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

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

Successful Visna/maedi control in a highly infected ovine dairy flock using serologic segregation and management strategies.

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.

Visna/maedi virus serology in sheep: survey, risk factors and implementation of a successful control programme in Aragón (Spain).

A serological survey of Visna/maedi virus (VMV) infection involving 274,048 sheep from 554 flocks was undertaken during 2002-2007 in Aragón, North-East Spain. One hundred and two of these flocks enrolled in a VMV control programme to reduce seroprevalence by selecting replacement lambs from seronegative dams and gradual culling of seropositive sheep. Twenty-five flocks were also visited to collect flock management and housing data. All study flocks had seropositive animals and 52.8% of animals tested were seropositive. Among flocks that joined the control programme 66 adopted the proposed measures and reduced seroprevalence significantly by between 26.1% and 76.9% whereas the remaining 36 flocks did not apply the measures and seroprevalence significantly increased. Seroprevalence increased with flock size and the number of days the sheep were housed, and decreased with increasing weaning age and shed open area, suggesting a reduced risk of VMV infection in sheep associated with better ventilation. At the end of the period, 24 flocks were certified as VMV-controlled with a seroprevalence <5%, and seven as VMV-free with 0% seroprevalence. These are the first officially recognised VMV-free flocks in Spain and represent a nucleus of VMV-free replacement animals for other flocks. Moreover, they are evidence of the possibility of eliminating VMV infection without resorting to whole-flock segregation or culling of seropositive sheep.

Phylogenetic analysis of SRLV sequences from an arthritic sheep outbreak demonstrates the introduction of CAEV-like viruses among Spanish sheep.

Small ruminant lentiviruses (SRLVs) cause different clinical forms of disease in sheep and goats. So far in Spain, Maedi visna virus-like (MVV-like) sequences have been found in both species, and the arthritic SRLV disease has never been found in sheep until a recent outbreak. Knowing that arthritis is common in goats, it was of interest to determine if the genetic type of the virus involved in the sheep arthritis outbreak was caprine arthritis encephalitis virus-like (CAEV-like) rather than MVV-like. Alignment and phylogenetic analyses on nucleotide and deduced amino acid sequences from SRLV of this outbreak, allowed a B2 genetic subgroup assignment of these SRLV, compatible with a correspondence between the virus genetic type and the disease form. Furthermore, an isolate was obtained from the arthritic outbreak, its full genome was CAEV-like but the pol integrase region was MVV-like. Although its LTR lacked a U3 repeat sequence and had a deletion in the R region, which has been proposed to reduce viral replication rate, its phenotype in sheep skin fibroblast cultures was rapid/high, thus it appeared to have adapted to sheep cells. This outbreak study represents the first report on CAEV-like genetic findings and complete genome analysis among Spanish small ruminants.

AA amyloidosis induced in sheep principally affects the gastrointestinal tract.

AA amyloidosis was initiated experimentally in adult sheep by induction of gangrenous pneumonia, an inflammatory process known to be associated with amyloid formation. A vegetable fragment contaminated with rumen content was instilled into the lungs of 4 experimental animals. A fifth animal was not inoculated and served as control. The animals were examined daily and blood and urine were sampled biweekly post-inoculation. One sheep was killed 18 days post-inoculation (dpi), another 49dpi, and the remaining two (as well as the control animal) 63dpi. Respiratory signs, diarrhoea and/or soft, unformed stool were observed in all inoculated sheep. All experimental animals developed gangrenous pneumonia with hypoalbuminaemia and hypergammaglobulinaemia, and elevated urinary protein, creatinine, gamma glutamyl transferase and ss-glucuronidase. Amyloid deposition was most pronounced in the gastrointestinal tract and was evident from 18dpi. Amyloid was present from the tongue to the rectum, but was most prominent in the duodenum where the deposits disrupted the normal mucosal architecture. Other body organs had only mild amyloid deposition. Immunohistochemistry confirmed that the deposits were AA amyloid. These findings suggest that the gastrointestinal tract is the main target organ for AA amyloid deposition in sheep. The observations in this experimental model must now be confirmed in animals with spontaneously arising AA amyloidosis.

Systemic DNA immunization against ovine lentivirus using particle-mediated epidermal delivery and modified vaccinia Ankara encoding the gag and/or env genes.

