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.

Abortion in goats by Caprine alphaherpesvirus 1 in Spain.

An abortion outbreak occurred in a goat herd of Murciano-Granadina breed in Almeria Region in Spain where 80 pregnant females aborted. All bacteriological and parasitological examinations resulted negative, whereas virological investigations and real-time PCR assay showed the presence of Caprine alphaherpesvirus 1 DNA in the pathological specimens from aborted foetuses. Nucleotide sequence analysis revealed that the DNA was highly close related to the Swiss strain E-CH (99.7%) and a little less extent to the Italian BA.1 strain (99.4%). Histopathological examination revealed multifocal, well-circumscribed, 50- to 200-µm-diameter foci of coagulative necrosis in the liver, lungs and kidneys of three foetuses. In the periphery of the necrosis, there were frequently epithelial cells with the chromatin emarginated by large, round, amphophilic intranuclear viral inclusion bodies. The source of the infection in the herd could not clearly find out even some hypothesis were formulated. This seems to be the first report of an abortion outbreak due to Caprine alphaherpesvirus 1 in a goat herd in Spain.

Coexistence of granulomatous enteric inflammation and neoplasia in an adult sheep.

A 7-year-old dairy sheep suffering from chronic loss of weight without diarrhea or anorexia was euthanized after failing to respond to any treatment (antibiotic and antiparasitic). The main findings at the necropsy of this animal were multifocal miliary nodules in several organs, mainly in the Peyer's patches of the small intestine, and a segmental thickening of the jejunal wall. Histologic examination of the samples taken at the necropsy showed a multifocal chronic granulomatous inflammation, with mineralization and caseous necrosis at the core of the larger granulomas and scarce intrahistiocytic acid-fast bacilli consistent with a disseminated digestive tuberculosis. Polymerase chain reaction and bacteriological culture from these samples confirmed Mycobacterium avium subsp avium to be the etiologic agent of this infection. Histologically, the cause of the segmental thickening of the jejunal wall was found to be a small intestine adenocarcinoma, which in some areas coexisted with the granulomatous lesion.

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.

Histopathological classification of lesions observed in natural cases of paratuberculosis in free-ranging fallow deer (Dama dama).

Ninety-five adult fallow deer, legally hunted in the Regional Hunting Reserve of El Sueve (Northern Spain), were subjected to a post-mortem examination for paratuberculosis, samples being taken from the proximal and distal jejunum, proximal and distal ileum, ileocaecal valve and associated lymph nodes. The lesions were divided into four categories. Focal lesions (n=19 cases) consisted of small granulomas, mainly in the jejunal and ileal lymph nodes. Multifocal lesions (n=4) consisted of well-demarcated granulomas in the intestinal lymphoid tissue and also in the intestinal lamina propria. Diffuse multibacillary lesions (n=2) were characterized by a severe granulomatous enteritis and lymphadenitis. Macrophages and numerous Langhans giant cells containing many mycobacteria were present, resulting in macroscopical changes in the normal gut morphology. These changes were found from the proximal jejunum to the ileocaecal valve, but lesions were always particularly severe in the distal jejunum. In diffuse intermediate (multibacillary-lymphocytic) lesions (n=3) the infiltrate consisted of lymphocytes, macrophages and Langhans giant cells, with small numbers of mycobacteria. Mycobacterium avium subspecies paratuberculosis was identified by a polymerase chain reaction technique. The widespread occurrence of paratuberculosis in fallow deer in this Reserve represents a potential source of infection for other susceptible species.

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.

Lambs are Susceptible to Experimental Challenge with Spanish Goat Encephalitis Virus.

