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Visna/maedi (VM) is a multisystemic disease of sheep characterized by persistent lentiviral infection, slow progression and eventually death. The VM virus (VMV) affects sheep throughout the world and is the focus of national eradication campaigns. A major host gene associated with infection has been identified in North American sheep (TMEM154); however, its effect is unknown in Turkish sheep. Our aim was to determine VMV seroprevalence in naturally infected Turkish sheep, characterize their TMEM154 alleles, and test for association with infection. A 2017 serological census was taken of 2266 ewes from 11 flocks, at six locations, comprising seven native and four composite Turkish breeds. VMV serum antibodies were measured with an enzyme linked immunosorbent assay. Eight of 11 flocks were infected with VMV (2 to 83% seroprevalence). TMEM154 variants were typed by sequencing exon 2 from all 287 seropositive ewes and a subset of their seronegative flockmates (1059 total). TMEM154 sequencing revealed five of 12 known haplotypes encoding missense mutations, and one previously unreported variant (G38R) in Kivircik sheep. VMV seroprevalence in ewes with highly-susceptible TMEM154 haplotypes (full length E35 variants) was higher than the overall flock seroprevalence and independent of breed type. Genetic association was tested in 76 matched case-control pairs from 751 comingled ewes from a research flock. Pairwise analyses showed the risk of infection was 3-fold greater for ewes with one or two copies of highly-susceptible TMEM154 haplotypes compared to those with combinations of K35 and deletion variants (CI95 1.3–8.7, p-value 0.009). Allelic combinations of TMEM154 K35 and deletion variants had an apparent protective effect against VMV strains in Turkish sheep. The low frequencies of K35 and deletion alleles in native Turkish breeds suggests selective breeding may help reduce the seroprevalence in affected flocks and decrease the risk of outbreaks in VMV-free flocks.

Visna/maedi (VM) is a multisystemic lentivirus infection of sheep that affecting sheep industry across the globe. TMEM154 gene has been identified to be a major VM-associated host gene, nevertheless, a recent study showed that the frequency of the VM-resistant TMEM154 haplotypes was very low or absent in indigenous sheep. Thus, the present study was designed to determine other possible co-receptors associated with VM. For this purpose, DRB1 gene, which is renowned for its role in host immune response against various diseases was targeted. A total number of 151 case-control matched pairs were constructed from 2266 serologically tested sheep. A broad range of DRB1 haplotype diversity was detected by sequence-based genotyping. Moreover, a novel 2 bp deletion (del) in the DRB1 intron 1 was identified. For the final statistic, the sheep carrying VM-resistant TMEM154 diplotypes were removed and a McNemar's test with a matched pairs experimental design was conducted. Consequently, it was identified for the first time that the 2 bp del variant is a genetic risk factor for VM (p value 0.002; chi-square 8.31; odds ratio 2.9; statistical power 0.90) in the dominant model. Thus, negative selection for 2 bp del variant could decrease VM infection risk in Turkish sheep., Republic of Turkey Ministry of Agriculture and Forestry, General Directorate of Agricultural Research and Policies (TAGEM) [TAGEM/HAYSUD/15/A01/P02/02-02], Funding for this research was provided by the Republic of Turkey Ministry of Agriculture and Forestry, General Directorate of Agricultural Research and Policies (TAGEM) (Project No. TAGEM/HAYSUD/15/A01/P02/02-02).

Background Maedi/Visna virus (MVV) is a contagious viral pathogen that causes considerable economic losses to the sheep industry worldwide. Objectives In China, MVV has been detected in several regions, but its molecular characteristics and genetic variations were not thoroughly investigated. Methods Therefore, in this study, we conducted next-generation sequencing on an MVV strain obtained from northwest China to reveal its genetic evolution via phylogenetic analysis. Results A MVV strain obtained from Inner Mongolia (NM) of China was identified. Sequence analysis indicated that its whole-genome length is 9193 bp. Homology comparison of nucleotides between the NM strain and reference strains showed that the sequence homology of gag and env were 77.1%–86.8% and 67.7%–75.5%, respectively. Phylogenetic analysis revealed that the NM strain was closely related to the reference strains isolated from America, which belong to the A2 type. Notably, there were 5 amino acid insertions in variable region 4 and a highly variable motif at the C-terminal of the surface glycoprotein (SU5). Conclusions The present study is the first to show the whole-genome sequence of an MVV obtained from China. The detailed analyses provide essential information for understanding the genetic characteristics of MVV, and the results enrich the MVV library.

Visna/Maedi virus (VMV) is a lentivirus that infects the cells of the monocyte/macrophage lineage in sheep, goats and wild ruminants. Infection with VMV causes a multisystemic inflammatory disorder, which includes pneumonia, encephalitis, mastitis or arthritis. The immune response to VMV infection is complex, and the infection and pathogenesis of this virus are not totally characterized yet. In this work, a gene expression microarray was used to identify the differentially ex-pressed genes in VMV infection and disease development by comparing sheep with different serologic status and with presence of VM-characteristic clinical lesions. The expression profile analysis has revealed many interesting genes that may be associated with the viral infection process. Among them, the OXT gene appeared significantly up-regulated, so the oxytocin-secreting system could play an essential role in VM disease. Moreover, some of the most significantly enriched functions in up-regulated genes appeared the complement pathway, which (in combination with the Toll-like receptor signaling network) could compose a mechanism in the VMV pathogenesis. Identifying the host genetic factors associated with VMV infection can be applied to develop strategies for preventing infection and develop effective vaccines that lead to therapeutic treatments., Financial support for this work was provided by the UPV/EHU (grants GIU14/23, UFI11/20 and PPGA18/11 to BMJugo) and a postdoctoral grant to Naiara Abendaño (ESP-DOC16/43).

The study was conducted with the objectives of determining the possible causes of pneumonia in the sheep and to characterize the microscopic lesions of concurrent infection of Maedi-Visna with lung worms and other infectious pneumonia. Detailed gross and microscopic examinations were employed on 35 Maedi-Visna seropositive sheep. Out of 35 sheep slaughtered and examined for gross lesions on their lungs, lung worms, fibrinous adhesions, and mottling on the cranioventral lobes were observed with decreasing order of frequency. Histopathologically, the pneumonic lungs (n = 35) revealed Maedi (100%), Maedi-Verminous pneumonia complex (28.57%, n = 10), Maedi bronchopneumonia complex (17.14%, n = 6), and Maedi-pulmonary adenomatosis complex (2.86%, n = 1). The occurrence of high proportion of concurrent infection of Maedi-Visna with lung worms and bacteria strongly suggest synergy of the pathogens and the presence of several stress factors including wind draft, unusually chilly weather and poor management in the area that are known to predispose the improved sheep breeds to respiratory disease complex. On top of culling by slaughter of Maedi-Visna seropositive sheep, regular deworming, redesigning the house, and vaccination of sheep with the available vaccines should be carried out timely at least to reduce the severity of Maedi-Visna induced loss.

Ovine visna/maedi (VM) infection is characterized by the development of chronic inflammatory lesions in different organs, mainly in the lung, mammary gland and central nervous system (CNS), with either histiocytic or lymphocytic pattern predominance being described in the CNS. To help to understand the role of host immune response in the development of these patterns, 50 naturally-infected sheep and eight non-infected sheep from intensive milk-producing flocks were studied. The histological lesion patterns in the three main target organs in each sheep were characterized. Lesion severity was determined, including minimal lesions. A histiocytic pattern was observed in 23 sheep (46%), a lymphocytic inflammatory pattern in 19 sheep (38%) and a mixed inflammatory pattern in eight sheep (16%). Forty animals showed moderate or severe lesions (80%), while 10 had minimal lesions (20%). Moderate or severe lesions affected only one target organ in 20 sheep (50%), two organs in 14 sheep (35%) and all three target organs in six sheep (15%). Infection was confirmed by immunohistochemistry (IHC) using an antibody specific for p28 of VM virus/caprine arthritis and encephalitis virus and by polymerase chain reaction (PCR) in all sheep. Minimal inflammatory lesions associated with positive IHC and PCR were observed. The results suggest that the development of a predominant inflammatory pattern in different organs within the same animal may be related to the host immune response. Minimal and focal lesions, not considered previously, should be taken into account when formulating a differential diagnosis in affected sheep.

