A. Ortín

Lack of a specific immune response against a recombinant capsid protein of Jaagsiekte sheep retrovirus in sheep and goats naturally affected by enzootic nasal tumour or sheep pulmonary adenomatosis

Enzootic nasal tumour (ENT) and sheep pulmonary adenomatosis (SPA) are two contagious adenocarcinomas of the respiratory tract of sheep and goats. Both diseases are associated with related, but distinct, type-D-retroviruses (ENTV and JSRV respectively). No evidence of circulating antibodies has been described in animals affected by either ENT or SPA using antigens from natural sources. We evaluated the usefulness of a recombinant JSRV capsid protein (JSRV-CA) as antigen to study the antibody responses of animals naturally affected by ENT or SPA, using immunoblotting. Positive reactions were detected in the sera of both affected and unaffected sheep and goats. The reactivity was abolished completely by absorption with the GST fusion partner but not by JSRV-CA, suggesting that it was not specific. The results support prior observations indicating that sheep and goats infected by JSRV and ENTV do not develop specific humoral responses to these retroviruses.

Roles of the Ras-MEK-Mitogen-Activated Protein Kinase and Phosphatidylinositol 3-Kinase-Akt-mTOR Pathways in Jaagsiekte Sheep Retrovirus-Induced Transformation of Rodent Fibroblast and Epithelial Cell Lines

ABSTRACT Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA), a transmissible lung cancer of sheep. The virus can induce tumors rapidly, and we previously found that the JSRV envelope protein (Env) functions as an oncogene, because it can transform mammalian and avian fibroblast cell lines. (N. Maeda, Proc. Natl. Acad. Sci. USA 98:4449-4454, 2001). The molecular mechanisms of JSRV Env transformation are of considerable interest. Several reports suggested that the phosphatidylinositol 3-kinase/Akt pathway is important for transformation of mammalian fibroblasts but not for chicken fibroblasts. In this study, we found that Akt/mTOR is involved in JSRV transformation of mouse NIH 3T3 fibroblasts, because treatment with the mTOR inhibitor rapamycin reduced transformation. We also found that H/N-Ras inhibitor FTI-277 and MEK1/2 inhibitors PD98059 and U0126 strongly inhibited JSRV transformation of NIH 3T3 fibroblasts, suggesting that the H/N-Ras-MEK-mitogen-activated protein kinase (MAPK) p44/42 pathway is necessary for the transformation. In RK3E epithelial cells, the MEK1/2 inhibitors also eliminated transformation, but FTI-277 only partially inhibited transformation. It was noteworthy that p38 MAPK inhibitors enhanced JSRV transformation in both fibroblasts and epithelial cells. Treatment of transformed cells with p38 inhibitors both increased levels of phospho-MEK1/2 and phospho-p44/42 and induced rapid enhancement of the transformed phenotype. Immunohistochemical staining of tumor tissues from naturally and experimentally induced OPA and naturally occurring enzootic nasal adenocarcinoma revealed strong activation of MAPK p44/42 in all cases examined. However, p38 activation was not generally observed. These results indicate that signaling through two pathways (in particular, H/N-Ras-MEK-MAPK and, to a lesser extent, Akt-mTOR) is important for JSRV-induced transformation and that p38 MAPK has a negative regulatory effect on transformation, perhaps via MEK1/2 and p44/42.

Enzootic Nasal Adenocarcinoma of Sheep and Goats

Enzootic nasal adenocarcinoma is a contagious tumour of the mucosal nasal glands affecting young adult sheep or goats. The disease occurs naturally in all continents except Australia and New Zealand. Clinical signs include continuous nasal discharge, respiratory distress, exophthalmos and skull deformations. The tumour is classified histologically as a low-grade adenocarcinoma. Nasal glands of both respiratory and olfactory muosal glands seem to be the origin of the neoplasia. It has been experimentally transmitted in sheep and goats using either tumour extracts or concentrated nasal fluids. Two distinct retroviruses are implicated in the aetiology of the neoplasia one in sheep (ONAV) and one in goats (CNAV). We suggest that jaagsiekte sheep retrovirus (JSRV), ONAV, CNAV, and their endogenous counterparts represent a unique family of retroviruses. The similarities between these viruses suggests that any control strategies, including vaccination, may be appropriate to both diseases. The differences, however, represent a unique resource for delineating the function of individual regions of the virus. It is intriguing that whilst ONAV and CNAV appear to be as different to each other as they are to JSRV, that they have very similar disease pathologies, distinct from that of OPA. Additionally, all three exogenous viruses manage to avoid instigating any apparent immune response. Whether this is indeed a result of tolerance induced by the endogenous counterparts or whether the viruses themselves have unique immunosuppressive properties will be an important finding.

