L. Luján

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.

Early detection of maedi-visna (ovine progressive pneumonia) virus seroconversion in field sheep samples.

The aim of this work was to investigate whether an enzyme-linked immunosorbent assay (ELISA) was useful for early detection of maedi-visna virus (MVV) infection in sheep under field conditions. An ELISA based on p25 recombinant protein and a gp46 synthetic peptide was used. Sequentially obtained serum samples (n = 1,941) were studied for 4 years. ELISA results were compared with those of the agar gel immunodiffusion (AGID) test, and results of both tests were compared with a reference result established using consensus scores for at least 2 of 3 serologic techniques (AGID, ELISA, and western blotting, which was used to resolve result discrepancies between the other 2 techniques). A total of 247 discrepancies were observed between ELISA and AGID. Of these, 131 were due to an earlier detection of 120 sera by the ELISA and 11 sera by AGID. The remaining discrepancies (116) were due to the presence of false reactions in both tests. Fewer false-negative results were found by ELISA than with AGID (6 vs. 69 sera, respectively), whereas the number of false-positive results was virtually the same for ELISA and AGID (21 vs. 20, respectively). In relation to the reference result, ELISA sensitivity and specificity were 97.8% and 98.2%, respectively, whereas values for AGID were 76.3% and 98.3%, respectively. The agreement between ELISA and the reference result was higher than that between AGID and the reference result (κ value: 0.96 and 0.77, respectively). A variation in the ELISA signal (based on optical density) was observed during the study period, suggesting different antibody levels throughout the animals life. The ELISA was useful for detecting MVV-infected sheep in field conditions and has potential for use in control and eradication programs.

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.

Ovine TRIM5α Can Restrict Visna/Maedi Virus

ABSTRACT The restrictive properties of tripartite motif-containing 5 alpha (TRIM5α) from small ruminant species have not been explored. Here, we identify highly similar TRIM5α sequences in sheep and goats. Cells transduced with ovine TRIM5α effectively restricted the lentivirus visna/maedi virus DNA synthesis. Proteasome inhibition in cells transduced with ovine TRIM5α restored restricted viral DNA synthesis, suggesting a conserved mechanism of restriction. Identification of TRIM5α active molecular species may open new prophylactic strategies against lentiviral infections.

Effects of housing on the incidence of visna/maedi virus infection in sheep flocks

The incidence of seroconversion to visna/maedi virus (VMV) infection and its relationship with management and sheep building structure was investigated in 15 dairy sheep flocks in Spain during 3-7years. Incidence rates were 0.09 per sheep-year at risk in semi-intensive Latxa flocks and 0.44 per sheep-year at risk in intensive Assaf flocks and was greatest for the one year old Assaf replacement flock. Separate multivariable models developed for replacement and adult flocks indicated that in both cases seroconversion was strongly associated to direct contact exposure to infected sheep and to being born to a seropositive dam. The latter effect was independent of the mode of rearing preweaning and the risk of seroconversion was similar for sheep fed colostrum and milk from a seropositive or a seronegative dam. These results are further evidence of the efficiency of horizontal VMV transmission by close contact between sheep and also suggest a inheritable component of susceptibility and resistance to infection. In contrast, indirect aerogenous contact with seropositive sheep was not associated with seroconversion as evidenced in replacement sheep housed in separate pens in the same building as adult infected sheep for one year. Consequently, VMV may not be efficiently airborne over short distances and this is important for control of infection. Moreover, there was no relationship between seroconversion and shed open areas. The latter could be related to having examined few flocks in which high infection prevalence dominated the transmission process while ventilation, may depend on a variety of unrecorded factors whose relationship to infection needs to be further investigated.

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.

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.

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.

