Small ruminant macrophage polarization may play a pivotal role on lentiviral infection

Small ruminant lentiviruses (SRLV) infect the monocyte/macrophage lineage inducing a long-lasting infection affecting body condition, production and welfare of sheep and goats all over the world. Macrophages play a pivotal role on the host’s innate and adaptative immune responses against parasites by becoming differentially activated. Macrophage heterogeneity can tentatively be classified into classically differentiated macrophages (M1) through stimulation with IFN-γ displaying an inflammatory profile, or can be alternatively differentiated by stimulation with IL-4/IL-13 into M2 macrophages with homeostatic functions. Since infection by SRLV can modulate macrophage functions we explored here whether ovine and caprine macrophages can be segregated into M1 and M2 populations and whether this differential polarization represents differential susceptibility to SRLV infection. We found that like in human and mouse systems, ovine and caprine macrophages can be differentiated with particular stimuli into M1/M2 subpopulations displaying specific markers. In addition, small ruminant macrophages are plastic since M1 differentiated macrophages can express M2 markers when the stimulus changes from IFN-γ to IL-4. SRLV replication was restricted in M1 macrophages and increased in M2 differentiated macrophages respectively according to viral production. Identification of the infection pathways in macrophage populations may provide new targets for eliciting appropriate immune responses against SRLV infection.     

Impact of natural sheep-goat transmission on detection and control of small ruminant lentivirus group C infections

Dissemination of small ruminant lentivirus (SRLV) infections in Norway is affected by the different control strategies used for maedi-visna virus (MVV) infections in sheep and caprine arthritis-encephalitis virus (CAEV) infections in goats. Here we investigated SRLV phylogenetic group variants in sheep. CAEV-like isolates, belonging to phylogenetic group C, were found among both seropositive sheep and goats in mixed flocks, in which sheep and goats are kept together. Intra-herd clustering confirmed that mixed flock animals were infected by the same virus variant, suggesting ongoing interspecies transmission. Few sheep flocks were found to be infected with the MVV-like phylogenetic group A. The apparent absence of SRLV group A type in goats is probably due to the MVV control programme and animal management practices. SRLV group C targets lungs and mammary glands in sheep, and induces typical SRLV pathological lesions. SRLV group C isolated from the sheep mammary glands suggested a productive infection and potential for transmission to offspring. SRLV group C was most prevalent among goats. A lower PCR sensitivity in seropositive sheep suggested a lower load of SRLV group C provirus in sheep than in goats. Higher genetic divergence of group C than in other SRLV groups and extensive heterogeneity among group C isolates in the matrix C-terminal region demonstrate the need for identifying conserved target regions when developing PCR protocols for SRLV detection. As sheep and goats may serve as reservoirs for all SRLV genogroup types, successful control programmes require inclusion of both species.     

In vivo kinetics of peripheral cellular immune responses in Mycobacterium avium subspecies paratuberculosis (MAP) infected and vaccinated goats

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of paratuberculosis (ParaTB) also known as Johne’s disease (JD) in ruminants, which is characterized by chronic intestinal inflammation. A similar counterpart has been observed in the form of Crohn's disease in humans. The present study is the first trail in goats to understand the peripheral cellular immune responses following experimental MAP infection and vaccination. Fifteen apparently healthy male kids (3–6 months old) of Barbari breed were included in this study. In the experimental study, 5 kids were infected with ‘S 5’ strain of MAP (“Indian Bison Type”), 5 were vaccinated (Indigenous Vaccine) against MAP infection (Singh et al., 2007) and the remaining 5 kids were uninfected and non-vaccinated controls. Kids were observed for a period of 180 days post exposure (infection and vaccination) and were tested for development of infection. Cellular immune responses (in blood) were recorded post-exposure by three assays. We measured the frequencies of CD4 and CD8T cells, estimated plasma IFNγ and TNα and in the third assay, in vitro cytokine production by peripheral blood mononuclear cells (PBMCs) from vaccinated, infected and controls were examined in response to polyclonal stimulation. The frequencies of peripheral CD4 and CD8T cells were comparable in control, infected and vaccinated animals except around day 49 post-infection where MAP infected animals showed a trend towards significantly reduced frequencies of CD4 T cells compared to apparently healthy controls. Significantly reduced plasma TNFα levels were also observed in infected animals compared to vaccinated animals,during the course of infection. Diminished levels (although non significant) of TNFα were observed in the supernatants from polyclonally stimulated PBMCs at around day 49 post infection. It is conceivable that the diminished cellular immune responses may coincide with an impairment (immune exhaustion) of perhaps antigen-specific CD4T cells that might, in the course of infection, contribute to the progressive nature of caprine paratuberculosis.     

