Investigation, management and control of a maedi outbreak in Norway in 2019-2020.

BACKGROUND: Visna-maedi is a notifiable disease in Norway, and eliminating the disease is a national goal. The import of sheep into Norway is very limited, and strict regulations apply to the movement of small ruminants between flocks and within defined geographical regions. Several outbreaks have occurred in the last 50 years, and the most recent before 2019 occurred in Trøndelag county in Central Norway in 2002. A national surveillance programme for small ruminant lentivirus infection exists since 2003. RESULTS: In 2019, the national surveillance programme detected seropositive animals for small ruminant lentivirus in a sheep flock in Trøndelag. Based on the result of polymerase chain reaction analysis and histopathological findings, the Norwegian Food Safety Authority concluded the diagnosis of maedi. Further investigations detected maedi in eight additional sheep flocks in the same county. The flocks were placed under restrictions, and the authorities also imposed restrictions on 82 contact flocks. Sequencing of partial gag genes indicated that the virus in the current outbreak was related to the small ruminant lentivirus detected in the same area between 2002 and 2005. CONCLUSIONS: The outbreak investigation shows the need for sensitive and specific diagnostic methods, and an improved and more targeted surveillance strategy. It also demonstrates the risk of disease spreading between flocks through animal movements, and highlights the importance of biosecurity and structured livestock trade. In addition to allowing livestock trade only from flocks documented free from maedi, it may be necessary to monitor sheep flocks over many years, when aiming to eliminate maedi from the Norwegian sheep population.

Differential gene expression and immune cell infiltration in maedi-visna virus-infected lung tissues.

BACKGROUND: Maedi-visna virus (MVV) is a lentivirus that infects monocyte/macrophage lineage cells in sheep, goats, and wild ruminants and causes pneumonia, mastitis, arthritis, and encephalitis. The immune response to MVV infection is complex, and a complete understanding of its infection and pathogenesis is lacking. This study investigated the in vivo transcriptomic patterns of lung tissues in sheep exposed to MVV using the RNA sequencing technology. RESULT: The results indicated that 2,739 genes were significantly differentially expressed, with 1,643 downregulated genes and 1,096 upregulated genes. Many variables that could be unique to MVV infections were discovered. Gene Ontology analysis revealed that a significant proportion of genes was enriched in terms directly related to the immune system and biological responses to viral infections. Kyoto Encyclopedia of Genes and Genomes analysis revealed that the most enriched pathways were related to virus-host cell interactions and inflammatory responses. Numerous immune-related genes, including those encoding several cytokines and interferon regulatory factors, were identified in the protein-protein interaction network of differentially expressed genes (DEGs). The expression of DEGs was evaluated using real-time polymerase chain reaction and western blot analysis. CXCL13, CXCL6, CXCL11, CCR1, CXCL8, CXCL9, CXCL10, TNFSF8, TNFRSF8, IL7R, IFN-γ, CCL2, and MMP9 were upregulated. Immunohistochemical analysis was performed to identify the types of immune cells that infiltrated MVV-infected tissues. B cells, CD4+ and CD8+ T cells, and macrophages were the most prevalent immune cells correlated with MVV infection in the lungs. CONCLUSION: Overall, the findings of this study provide a comprehensive understanding of the in vivo host response to MVV infection and offer new perspectives on the gene regulatory networks that underlie pathogenesis in natural hosts.

Effect of TMEM154 E35K variant (haplotypes 1 and 3) on the incidence of ovine lentivirus infection and ewe productivity during lifetime exposure.

