Interspecific transmission of small ruminant lentiviruses from goats to sheep.

This study was conducted in order to evaluate the transmission of caprine lentivirus to sheep using different experimental groups. The first one (colostrum group) was formed by nine lambs receiving colostrum from goats positive for small ruminant lentiviruses (SRLV). The second group (milk group) was established by nine lambs that received milk of these goats. Third was a control group, consisting of lambs that suckled colostrum and milk of negative mothers. Another experimental group (contact group) was formed by eight adult sheep, confined with two naturally infected goats. The groups were monitored by immunoblotting (IB), enzyme-linked immunosorbent assay (ELISA), agar gel immunodiffusion (AGID) and nested polymerase chain reaction (nPCR). All lambs that suckled colostrum and milk of infected goats and six sheep of the contact group had positive results in the nPCR, although seroconversion was detected only in three of the exposed animals, with no clinical lentiviruses manifestation, in 720 days of observation. There was a close relationship between viral sequences obtained from infected animals and the prototype CAEV-Cork. Thus, it was concluded that SRLV can be transmitted from goats to sheep, however, the degree of adaptation of the virus strain to the host species probably interferes with the infection persistence and seroconversion rate.

Interspecific transmission of small ruminant lentiviruses from goats to sheep

This study was conducted in order to evaluate the transmission of caprine lentivirus to sheep using different experimental groups. The first one (colostrum group) was formed by nine lambs receiving colostrum from goats positive for small ruminant lentiviruses (SRLV). The second group (milk group) was established by nine lambs that received milk of these goats. Third was a control group, consisting of lambs that suckled colostrum and milk of negative mothers. Another experimental group (contact group) was formed by eight adult sheep, confined with two naturally infected goats. The groups were monitored by immunoblotting (IB), enzyme-linked immunosorbent assay (ELISA), agar gel immunodiffusion (AGID) and nested polymerase chain reaction (nPCR). All lambs that suckled colostrum and milk of infected goats and six sheep of the contact group had positive results in the nPCR, although seroconversion was detected only in three of the exposed animals, with no clinical lentiviruses manifestation, in 720 days of observation. There was a close relationship between viral sequences obtained from infected animals and the prototype CAEV-Cork. Thus, it was concluded that SRLV can be transmitted from goats to sheep, however, the degree of adaptation of the virus strain to the host species probably interferes with the infection persistence and seroconversion rate.

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.

Compartmentalization of small ruminant lentivirus between blood and colostrum in infected goats.

The compartmentalization of small ruminant lentivirus (SRLV) subtype A (Maedi-Visna virus) and B (caprine arthritis-encephalitis virus) variants was analyzed in colostrum and peripheral blood mononuclear cells of four naturally infected goats. Sequence analysis of DNA and RNA encompassing the V4-V5 env regions showed a differential distribution of SRLV variants between the two compartments. Tissue-specific compartmentalization was demonstrated by phylogenetic analysis in three of the four cases. In these animals colostrum proviral sequences were clustered relative to the blood viral sequences. In one goat, the blood and colostrum-derived provirus sequences were intermingled, suggesting trafficking of virus between the two tissues or mirroring a recent infection. Surprisingly, the pattern of free virus variants in the colostrum of all animals corresponded only partially to that of the proviral form, suggesting that free viruses might not derive from infected colostral cells. The compartmentalization of SRLV between peripheral blood and colostrum indicates that lactogenic transmission may involve specific viruses not present in the proviral populations circulating in the blood.

PCR detection of colostrum-associated Maedi-Visna virus (MVV) infection and relationship with ELISA-antibody status in lambs.

A recent large-scale experimental study showed that bottle-feeding ovine colostrum from seropositive ewes results in high MVV-seroconversion in lambs. In contrast, relatively few lambs that naturally suckled colostrum from seropositive dams seroconverted as a result of it. Furthermore, lambs fed uninfected bovine colostrum readily seroconverted when mixed with ovine-colostrum lambs indicating that horizontal MVV transmission between lambs was efficient. MVV-infection was further investigated in the same samples using two PCR tests targeting sequences in the long-terminal repeats (LTR) and POL MVV genes. PCR-tests confirmed previous serological findings. However, the LTR-PCR was more sensitive and allowed detecting infection earlier than the other tests, including 5-8% of new-born lambs from seropositive dams, providing more evidence that prenatal MVV-infection may be more important than considered. The degree of agreement between PCR and antibody tests in individual samples was low up to 6 months of age and moderate at 10 months-old. Nine percent of lambs were always PCR-negative but seroconverted and 19% of lambs were PCR-positive at least once and did not seroconvert. However, seroconversion was associated with increasing number of times lambs were PCR-positive and ovine colostrum-fed lambs were more frequently PCR-positive than other lambs. The significance of these findings in terms of MVV-infection, epidemiology and control is discussed.

