RESUMO
Introduction: Cryptosporidiosis is a poorly controlled zoonosis caused by an intestinal parasite, Cryptosporidium parvum, with a high prevalence in livestock (cattle, sheep, and goats). Young animals are particularly susceptible to this infection due to the immaturity of their intestinal immune system. In a neonatal mouse model, we previously demonstrated the importance of the innate immunity and particularly of type 1 conventional dendritic cells (cDC1) among mononuclear phagocytes (MPs) in controlling the acute phase of C. parvum infection. These immune populations are well described in mice and humans, but their fine characterization in the intestine of young ruminants remained to be further explored. Methods: Immune cells of the small intestinal Peyer's patches and of the distal jejunum were isolated from naive lambs and calves at different ages. This was followed by their fine characterization by flow cytometry and transcriptomic analyses (q-RT-PCR and single cell RNAseq (lamb cells)). Newborn animals were infected with C. parvum, clinical signs and parasite burden were quantified, and isolated MP cells were characterized by flow cytometry in comparison with age matched control animals. Results: Here, we identified one population of macrophages and three subsets of cDC (cDC1, cDC2, and a minor cDC subset with migratory properties) in the intestine of lamb and calf by phenotypic and targeted gene expression analyses. Unsupervised single-cell transcriptomic analysis confirmed the identification of these four intestinal MP subpopulations in lamb, while highlighting a deeper diversity of cell subsets among monocytic and dendritic cells. We demonstrated a weak proportion of cDC1 in the intestine of highly susceptible newborn lambs together with an increase of these cells within the first days of life and in response to the infection. Discussion: Considering cDC1 importance for efficient parasite control in the mouse model, one may speculate that the cDC1/cDC2 ratio plays also a key role for the efficient control of C. parvum in young ruminants. In this study, we established the first fine characterization of intestinal MP subsets in young lambs and calves providing new insights for comparative immunology of the intestinal MP system across species and for future investigations on host-Cryptosporidium interactions in target species.
Assuntos
Criptosporidiose , Cryptosporidium parvum , Homeostase , Animais , Criptosporidiose/imunologia , Criptosporidiose/parasitologia , Cryptosporidium parvum/imunologia , Ovinos , Bovinos , Homeostase/imunologia , Células Dendríticas/imunologia , Células Dendríticas/parasitologia , Fagócitos/imunologia , Fagócitos/parasitologia , Animais Recém-Nascidos , Doenças dos Ovinos/parasitologia , Doenças dos Ovinos/imunologia , Nódulos Linfáticos Agregados/imunologia , Nódulos Linfáticos Agregados/parasitologia , Macrófagos/imunologia , Macrófagos/parasitologia , Intestinos/parasitologia , Intestinos/imunologia , Ruminantes/parasitologia , Ruminantes/imunologiaRESUMO
Influenza D virus (IDV) was first isolated in 2011 in Oklahoma, USA from pigs presenting with influenza-like symptoms. IDV is known to mainly circulate in ruminants, especially cattle. In Africa, there is limited information on the epidemiology of IDV, although the virus has likely circulated in the region since 2012. In the present study, we investigated the seropositivity of IDV among domestic ruminants and swine in West and East Africa from 2017 to 2020. Serum samples were analyzed using the hemagglutination inhibition (HI) assay. Our study demonstrated that IDV is still circulating in Africa, with variations in seropositivity among countries and species. The highest seropositivity was detected in cattle (3.9 to 20.9%). Our data highlights a need for extensive surveillance of IDV in Africa in order to better understand the epidemiology of the virus in the region.
