene expression profiles of the innate immune system in the conjunctival membrane of buffalo. Ocular conjunctival tissue samples were collected from 60 clinically healthy slaughtered animals in the northern Brazilian state of Amapá. The samples were histologically processed for classification into three groups according to the quantitative degree of lymphoid tissue associated with the conjunctiva (discrete, G1; slight, G2; and moderate, G3 presence of lymphoid tissue). RT-PCR was used to quantify gene expression of inflammatory cytokine (IL6, IL10, TNFA, IFNG), Toll-like receptor 4 (TLR4), and Defensin beta 110 (DEFB110), relative to the endogenous GAPDH gene. G1 animals presented low expression for IL6, IL10, TNFA, and DEFB110, while G2 exhibited high expression for IL6, IL10, IFNG, and TLR4. All G3 animals showed high expression for all tested genes. These results suggest a greater resistance to pathogenic microorganisms of buffalos in the G3 group, and the proportion of lymphoid tissue associated with the conjunctiva may be related to the immune resistance of individuals.(AU)

A produção de búfalos domésticos desempenha um papel economicamente importante na Amazônia brasileira, mas eles são suscetíveis a muitas doenças favorecidas pelo clima tropical e habitats inundados anualmente, incluindo doenças oculares. Nesse contexto, é importante selecionar genótipos que maximizem a imunidade ocular inata em rebanhos amazônicos. Objetivamos caracterizar, pela primeira vez, perfis de expressão gênica do sistema imune inato na membrana conjuntival de búfalos. Amostras de tecido conjuntival ocular foram coletadas de 60 animais clinicamente saudáveis abatidos no estado do Amapá, norte do Brasil. As amostras foram processadas histologicamente para classificação em três grupos de acordo com o grau quantitativo de tecido linfoide associado à conjuntiva (discreta, G1; leve, G2; e moderada, G3 presença de tecido linfoide). RT-PCR foi utilizado para quantificar a expressão gênica de citocinas inflamatórias (IL6, IL10, TNFA, IFNG), receptor Toll-like 4 (TLR4) e Defensina beta 110 (DEFB110), em relação ao gene GAPDH endógeno. Os animais do G1 apresentaram baixa expressão para IL6, IL10, TNFA e DEFB110, enquanto G2 exibiu alta expressão para IL6, IL10, IFNG e TLR4. Todos os animais do G3 apresentaram alta expressão para todos os genes testados. Esses resultados sugerem maior resistência aos microrganismos patogênicos dos búfalos do grupo G3, e a proporção de tecido linfoide associado à conjuntiva pode estar relacionada à resistência imunológica dos indivíduos.(AU)

Effects of broiler breeder age on immune system development of progeny

The objective of this study was to determine the effects that breeder age has on digestive and immune system development; the transfer of immunoglobulins to egg yolk, yolk sac, and neonate chicks; and the immune response of chicks up to 35 days old. Three ages (32, 42, and 52 weeks) of Hubbard breeders were studied with ages as treatments. A total of 425 eggs were weighed for each of the three treatments and incubated. After hatching, a total of 300 1-day-old chicks were used in each treatment. We studied the development of the gastrointestinal tract and immune system of progeny and IgY transfer from breeder to progeny. Chicks from 52-week-old breeders had greater gastrointestinal tract growth up to seven days of life and greater body weight at 14 days. Older breeders (52 weeks) had higher amounts of IgY in serum and egg yolk. Chicks from the youngest breeders (32-weeks-old) had a better immune response at two weeks post-vaccination. It can be concluded that the older breeders have a greater capacity to immunize progeny up to 14 days. Strategies can be developed to increase IgY in the serum of young breeders and, consequently, increase the innate immunity of the newly-hatched chicks.(AU)

Vector saliva controlled inflammatory response of the host may represent the Achilles heel during pathogen transmission