Infection of central nervous system endothelial cells by cell-associated EHV-1

Infection with equine herpesvirus-1 (EHV-1) causes respiratory disease, late-term abortions and equine herpesvirus myeloencephalitis (EHM). Our understanding of EHM pathogenesis is limited except for the knowledge that EHV-1 infected, circulating peripheral blood mononuclear cells (PBMC) transport virus to the central nervous system vasculature causing endothelial cell infection leading to development of EHM. Our objective was to develop a model of CNS endothelial cell infection using EHV-1 infected, autologous PBMC. PBMCs, carotid artery and brain endothelial cells (EC) from 14 horses were harvested and grown to confluency. PBMC or ConA-stimulated PBMCs (ConA-PBMCs) were infected with EHV-1, and sedimented directly onto EC monolayers ('contact'), or placed in inserts on a porous membrane above the EC monolayer ('no contact'). Cells were cultured in medium with or without EHV-1 virus neutralizing antibody. Viral infection of ECs was detected by cytopathic effect. Both brain and carotid artery ECs became infected when cultured with EHV-1 infected PBMCs or ConA-PBMCs, either in direct contact or no contact: infection was higher in carotid artery than in brain ECs, and when using ConA-PBMCs compared to PBMCs. Virus neutralizing antibody eliminated infection of ECs in the no contact model only. This was consistent with cell-to-cell spread of EHV-1 infection from leucocytes to ECs, demonstrating the importance of this mode of infection in the presence of antibody, and the utility of this model for study of cellular interactions in EHV-1 infection of ECs.     

Serological characterization of the new genotype E of small ruminant lentivirus in roccaverano goat flocks

Maedi visna virus (MVV) and caprine arthritis encephalitis virus (CAEV) are a heterogeneous group of infectious agents affecting sheep and goats. Due to their natural cross-species infection they are referred to as small-ruminant lentiviruses (SRLV). Recently a new genetic cluster, highly divergent from MVV and CAEV was identified in the north-west part of Italy. A panel of genotype E specific antigens was developed and evaluated in flocks infected with B1 and E strains. The results clearly indicate that a strain specific antigen is required to correctly identify animals infected with different genotypes.

     

First partial characterisation of small ruminant lentiviruses from Greece

Small ruminant lentivirus (SRLV) infections are widespread in Greece, but SRLVs have never been isolated and characterized. In this study, we present the sequence of a 574-nucleotide (191-amino acid) region of the gag gene of SRLV strains from four sheep and one goat from a single geographic area of Greece. All five sequences appeared to be closely related at both nucleotide (2.1-14.2% variation) and deduced amino acid (1.6-4.2% variation) level. Greek SRLV strains were closer to ovine prototypic strains (average divergence 16.8%) than to the caprine strain CAEV-Co (21% divergence). By amino acid composition, the Greek SRLVs were on the average more than twice as distant from CAEV-Co as from other ovine strains. Phylogenetic analysis suggested that Greek strains segregate into a unique group, separate from, but related to, other ovine prototype sequences.     

Serological characterization of the new genotype E of small ruminant lentivirus in roccaverano goat flocks

Maedi visna virus (MVV) and caprine arthritis encephalitis virus (CAEV) are a heterogeneous group of infectious agents affecting sheep and goats. Due to their natural cross-species infection they are referred to as small-ruminant lentiviruses (SRLV). Recently a new genetic cluster, highly divergent from MVV and CAEV was identified in the north-west part of Italy. A panel of genotype E specific antigens was developed and evaluated in flocks infected with B1 and E strains. The results clearly indicate that a strain specific antigen is required to correctly identify animals infected with different genotypes.     

Proteomic analysis of chicken peripheral blood mononuclear cells after infection by Newcastle disease virus

Characteristic clinical manifestations of Newcastle disease include leukopenia and immunosuppression. Peripheral blood mononuclear cells (PBMCs) are the main targets of Newcastle disease virus (NDV) infection. To survey changes in proteomic expression in chicken PBMCs following NDV infection, PBMC proteins from 30 chickens were separated using two-dimensional electrophoresis (2-DE) and subjected to mass spectrometry analysis. Quantitative intensity analysis showed that the expression of 78 proteins increased more than two-fold. Thirty-five proteins exhibited consistent changes in expression and 13 were identified as unique proteins by matrix assisted laser desorption ionization-time of flight mass spectrometer/mass spectrometer including three that were down-regulated and 10 that were up-regulated. These proteins were sorted into five groups based on function: macromolecular biosynthesis, cytoskeleton organization, metabolism, stress responses, and signal transduction. Furthermore, Western blot analysis confirmed the down-regulation of integrin-linked kinase expression and up-regulation of lamin A production. These data provide insight into the in vivo response of target cells to NDV infection at the molecular level. Additionally, results from this study have helped elucidate the molecular pathogenesis of NDV and may facilitate the development of new antiviral therapies as well as innovative diagnostic methods.     