Ovine progressive pneumonia virus (OPPV) is a small ruminant lentivirus that is widespread throughout U.S. sheep flocks. Infections with OPPV are lifelong and effects are multi-systemic with significant implications for animal well-being and productivity. A protein isoform with lysine at position 35 (K35, haplotype "1") encoded by the ovine transmembrane protein 154 (TMEM154) gene has been associated with reduced susceptibility to infection when two copies are present (i.e., diplotype "1,1"). Conversely, the ancestral protein isoform with glutamate at position 35 (E35, haplotype "3") is associated with high susceptibility to infection when at least one copy is present. The beneficial effect of TMEM154 K35 alleles on ewe productivity has not been previously measured in controlled challenge experiments and was a major objective of this study. Ewes with TMEM154 diplotypes "1,1"; "1,3"; and "3,3" (n = 31, 47, and 30, respectively) were born and reared by OPPV-infected dams and managed under continual natural exposure to OPPV. Ewes were tested for serological status at 4-mo intervals for up to 5.5 yr. The incidence of infection in ewes with diplotype "1,1" was 6.5% to 9.7% and significantly lower (P < 0.001) than ewes with diplotype "1,3" (60.5 to 97.3%) or "3,3" (64.0 to 91.4%). Furthermore, the incidence among ewes with diplotype "1,1" did not increase from 10 to 67 mo of age (P > 0.99), whereas the incidence among diplotype "1,3" and "3,3" ewes increased steadily until reaching an asymptote at approximately 52 mo of age. Total number and weight of lamb weaned per ewe exposed through 5.5 yr from ewes with diplotype "1,1" far exceeded (P ≤ 0.05) those with diplotypes "1,3" and "3,3" by, on average, 2.1 lambs and 40 kg, respectively. The present study confirmed that TMEM154 diplotype "1,1" animals have reduced incidence of OPPV infection and, correspondingly, improved productivity. In flocks with a high frequency of TMEM154 haplotype "3," selection for haplotype "1" appears to be a cost-effective approach to mitigate the impact of this economically important disease.

Update on Small Ruminant Lentiviruses.

Small ruminant lentiviruses (SLRVs) have been recognized throughout the world for decades. SLRVs are a heterogenous group of viruses that can infect sheep, goats, and wild ruminants. Evidence supports cross-species infection. These viruses cause lifelong infections where they target specific organs, which can result in production losses due to diminished milk production, consequential increases in neonatal death and diminished growth, and premature culling of prime age animals. No vaccine or treatments have proved effective. Control programs rely on an understanding of viral transmission and application of highly sensitive, specific, and frequent testing regimens.

Etiology, Epizootiology and Control of Maedi-Visna in Dairy Sheep: A Review.

Maedi-visna (MV) in sheep is caused by maedi-visna virus (MVV), a small ruminant lentivirus (SRLV) that causes chronic infection and inflammatory lesions in infected animals. Pneumonia and mastitis are its predominant clinical manifestations, and the tissues infected by MVV are mainly the lungs, the mammary gland, the nervous system and the joints. MV has a worldwide distribution with distinct MVV transmission patterns depending on circulating strains and regionally applied control/eradication schemes. Nevertheless, the prevalence rate of MV universally increases. Currently, gaps in understanding the epizootiology of MV, the continuous mutation of existing and the emergence of new small ruminant lentiviruses (SRLVs) strains, lack of an effective detection protocol and the inefficiency of currently applied preventive measures render elimination of MV an unrealistic target. Therefore, modifications on the existing MV surveillance and control schemes on an evidentiary basis are necessary. Updated control schemes require the development of diagnostic protocols for the early and definitive diagnosis of MVV infections. The objectives of this review are to summarize the current knowledge in the epizootiology and control of MV in dairy sheep, to describe the research framework and to cover existing gaps in understanding future challenges regarding MV.

Classification of small ruminant lentivirus subtype A2, subgroups 1 and 2 based on whole genome comparisons and complex recombination patterns.

Background: Small ruminant lentiviruses (SRLVs) cause a multisystemic chronic wasting disease in sheep across much of the world. SRLV subtype A2 is prevalent in North America and further classified into multiple subgroups based on variation in the group antigens gene (gag) and envelope (env) genes. In sheep, the ovine transmembrane protein 154 (TMEM154) gene is associated with SRLV susceptibility. Ewes with at least one copy of TMEM154 encoding a full-length protein with glutamate at position 35 (E35; haplotypes 2 and 3), are highly susceptible to SRLV infection while ewes with any combination of TMEM154 haplotypes which encodes lysine (K35; haplotype 1), or truncated proteins (haplotypes 4 and 6) are several times less so. A2 subgroups 1 and 2 are associated with host TMEM154 genotypes; subgroup 1 with the K35/K35 genotype and subgroup 2 with the E35/E35 genotype. Methods:  Sequence variation within and among full-length assemblies of SRLV subtype A2 subgroups 1 and 2 was analyzed to identify genome-scale recombination patterns and subgroup-specific variants. Results:  Consensus viral genomes were assembled from 23 infected sheep, including animals of assorted TMEM154 genotypes comprised of haplotypes 1, 2, or 3. Viral genome analysis identified viral subgroups 1 and 2 among the samples, and revealed additional sub-structure within subgroup 2 based on models predicting complex patterns of recombination between the two subgroups in several genomes. Animals with evidence of dual subgroup infection also possessed the most diverse quasi-species and the most highly recombined consensus genomes. After accounting for recombination, 413 subgroup diagnostic single nucleotide polymorphisms (SNPs) were identified. Conclusions:  The viral subgroup framework developed to classify SRLV consensus genomes along a continuum of recombination suggests that animals with the TMEM154 E35/K35 genotype may represent a reservoir for producing viral genomes representing recombination between A2 subgroups 1 and 2.