Spanish goat encephalitis virus (SGEV) is a member of the genus Flavivirus, family Flaviviridae, and causes encephalomyelitis in goats. The aim of this study was to determine whether sheep are susceptible to experimental challenge with SGEV by two different routes. The results show that SGEV can infect sheep by both the subcutaneous and intravenous routes, resulting in neurological clinical disease with extensive and severe histological lesions in the central nervous system. Lambs challenged subcutaneously developed more severe lesions on the ipsilateral side of the brain, but the lesion morphology was similar irrespective of the route of challenge. The clinical presentation, pathogenesis, lesion morphology and distribution shows that SGEV is very similar to louping ill virus (LIV) and therefore any disease control plan must take into account any host species and SGEV vectors as potential reservoirs. Furthermore, discriminatory diagnostics need to be applied to any sheep or goat suspected of disease due to any flavivirus in areas where SGEV and LIV co-exist.

Vaccination against Louping Ill Virus Protects Goats from Experimental Challenge with Spanish Goat Encephalitis Virus.

Spanish goat encephalitis virus (SGEV) is a recently described member of the genus Flavivirus belonging to the tick-borne encephalitis group of viruses, and is closely related to louping ill virus (LIV). Naturally acquired disease in goats results in severe, acute encephalitis and 100% mortality. Eighteen goats were challenged subcutaneously with SGEV; nine were vaccinated previously against LIV and nine were not. None of the vaccinated goats showed any clinical signs of disease or histological lesions, but all of the non-vaccinated goats developed pyrexia and 5/9 developed neurological clinical signs, primarily tremors in the neck and ataxia. All non-vaccinated animals developed histological lesions restricted to the central nervous system and consistent with a lymphocytic meningomyeloencephalitis. Vaccinated goats had significantly (P <0.003) greater concentrations of serum IgG and lower levels of IgM (P <0.0001) compared with unvaccinated animals. SGEV RNA levels were below detectable limits in the vaccinated goats throughout the experiment, but increased rapidly and were significantly (P <0.0001) greater 2-10 days post challenge in the non-vaccinated group. In conclusion, vaccination of goats against LIV confers highly effective protection against SGEV; this is probably mediated by IgG and prevents an increase in viral RNA load in serum such that vaccinated animals would not be an effective reservoir of the virus.

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.

Sheep as a Potential Source of Bovine TB: Epidemiology, Pathology and Evaluation of Diagnostic Techniques.

Bovine tuberculosis (TB) infection is infrequently diagnosed in sheep. Most reports are from single individual cases or flock outbreaks. However, in Spain several outbreaks have been reported recently, all of which had epidemiological links with TB-infected cattle herds. A total of 897 sheep suspected of being infected with TB and belonging to 23 flocks cohabiting with TB-infected cattle herds and/or goats were tested between 2009 and 2013 in Galicia (north-western Spain), using pathological, immunological and molecular techniques. Of these, 50.44% were positive by culture, 83.23% by histopathology and 24.92%, 4.86% and 59.42% by single intradermal tuberculin test (SITT), interferon-γ and ELISA, respectively. Results suggest that in circumstances akin to those in our study, sheep may be considered as a potential source of TB. We conclude that under similar conditions, serious consideration should be given to TB testing sheep, as they may represent a potential risk to other susceptible co-habiting species. The SITT and ELISA are recommended as the simplest and most cost-effective initial approaches for the diagnosis of TB in sheep under field conditions. However, when possible, interferon-γ should be applied to increase sensitivity.

Histopathological classification of lesions associated with natural paratuberculosis infection in cattle.