Infection with vector-borne pathogens starts with the inoculation of these pathogens during blood feeding. In endemic regions, the population is regularly bitten by naive vectors, implicating a permanent stimulation of the immune system by the vector saliva itself (pre-immune context). Comparatively, the number of bites received by exposed individuals from non-infected vectors is much higher than the bites from infected ones. Therefore, vector saliva and the immunological response in the skin may play an important role, so far underestimated, in the establishment of anti-pathogen immunity in endemic areas. Hence, the parasite biology and the disease pathogenesis in "saliva-primed" and "saliva-unprimed" individuals must be different. This integrated view on how the pathogen evolves within the host together with vector salivary components, which are known to be endowed with a variety of pharmacological and immunological properties, must remain the focus of any investigational study dealing with vector-borne diseases. Considering this three-way partnership, the host skin (immune system), the pathogen, and the vector saliva, the approach that consists in the validation of vector saliva as a source of molecular entities with anti-disease vaccine potential has been recently a subject of active and fruitful investigation. As an example, the vaccination with maxadilan, a potent vasodilator peptide extracted from the saliva of the sand fly Lutzomyia longipalpis, was able to protect against infection with various leishmanial parasites. More interestingly, a universal mosquito saliva vaccine that may potentially protect against a range of mosquito-borne infections including malaria, dengue, Zika, chikungunya and yellow fever. In this review, we highlight the key role played by the immunobiology of vector saliva in shaping the outcome of vector-borne diseases and discuss the value of studying diseases in the light of intimate cross talk among the pathogen, the vector saliva, and the host immune mechanisms.(AU)

Vector saliva controlled inflammatory response of the host may represent the Achilles heel during pathogen transmission

Infection with vector-borne pathogens starts with the inoculation of these pathogens during blood feeding. In endemic regions, the population is regularly bitten by naive vectors, implicating a permanent stimulation of the immune system by the vector saliva itself (pre-immune context). Comparatively, the number of bites received by exposed individuals from non-infected vectors is much higher than the bites from infected ones. Therefore, vector saliva and the immunological response in the skin may play an important role, so far underestimated, in the establishment of anti-pathogen immunity in endemic areas. Hence, the parasite biology and the disease pathogenesis in "saliva-primed" and "saliva-unprimed" individuals must be different. This integrated view on how the pathogen evolves within the host together with vector salivary components, which are known to be endowed with a variety of pharmacological and immunological properties, must remain the focus of any investigational study dealing with vector-borne diseases. Considering this three-way partnership, the host skin (immune system), the pathogen, and the vector saliva, the approach that consists in the validation of vector saliva as a source of molecular entities with anti-disease vaccine potential has been recently a subject of active and fruitful investigation. As an example, the vaccination with maxadilan, a potent vasodilator peptide extracted from the saliva of the sand fly Lutzomyia longipalpis, was able to protect against infection with various leishmanial parasites. More interestingly, a universal mosquito saliva vaccine that may potentially protect against a range of mosquito-borne infections including malaria, dengue, Zika, chikungunya and yellow fever. In this review, we highlight the key role played by the immunobiology of vector saliva in shaping the outcome of vector-borne diseases and discuss the value of studying diseases in the light of intimate cross talk among the pathogen, the vector saliva, and the host immune mechanisms.(AU)

Lysozyme activity as an indicator of innate immunity of tilapia (Oreochromis niloticus) when challenged with LPS and Streptococcus agalactiae

The objective of this study was to fully describe the protocol with standardized modifications and evaluate the lysozyme activity, an indicator of innate immunity in tilapia, to compare lipopolysaccharide (LPS) with Streptococcus agalactiae injections. Lysozyme was determined in serum using the turbidimetric method, in which lysozyme activity was evaluated by Micrococcus lysodeikticus lysis, with modifications for microplate assay. The experiment was conducted in a completely randomized design. Juvenile tilapia was divided in the following six treatments: challenged with phosphate buffer PBS (control) and 100, 250, 500, and 600 µg kg−1 LPS and S. agalactiae. All treatments were challenged for 72 h and seven days and then sampled to determine lysozyme activity. After 72 h or seven days, concentrations of LPS promoted changes in lysozyme production, either lesser or equal, depending on concentration when compared with fish injected with S. agalactiae. It was possible to standardize the analysis and determine that the treatment with LPS promotes immunomodulation at a concentration of 250 µg kg−1 LPS, this response being similar to challenge with S. agalactiae.

Innate immunity in chicken lines developed by EMBRAPA Suínos e Aves: antimicrobial activity of macrophages and serum

The objective of this study was to compare the antimicrobial activity of macrophages and serum in laying hen (MM, CC, and CCc) and broiler chicken lineages (TT and LL). Macrophages were evaluated for phagocytic and antimicrobial activity. Microbicidal serum activity was evaluated by the resistance test for serum and the agar test. The results showed that phagocytic activity was higher in males of the MM strain, with 13% of macrophages presenting phagocytosis, while the other lineages studied, and even female MM, presented a rate of 6% of phagocytic cells. However, antimicrobial activity in macrophages from males of CCc lineage and females of TT lineage were higher, eliminating more than 30% of the Salmonella enterica inoculum, while in the other strains, the results were similar, with inoculum reduction below 30%. In the serum resistance assay, female laying lines presented higher antibacterial activity than female broiler lines. In the trials to evaluate the microbicide activity of the serum, females of both broiler and laying lineages presented higher performance when compared with males of the same lineage. Females of laying hen lines (MM and CC) present a greater antibacterium activity than males. These results can contribute to a better understanding of the immune response in broiler chicken and laying hen lineages, to aid development of lineages of birds more resistant to pathogens.