Pathology and polymerase chain reaction detection of ovine progressive pneumonia (maedi) cases in slaughtered sheep in India.

AIM: The small ruminant lentiviruses are known to cause maedi-visna (MV) and caprine arthritis - encephalitis in sheep and goats, typically affecting joints, udder, lungs, and the central nervous system. The diagnosis usually involves serology, clinical signs, immunohistochemistry, and polymerase chain reaction (PCR). In the present study, the histopathologically positive pneumonia cases of MV were confirmed by PCR in lung tissue probably for the first time in India. MATERIALS AND METHODS: A total of 888 lungs of adult sheep, aged between 2 and 5 years, were screened during slaughter, of which 121 were found to have pneumonic lesions. The tissues from each pneumonic lung including associated lymph nodes were collected in 10% neutral buffered formalin for histopathology. The frozen tissues of the same were also collected and stored at -20°C for PCR confirmation. RESULTS: Three of 121 cases of pneumonic lungs of sheep revealed gross and histopathological lesions suggestive of maedi or ovine progressive pneumonia infection. These 3 cases were further confirmed by PCR technique that amplified 291-base pair DNA in the long terminal repeat sequence of MV provirus. CONCLUSION: This study suggests the low occurrence of MV virus (MVV) infection in India in naturally affected sheep based on pathomorphological lesions and using the molecular tool of PCR detection of the virus in tissues. Further, a combination of pathomorphology or/and PCR testing might be optimal for detecting the animals infected with MVV.

Small Ruminant Lentiviruses in Sheep: Pathology and Tropism of 2 Strains Using the Bone Marrow Route.

The objective of this work was to comparatively study the tissue tropism and the associated pathology of 2 autochthonous small ruminant lentivirus (SRLV) field strains using an experimental infection in sheep through the bone marrow. Fifteen male, SRLV-free lambs of the Rasa Aragonesa breed were inoculated with strain 697 (nervous tissue origin, animals A1-A6), with strain 496 (articular origin, animals B1-B6), or with uninfected culture medium (C1-C3). Clinical, serologic, and polymerase chain reaction (PCR) evaluations were performed periodically. Two lambs from each infected group and a control animal were euthanized at 134, 273, and 319 days postinfection. Tissues were analyzed by gross and histopathologic evaluation; immunohistochemistry for CD3, CD4, CD8, CD68, and FoxP3 cell markers; lung morphometric evaluation; and tissue proviral quantification by PCR. All infected animals became positive either by enzyme-linked immunosorbent assay and/or PCR, with group B lambs showing the highest serologic values and more consistently positive PCR reactions. Group A lambs showed representative lung lesions but only mild histopathologic changes in the central nervous system (CNS) or in carpal joints. Contrarily, group B lambs demonstrated intense carpal arthritis and interstitial pneumonia but an absence of lesions in the CNS. Proviral copies in tissues were detected only in group B lambs. Experimental infection with these SRLV strains indicates that strain 496 is more virulent than strain 697 and more prone to induce arthritis, whereas strain 697 is more likely to reproduce encephalitis in Rasa Aragonesa lambs. Host factors as well as viral factors are responsible for the final clinicopathologic picture during SRLV infections.