Assuntos
Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/imunologia , Ruminantes/imunologia , Ruminantes/virologia , Thogotovirus/imunologia , Thogotovirus/patogenicidade , África Oriental/epidemiologia , África Ocidental/epidemiologia , Animais , Bovinos , Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/virologia , Feminino , Masculino , Estudos Soroepidemiológicos , Suínos , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/imunologia , Doenças dos Suínos/virologiaRESUMO
Bluetongue (BT) is a severe and economically important disease of ruminants that is widely distributed around the world, caused by the bluetongue virus (BTV). More than 28 different BTV serotypes have been identified in serum neutralisation tests (SNT), which, along with geographic variants (topotypes) within each serotype, reflect differences in BTV outer-capsid protein VP2. VP2 is the primary target for neutralising antibodies, although the basis for cross-reactions and serological variations between and within BTV serotypes is poorly understood. Recombinant BTV VP2 proteins (rVP2) were expressed in Nicotiana benthamiana, based on sequence data for isolates of thirteen BTV serotypes (primarily from Europe), including three 'novel' serotypes (BTV-25, -26 and -27) and alternative topotypes of four serotypes. Cross-reactions within and between these viruses were explored using rabbit anti-rVP2 sera and post BTV-infection sheep reference-antisera, in I-ELISA (with rVP2 target antigens) and SNT (with reference strains of BTV-1 to -24, -26 and -27). Strong reactions were generally detected with homologous rVP2 proteins or virus strains/serotypes. The sheep antisera were largely serotype-specific in SNT, but more cross-reactive by ELISA. Rabbit antisera were more cross-reactive in SNT, and showed widespread, high titre cross-reactions against homologous and heterologous rVP2 proteins in ELISA. Results were analysed and visualised by antigenic cartography, showing closer relationships in some, but not all cases, between VP2 topotypes within the same serotype, and between serotypes belonging to the same 'VP2 nucleotype'.
Assuntos
Vírus Bluetongue/classificação , Vírus Bluetongue/genética , Proteínas do Capsídeo/classificação , Proteínas do Capsídeo/genética , Reações Cruzadas/imunologia , Sorogrupo , Animais , Antígenos Virais/imunologia , Bluetongue/imunologia , Bluetongue/virologia , Vírus Bluetongue/imunologia , Proteínas do Capsídeo/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Coelhos/imunologia , Ruminantes/imunologia , Sorotipagem , Ovinos/imunologia , Nicotiana/genéticaRESUMO
Viral infections have long provided a platform to understand the workings of immunity. For instance, great strides towards defining basic immunology concepts, such as MHC restriction of antigen presentation or T-cell memory development and maintenance, have been achieved thanks to the study of lymphocytic choriomeningitis virus (LCMV) infections. These studies have also shaped our understanding of antiviral immunity, and in particular T-cell responses. In the present review, we discuss how bluetongue virus (BTV), an economically important arbovirus from the Reoviridae family that affects ruminants, affects adaptive immunity in the natural hosts. During the initial stages of infection, BTV triggers leucopenia in the hosts. The host then mounts an adaptive immune response that controls the disease. In this work, we discuss how BTV triggers CD8+ T-cell expansion and neutralizing antibody responses, yet in some individuals viremia remains detectable after these adaptive immune mechanisms are active. We present some unpublished data showing that BTV infection also affects other T cell populations such as CD4+ T-cells or γδ T-cells, as well as B-cell numbers in the periphery. This review also discusses how BTV evades these adaptive immune mechanisms so that it can be transmitted back to the arthropod host. Understanding the interaction of BTV with immunity could ultimately define the correlates of protection with immune mechanisms that would improve our knowledge of ruminant immunology.
Assuntos
Imunidade Adaptativa , Anticorpos Antivirais/sangue , Vírus Bluetongue/imunologia , Bluetongue/imunologia , Linfócitos T/imunologia , Animais , Anticorpos Neutralizantes/sangue , Apresentação de Antígeno , Bluetongue/virologia , Ruminantes/imunologia , Ovinos/imunologia , Linfócitos T/classificaçãoRESUMO
Dugbe orthonairovirus (DUGV) and Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) are tick-borne arboviruses within the order Bunyavirales. Both viruses are endemic in several African countries and can induce mild (DUGV, BSL 3) or fatal (CCHFV, BSL 4) disease in humans. Ruminants play a major role in their natural transmission cycle. Therefore, they are considered as suitable indicator animals for serological monitoring studies to assess the risk for human infections. Although both viruses do not actually belong to the same serogroup, cross-reactivities have already been reported earlier-hence, the correct serological discrimination of DUGV and CCHFV antibodies is crucial. In this study, 300 Nigerian cattle sera (150 CCHFV seropositive and seronegative samples, respectively) were screened for DUGV antibodies via N protein-based ELISA, indirect immunofluorescence (iIFA) and neutralization assays. Whereas no correlation between the CCHFV antibody status and DUGV seroprevalence data could be demonstrated with a newly established DUGV ELISA, significant cross-reactivities were observed in an immunofluorescence assay. Moreover, DUGV seropositive samples did also cross-react in a species-adapted commercial CCHFV iIFA. Therefore, ELISAs seem to be able to reliably differentiate between DUGV and CCHFV antibodies and should preferentially be used for monitoring studies. Positive iIFA results should always be confirmed by ELISAs.
