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This study sought to determine if T-cell cytokine responses to mycobacterial infections in sheep were similar to those in other species and if such responses correlated with prevailing gut pathology. Lymphocytes were isolated from the blood (PBL), mesenteric lymph nodes (MLN) and ileal lamina propria (LPL) of control sheep and of sheep with clinical Johne's disease due to infection with Mycobacterium avium ssp. paratuberculosis (M.a. paratuberculosis). These animals had previously been categorised into two groups exhibiting either the 'tuberculoid' (paucibacillary) form of lesion or the 'lepromatous' (multibacillary) form. Lymphocytes were examined for their capacity, following stimulation with johnin-PPD, to release interferon-gamma (IFN-gamma) and interleukin 2 (IL-2) characteristic of the Th1 subset of MHC Class II-restricted CD4+ (helper) T-cells in other species. The expression of the two cytokines appeared related to the type of histological lesion observed. Antigen-stimulated lymphocytes from the tuberculoid group exhibited greater release of IFN-gamma and IL-2 than lymphocytes from the lepromatous group suggesting a Th1-type of response in the former in which expression of IFN-gamma by PBL showed a significant positive correlation with that expressed by MLN and LPL. Lymphocytes from animals with lepromatous lesions released lesser mycobacterium-induced IFN-gamma and IL-2 indicating a diminished role for a Th1 subset in this group of sheep. Differences in cytokine expression were much more apparent with lymphocytes which were derived from MLN.

The sequence of the complete genome of ovine enzootic nasal tumor virus, an exogenous retrovirus associated exclusively with contagious intranasal tumors of sheep, was determined. The genome is 7,434 nucleotides long and exhibits a genetic organization characteristic of type B and D oncoviruses. Enzootic nasal tumor virus is closely related to the Jaagsiekte sheep retrovirus and to sheep endogenous retroviruses.

Nineteen adult sheep diagnosed as having clinical paratuberculosis (Johne's disease) and 16 unaffected controls were examined in this study. Animals were tested for the presence of circulating antibodies of Mycobacterium avium ssp. paratuberculosis (M. a. paratuberculosis) and lymphocytes derived from the blood, mesenteric lymph nodes and intestines were examined for cell-mediated immune (CMI) responses to Johnin pure protein derivative (Johnin-PPD: J-PPD). Bacteriological examinations were carried out on faeces and tissues and any mycobacterial isolates identified as M. a. paratuberculosis (IS900+) or M. avium ssp. silvaticum (M. a. silvaticum) (IS901+) by polymerase chain reaction (PCR). Full necropsy and histopathological studies were performed and diseased animals were categorised on the basis of having a lepromatous or tuberculoid form of intestinal pathology. Unaffected control sheep were generally antibody-negative and demonstrated varying CMI responses to J-PPD. Clinically-affected animals were almost always antibody-positive with variable CMI responses. A correlation was observed between the histological lesion type in the intestine and the cellular immune response. Tuberculoid-type lesions were associated with strong CMI responses in lymphocytes derived from the peripheral blood, mesenteric lymph node and intestine, whereas lepromatous-type lesions were associated with weak CMI responses in all tissues examined.

Molecular biology has contributed to our knowledge and understanding of the structure of Mycobacterium avium subspecies paratuberculosis and has been particularly useful in determining those components that elicit immune responses in the host or discriminate M. avium paratuberculosis from other closely related environmental mycobacteria. As such, it has made a significant impact in the field of diagnosis, and has been instrumental in the development of specific and sensitive diagnostic tests. The next decade will see exciting new developments in paratuberculosis research as a consequence of substantial advances made in the construction of gene transfer systems in mycobacteria. These will provide opportunities for applying new strategies to determine the genetic basis for pathogenesis and the mechanisms of drug resistance and will offer new prospects for the rational design of efficient vaccines.

