Characterization of minimal lesions related to the presence of visna/maedi virus in the mammary gland and milk of dairy sheep.

BACKGROUND: In order to characterize the complete range of lesions, especially minimal, affecting mammary gland and viral antigen distribution and target cells using immunohistochemistry in naturally Visna/maedi (VM) 84 infected sheep were studied, forty-four from flocks with clinical cases (A) and 35 randomly sampled from two abattoirs (B) together with five negative controls (C). An immunocytochemistry technique was developed and further milk samples (n = 39) were used to study viral excretion, carrier cells and the role of milk and colostrum in the transmission of the disease. RESULTS: All sheep from group C and three sheep from group B were negative to VM in tissue sections by histopathology, immunohistochemistry and PCR, and also in serum using ELISA. Several degrees of CD3 + lymphocytic interstitial mastitis were observed in groups A and B: minimal (+) n = 26 sheep; moderate (++), n = 32 and severe (+++), n = 12. No differences in lesion distribution were observed between groups A and B. Viral presence was confirmed by immunohistochemistry using two different antibodies and/or PCR in every tissue with lesions while serology was negative in six sheep with lesions. Two milk samples taken from milk tanks from two flocks from group A and fourteen milk samples from 29 infected sheep from group B were positive to VM (most of them from animals with moderate and severe lesions). Positivity was only found in macrophages, even in focal and minimal lesions, while no positivity was observed in epithelial or any other cells in either tissue and milk samples. CONCLUSIONS: This new observation of the minimal lesions described in this work increased the prevalence of VM lesions in mammary gland up to 90.9% and VM should be considered as a differential diagnosis when minimal interstitial lesions are detected. A high prevalence of VM was observed in intensive milk-producing sheep, ELISA serology did not detect as positivity all infected animals, while histology, IHC or PCR showed higher sensitivity. The cytological technique developed was very useful in milk-cell studies using hematoxylin and eosin and immunocytochemistry. Viral detection in milk samples (16/39) confirms a potential but limited role of milk/colostrum in viral transmission.

Perivascular inflammatory cells in ovine Visna/maedi encephalitis and their possible role in virus infection and lesion progression.

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.

Pulmonary adenomatosis and maedi-visna in Ethiopian central highland sheep: a microscopic study.

Microscopic examination of pneumonic lungs of the Ethiopian highland sheep (n = 35) was made and compared with the pneumonic lungs from ten sheep and 66 goats from the lowlands. Lesions compatible with sheep pulmonary adenomatosis (SPA; 8/35, 22.8%), and maedi-visna (MV; 9/35, 25.7%) were recorded only in sheep from the central highlands. Interstitial pneumonia (43.2%), bronchopneumonia (35.1%), and verminous pneumonia (6.3%) were recorded in both sheep and goats from the high- and the lowlands. SPA was documented for the first time in sheep from Ethiopia in this report. We believe that MV and SPA were introduced into Ethiopia through importation of exotic sheep. These infections should be considered in dealing with the diagnosis of respiratory diseases in all the sheep breeds in the central highlands and in the exotic and the crossbred sheep in the other parts of the country.

Maedi-visna: the meningoencephalitis in naturally occurring cases.

Lesions were examined at different levels of the central nervous system (CNS) in 64 sheep with natural maedi-visna (MV) meningoencephalitis. All animals showed lesions in more than one of the CNS locations examined; the lesions in the cranial regions were periventricular, while those in the spinal cord affected the white matter funicles. Lesions were found particularly in the cerebellar peduncles (non-suppurative meningoencephalitis), followed by the corpus callosum, hippocampus and thoracic spinal cord. Vascular, infiltrative and malacic histopathological patterns were recognized. One pattern predominated in each section examined, although mixed forms occurred. Vascular lesions occurred with similar frequency at all CNS levels, but infiltrative and malacic lesions predominated at rostral and caudal levels, respectively. Cells consistent with macrophages and shown immunohistochemically to be associated with MV virus were seen in malacic and infiltrative lesions, at the periphery of damaged areas.

Inflammatory Lesion Patterns in Target Organs of Visna/Maedi in Sheep and their Significance in the Pathogenesis and Diagnosis of the Infection.

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.

Natural cases of visna in sheep with myelitis as the sole lesion in the central nervous system.

Of 118 sheep with visna, 12 showed myelitis as the only nervous lesion. They were ovine lentivirus (OvLV)-seropositive and provirus DNA was demonstrated by LTR-PCR in all the samples with lesions. Clinically, all showed hindlimb paralysis and some were completely recumbent. Grossly, a swollen and discoloured area was identified in the white matter in 10 sheep. Microscopical changes consisted of a wedge-shaped area of non-suppurative leucomyelitis with mononuclear perivascular cuffing, demyelination and white matter degeneration. Except for two samples, grey matter was affected adjacent to severe white matter lesions. Three different microscopical patterns of lesion were identified, all having in common the presence of perivascular inflammation: the so-called vascular pattern was characterized by perivascular cuffs with minimal lesions in the adjacent neuroparenchyma; the malacic pattern, which was the commonest type, was characterized by severe white matter destruction and small numbers of macrophages; and the infiltrative pattern was characterized by a severe infiltrate of histiocytes in the parenchyma. Maedi-visna virus antigen was detected immunohistochemically only in areas with lesions, and the degree of immunolabelling was unrelated to the severity of the damage. Diagnosticians should bear in mind that a considerable number of visna cases show only spinal cord lesions. Examination of paraffin wax-embedded samples by LTR-PCR and immunohistochemistry would seem useful in confirming a histopathological diagnosis of visna from spinal cord samples.

