Small Ruminant Lentivirus Infection Research Articles

Small Ruminant Lentiviruses: Genetic Variability, Tropism and Diagnosis

Small ruminant lentiviruses (SRLV) cause a multisystemic chronic disease affecting animal production and welfare. SRLV infections are spread across the world with the exception of Iceland. Success in controlling SRLV spread depends largely on the use of appropriate diagnostic tools, but the existence of a high genetic/antigenic variability among these viruses, the fluctuant levels of antibody against them and the low viral loads found in infected individuals hamper the diagnostic efficacy. SRLV have a marked in vivo tropism towards the monocyte/macrophage lineage and attempts have been made to identify the genome regions involved in tropism, with two main candidates, the LTR and env gene, since LTR contains primer binding sites for viral replication and the env-encoded protein (SU ENV), which mediates the binding of the virus to the host’s cell and has hypervariable regions to escape the humoral immune response. Once inside the host cell, innate immunity may interfere with SRLV replication, but the virus develops counteraction mechanisms to escape, multiply and survive, creating a quasi-species and undergoing compartmentalization events. So far, the mechanisms of organ tropism involved in the development of different disease forms (neurological, arthritic, pulmonary and mammary) are unknown, but different alternatives are proposed. This is an overview of the current state of knowledge on SRLV genetic variability and its implications in tropism as well as in the development of alternative diagnostic assays.

Influence of Small Ruminant Lentivirus Infection on Reproductive Traits in Ewes

Abstract The study included a sheep flock comprising five genetic groups. The ELISA was applied to perform constant monitoring (every six months) for the infection of ewes with small ruminant lentivirus (SRLV). The research results demonstrated a negative effect of SRLVs infection on lamb rearing that, depending on the genetic group, proved to be lower 1.3%-1.4% compared to the seronegative mothers. At relatively equal fertility (94%-100%) and more differentiated prolificacy (179%-198%) in all the examined groups (except the Suffolk breed), a rearing index was higher in the seronegative animals 6.8%-24.1% compared to the seropositive mothers. The Suffolk breed proved to be the genetic group most susceptible to SRLV infection. A prolificacy of infected ewes was 10% lower, a lamb rearing rate was 13% lower , and a general reproductive performance was 18% lower in comparison to healthy ewes.

Molecular and genetic characteristics of small ruminant lentiviruses in Slovenia

Small ruminant lentiviruses (SRLV) are spread throughout the world, including Slovenia, where the first evidence of caprine arthritis encephalitis virus (CAEV) infection was found in 1996. This study was conducted to investigate the molecular and genetic characteristics of SRLV infection in Slovenia in order to classify our strains in relation to other known SRLV strains worldwide as well as to establish molecular techniques in concordance with serology. In this study, 340 goats and sheep were tested. Serological examination revealed that 57% of the goats and only 14% of the sheep were seropositive. The results of this study also show that the polymerase chain reaction (PCR) used in this study is less reliable than ELISA, with only 60.6% of the seropositive animals being PCR positive. Thirty-eight nucleotide sequences of the gag region encoding the matrix protein were determined and compared to sequences derived from the GenBank, revealing that Slovenian SRLV strains belong to sequence groups A and B, being maedivisna virus (MVV) and CAEV-like, respectively. In one goat herd, the presence of more than one genotype was confirmed and the majority of goat SRLV sequences were more closely related to MVV than to CAEV prototype strains.

An insight into a combination of ELISA strategies to diagnose small ruminant lentivirus infections

A single broadly reactive standard ELISA is commonly applied to control small ruminant lentivirus (SRLV) spread, but type specific ELISA strategies are gaining interest in areas with highly prevalent and heterogeneous SRLV infections. Short (15-residue) synthetic peptides (n=60) were designed in this study using deduced amino acid sequence profiles of SRLV circulating in sheep from North Central Spain and SRLV described previously. The corresponding ELISAs and two standard ELISAs were employed to analyze sera from sheep flocks either controlled or infected with different SRLV genotypes. Two outbreaks, showing SRLV-induced arthritis (genotype B2) and encephalitis (genotype A), were represented among the infected flocks. The ELISA results revealed that none of the assays detected all the infected animals in the global population analyzed, the assay performance varying according to the genetic type of the strain circulating in the area and the test antigen.Five of the six highly reactive (57–62%) single peptide ELISAs were further assessed, revealing that the ELISA based on peptide 98M (type A ENV-SU5, consensus from the neurological outbreak) detected positives in the majority of the type-A specific sera tested (Se: 86%; Sp: 98%) and not in the arthritic type B outbreak. ENV-TM ELISAs based on peptides 126M1 (Se: 82%; Sp: 95%) and 126M2 0,65 0.77 (Se: 68%; Sp: 88%) detected preferentially caprine arthritis encephalitis (CAEV, type B) and visna/maedi (VMV, type A) virus infections respectively, which may help to perform a preliminary CAEV vs. VMV-like typing of the flock. The use of particular peptide ELISAs and standard tests individually or combined may be useful in the different areas under study, to determine disease progression, diagnose/type infection and prevent its spread.