Assuntos
Anticorpos Antivirais/sangue , Coinfecção/veterinária , Coinfecção/virologia , Ensaio de Imunoadsorção Enzimática/normas , Vírus da Febre Hemorrágica da Crimeia-Congo/imunologia , Vírus da Doença do Carneiro de Nairobi/imunologia , Doenças Transmitidas por Carrapatos/veterinária , Animais , Bovinos , Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/virologia , Coinfecção/epidemiologia , Coinfecção/imunologia , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática/métodos , Imunoglobulina G/sangue , Nigéria/epidemiologia , Ruminantes/imunologia , Ruminantes/virologia , Estudos Soroepidemiológicos , Doenças Transmitidas por Carrapatos/epidemiologia , Doenças Transmitidas por Carrapatos/imunologia , Doenças Transmitidas por Carrapatos/virologia , Carrapatos/virologiaRESUMO
This review provides insight into the importance of understanding NETosis in cows, sheep, and goats in light of the importance to their health, welfare and use as animal models. Neutrophils are essential to innate immunity, pathogen infection, and inflammatory diseases. The relevance of NETosis as a conserved innate immune response mechanism and the translational implications for public health are presented. Increased understanding of NETosis in ruminants will contribute to the prediction of pathologies and design of strategic interventions targeting NETs. This will help to control pathogens such as coronaviruses and inflammatory diseases such as mastitis that impact all mammals, including humans. Definition of unique attributes of NETosis in ruminants, in comparison to what has been observed in humans, has significant translational implications for one health and global food security, and thus warrants further study.
Assuntos
Armadilhas Extracelulares/imunologia , Imunidade Inata , Neutrófilos/imunologia , Ruminantes/imunologia , Animais , Humanos , Neutrófilos/citologiaRESUMO
Foot-and-mouth disease (FMD) is endemic in Kenya affecting cloven-hoofed ruminants. The epidemiology of the disease in small ruminants (SR) in Kenya is not documented. We carried out a cross-sectional study, the first in Kenya, to estimate the sero-prevalence of FMD in SR and the associated risk factors nationally. Selection of animals to be sampled used a multistage cluster sampling approach. Serum samples totaling 7564 were screened for FMD antibodies of non-structural-proteins using ID Screen® NSP Competition ELISA kit. To identify the risk factors, generalized linear mixed effects (GLMM) logistic regression analysis with county and villages as random effect variables was used. The country animal level sero-prevalence was 22.5% (95% CI: 22.3%-24.3%) while herd level sero-prevalence was 77.6% (95% CI: 73.9%-80.9%). The risk factor that was significantly positively associated with FMD sero-positivity in SR was multipurpose production type (OR = 1.307; p = 0.042). The risk factors that were significantly negatively associated with FMD sero-positivity were male sex (OR = 0.796; p = 0.007), young age (OR = 0.470; p = 0.010), and sedentary production zone (OR = 0.324; p<0.001). There were no statistically significant intra class correlations among the random effect variables but interactions between age and sex variables among the studied animals were statistically significant (p = 0.019). This study showed that there may be widespread undetected virus circulation in SR indicated by the near ubiquitous spatial distribution of significant FMD sero-positivity in the country. Strengthening of risk-based FMD surveillance in small ruminants is recommended. Adjustment of husbandry practices to control FMD in SR and in-contact species is suggested. Cross-transmission of FMD and more risk factors need to be researched.