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.

In Switzerland, viruses belonging to two different phylogenetic groups of small ruminant lentiviruses (SRLV) are currently circulating: the caprine arthritis-encephalitis virus (CAEV) and visna/maedi virus (VMV). In the past two decades, a mandatory national control program has led to a very low prevalence of seropositivity, while completely eliminating CAE as a clinical manifestation. However, in order to reduce the high costs and effort associated with this program, adjustments based on the most recent epidemiological knowledge are needed. The purpose of this study was to estimate the seroprevalence of CAEV and VMV using the newest diagnostic tools available, and to identify potential risk factors for infection with these viruses in Switzerland. For the prevalence estimation, a census was carried out including 10,696 farms with a total of 85,454 goats. Blood samples were analysed using a 3-step serological testing algorithm consisting of Chekit ELISA, Western Blot and SU5 ELISA. A risk factor analysis was conducted using logistic regression models built with data obtained from a mail questionnaire, and serological results from the census. The apparent herd-level prevalences were 0.38%, 2.77%, and 3.04% for CAEV, VMV and SRLV, respectively. Animal-level prevalences were 0.06% for CAEV, 0.55% for VMV, and 0.61% for SRLV. No statistically significant risk factors associated with CAEV or VMV infection were identified. However, the proportional high number of CAEV seropositive dwarf goats, in relation to their population size, could indicate that these hobby breeds may slip through some of the official controls. For an infection with SRLV, a medium herd size (7-40 goats) was found to be protective, compared with smaller (OR=1.90, p=0.034) and larger herds (OR=1.95, p=0.038). In conclusion, considering that all CAEV positive animals were culled, these results imply that CAEV is no longer actively spreading and has successfully been controlled in Switzerland. However, given the uncertain pathogenic potential of VMV in goats, future surveillance should also be taking into account the not insignificant number of VMV circulating in the Swiss goat population.

U Licinijanovom prikazu rimskog ratovanja u zaleđu Makedonije (Gran. Licin. 35.79-81 Criniti), 85. godine pre Hrista, spominju se dve kampanje protiv balkanskih plemena. Tekst nije sačuvan u potpunosti, stoga dopušta različita čitanja. U skladu sa interpretacijom Licinijanovog teksta koja je predložena u ovom radu, razmotreni su odnosi između Rima i pojedinih balkanskih varvara, naročito u vreme Prvog Mitridatovog rata. Osim toga, analizirano je hronološko određenje koje stoji uz Hortenzijevu kampanju. In Granius Licinianus' description (35.79-81 Criniti) of the Roman war in the hinterland of Macedonia in 85 B.C., two independent Roman campaigns against the Balkan tribes are mentioned. The fragmentary text has given rise to emendations, thus admits different readings. It is proposed here an interpretation of the text that seems the most plausible, on both historical and linguistic grounds. Relying on this interpretation, it seems proper to reconsider some common observations regarding certain Balkan tribes and their part in the First Mithridatic war, as well as the timing of the Hortensius' military campaign.

Visna/Maedi is a disease of sheep caused by small ruminant lentivirus (SRLV) infection that is widespread throughout the world and that has been recognized to be present in the Basque Country (Spain) since the early 1980′s. Nearly seven decades of studies have improved the knowledge on its clinical signs and epidemiology. However, its slow progressive nature, subclinical most of the time, makes difficult to assess its real impact on productive traits, a question of critical importance to balance out the economic costs it causes and the benefits of designing and deploying an eradication program. Development of a dairy breeding program since the 90 s in the local Latxa sheep population has provided data on milk productivity in several flocks where SRLV infection prevalence has been continuously monitored. This study analyses retrospectively the association between SRLV prevalence and production variables during ten yearly lactations in three Latxa dairy flocks with medium-high SRLV seroprevalence. Our results indicate that average standard lactation of seropositive sheep was 6.7 % lower than controls. The largest differences (p < 0.001) were observed at the ewe lifetime peak of production between second and fourth lactations. Lifelong milk and lamb production data indicated even a higher impact, with costs rising up to nearly 50 €/ewe/year. This substantial production decrease associated with subclinical SRLV infection in Latxa dairy sheep supports the benefit of establishing a SRLV control program. A rough cost-benefit analysis indicated that even in a medium-yielding breed, testing expenses would be largely covered by milk production improvement. © 2020 Elsevier B.V. &nbsp

A total of 1425 ovine lungs were grossly examined at the local abattoir during a 6-month survey on the aetiology and pathology of pneumonia in slaughtered sheep at the Al-Qassim region, Kingdom of Saudi Arabia. The results of the survey demonstrated the occurrence of several types of pneumonic and related lesions in 140 specimens of condemned lungs. However, more detailed microscopic examination of the pneumonic lungs incidentally revealed the coexistence of some other significant pathological alterations indicative of sheep pulmonary adenomatosis (Jaagziekte), ovine syncytial virus infection, ovine progressive pneumonia (maedi) and verminous pneumonia. In addition, pulmonary corpora amylacea (PCA) were also detected in the bronchial lumen of some pneumonic lungs initially diagnosed as fibrinous pneumonia. It is worth mentioning that none of the abovementioned cases was previously recognized or diagnosed in the Al-Qassim region or in any other parts of the Saudi Kingdom.

Visna/Maedi virus (VMV), a small ruminant lentivirus responsible for lymphoproliferative pneumonia, encephalitis, arthritis and/or mastitis in sheep, has been detected in different non-lymphoid organs. However, only a few investigations have been carried out in lymphoid tissues. In this study, some lymphoid tissues and lymph node draining or non-draining VMV target organs from five sheep infected experimentally by the respiratory route three years previously were investigated. Archival samples of spleen, red bone marrow, caudal mediastinal lymph nodes, mammary lymph nodes, popliteal lymph nodes and mesenteric lymph nodes were tested by PCR for the presence of proviral DNA. Popliteal and mesenteric lymph node samples were tested also by immunohistochemi - cal staining of the viral capsid antigen p28. The proviral DNA was detected by PCR in all the lymphoid tissue samples from the infected sheep. The viral antigen was stained in mononuclear cells in popliteal and mesenteric lymph nodes of the infected sheep. Although the lymph nodes draining the classical target organs seem to be more infected than the others, both the viral capsid antigen and the proviral DNA were present also in lymph nodes draining non-target organs, such as the mesenteric lymph nodes. These findings show the presence of VMV in different lymphoid tissues in the late stages of infection and suggest a potential role of these tissues as a site for viral reservoir and replication, even three years after infection.

Small ruminant lentiviruses include members that infect sheep (ovine lentivirus [OvLV]; also known as ovine progressive pneumonia virus/maedi-visna virus) and goats (caprine arthritis encephalitis virus [CAEV]). Breed differences in seroprevalence and proviral concentration of OvLV had suggested a strong genetic component in susceptibility to infection by OvLV in sheep. A genetic marker test for susceptibility to OvLV has been developed recently based on the TMEM154 gene with validation data from over 2,800 sheep representing nine cohorts. While no single genotype has been shown to have complete resistance to OvLV, consistent association in thousands of sheep from multiple breeds and management conditions highlight a new strategy for intervention by selective breeding. This genetic marker-assisted selection (MAS) has the potential to be a useful addition to existing viral control measures. Further, the discovery of multiple additional genomic regions associated with susceptibility to or control of OvLV suggests that additional genetic marker tests may be developed to extend the reach of MAS in the future. This review will cover the strengths and limitations of existing data from host genetics as an intervention and outline additional questions for future genetic research in sheep, goats, small ruminant lentiviruses, and their host-pathogen interactions.