Ocimum gratissimum essential oil improved the health, innate immunity and resistance to Aeromonas hydrophila infection in Pseudoplatystoma reticulatum / Óleo essencial de Ocimum gratissinum melhorou a saúde, imunidade inata e resistência à infecção por Aeromonas hydrophila em Pseudoplatystoma reticulatum

The essential oil of alfavaca (Ocimum gratissimum) contains important compounds, such as 1.8-cineol and eugenol. This study evaluated the effects of diets containing alfavaca essential oil on the zootechnical performance, plasma glucose, leukocyte respiratory activity, haematology, and intestinal histomorphometry in "cachara" (Pseudoplatystoma reticulatum) challenged with Aeromonas hydrophila. A total of 300 juvenile fish, with an average total length of 14.98 ± 0.28 cm and average weight of 18.84 ± 1.22 g, distributed in 20 tanks of 80 L, were fed twice a day with a diet containing essential oil at 0.5, 1.0, or 1.5% or without its inclusion in five replicates. After 45 days, blood collection and mid-intestinal bowel were sampled, before a challenge with Aeromonas hydrophila was performed. The fish supplemented with 1.0% of essential oil presented better weight gain, mean corpuscular volume, concentration of thrombotic and neutrophils, respiratory activity of leukocytes, and height of villi (p < 0.05) when compared to other groups. The cumulative mortality response was lower for fish fed 1.5% essential oil of alfavaca diets. Thus, the use of alfavaca essential oil is recommended in the diet of cachara catfish (P. reticulatum) at the level of up to 1.0% for 45 days for improvements in the zootechnical, haematological, and intestinal histomorphometric parameters.(AU)

O óleo essencial de alfavaca (Ocimum gratissimum) contém componentes importantes, como por exemplo o 1.8-cineol e o eugenol. Este estudo avaliou os efeitos de dietas contendo óleo essencial de alfavaca sobre o desempenho zootécnico, glicose plasmática, atividade respiratória leucocitária, hematologia e histomorfometria intestinal em cachara (Pseudoplatystoma reticulatum) desafiado com Aeromonas hydrophila. Um total de 300 peixes juvenis, com comprimento total médio de 14,98 ± 0,28 cm e peso médio de 18,84 ± 1,22 g, distribuídos em 20 tanques de 80 L, alimentados duas vezes ao dia com dieta contendo o óleo essencial a 0,5, 1,0 ou 1,5 % ou sem inclusão de óleo em cinco repetições. Após 45 dias, foi realizada a biometria final, coleta de sangue, amostra do intestino médio e desafio com Aeromonas hydrophila. Após o período experimental, a dieta suplementada com 1,0% do óleo essencial apresentou melhor ganho de peso, volume corpuscular médio, concentração de trombóticos e neutrófilos, atividade respiratória de leucócitos e altura de vilosidades (p < 0,05) quando comparada aos demais grupos. A resposta de mortalidade cumulativa foi menor para peixes alimentados com dietas com o óleo essencial de alfavaca a 1,5%. Assim, o uso de óleo essencial de alfavaca é recomendado na dieta do bagre cachara (P. reticulatum) no nível de até 1,0% por 45 dias para melhorias nos parâmetros zootécnicos, hematológicos e histomorfométricos intestinais.(AU)