Assuntos
Febre Aftosa/epidemiologia , Ruminantes/virologia , Animais , Anticorpos Antivirais/imunologia , Estudos Transversais , Estudos Epidemiológicos , Feminino , Febre Aftosa/imunologia , Febre Aftosa/virologia , Vírus da Febre Aftosa/imunologia , Vírus da Febre Aftosa/patogenicidade , Quênia/epidemiologia , Masculino , Prevalência , Fatores de Risco , Ruminantes/imunologia , Estudos SoroepidemiológicosRESUMO
γδ T cells constitute a major portion of lymphocytes in the blood of both ruminants and swine. Subpopulations of swine γδ T cells have been distinguished by CD2 and CD8α expression. However, it was not clear if they have distinct expression profiles of their T-cell receptor (TCR) or WC1 genes. Identifying receptor expression will contribute to understanding the functional differences between these subpopulations and their contributions to immune protection. Here, we annotated three genomic assemblies of the swine TCRγ gene locus finding four gene cassettes containing C, J and V genes, although some haplotypes carried a null TRGC gene (TRGC4). Genes in the TRGC1 cassette were homologs of bovine TRGC5 cassette while the others were not homologous to bovine genes. Here we evaluated three principal populations of γδ T cells (CD2+/SWC5-, CD2-/SWC5+, and CD2-/SWC5-). Both CD2- subpopulations transcribed WC1 co-receptor genes, albeit with different patterns of gene expression but CD2+ cells did not. All subpopulations transcribed TCR genes from all four cassettes, although there were differences in expression levels. Finally, the CD2+ and CD2- γδ T-cell populations differed in their representation in various organs and tissues, presumably at least partially reflective of different ligand specificities for their receptors.
Assuntos
Bovinos/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/genética , Ruminantes/imunologia , Suínos/imunologia , Linfócitos T/imunologia , Animais , Antígenos CD2/metabolismo , Genes Codificadores dos Receptores de Linfócitos T/genética , Glicoproteínas de Membrana/metabolismoRESUMO
Mycobacterium avium subsp. paratuberculosis (MAP) is a causative agent of Johne's disease, which is a chronic and debilitating disease in ruminants. MAP is also considered to be a possible cause of Crohn's disease in humans. However, few studies have focused on the interactions between MAP and human macrophages to elucidate the pathogenesis of Crohn's disease. We sought to determine the initial responses of human THP-1 cells against MAP infection using single-cell RNA-seq analysis. Clustering analysis showed that THP-1 cells were divided into seven different clusters in response to phorbol-12-myristate-13-acetate (PMA) treatment. The characteristics of each cluster were investigated by identifying cluster-specific marker genes. From the results, we found that classically differentiated cells express CD14, CD36, and TLR2, and that this cell type showed the most active responses against MAP infection. The responses included the expression of proinflammatory cytokines and chemokines such as CCL4, CCL3, IL1B, IL8, and CCL20. In addition, the Mreg cell type, a novel cell type differentiated from THP-1 cells, was discovered. Thus, it is suggested that different cell types arise even when the same cell line is treated under the same conditions. Overall, analyzing gene expression patterns via scRNA-seq classification allows a more detailed observation of the response to infection by each cell type.
Assuntos
Imunidade/imunologia , Mycobacterium avium subsp. paratuberculosis/imunologia , Paratuberculose/imunologia , RNA/imunologia , Células THP-1/imunologia , Animais , Células Cultivadas , Doença de Crohn/imunologia , Doença de Crohn/microbiologia , Citocinas/imunologia , Expressão Gênica/imunologia , Humanos , Macrófagos/imunologia , Macrófagos/microbiologia , Paratuberculose/microbiologia , Ruminantes/imunologia , Ruminantes/microbiologia , Análise de Sequência de RNA/métodos , Células THP-1/microbiologiaRESUMO
Ruminant γδ T cells were discovered in the mid-1980's shortly after a novel T cell receptor (TCR) gene from murine cells was described in 1984 and the murine TCRγ gene locus in 1985. It was possible to identify γδ T cell populations early in ruminants because they represent a large proportion of the peripheral blood mononuclear cells (PBMC). This null cell population, γδ T cells, was designated as such by its non-reactivity with monoclonal antibodies (mAb) against ovine and bovine CD4, CD8 and surface immunoglobulin (Ig). γδ T cells are non-conventional T cells known as innate-like cells capable of using both TCR as well as other types of receptor systems including pattern recognition receptors (PRR) and natural killer receptors (NKR). Bovine γδ T cells have been shown to respond to stimulation through toll-like receptors, NOD, and NKG2D as well as to cytokines alone, protein and non-protein antigens through their TCR, and to pathogen-infected host cells. The two main populations of γδ T cells are distinguished by the presence or absence of the hybrid co-receptor/PRR known as WC1 or T19. These two populations not only differ by their proportional representation in various tissues and organs but also by their migration into inflamed tissues. The WC1+ cells are found in the blood, skin and spleen while the WC1- γδ T cells predominate in the gut, mammary gland and uterus. In ruminants, γδ T cells may produce IFNγ, IL-17, IL-10 and TGFß, have cytotoxic activity and memory responses. The expression of particular WC1 family members controls the response to particular pathogens and correlates with differences in cytokine responses. The comparison of the WC1 gene families in cattle, sheep and goats is discussed relative to other multigenic arrays that differentiate γδ T cells by function in humans and mice.
