Ovine Origin Research Articles

Cross-species PCR cloning of gerbil ( Meriones unguiculatus) interleukin-2 cDNA and its expression in COS-7 cells

A cDNA clone of the gerbil interleukin-2 (IL-2) gene was isolated by cross-species PCR cloning, and demonstrated to produce a functional gerbil IL-2 protein when inserted in the eucaryotic expression vector pSV-SPORT1 and transfected into COS-7 monkey cells. The open reading frame codes for a polypeptide of 155 amino acid residues with a molecular weight (MW) of 17,601 which includes a putative signal peptide. The mature gerbil IL-2 is deduced to contain 135 amino acid residues and has a calculated MW of 15,496. Culture supernatant of COS-7 cells transfected with pSV-SPORT1-GIL-2, but not pSV-SPORT1 stimulates the proliferation of the IL-2 dependent murine CTLL-2 cells. Molecular characteristics of gerbil IL-2 have been compared with IL-2 of mouse, rat, human, bovine, ovine and porcine origin. The mature form of gerbil IL-2 is similar in molecular weight to all species except the mouse. A N-glycosylation site present in bovine, ovine and porcine IL-2 respectively, is absent in gerbil. Three Cys residues are conserved in all compared mature IL-2 molecules. In these comparisons, gerbil IL-2 has highest identity with rat IL-2 for both nucleotide and amino acid sequence.

Identification of the activation domain of equine infectious anemia virus rev

Several members of the lentivirus family of complex retroviruses have been shown to encode proteins that are functionally equivalent to the Rev posttranscriptional regulatory protein of human immunodeficiency virus type 1 (HIV-1). Furthermore, the domain organization of HIV-1 Rev, featuring a highly basic N-terminal RNA binding domain and a leucin-rich C-terminal effector domain, has also been shown to be highly conserved among Rev proteins derived from not only the primate but also the ovine and caprine lentiviruses. Although it has therefore appeared highly probable that the lentivirus equine infectious anemia virus (EIAV) also encodes a Rev, the predicted amino acid sequence of this putative EIAV regulatory protein does not display any evident homology to the basic and leucine-rich motifs characteristic of other known Rev proteins. By fusion of different segments of the proposed EIAV Rev protein to the well-defined RNA binding domain of either HIV-1 or visna virus Rev, we have identified a segment of this EIAV protein that can efficiently substitute in cis for the otherwise essential activation motif. Interestingly, the minimal EIAV Rev activation motif identified in this study comprises approximately 18 amino acids located toward the protein N terminus that lack any evident similarity to the leucine-rich activation domains found in these other lentivirus Rev proteins. It therefore appears that the Rev protein of EIAV, while analogous in function to Rev proteins defined in lentiviruses of primate, ovine, and caprine origin, is nevertheless distinguished by an entirely novel domain organization.

Hormonal Induction of Male Courtship Behavior in the Japanese Newt, Cynops pyrrhogaster

In the breeding season, the sexually mature male newt, Cynops pyrrhogaster, vibrates the tail in front of the female at an early stage of courtship. Effects of prolactin (PRL), gonadotropin (GTH), and sex steroids on this behavior were investigated in the male paired with the female receiving PRL and GTH. The behavior was elicited in the sexually inert male by injections of PRL of bovine, ovine, or bullfrog origin and human chorionic gonadotropin or bullfrog LH and FSH in combination. The effect of PRL or GTH alone was less marked than that of PRL plus GTH, especially in terms of frequency of the behavior. In the hypophysectomized male, combination of PRL and GTH significantly increased both the incidence and frequency of the behavior. However, PRL alone was not effective, and the effect of GTH alone was less pronounced than that in the intact animal receiving GTH injections. The effect of GTH was nullified by castration. In the PRL-treated castrated animal, testosterone or dihydrotestosterone, but not estradiol, was effective in inducing the behavior.

Ovine enzootic abortion

Ovine enzootic abortion (OEA) is caused by a member of the chlamydia, Chlamydia psittaci. It was some 25 years ago that the potential significance for the pregnant woman of infection with Chlamydia psittaci of ovine origin was first recognised. It was only in the 1980's, however, that following a spate of case reports of maternal illness and abortion due to OEA that it gained more widespread attention, and even elicited questions in the House of Lords. Ovine enzootic abortion also presents major problems to sheep farming in the UK as it is the most frequent cause of abortion of ewes in the UK, and its spread has proved resistant to available vaccines.