Soroprevalência de Maedi-visna em rebanhos ovinos do estado do Maranhão, Brasil / Seroprevalence of Maedi-Visna in sheep flocks State of Maranhão, Brazil

Apesar de ter sido relatada em vários estados, não há informação sobre o Vírus da Maedi Visna (MVV) no Maranhão, e com o crescimento de sua ovinocultura, aumenta o fluxo de animais de outras regiões. Com isso objetivou-se determinar a soroprevalência do MVV em rebanhos ovinos das três principais mesorregiões produtoras do estado do Maranhão, através da pesquisa das 1.495 amostras sanguíneas de ovinos, com idade superior a seis meses, pertencentes a 83 rebanhos de 23 municípios das mesorregiões Cento, Leste e Norte. O diagnóstico sorológico da infecção pelo vírus MVV foi realizado por meio do teste de imunodifusão em gel de ágar (micro-IDGA). Constatou-se uma prevalência geral de 0,7% (IC95%:0,4-1,3%) de ovinos soropositivos e prevalências nas mesorregiões Centro, Leste e Norte de 0,5% (IC95%:0,1-1,4%), 0,7% (IC95%:0,2-1,8%) e 1% (IC95%:0,3-2,4%) respectivamente. Em relação à variável sexo, não foi observada diferença significativa (P>0,05) entre machos (0,5%, IC95%:0-2,7%) e fêmeas (0,8%, IC95%:0,4-1,4%), assim como quanto a genética comparando-se ovinos de raças puras (1,5%, IC95%: 0,4-8,1%), mestiços (1%, IC95%:0,4-2,0%) e SRD (0,3%, IC95%:0,04-1,1%). A análise quanto à idade não demonstrou diferença significante (P>0,05). Conclui-se que a infecção pelo MVV está presente em ovinos das mesorregiões estudadas, sendo este o primeiro registro desta enfermidade no estado do Maranhão. Considerando a baixa prevalência, é necessário evitar a introdução e a propagação do vírus entre os rebanhos, através da exigência de testes negativos para MVV e descarte dos ovinos positivos.

Although it has been reported in several states, no information about Maedi-Visna (MV) in the state of Maranhão is available. The aim of the present study was to determine the seroprevalence of Maedi Visna Virus (MVV) in sheep flocks of the three most important sheep rearing areas from Maranhão State, Brazil. We surveyed 1.495 blood samples from sheep older than six months, of both sexes and various breeds. The samples were collected from 83 herds of 23 municipalities present in the Central, East and North regions of Maranhão. The immunodifusion agar gel (micro-AGID) performed serological diagnosis of infection MVV. The statistical analysis was performed by Fisher's test, using Epi Info. It was found an overall prevalence of MVV infection of 0,7% (CI95%:0,4-1,3%) the ovines and prevalence of 0,5% (CI95%:0,1-1,4%), 0,7% (CI95%:0,2-1,8%) e 1% (CI95%:0,3-2,4%) in the Central, East and North regions, respectively. In relation to sex, there wasn't a significant difference (P>0.05) between males (0,5,%, CI95%:0-2,7%) and females (0,8%, CI95%:0,4-1,4%), as well as in relation to genetic comparing sheep purebreds (1,5%, CI95%:0,4-8,1%), crossbred (1%, CI95%:0,4-2,0%) and SRD (0,3%, IC95%:0,04-1,1%). In relation to age wasn't observed significant difference. It has concluded that infection with MVV is present in the studied population in low prevalence. This is the first record of MVV in sheep in the State of Maranhão. Considering the low prevalence is necessary to prevent the introduction and spread of the virus between flocks by requiring negative tests for MVV and disposal of positive sheep.

Incidence of infection in 39-month-old ewes with TMEM154 diplotypes "1 1," "1 3," and "3 3" after natural exposure to ovine progressive pneumonia virus.