Desarrollo de una técnica de PCR para detectar virus maedi-vinsa (VMV) libre e integrado en células y aplicación al estudio de la infecci´n calostral en corderos / Development of a PCR test to detect cell-free and -integrated maedi-visna virus (mvv) and it’s use to investigate colostral infection in lambs

Se desarrolló una técnica de PCR para detectar virus maedi-visna (VMV) libre e integrado en células, seestudio su concordancia con una PCR-LTR y un ELISA de anticuerpos de probada eficacia en muestras decalostro de ovejas infectadas y se emplearon los tres ensayos para investigar la infección calostral por VMV encorderos. Los cebadores se diseñaron en una región conservada en las seis secuencias de VMV disponibles enGenBank y amplifican un producto de 744 pares de bases (pb). Se realizaron 856 ensayos incluidos 283 con lagag, e independientemente de la PCR empleada se observó una buena correlación entre la presencia de viruslibre e integrado y esto es novedoso y plantea la importancia relativa de ambas formas víricas en la infección porVMV. En cambio, la concordancia entre las PCRs y el ELISA fue solo moderada y se corroboró que a menudono se detecta VMV en animales seropositivos. Las PCRs detectaron VMV en la mayoría de calostros ingeridospor corderos que posteriormente a los 10 meses de edad fueron seropositivos y además la gag y en menormedida la LTR, también en algunos calostros de corderos que fueron seronegativos, probablemente porquetomaron menos cantidad de calostro que los seropositivos. Además de aportar mas evidencia de la asociaciónpositiva entre la infección por VMV y el volumen de calostro con VMV ingerido, este resultado sugiere que lagag es más sensible que la LTR en esta matriz

A PCR test was developed to detect cell-free and cell-integrated maedi-visna virus (MVV), its degree ofagreement with an LTR-PCR and an antibody ELISA of proven efficacy was analysed in colostrum samplesand all three tests were employed to investigate colostral MVV infection in lambs. Primers were designed fromgag gene sequences homologous in the six MVV sequences presently in GenBank, and amplify a 744bp fragment.856 assays were carried out including 283 with the new gag-PCR and a good correlation was observedbetween the presence of cell-free and -integrated MVV. This is novel and questions the relative role of the twoviral forms in MVV infection. Instead, the correlation between PCR and ELISA results was only moderateand provided further evidence that MVV detection may fail in infected animals. The PCRs detected MVV incolostrum ingested by most lambs that later tested seropositive at 10 months-old and additionally, the gag-PCRand to a lesser extent the LTR-PCR, detected MVV in colostrum taken by lambs seronegative at 10 months-oldmost likely because they ingested less colostrum. As well as providing further evidence of the positive associationbetween MVV infection and volume of MVV-containing colostrum ingested, this result suggest that thegag-PCR developed is more sensitive than the LTR-PCR used in this study

Relative contribution of colostrum from Maedi-Visna virus (MVV) infected ewes to MVV-seroprevalence in lambs.

Maedi-visna virus (MVV) seroprevalence associated with consumption of colostrum from seropositive ewes was investigated in 276 housed lambs from birth to 300 days-old. At birth, lambs were allocated to five experimental groups according to the maternal MVV-serological status, source and mode of feeding colostrum (bovine or ovine and bottle fed or suckled from the dam) and type of horizontal MVV-exposure (raised with the dam or separately with other lambs). The risk of being seropositive at 300 days-old was associated with feeding ovine colostrum from seropositive ewes and increased with intake of bottle-fed ovine colostrum and was higher in lambs separated from their dams and raised with other experimental lambs compared to lambs raised with their dams. Approximately 75-87% of ELISA-positive results in lambs that had ovine colostrum was attributable to colostrum itself. However, approximately only 16% of naturally raised and 29-61% of bottle-fed ovine colostrum lambs were ELISA-positive as a result feeding ovine colostrum. These results confirm that ovine colostrum from seropositive ewes can be a major source of MVV but its overall contribution to seroprevalence in natural conditions is relatively low, and shows that horizontal MVV transmission can be an important source of infection in new-born lambs.

