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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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

There are few reports on the pathogenesis of scrapie (Sc) and Visna/maedi virus (VMV) coinfections. The aim of this work was to study in vivo as well as post mortem both diseases in 91 sheep. Diagnosis of Sc and VMV infections allowed the distribution of animals into five groups according to the presence (+) or absence (-) of infection by Sc and VMV: Sc-/VMV-, Sc-/VMV+, Sc+/VMV- and Sc+/VMV+. The latter was divided into two subgroups, with and without VMV-induced lymphoid follicle hyperplasia (LFH), respectively. In both the lung and mammary gland, PrPSc deposits were found in the germinal center of hyperplasic lymphoid follicles in the subgroup of Sc+/VMV+ having VMV-induced LFH. This detection was always associated with (and likely preceded by) PrPSc observation in the corresponding lymph nodes. No PrPSc was found in other VMV-associated lesions. Animals suffering from scrapie had a statistically significantly lower mean age than the scrapie free animals at the time of death, with no apparent VMV influence. ARQ/ARQ genotype was the most abundant among the 91 ewes and the most frequent in scrapie-affected sheep. VMV infection does not seem to influence the scrapie risk group distribution among animals from the five groups established in this work. Altogether, these data indicate that certain VMV-induced lesions can favor PrPSc deposits in Sc non-target organs such as the lung and the mammary gland, making this coinfection an interesting field that warrants further research for a better comprehension of the pathogenesis of both diseases., (© The authors, INRA/EDP Sciences, 2010.)

The main routes of transmission of Visna/maedi virus (VMV), an ovine lentivirus, are thought to be through ingestion of infected colostrum and/or milk or through inhalation of respiratory secretions. Whereas oral transmission appears to be mediated via epithelial cells within the small intestine, the mechanism of virus uptake in the respiratory tract is unknown. In addition, it is not known whether infection is mediated by cell-associated or cell-free VMV, previous studies having not addressed this question. Intratracheal (i.t.) injection of VMV is known to be a highly efficient method of experimental infection, requiring as little as 10(1) TCID(50) VMV for successful infection. However, using a tracheal organ culture system, we show here that ovine tracheal mucosa is relatively resistant to VMV, with detectable infection only seen after incubation with high titres of virus (> or =10(5) TCID(50) ml(-1)). We also demonstrate that i.t. injection results in exposure of both trachea and the lower lung and that the time taken for viraemia and seroconversion to occur after lower lung instillation of VMV was significantly shorter than that observed for tracheal instillation of an identical titre of virus (P=0.030). This indicates that lower lung and not the trachea is a highly efficient site for VMV entry in vivo. Furthermore, cell-free virus was identified within the lung-lining fluid of naturally infected sheep for the first time. Together, these results suggest that respiratory transmission of VMV is mediated by inhalation of aerosols containing free VMV, with subsequent virus uptake in the lower lung.

A model of experimental infection with EV1, a British isolate of maedi-visna virus (MVV), has been developed. Twelve male Texel sheep were allocated to three groups and inoculated by the respiratory route with different inocula. Six of the animals received 10(7.2) tissue culture infective dose (TCID50) of MVV EV1 strain. Two sheep were inoculated with the same dose of heat inactivated MVV EV1 strain. An additional group of four sheep was sham-inoculated with identically prepared virus-free culture media. Experimental infection was followed for 16 weeks. Prior to inoculation, routine haematology, bronchoalveolar lavage (BAL) and flow cytometric analysis of bronchoalveolar lavage fluid (BALF) lymphocytes were performed in all animals to provide baseline parameters. Flow cytometric analysis of BALF lymphocytes and differential BALF cell counts were performed. Precipitating antibodies to MVV developed in all MVV-inoculated animals during the first 4 weeks post-inoculation, while the rest remained seronegative to MVV. MVV-infected animals had significantly decreased (P < 0.05) percentages of macrophages and significantly increased (P < 0.05) percentages of lymphocytes in BALF 4 weeks post-inoculation. Phenotypic changes in BALF T lymphocytes from MVV-inoculated animals, compared with the other two groups, showed significantly decreased (P < 0.05) percentages of CD4+ and gamma delta + T lymphocytes, significantly increased (P < 0.05) percentages of CD8+ lymphocytes and significant inversion (P < 0.05) of the CD4+/CD8+ ratio at different sampling times, but between 2 and 12 weeks post-inoculation. These findings indicate that during experimental MVV-infection an early, short-term cellular reaction occurs in the lung, that is characterised by T lymphocyte phenotypic changes that are very similar, if not identical, to those observed in natural MVV infection.