Antigenic and morphological differentiation of placental and intestinal isolates of Chlamydia psittaci of ovine origin

Ewe placental and lamb intestinal isolates of Chlamydia psittaci recovered from flocks affected with ovine enzootic abortion were examined by inclusion morphology, indirect immunofluorescence (IIF) and immunoblot analysis. Chlamydiae recovered from the faeces of sheep from two flocks free of clinical disease were also examined. In cell culture ovine abortion (OA) and intestinal isolates were distinguishable by inclusion development and morphology. Similarly, in two-way IIF tests with one week mouse antisera isolates fell into two distinct groups: abortion or intestinal. Immunoblotting with convalescent sheep abortion antiserum identified 30 out of at least 40 silver staining polypeptides as antigenic both in OA and intestinal isolates. The serum produced a similar reaction pattern to the resolved proteins of each OA isolate, indicating a higher degree of antigenic conservation among these isolates. Considerable cross reactivity between the OA and intestinal isolates was identified, but the serum also showed apparent molecular weight differences between antigens of the two types in the 87-116 kDa, 38-44 kDa and 26-28 kDa regions. Furthermore, the immunoblotting analysis revealed heterogeneity among the intestinal isolates, particularly in antigens between 87-116 kDa and 38-44 kDa.

Identification of cell membrane proteins that bind visna virus.

Visna virus infects cells of ovine origin by attaching to a cell surface receptor via its envelope glycoprotein. The identity of the visna virus receptor is not known. To identify the molecule responsible for binding the virus to target cells, virus overlay protein blot assays were used to examine the molecular weights of cell surface molecules which bind purified virus. Molecules on the surface of goat synovial membrane (GSM) cells and sheep choroid plexus (SCP) cells of approximately 15, 30, and 50 kDa bound to visna virus. The binding of visna virus to these proteins was reduced by preincubating virus with neutralizing antibodies. 125I-labeled cell membrane preparations of GSM and SCP cells were used to affinity purify these virus-binding proteins. These proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had molecular masses of 15, 30, and 50 kDa. Antibodies to the 50-kDa protein bound to the surface of both live SCP and GSM cells in immunofluorescence assays. In addition, antibodies to the 50-kDa protein blocked the binding of [35S]methionine-labeled visna virus to SCP cells in culture. Antibodies raised against the 15- and 30-kDa proteins did not block virus binding to cells. The blocking activity of antibody of the 50-kDa protein provided data that this protein is the molecule which visna virus recognizes and binds to on the surface of target cells.

Ovine psittacosis and sarcoidosis in a pregnant woman.

A woman in the first trimester of pregnancy presented with pneumonia and hilar lymphadenopathy after exposure to lambing ewes. She subsequently aborted. Infection with Chlamydia psittaci of ovine origin was confirmed. Pregnant women are susceptible to this infection, which may cause life threatening disease. The patient also had features of sarcoidosis, and the two conditions ran a similar time course. There is a possibility that ovine psittacosis caused an illness indistinguishable from sarcoidosis.

Comparison of isolates of Chlamydia psittaci of ovine, avian and feline origin by analysis of polypeptide profiles form purified elementary bodies

The single species Chlamydia psittaci is a diverse grouping which contains several different types of chlamydial strain for which there is no generally accepted typing method. The results obtained when profiles of polypeptides from purified elementary bodies are compared are consistent with type designations obtained using other criteria. However, the method still requires large scale culture and extensive purification of the chlamydial cells.