Assuntos
Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Ruminantes/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Bovinos , Humanos , Glicoproteínas de Membrana/imunologiaRESUMO
Dugbe orthonairovirus (DUGV) is a tick-borne arbovirus within the order Bunyavirales. DUGV was first isolated in Nigeria, but virus isolations in ten further African countries indicate that DUGV is widespread throughout Africa. Humans can suffer from a mild febrile illness, hence, DUGV is classified as a biosafety level (BSL) 3 agent. In contrast, no disease has been described in animals, albeit serological evidence exists that ruminants are common hosts and may play an important role in the transmission cycle of this neglected arbovirus. In this study, young sheep and calves were experimentally inoculated with DUGV in order to determine their susceptibility and to study the course of infection. Moreover, potential antibody cross-reactivities in currently available diagnostic assays for Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) were assessed as DUGV is distantly related to CCHFV. Following subcutaneous inoculation, none of the animals developed clinical signs or viremia. However, all ruminants seroconverted, as demonstrated by two DUGV neutralization test formats (micro-virus neutralization test (mVNT), plaque reduction (PRNT)), by indirect immunofluorescence assays and in bovines by a newly developed DUGV recombinant N protein ELISA. Sera did not react in commercial CCHFV ELISAs, whereas cross-reactivities were observed by immunofluorescence and immunoblot assays.
Assuntos
Infecções por Arbovirus/imunologia , Arbovírus/imunologia , Vírus da Febre Hemorrágica da Crimeia-Congo/imunologia , Febre Hemorrágica da Crimeia/imunologia , Animais , Anticorpos Antivirais/imunologia , Infecções por Arbovirus/virologia , Bovinos , Técnica Indireta de Fluorescência para Anticorpo/métodos , Febre Hemorrágica da Crimeia/virologia , Testes de Neutralização/métodos , Nigéria , Ruminantes/imunologia , Ruminantes/virologia , Testes Sorológicos/métodos , Ovinos , Carrapatos/imunologia , Carrapatos/virologiaRESUMO
Arthropod-borne Batai virus (BATV) is an Orthobunyavirus widely distributed throughout European livestock and has, in the past, been linked to febrile diseases in humans. In Germany, BATV was found in mosquitoes and in one captive harbor seal, and antibodies were recently detected in various ruminant species. We have, therefore, conducted a follow-up study in ruminants from Saxony-Anhalt, the most affected region in Eastern Germany. A total of 325 blood samples from apparently healthy sheep, goats, and cattle were tested using a BATV-specific qRT-PCR and SNT. Even though viral RNA was not detected, the presence of antibodies was confirmed in the sera of all three species: sheep (16.5%), goats (18.3%), and cattle (41.4%). Sera were further analyzed by a glycoprotein Gc-based indirect ELISA to evaluate Gc-derived antibodies as a basis for a new serological test for BATV infections. Interestingly, the presence of neutralizing antibodies was not directly linked to the presence of BATV Gc antibodies. Overall, our results illustrate the high frequency of BATV infections in ruminants in Eastern Germany.