Diagnosis of the nervous form of Maedi-Visna infection with a high frequency in sheep in Castilla y Leon, Spain.

Between 1997 and March 2004, the nervous form, or visna, of maedi-visna infection was diagnosed in 71 of 1631 sheep (4.35 per cent) examined in the Castilla y León region of Spain, of which 634 had shown nervous signs. The presence of the virus was confirmed by immunohistochemistry and in some cases by pcr on frozen-thawed or paraffin-embedded tissue samples. The main clinical signs were hindleg ataxia and paresis, but blindness or nystagmus were also observed. Thirty-three of the affected sheep (46.5 per cent) were two years old or younger. The affected sheep showed variable degrees of a non-suppurative meningoencephalitis, and immunohistochemistry identified positive cells in all cases, with no relation to the intensity of the inflammatory lesion.

Ovine lentivirus-associated leucomyelitis in naturally infected North American sheep.

Leucomyelitis was the predominant feature in four North American adult sheep (cases 1-4) with ovine lentivirus (OvLV) infection. All four animals were OvLV-seropositive and a syncytogenic virus consistent with OvLV was isolated from the brain of case 3 and the lungs of case 4. Clinically, the sheep had dyspnoea and neurologic signs of varying severity. Changes in the central nervous system included asymmetrical meningoleucomyelitis with white matter degeneration in all four sheep and scattered foci of leucoencephalitis in periventricular, subependymal and other white matter areas of the brain of the three animals (cases 1, 2 and 4) for which the brain was examined. In the lungs of two sheep (cases 3 and 4), there was lymphoid interstitial pneumonia with marked lymphoid hyperplasia. The viral capsid antigen (p25) was detected by immunohistochemistry (IHC) in sections of lung, brain and spinal cord of the four sheep and OvLV RNA was detected by in-situ hybridization (ISH) in lung and spinal cord samples. The results confirm the usefulness of the IHC and ISH for differential diagnosis of visna.

Pathogenesis of central nervous system lesions in visna: cell-mediated immunity and lymphocyte subsets in blood, brain and cerebrospinal fluid.

There are several indications that central nervous system (CNS) lesions in visna are immune-mediated and that cell-mediated immunity (CMI) may be of importance in the initiation of the lesions. To study the role of CMI in the pathogenesis of CNS lesions, five sheep were infected by intracerebral inoculation with visna virus and observed for 1 year. The following parameters were monitored at regular intervals: (1) neutralizing and ELISA antibodies; (2) visna virus-specific stimulation of lymphocytes from peripheral blood; (3) lymphocyte subpopulations in peripheral blood, cerebrospinal fluid (CSF) and brain at sacrifice. The CNS lesions were graded and compared with other parameters. The time course and titers of antibodies did not correlate with the severity of CNS lesions whereas the CMI did, indicating that CMI may play an important role in lesion development. The correlation of the number of CD8-positive cells in the CSF with the severity of lesions and the reversed ratio of CD4/CD8-positive cells in the diffusely infiltrated neuroparenchyma indicates that the CD8-positive T lymphocyte may be an important effector cell in the induction of CNS lesions.

Pathogenesis of central nervous system lesions in visna cell-mediated immunity and lymphocyte subsets in blood, brain, and cerebrospinal fluid.

The time course and titers of antibodies did not correlate with the severity of CNS lesions whereas the CMI did, indicating that CMI may play an important role in lesion development. The correlation of the number of CD8 positive cells in the CSF with the severity of lesions and the reversed ratio of CD4/CD8 positive cells in the diffusely infiltrated neuroparenchyma indicates that the CD8 positive T cells may be an important effector cell in the induction of CNS lesions.

Clinicopathological investigation of primary, uncomplicated maedi-visna virus infection.

Maedi-visna virus infection in a flock of sheep in Scotland was associated with respiratory disease, neurological disease, mastitis and lameness. The major clinical signs were dyspnoea (particularly on exercise), progressive fore- and hindlimb ataxia and balance defects, mammary induration and multilimb lameness, occasionally with enlarged carpal joints. Pathological examinations revealed lesions in the lungs, central nervous system, mammary glands and joints which were consistent with those induced by maedi-visna virus. The was no clinical or pathological evidence of concurrent sheep pulmonary adenomatosis, and pulmonary bacterial infections, when they occurred, were superimposed on the lesions due to maedi-visna virus.

[Suspicion of visna in a sheep from Graubünden Canton]. / Visnaverdacht bei einem Schaf aus dem Kanton Graubünden.

A lentivirus belonging to the group of retroviridae causes a chronic progressive interstitial pneumopathy (maedi) or a demyelinating encephalo-myelitis (visna) in sheep and goats. Pulmonary lesions of maedi as well as sero-positive, clinically healthy animals can be observed in Switzerland; visna, which even in countries with endemic infection does not occur frequently, is extremely rare. The head and cervical spine of an ewe with severe nervous troubles suspected of scrapie were submitted for postmortem examination. We found a demyelinating myelitis resembling visna. Immunocytochemical staining (PAP) to demonstrate the presence of the virus in paraffin sections was negative, but several animals of the flock proved to be seropositive for maedi/visna virus, which supports the histological diagnosis. Although there is no risk of an epidemic outbreak of maedi/visna in Switzerland, modes of transmission, eradication programmes and prophylactic measures successfully employed in countries with endemic maedi/visna are discussed.