Maedi and visna are contagious sheep diseases which were introduced into Iceland in 1933 by imported sheep of Karakul breed. Maedi, a slowly progressing pneumonia, and the central nervous system disease visna were shown to be transmissible in sheep and most likely caused by a virus. In 1957, visna virus was isolated in tissue culture from sheep brain and maedi virus was isolated the following year from sheep lungs. Both viruses showed similar cytopathic effect in tissue culture. Electron microscope studies of ultrathin sections from visna virus infected cells demonstrated spherical particles, 70-100 nm in diameter, which were formed by budding from the cell membrane. Later studies showed identical particles in maedi virus infected cultures. These, and several other comparative studies, strongly indicated that maedi and visna were caused by strains of the same virus, later named maedi-visna virus (MVV). Comparative studies in tissue culture suggested that MVV was related to RNA tumor viruses of animals, the oncornaviruses. This was later supported by the finding that MVV is an RNA virus. A few months after reverse transcriptase was demonstrated in oncornaviruses, the enzyme was also found in MVV virions. Thus, MVV was classified as a retrovirus together with the oncornaviruses. However, MVV is not oncogenic in vivo or in vitro and was in 1975 placed in a subgroup of retroviruses named lentiviruses, which cause cytopathic effect in vitro and slowly progressing inflammatory disease in animals, but are nononcogenic. In the early 1980s, the causative agent of AIDS was found to be a non-oncogenic retrovirus and was classified as a lentivirus. Thus, HIV became the first human lentivirus.

The aim of the present transversal descriptive study was to determine the exposure and risk factors associated with caprine arthritis-encephalitis virus (CAEV) in southern Spain. A total of 3,312 serum samples were collected from goat belonging to three different breeds coming from 48 flocks located in different geographic areas from southern Spain. In addition, health and productive parameters were surveyed during the visit to the herds. Serum samples were analysed by INgezim Maedi Screening (Ingenasa®) ELISA kit. The total percentage of herds exposed to CAEV was 87.71% (CI95 78.42–97.00). A total of 733 goats were seropositive with overall seroprevalence of 23.22% (CI95 21.78–24.65). The intraherd seroprevalence was 20.82%±24.07. Multivariate logistic regression showed significant association between CAEV and the next variables: (i) herd size (P

Highlights ► Mammalian review. ► Important veterinary viral immunodiseases. ► Immunoprophylaxis. ► Other control measures. ► Current status of vaccine research., This paper offers an overview of important veterinary viral diseases of mammals stemming from aberrant immune response. Diseases reviewed comprise those due to lentiviruses of equine infectious anaemia, visna/maedi and caprine arthritis encephalitis and feline immunodeficiency. Diseases caused by viruses of feline infectious peritonitis, feline leukaemia, canine distemper and aquatic counterparts, Aleutian disease and malignant catarrhal fever. We also consider prospects of immunoprophylaxis for the diseases and briefly other control measures. It should be realised that the outlook for effective vaccines for many of the diseases is remote. This paper describes the current status of vaccine research and the difficulties encountered during their development.

FIRST ISOLATION OF SMALL RUMINANT LENTIVIRUS FROM A NATURALLY INFECTED GOAT IN A FLOCK IN THE STATE OF RIO GRANDE DO NORTE, BRAZIL. Caprine Arthritis Encephalitis virus (CAEV) and Visna/maedi virus (VISNA), belong to the genus Lentivirus family Retroviridae. The aim of the present study was to isolate the CAE virus from a positive goat when tested by agarose gel immunodifusion (AGID) by cocultivation techniques of infected peripheral blood leukocytes in goat synovial membrane (GSM). In this study, one flock of 16 goats, obtained from a flock of Rio Grande do Norte, Brazil, with clinical suspicions for CAEV, was screened using agar-gel immunodiffusion (agid) and Western blotting. The result was positive for one animal, confirmed by Western blotting. For virus isolation, peripheral blood leukocytes (PBL) were isolated from the blood for co-culture on goat sinovial membrane (GSM) cells. Monocytes/ macrophages collected were inoculated in a monolayer of 90% semiconfluent cells in the A25 culture bottles. After 50 days of co-culture, the monolayer was stained with crystal violet 0.1% for viewing viral cytopathic effects (CPE) characteristic of viruses, and nested-PCR was performed with the supernatant of co-culture for confirmation of CPE. The isolate, named BrRN-CNPC.G1, was considered the first isolation of small ruminant lentivirus from a naturally infected goat in a flock in Rio Grande do Norte, Brazil. This study will soon allow for the molecular characterization of the isolated virus genome, by analysis of its different structural genes and their comparison with other isolated viral sequences to identify the likely source of infection of that animal and establish the possible differences between strains of lentiviruses and circulating regional strains.

Maedi Visna virus (MVV) causes progressive degenerative inflammatory disease in multiple organs including the lungs (pneumonia, 'maedi'), mammary gland, joints and nervous system (meningoencephalomyelitis, 'visna') in sheep. Maedi Visna Virus has been detected in macrophages of several tissues and epithelial cells in vivo: bone marrow, cells of the central nervous system, lung and bronchial tissues, milk epithelial cells recovered from milk samples and epithelial cells of mammary tissue. However, the presence of MVV in the genital tracts of naturally infected ewes has not previously been studied. The aim of this study was to use nested-PCR, targeting the gag gene, to determine whether genital tissues (ovaries, oviducts and uterus) from 83 ewes originating from various breeding herds in the South-East of France were positive for MVV-proviral DNA. Peripheral blood mononuclear cells (PBMC) tested positive for MVV-proviral DNA, using nested-PCR analysis, in 57.8% of ewes (48/83). The provirus was also identified in 47% (78/166) of the ovaries, 38.6% (64/166) of the oviducts and 45.8% (38/83) of the uteri sampled. These findings clearly demonstrate, for the first time, that tissue samples from the genital tract of ewes (ovary, oviduct and uterus) can be infected with MVV. This suggests that there is a risk of vertical and/or horizontal transmission of MVV during embryo transfer from embryos produced in vivo or in vitro.

Caprine Arthritis Encephalitis virus (CAEV) and Visna/maedi virus (VISNA), are considered to be genetically distinct but antigenically related pathogens of goats and sheep. In this study, one flock of 250 goats was screened using Agar-gel Immunodiffusion (AGID), and the level of seroprevalence observed was 11 animals (4.4%) of positive tested serum. Four goats positive to AGID were analyzed using a Polymerase Chain Reaction (PCR), which amplifies part of the gag gene. All the animals were found to be positive. The nucleotide sequences of gag gene were determined from four BR/CNPC lentivirus isolates from naturally infected goats. The four gag gene sequences BR/CNPC small ruminant lentivirus (SRLV) were compared to GenBank, and the results demonstrated that these sequences were more related to the caprine rather than the ovine strains. Further phylogenetic analysis of the proviral gag sequences showed that they constituted subtype B1 of the CAEV group. The analyses of the sequences also showed that the viruses were genetically stable.