Assuntos
Vírus Bunyamwera/genética , Vírus Bunyamwera/imunologia , Infecções por Bunyaviridae/imunologia , Infecções por Bunyaviridae/virologia , Ruminantes/imunologia , Ruminantes/virologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Bovinos , Chlorocebus aethiops , Ensaio de Imunoadsorção Enzimática/métodos , Seguimentos , Alemanha , Cabras/imunologia , Cabras/virologia , RNA Viral/genética , Ovinos/imunologia , Ovinos/virologia , Células VeroRESUMO
Numerous infectious diseases impacting livestock impose an important economic burden and in some cases also represent a threat to humans and are classified as zoonoses. Some zoonotic diseases are transmitted by vectors and, due to complex environmental and socio-economic factors, the distribution of many of these pathogens is changing, with increasing numbers being found in previously unaffected countries. Here, we developed a multiplex assay, based on a suspension microarray, able to detect specific antibodies to five important pathogens of livestock (three of them zoonotic) that are currently emerging in new geographical locations: Rift Valley fever virus (RVFV), Crimean-Congo haemorrhagic fever virus (CCHFV), Schmallenberg virus (SBV), Bluetongue virus (BTV) and the bacteria complex Mycobacterium tuberculosis. Using the Luminex platform, polystyrene microspheres were coated with recombinant proteins from each of the five pathogens. The mix of microspheres was used for the simultaneous detection of antibodies against the five corresponding diseases affecting ruminants. The following panel of sera was included in the study: 50 sera from sheep experimentally infected with RVFV, 74 sera from calves and lambs vaccinated with SBV, 26 sera from cattle vaccinated with Mycobacterium bovis, 30 field sera from different species of ruminants infected with CCHFV and 88 calf sera infected with BTV. Finally, to determine its diagnostic specificity 220 field sera from Spanish farms free of the five diseases were assessed. All the sera were classified using commercial ELISAs specific for each disease, used in this study as the reference technique. The results showed the multiplex assay exhibited good performance characteristics with values of sensitivity ranging from 93% to 100% and of specificity ranging from 96% to 99% depending on the pathogen. This new tool allows the simultaneous detection of antibodies against five important pathogens, reducing the volume of sample needed and the time of analysis where these pathogens are usually tested individually.
Assuntos
Anticorpos Antibacterianos/sangue , Anticorpos Antivirais/sangue , Mycobacterium tuberculosis/imunologia , Infecções por Vírus de RNA/veterinária , Vírus de RNA/imunologia , Ruminantes/imunologia , Testes Sorológicos/veterinária , Tuberculose/veterinária , Animais , Vírus Bluetongue/imunologia , Bovinos , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/epidemiologia , Ensaio de Imunoadsorção Enzimática/veterinária , Vírus da Febre Hemorrágica da Crimeia-Congo/imunologia , Infecções por Vírus de RNA/diagnóstico , Infecções por Vírus de RNA/epidemiologia , Febre do Vale de Rift/diagnóstico , Febre do Vale de Rift/epidemiologia , Vírus da Febre do Vale do Rift/imunologia , Ruminantes/virologia , Ovinos/imunologia , Doenças dos Ovinos/diagnóstico , Doenças dos Ovinos/epidemiologia , Tuberculose/diagnóstico , Tuberculose/epidemiologia , ZoonosesRESUMO
Peripheral blood from healthy sheep (n = 3) and goats (n = 3) were employed to establish an efficient method for simultaneous isolation of peripheral blood mononuclear cells (PBMCs) and neutrophils and to standardize protocols for monocyte purification and generation of monocyte-derived macrophages (MDMs). In both species, a significantly enriched population of PBMCs, with higher purity and number of cells determined by flow cytometry, was achieved when processing through a density gradient a mixture of buffy-coat and red blood cell layer (RBC) in comparison to the use of just the buffy-coat (p < 0.05). Neutrophils could be subsequently isolated from the layer, located underneath PBMCs fraction with significant higher purity rates, higher than 85 % determined by flow cytometry, than those obtained with protocols without density gradients (< 60 %) (p < 0.05). This technique would allow the isolation of both cell populations from the same sample of blood. A pure cell population of monocytes, CD14+ cells, was purified from PBMCs when using immunomagnetic columns, which allow for 17 % (nº monocytes/nº PBMCs) of yield and high percentages of expression of CD14+ (88 %), MHC-II+ (91.5 %) and CD11b+ (94 %) established by flow cytometry. On the other hand, the classical and non-expensive purification of monocytes from PBMCs based on the adherence capacity of the former, allowed significantly lower yield of monocytes (4.6 %), with percentages of surface markers expression that dropped to 35 %, 65 % and 55 %, respectively (p < 0.001), suggesting the isolation of a mixed population of cells. The addition of GM-CSF to the culture, at concentration from 25 to 125 ng/mL, enhanced proportionally the number of MDMs generated compared to the absence of supplementation or the use of autologous serum from 5% to 20 %. However, purification of monocytes through the adherence method achieved higher yields of MDMs than those isolated through immunomagnetic columns in both species (p < 0.001). Under the conditions of this study, the use of centrifugation in density gradients allow for the simultaneous purification of PBMCs and neutrophils, with high purity of both populations, from the same sample of blood. The isolation of monocytes could be subsequently achieved through two different methods, i.e. based on immunomagnetic columns or adherence. The preference between both methods would depend on the necessities of the experiment, the initial sample with high purity of monocytes or a final population of MDMs required.
