Jaagsiekte Sheep Retrovirus Can Reach Peyer's Patches and Mesenteric Lymph Nodes of Lambs Nursed by Infected Mothers.

Ovine pulmonary adenocarcinoma (OPA) is a contagious lung cancer of sheep caused by jaagsiekte sheep retrovirus (JSRV). It is generally accepted that transmission by the respiratory route occurs under natural conditions. However recent studies strongly indicate that JSRV can also be transmitted to lambs perinatally via colostrum and milk (C/M). The aim of this work was to confirm that C/M can transmit JSRV infection to lambs under natural conditions and investigate the initial events associated with this transmission route. We have analyzed the presence of JSRV in C/M samples from 22 naturally infected, asymptomatic ewes throughout a lactation period, and in various tissues collected from a group of 36 of their lambs that were fed naturally. The lambs were euthanized at 12, 24, 48, and 72 hours and at 5 and 10 days after birth. We detected JSRV-provirus by PCR in the somatic C/M cells from 10/22 ewes (45.45%). The virus was also detected in 9/36 lambs (25%). JSRV-infected cells, with lymphoreticular-like morphology, were observed by immunohistochemistry (IHC) and in situ hybridization (ISH) in Peyer's patches (PP) from the small intestine of the youngest lambs and in mesenteric lymph nodes (MLN) from lambs older than 72 hours. The virus was also detected by PCR in white blood cells (WBC) in 2/36 lambs (5.5%). These results confirm colostral transmission of JSRV to lambs under natural conditions. Infected lymphoreticular cells contained in C/M appear to be involved. These cells can cross the intestinal barrier of newborn lambs, reach the MLN and enter into circulation.

Colostrum and milk can transmit jaagsiekte retrovirus to lambs.

Ovine pulmonary adenocarcinoma (OPA) is a contagious disease caused by jaagsiekte sheep retrovirus (JSRV). In the three studies performed, we have obtained data of the importance of colostrum/milk (C/M) in the transmission of JSRV. In the first study, a group of sheep from a flock with a long history of OPA, samples from colostrum and peripheral blood leucocytes (PBLs) were collected. Two specific PCRs (U3-LTR and env of the JSRV) were carried out. Using U3PCR 8/34 sheep were positive in colostrum whereas with envPCR 7/34 were positive. From these animals only one was positive with U3PCR in the PBLs. Evidence of the transmission of JSRV infection by C/M was obtained in two more separate studies. In the second study, PBLs from five lambs from JSRV+ ewes and two from JSRV-ewes were tested by the U3PCR. They were fed C/M by their mothers during 3 months and slaughtered 7 months after birth. Three out of five lambs from the JSRV+ sheep become PBL positive at 3-4 months old and the other two were also positive at 4-6 months of age. One lamb of the JSRV-sheep became also PBL positive at an age of 3 months. In the third study, a group of lambs from JSRV negative mothers were fed with C/M from JSRV+ sheep and housed in separate unit. For comparison, another group of the same origin and maintained in another different unit, were fed with C/M containing a JSRV virus preparation. All lambs were blood sampled monthly and JSRV infection was detected as early as 15 days and several times onwards in both groups. Control groups fed with C/M from JSRV free flock and JSRV blood test negative sheep were always negative. Together these results indicate that suckling is an important natural transmission route for JSRV.

Eradication of ovine pulmonary adenocarcinoma by motherless rearing of lambs.

The principles of maedi-visna eradication programmes were applied to a field trial for the eradication of ovine pulmonary adenocarcinoma (OPA). In two maternal flocks the prevalence of gross and histological lesions in slaughtered animals was 18.3 per cent and 29.8 per cent, respectively. The lambing period was supervised for three consecutive years from 1999 to 2001, during which the lambs were taken away from their mothers at birth, deprived of maternal colostrum, and hand-reared away from other sheep. Over the three-year period, 322 hand-reared animals, mainly male lambs between 10 and 14 months old, were slaughtered; their lungs were examined grossly, 52.5 per cent of them were examined histologically, and 105 samples of caudal mediastinal lymph nodes were examined by PCR. No OPA tumours were detected in the slaughter specimens from the derived flock, but one lamb had histological lesions in one lung location; intrauterine transmission was ruled out in this case. No clinical OPA has subsequently been observed in the hand-reared flock. Bronchoalveolar lavage samples from the breeding stock were examined by PCR in order to rule out further subclinical cases of OPA. No Jaagsiekte retrovirus was detected in any of the 488 samples.

Infection of lung epithelial cells and induction of pulmonary adenocarcinoma is not the most common outcome of naturally occurring JSRV infection during the commercial lifespan of sheep.

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA). In this study, we followed over a 31-month period the natural transmission of JSRV in adult sheep and in their offspring. We established groups derived from flocks with either a high or low incidence of OPA and monitored virus transmission, clinical disease and macroscopic/microscopic lung lesions at necropsy. Results obtained show that (i) JSRV infection can occur perinatally or in the first few months of life in lambs and in adult sheep; (ii) only a minority of JSRV-infected animals develop clinical disease during their commercial lifespan; and (iii) JSRV is more readily detectable in peripheral blood leucocytes and lymphoid organs than in the lungs. These data support a model of opportunistic JSRV infection and tumorigenic conversion of type II pneumocytes/Clara cells in the lungs, while lymphoreticular cells serve as the principal virus reservoir.