Zusammenfassung In 10 Texelschafbestanden wurden pathologisch-anatomische und histologische Untersuchungen uber die Maedi-Pneumonie durchgefuhrt. In einem dieser Bestande, in dem neben den Texelschafen auch die Heidschnucken erkrankt waren, erfolgte die Isolierung und Identifizierung des Virus durch Straub (19). Die Mehrzahl der anderen Bestande hatte von dieser Herde Zuchttiere erworben. Die typischen klinischen Symptome fortschreitender Dyspnoe und Abmagerung werden wegen der langen “praeklinischen Phase” uberwiegend bei mehrjahrigen Tieren beobachtet, aber auch 1jahrige Schafe konnen schon erkranken. Markoskopisch findet man in den Spitzen-, Mittel-und Hauptlappen der Lungen graue konfluierende, feste Infiltrate. Die auch in fortgeschrittenem Stadium noch dazwischen liegenden lufthaltigen Gewebsinseln erklaren die lange klinische Symptomlosigkeit. Das histologische Bild ist gekennzeichnet durch Transformation der Alveolarzellen, Bildung peribronchialer Lymphfollikel und interalveolare Zellinfiltration. Diese Prozesse sollten ihrem Wesen nach als “progressive” Pneumonie definiert werden. Differentialdiagnostisch mussen besonders die Lungenadenomatose und chronische Lungenwurmpneumonien von der Maedi-Pneumonie abgegrenzt werden. Summary The picture in chronic progressive pneumonia (Maedi) in sheep In 10 breeding flocks of Texel sheep, pathological and histological studies were carried out on Maedi pneumonia. In one flock in which not oonly Texel sheep but also Heidschnucken sheep were affected, the virus was isolated and identified by Straub. Most of the other flocks had received breeding animals from this flock. The typical clinical symptoms of progressive dyspnoea and emaciation were observed mainly in older sheep because of the long “pre-clinical” phase, but one-year old sheep were also affected. Macroscopically there was a grey confluent solid infiltration of the aqical, middle and chief lobes of the lungs. The surviving air-filled islets of lung between the affected areas, which were present even in advance stages, explain the long period free from clinical symptoms. The serological picture is characterised by transformation of alveolar cells, formation of peribronchial lymph follicles and inter-alveolar cellular infiltrations. The nature of these processes designates the condition as a “progressive” pneumonia. Differential diagnosis must take into account especially pulmonary adenomatosis and chronic parasitic pneumonia. Resume Contribution a la pneumonie progressive chronique (Maedi) du mouton On a entrepris des examens anatomo-pathologiques et histologiques sur la pneumonie de Maedi dans 10 exploitations de moutons du Texel. Dans une de ces exploitations, dans laquelle non seulement les moutons du Texel, mais aussi les brebis des landes etaient atteints de la maladie, Straub (19) est parvenu a isoler et identifier le virus. La plupart des autres exploitations avaient acquis dans ce troupeau des animaux reproducteurs. On observe les symptomes cliniques typiques, dyspnee progressive et amaigrissement, principalement chez des animaux plus ages, a cause de la longue phase preclinique, mais des moutons ages d'une annee peuvent deja tomber malades. A l'examen macroscopique, on trouve des infiltrations grises confluentes denses dans les lobes du sommet et les lobes moyens et principaux des poumons. Les ilots tissulaires remplis d'air intercales entre ces infiltrations, měme dans un stade avance, expliquent la longue phase exempte de symptomes cliniques. L'image histologique est caracterisee par une transformation des cellules alveolaires, une formation de follicules lymphatiques peribronchiques et une infiltration cellulaire interalveolaire. D'apres leur essence měme, ces processus peuvent ětre definis comme pneumonie «progressive». Un diagnostic differentiel doit distinguer la pneumonie de Maedi de l'adenomatose pulmonaire et des pneumonies vermineuses chroniques. Resumen En 10 rebanos de ovejas Texel se efectuaron estudios anatome e histopatologicos sobre la neumonia Maedi. En uno de estos rebanos, en el cual al lado de ovejas Texel tambien habian enfermado algunas pertenecientes a la raza de Lueneburgo, se verifico el aislamiento e identificacion del virus por Straub (19). La mayoria de los rebanos restantes habian adquirido animales de cria en esta explotacion. Los sintomas clinicos tipicos de disnea progresiva y emaciacion se observan, a causa de la «fase preclinica» prolongada, en los animales que cuentan varios anos de edad, aunque tambien pueden enfermar ovejas de 1 ano de edad. Macroscopicamente, se hallan en los lobulos apicales, cardiacos y diafragmaticos de los pulmones unas infiltraciones grises, confluentes y compactas. Las insulas histicas, que engloban aire y las que aun se encuentran entremedias en el estadio progresivo, explican la falta prolongada de sintomas clinicos. La imagen histopatologica se caracteriza por la transformacion de las celulas alveolares, formacion de foliculos linfaticos peribronquiales e infiltracion celular interalveolar. Estos procesos deberian definirse con arreglo a su esencia como neumonia «progresiva». Desde el punto de vista del diagnostico diferencial, se tienen que delimitar sobre todo la adenomatosis pulmonar y las neumonias verminosas cronicas con relacion a la neumonia Maedi.

Ovine pulmonary adenocarcinoma (OPA) and Maedi-Visna (Maedi) are two chronic respiratory diseases of retroviral origin which occur worldwide. It is known that different host genetic factors influence the outcome of viral infections. To determine if variation in the Mhc-DRB1 gene was associated with progression to these ovine diseases, sheep lungs with and without OPA and Maedi lesions were collected. A sequence-based method was applied and 40 different alleles were detected in the sample analysed. In the allele-by-allele association analysis, allele DRB1*0325 had a significant association with susceptibility to Maedi (P = 0.045). For OPA, DRB1*0143 and DRB1*0323 were significantly associated with susceptibility (P = 0.024 and P = 0.029), and allele DRB1*0702 was significantly associated with resistance (P = 0.012). Based on these results, the Mhc-DRB1 alleles were classified by effect in three categories-susceptible (S), resistant (R) and neutral (N)-and animals were reassigned the genotypes as S/S, S/R, S/N, R/R, R/N and N/N. In a second analysis, penalised logistic regression models including a flock effect were run. In Maedi, significant association was detected for the N/S heterozygote (P = 0.0007), but not for the S/S homozygote, probably as a result of the low number of S/S animals. In OPA, association was detected for both the S/S and R/R homozygotes (P = 0.005 and P = 0.047). This allele grouping method may be applied in association studies with highly variable genes. This is the first study demonstrating significant associations between sheep Mhc-DRB1 alleles and susceptibility to OPA and Maedi. Therefore, both diseases are suitable candidates for more comprehensive genetic studies.

International audience; Small ruminant lentiviruses (SRLV) are widespread amongst domesticated goats and sheep worldwide, but have not been clearly identified in wild small ruminants, where they might constitute an animal health risk through contamination from local domesticates. SRLV proviruses from three ibexes from the French Alps are described and sequences from their gag gene and long terminal repeats (LTRs) were compared with sequences from local goats and goat/ibex hybrids. The ibex and hybrid proviruses formed a closely related group with

A major route of transmission ofVisna/maedi virus(VMV), an ovine lentivirus, is thought to be via the respiratory tract, by inhalation of either cell-free or cell-associated virus. In previous studies, we have shown that infection via the lower respiratory tract is much more efficient than via upper respiratory tissues (T. N. McNeilly, P. Tennant, L. Lujan, M. Perez, and G. D. Harkiss, J. Gen. Virol.88:670-679, 2007). Alveolar macrophages (AMs) are prime candidates for the initial uptake of virus in the lower lung, given their in vivo tropism for VMV, abundant numbers, location within the airways, and role in VMV-induced inflammation. Furthermore, AMs are the most likely cell type involved in the transmission of cell-associated virus. In this study, we use an experimental in vivo infection model that allowed the infection of specific segments of the ovine lung. We demonstrate that resident AMs are capable of VMV uptake in vivo and that this infection is associated with a specific up-regulation of AM granulocyte-macrophage colony-stimulating factor mRNA expression (P< 0.05) and an increase in bronchoalveolar lymphocyte numbers (P< 0.05), but not a generalized inflammatory response 7 days postinfection. We also demonstrate that both autologous and heterologous VMV-infected AMs are capable of transmitting virus after lower, but not upper, respiratory tract instillation and that this transfer of virus appears not to involve the direct migration of virus-infected AMs from the airspace. These results suggest that virus is transferred from AMs into the body via an intermediate route. The results also suggest that the inhalation of infected AMs represents an additional mechanism of virus transmission.