Colostral transmission of maedi visna virus: sites of viral entry in lambs born from experimentally infected ewes.

Maedi visna virus (MVV) vertical transmission in sheep via infected colostrums is a very important route of infection in lambs. To verify colostral transmission and to study early viral entry in lambs, colostrum samples, and small intestine and mesenteric lymph nodes of lambs born from experimentally infected ewes were examined by histopathology, immunohistochemistry (IHC) and in situ hybridisation (ISH) studies. In particular, newborn lambs were naturally fed maternal colostrum and humanely killed at 10, 24, 48, 72, 96 h and 7 and 10 days after birth; two caesarian-derived lambs served as uninfected controls. No lesions suggestive of MVV infection were found, but marked immunoreactions for MVV capsid antigen (CA, p28) were detected in lambs fed maternal colostrum and in macrophages cultured from colostrum. IHC results in lambs suggest an initial viral absorption by intestinal epithelial cells at the tip of the villi, passage to mononuclear cells in the lamina propria and involvement of ileum Peyers' patches and mesenteric lymph nodes, with different staining patterns depending on infection times. ISH on intestinal sections of the 72 h lamb revealed the presence of proviral DNA in epithelial cells at the tip of the villi, suggesting a role for these cells in early MVV replication. The results contribute to knowledge about the pathogenesis of ovine lentivirus infection suggesting that the small intestine and mesenteric nodes are the sites of entry and propagation of MVV in lambs fed colostrums from infected ewes.

Transmission and control implications of seroconversion to Maedi-Visna virus in Basque dairy-sheep flocks.

A retrospective analysis of seroconversion to Maedi-Visna virus (MVV) was carried out for 10 infected semi-intensively reared dairy-sheep flocks that were tested annually between 1994 and 1999. Four of the flocks raised replacement lambs artificially with bovine colostrum and milk replacement to avoid lactogenic MVV infection but did not prevent aerosol contact between replacements and other sheep in the flock. Flock culling percentages ranged between 14 and 25% and in eight flocks the number of sheep that seroconverted was similar to or lower than the number of sheep culled--suggesting that incidence could be reduced by culling seropositive sheep without increasing average culling percentages. Random-effects logistic regression indicated that seroconversion was associated positively with increasing contact with infected sheep and with lifetime MV-serological status of the dam (used as a proxy measure of genetic susceptibility), but not with mode of rearing pre-weaning (artificially or with a seropositive or seronegative dam). Our results indicate that when conditions allow efficient horizontal transmission, there is no evidence that lactogenic infection increases the risk of MV infection and that there is an important inheritable component of disease resistance or susceptibility.

A comparative study on the diagnosis of maedi-visna infection in serum and colostrum samples using agar gel immunodiffusion (AGID) technique.

Serum/colostrum pairs were collected from 245 ewes in 6 sheep herds which had been determined previously to be infected with MV virus and were tested against maedi-visna infection using AGID test. Positive rates were detected as 3.8-41.2% in tested flocks. Serum and colostrum samples obtained from 53 sheep were positive for MV virus specific antibodies by AGID test. 16 colostrum samples were negative although serum samples obtained from the same animals were found to be positive for MV antibodies. Of the 245 sera and colostrum pairs tested, there was total agreement of results (+ or -) in 229 and disagreement in the results with the other 16 serum/colostrum pairs. Of the latter, all serum samples were positive and all colostrum samples were negative for MV antibodies. This study compared colostrum and serum samples for the determination of MV antibodies using AGID test under field conditions on naturally infected animals and on healthy animals. The results show that colostrum antibodies can be detected using AGID test and that colostrum is a reliable material to determine anti-MV virus antibodies. The procedure can be used for herd diagnosis.

Ovine lentivirus antibody detection in serum, colostrum and milk using a recombinant transmembrane protein ELISA.

An enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies against ovine lentivirus (OLV) in serum, colostrum, and milk from naturally infected sheep. The assay used OLV recombinant transmembrane envelope protein (rTM) as a test antigen. Matched serum/colostrum and serum/milk samples were collected at 24h, 4 weeks (mid-lactation), and 8 weeks (weaning) post-lambing. Among 129 paired samples collected at 24 h post-lambing, there was overall test agreement (concordance) of 82.9% and a kappa value of 0.658 between serum and colostrum rTM ELISA results. Among 130 mid-lactation samples, the milk ELISA had 100% specificity and 64.9% sensitivity relative to the serum ELISA, there was concordance of 79.2%, and a kappa value of 0.602. At mid-lactation, the serum agar gel immunodiffusion test had a sensitivity of 0.390 and 0.560 relative to the serum and milk rTM ELISAs, respectively. Matched serum and milk rTM ELISA results at weaning were very similar to those at mid-lactation. Finally, increased occurrence and severity of subclinical mastitis at weaning was found in ELISA-seropositive compared with ELISA-seronegative ewes. Both subclinical mastitis and ewe OLV infection had a negative impact on lamb growth and weaning weights. Compared with blood, colostrum and milk are easier and less expensive to sample and store. These results suggest that rTM ELISA testing of colostrum and milk could be used to supplement serologic testing in OLV screening or eradication programs.

Inhibitory effect of 9-(2-phosphonylmethoxyethyl)adenine on visna virus infection in lambs: a model for in vivo testing of candidate anti-human immunodeficiency virus drugs.

The acyclic nucleoside phosphonate analog 9-(2-phosphonylmethoxyethyl)adenine (PMEA) was recently found to be effective as an inhibitor of visna virus replication and cytopathic effect in sheep choroid plexus cultures. To study whether PMEA also affects visna virus infection in sheep, two groups of four lambs each were inoculated intracerebrally with 10(6.3) TCID50 of visna virus strain KV1772 and treated subcutaneously three times a week with PMEA at 10 and 25 mg/kg, respectively. The treatment was begun on the day of virus inoculation and continued for 6 weeks. A group of four lambs were infected in the same way but were not treated. The lambs were bled weekly or biweekly and the leukocytes were tested for virus. At 7 weeks after infection, the animals were sacrificed, and cerebrospinal fluid (CSF) and samples of tissue from various areas of the brain and from lungs, spleen, and lymph nodes were collected for isolation of virus and for histopathologic examination. The PMEA treatment had a striking effect on visna virus infection, which was similar for both doses of the drug. Thus, the frequency of virus isolations was much lower in PMEA-treated than in untreated lambs. The difference was particularly pronounced in the blood, CSF, and brain tissue. Furthermore, CSF cell counts were much lower and inflammatory lesions in the brain were much less severe in the treated lambs than in the untreated controls. The results indicate that PMEA inhibits the propagation and spread of visna virus in infected lambs and prevents brain lesions, at least during early infection. The drug caused no noticeable side effects during the 6 weeks of treatment.

The basic domain of the lentiviral Tat protein is responsible for damages in mouse brain: involvement of cytokines.

The HIV and visna lentiviruses induce an inflammatory reaction in the central nervous system (CNS) of the infected hosts leading to dysmyelination, demyelination, and neuronal loss. The basic domain of the transactivating Tat protein has been involved in CNS damage. Infusion of basic containing domain Tat peptides in the lateral ventricle (systemic injection) or in the grey matter, i.e., hippocampus and thalamus (local injection), induced an inflammatory process characterized by the formation of an edema and invasion of macrophage accompanied by reactive astrogliosis. Control peptides originating from either lentiviral proteins or irrelevant protein as ovalbumin did not lead to any inflammatory reaction or cell death. The inflammation led to the loss of ependymal cells in the lateral ventricles and neurons in the grey matter. RNA extracted from the Tat-injected hemisphere reacted with TNF-alpha, IL-1 alpha and beta, and IL-6 probes. The macrophage/microglia inducible nitric oxyde synthase was also expressed. Blockade of TNF-alpha by a pentoxifylline treatment led to the decrease of IL-1 and iNOS expression accompanied by a reduction of the volume of the lesions indicating that the Tat-induced lesions might be mediated by TNF production.

[Expression of visna-maedi virus in the mammary secretions of a seropositive ewe during gestation and then an artificial induction of lactation]. / Expression du virus Visna-Maedi dans les sécrétions mammaires d'une brebis séropositive lors d'une gestation puis d'une induction artificielle de lactation.