Effects of venoms on neutrophil respiratory burst: a major inflammatory function

Neutrophils play a pivotal role in innate immunity and in the inflammatory response. Neutrophils are very motile cells that are rapidly recruited to the inflammatory site as the body first line of defense. Their bactericidal activity is due to the release into the phagocytic vacuole, called phagosome, of several toxic molecules directed against microbes. Neutrophil stimulation induces release of this arsenal into the phagosome and induces the assembly at the membrane of subunits of the NAPDH oxidase, the enzyme responsible for the production of superoxide anion that gives rise to other reactive oxygen species (ROS), a process called respiratory burst. Altogether, they are responsible for the bactericidal activity of the neutrophils. Excessive activation of neutrophils can lead to extensive release of these toxic agents, inducing tissue injury and the inflammatory reaction. Envenomation, caused by different animal species (bees, wasps, scorpions, snakes etc.), is well known to induce a local and acute inflammatory reaction, characterized by recruitment and activation of leukocytes and the release of several inflammatory mediators, including prostaglandins and cytokines. Venoms contain several molecules such as enzymes (phospholipase A2, L-amino acid oxidase and proteases, among others) and peptides (disintegrins, mastoporan, parabutoporin etc.). These molecules are able to stimulate or inhibit ROS production by neutrophils. The present review article gives a general overview of the main neutrophil functions focusing on ROS production and summarizes how venoms and venom molecules can affect this function.(AU)

Molecular identification, characterization, and expression analysis of a serine protease inhibitor gene from cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae) / Identificação molecular, caracterização e análise de expressão de um gene inibidor de serina protease da lagarta Helicoverpa armigera (Lepidoptera: noctuidae) na cultura de algodão

Serine protease inhibitors (serpins), a superfamily of protease inhibitors, are known to be involved in several physiological processes, such as development, metamorphosis, and innate immunity. In our study, a full-length serpin cDNA, designated Haserpin1, was isolated from the cotton bollworm Helicoverpa armigera. The cDNA sequence of Haserpin1 is 1176 nt long, with an open reading frame encoding 391 amino acids; there is one exon and no intron. The predicted molecular weight of Haserpin1 is 43.53 kDa, with an isoelectric point of 4.98. InterProScan was employed for Haserpin1 functional characterization, which revealed that Haserpin1 contains highly conserved signature motifs, including a reactive center loop (RCL) with a hinge region (E341N350), the serpin signature, (F367F375) and a predicted P1P1 cleavage site (L357S358), which are useful for identifying serpins. Transcripts of Haserpin1 were constitutively expressed in the fat body, suggesting that it is the major site for serpin synthesis. During the developmental stages, a fluctuation in the expression level of Haserpin1 was observed, with low expression detected at the 5th-instar larval stage. In contrast, relatively high expression was detected at the prepupal stage, suggesting that Haserpin1 might play a critical role at the H. armigera wandering stage. Although the detailed function of this serpin (Haserpin1) needs to be elucidated, our study provides a perspective for the functional investigation of serine protease inhibitor genes.(AU)

Sabe-se que os inibidores de serina protease (serpinas), uma superfamília de inibidores de protease, estão envolvidos em vários processos fisiológicos, como desenvolvimento, metamorfose e imunidade inata. Neste estudo, um cDNA de serpina de comprimento total, denominado Haserpin1, foi isolado da lagarta Helicoverpa armigera na cultura de algodão. A sequência de ADNc de Haserpin1 tem 1.176 nt de comprimento, com uma grelha de leitura aberta que codifica 391 aminoácidos; existe um éxon, mas nenhum íntron. O peso molecular previsto de Haserpin1 é de 43,53 kDa, com um ponto isoelétrico de 4,98. O InterProScan foi empregado para a caracterização funcional do Haserpin1, que revelou que o Haserpin1 contém motivos de assinatura altamente conservados, incluindo um loop central reativo (RCL) com uma região de dobradiça (E341-N350), a assinatura da serpina (F367-F375) e um local de clivagem previsto de P1-P1' (L357-S358), que são úteis para identificar serpinas. As transcrições de Haserpin1 foram expressas constitutivamente no corpo gordo, sugerindo que é o principal local para a síntese de serpinas. Durante os estágios de desenvolvimento, observou-se uma flutuação no nível de expressão de Haserpin1, com baixa expressão detectada no estágio larval do 5º ínstar. Por outro lado, detectou-se uma expressão relativamente alta no estágio pré-pupal, sugerindo que o Haserpin1 pode desempenhar um papel crítico no estágio errante de H. armigera. Embora a função detalhada dessa serpina (Haserpin1) precise ser elucidada, este estudo fornece uma perspectiva para a investigação funcional dos genes inibidores da serina protease.(AU)