Production and well-being of sheep and goats in many countries are harmfully impacted by small ruminant lentiviruses (SRLV) that cause incurable, progressive diseases. Susceptibility to ovine progressive pneumonia virus (OPPV), the North American form of SRLV, is influenced by variants of the ovine transmembrane protein 154 gene (TMEM154). The experimental objective was to estimate additive and dominance effects of TMEM154 haplotypes 1 and 3 on susceptibility of breeding ewes to infection after natural exposure to OPPV from birth to 39 mo of age. Sires and dams were heterozygous for TMEM154 haplotypes 1 and 3, producing ewe lambs with diplotypes "1 1," "1 3," and "3 3." These lambs were raised by mature, infected dams to ensure natural, maternal exposure to OPPV. Ewe lambs (n = 108) were kept for breeding and joined an infected flock of ewes to guarantee natural, nonmaternal exposure to OPPV. Ewes were bred to lamb at 1, 2, and 3 yr of age. Serum samples were collected at breeding, 1 mo before lambing and shortly after weaning each year to monitor infection status to 39 mo of age. During the experiment, 9 of the 108 ewes died while uninfected and data collected on these ewes were not analyzed. Infection status of the remaining 99 ewes at 39 mo of age was analyzed using logistic regression procedures. Effects of ewe type of birth, ewe type of rearing, and breed type of dam were not detected (P > 0.10), and the estimated sire variance component was nil. Ewe diplotype affected infection status (P < 0.0001), as did additive (P < 0.0001) and dominance (P < 0.0022) effects. Predicted probabilities of infection for ewes with diplotypes "1 1," "1 3," and "3 3" were 0.10, 0.88, and 0.89, respectively, and confidence intervals for diplotypes "1 3" and "3 3" were distinct from "1 1." Haplotype 3 was completely dominant to haplotype 1 at 39 mo of age. The probability of infection for ewes with either diplotype "1 3" or "3 3" averaged 8.5 times that of ewes with diplotype "1 1." Diplotype "1 3" and "3 3" ewes were highly susceptible to nonmaternal transmission of OPPV, in contrast to diplotype "1 1" ewes. Therefore, the distribution of ewes with diplotypes "1 1," "1 3," and "3 3" within a flock will influence the number of infections caused by each route of transmission. Selection and mating strategies can be implemented to produce sheep that are genetically less susceptible to OPPV infection.

Small ruminant lentivirus-induced arthritis: clinicopathologic findings in sheep infected by a highly replicative SRLV B2 genotype.

We describe the clinicopathologic features of an arthritis outbreak in sheep induced by small ruminant lentivirus (SRLV), linked to the presence of a new SRLV isolate phylogenetically assigned to caprine arthritis encephalitis virus-like subgroup B2. Thirteen SRLV seropositive Rasa Aragonesa adult ewes were selected from 5 SRLV highly infected flocks (mean seroprevalence, 90.7%) for presenting uni- or bilateral chronic arthritis in the carpal joint. A complete study was performed, including symptomatology, histopathology, immunocytochemistry, immunohistochemistry, in situ hybridization, and microbiology. The carpus was the joint almost exclusively affected, with 10 sheep (76%) showing a moderate increase in carpal joint size (diameter range, 18-20 cm; normal range, 15-16 cm) without signs of locomotion problems and with 3 ewes (23%) showing severe inflammation with marked increase in diameter (21-24 cm), pain at palpation, and abnormal standing position. Grossly, chronic proliferative arthritis was observed in affected joints characterized by an increased thickness of the synovial capsule and synovial membrane proliferation. Microscopically, synovial membrane inflammation and proliferation and hyperplasia of synoviocytes were observed. More positive cases of SLRV infection were detected by immunocytochemistry of articular fluid than of bronchoalveolar lavage fluid. Immunohistochemistry and in situ hybridization also detected positive cells in the subsynovial connective tissue, lung, mediastinal lymph node, mammary gland, and mammary lymph node. All animals were negative for the presence of Mycoplasma or other bacteria in the articular space. The present outbreak likely represents an adaptation of a caprine virus to sheep. Our results underline the importance of the arthritis induced by SRLV in sheep, a clinical form that might be underestimated.

Mutations in Ovis aries TMEM154 are associated with lower small ruminant lentivirus proviral concentration in one sheep flock.

Small ruminant lentivirus (SRLV), also called ovine progressive pneumonia virus or maedi-visna, is present in 24% of US sheep. Like human immunodeficiency virus, SRLV is a macrophage-tropic lentivirus that causes lifelong infection. The production impacts from SRLV are due to a range of disease symptoms, including pneumonia, arthritis, mastitis, body condition wasting and encephalitis. There is no cure and no effective vaccine for preventing SRLV infection. However, breed differences in prevalence and proviral concentration indicate a genetic basis for susceptibility to SRLV. Animals with high blood proviral concentration show increased tissue lesion severity, so proviral concentration represents a live animal test for control post-infection in terms of proviral replication and disease severity. Recently, it was found that sheep with two copies of TMEM154 haplotype 1 (encoding lysine at position 35) had lower odds of SRLV infection. In this study, we examined the relationship between SRLV control post-infection and variants in two genes, TMEM154 and CCR5, in four flocks containing 1403 SRLV-positive sheep. We found two copies of TMEM154 haplotype 1 were associated with lower SRLV proviral concentration in one flock (P < 0.02). This identified the same favorable diplotype for SRLV control post-infection as for odds of infection. However, frequencies of haplotypes 2 and 3 were too low in the other three flocks to test. The CCR5 promoter deletion did not have consistent association with SRLV proviral concentration. Future work in flocks with more balanced allele frequencies is needed to confirm or refute TMEM154 association with control of SRLV post-infection.