Antigen-specific IFN-gamma and IL-4 production in caprine herpesvirus infected goats

The analysis of cytokines secreted by antigen-specific lymphocytes is hampered in goats by the paucity of species-specific reagents yet it is crucial to study immune responses to infections. To overcome this limit, two commercial kits designed to measure soluble bovine IL-4 (by ELISA) and frequencies of bovine IFN-gamma secreting cells (by ELISPOT) were tested for cross-reactivity in goats. In addition, an ELISA specific to bovine/ovine IL-4 and employing two monoclonal antibodies, clones CC313 and CC314, was tested as well. Concanavalin A-stimulated caprine peripheral blood mononuclear cells (PBMCs) cultures were studied and they exhibited high levels of soluble IL-4 and high frequencies of IFN-gamma secreting cells. In addition, the two IL-4 ELISAs detected similar amounts of cytokine. To start defining the cytokine response triggered by caprine herpesvirus 1 (CpHV-1) infection, PBMC cultures were setup from goats naturally or experimentally infected with CpHV-1. High frequencies of IFN-gamma producing cells and low, when detectable, levels of soluble IL-4 were observed in CpHV-1-specific PBMC cultures from both groups of infected goats. Thus, the availability of cross-reactive research tools can expand cytokine studies in goats and can implement the research on immunity to other caprine infections.     

Phylogenetic analysis of SRLV sequences from an arthritic sheep outbreak demonstrates the introduction of CAEV-like viruses among Spanish sheep

Small ruminant lentiviruses (SRLVs) cause different clinical forms of disease in sheep and goats. So far in Spain, Maedi visna virus-like (MVV-like) sequences have been found in both species, and the arthritic SRLV disease has never been found in sheep until a recent outbreak. Knowing that arthritis is common in goats, it was of interest to determine if the genetic type of the virus involved in the sheep arthritis outbreak was caprine arthritis encephalitis virus-like (CAEV-like) rather than MVV-like. Alignment and phylogenetic analyses on nucleotide and deduced amino acid sequences from SRLV of this outbreak, allowed a B2 genetic subgroup assignment of these SRLV, compatible with a correspondence between the virus genetic type and the disease form. Furthermore, an isolate was obtained from the arthritic outbreak, its full genome was CAEV-like but the pol integrase region was MVV-like. Although its LTR lacked a U3 repeat sequence and had a deletion in the R region, which has been proposed to reduce viral replication rate, its phenotype in sheep skin fibroblast cultures was rapid/high, thus it appeared to have adapted to sheep cells. This outbreak study represents the first report on CAEV-like genetic findings and complete genome analysis among Spanish small ruminants.     

Prevention strategies against small ruminant lentiviruses: an update

Small ruminant lentiviruses (SRLVs), including maedi-visna virus (MVV) of sheep and caprine arthritis-encephalitis virus (CAEV), are widespread, cause fatal diseases and are responsible for major production losses in sheep and goats. Seventy years after the legendary maedi-visna sheep epidemic in Iceland, which led to the first isolation of a SRLV and subsequent eradication of the infection, no vaccine or treatment against infection has been fully successful. Research during the last two decades has produced sensitive diagnostic tools, leading to a variety of approaches to control infection. The underlying difficulty is to select the strategies applicable to different epidemiological conditions. This review updates the knowledge on diagnosis, risk of infection, immunisation approaches and criteria for selecting the different strategies to control the spread of SRLVs.     

Diagnostic tests for small ruminant lentiviruses

Maedi visna virus and caprine arthritis encephalitis virus are closely related retroviruses that cause chronic inflammatory disease in small ruminants. The infections are characterised by insidious onset and slow progression. Diagnosis of infection is usually by serological testing. A variety of assays are available for this purpose, though the relative sensitivity and specificity of these assays has not been compared systematically. Here we review recent developments in laboratory diagnostic methods and their use in field diagnosis. The results suggest that a combination of ELISA and PCR might afford optimal detection of SRLV infection.     