A study to determine the putative relationship between lymphocyte phenotypic alterations in bronchoalveolar lavage fluid and stage of lung pathology in maedi-visna infected sheep has been carried out. Twenty-one ewes (16 Texel and five Scottish blackface) naturally infected by maedi-visna virus and three Oxford controls were used. Animals were killed, lungs were removed, bronchoalveolar lavage was performed and pathological studies were completed. Blood samples were also obtained from 16 animals. Lymphocytes in both bronchoalveolar lavage and peripheral blood were labelled with monoclonal antibodies against the main T lymphocyte subsets (CD4, CD8, CD5 and gamma delta TCR) in order to perform flow cytometric studies. Three aspects of pathology were studied: lymphoid interstitial pneumonia, lymphoid follicular hyperplasia and smooth muscle hyperplasia. Percentages of CD4+, CD5+, gamma delta + T cells and the value for the CD4+ / CD8+ ratio in bronchoalveolar lavage of maedi-visna infected animals were significantly decreased (P < 0.05) when compared to controls, while percentages of CD8+ lymphocytes were increased in bronchoalveolar lavage of infected sheep and they were very close to being significant (P = 0.07) when compared to controls. Lesions were evaluated and simple least-squares regression tests demonstrated that there were several significant correlations between various lymphocyte subsets and pathological parameters studied in this work. However, when a multiple regression test was applied to the data, it was observed that only the CD8+ T cell subset both in bronchoalveolar lavage and in blood was significantly correlated with severity of lung pathology. It is concluded that CD8+ lymphocytes are key cells in the development of the interstitial reaction and the lymphocytic alveolitis observed in maedi-visna infected ewes and that the CD8+ alveolitis is a parallel feature to the intensity of lung lesions. It is further suggested that the percentage of CD8+ lymphocytes in bronchoalveolar lavage and in blood may act as in vivo indicators of lung pathology in maedi-visna infected sheep.

A model of experimental infection with EV1, a lytic British isolate of maedi-visna virus (MVV), was developed. Ten Texel sheep were allocated to two groups and inoculated by the respiratory route with different inocula. Six of the animals received 10(7.2) TCID50 (tissue culture infective dose) of EV1 strain, while four sheep were sham-inoculated with identically prepared virus-free buffer solution. Experimental infection was followed for 8 weeks post-inoculation (PI), with development of precipitating antibodies to MVV developed in the MVV-inoculated animals during the first 4 weeks PI. Transforming growth factor-beta (TGF-beta) levels, in both bronchoalveolar lavage fluid supernatant and plasma samples, were measured. Concentrations of pulmonary epithelial lining fluid (PELF) TGF-beta were calculated. TGF-beta concentrations in PELF were approximately 165-fold higher than in plasma. No significant differences in the concentrations of plasma or PELF TGF-beta, either within or between groups, were observed.

Static pressure-volume curves were generated from data obtained from 18 normal anaesthetized adult sheep. Lung volumes were determined by helium dilution. An exponential curve of the form V = Vmax - Ae-KP was fitted to the pressure-volume data from each sheep where P is the static recoil pressure, Vmax represents the volume asymptote, A is the difference between Vmax and the intercept on the volume axis and K defines the slope and hence the shape of the P-V curve. Quality of fit of the data was assessed visually, by means of a sign test and a runs test and by the coefficient of determination (r2). Exponential equations were found to adequately describe the shape of the pressure-volume curve in sheep. The exponent K was not correlated with effective alveolar volume (VAeff) (rs = 0.183; P > 0.05). Static lung compliance was determined over a volume range from the end-expiratory level (VEEL) to VEEL plus 400 ml. Measurements of static lung compliance were significantly correlated with measurements of effective alveolar volume (VAeff) (rs = 0.505; P < 0.025). In the ovine, the exponent K, an index of distensibility, is independent of lung volume and offers a means of assessing lung distensibility in this species.

Maedi-visna, a multisystemic disease of adult sheep, was first described in Spain in 1984. To get an idea of the seroprevalence of the disease locally and to estimate the number of seropositive animals with lesions, samples of blood, lungs and mammary glands were taken from 124 randomly selected sheep killed in the main slaughterhouse of Zaragoza. In the agar gel immunodiffusion test, 74 (59.7 per cent) of the sheep were positive and 50 were negative. Among the 74 seropositive animals, 19 (25.6 per cent) had no lesions in any organ, 12 (16.2 per cent) had lesions in the lungs only, 15 (20.2 per cent) had lesions in the mammary glands and 28 (37.8 per cent) had lesions in both organs. In the lungs hyperplasia of lymphoid follicles was more evident than an interstitial infiltrate but in the mammary glands this relationship was not observed. Even when the lesions occurred in both organs, they did not show the expected proportion in terms of either type or severity. Among the 50 seronegative sheep, eight (16 per cent) showed maedi-like lesions, formed exclusively by the hyperplasia of lymphoid follicles.