Development and validation of TaqMan probe based real time PCR assays for the specific detection of genotype A and B small ruminant lentivirus strains

BackgroundSmall ruminant lentiviruses (SRLV) are members of the Retroviridae family and infect goats and sheep worldwide. Detection of specific antibodies using AGID and ELISA is the most commonly used means of diagnosing SRLV infection. The most frequent molecular method for detecting the provirus genome is PCR, using peripheral blood leucocytes as target cells. Real time PCR has also recently been used. The aim of this study was to develop a real time PCR for detection of SRLV in order to improve molecular diagnostics of SRLV infections in sheep and goats.ResultsTwo new real time PCR assays using TaqMan probes for the specific detection of genotype A (MVV assay) and genoptype B (CAEV assay) SRLV strains and differentiation between them were developed and validated at both analytical and diagnostic levels following MIQE guidelines. The validation results showed that the new real time PCR is 100% specific, with a reliable limit of detection of 26 (CAEV assay) and 72 (MVV assay) plasmid DNA copies, while compared to ELISA the diagnostic sensitivity of both assays was 79% when tested with Slovenian SRLV field samples. Intra-assay and inter-assay coefficients of variation showed overall good repeatability and reproducibility of the new real time PCR assays, except for the highest dilutions.ConclusionsTwo new TaqMan probe based real time PCR assays for the specific detection of genotype A and B SRLV strains and differentiation between them were developed and validated. They can serve as an additional tool for confirming infection with SRLV and may also be useful for early detection of infected animals prior to seroconversion.

Control of brucellosis and of respiratory Small Ruminant Lentivirus infection in small ruminants in the Basque country, Spain

A compulsory control program to control-eradicate Brucella infections was initiated 1985 in the Basque Country in Northern Spain. The program included both Brucella melitensis infection (ovine/caprine brucellosis), which were later included in a national plan, and Brucella ovis infection (ovine epididymitis), which was promoted by the local animal health authorities. Prevalences of these infections in small ruminant farms at that time were 10% and 60% of flocks, respectively. Since then, the flocks/herds have been tested annually and animals found to be infected by means of the tests employed were slaughtered. At the same time, a voluntary Small Ruminant Lentivirus infection control program also started in some sheep flocks of the region. Currently, prevalences of brucellosis have dropped to 0% for B. melitensis infection and <0.1% for B. ovis. This implies an overall rate reduction of 100% and 99.9%, respectively. The results for Small Ruminant Lentivirus infection have not been so good and only three of the eight flocks into the scheme have reached eradication. Good results have been obtained by local and national animal health authorities and farmers during the past 25 years for brucelosis control. Use of simple serological testing and culling has allowed reduction-eradication of the infection. Once advanced control is reached, an economic balance will be necessary, in order to optimize best use of resources, including allocating them to control other small ruminant problems.

Diverse host–virus interactions following caprine arthritis-encephalitis virus infection in sheep and goats

Interspecies transmissions substantially contribute to the epidemiology of small ruminant lentiviruses (SRLVs), including caprine arthritis encephalitis virus (CAEV) and visna-maëdi virus. However, comprehensive studies of host-virus interactions during SRLV adaptation to the new host are lacking. In this study, virological and serological features were analysed over a 6 month period in five sheep and three goats experimentally infected with a CAEV strain. Provirus load at the early stage of infection was significantly higher in sheep than in goats. A broad antibody reactivity against the matrix and capsid proteins was detected in goats, whereas the response to these antigens was mostly type-specific in sheep. The humoral response to the major immunodominant domain of the surface unit glycoprotein was type-specific, regardless of the host species. These species-specific immune responses were then confirmed in naturally infected sheep and goats using sera from mixed flocks in which interspecies transmissions were reported. Taken together, these results provide evidence that SRLV infections evolve in a host-dependent manner, with distinct host-virus interactions in sheep and goats, and highlight the need to consider both SRLV genotypes in diagnosis, particularly in sheep.