Assuntos
Contagem de Células/métodos , Separação Celular/métodos , Separação Celular/normas , Leucócitos Mononucleares/fisiologia , Macrófagos/fisiologia , Ruminantes/imunologia , Animais , Contagem de Células/normas , Diferenciação Celular , Células Cultivadas/imunologia , Células Dendríticas/imunologia , Cabras/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Leucócitos/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Monócitos/imunologia , Ovinos/imunologiaRESUMO
Two types of cholinesterases (ChEs) are present in mammalian blood and tissues: acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). While AChE regulates neurotransmission by hydrolyzing acetylcholine at the postsynaptic membranes and neuromuscular junctions, BChE in plasma has been suggested to be involved in detoxifying toxic compounds. This study was undertaken to establish the identity of circulating ChE activity in plasmas from domestic animals (bovine, ovine, caprine, porcine and equine) by assessing sensitivity to AChE-specific inhibitors (BW284c51 and edrophonium) and BChE-specific inhibitors (dibucaine, ethopropazine and Iso-OMPA) as well as binding to anti-FBS AChE monoclonal antibodies (MAbs). Based on the inhibition of ChE activity by ChE-specific inhibitors, it was determined that bovine, ovine and caprine plasma predominantly contain AChE, while porcine and equine plasma contain BChE. Three of the anti-FBS AChE MAbs, 4E5, 5E8 and 6H9, inhibited 85-98% of enzyme activity in bovine, ovine and caprine plasma, confirming that the esterase in these plasmas was AChE. These MAbs did not bind to purified recombinant human or mouse AChE, demonstrating that these MAbs were specific for AChEs from ruminant species. These MAbs did not inhibit the activity of purified human BChE, or ChE activity in porcine and equine plasma, confirming that the ChE in these plasmas was BChE. Taken together, these results demonstrate that anti-FBS AChE MAbs can serve as useful tools for distinguishing between AChEs from ruminant and non-ruminant species and BChEs.
Assuntos
Acetilcolinesterase/imunologia , Anticorpos Monoclonais/sangue , Butirilcolinesterase/imunologia , Acetilcolinesterase/sangue , Animais , Animais Domésticos/imunologia , Butirilcolinesterase/sangue , Bovinos , Inibidores da Colinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Sangue Fetal/imunologia , Humanos , Camundongos , Ruminantes/imunologiaRESUMO
A total of 1,337 serum and plasma specimens (939, 393 and 15 from cattle, sheep and goats, respectively) were collected monthly for one a year from ruminant species slaughtered in three Turkish cities endemic for Crimean-Congo hemorrhagic fever virus (CCHFV), Samsun, Sivas and Tokat. The serum samples were tested by commercial indirect ELISA to detect CCHFV antibodies, and positive or equivocal samples were later confirmed by a virus neutralization test (VNT). The seroprevalence in cattle, sheep, and goats was 36.21% (340/939), 6.27% (24/383), and 6.67% (1/15), respectively. Quantitative real-time RT-PCR was employed to detect viraemic animals at slaughter time. The percentage of CCHFV-viraemic animals was 0.67% (9/1337). The virus load varied between 4.1 x 101 and 2.4 x 103 RNA equivalent copies/mL in viraemic animals. The plasma samples that were positive for CCHFV genomic RNA were collected between April and May, when Hyalomma ticks are active. This study presents quantitative CCHFV load data in ruminant species at slaughter and interprets the likelihood of transmission for employees working in slaughterhouses in CCHFV-endemic regions.