Clinical characterisation of natural scrapie in a native Spanish breed of sheep

SCRAPIE is the prototype of a group of diseases referred to as transmissible spongiform encephalopathies; it affects sheep and goats and is caused by the accumulation of an abnormal protease-resistant isoform (PrPSc) of cellular prion protein (PrPc) in tissues of the central nervous system (Prusiner 1982). The clinical signs of scrapie may differ between countries: in the UK pruritus is the most common sign; in the USA, wasting, debility, tremor and incoordination are distinctive signs of scrapie, with pruritus, if observed at all, as a subtle sign (Detwiler 1992). In Spain, the disease has been reported as having a slow and progressive course of three to six months’ duration from the onset of clinical signs (Garcia de Jalon and others 1987). This short communication describes a study to characterise the clinical features observed in sheep naturally affected with scrapie in Aragon, Spain, and to compare the results with previously described findings. A group of 24 adult sheep of the Rasa Aragonesa breed with clinical signs compatible with scrapie were selected from five flocks located in the Aragon region of Spain in which laboratory confirmation of the disease had been made. Another 26 adult sheep without clinical signs, or showing clinical signs not compatible with scrapie, were selected from the same flocks to make up a control group. All the scrapie-affected sheep were between two and five years old and they were homozygous for A136R154Q17 (ARQ/ARQ), the genotype associated with a high risk of suffering scrapie (Dawson and others 1998). The control animals also had genotypes conferring a high or intermediate risk for suffering the disease (ARQ/ARQ, ARQ/ARH, ARR/ARQ, ARQ/AHQ and ARQ/VRQ) (Monleon and others 2004). A complete clinical examination of all the animals was carried out, including their clinical history, a general examination and special examination of all systems, with particular attention to the nervous system (Lisle 1996). The animals’ body condition score (BCS) was evaluated on a scale of 1 to 5: 1 Emaciated, 2 Thin, 3 Average, 4 Fat, 5 Obese (Russel 1991). Electrocardiography was carried out using a bipolar lead base-apex electrocardiogram (Cardiofax ECG 6511; Nihon Kohden) (Reef and McGuirk 1996). The animals were euthanased by an overdose of barbiturates, according to ethical recommendations. Routine histopathological, immunohistochemical and Western blotting analysis of the brain tissues were used to make a definitive diagnosis of scrapie by previously described methods (Schaller and others 1999, Monleon and others 2003). Statistical analysis was performed by the Pearson chi-squared test or Fisher’s exact test, using SSPS software; the level of significance was established as P<0·05. If possible, the odds ratio (OR) was calculated as an indicator of the relationship grade between the clinical signs and the presence of the disease (Daniel 1997). The course of the disease lasted on average three months (range one to six months) from the onset of clinical signs to euthanasia when the animal was in the final stages of the disease. The most important clinical features observed could be classified as alteration of the animal’s mental status, alteration of superficial sensitivity or alteration of deep sensation and motor functions. Other clinical features observed in the scrapie-affected animals were alteration of cranial nerve function, cardiac rhythm and BCS. The first clinical sign observed in 70·8 per cent of the affected animals (P=0·251) (Fig 1) was alteration of mental status, a feature which has also been observed by other researchers (Stamp 1980, Parry 1983, Detwiler 1992, Capucchio and others 2001). Alterations were mostly characterised by excitatory signs such as restlessness, anxiety and a tendency to avoid restraint. However, hyperexcitability to external stimuli, such as a hand clap and/or approach, were observed in 50 per cent of affected animals (P<0·0001) (Fig 1), and teeth grinding was also observed in 41·7 per cent of them (OR=3·93, P=0·039) (Fig 1), this finding being in accordance with that of Healy and others (2003). Alteration of superficial sensitivity was characterised by hypoaesthesia or total anaesthesia at the distal extremes of the hindlimbs and sometimes the forelimbs; this was observed in 54·2 per cent of the scrapie-positive animals (OR=29·54, P=0·0001) (Fig 1). Pruritus was also observed in 70·8 per cent of them (P<0·0001) (Fig 1), mainly on the rump and tail areas, but sometimes also on the head, thorax and both the internal and external sides of the hindlimbs (Detwiler 1992, Schreuder 1998, Ulvund 1999, Capucchio and others 2001). However, pruritus was mostly a subtle sign, which sometimes could only be observed indirectly, either by wool loss (OR=13·36, P=0·0001) (Fig 1) in all the abovementioned areas or by inducing the nibbling reflex, which was only observed in 45·8 per cent of the affected animals (P=0·0001) (Fig 1). Alteration of deep sensation and motor functions were characterised by hyporeflexia, which was observed in 41·7 per cent of the scrapie-affected sheep (OR=5·48, P=0·017) (Fig 1), mainly in the hindlimbs (patellar reflex) but sometimes in the forelimbs (carporadial extensor reflex). Head tremors were observed in 41·7 per cent of the positive animals (OR=17·86, P=0·001) (Fig 1) and were more evident when the animal was stressed. Ataxia and gait abnormalities were observed in 54·2 per cent of the positive animals (OR=4·96, P=0·011) (Fig 1) and were characterised by a high-stepping gait (trotting) of the forelimbs, jumping of the hindlimbs (bunny hop) and hypermetria, which progressed to ataxia of the hindlimbs, generalised ataxia, falls, difficulty in rising and total recumbency, as has been described by other authors (Stamp 1980, Detwiler 1992). Short Communications

An insight into a combination of ELISA strategies to diagnose small ruminant lentivirus infections.

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.