Virological and phylogenetic characterization of attenuated small ruminant lentivirus isolates eluding efficient serological detection

Three field isolates of small ruminant lentiviruses (SRLVs) were derived from a mixed flock of goats and sheep certified for many years as free of caprine arthritis encephalitis virus (CAEV). The phylogenetic analysis of pol sequences permitted to classify these isolates as A4 subtype. None of the animals showed clinical signs of SRLV infection, confirming previous observations which had suggested that this particular subtype is highly attenuated, at least for goats. A quantitative real time PCR strategy based on primers and probes derived from a highly variable env region permitted us to classify the animals as uninfected, singly or doubly infected. The performance of different serological tools based on this classification revealed their profound inadequacy in monitoring animals infected with this particular SRLV subtype. In vitro, the isolates showed differences in their cytopathicity and a tendency to replicate more efficiently in goat than sheep cells, especially in goat macrophages. By contrast, in vivo, these viruses reached significantly higher viral loads in sheep than in goats. Both env subtypes infected goats and sheep with equal efficiency. One of these, however, reached significantly higher viral loads in both species. In conclusion, we characterized three isolates of the SRLV subtype A4 that efficiently circulate in a mixed herd of goats and sheep in spite of their apparent attenuation and a strict physical separation between goats and sheep. The poor performance of the serological tools applied indicates that, to support an SRLV eradication campaign, it is imperative to develop novel, subtype specific tools.

“Milk-drop syndrome of ewes”: Investigation of the causes in dairy sheep in Greece

“Milk-drop syndrome of ewes” is defined as a pathological entity at flock level and a syndrome characterized by reduced milk yield of lactating ewes, with no clinical signs specific to a disease. Concurrent presence of the following two criteria was necessary to diagnose the syndrome in a flock: (i) >30% reduction of flock milk production and (ii) >25% of ewes affected, each ewe with >25% reduction in milk yield. An investigation into the causes of the syndrome was carried out in 22 flocks in Greece. Reduction in milk production in the flocks ranged from 30% to 90% and proportion of affected animals ranged from 27% to 100%. Subclinical mastitis was the primary cause of the syndrome (11 flocks as sole cause/3 flocks co-existing with other disorders). Contagious agalactia was another important cause of the syndrome (3 flocks as sole cause/1 flock co-existing with other disorders). Other causes of the syndrome included: inappropriate feeding regime (2/4 flocks), small ruminant lentivirus infection (2/2 flocks), ruminal acidosis (0/3 flocks) and increased burdens of gastrointestinal nematodes (0/1 flock).

Study of compartmentalization in the visna clinical form of small ruminant lentivirus infection in sheep

BackgroundA central nervous system (CNS) disease outbreak caused by small ruminant lentiviruses (SRLV) has triggered interest in Spain due to the rapid onset of clinical signs and relevant production losses. In a previous study on this outbreak, the role of LTR in tropism was unclear and env encoded sequences, likely involved in tropism, were not investigated. This study aimed to analyze heterogeneity of SRLV Env regions - TM amino terminal and SU V4, C4 and V5 segments - in order to assess virus compartmentalization in CNS.ResultsEight Visna (neurologically) affected sheep of the outbreak were used. Of the 350 clones obtained after PCR amplification, 142 corresponded to CNS samples (spinal cord and choroid plexus) and the remaining to mammary gland, blood cells, bronchoalveolar lavage cells and/or lung. The diversity of the env sequences from CNS was 11.1-16.1% between animals and 0.35-11.6% within each animal, except in one animal presenting two sequence types (30% diversity) in the CNS (one grouping with those of the outbreak), indicative of CNS virus sequence heterogeneity. Outbreak sequences were of genotype A, clustering per animal and compartmentalizing in the animal tissues. No CNS specific signature patterns were found.ConclusionsBayesian approach inferences suggested that proviruses from broncoalveolar lavage cells and peripheral blood mononuclear cells represented the common ancestors (infecting viruses) in the animal and that neuroinvasion in the outbreak involved microevolution after initial infection with an A-type strain. This study demonstrates virus compartmentalization in the CNS and other body tissues in sheep presenting the neurological form of SRLV infection.