To determine whether systemic immunization with plasmid DNA and virus vector against visna/maedi virus (VMV) would induce protective immune responses, sheep were immunized with VMV gag and/or env sequences using particle-mediated epidermal bombardment and injection of recombinant modified vaccinia Ankara. The results showed that immunization induced both humoral and cell-mediated responses prior to and after virus challenge. The vaccination protocol did not prevent infection, but immunization with the gag gene or a combination of gag and env genes resulted in significantly reduced provirus loads in blood and mediastinal lymph node, respectively. Provirus loads in lung and draining lymph node were unaffected, but p25 expression was undetectable in lungs of animals immunized with a combination of gag and env genes. Analysis of target tissues for lesions at post-mortem showed that immunization with the env gene caused a significant increase in lesion score, while the gag gene or a combination of gag and env genes had no effect. Inclusion of the ovine interferon-gamma gene in the initial priming mixture had minimal effect on immune responses, provirus load, or lesion development, although it resulted in a decreased p25 expression in the lung. The results thus show that systemic immunization with gag or a combination of gag and env genes reduces provirus load in blood and lymphoid tissue, respectively whereas env immunization has no effect on provirus load but increased lesion development.

Mucosal immunization against ovine lentivirus using PEI-DNA complexes and modified vaccinia Ankara encoding the gag and/or env genes.

Sheep were immunized against Visna/Maedi virus (VMV) gag and/or env genes via the nasopharynx-associated lymphoid tissue (NALT) and lung using polyethylenimine (PEI)-DNA complexes and modified vaccinia Ankara, and challenged with live virus via the lung. env immunization enhanced humoral responses prior to but not after VMV challenge. Systemic T cell proliferative and cytotoxic responses were generally low, with the responses following single gag gene immunization being significantly depressed after challenge. A transient reduction in provirus load in the blood early after challenge was observed following env immunization, whilst the gag gene either alone or in combination with env resulted in significantly elevated provirus loads in lung. However, despite this, a significant reduction in lesion score was observed in animals immunized with the single gag gene at post-mortem. Inclusion of IFN-gamma in the immunization mixture in general had no significant effects. The results thus showed that protective effects against VMV-induced lesions can be induced following respiratory immunization with the single gag gene, though this was accompanied by an increased pulmonary provirus load.

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.

Clinicopathological features in ovine AA amyloidosis.

This paper describes the clinicopathological findings in sheep with AA amyloidosis. Serum samples from 12 AA amyloid-affected sheep and urine samples from 5 of these ewes were analyzed. In sera, the most important alteration was reflected in hypoalbuminemia, high concentration in beta and gamma-globulins and high levels of serum BUN, phosphorous and potassium. Serum creatinine, cholesterol and calcium concentrations showed no alterations. Urinary analysis showed proteinuria and a high protein/creatinine ratio. In three urine samples, high activities of urinary enzymes gamma-glutamyl transferase (GGT), beta-glucuronidase (GRS) and N-acetyl-beta-D-glucosaminidase (NAG) were observed, their ratios with urinary creatinine being increased for GGT and NAG and decreased for GRS. In conclusion, important alterations in biochemical and urinary parameters were observed in ovine affected by systemic AA amyloidosis. Those related to the activities of urinary enzymes could constitute reliable parameters for assessing renal injury in ovine AA amyloidosis.

Pathology of AA amyloidosis in domestic sheep and goats.

We describe the main pathologic changes in small ruminants affected by AA amyloidosis, together with the partial sequence of the protein involved. Twenty-one sheep and one goat were selected for presenting macroscopic kidney lesions compatible with systemic amyloidosis. Available tissue samples were studied by histologic, immunopathologic, and ultrastructural means. Renal lesions were characterized grossly by pale cortical surfaces with scattered, miliary, whitish-yellow foci and on cut cortical surfaces by straight, whitish-yellow striations. Gangrenous pneumonia was observed in 16 out of 21 affected sheep (76.2%), although other chronic inflammations were also observed. Amyloid was detected in all grossly affected kidneys using Congo red staining, lesions being most remarkable in glomeruli, affecting 95.5% of animals studied. Congophilic deposits were also observed in intertubular interstitium (68.2%) and medulla (57.1%). All amyloid-affected animals presented proximal convoluted tubule lesions, mostly characterized by an increase in diameter and by hyaline granular degeneration that were responsible for the macroscopic appearance of the kidney. Histologically, amyloid was also seen in blood vessels, spleen, liver, lymph nodes, gastrointestinal tract, and adrenal glands. All amyloid deposits demonstrated greenish-yellow birefringence with polarized light, and the antisera prepared against goat amyloid extracts specifically reacted with birefringent congophilic deposits of both sheep and goats. Ultrastructurally, these deposits were formed by masses of straight, nonbranching fibrils located predominantly in the basement membranes of glomerular capillaries and in the mesangium. Partial sequence of the protein in sheep and goats indicated a high degree of homology with the previously reported sequence of sheep Serum Amyloid A.