Comparison of Surface Antigens of Some Campylobacter fetus subsp. fetus Strains of Ovine Origin by Polyacrylamide Gel Electrophoresis and Immunoblotting

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were used to identify and to compare the surface antigens of eight C. fetus subsp. fetus strains. Seven strains (one of serogroup A and six of serogroup B) were isolated from aborted ovine fetuses, while one strain (serogroup A) originated from an aborted calf fetus. Saline extracts at 56 degrees C and 100 degrees C were used as antigens. Antisera were produced in rabbits. In saline extracts (56 degrees C) of the strains at least 19 fractions were identified by SDS-PAGE, with molecular masses ranging from approx. 4,800 to 205,000. The major bands appeared at 205,000, 66,000, 31,500, 25,000, 21,000 and 17,500. Despite the fact that the strains were cultured from 4 different sheep flocks and belonged to serogroup A or B, the SDS-PAGE profiles of the strains were very similar. When boiled (100 degrees C) extracts were used, a band migrating at 32,500 in sheep strains and a band at 97,500 in the calf isolate were missing. Most of the bands obtained by SDS-PAGE could be identified also by the immunoblot procedure. A or B type specificity of the ovine isolates was due to an LPS fraction, migrating at approx. 21,000, while the other LPS fractions appearing under this region although reacted with antisera did not influence the type specificity. Using alkaline extracts (pH 12) in SDS-PAGE, LPS fractions gave more pronounced profiles. In two of our C. fetus subsp. fetus isolates, plasmids with a molecular mass of 31,500 were identified.

Characterization of Mycobacterium paratuberculosis and organisms of the Mycobacterium avium complex by restriction polymorphism of the rRNA gene region

Nineteen Mycobacterium paratuberculosis strains, including strains of bovine, caprine, ovine, cervid, subhuman primate, and human origins, were compared with organisms of the M. avium complex by restriction fragment length polymorphism with a 5S rRNA gene probe as the reference DNA. Mycobacterial DNA was extracted, digested with several restriction enzymes, subjected to electrophoresis and Southern blotting, and then hybridized with a 5S rRNA gene probe from Escherichia coli. Hybridizing bands were visualized by autoradiography, and the sizes of the resulting rRNA fragments in kilobases were determined. Base substitutions were calculated on the basis of the number of shared fragments between species and strains. It was determined that M. paratuberculosis and the M. avium complex possess a single copy of the rRNA genes within their genomes and that the M. avium complex and M. paratuberculosis are a group of closely related organisms, likely with a common ancestral link. In proximity to the 5S rRNA gene exists a region or regions which display polymorphisms that are capable of species and subspecies differentiation. M. paratuberculosis strains isolated from humans, subhuman primates, and animals were found to be genetically identical to each other. M. paratuberculosis strains lacked the genetic heterogeneity (restriction fragment length polymorphisms) characteristic of most species, suggesting that this organism has unidirectional genetic selection. It is therefore assumed to be biologically isolated, occupying a unique and specific biological niche. This homogeneity was present in all strains, including those of animal and primate (subhuman and human) origin and strains isolated from different parts of the world.

Regulation of endometrial prostaglandins during the menstrual cycle and in early pregnancy

Prostaglandins are important regulators of endometrial function. In turn, their secretion is controlled by endocrine and paracrine mediators. Cyclical effects of ovarian oestrogen and progesterone throughout the menstrual or oestrous cycle result in overall higher levels of prostaglandin release during the secretory or luteal phase of the cycle than during the proliferative phase. Potential paracrine regulators of endometrial origin include cytokines, growth factors and histamine, some of which may arise from infiltrating cells. Embryo-derived factors can regulate endometrial prostaglandin release in early pregnancy. Both platelet-activating factor and ovine trophoblast protein-1 (an embryonic interferon) modify prostaglandin release from primary cultures of endometrial cells of human and ovine origin respectively. Manipulation of such mediators may provide new means for fertility control.

Qualitative variation in the immune response to ovarian follicular fluid proteins in cattle

In two experiments 44 heifers were immunised with inhibin preparations of partially purified ovarian follicular fluid (PPFF) of ovine, porcine and equine origin in non-ulcerative Freund's adjuvant. In the first experiment 20 cattle were immunised with ovine PPFF1. In the second, a further 24 were immunised with either a different sheep PPFF (ovine PPFF2) or PPFF from pigs or horses. Large molecules of over 30 kDa initiated a strong immune response in some animals, but none in others, although there was no obvious relationship with the MHC Class I (BoLA) phenotype. Immunisation with ovine partly purified follicular fluid affected ovulation rate in some cattle, and only these cows produced detectable antibodies to an approximately 40 kDa protein. These data show that the immunological response of cattle to a mixture of antigens is highly variable between individuals and may explain the variation in the ovarian response to immunological manipulations of ovulation rate seen in this species.