Genetic and antigenic characterization of small ruminant lentiviruses circulating in Poland

Small ruminant lentivirus (SRLV) infections are widespread in Poland, but the genetic features of sheep viruses are still lacking and limited to partial gag sequences for goat viruses. In this study, segments from the gag and env genes of Polish SRLV strains screened by heteroduplex mobility assay were subjected to genetic analyses. Subtype A1 was found in both sheep and goats, while subtypes B1 and B2 were found in goats and sheep, respectively. In addition, two novel subtypes (named A12 and A13) were found in sheep. Their close phylogenetic relatedness with SRLV strains previously isolated from Polish goats indicated that these new subtypes are predominant and circulate in both species. The antigenic relationships of subtypes A12 and A13 with other SRLV subtypes were tested in an ELISA assay based on recombinant antigens carrying the immunodominant domains of structural proteins (MA, CA and SU). Antigenic cross-reactivity in the Gag epitopes was evident among genotype A subtypes and, to a lower extent, between genotypes A and B. In contrast, a subtype-specific immunoresponse was detected in the SU epitopes. These results emphasize the broad genetic and antigenic diversity of SRLV strains circulating in Europe and confirmed the need to consider all viral genotypes to choose the antigens in serological tests in order to avoid misdiagnosis in control and eradication programs.

Interference of Vaccination against Bluetongue Virus Serotypes 1 and 8 with Serological Diagnosis of Small-Ruminant Lentivirus Infection

The effects of the recent vaccinations against bluetongue virus serotype 1 (BTV-1) and BTV-8 in Europe on the reliability of enzyme-linked immunosorbent assays (ELISAs) currently used for diagnosis of small-ruminant lentivirus (SRLV) infection were examined. Primary vaccination against BTV-8 in goats induced an increase in reactivity that did not exceed 3 months in a whole-virus indirect ELISA and a competitive ELISA based on the gp135 glycoprotein. Subsequent BTV-1/8 vaccination extended the time scale of false-positive reactivity for up to 6 months. These results are of relevance for SRLV-monitoring programs.

Elimination of small ruminant lentivirus infection from sheep flocks and goat herds aided by health schemes in Great Britain

Over a period of 11 years, 33 sheep or goat holdings lost their maedi-visna or caprine arthritis-encephalitis accredited status (mean 2.8 per year [0.09 per cent] of the accredited holdings...

Diagnostic Assays Used to Control Small Ruminant Lentiviruses

The serologic diagnostic tests, such as the agar gel immunodiffusion assay and various types of enzyme-linked immunosorbent assays (ELISAs), have contributed to the reduction of small ruminant lentivirus (SRLV) infections worldwide. Because there are no treatments or efficacious vaccines, the serologic diagnostic tests have supported most of the eradication efforts by testing and removal or separation of adult animals that generate antibodies to SRLVs. With the advent of molecular diagnostics, standard and quantitative polymerase chain reaction (PCR)-based assays for the detection of provirus in peripheral blood cells are becoming more common and aid in the detection of infected goats and sheep before antibody detection by ELISA in some animals. Performance of the serologic and molecular diagnostic tests is dependent upon a number of factors, including the format of the assay, the percentage of identity between the viral nucleotide sequences in a flock or herd of a certain geographic region and the sequences used to generate SRLV test reagents, and the intrinsic pathogenesis or amount of provirus and SRLV antibody generated in a species or individual small ruminant. In addition, small ruminant genomics may help with establishing genetic markers of SRLV infection and disease, which could also aid eradication or reduction of SRLVs from herds and flocks throughout the world.

Multinucleated giant cells with an osteoclast phenotype derived from caprine peripheral blood mononuclear cells

Formation of multinucleated giant cells (MGCs) by macrophage fusion is a typical cytopathic effect of lentiviral replication in caprine monocytes and MGC formation from cultured caprine peripheral blood mononuclear cells (PBMCs) has been considered to be diagnostic for small ruminant lentivirus (SRLV) infection. In this study, formation of MGCs was observed after 7-14 days when PBMCs were cultured from healthy goats free from SRLV infection. These MGCs expressed tartrate-resistant acid phosphatase, calcitonin receptor, integrin αVβ3, cathepsin K and matrix metalloproteinase 9 and were able to resorb bone in vitro in the absence of RANKL and macrophage colony stimulating factor, consistent with an osteoclast phenotype.