Transformation and oncogenesis by jaagsiekte sheep retrovirus.

Jaagsiekte sheep retrovirus (JSRV) is an exogenous retrovirus of sheep that induces a contagious lung cancer, ovine pulmonary adenocarcinoma (OPA). JSRV is a potent carcinogen in the experimental setting, inducing end-stage tumors at around 6 weeks of age when newborn lambs are inoculated intratracheally. Despite this rapid oncogenesis, inspection of the JSRV genome sequence does not reveal any obvious viral oncogenes. In this review, recent advances in studies of JSRV oncogenic transformation are described. Molecular cloning of an infectious and oncogenic JSRV provirus was instrumental in the studies. DNA transfection of JSRV proviral DNA into mouse NIH3T3 cells results in morphological transformation, indicating that the JSRV genome carries an oncogene. Further experiments identified the JSRV envelope protein as the transforming gene, and a PI3 kinase docking site in the cytoplasmic tail of the transmembrane (TM) protein was shown to be necessary for transformation. Avian DF-1 cells infected with an avian retroviral vector (RCAS) expressing the JSRV envelope protein also undergo tumorigenic transformation. Possible mechanisms of transformation are discussed, and a cooperating role for insertional activation of proto-oncogenes in tumorigenesis is also considered. The transforming potential of the JSRV envelope protein may be necessary for JSRV infection and replication in vivo.

Prevention of transmission of sheep pulmonary adenomatosis by embryo transfer.

Two-hundred-and-fifteen embryos recovered from 76 donor ewes from flocks endemically infected with sheep pulmonary adenomatosis (SPA) and mated with uninfected rams were transferred to 131 uninfected recipients under strict sanitary conditions using International Embryo Transfer Society protocols. The recipients and their progeny were kept in a closed, isolated SPA-free flock. Thirty-eight of 51 progeny from SPA-positive donors and 55 of 74 progeny from donors in which no lesions of SPA were detected survived for at least five years after birth. In a similar study 11 embryos from four uninfected donors mated to an SPA-infected ram were transferred to seven recipients, and four of five progeny born to four recipients survived for at least five years. No evidence of SPA was found in the recipients or their progeny by embryo transfer in either study. On the basis of clinical and pathological criteria, it is concluded that embryo transfer can be used to provide an effective barrier against the transmission of SPA from donors from infected flocks, whether or not the parents show clinical signs of the disease.

The exogenous form of Jaagsiekte retrovirus is specifically associated with a contagious lung cancer of sheep.

Sheep pulmonary adenomatosis ([SPA] ovine pulmonary carcinoma) is a transmissible lung cancer of sheep that has been associated etiologically with a type D- and B-related retrovirus (jaagsiekte retrovirus (JSRV]). To date it has been impossible to cultivate JSRV in vitro and therefore to demonstrate the etiology of SPA by a classical approach. In addition, the presence of 15 to 20 copies of endogenous JSRV-related sequences (enJSRV) has hampered studies at the molecular level. The aim of this study was to investigate whether the expression of exogenous JSRV was specifically associated with neoplasia in SPA-affected animals. Initially, we found that enJSRVs were transcribed in a wide variety of normal sheep tissues. Then, by sequencing part of the gag gene of enJSRV we established a ScaI restriction site in gag as a molecular marker for the exogenous form of JSRV. Restriction enzyme digestion of PCR products obtained from the amplification of cDNA from a total of 65 tissues collected from SPA-affected and unaffected control sheep revealed that the exogenous form of JSRV was exclusively and consistently present in tumor tissues and lung secretions of the affected animals. In addition, exogenous JSRV provirus was detected only in DNA from SPA tumors and not from nontumor tissues of the same animals. This study has demonstrated clearly that the exogenous form of JSRV is specifically associated with SPA tumors.

Experimental transmission of jaagsiekte (ovine pulmonary adenomatosis) to goats.

Jaagsiekte was successfully transmitted to at least 2 out of 6 goats inoculated intratracheally with partially purified jaagsiekte retrovirus. Multiple, small, well circumscribed nodules found in the lungs consisted of typical papilliform proliferations of neoplastic Type II epithelial cells. Histological evidence of a mild interstitial pneumonia in 4 of the experimental animals can probably be attributed to a contaminating lentivirus in the jaagsiekte retrovirus preparation, as suggested by the seroconversion of the animals.

Sheep pulmonary adenomatosis: a contagious tumour and its cause.

Sheep pulmonary adenomatosis (SPA) is a contagious lung tumour that can be transmitted experimentally. Two viruses have been associated with the disease, a herpesvirus and a retrovirus. All ovine herpesviruses are related antigenically and have been isolated only from SPA tumour tissues. They do not appear to cause the tumour and their association with SPA appears to arise from reactivation of latent virus in the respiratory tract. SPA tumour tissue and lung fluid contain a retrovirus that has properties similar to those of type B and type D retroviruses. Homogenates of tumour that contain this retrovirus can transmit SPA to experimentally inoculated sheep. Various retroviruses have been cultured from such tumours. Only one of these has properties similar to that of the retrovirus detected in the tumour and it can transmit SPA experimentally. The others appear to be isolates of the non-oncogenic ovine lentivirus, maedi-visna virus, and play no part in the aetiology of SPA.

