Oat cells in the pathology of ovine pneumonia-pleurisy

Light microscope observations on oat cells in the ovine pneumonia-pleurisy complex are presented. This study is based on the experimental production of the disease by viruses and Pasteurella haemolytica. Oat cells appeared only in necrotic lesions associated with large numbers of P. haemolytica in thi pneumonic lung. It is suggested that oat cells originate from blood monocytes, which transform into the oat shape when developing in the necrotic, hypoxic environment created by P. haemolytica. They were not, however, observed to be phagocytic. Oat cells are characteristic of pneumonic pasteurellosis but are not pathognomonic because they can also be found in extrapulmonary locations and in other pathological conditions of the lungs.     

Oat cells in the pathology of ovine pneumonia-pleurisy

Light microscope observations on oat cells in the ovine pneumonia-pleurisy complex are presented. This study is based on the experimental production of the disease by viruses and Pasteurella haemolytica. Oat cells appeared only in necrotic lesions associated with large numbers of P. haemolytica in the pneumonic lung. It is suggested that oat cells originate from blood monocytes, which transform into the oat shape when developing in the necrotic, hypoxic environment created by P. haemolytica. They were not, however, observed to be phagocytic. Oat cells are characteristic of pneumonic pasteurellosis but are not pathognomonic because they can also be found in extrapulmonary locations and in other pathological conditions of the lungs.     

Distribution of the Shadoo protein in the ovine brain assessed by immunohistochemistry

Shadow of prion protein is a gene potentially involved in the pathogenesis of prion diseases. However, the Shadoo protein encoded by this gene has not yet been studied in sheep, an important species in prion matters. Therefore, we developed a polyclonal antibody against ovine Shadoo and assessed the presence and distribution of this protein in the ovine brain by immunohistochemistry. The strongest staining level was found in the cerebellum (especially in the Purkinje cells) and in the pons, but cerebrum, hippocampus, pituitary gland, medulla oblongata, thalamus and hypothalamus were also immunopositive. Remarkably, a typical granular pattern was seen in most of the tested brain tissues, which might indicate that Shadoo is primarily expressed at synapses. The results of this study and the availability of an ovine anti-Shadoo antibody can contribute to future research on the function of Shadoo and on its potential involvement in prion diseases.     

Comparison of brain PrPd distribution in ovine BSE and scrapie

Scrapie and bovine spongiform encephalopathy (BSE) are both prion diseases affecting ruminants, and these diseases do not share the same public health concerns. Surveillance of the BSE agent in small ruminants has been a great challenge, and the recent identification of diverse prion diseases in ruminants has led to the development of new methods for strain typing. In our study, using immunohistochemistry (IHC), we assessed the distribution of PrP(d) in the brains of 2 experimentally BSE-infected sheep with the ARQ/ARQ genotype. Distribution of PrP(d) in the brain, from the spinal cord to the frontal cortex, was remarkably similar in the 2 sheep despite different inoculation routes and incubation periods. Comparatively, overall PrP(d) brain distribution, evaluated by IHC, in 19 scrapie cases with the ARQ/ARQ, ARQ/VRQ, and VRQ/VRQ genotypes, in some cases showed similarities to the experimentally BSE-infected sheep. There was no exclusive neuroanatomical site with a characteristic and specific PrP(d) type of accumulation induced by the BSE agent. However, a detailed analysis of the topography, types, and intensity of PrP(d) deposits in the frontal cortex, striatum, piriform cortex, hippocampus, mesencephalon, and cerebellum allowed the BSE-affected sheep group to be distinguished from the 19 scrapie cases analyzed in our study. These results strengthen and emphasize the potential interest of PrP(d) brain mapping to help in identifying prion strains in small ruminants.     

Growth of differentiated ovine tracheal epithelial cells in vitro

Culture of ovine tracheal epithelial cells is a useful tool for conducting various in vitro studies. We describe herein an in vitro technique and the conditions for culturing primary epithelial cells derived from tracheas of adult sheep. Ovine tracheas were surgically removed from 2- to 3-month-old healthy sheep and tracheal epithelial cells were isolated by 0.15% pronase digestion. After epithelial cells isolation, a Millicell insert with porous membrane was coated with 0.05% human placental collagen and the epithelial cells were added to the membrane. To create an air-liquid interface environment for the cells, the apical compartment of the membrane containing the tracheal epithelial cells was left exposed to 5% CO(2) at 37 degrees C for 2 days then increased to 9% CO(2) while cells in the basolateral compartment underneath the membrane contained the growth medium necessary for cells nourishment. Pepsin digestion was more effective in reducing the number of fibroblasts than other procedures. Cells were allowed to grow for 6-7 days to form a confluent monolayer and nearly 21 days for cilia formation on the apical surface as determined by light microscopy of haematoxylin and eosin-stained sections of membranes. In order to further confirm the epithelial origin of cells, cells were stained for cytokeratin antigen by immunohistochemistry. Most ciliated epithelial cells were immunoreactive for cytokeratin. This is the first report of differentiated ovine tracheal epithelial cells growth and isolation. This technique can be used in numerous in vitro investigative studies in ovine species as an animal model for human disease.     