PREVALÊNCIA E FATORES ASSOCIADOS À INFECÇÃO POR LENTIVÍRUS DE PEQUENOS RUMINANTES EM CAPRINOS NO ESTADO DO TOCANTINS

The aim of this study was to estimate the prevalence of caprine blood reagents to small-ruminant lentiviruses (SRLV), through a non probabilistic sampling of herds in Tocantins State. In order to detect the anti-SRLV antibodies, 843 blood samples were analyzed using the microimmunodiffusion test in agarose gel – MICRO-IDGA. The frequency of blood reagent goats was 2.7% (23/843). Five focus, being two in the city of Araguaina and one in each of the cities of Babaculândia, Filadelfia and Miracema do Tocantins had been identified. The results were distributed according to micro-region in the following manner: 10.0 (20/200) and 1.4% (3/207) for the North and Miracema micro-regions, respectively. The Saanen was the breed with the highest percentage of blood reagent animals (11.7%; 7/60), the undefined breed animals 0.6% (2/310) and Anglo-Nubian (3.0%; 14/466). Regarding age, 1.9% (6/314) and 3.2% (17/529) of animals under and over 24 months of age, respectively, tested positive. 2.4% (4/166) of males and 2.8% (19/677) of females tested positive. The SRLV infection occurs at a low prevalence among goats in Tocantins State, affecting mainly animal of the Saanen breed and from the north region. KEY WORDS: Brazil, CAEV, epidemiology, microimmunodiffusion.

Antibody-based diagnosis of small ruminant lentivirus infection in seminal fluid

Antibody-based diagnosis of small ruminant lentiviruses (SRLVs) has been efficiently achieved using serum and milk, but not semen, for which polymerase chain reaction (PCR) has been proposed as a confirmatory technique. This work, involving 296 ovine ( Ovis aries) and caprine ( Capra hircus) semen donors, investigates whether seminal fluid (SF) can be reliably used in antibody-based SRLV diagnosis. First, a gold standard was established to assess the infection status and determine the sensitivity and specificity of three commercial enzyme-linked immunosorbent assays (ELISAs) in serum testing using Western blot and PCR as confirmatory tests. For SF testing, both gold standard and serum testing results were used as reference. The performance of SF testing was affected not only by the ELISA assay sensitivity (related to antigen spectrum) compared with that of the gold standard (as it occurred in serum testing) but also by SF sample quality and SF working dilution. Nonturbid SF samples, commonly collected in artificial insemination centers (AICs), were required. Compared with serum, SF testing had a decreased sensitivity in two of the ELISA assays (with original serum working dilutions ≤1/20 in serum testing) but reached a similar sensitivity (and specificity) in the assay designed to work at the highest serum dilution (1/500). A SF concentration of about 1/2 (250-fold that used in serum testing) was found optimal in this assay, yielding highly repeatable results that were in almost perfect agreement with those of serum testing (κ ± SE, 0.91 ± 0.81). Thus, SF ELISA can be reliably applied in antibody-based SRLV diagnosis. This information may be useful to control infection in AICs and animal and semen trade programs requiring health-certified quality of semen donors.

Use of serology and polymerase chain reaction for the rapid eradication of small ruminant lentivirus infections from a sheep flock: A case report

A SRLV-free sheep flock incurred infection which led to an SRLV infection rate of over 50% of the ewes (34/64) within a 30 months period, indicating that environmental conditions were favourable to transmission. An intensive regimen of sampling at short intervals and testing for SRLV antibodies and proviral DNA combined with strict management was implemented for the entire flock, lambs and yearlings included. This resulted in eradication of the infection within two testing and culling rounds with a 3 months interval. The additional value of the proviral DNA detection by PCR in identifying infected animals was clear in that nine infected animals were found that would have been missed if tested by serology alone. PCR also saved two lambs from being culled; they were sero-positive probably due to maternal antibodies, but not infected.

Improvements in the detection of small ruminant lentivirus infection in the blood of sheep by PCR

The polymerase chain reaction on blood samples has been considered a complement to serological methods for the detection of small ruminant lentiviruses (SRLV) infections in sheep and goats. This is a report on the results of a study to evaluate the use of the same blood sample for the detection of infected animals by ELISA and PCR. A comparison between the results obtained by applying PCRs targeting LTR and gag sequences on blood clot, serum and peripheral blood leucocytes was made. In addition to simplifying sampling and laboratory work, the use of blood clot samples with the gag-PCR improved remarkably the detection of infected animals. Finally, this study has shown the existence of a cell-free viremia in the serum of SRLV-infected sheep.

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.