Although it has long been thought that a retrovirus is the responsible agent for ovine pulmonary carcinoma (OPC), identification of a replicative viral agent has proven difficult. Recently, the genome of a new retrovirus, jaagsiekte sheep retrovirus (JSRV), found in the lung-wash of affected sheep lung, has been cloned and sequenced; characterization of this virus and its consistent presence in tumor cells argue for its role as the aetiologic agent of OPC. Analysis of the nucleic acid sequence of the JSRV genome, suggests a new class of retrovirus, one that is chimeric according to the morphological classification scheme used for retroviruses. The genome of this virus does not appear to contain an oncogene, and the mechanism by which it causes disease is still unknown. The presence of multiple copies of endogenous retroviruses related to JSRV in DNA of OPC-affected and unaffected sheep further complicates investigation of oncogenesis in OPC. This review examines the evidence for a retrovirus as the causative agent for OPC, with particular emphasis on the viruses studied to date. The significance of endogenous JSRV-related sequences is considered. The mechanisms by which a retrovirus such as JSRV might induce lung tumours in sheep, and which of these are most likely, are discussed in light of these developments, as are the prospects for new means of diagnosis and treatment of this disease.

A multiplex polymerase chain reaction has been developed which is able to distinguish Mycobacterium tuberculosis from other members of the M. tuberculosis complex. The assay is based on the simultaneous amplification of two different targets: a 396bp region from the mtp40 species-specific gene sequence of M. tuberculosis and a 245bp fragment from the M. tuberculosis complex insertion sequence IS986. Results have been obtained for 54 mycobacterial strains including five non-M. tuberculosis complex isolates. All 49 strains of the M. tuberculosis complex were positive for IS986 but only the 27 M. tuberculosis isolates were positive for both IS986 and mtp40.

A pool of five oligonucleotides has been used to detect the pathogenic organism Mycobacterium avium subspecies paratuberculosis in PCR-amplified DNA from ruminants. The oligonucleotides were labelled at the 5'-end with three dinitrophenyl reporter groups and hybridised to the target DNA, which was fixed to a nylon membrane by ultraviolet irradiation. Colourimetric detection of the PCR product was carried out using an anti-DNP antibody conjugated to horseradish peroxidase or to alkaline phosphatase. Detection with alkaline phosphatase was more sensitive than with horseradish peroxidase but, in both cases, the PCR product could be easily detected. The DNP labelling system offers an economic and effective alternative to biotin, digoxigenin or fluorescein for the detection of PCR-amplified DNA.

An outbreak of enzootic intranasal tumour (EIT) in a goat herd in Central Italy is described. From October 1990 to September 1992 41 tumors were diagnosed in goats ranging in age from 6 months to 6 years and of both the Alpine and Saanen breeds. Clinically the affected goats showed nasal discharge, facial swelling, sneezing and snoring respiration and weight loss. Post-mortem examinations revealed uni- or bilateral tumours of the olfactory mucous membrane. The neoplasms were classified as low grade adenocarcinomas. Ultrastructurally, retrovirus-like particles were observed in 10/10 tumours examined. Epidemiological and ultrastructural findings strongly suggest a viral aetiology for intranasal enzootic adenocarcinoma in the goats examined.

Vaccination of pregnant ewes with a Pasteurella haemolytica adjuvanted vaccine which contained serotypes A1, A2 and A6 antigens caused significant increases in serum antibody titres to A1 and A6 measured by the indirect haemagglutination test (IHA). The response to vaccination with the serotype A2 antigen contained in this vaccine cannot be measured by the IHA test. There were also increased antibody titres in the colostrum from the vaccinated ewes and in the serum of their lambs after sucking compared with the corresponding titres in unvaccinated ewes and their lambs. The inoculation of either 10 +/- 2- or 18 +/- 2-day-old lambs from both vaccinated or unvaccinated ewes with the same vaccine induced the active production of antibodies to serotypes A1 and A6 despite the presence of passively acquired antibodies. By four weeks after vaccination the group geometric mean serum antibody titres of lambs from both vaccinated and unvaccinated were similar whether the lambs were vaccinated at 10 or 18 days of age. Successful vaccination of young lambs with this type of vaccine is therefore possible. Optimum protection would be obtained by vaccinating ewes in late pregnancy and their lambs within the first two weeks of life.