Site-directed mutagenesis of the myostatin gene in ovine fetal myoblast cells in vitro

Myostatin is an important negative regulator of muscle growth and development. Natural mutations of the myostatin gene cause a double muscling phenotype in beef cattle, pigs and sheep. Therefore, it is feasible to produce a high growth domestic breed by generating a transgenic animal with a mutation, deletion or knockout of the myostatin gene. Our objective was to introduce a subtle mutation of G to A 281-bp upstream of the 3' untranslated region (3'UTR) end of the myostatin gene in Poll Dorset fetal myoblast cells in vitro. Fetal myoblast cells were isolated from fetuses at day 50 of gestation from Poll Dorset sheep and transfected with linear gene-targeting vector pMSTN-A using electroporation. We obtained seven gene-targeted cell colonies with homologous recombination, which were positive as confirmed by PCR, Southern blot. The Western blot analysis result demonstrated that the myostatin protein expression in positive colonies is lower than that of negative ones. These results strongly suggest that we successfully mutated the myostatin gene of Poll Dorset ovine fetal myoblast cells and the mutation can effectively downregulate the myostatin protein expression.     

Maedi-visna virus infection of ovine mammary epithelial cells

The aim of this work was to perform a complete study of maedi-visna virus (MVV) infected mammary glands of naturally-infected sheep, and to determine if cells other than macrophages undergo a productive viral infection in this organ. Fifteen seropositive and two seronegative ewes were selected from MVV-infected flocks on the basis of clinical indurative mastitis and three sheep from an MVV-free flock. Within the mammary gland, MVV-positive cells were located by immunohistochemistry in the stroma and the epithelial alveolar barrier, most likely the ovine mammary epithelial cells (OMEC) of the acini. In situ hybridization confirmed these findings. Ultrastructural studies showed the presence of lentivirus-like particles budding off the cell surface in the alveolar barrier and also free in the acinar lumen. The presence of mammary histopathological lesions and MVV together with clear indications of productive infection (demonstration of a cytopathic effect in OMEC cultures and infection of co-cultures) were observed in the 15 seropositive and one of the seronegative sheep from the infected flock. These findings demonstrate that the OMEC were infected in vivo and probably underwent productive infection when studied ex-vivo. The OMEC of MVV-free sheep, which had subsequently been infected in vitro with MVV, also showed productive infection when challenged in vitro, confirming the replication of MVV in OMEC in vitro. The presence of MVV-infected OMEC in the mammary gland from infected animals, the productive infection in these OMEC and the release of lentiviral particles to the acinar lumen may have relevance in the pathogenesis and transmission of MVV infection.     

Co-culture of ovine ova with oviductal cells in medium 199

Three experiments were conducted to test the suitability of medium 199 supplemented with 10% fetal calf serum (M199FCS) as a medium for co-culture of one-cell sheep ova with sheep oviductal cells. In Exp. 1, ova were co-cultured for 5 d in 5 ml of M199FCS or in Ham's F10 medium supplemented with 10% fetal calf serum (F10FCS). Co-culture did not increase the number of cleavages at the end of 5 d of culture, but M199FCS supported more cleavages than did F10FCS (P = .016). In Exp. 2, ova were cultured for 1 to 3 d in M199FCS alone or on oviductal, uterine or kidney cell monolayers from ewes 2 d postestrus and transferred to recipients from which they were recovered at 8 d postestrus. Co-culture with oviductal cells improved (P less than .001) the cleavage index of recovered embryos compared with culture in medium alone or co-culture with other cell types. In Exp. 3, monolayers of oviductal cells from ewes 2 d postestrus and from luteal-phase ewes were cultured as in Exp. 2. No difference was observed between the two sources of oviductal cells for their ability to support in vitro development of one-cell sheep eggs for 1 or 2 d. These studies suggest that M199FCS may be a good medium to use in an oviductal cell co-culture system for one-cell sheep ova. Results further suggest that specific secretions of oviductal cells may be important for early embryo development in vivo.     