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.

Vaccination schedules in small ruminant farms.

Development and implementation of health management plans is the cornerstone of profitable farms; prevention of microbial diseases by means of vaccination is an integral part of such a plan. In every production type and management system in small ruminants, microbial diseases have a major significance, hence their proper control must be based in good health management practices, including use of effective and safe vaccines. Development of various types of vaccines is evolving very quickly in recent years and the improvement of new type of vaccines offers prospects. The article reviews and discusses vaccination programs and latest advances in development of vaccines against diseases that cause major economic losses in small ruminants. Specifically, vaccination schedules for the following diseases are reviewed: bacterial abortion (abortion associated with Brucella melitensis, Campylobacter spp., Chlamydophila abortus, Coxiella burnetii, Salmonella abortus ovis or Salmonella brandenburg), caseous lymphadenitis, clostridial diseases, colibacillosis, contagious echtyma, epididymitis caused by Brucella ovis, footrot, mammary diseases (contagious agalactia, mastitis), paratuberculosis and respiratory diseases (respiratory disease caused by Mannheimia haemolytica or other Pasteurellaceae).

The distribution of immune cells in the lungs of classical and atypical ovine pulmonary adenocarcinoma

Ovine pulmonary adenocarcinoma (OPA) is a contagious tumour caused by infection of sheep with Jaagsiekte sheep retrovirus. Two forms of OPA have been identified, classical and atypical, which can be distinguished clinically and pathologically. Most notably classical OPA is progressive until death, while atypical OPA remains subclinical. In the present study the local immune responses in the lungs of cases of atypical OPA were compared with those from classical cases by immunohistochemistry using a panel of mouse anti-sheep mAbs. Distinct differences in the distribution of immune cell subsets in the two forms of OPA were observed. In particular there was an intratumoural influx of T cell subsets and MHC Class II expression on the tumour cells in atypical OPA, neither of which was seen in classical OPA. It is possible that these differences may contribute, at least in part, to determining the progressive course of classical OPA compared with the subclinical nature of atypical OPA.

Photosensitisation in sheep grazing alfalfa infested with aphids and ladybirds

FIG 1: Lesions of photosensitisation in sheep. (a) Sheep with generalised oedema of the head, showing pendulous ears and oedema of the eyelids and lips; (b) severe oedema and erythema in the udder and teat of a lactating ewe FIG 2: (a) Alfalfa plant heavily infested with aphids. (b) Seven-spot ladybird (Coccinella septempuncta) larvae (arrowheads) and one chrysalis (arrow) attached to the alfalfa leaves (a)

Coexistence of pituitary adenocarcinoma and intraocular melanoma in a sheep.