Determination of the minimum time of praziquantel therapy required for the in vitro treatment of protoscoleces of Echinococcus granulosus.

Ovine and equine protoscoleces of Echinococcus granulosus were cultured for 26 days with our without praziquantel and viability assessed, by eosin exclusion, for cultures in various drug concentrations (50, 250 and 500 micrograms/l) and periods of exposure (1, 3 or 7 days (d] before removing/'rescuing' to drug-free medium. Drug efficacy was proportional to drug concentration and to length of exposure. At higher drug concentrations shorter exposures were required to produce the effect of continuous drug treatment, 1d therapy at 500 micrograms/l killing 96% ovine protoscoleces by day 14 whereas 7d therapy at 50 micrograms/l was required to produce a similar effect. Equine protoscoleces appeared marginally less susceptible than those of ovine origin. The relevance of the results in the need for peri-operative prophylaxis against spilled protoscoleces in man is discussed.

Characterization of Mycobacterium paratuberculosis by gas-liquid and thin-layer chromatography and rapid demonstration of mycobactin dependence using radiometric methods.

Thirty-six Mycobacterium paratuberculosis isolates of bovine, caprine, and ovine origins were evaluated by using gas-liquid chromatography (GLC), thin-layer chromatography (TLC), and BACTEC 7H12 Middlebrook TB medium (12A; Johnston Laboratories, Inc., Towson, Md.) in an effort to more rapidly differentiate this group of organisms from other mycobacteria. Bacterial suspensions (0.1 ml) were inoculated by syringe into 7H12 broth containing 2 micrograms of mycobactin P per ml and control broth without mycobactin P. Cultures were incubated at 37 degrees C and read daily with a BACTEC Model 301 (Johnston Laboratories). After 8 days of incubation, the growth index readings for the test broths containing mycobactin P were twice those of the control broths without mycobactin P. Sixty-five isolates of mycobacteria other than M. paratuberculosis were also examined. No difference was noted between the growth index readings of control and mycobactin-containing broths. Except for Mycobacterium avium-Mycobacterium intracellulare, TLC studies differentiated M. paratuberculosis from the other mycobacterial species tested. The GLC data reveal that all M. paratuberculosis isolates had a distinctive peak (14A) which was not found among M. avium-M. intracellulare complex organisms. These data indicate that 7H12 radiometric broth was able to rapidly demonstrate the mycobactin dependence of M. paratuberculosis and GLC and TLC procedures were capable of rapidly differentiating this organism from the other mycobacteria studied.

Characterization of a Bioeffective Monoclonal Antibody against Thyrotropin β-Subunit*

To describe a region of the TSH molecule participating in binding to receptor, a monoclonal antibody specific for a TSH epitope shared by beta-subunits of bovine (b), ovine (o), and human (h) TSH was obtained by immunization with mixtures of purified bTSH and hTSH. RIAs showed that the antibody also bound the beta-subunits of bLH, oLH, hLH, and hCG, but not the beta-subunits of porcine LH and TSH. Preincubation of [125I]iodo-bTSH with the antibody completely inhibited binding of the hormone to the TSH receptor of bovine thyroid membrane preparations at pH 7.4 in 50 mM NaCl (ED50 = 10 nM). The antibody also inhibited TSH-induced mitogenesis of FRTL-5 cells (ED50 = 50 nM). We conclude that the antibody binds to a site on the bTSH molecule that participates in high affinity binding of hormone to physiological TSH receptor. The target epitope includes a conserved structural determinant in beta-subunits of the glycoprotein hormones as well as a feature that allows discrimination of porcine hormones from those of bovine, ovine, and human origin.

Observations on muscle press juice from bovine, ovine and porcine muscles

Differences in the amounts and protein concentrations of muscle press juices from fresh and frozen psoas and l. dorsi muscles of bovine, ovine and porcine origin suggest the value of species and anatomical selection for functional applications in meat products.

Sexual dimorphism in immunoneutralization of bioactivity of rat and ovine inhibin.