Paratuberculosis-associated lesions in 116 naturally infected adult cows, with or without clinical signs, were classified histopathologically. Tissue samples obtained focused on gut-associated lymphoid tissue. Lesions were divided into five categories. Focal lesions (n=68 cases), consisted of small granulomas in the ileal and jejunal lymph nodes or the ileocaecal lymphoid tissue. In the multifocal type (n=13 cases), small granulomas or scattered giant cells appeared in some intestinal villi, as well as in the lymph nodes. Diffuse multibacillary lesions (n=15 cases), associated with severe granulomatous enteritis affecting different intestinal locations and lymph nodes, were formed by macrophages containing large numbers of acid-fast bacilli. In diffuse lymphocytic lesions (n=3 cases), lymphocytes were the main inflammatory cells, with some macrophages or giant cells containing few if any mycobacteria. In diffuse intermediate forms (n=17 cases), the infiltrate was formed by abundant lymphocytes and macrophages, and mycobacteria were present to varying degrees related to the number of macrophages. Clinical signs and gross lesions were mainly associated with diffuse forms. Thickening of the intestinal wall, which was the most common macroscopical finding, was related to the degree of submucosal change. Mycobacterium avium subsp. paratuberculosis was identified by culture or PCR in all cows with diffuse lesions, and in 55.5 and 37% of those with multifocal or focal forms, respectively. The importance of sampling the ileal and caudal jejunal lymph nodes to find histological lesions of paratuberculosis in cattle is emphasized.

First Confirmation of Schmallenberg Virus in Cattle in Spain: Tissue Distribution and Pathology.

Between January and June 2013, nine stillborn bovine foetuses with congenital malformations from nine cattle herds located in Salamanca (central Spain) were detected. Necropsy was performed on two calves. Pathological lesions together with molecular genetics and serological results allowed a definitive diagnosis: first confirmation of Schmallenberg virus (SBV) infection in cattle in Spain. SBV was detected in different tissues and organic fluids in both animals including blood, suggesting a possible viraemia. The umbilical cord was also positive for the presence of SBV in both animals. The former tissue provides an easy to obtain sample and might be a sample of choice when necropsy is carried out in the field.

Small ruminant lentivirus infections and diseases.

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 programmes. 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.

Evaluation of cellular and serological diagnostic tests for the detection of Mycobacterium bovis-infected goats.

We have evaluated the comparative intradermal skin (CID) test, the interferon gamma (IFN-gamma) assay, two ELISAs with bovine PPD antigen, the standard and the anamnestic using sera obtained, respectively, at the time of the tuberculin injection and 15 days later, for the diagnosis of caprine tuberculosis (TB). The sensitivity and specificity results were high for the CID test (83.7%, 100%), the IFN-gamma assay (83.7%, 96%) and the anamnestic ELISA (88.6%, 95.8%). In contrast, they were comparatively low for the standard ELISA (54.9%, 88%). However, test results with the standard ELISA were positive in a group of goats with cavitating TB (100%). A combination of the CID test and the IFN-gamma assay offered the highest sensitivity, 95.8%, and also high specificity, 96%. In spite of this, the evidence that the serological tests were most sensitive for the detection of goats with severe lesions (100% positivity) suggested that a combination of CID test and anamnestic ELISA may be most useful as part of an eradication campaign against caprine TB.

Differences in the immune responses in lambs and kids vaccinated against paratuberculosis, according to the age of vaccination.

In order to evaluate and compare the peripheral immune responses induced by the vaccination against paratuberculosis in relation with the age of immunization, two groups of lambs and goat kids were vaccinated at 15 days and 5 months old, respectively. A heat-killed commercial vaccine was inoculated subcutaneously and humoral and cellular immune responses were measured by an ELISA and IFN-gamma assay, respectively, at 0, 30, 90, 180, 270 and 360 dpv in the lambs and 0, 30, 90 and 180 dpv in the caprine. IFN-gamma values did not show statistically significant differences between both groups, but when compared to the unvaccinated controls, this cytokine response tend to disappear earlier in animals vaccinated at 15 days old. The antibody response was always higher and more persistent in animals vaccinated at 5 months. The possibility of the incomplete degree of maturation of the immune system in 15 days old animals as the cause of the differences in the immune response to vaccination is suggested.

Immunological detection of sheep experimentally infected with strains of Mycobacterium avium subspecies containing insertion sequence IS901/IS902 and a 40 kDa protein.