PITUITARY adenocarcinomas and intraocular melanomas are very rare tumours in sheep. This short communication describes the coexistence of both these tumour types in a sheep that also showed lesions of ovine pulmonary adenocarcinoma, another more frequently diagnosed neoplasia. A 14-year-old Rasa Aragonesa ewe was presented to the Veterinary Faculty Hospital, University of Zaragoza, Spain, with a history of neurological signs. Its rectal body temperature was 38·5°C, heart rate 102 bpm and respiratory rate 16 breaths/minute. Examination of the cardiovascular, respiratory and digestive systems detected no abnormalities. The animal was very depressed, with reduced conscious proprioception and slow spinal reflexes, and showed a tendency to head press against the walls of the cage. Pupillary light reflexes were reduced on the left side and absent on the right side. Ocular examination of the left eye was within normal limits, but ocular disorders, including microphthalmos and severe enophthalmos with the eyelid detached from the ocular globe, were observed in the right eye. A deeper ocular examination of this eye by ultrasound (HDI 3500 system; Advanced Technology Laboratories) with a high frequency probe, showed an internal mass invading the posterior chamber, and several calcification foci were also evident (Fig 1a). The animal was euthanased due to its advanced age and ongoing deterioration. At postmortem examination, a large neoplastic mass (4·0 mm x 2·5 mm x 3·0 mm) replaced the normal pituitary gland, and compressed the adjacent brain structures (Fig 2). Histopathologically, the mass was composed of neoplastic cells polyhedral in shape, very pleomorphic with a lightly eosinophilic cytoplasm. The shape of the nuclei was very variable, but the majority were large and vesiculated. Multinucleated cells were occasionally observed and mitotic figures were very common, as were haemosiderin -laden macrophages (Fig 3a). Internally, the mass had large areas of necrosis, with many haemorrhages and calcified foci evident. The tumour had destroyed the pars distalis of the pituitary gland, and infiltrated the neurohypophysis and adjacent brain structures. These findings were histopathological features strongly suggestive of malignancy (Capen 2002) and the tumour was diagnosed as a pituitary chromophobe adenocarcinoma. Macroscopically, the right ocular globe was atrophied. Calcified foci were found in the posterior chamber and a large, deeply pigmented mass filled this chamber producing lens dislocation and a rupture into the anterior chamber (Fig 1b). Histologically, the tumour appeared to originate from the iris and ciliary body and extended into the retina producing degeneration and detachment. The tissue consisted of spindle-shaped cells with abundant intracytoplasmic melanin granules that obscured the nuclear morphology (Fig 3b). Intraocular melanoma was diagnosed. Another lesion was found in the right lung. A small grey nodule was located on the surface of the diaphragmatic lobe in a ventral position. Histologically, this nodule was consistent with a preclinical lesion of classical ovine pulmonary adenocarcinoma (OPA), a lung neoplasia associated aetiologically with a Jaagsiekte sheep retrovirus (JSRV) infection (De las Heras and others 1992, 1995, Garcia-Goti and others 2000). The presence of this virus was confirmed by JSRVspecific PCR (Palmarini and others 1996). Metastases of these three tumours were not observed and the only other lesions detected were grossly visible sarcocysts in the oesophagus. The most common pituitary tumours in domestic animals are found in the dog and horse, and reports in ruminants are very rare, especially in sheep. The majority of the ovine descriptions are adenomas (Olson and others 1981, Gonzalez and others 1994, Oda and others 1997), but more recently an adenocarcinoma was documented (Zanolari and others 2004). In the sheep in this report, the malignant histological features of the pituitary tumour favoured its diagnosis as an adenocarcinoma, even though metastases were not detected. However, other primary tumours were found in this animal. To the authors’ knowledge, this is the first evidence for the coexistence of a pituitary chromophobe adenocarcinoma, an intraocular melanoma and an OPA nodule within the same sheep. Pigmented ocular tumours are also extremely rare in sheep, and reports are limited to an intraocular malignant melanoma (Saunders and Barron 1958) and a melanotic tumour of the eye (Head 1990). In contrast, OPA is an ovine lung cancer that has been recorded on many occasions in many countries (Sharp and DeMartini 2003). The advanced age of this animal could have contributed to the emergence of the multiple tumours, increasing the opportunity for the development of several primary tumours. Reported cases of pituitary tumours to date have Veterinary Record (2006) 159, 718-719

Staphylococcus chromogenes-induced folliculitis in goat kids

THE haemolytic, coagulase-negative bacterium Staphylococcus chromogenes is a common component of the skin flora in some species of farm animals. It is also a common cause of mastitis in dairy cattle, but is rarely described as the aetiological agent of skin diseases. This short communication

Bronchioloalveolar Carcinoma Not Related to Jaagsiekte Sheep Retrovirus in a Goat

A spontaneous lung tumor in a 5–year-old goat of the Murciano-Granadina breed is described in this paper. Clinical signs of cachexia and tachypnoea were evident, and a considerable amount of white mucous foamy fluid was discharged from the nostrils when the animals head was lowered. A lung tumor with the characteristics of bronchioloalveolar carcinoma was detected during histopathologic examination. The tumor cells were positive for surfactant proteins C and B, confirming that alveolar type II cells were the origin of the neoplasia. Tumor samples were tested by polymerase chain reaction, immunoblotting, and immunohistochemistry for the presence of Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV), another retrovirus very closely related to JSRV, but all tests were negative. Therefore, this is the first reported case of spontaneous bronchioloalveolar carcinoma not related to JSRV or ENTV infection in a goat.