Two groups of 10, specific pathogen free lambs were injected with a long acting oxytetracycline preparation at a dose rate of 20 mg/kg either 24 hours before or 24 hours after exposure to an aerosol of Pasteurella haemolytica. When compared with the response of similarly infected but untreated lambs, the effect of pretreatment was to postpone the appearance of clinical signs of pneumonia for four days and the deaths of five lambs for five to six days post infection, by which time seven untreated lambs had died. Treatment 24 hours after infection caused a rapid clinical recovery which persisted until six days after infection but two treated lambs died seven days after infection. Lung lesions in the group treated after infection were significantly less extensive than those in the untreated lambs.

The infective agent of jaagsiekte was shown to be present in the fluid which accumulates in the respiratory tract of sheep during the terminal stages of the disease. The fluid also contained reverse transcriptase (RT) activity which showed a clear preference for a ribonucleic acid synthetic template over the corresponding deoxyribonucleic acid template and which utilised the RT specific template/primer poly (2'-0-methylcytidylate) oligodeoxyguanylate. This enzyme activity was associated with a particle which had typical retroviral buoyant densities in a range of gradient media.

Methods of immunising lambs against pneumonic pasteurellosis, caused by several serotypes of Pasteurella haemolytica, were assessed in specific pathogen free lambs. Lambs were vaccinated intramuscularly with sodium salicylate extract (SSE) of P haemolytica, either alone or in combination with heat-killed organisms (HKO). SSE of P haemolytica type A1 protected vaccinated lambs against pneumonia resulting from challenge with the homologous serotype. SSE of type A2 also provided some protection but this was improved by vaccination with a combination of SSE and HKO.

An efficient and reproducible technique is described for the isolation of transformed sheep pulmonary adenomatosis cells. It includes three basic steps: prolonged trypsinisation to kill fibroblasts, magnetic removal of macrophages and adherence to remove the rapidly adherent cells. The resultant preparations of lung cells were enriched to 96.6 per cent type 2 pneumocytes.

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.

Sentinel cattle at a number of localities in northern and central coastal New South Wales were sampled over the summer and autumn seasons of the years 1979, 1980 and 1981. A total of 118 orbiviruses were isolated; 99 were of the Palyam group, 15 were of the epizootic haemorrhagic disease (EHD) of deer group, and 4 of the bluetongue group. The Palyam group viruses were identified by serotype as 68 Bunyip Creek, 23 CSIRO Village, 7 D'Aguilar and one was not typed. The EHD viruses were identified as 13 type 5 and 2 type 6. All 4 bluetongue viruses were type 21. There was also convincing serological evidence that bluetongue type 1 infection occurred in 1980. Antibody to the bluetongue group, as demonstrated in a gel diffusion precipitin test, was often transient. It appeared to be mostly cross-reactive with, and induced by, other orbivirus infections, particularly those of the EHD group. Viruses of the Palyam group also seemed to be implicated in some circumstances. Where infections by viruses of the bluetongue group were demonstrated, the precipitating antibody responses to a bluetongue group antigen were not noticeably stronger than many which followed EHD virus infection. The results generally confirm previous conclusions, deduced from serological surveys, regarding the frequency of orbivirus infections, the presence of bluetongue viruses, and the transient nature of many bluetongue group antibody reactions.

The efficacy of a multicomponent clostridial vaccine containing Pasteurella haemolytica antigens was tested in specific pathogen free or conventionally reared lambs exposed to experimental infection with P haemolytica serotypes A1, A2 or A6. In four experiments assessment was based upon the findings of clinical, pathological and bacteriological examinations. Three experiments carried out in conventionally reared lambs demonstrated protection against challenge infection with P haemolytica serotypes A1, A2 and A6 in vaccinated lambs. However, the inconsistency of the disease induced in these experiments emphasised the need to perform definitive studies in specific pathogen free conditions. The final experiment was carried out with specific pathogen free lambs and confirmed the efficacy of the multicomponent clostridial vaccine containing P haemolytica antigen in protecting against the effects of infection with P haemolytica serotype A6. In addition, this experiment indicated that the inclusion of several components in a vaccine did not affect the efficacy of an individual antigenic component.