Effects of venoms on neutrophil respiratory burst: a major inflammatory function

Neutrophils play a pivotal role in innate immunity and in the inflammatory response. Neutrophils are very motile cells that are rapidly recruited to the inflammatory site as the body first line of defense. Their bactericidal activity is due to the release into the phagocytic vacuole, called phagosome, of several toxic molecules directed against microbes. Neutrophil stimulation induces release of this arsenal into the phagosome and induces the assembly at the membrane of subunits of the NAPDH oxidase, the enzyme responsible for the production of superoxide anion that gives rise to other reactive oxygen species (ROS), a process called respiratory burst. Altogether, they are responsible for the bactericidal activity of the neutrophils. Excessive activation of neutrophils can lead to extensive release of these toxic agents, inducing tissue injury and the inflammatory reaction. Envenomation, caused by different animal species (bees, wasps, scorpions, snakes etc.), is well known to induce a local and acute inflammatory reaction, characterized by recruitment and activation of leukocytes and the release of several inflammatory mediators, including prostaglandins and cytokines. Venoms contain several molecules such as enzymes (phospholipase A2, L-amino acid oxidase and proteases, among others) and peptides (disintegrins, mastoporan, parabutoporin etc.). These molecules are able to stimulate or inhibit ROS production by neutrophils. The present review article gives a general overview of the main neutrophil functions focusing on ROS production and summarizes how venoms and venom molecules can affect this function.(AU)

Leukocytes and albumin in canine leishmaniasis

Canine Leishmaniasis (CanL) is a multisystemic and chronic inflammatory disease characterized by nonspecific clinical manifestations. In CanL, inflammatory cells and chemical mediators released in response to the parasite play a role in disease development and progression. Alterations on hematological parameters have been documented in CanL. These changes can also be assessed in relation to systemic inflammation caused by this disease. The circulating leukocyte counting, such as neutrophils, as well as the albumin level, are considered direct indicators of an inflammatory host environment. Several studies point to the use of biomarkers on the assistance in diagnosis and prognosis of several canine pathologies. The present study investigated the Neutrophils to Lymphocyte Ratio (NLR), Albumin to Globulin Ratio (AGR), and Neutrophils to Albumin Ratio (NAR) on systemic inflammatory response induced by Canine Leishmaniasis (CanL). For this purpose, adult dogs with confirmed diagnosis to CanL were divided into symptomatic (SD, n = 33) and asymptomatic (AD, n = 20) dogs for L. infantum and control dogs (CD, n = 20). Routine hematological and biochemical parameters were determined in blood samples using a veterinary automatic hematology and biochemical analyzers. Asymptomatic dogs (AD) had a higher number of white blood cells and neutrophils (16.48 ± 4.93; 13.41 ± 3.60, respectively) in relation to symptomatic dogs (SD) (13.54 ± 5.13; 10.42 ± 3.69, respectively) (P = 0.015 and P < 0.0001, respectively). Neutrophils to Lymphocyte Ratio (NLR) was higher in dogs with leishmaniasis (9.45 ± 3.76) than in healthy dogs (3.39 ± 1.19) (P < 0.0001). Serum total proteins (STP) and globulins increased in CanL, while albumin and AGR decreased in CanL, when compared to CD and references values to canine species. Neutrophils to Albumin Ratio (NAR) was higher in AD and SD (5.02 ± 1.14; 4.79 ± 1.07, respectively) when compared to CD (2.36 ± 0.55) (P < 0.0001). As reported in scientific researches, dogs with Leishmaniasis present alterations in circulating cell counts. Based on these data, we decided to expand this information using the NLR as a parameter in an attempt to better clarify the changes in these cells in CanL. We observed that NLR was increased on CanL in relation to healthy dogs, which could be a consequence of relative neutrophilia rather than lymphopenia. Neutrophils to Lymphocyte Ratio (NLR) is a biomarker that conveys information about inflammatory conditions. An elevated NLR can reflect an upregulated innate immune response, since neutrophils are effector cells of innate immunity and are involved in several acute and chronic inflammatory processes. Albumin is an acute phase protein that is considered an immune-inflammatory biomarker, which can be found reduced systemically in progressive inflammatory response. Serum total proteins (STP) and globulins were increased in CanL. These data are already well documented in CanL, which serum globulins are mainly associated with the increase of acute phase proteins, cytokines, and increase of specific antibodies to Leishmania infantum. Our results showed neutrophilia with hypoalbuminemia in CanL. So, in an attempt to assess the relationship of these two available markers, we used NAR calculation in order to evaluate the changes induced by CanL. In this study NAR was higher in CanL when compared to control dogs. Thus, our data indicate that NLR and NAR could be used as biomarkers in veterinary medical clinics in order to assess inflammatory profile in CanL, mainly in asymptomatic dogs. These parameters obtained from routine blood tests might be useful as cost-effective, easily accessible and helpful markers in order to distinguish the inflammatory response intensity in CanL.(AU)