Lentiviral transmission by transfer of infected colostrum and/or milk is considered to be highly efficient. In this study, postpartum transmission of ovine progressive pneumonia virus (OPPV) from 10 naturally infected ewes to their 23 lambs was followed from the perinatal period throughout a four-year period. The lambs were allowed to suckle from their dam from birth through 32 weeks of age. Virus was tracked by virus isolation, quantitative PCR (qPCR), and anti-OPPV antibody responses as measured by cELISA. Cell-associated OPPV was isolated from colostrum/milk cells in 7 out of 10 ewes and provirus envelope (env) loads ranged 8 to 105 copies/μg DNA in colostrum/milk cells from the 10 ewes using qPCR. Provirus env loads were also detected in the peripheral circulation of 21 lambs at 8 weeks and two lambs at 22 weeks. The qPCR product at 8 weeks was confirmed as the transmembrane (tm) gene of OPPV by cloning and sequencing. Both cELISA titers ranging from 325 to 3125 and cross-neutralizing antibody titers ranging from 6 to 162 to seven different OPPV strains were found in the colostrum of the 10 ewes. Furthermore, cELISA titers in serum from lambs remained detectable through 32 weeks following the clearance of provirus at 24 weeks. After 32 weeks, both provirus and anti-OPPV antibody responses have subsequently remained undetectable through 4 years of age. These data suggest the clearance of cell-associated lentiviruses from lamb circulation after passive transfer of antibody via colostrum.

Summary Objective: This study summarizes the clinical, serological, pathomorphological and laboratory findings in 19 sheep with spontaneous Visna in Germany. The cases occurred over a period of 13 years in 13 flocks. In all affected sheep cytologic examination of cerebrospinal fluid (CSF) was performed. In addition in six flocks from which 12 of the Visna cases came the whole flock was tested for Maedi/Visna virus (MVV) antibodies. Material and methods: The diagnosis of spontaneous Visna with typical clinical signs was proven by serological and histopathological investigations. Serological screening for MVV antibodies was performed with the agargel-immuno- precipitation-test (AGIDT). Additionally, cerebrospinal fluid (CSF) was obtained by lumbosacral puncture and examined cytologically. Results: Nine of the 19 cases were Texels and six East Frisian Milk sheep. Six out of nine Texels came from the same flock with a MVV-antibody intra-flock prevalence of 0.94. The mean age of the sheep was 3.9 ± 2.3 years (0.5–10 years). Time between onset of clinical symptoms until euthanasia due to recumbency was 35 ± 23 days (6–102 days). In most cases clinical signs started with ataxia, staggering and circling, followed by increasing paresis of the hind legs. In the late stages of the disease 14 sheep suffered from tetraparesis. Trembling of lips or eye lids, blindness and scratching were rare signs. In all cases pleocytosis (11–876 M/l, mean = 167 M/l) with mononuclear cells, predominantly macrophages, was found. This finding provides an additional diagnostic aid to support the clinical differentiation from other infectious diseases with neurological manifestation in sheep. Histopathology of the CNS revealed in most cases a severe leukoencephalitis and demyelinisation with perivascular lymphocytic infiltration. Conclusion and clinical relevance: Visna is an important differential diagnosis in CNS disorders of sheep. It also occurs in animals younger than one year. Visna occurs especially in highly infected flocks of susceptible sheep breeds. CSF cytology can help to differentiate Visna from other CNS diseases. Clinically, Visna may be an important differential diagnosis to Scrapie.

15 páginas, 7 figuras, 1 tabla., Small ruminant lentiviruses include viruses with diverse genotypes that frequently cross the species barrier between sheep and goats and that display a great genetic variability. These characteristics stress the need to consider the whole host range and to perform local surveillance of the viruses to opt for optimum diagnostic tests, in order to establish control programes. In the absence of effective vaccines, a comprehensive knowledge of the epidemiology of these infections is of major importance to limit their spread. This article intends to cover these aspects and to summarise information related to characteristics of the viruses, pathogenesis of the infection and description of the various syndromes produced, as well as the diagnostic tools available, the mechanisms involved in transmission of the pathogens and, finally, the control strategies that have been designed until now, with remarks on the drawbacks and the advantages of each one. We conclude that there are many variables influencing the expected cost and benefits of control programs that must be evaluated, in order to put into practice measures that might lead to control of these infections.

In doubtful cases, the histopathological diagnosis of lesions induced by Maedi Visna virus (MVV), a chronic multisystemic lentiviral disease of sheep, needs to be confirmed by the demonstration of MVV in the tissues. The influence of fixatives and the duration of fixation on the detection of MVV by immunohistochemistry (IHC) and PCR in paraffin-embedded tissues was assessed in lung samples with lesions in different degree, from five sheep serologically positive. Samples were fixed in 10% neutral buffered formalin (NBF), Bouin's solution (BS) and a zinc salts-based fixative (ZSF), for different periods of time between 24 h and 30 days. The three fixatives preserved the morphology of the tissues, although in ZSF-fixed samples an increase in the number of desquamated cells was seen in the alveoli. Tissues showed a similar degree of immunolabelling, irrespective of the duration of fixation using ZSF and NBF fixatives. MVV nucleic acids could be detected in samples fixed up to 14 days in NBF and 30 days in ZSF. However, in BS fixed tissues, immunostaining was weak and non-specific signals were observed after 4 days of fixation. Amplification of proviral DNA could not be obtained by PCR in these samples. IHC detected viral antigens in all sheep whereas one sheep with mild lesions was always negative by PCR.

Bronchoalveolar lavage fluid was collected postmortem from the lungs of 113 sheep, and total and differential cell counts were analysed in relation to the presence of gross and microscopic lung pathology. The diffuse lung diseases, maedi and adenomatosis, were both characterised by an increase in overall cellularity and by increases in the percentages of lymphocytes and neutrophils, respectively. Focal parasitic lung disease was characterised by an increase in the percentage of eosinophils and mast cells. Consolidated lung lesions were characterised by a slight increase in cellularity but no change in the differential cell profile. In regions of parasitised and consolidated lungs without lesions the differential cell profile was consistent with focal lung pathology, although the slight increase in cellularity observed in the consolidated regions was not observed in the regions without lesions. A decision tree was developed to facilitate the interpretation and indicate the likely predictive capacity of the differential cytology of the fluid.

Four sheep were infected intratracheally with an attenuated molecular clone of maedi-visna virus (MVV). All four became infected. Ten months later these sheep were challenged intratracheally with a genetically similar but pathogenic clone of MVV. Four unvaccinated sheep were infected simultaneously. All sheep became infected by the challenge virus. The vaccinated sheep were not protected against superinfection with the challenge clone. However, virus was isolated more frequently from the blood of the unvaccinated controls than of the vaccinated animals and ten times more frequently from lungs of unvaccinated sheep than from lungs of vaccinated sheep at sacrifice, indicating partial protection.

The long terminal repeats (LTR) sequence divergence among Maedi Visna virus (MVV) isolates leads to LTRs with distinct transcriptional activities, which may result in distinct biological behaviours. The genetic heterogeneity, as well as basal and Tat-induced transcriptional activity of the LTRs from P1OLV and WLC-1 MVV viruses, slow/low and rapid/high isolates, respectively, have been examined and compared with LTRs from other strains of small ruminant lentiviruses (SRLV). Transfection assays using a reporter construct containing the LTR fused to a luciferase gene demonstrated that the LTR from P1OLV virus had the weakest promoter activity, suggesting a correlation between the level of promoter activity and the viral replication rate. To confirm this hypothesis, the promoter of P1OLV was cloned into infectious molecular clone KV1772kv72/67 and the resulting chimeric virus was tested for growth in various cell types. Compared to the parental KV1772, the LTR-chimeric virus KV1772/P1OLV exhibited a drastic reduction in replication rate in sheep choroid plexus (SCP) and lung cells, while in ovine macrophages and goat synovial membrane cells (GSM), chimeric virus showed a growth rate similar to that of parental virus. These observations suggest that the LTR is responsible for the slow/low in vitro phenotype presented by P1OLV in SCP and lung cells.