Deletion variant near ZNF389 is associated with control of ovine lentivirus in multiple sheep flocks.

Ovine lentivirus (OvLV) is a macrophage-tropic lentivirus found in many countries that causes interstitial pneumonia, mastitis, arthritis and cachexia in sheep. There is no preventive vaccine and no cure, but breed differences suggest marker-assisted selective breeding might improve odds of infection and control of OvLV post-infection. Although variants in TMEM154 have consistent association with odds of infection, no variant in any gene has been associated with host control of OvLV post-infection in multiple animal sets. Proviral concentration is a live-animal diagnostic measure of OvLV control post-infection related to severity of OvLV-induced lesions. A recent genome-wide association study identified a region including four zinc finger genes associated with proviral concentration in one Rambouillet flock. To refine this region, we tested additional variants and identified a small insertion/deletion variant near ZNF389 that showed consistent association with proviral concentration in three animal sets (P < 0.05). These animal sets contained Rambouillet, Polypay and crossbred sheep from multiple locations and management conditions. Strikingly, one flock had exceptionally high prevalence (>87%, including yearlings) and mean proviral concentration (>950 copies/µg), possibly due to needle sharing. The best estimate of proviral concentration by genotype, obtained from all 1310 OvLV-positive animals tested, showed insertion homozygotes had less than half the proviral concentration of other genotypes (P < 0.0001). Future work will test additional breeds, management conditions and viral subtypes, and identify functional properties of the haplotype this deletion variant tracks. To our knowledge, this is the first genetic variant consistently associated with host control of OvLV post-infection in multiple sheep flocks.

Prevalência de anticorpos contra os agentes da maedi-visna e clamidofilose em ovinos no município de Uberlândia, MG / Prevalence of antibodies against the agents of maedi-visna and chlamydophilosis in sheep from Uberlândia county, MG, Brazil

Objetivou-se com este estudo realizar o primeiro inquérito soro-epidemiológico para o vírus da maedi-visna e Chlamydophila spp. em 12 rebanhos de ovinos do Município de Uberlândia, MG. Foram utilizadas 334 amostras de soro sanguíneo de ovinos e aplicou-se um inquérito epidemiológico a cada propriedade. Os testes realizados para a pesquisa de anticorpos contra o vírus da maedi-visna e Chlamydophila spp. foram imunodifusão em gel de ágar (IDGA) e reação de fixação do complemento (RFC), respectivamente. Não foram detectados ovinos reagentes para maedi-visna. Verificou-se uma prevalência de 3,3% para Chlamydophila spp., com títulos variando de 32 a 64. Não houve diferença estatística significativa (p > 0,05) para os fatores de risco analisados. Ressalta-se a importância dos sistemas de vigilância epidemiológica para atuar no controle dessas infecções, evitando a introdução do vírus da maedi-visna e uma maior propagação da Chlamydophila spp. neste município.

The aim of this study was to carry out the first investigation into the serological prevalence of maedi-visna virus and Chlamydophila spp. on 12 sheep breeding farms in Uberlândia County, MG, Brazil. A total of 334 blood serum samples were used and an epidemiological survey was completed by each farm. The tests to detect maedi-visna and Chlamydophila spp. antibodies were an agar gel immunodiffusion (AGID) and a complement fixation test (CFT), respectively. None of the sheep were reactive to maedi-visna. The detection of antibodies against Chlamydophila spp. was 3.3%, with titers varying from 32 to 64. There was no statistically significant difference (p > 0.05) in regard to the risk factors analyzed. The importance of epidemiological surveillance systems to aid in the control of these infections is emphasized, in order to avoid the introduction of maedi-visna virus and a wider spread of Chlamydophila spp. in this county.