Mini review: immunologic functions of dual oxidases in mucosal systems of vertebrates / Minirrevisão: funções imunológicas de oxidases duplas em sistemas mucosos de vertebrados

Mucosal epithelial cells act as the first immunologic barrier of organisms, and contact directly with pathogens. Therefore, hosts must have differential strategies to combat pathogens efficiently. Reactive oxygen species (ROS), as a kind of oxidizing agents, participates in the early stage of killing pathogens quickly. Recent reports have revealed that dual oxidase (DUOX) plays a key role in mucosal immunity. And the DUOX is a transmembrane protein which produces ROS as their primary enzymatic products. This process is an important pattern for eliminating pathogens. In this review, we highlight the DUOX immunologic functions in the respiratory and digestive tract of vertebrates.(AU)

As células epiteliais da mucosa atuam como a primeira barreira imunológica dos organismos e entram em contato direto com os patógenos. Portanto, os hospedeiros devem ter estratégias diferenciadas para combater os patógenos de forma eficiente. Trabalhos recentes revelaram que a oxidase dupla (DUOX) desempenha um papel fundamental para a imunidade da mucosa. A DUOX é uma proteína transmembrana geradora de espécies reativas de oxigênio (EROs) como seus principais produtos enzimáticos. Nesta revisão, apresentaremos as funções imunológicas da DUOX no trato respiratório e digestivo dos vertebrados.(AU)

Molecular identification, characterization, and expression analysis of a serine protease inhibitor gene from cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae)

Abstract Serine protease inhibitors (serpins), a superfamily of protease inhibitors, are known to be involved in several physiological processes, such as development, metamorphosis, and innate immunity. In our study, a full-length serpin cDNA, designated Haserpin1, was isolated from the cotton bollworm Helicoverpa armigera. The cDNA sequence of Haserpin1 is 1176 nt long, with an open reading frame encoding 391 amino acids; there is one exon and no intron. The predicted molecular weight of Haserpin1 is 43.53 kDa, with an isoelectric point of 4.98. InterProScan was employed for Haserpin1 functional characterization, which revealed that Haserpin1 contains highly conserved signature motifs, including a reactive center loop (RCL) with a hinge region (E341N350), the serpin signature, (F367F375) and a predicted P1P1 cleavage site (L357S358), which are useful for identifying serpins. Transcripts of Haserpin1 were constitutively expressed in the fat body, suggesting that it is the major site for serpin synthesis. During the developmental stages, a fluctuation in the expression level of Haserpin1 was observed, with low expression detected at the 5th-instar larval stage. In contrast, relatively high expression was detected at the prepupal stage, suggesting that Haserpin1 might play a critical role at the H. armigera wandering stage. Although the detailed function of this serpin (Haserpin1) needs to be elucidated, our study provides a perspective for the functional investigation of serine protease inhibitor genes.

Resumo Sabe-se que os inibidores de serina protease (serpinas), uma superfamília de inibidores de protease, estão envolvidos em vários processos fisiológicos, como desenvolvimento, metamorfose e imunidade inata. Neste estudo, um cDNA de serpina de comprimento total, denominado Haserpin1, foi isolado da lagarta Helicoverpa armigera na cultura de algodão. A sequência de ADNc de Haserpin1 tem 1.176 nt de comprimento, com uma grelha de leitura aberta que codifica 391 aminoácidos; existe um éxon, mas nenhum íntron. O peso molecular previsto de Haserpin1 é de 43,53 kDa, com um ponto isoelétrico de 4,98. O InterProScan foi empregado para a caracterização funcional do Haserpin1, que revelou que o Haserpin1 contém motivos de assinatura altamente conservados, incluindo um loop central reativo (RCL) com uma região de dobradiça (E341-N350), a assinatura da serpina (F367-F375) e um local de clivagem previsto de P1-P1' (L357-S358), que são úteis para identificar serpinas. As transcrições de Haserpin1 foram expressas constitutivamente no corpo gordo, sugerindo que é o principal local para a síntese de serpinas. Durante os estágios de desenvolvimento, observou-se uma flutuação no nível de expressão de Haserpin1, com baixa expressão detectada no estágio larval do 5º ínstar. Por outro lado, detectou-se uma expressão relativamente alta no estágio pré-pupal, sugerindo que o Haserpin1 pode desempenhar um papel crítico no estágio errante de H. armigera. Embora a função detalhada dessa serpina (Haserpin1) precise ser elucidada, este estudo fornece uma perspectiva para a investigação funcional dos genes inibidores da serina protease.