Maedi-visna is a systemic disease of sheep caused by a lentivirus, maedi-visna virus (MVV), which mainly affects the lungs and central nervous system but may also affect the mammary glands, joints and other tissues. The aim of the present study was to determine whether the third eyelid was affected in cases of systemic infection. Third eyelid and lung samples from sheep naturally infected with maedi were used. Total DNA was extracted from paraffin-wax-embedded tissues, and a nested polymerase chain reaction (PCR) was performed to amplify MVV proviral DNA. The samples were also tested by in-situ PCR and immunohistochemical methods specific for the detection of MVV proviral DNA and p25, respectively. All sheep showed moderate to severe chronic lymphoproliferative inflammation in the third eyelids. Products of the expected size were obtained by PCR from both lung and third eyelid tissue. In the nictitating membrane, MVV proviral DNA was detected in situ within macrophages, and glandular, ductal and surface epithelia. Immunohistochemistry demonstrated that the infection was productive. Taken together, these results indicate that the third eyelid may represent a target for natural MVV infection and may play a role in disease transmission.

A novel, simple polymerase chain reaction (PCR) protocol based on the amplification of a 291 base pair DNA fragment in the long terminal repeat (LTR) region of the Maedi-Visna (MV) provirus has been evaluated on samples collected from 115 sheep at the time of slaughter. The sheep came from Maedi-Visna virus (MVV) non-infected ( n =18) or MVV infected ( n =97) flocks, and the samples examined included peripheral blood leukocytes (PBLs; 115 sheep), milk cells (MCs; 64 sheep) and several tissue samples (TSs; 91 sheep). The LTR-PCR results were compared with the results of an indirect enzyme-linked immunosorbent assay (ELISA) and an agar-gel immunodiffusion test (AGIDT) performed on sera from the same animals obtained not only at the time of slaughter, but also in previous samplings. The LTR-PCR showed 100% specificity and an overall sensitivity of 98% in comparison with the two serological methods combined. Its sensitivity was lower when single types of samples were considered (84% in PBLs, 67% in MCs and 88% in TSs). It is concluded that this LTR-PCR can be used to complement serological tests and to perform studies of the pathogenesis of this lentiviral infection.

The present study was aimed for documenting various pathological conditions affecting the lungs of sheep and goats. A total of 1350 lungs were screened, out of which, 270 lungs (20%) exhibited pneumonic lesions; 121 in sheep (44.1%) and 149 in goats (55.1%). Out of 270 lungs selected for the study, 111/270 cases (41.1%) belonged to sheep and 55/270 cases (20.3%) to goats from slaughtered cases, and 10/270 cases (3.70%) to sheep and 95/270 cases (35.18%) to goats from fallen animals. The pneumonic lesions were classified as: acute bronchopneumonia (90 cases, 33.33%), sub-acute bronchopneumonia (18 cases, 6.66%), fibrinous bronchopneumonia (30 cases, 11.1%), suppurative bronchopneumonia (10 cases, 3.70%), chronic bronchopneumonia (15 cases, 5.5%), Peste des petits ruminants (PPR) (25 cases, 9.25%), broncho-interstitial pneumonia (5 cases, 1.85%), Maedi or ovine progressive pneumonia (3 cases, 1.11%), ovine pulmonary adenocarcinoma (31 cases, 11.48%), Hydatidosis (20 cases, 7.40%), atelectasis and emphysema (7 cases, 2.60%) and other nonspecific lesions (16 cases, 5.92%).

BACKGROUND Small ruminant lentiviruses escaping efficient serological detection are still circulating in Swiss goats in spite of a long eradication campaign that essentially eliminated clinical cases of caprine arthritis encephalitis in the country. This strongly suggests that the circulating viruses are avirulent for goats.To test this hypothesis, we isolated circulating viruses from naturally infected animals and tested the in vitro and in vivo characteristics of these field isolates. METHODS Viruses were isolated from primary macrophage cultures. The presence of lentiviruses in the culture supernatants was monitored by reverse transcriptase assay. Isolates were passaged in different cells and their cytopathogenic effects monitored by microscopy. Proviral load was quantified by real-time PCR using customized primer and probes. Statistical analysis comprised Analysis of Variance and Bonferroni Multiple Comparison Test. RESULTS The isolated viruses belonged to the small ruminant lentiviruses A4 subtype that appears to be prominent in Switzerland. The 4 isolates replicated very efficiently in macrophages, displaying heterogeneous phenotypes, with two isolates showing a pronounced cytopathogenicity for these cells. By contrast, all 4 isolates had a poor replication capacity in goat and sheep fibroblasts. The proviral loads in the peripheral blood and, in particular, in the mammary gland were surprisingly high compared to previous observations. Nevertheless, these viruses appear to be of low virulence for goats except for the mammary gland were histopathological changes were observed. CONCLUSIONS Small ruminant lentiviruses continue to circulate in Switzerland despite a long and expensive caprine arthritis encephalitis virus eradication campaign. We isolated 4 of these lentiviruses and confirmed their phylogenetic association with the prominent A4 subtype. The pathological and histopathological analysis of the infected animals supported the hypothesis that these A4 viruses are of low pathogenicity for goats, with, however, a caveat about the potentially detrimental effects on the mammary gland. Moreover, the high proviral load detected indicates that the immune system of the animals cannot control the infection and this, combined with the phenotypic plasticity observed in vitro, strongly argues in favour of a continuous and precise monitoring of these SRLV to avoid the risk of jeopardizing a long eradication campaign.

Paratuberculosis as well as the slow virus infections maedi/visna and jaagsiekte came to Iceland in 1933 when 20 sheep of the Karakul breed were imported from Halle, Germany. At least five of these sheep were subclinical carriers of paratuberculosis. Within 16 years paratuberculosis together with the other Karakul diseases (maedi/visna and jaagsiekte) almost ruined sheep farming, the main agricultural industry in Iceland. The first clinical case of paratuberculosis in sheep was confirmed in 1938, and in cattle in 1944. The first cattle cases of paratuberculosis appeared on farms where the disease had been prevalent in sheep for years. The virulence in cattle appeared to be considerably lower than in sheep. Extensive measures were used to control the spread of paratuberculosis in sheep. Hundreds of kilometres of fences were put up and used together with natural geographic borders to restrict the movement of sheep from infected areas. Serological and other immunological tests were also used to detect and dispose of infected individuals. These measures proved inadequate and the disease could not be eradicated. Culling and restocking of uninfected sheep in endemic areas eradicated maedi/visna and jaagsiekte but not paratuberculosis. Experiments showed that vaccination against paratuberculosis could reduce mortality in sheep by 94%. Vaccination of sheep in endemic areas has been compulsory in Iceland since 1966 and as a result losses have been reduced considerably. Today, serology is used to detect and control infection in cattle herds. Furthermore, serology is used to control vaccination of sheep and screen for infection in non-endemic areas. The complement fixation (CF) test for paratuberculosis has been used until now, but recently we have started comparing the CF test with the CSL absorbed ELISA test.