Inhibin was partially purified from bovine follicular fluid using chromatography on immobilized Procion Red 3B and anion-exchange chromatography. Ovariectomized Texel ewes were immunized against the inhibin-containing fraction from the Procion Red 3B column and the immune response was subsequently boosted with similar fractions or with the preparation obtained from the anion-exchange column. The potencies of the resulting antisera were evaluated in an in-vitro bioassay system for estimating inhibin activity, using dispersed rat pituitary cells. The antisera were found to inhibit the bioactivity of inhibin preparations from ovarian follicular fluid of bovine, porcine, ovine or human origin, as well as inhibin activity in ovine testicular lymph and rete testis fluid, in culture media from rat granulosa and rat Sertoli cells and in homogenates of rat ovaries and testes. These results indicate that the inhibin molecules from several species contain a common bioactive moiety. The results also showed that the antiserum was more effective in neutralizing inhibin activity from ovarian than from testicular sources in both sheep and rat, indicating a sex-related difference in the inhibin molecules within a species.

Antigenic diversity of Chlamydia psittaci of mammalian origin determined by microimmunofluorescence.

A group of twenty-five isolates of Chlamydia psittaci representing at least seven different biotypes of bovine, ovine, caprine, equine, feline, porcine, and guinea pig origin were immunotyped by an indirect microimmunofluorescence test. Different groups of chlamydia-free BALB/c mice received two weekly intravenous inoculations with chicken embryo-propagated, partially purified elementary bodies of each strain. Antisera for immunotyping were collected 4 days after the first inoculation and 3 to 4 days after the second inoculation and tested for antichlamydial immunoglobulin M and immunoglobulin G antibodies by the indirect microimmunofluorescence test with cell culture-propagated, partially purified homologous and heterologous antigens. Nine immunotypes of C. psittaci were distinguished. The correlation between immunotypes and biotypes was close, and a pattern of either disease or host specificity could be associated with each immunotype. Most immunotypes identified induced cross-reacting antibodies against each other, but no significant cross-reactions were observed with elementary bodies of the mouse pneumonitis strain of C. trachomatis. Findings from this study should provide the necessary background for the rational selection of prototype strains of C. psittaci for further antigenic analysis at the molecular level.

Reevaluation of lipolytic activity of growth hormone in rabbit adipocytes

The lipolytic activities of porcine pituitary fractions and purified growth hormone (GH) from human (h), porcine (p), ovine (o) and rabbit (Rb) origin as well as ovine placental lactogen (oPL), were compared to that of ACTH on rabbit adipocytes. All the GH preparations and oPL were equivalent in inhibiting the binding of labelled oGH to liver plasma membranes from pregnant rabbits. ACTH, and to a lesser extent porcine pituitary fractions and hGH, stimulated free fatty acid production by isolated adipocytes. The sensitivity of the adipocytes to these factors was increased when adenosine deaminase was added to the incubation medium. But, RbGH, pGH, oGH and oPL had no effect. We conclude that GH is not directly involved in the control of lipolysis in rabbit adipocytes and that the effect of hGH is rather due to a contamination of this preparation by other pituitary factors.

The ketogenic effect of glucose in rumen epithelium of ovine ( ovis aries) and bovine ( bos taurus) origin

In experiments with rumen epithelium incubated in vitro the ratio of 3-hydroxybutyrate: acetoacetate produced was similar to the ratio reported for portal blood, and the ratio ketogenesis: oxidized to CO2 of butyrate was also close to values reported in vivo. Ovine and bovine epithelium incubated with butyrate differed significantly by the values of about 12-17 and 4-7 obtained for the ratio of 3-hydroxybutyrate: acetoacetate. Increasing levels of butyrate in the incubation medium resulted in a decreasing proportion of butyrate oxidized to CO2 and an increasing proportion of ketogenesis. The addition of glucose to butyrate in the incubation medium significantly increased the rate of ketogenesis from butyrate by ovine and bovine tissues. The addition of glucose to butyrate in the incubation medium significantly decreased the rate of butyrate oxidation to CO2 by ovine and bovine tissues. The ketogenic effect of glucose was also apparent in perfused rumen epithelium with butyrate at the mucosal side and glucose at the serosal side.