Virus particles, morphologically indistinguishable from maedi-visna virus or caprine arthritis-encephalitis virus, were detected in tissue explant cultures prepared post mortem from a goat with joint, lung and kidney lesions similar to those associated with the caprine arthritis-encephalitis complex. Serum from this goat and others in its herd of origin gave positive reactions in an agar gel immunodiffusion test for maedi-visna virus antibodies.

Two retrovirus-associated pulmonary diseases of sheep [ovine pulmonary carcinoma (OPC); sheep pulmonary adenomatosis], a bronchoalveolar carcinoma, and lymphoid interstitial pneumonia (LIP) were induced simultaneously in 9 of 9 neonatal lambs. The lambs were killed 8-28 weeks after intratracheal injection of lung tumor homogenate or lung fluid derived from sheep with naturally occurring OPC and ovine lentivirus (OvLV) infection. The inoculated lambs developed multifocal neoplasms of alveolar type II cells or nonciliated bronchiolar epithelial cells, LIP, and pulmonary lymph node hyperplasia, and all produced antibody to OvLV. OvLV was isolated from 6 to 7 lambs tested, and infectious center assay of pulmonary lavage cells from 3 lambs revealed that approximately 1 in 1,000 pulmonary lavage cells contained infectious lentivirus. Neither contact control lambs nor control lambs that received ultrafiltered lung fluid developed evidence of either disease or of OvLV infection. Lung fluid or tumor tissue of lambs with OPC contained a 26,000-dalton protein that cross-reacted with antiserum to p27 to Mason-Pfizer monkey virus, a type D retrovirus. The fact that no antigenic cross-reaction between OvLV and type D retroviruses has been demonstrated supports the presence of two retroviruses in sheep with OPC. Although the contributions of each agent to oncogenesis in this model are difficult to evaluate, the rapid development of two retrovirus-induced pulmonary diseases in experimentally inoculated lambs suggests an etiologic or pathogenetic synergism between these two members of the family Retroviridae.

Using antigens prepared from cell cultures infected by bluetongue (BLU) virus type 20 (BLU-20), and sera from cattle which had recovered from experimental infection by that virus, two distinct precipitin reactions were demonstrated by immunodiffusion. Two distinct gel diffusion precipitin tests were developed based on these reactions. The antigen of one was common to BLU-20 and two other Australian BLU isolates, CSIRO 154 (BLU-21) and CSIRO 156 (BLU-1). It was therefore concluded to be a group-specific test. The antigen of the second appeared to be unique to BLU-20. The test based on this antigen correlated well with the virus neutralization test for BLU-20 and it was therefore concluded to be type-specific. Similar methods applied to a virus of the Palyam (PAL) group demonstrated two precipitin reactions of similar broad (group) and narrow (type) specificity.

A retrovirus that causes pulmonary adenomatosis, a contagious lung tumour of sheep, contains a 25 000 mol. wt. polypeptide which cross-reacts with the major core protein (p27) of Mason-Pfizer monkey virus and mouse mammary tumour virus.

Measurements of pulmonary function were made on patients within 1 wk, 3 wk, 5 wk, 3 months, and 5 months of spinal cord injury. In patients with functionally complete transection of the cord between segment C5 and C6, the vital capacity was 30% of predicted in the first week after injury. Patients with injuries at C4 had smaller vital capacities. A significant increase in vital capacity occurred within 5 wk of injury with an approximate doubling of vital capacity 3 months after injury. Expiratory flow rates were directly dependent upon vital capacity. There was a high incidence of arterial hypoxemia, in the acute stage, even in patients with adequate ventilatory ability and normocarbia.