Viral load may be an important indicator of disease progression in sheep infected with maedi-visna virus (MVV). To assess this variable accurately in MVV-infected sheep, a quantitative competitive-polymerase chain reaction (QC-PCR) was developed. A conserved region of the MVV pol gene was selected. The RT-PCR MVV pol product was cloned and mutagenised in vitro by PCR to produce a competitor template reduced in length from 217 to 192 bp, but which retained the original flanking MVV pol PCR primers. The competitor template was quantified accurately and in an optimised QC-PCR protocol serial dilutions of this template were co-amplified with known amounts of sample DNA. MVV DNA levels in peripheral blood monocytes and alveolar macrophages from MVV-infected sheep (n=12) were assessed by QC-PCR. Viral DNA load in alveolar macrophages was significantly higher than that in peripheral blood monocytes when the animals were compared overall. A comparison was also made between alveolar macrophages from the lungs of seropositive animals with or without histopathological evidence of pulmonary lesions. The load of MVV DNA in alveolar macrophages was low in sheep without histopathological evidence of lesions in the lung. In contrast, in alveolar macrophages from sheep with histopathological lesions in the lung, there was a significantly higher level of MVV DNA. The correlation of MVV load with pulmonary lesions suggests that infected alveolar macrophages play a key role in the pathogenesis of this lymphoid interstitial pneumonia.

Visna virus is the prototype of the lentivirus subgroup of retroviruses. Lentivirus means slow virus; this refers to the fact that visna virus causes a slow infection in sheep which leads either to the lung disease maedi or more rarely to the central nervous system (CNS) disease visna. Maedi, meaning shortness of breath, is a slow progressive lymphoid interstitial pneumonia which was first described in South Africa and in Montana in the USA 80 years ago. Since then it has been found to be endemic in many countries in Europe and Asia [l]. Visna, which is a slowly progressing leukoencephalomyelitis, is rarely seen even in areas where the lung disease is prevalent. Thus, in a flock of sheep with endemic slow progressive pneumonia in the Rocky Mountains, where 76% of the sheep were affected by the lung disease, only 7% showed lesions in the CNS

During the epidemic caused by maedi-visna virus (MVV) of sheep in Iceland, the pulmonary affection, maedi, was the predominant clinical manifestation. In some flocks, however, a central nervous system (CNS) affection, visna, was the main cause of morbidity and mortality. As there is only one breed of sheep in the country, host factors did apparently not play an important role in the different clinical manifestations. To obtain some information on possible viral genetic determinants of neurotropism and neurovirulence we studied both phenotypic and genotypic properties of two maedi-visna virus strains; a strain that was originally isolated from the brain of sheep with encephalitis (visna), and another strain isolated from the lungs of a sheep suffering from pneumonia (maedi). The brain isolate was found to grow faster in sheep choroid plexus cells than the lung isolate, whereas the growth rate in macrophages was similar for the maedi and visna virus strains. Intracerebral inoculation indicated that the visna virus isolate induced more severe brain lesions than the maedi isolate. In addition, a pathogenic molecular clone derived from a visna strain (KV1772kv72/67) was tested for growth in sheep choroid plexus cells and macrophages. The molecularly cloned virus retained the fast growth rate in choroid plexus cells. The nucleotide sequence of the env gene and the U3 of the LTR was determined for the maedi strain and compared to that of the visna strains. There was an 11.7% difference in deduced amino acid sequence in the Env protein and a 6% difference in the LTR. The molecular clone KV1772kv72/67 will be a useful reagent for characterization of viral determinants of cell tropism in vitro and possibly neurovirulence in vivo.

Lentiviruses, a genus of retroviruses, include the agents that cause ovine progressive pneumonia or maedi. Infection is characterized by long incubation periods and insidious, slowly progressive clinical courses resulting in chronic degenerative diseases. The ovine lentiviruses are widespread among sheep in North America, yet their significance to livestock production is currently not well defined. Lentiviruses persist and replicate in the presence of host specific immune responses and cause immune-mediated lesions in several organ systems. Due to this unusual relationship with their host, the diagnosis, control and treatment of these infections is difficult and expensive. Scientific studies on the biology of ovine lentiviruses (OvLV) and their complex relationship with the host are yielding new approaches to the detection of infected animals and methods for study of the epizootiology and control of OvLV-related diseases. We summarize some of the known biological properties of the virus, spectrum of clinical features of the diseases, current concepts of disease pathogenesis, economic importance, and strategies used to diagnose and control OvLV infections.

Sheep naturally infected by visna-maedi virus often develop a chronic interstitial lung disease characterized by an alveolitis comprising lymphocytes, neutrophils and macrophages. The alpha chemokin interleukin-8 (IL-8) was detected in cell free bronchoalveolar lavage fluid from naturally infected animals, confirmed by RT-PCR, presenting typical lesions of maedi and elevated total alveolar cell counts. No detectable IL-8 was found in the fluid obtained from uninfected animals. IL-8 concentration in alveolar fluid is correlated with alveolar neutrophil counts. Bronchoalveolar lavage cells from infected animals were found to contain a large amount of IL-8 mRNA and may contribute to IL-8 production. In situ hybridization showed that macrophages were the predominant cell type expressing IL-8 mRNA. Sustained production of IL-8 by alveolar macrophages during visna-maedi infection could suffice for neutrophil attraction to the alveoli, and may contribute to the development of lesions.

This work was aimed at studying the effect of maedi-visna virus (MVV) infection in vitro on the ability of sheep cells to adhere to staphylococci (Staphylococcus aureus and Staphylococcus epidermidis), and phagocytose these bacteria. Adherence was studied in sheep choroid plexus cells (SCPC) using an ELISA test and phagocytosis was studied in pulmonary alveolar macrophages (PAM) by chemiluminescence. A 5- and 7-day of in vitro MVV infection resulted in syncytium formation and a significant increased adherence (P < 0.01) of SCPC to bacteria. SCPC endogenous fibronectin was significantly higher (P < 0.01) on days 5 and 7 than on day 0 of MVV infection. A significantly decreased phagocytosis (P < 0.05) was also observed on days 5 and 7 of MVV infection in PAM when compared to MVV-free controls. Comparatively, phagocytosis was highest for S. aureus non-slime producing strains, followed by S. epidermidis, and S. aureus slime producing strains, in that order. Finally, increased expression of both, class I and class II major histocompatibility antigens was also observed in MVV-infected PAM on days 5 and 7, whereas SCPC only demonstrated upregulation of MHC class I. These results, indicative of an alteration of some cell functions in MVV-infected cells, may help to understand interactions between MVV-infected cells and bacteria in simultaneous infections and may provide clues to the possible in vivo interactions of both pathogens.

Maedi/visna (MV) is a lentiviral disease of sheep caused by the maedi/visna virus (MVV). Although MV is prevalent in many countries, it had not been reported in Japan. In 2011, however, three sheep in northern Japan were reported to be seropositive against the MVV antigen, indicating a persistent MVV infection. In the present study, we isolated MVV from one sheep to confirm MVV infection and conducted genomic classification of the virus. The co-culture of leukocytes from a seropositive sheep with fetal goat lung cells resulted in the formation of syncytial cells and the amplification of a long terminal repeat sequence of MVV by polymerase chain reaction. The isolate was confirmed as being MVV, rather than the caprine arthritis-encephalitis virus based on phylogenetic analysis of the gag gene sequence. Although the sheep was asymptomatic, nonpurulent meningitis and demyelination were found in the spinal cord. These were considered to be early lesions associated with pathogenic MVV infection. Therefore, the present study demonstrated that MVV is distributed in Japan.