Assuntos
Vírus da Febre Hemorrágica da Crimeia-Congo/isolamento & purificação , Febre Hemorrágica da Crimeia/virologia , Ruminantes/virologia , Matadouros , Animais , Anticorpos Antivirais/imunologia , Células Cultivadas , Chlorocebus aethiops/imunologia , Chlorocebus aethiops/virologia , Vírus da Febre Hemorrágica da Crimeia-Congo/imunologia , Febre Hemorrágica da Crimeia/epidemiologia , Febre Hemorrágica da Crimeia/imunologia , Testes de Neutralização/métodos , RNA Viral/genética , RNA Viral/imunologia , Ruminantes/imunologia , Estudos Soroepidemiológicos , Carrapatos/imunologia , Carrapatos/virologia , Turquia/epidemiologia , Células VeroRESUMO
Cattle possess the most diverse repertoire of NK cell receptor genes among all mammals studied to date. Killer cell receptor genes encoded within the NK complex and killer cell Ig-like receptor genes encoded within the leukocyte receptor complex have both been expanded and diversified. Our previous studies identified two divergent and polymorphic KLRA alleles within the NK complex in the Holstein-Friesian breed of dairy cattle. By examining a much larger cohort and other ruminant species, we demonstrate the emergence and fixation of two KLRA allele lineages (KLRA*01 and -*02) at a single locus during ruminant speciation. Subsequent recombination events between these allele lineages have increased the frequency of KLRA*02 extracellular domains. KLRA*01 and KLRA*02 transcription levels contrasted in response to cytokine stimulation, whereas homozygous animals consistently transcribed higher levels of KLRA, regardless of the allele lineage. KLRA*02 mRNA levels were also generally higher than KLRA*01 Collectively, these data point toward alternative functional roles governed by KLRA genotype and allele lineage. On a background of high genetic diversity of NK cell receptor genes, this KLRA allele fixation points to fundamental and potentially differential function roles.
Assuntos
Subfamília A de Receptores Semelhantes a Lectina de Células NK/genética , Ruminantes/genética , Transcrição Gênica/genética , Alelos , Animais , Bovinos , Frequência do Gene/genética , Frequência do Gene/imunologia , Genótipo , Células Matadoras Naturais/imunologia , Subfamília A de Receptores Semelhantes a Lectina de Células NK/imunologia , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Ruminantes/imunologia , Transcrição Gênica/imunologiaRESUMO
Vaccination is a critical tool in modern animal production and key to maintaining animal health. Adjuvants affect the immune response by increasing the rate, quantity, or quality of the protective response generated by the target antigens. Although adjuvant technology dates back to the nineteenth century, there was relatively little improvement in adjuvant technology before the late twentieth century. With the discovery of molecular pathways that regulate the timing, quantity, and quality of the immune response, new technologies are focused on bringing safer, more effective, and inexpensive adjuvants to commercial use.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Doenças dos Animais/prevenção & controle , Ruminantes/imunologia , Vacinação/veterinária , Adjuvantes Imunológicos/farmacologia , Doenças dos Animais/imunologia , AnimaisRESUMO
Infectious diseases are the outcome of complex interactions between the host, pathogen, and environment. After exposure to a pathogen, the host immune system uses various mechanisms to remove the pathogen. However, environmental factors and characteristics of pathogens can compromise the host immune responses and subsequently alter the outcome of infection. In this article, genetic and epigenetic factors that shape the individual variation in mounting protective responses are reviewed. Different approaches that have been used by researchers to investigate the genetic regulation of immunity in ruminants and various sources of genetic information are discussed.
Assuntos
Infecções/veterinária , Ruminantes/genética , Ruminantes/imunologia , Animais , Animais Domésticos , Bovinos , Doenças dos Bovinos/genética , Doenças dos Bovinos/imunologia , Resistência à Doença , Epigênese Genética , Infecções/genética , Infecções/imunologiaRESUMO
Gamma delta (γδ) T cells constitute a major lymphocyte population in peripheral blood and epithelial surfaces. They play nonredundant roles in host defense against diverse pathogens. Although γδ T cells share functional features with other cells of the immune system, their distinct methods of antigen recognition, rapid response, and tissue tropism make them a unique effector population. This review considers the current state of our knowledge on γδ T cell biology in ruminants and the important roles played by this nonconventional T cell population in protection against several infectious diseases of veterinary and zoonotic importance.