A monoclonal antibody raised against a 40 kDa protein present in certain M. avium strains (IS901/IS902 positive) was used for developing a blocking ELISA. Sera from experimentally infected sheep were evaluated by indirect ELISA, AGID and blocking ELISA. The blocking assay proved to be highly specific for differentiation of sheep infected with different subspecies of M. avium.

Proximal aortic dissection (dissecting aortic aneurysm) in a mature ostrich.

The gross and histopathologic lesions observed in a case of spontaneous proximal aortic dissection (dissecting aortic aneurysm) in a mature ostrich are reported. At necropsy, a dissecting intramural hematoma was seen in the proximal aorta, extended about 12 cm distally from the aortic valves. Histopathologic changes in aortic dissection included fragmentation and disruption of elastic laminae, presence of cystic extracellular spaces, and pooling of ground substance in the tunica media. Hepatic copper levels were measured, and the low concentration found suggested that a copper deficiency together with other risk factors such as the elevation of blood pressure may have been implicated in the development of the aortic dissection seen in this ostrich.

Comparison of Ziehl-Neelsen staining and immunohistochemistry for the detection of Mycobacterium bovis in bovine and caprine tuberculous lesions.

The avidin-biotin complex peroxidase (ABC-P) method was used to detect Mycobacterium bovis, and the results were compared with those obtained by the Ziehl-Neelsen (ZN) technique. Lesions were examined from 18 cows and 24 goats with tuberculosis. All animals showed pulmonary lesions, which in the cattle were mainly minor (i.e. primary complex) but in the goats were sometimes minor and sometimes severe. Microscopically, typical granulomas were seen in the lungs and lymph nodes, with central necrosis and the cellular components of chronic inflammation, but mycobacteria were either seen in small numbers or were not detectable. The ABC-P technique was more sensitive than the ZN method, as shown by the number of positive animals detected, the intensity of staining, and the successful use of low magnification. Caprine lesions, although more severe than bovine lesions, appeared to contain fewer organisms.

Description and classification of different types of lesion associated with natural paratuberculosis infection in sheep.

Paratuberculosis lesions in naturally infected sheep were classified histologically. Adult sheep (n = 166) culled for various reasons, from four flocks in which clinical cases of the disease had occurred, were studied. Eight-two sheep (49.4%) showed lesions that could be divided into three main categories. Type 1 lesions, found in 24.1% of these animals, consisted of small granulomata formed by macrophages and were located exclusively in the ileocaecal Peyer's patch. In type 2 lesions, found in 4.8% of the sheep, granulomata were also observed in the mucosa associated with Peyer's patches. Type 3 lesions were characterized by granulomata in areas of the mucosa associated with, and also distinct from, the Peyer's patches. Three subtypes of type 3 lesions were recognized. In subtype 3a, found in 4.2% of the sheep, multifocal granulomata appeared in different areas of the lamina propria; they were not apparently associated with lymphoid tissue, and neither did they modify the morphology of the affected areas. Subtype 3b, found in 13.9% of animals, consisted of large numbers of macrophages, widespread in the lamina propria; in subtype 3c (2.4% of sheep) lymphocytes were the main inflammatory cell, with some macrophages scattered amongst them. In subtypes 3a and b, villi were distended and the mucosa appeared thickened. Mycobacteria could be demonstrated in tissue sections from all the samples with subtype 3b lesions and in almost all of those with type 2 and 3a lesions; these organisms were absent or sparse, however, in type 1 and 3c lesions. Macroscopical lesions were clearly visible only in sheep with type 3b and 3c lesions. Mycobacterium paratuberculosis was cultured from all the animals with type 3 lesions, from 87.5% of those with type 2 lesions, and from 47.5% of those with type 1. Type 3b lesions resembled the "borderline-lepromatous" form of mycobacterial lesions, whereas type 3c lesions resembled the "borderline-tuberculoid" form. The relationship between intestinal lymphoid tissue and paratuberculosis lesions is discussed, and the diagnostic importance of histological examination of the ileocaecal valve emphasized.