Small ruminant lentiviruses (SRLV) are members of the Retrovirus family comprising the closely related Visna/Maedi Virus (VMV) and the Caprine Arthritis-Encephalitis Virus (CAEV), which infect sheep and goats. Both infect cells of the monocyte/macrophage lineage and cause lifelong infections. Infection by VMV and CAEV can lead to Visna/Maedi (VM) and Caprine Arthritis-Encephalitis (CAE) respectively, slow progressive inflammatory diseases primarily affecting the lungs, nervous system, joints and mammary glands. VM and CAE are distributed worldwide and develop over a period of months or years, always leading to the death of the host, with the consequent economic and welfare implications. Currently, the control of VM and CAE relies on the control of transmission and culling of infected animals. However, there is evidence that host genetics play an important role in determining Susceptibility/Resistance to SRLV infection and disease progression, but little work has been performed in small ruminants. More research is necessary to understand the host-SRLV interaction. The research of Genomics and Health group is funded by the Basque Government Industry Dpt. (grant SA-10/00120 to B.M.J.) and UPV/EHU (grants GIU07/62 and GIU10/22 to B.M.J. and UFI 11/20)

Visna/Maedi virus (VMV) is a lentivirus that infects cells of the monocyte/macrophage lineage in sheep. Infection with VMV may lead to Visna/Maedi (VM) disease, which causes a multisystemic inflammatory disorder causing pneumonia, encephalitis, mastitis and arthritis. The role of ovine immune response genes in the development of VM disease is not fully understood. In this work, sheep of the Rasa Aragonesa breed were divided into two groups depending on the presence/absence of VM-characteristic clinical lesions in the aforementioned organs and the relative levels of candidate gene expression, including cytokines and innate immunity loci were measured by qPCR in the lung and udder.Sheep with lung lesions showed differential expression in five target genes: CCR5, TLR7, and TLR8 were up regulated and IL2 and TNFα down regulated. TNFα up regulation was detected in the udder. © 2013 Elsevier Ltd., Financial support was provided by the UPV/EHU (Projects GIU 07/62 and GIU 10/22 to BM Jugo, and UFI 11/20).

A control system for Visna/maedi virus (VMV) infection based on serologic segregation and management strategies was applied in an infected Spanish dairy Manchega breed sheep flock (n = 670) that was affected by a severe respiratory process associated to VMV. The control started in 2004 and consisted on the serological study of animals, segregation in two different flocks (seropositive and seronegative), separate management of flocks, selection of young female lambs for replacement only from seronegative ewes offspring, immediate removal of seropositive animals detected in the seronegative flock and a management tending toward the reduction and final culling of the seropositive flock. The serological control was repeated yearly or twice a year, approximately. Initial VMV seroprevalence of the undivided flock was 66.4% (January 2004) that descended to 47.3%, 12.8%, 2.2% and 0.2% between July 2004 and May 2006. Residual seroprevalence fluctuated slightly thereafter with a peak of 2.2% in April 2008. After segregation, number of animals in the seronegative flock was 378 that descended to 323 in October 2005. Since then, this number has increased steadily reaching 650 sheep in December 2011. The seropositive flock was progressively reduced by culling until its total disappearance in June 2010. This work presents the detailed results obtained in the control strategy against VMV in a single dairy sheep flock by implementing a segregation system based on serologic testing. The system is highly successful, as it reduces to residual levels VMV infection in about two years without the need of culling a high number of animals, as required by other methods. Moreover, the original size flock was been recovered within 8 years and has led to a subjective improvement of animal health and welfare in the flock. The residual seroprevalence could be eliminated at this stage by applying more sensitive molecular or other serological techniques to reach eradication., We also thank the autonomous Government of Aragón (Spain) for funding technicians and providing materials and reagents. This work has been partially supported by a grant from CICYT AGL2010-22341-C04-02.

Macrophages from maedi-visna virus (MVV) infected sheep have been shown to have an activated phenotype from sites of lesions in vivo. Here we have looked at the direct effect of virus infection on macrophage phenotype and activity in vitro by flow cytometry. There was no significant difference in the expression of several surface markers (CD4, CD8, MHC Class I, MHC Class II, lymphocyte function associated antigen(LFA)-1 and LFA-3) on monocyte-derived macrophages (MDM) by 5 days post MVV infection. In contrast the phagocytic activity of MVV-infected MDM for the yeast Candida utilis and erythrocytes was decreased by 5 days p.i. although the surface binding of erythrocytes was not affected. Interestingly, an activated phenotype was seen on alveolar macrophages (AM) from sheep with maedi (surface expression of MHC Class I, Class II and LFA-1 was increased), but there was no difference in the binding and phagocytosis of erythrocytes by these cells. However the binding and phagocytosis of the bacterium, Pasteurella hemolytica was increased with AM from MVV-infected sheep without lesions. Similarly there was no significant difference in the phagocytic and erythrocyte rosetting activity between fresh monocytes from MVV-infected and uninfected control sheep. Therefore the phenotype of macrophages taken from sites of lesions caused by MVV does not correspond to a direct effect by the virus on these cells or to particular activities of the macrophages.

Jaagsiekte was studied in the lungs of 85 sheep of Sardi and mixed breeds ranging from 7 months to 8 years of age. Lungs were examined grossly, histologically and, in five cases, by transmission electron microscopy. Lesions in lungs with jaagsiekte were described: they consisted of multiple focal to coalescent neoplastic nodules. Microscopic architecture of the neoplasms was similar in all cases, and lesions were seen as foci of cells arranged in glandular-like patterns (acinar and/or papillary). Intraalveolar fibrosis was prominent in many cases, specially in chronic ones, and alveolar macrophages were consistently found within alveolar spaces. In some cases (38/85), nodules or masses of myxomatous-like tissue were seen in the vicinity of adenomatous areas. Associated verminous pneumonia, hydatid cysts, and secondary bacterial bronchopneumonia were frequently encountered. Ultrastructurally, the proliferating neoplastic cells appeared to result from the transformation of Type II pneumocytes. Neither metastatic lesions nor viral inclusions were observed during this investigation. Sheep in the present study were not tested for antibodies to ovine lentivirus, maedi/visna. This descriptive study provides a morphological background necessary for routine differentiation and indicates the necessity of initiating studies on viral etiology and pathogenesis of jaagsiekte in Morocco.

Sheep infected with maedi visna virus were tested for immune reactivity to recombinant HSP65 and tuberculin PPD from mycobacteria. The results showed that both naturally and experimentally infected animals had elevated IgM but not IgG or IgA antibodies to HSP65 from Mycobacterium leprae or M. bovis. In experimentally infected animals, the elevated IgM antibodies appeared in blood from about 3 to 4 weeks postinfection. Increased T cell proliferative responses to HSP65 and PPD were also found in both naturally and experimentally infected sheep. The T cell responses to HSP65 were substantially inhibited by antibodies to ovine major histocompatibility complex class II molecules, indicating that the responses were class II restricted. Increased expression of a putative HSP65 molecule was observed in synovial membranes from sheep infected with maedi visna virus and goats infected with the related, caprine arthritis encephalitis virus. The results thus show that lentivirus infection induces T and B cell anti-HSP65 immune responses and suggest that synovial inflammation may be due, at least in part, to T and B cell recognition of HSP65-like molecules expressed in joints.

Andrés, Ximena de et al., 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. © 2013 Elsevier B.V., Funded by CICYT (AGL2010-22341-C04-01) and Government of Navarra IIQ010449.RI1 and IIQ14064.RI1. H. Ramírez was supported by PASPA program, UNAM and Public University of Navarra. R. Reina was supported by CSIC JAE-Doc contract.

6 Páginas., We examined the distribution in the perivascular spaces of Visna/maedi antigen, T cells (CD3+, CD4+ and CD8+), B cells and macrophages by immunohistochemistry in 22 natural cases of Visna/maedi encephalitis. Sheep showed lymphocytic or histiocytic lesions. In mild lymphocytic lesions, the viral antigen was detected in perivascular cuffs where CD8+ T cells predominated, but in severe lymphocytic lesions, sparse antigen was identified, and CD8+/CD4+ T cells appeared in a similar proportion in multilayer perivascular sleeves. In histiocytic lesions, vessels were surrounded by macrophages with abundant viral antigen, with CD8+/CD4+ T cells and B cells in the periphery. These results could reflect different stages of virus neuroinvasion and clarify the neuropathogenesis of Visna/maedi encephalitis., Spanish Ministry of Education AGL2007-66874-CO4-04/GAN Junta of Castilla y Leon LE36A12-1 Ministry of Education AP2007-02007