Rapid transmission of sheep pulmonary adenomatosis (jaagsiekte) in young lambs. Brief report.

Jaagsiekte, a contagious lung tumour of sheep, was induced within 3-6 weeks in day-old lambs by intratracheal inoculation of SPA lung fluids concentrated by centrifugation. Electronmicroscopic examination of the tumour revealed retrovirus particles whose morphogenesis and morphology support biophysical and immunological findings that suggest a relationship with type B and type D retroviruses.

Aetiology of jaagsiekte: transmission by means of subcellular fractions and evidence for the involvement of a retrovirus.

Jaagsiekte (ovine pulmonary adenomatosis) was transmitted to new-born lambs by inoculation of the microsomal fraction of a cytoplasmic extract of cultured tumour cells or tumour tissue. Various treatments of the biologically active fraction were carried out to differentiate between various classes of possible aetiological agents. The results obtained suggested the involvement of a membrane-associated RNA containing virus. Reverse transcriptase activity dependent on Mg++ was subsequently demonstrated in these extracts and in lung exudate, and was shown to be associated with particles banding at a density 1,175 in sucrose gradients. These characteristics, as well as the appearance of the particles in the electron microscope, are similar to those reported for Type B and Type D retroviruses. Serial transmissions of jaagsiekte over a number of years, using cytoplasmic extracts and purified virus, strongly suggest that this virus is the aetiologic agent of jaagsiekte.

On the aetiology of Jaagsiekte.

A summary is given of the results obtained in experimental transmission of jaagsiekte by means transplantation of cell cultures. Evidence is also presented of transformation as the mechanism of oncogenesis and possibility of a viral aetiology is discussed briefly.

Aetiology of jaagsiekte: experimental transmission to lambs by means of cultured cells and cell homogenates.

Studies on the transmission of jaagsiekte (ovine pulmonary adenomatosis) both by subinoculation of cells of known sex and by cell homogenates into male and female lambs are reported. The results obtained indicate a thymocyte-dependent rejection of male cells in female recipients in contrast to the successful transplantation of male cells in male animals and female cells in both sexes. This suggests the presence of a surface antigen determined by the gamma-chromosome in the tumour cells. A second mechanism of transmission, dependent on the transformation of the recipient's cells, was demonstrated by 2 cases of heterologous transplantation and confirmed by inoculation of cellular homogenates.

On the etiological role of Herpesvirus ovis in jaagsiekte.

Serologically related ovine herpesviruses have been isolated independently by various workers in different countries from adenomatous lung tissue of sheep or cell cultures derived from it. Although the disease can be transmitted with lung homogenates and with cell cultures, transmission attempts with virus alone failed. IUDR treatment of tumour-cell cultures and co-cultivation or fusion with cells permissive for virus replication induced antigens which react with some sera from tumour-bearing animals. These antigens give positive immunofluorescence reactions with sera both positive and negative in neutralization assays against the virus, however. Hybridization studies failed to demonstrate the presence of viral genomes in 15.4 tumour cells.

Transmission of jaagsiekte (ovine pulmonary adenomatosis) by means of a permanent epithelial cell line established from affected lungs.

An epithelial cell line, designated JS-15,4, has been established in culture from jaagiekte lesions and subcultured in vitro for almost 2 years. It exhibits morphological and other features of transformed cells and has been shown by electron microscopy to consist of type B ovine alveolar epithelial cells. Jaagiekte was successfully transmitted to 3 new-born lambs by the intratracheal injection of cells following immunosuppressive treatment with either anti-thymocyte immunoglobulin alone or combined with anti-macrophage immunoglobulin. Incubation periods as short as 10 weeks were recorded. Evidence was also obtained that natural transmission may result from the inhalation of viable cells.

Vertical transmission of progressive pneumonia of sheep.

Forty-two lambs were derived by hysterectomy from 27 ewes. Eight ewes had lesions of chronic progressive pneumonia (CPP) and 19 did not. Eleven lambs were derived from ewes with lesions of CPP. These 11 lambs were maintained in isolators under germfree conditions until killed at 2 to 4 months of age. Lungs from each lamb were examined for gross and microscopic lesions of CPP and examined by microbiological cultural technique (blood, eosin methylene blue, and PPLO agar). The lungs of one 4-month-old lamb from a ewe infected with CPP had foci of gray consolidation in the apical and cardiac lobes. The microscopic changes were comparable to those in the dam. The lungs of a 2.5-month-old lamb from a ewe infected with CPP had lymphocytic accumulations in the alveolar walls and around blood vessels and bronchioles. The 9 other lambs were free of lesions. Neither bacteria nor mycoplasmas were isolated from any of the 11 lambs. Lesions of CPP were not found in 31 lambs from the 19 noninfected ewes.