Lentiviruses in small ruminants are specially harboured in the mononuclear phagocyte lineage. The virus expression in macrophages is strictly correlated with physiological events of the reproduction life of the host. In a pregnant ewe, Visna-Maedi seropositive, we cultivated infectious cells from mammary secretions, colostrum and milk. In the same animal, a hormonally induced artificial lactation produced the same increase of infectious macrophages in mammary secretions. The virus is more easily obtained by coculture of milk macrophages when the number of macrophages is higher in mammary secretions.

Hyaluronidase enhances cell fusion and synthesis of viral DNA during infection with caprine arthritis encephalitis virus.

Caprine arthritis encephalitis virus (CAEV) is a lentivirus which infects goats and causes chronic progressive arthritis after a prolonged incubation period. CAEV replicates productively in cultures of goat synovial membrane cells and causes cytopathic effects characterized by multinucleated giant cell formation. The enzyme hyaluronidase was found to accelerate this virus induced fusion of GSM cells. Hyaluronidase treatment also resulted in synthesis of increased levels of unintegrated viral DNA early after infection. However, there was no significant increase in viral RNA in the infected cells or in the amount of virus produced. These studies suggest that hyaluronidase facilitates the interaction of CAEV with the target cells. Further it suggests that only a few copies of viral DNA are required to achieve maximal levels of virus replication. Additional copies of viral DNA appear to be redundant not contributing to viral specific transcription or increased production of virus.

Efficiency of in situ hybridization as a function of probe size and fixation technique.

In an attempt to improve fixation technique for viral RNA detection by in situ hybridization, we have quantitatively compared the hybridization signal obtained when measles virus or visna virus infected cell cultures were fixed with eight different fixatives and hybridized with 35S-labeled virus-complementary DNA probes of several size ranges. Small probes (mean length, 70 bases) gave higher signals than larger probes (mean lengths 140, 350, and 780 bases) with all fixatives. This increase in signal was minimal with acetic ethanol or formalin, but was dramatic with fixatives containing glutaraldehyde; with these fixatives the signals with small probes were 6.5- to 22-fold greater than with large probes. The highest signals were obtained with periodate-lysine-paraformaldehyde-glutaraldehyde (PLPG) fixed cells hybridized with small probes, and were 1.5- to 6.7-fold greater than those obtained with the commonly used fixative acetic ethanol. PLPG and other glutaraldehyde based fixatives also greatly improved the preservation of cellular morphology compared to acetic ethanol.

Maedi-visna control in sheep. I. Artificial rearing of colostrum-deprived lambs.

A field trial to study the practicability and efficacy of maedi-visna control in sheep by artificial rearing of lambs was carried out during the lambing season of 1979. Lambs were immediately separated from the dams at birth, deprived of ovine colostrum, and reared isolated from the parent flock. Bovine colostrum was given instead of maternal colostrum. Eleven farms participated in the experiment. All flocks were severely infected with maedi-visna virus: 63-100% of the ewes were seropositive as demonstrated by ELISA. Artificially reared lambs were serologically tested and positives culled at the age of 6, 12, 18, 24, 30 and 36 months. Only very few positives were found: 1/389, 1/376, 0/337, 1/223, 1/192 and 0/144, respectively. The first two sero-positive lambs occurred in one flock, and it could be ascertained that both had mistakenly been given ovine colostrum probably containing maedi-visna virus. No explanation, other than sub-optimal hygiene and isolation, could be found for the two sero-positive sheep that turned up in another flock at 24 and 30 months of age although, transplacental infection cannot be entirely excluded. It is concluded that artificial rearing of ovine colostrum-deprived lambs is an effective and practicable method for the control of maedi-visna in sheep. The method appears particularly useful when valuable genetic material has to be salvaged.

Analysis of ovine colostrum to detect antibody against progressive pneumonia virus.

Immunoglobulins were isolated and purified from the colostrum and serum of progressive pneumonia virus infected sheep and also from non-infected control sheep. Four classes of immunoglobulins were isolated from sheep colostrum (IgG1, IgG2, IgA and Ig10s). Three classes of immunoglobulins were isolated from sheep serum (IgG1, IgG2 and IgM). Low levels of virus neutralizing activity were demonstrated only in the whole serum and purified serum IgG1 preparations. No complement fixing activity was detected in any of the antibody preparations from colostrum.