视黄酸诱导鸡胚胎干细胞向雄性生殖细胞分化的研究

体外胚胎干细胞（embryonic stem cells,ESCs）向生殖细胞分化可用于治疗不育症,同时也为揭示种系世代的分子机制提供最佳模型。试验旨在探讨视黄酸（retinoic acid,RA）诱导鸡胚胎干细胞（chicken embryonic stem cells,cESCs）向雄性生殖细胞（male germ cells,MGCs）分化的作用效果。利用胰蛋白酶消化法从新鲜种蛋X期鸡胚中分离胚胎干细胞,以鸡胚成纤维（chicken embryo fibroblast,CEF）细胞为滋养层,进行体外培养,利用形态法、碱性磷酸酶（alkaline phosphatase,AKP）染色和胚胎阶段特异性表面抗原（embryo specific surface antigen 1,SSEA-1）检测对获得的胚胎干细胞进行鉴定。结果表明,获得典型的呈巢状或岛状的cESCs克隆,细胞AKP染色呈蓝紫色,表明其具有较高的内源性AKP活性;SSEA-1鉴定结果呈阳性,显示cESCs克隆具有多能性。采用10^-5 mol/L RA诱导鸡胚胎干细胞向雄性生殖细胞分化,镜下观察细胞形态变化,分别于诱导第0、2、4、6、8、10天提取细胞总RNA,反转录成cDNA,用于实时荧光定量PCR检测生殖细胞标志基因的表达。结果表明,在此诱导过程中,作为胚胎干细胞标志基因Nanog、Sox2表达量持续显著下降,而生殖细胞特异性基因Dazl、Stra8、c-kit、integrin α6表达量呈持续上升趋势;免疫细胞化学检测可观察到特异基因相关蛋白的阳性克隆。本研究成功分离出cESCs,可体外培养并保持未分化状态及多能性。10^-5 mol/L RA能够促进cESCs向雄性生殖细胞方向分化,可以引起生殖细胞相应基因的表达,为进一步研究雄性生殖细胞的形成和调控机制提供参考。     

Immunomodulation of caprine lentiviral infection by interleukin-16

Interleukin-16 (IL-16) is a proinflammatory cytokine produced by a variety of cells including lymphocytes, macrophages, mast cells, and eosinophils. We have shown in our previous studies increased expression of IL-16 mRNA and protein in caprine arthritis-encephalitis virus (CAEV)-infected goats blood. In this study, we determined the immunomodulatory effects of IL-16 in vitro using cells derived from CAEV infected and uninfected goats. Human recombinant IL-16 (rhIL-16) significantly increased chemotaxis of peripheral blood mononuclear cells (PBMCs) of both control and CAEV-infected goats. Pretreatment of PBMC with anti-goat CD4 monoclonal antibody inhibited IL-16-induced chemotaxis of PBMC of control and infected goats suggesting that IL-16 exerts its action in goats primarily by binding to CD4. The CAEV proviral DNA was less in caprine monocytes treated with rhIL-16 infected in vitro with CAEV. These data suggest inhibitory effect of IL-16 on viral integration. Flow cytometric studies indicated a trend toward IL-16-induced increased expression of lymphocyte activation markers. Combined with our previously reported data, these experiments suggest that increased IL-16 expression during CAEV infection may inhibit viral integration.     

Diagnostic performance of PCR and ELISA on blood and milk samples and serological survey for small ruminant lentiviruses in central Spain

The diagnostic performance of an ELISA for the detection of antibodies to the small ruminant lentiviruses (SRLVs) maedi-visna virus and caprine arthritis-encephalitis virus in milk and corresponding blood samples was evaluated in 50 sheep. The agreement between ELISA results in blood and milk was 90 per cent, and the κ value was 0.79. In addition, a serological survey in the central zone of Spain was performed using milk samples from 413 animals (250 sheep and 163 goats) from 12 flocks/herds. All flocks/herds had some animals that were positive for SRLV. Among the animals, 60.0 per cent of the sheep and 8.0 per cent of the goats tested were seropositive. Each sample was also tested using a PCR technique, which increased the percentage of positive animals detected. Using a combination of ELISA and PCR gave a total of 72.2 per cent of sheep and 28.8 per cent of goats positive for SRLV.     

Phylogenetic analysis of small ruminant lentiviruses detected in Slovenia

Small ruminant lentiviruses (SRLV), which belong to the Retroviridae family, infect goats and sheep worldwide. The aim of this study was to characterize the SRLV strains circulating in Slovenia, by phylogenetic analysis of two genomic regions, 1.8 kb gag-pol fragment and 1.2 kb pol fragment. The results of our study revealed that Slovenian SRLV strains are highly heterogeneous, with ovine strains belonging to genotype A and caprine strains to genotypes A and B. The closest relatives of sheep virus sequences from two flocks that clustered together (SLO 35, 36) were found to be in subtype A5. A cluster composed of four sheep virus sequences (SLO 31) was clearly divergent from all other subtypes in group A and could not be assigned to any of them. The virus sequences from one goat flock belonged solely to subtype B1, whereas virus sequences from more than one genotype were found to circulate within the other two goat flocks, belonging to subtype B1 (SLO 1 and SLO 37) and to genotype A (SLO 2 and 78–88 g). Two goat virus sequences (SLO 2) were found to belong to genotype A and could not be assigned to existing subtypes. One goat virus sequence (37–88 g) from flock 37 was clearly different from other sequences of this flock and was more closely related to genotype A sequences. We propose two new subtypes within genotype A, subtype A14 (SLO 2) and A15 (SLO 31).