Specific binding of naloxone to ovine brain tissue: comparison of brain regions and endocrine states

Binding of [3H]naloxone ([3H]NAL) to brain membranes was quantified by Scatchard analysis using two methods of separating bound from free [3H]NAL. In the centrifugation method, membranes that were soluble at 1,000 x g, but sedimented at 20,000 x g, were incubated with [3H]NAL. For filtration, all membranes that sedimented at 20,000 x g were incubated and filtered through glass filter fibers. Nonspecific binding was estimated using greater than 500-fold excess of unlabeled naloxone (10(-6) M). Specific binding of [3H]NAL was used to generate linear multiple-point Scatchard plots, which indicated a single class of high-affinity sites. In Exp. 1, 10 ovariectomized (OVX) ewes were injected with estradiol-17 beta alone or in combination with progesterone. Compared with OVX controls, these hormonal treatments did not affect binding of [3H]NAL (centrifugation method) to combined hypothalamus (HYP) + preoptic (POA) tissues. In cyclic ewes (Exp. 2, filtration method), affinity constants (2.4 +/- .2 x 10(8) M-1) did not differ among HYP, POA and basal forebrain (BF) tissues, but BF had more sites (39 +/- 3 fmol/mg) than either HYP (14 +/- 1) or POA (17 +/- 1). Binding affinity and concentration of sites within each brain area (HYP, POA, BF) did not differ between d 8 and d 16 (preovulatory but after luteolysis) in normally cycling ewes. Overall, neural tissue dissected from BF had a greater concentration of binding sites than HYP or POA. Exogenous and endogenous fluctuations in ovarian steroids did not affect binding of [3H]NAL to these tissues.     

Pathology in the ovine foetus caused by an ovine pestivirus

Homogenised tissues or tissue culture supernatant fluid containing a noncytopathic pestivirus obtained from a lamb with a neurologic form of border disease, were inoculated into ewes at different stages of pregnancy. Foetal death occurred in 9 ewes of those inoculated between 19 and 47 days of pregnancy while 3 ewes did not lamb. Eight of the foetuses were aborted between 77 and 132 days of pregnancy; of these 6 were autolysed or mummified and one had arthrogryposis. The one full-term dead lamb had a hairy birth coat and lissencephalic micrencephaly. Foetal death occurred in only 7 of 14 ewes inoculated between 57 and 72 days of pregnancy. Four of these ewes aborted between 77 and 108 days of pregnancy and 3 gave birth to full-term, dead, hairy lambs. The remaining 7 ewes gave birth to live hairy lambs with severe inco-ordination. All lambs carried to term and aborted foetuses or lambs that could be examined had a range of intracranial malformations including focal leucomalacia, micrencephaly, hydranencephaly, porencephaly, lissencephaly and cerebellar hypoplasia. Some lambs also had skeletal abnormalities including arthrogryposis, scoliosis and brachygnathia inferior. The pestivirus isolate used in these trials produced more severe effects on the ovine foetus than previously observed in similar inoculation trials using pestivirus isolates from border disease lambs without nervous signs.     

Simple technique for the direct isolation of toxoplasma tissue cysts from fetal ovine brain

A simple technique is described for the isolation of Toxoplasma gondii tissue cysts from fetal ovine brain by centrifugation on a discontinuous density gradient of 30 per cent and 90 per cent Percoll (colloidal silica solution). Brain samples from 51 aborted ovine fetuses were examined by both the Percoll and mouse inoculation techniques; eight infections were detected by the Percoll technique compared to 12 by mouse inoculation. Possible reasons for this discrepancy and the scope for improving the Percoll technique are discussed.     

绵羊卵母细胞和卵丘细胞体外成熟过程中ghrelin mRNA的表达

运用实时定量RT-PCR技术检测了不同成熟培养时间绵羊卵母细胞和卵丘细胞ghrelin mRNA的相对表达量。结果表明,卵母细胞ghrelin mRNA相对表达量在16、24h呈现显著升高,而卵丘细胞ghrelin mRNA则随着成熟时间的延长表现显著降低。绵羊卵母细胞和卵丘细胞ghrelin mRNA的持续表达以及动力学变化提示这一新型分子在生殖功能上有潜在调控作用。     

NKp46 defines ovine cells that have characteristics corresponding to NK cells

ABSTRACT: Natural killer (NK) cells are well recognized as playing a key role in innate immune defence through cytokine production and cytotoxic activity; additionally recent studies have identified several novel NK cell functions. The ability to study NK cells in the sheep has been restricted due to a lack of specific reagents. We report the generation of a monoclonal antibody specific for ovine NKp46, a receptor which in a number of mammals is expressed exclusively in NK cells. Ovine NKp46+ cells represent a population that is distinct from CD4+ and γδ+ T-cells, B-cells and cells of the monocytic lineage. The NKp46+ cells are heterogenous with respect to expression of CD2 and CD8 and most, but not all, express CD16 - characteristics consistent with NK cell populations in other species. We demonstrate that in addition to populations in peripheral blood and secondary lymphoid organs, ovine NKp46+ populations are also situated at the mucosal surfaces of the lung, gastro-intestinal tract and non-gravid uterus. Furthermore, we show that purified ovine NKp46+ populations cultured in IL-2 and IL-15 have cytotoxic activity that could be enhanced by ligation of NKp46 in re-directed lysis assays. Therefore we conclude that ovine NKp46+ cells represent a population that by phenotype, tissue distribution and function correspond to NK cells and that NKp46 is an activating receptor in sheep as in other species.