Effect of protein supplementation on expression and distribution of urea transporter-B in lambs fed low-quality forage.

Two experiments were conducted to determine the effects of ruminal protein degradability, supplementation frequency, and increasing dietary protein on the expression and distribution of urea transporter-B (UT-B) in lambs fed low-quality forage (mature crested wheatgrass hay; 4.2 to 4.7% CP). In Exp. 1, 15 Dorset wether lambs (initial BW=45.8+/-1.3 kg) were blocked by initial BW and assigned to 1 of 3 treatments within a randomized complete block design for 28 d, with supplements fed to achieve 7, 10, or 13% total dietary CP. In Exp. 2, 13 Dorset wether lambs (initial BW=34+/-4 kg) were used in a completely randomized design and given 1 of 4 isonitrogenous supplements: 1) ruminally degradable protein (RDP) fed daily (n=3), 2) RDP fed on alternate days (n=3), 3) ruminally undegradable protein (RUP) fed on alternate days (n=3), or 4) a 50:50 mixture of RDP and RUP fed on alternate days (n=4) for 18 d. Alternate-day treatments were fed at twice that of daily supplementation. On the last day of both experiments, lambs were killed and samples taken for Western blot analyses for UT-B. Immunoblotting using a rabbit polyclonal antibody to UT-B confirmed the presence of distinct 32-kDa (consistent with a nonglycosylated UT-B protein) and 47-kDa (probable N-glycosylated form of UT-B) protein bands in all 9 tissues analyzed. In both experiments, the liver, dorsal rumen, reticulum, and ventral rumen displayed strong bands at 32 kDa and lighter bands at 47 kDa, whereas the cecum, large colon, spiral colon, and parotid salivary gland displayed slight 32-kDa bands and stronger, more visible bands at 47 kDa. Both protein bands were apparent in the kidney at similar visual intensities in Exp. 1, whereas the relative intensities of the 2 UT-B bands in the kidney were variable, and appeared somewhat reciprocal among animals in Exp. 2. Although the abundance of the 47-kDa UT-B band in the ventral rumen was greater (P=0.03) in lambs fed RDP daily in Exp. 2, no other treatment differences (P >or= 0.15 to 0.99) in the abundance of the 32- or 47-kDa UT-B proteins within tissues were observed in either experiment. Although protein supplementation strategy had little effect on UT-B expression in tissues other than the ventral rumen, differences in the degree of glycosylation of UT-B across tissues may provide insight into its regulation.

Immune response and serum immunoglobulin G concentrations in beef calves suckling cows of differing body condition score at parturition and supplemented with high-linoleate or high-oleate safflower seeds.

Two experiments were conducted to determine the effect of maternal lipid supplementation on the immune response to antigenic challenge in suckling calves. In Exp. 1, beginning 1 d postpartum, 18 primiparous crossbred beef cows were fed Foxtail millet hay and a low-fat (control) supplement or a supplement containing cracked, high-linoleate safflower seed in individual feeding stanchions until d 40 of lactation. The diets were formulated to provide similar quantities of N and TDN, and the linoleate diet was formulated to contain 5% of DMI as fat. Calves were injected s.c. with 15 mg of antigen (ovalbumin) at d 21 and again at d 35 of age. To measure the total serum antibody production in response to the antigen, blood samples were collected from the calves every 7 d via jugular venipuncture from d 14 to 42. Calves from linoleate-supplemented cows had a decrease (P = 0.04) in total antibody production in response to ovalbumin and appeared to have a delayed response to antigen challenge. Total antibody production increased (P < 0.001) after secondary exposure to ovalbumin. In Exp. 2, 36 Angus x Gelbvieh beef cows that were nutritionally managed to achieve a BCS of 4 or 6 at parturition were used to determine the effects of prepartum energy balance and postpartum lipid supplementation on the passive transfer of immunoglobulins and the immune response to antigenic challenge in their calves. Beginning at 3 d postpartum and continuing until d 60 of lactation, cows were fed hay and a low-fat control supplement or supplements consisting of either cracked, high-linoleate or high-oleate safflower seeds. Safflower seed supplements were formulated to provide 5% of DMI as fat. Calves were injected s.c. with 15 mg of ovalbumin at 21 d of age and again at 48 d of age. The antibody responses were determined in serum; cell-mediated immunity was assessed by intradermal antigen injection at 60 d of age. A trend was noted (P = 0.10) for calves suckling control-supplemented cows to have a greater response to antigen compared with calves from linoleate- and oleate-supplemented cows; however, no difference was observed among treatments (P = 0.86) in cell-mediated immune response. Postpartum oilseed supplementation in beef cows appears to decrease antibody production in response to antigenic challenge in suckling calves. However, BCS at parturition did not influence passive transfer of immunoglobulins in neonatal calves.

Expression of CA-125 by progestational bovine endometrium: prospective regulation and function.

Cancer antigen 125 (CA-125) is expressed by malignant human ovarian surface epithelial cells and derivatives of the Müllerian duct system. This study explored the expression, regulation, and function of CA-125 in the bovine uterus. CA-125 was localized by immunohistochemistry to the apical surfaces of epithelial cells lining the endometrium and proximal glands of the late luteal phase and early pregnancy; antigen was not detected during oestrus or the postpartum period. Production of CA-125 by bovine endometrial cells in vitro was upregulated by progesterone and interferon-tau. Immunopurified CA-125 from uterine flushes of dioestrous or pregnant cows was similar in biochemical composition (as determined by gel electrophoresis and amino acid content) to the human antigen isolated from incubation medium conditioned by the ovarian cancer cell line OVCAR-3. Bovine CA-125 inhibited complement-induced lysis of antibody-sensitized sheep erythrocytes. It is suggested that endometrial CA-125 exerts a progestational role in part by protecting maternal and embryonic cells from immune targeting and lysis.

Ingestion of Neospora caninum tissue cysts by Mustela species.

Dogs are a definitive host of Neospora caninum, a protozoal parasite that causes abortion in cattle. Mustelids were tested to determine if they could also be definitive hosts. The procedures used were the same as those previously used to test dogs. Ermine (Mustela erminea), weasels (Mustela frenata) and ferrets (Mustela putorius) were fed N. caninum-infected mice. Neospora caninum oocysts were not observed. Mustelid faeces were fed to mice. The mice did not seroconvert and N. caninum was not detected in murine brains using tissue culture and PCR. The hypothesis that Mustela spp. are definitive hosts of N. caninum is not supported.

Toxicologic evaluation of a high-selenium hay diet in captive pronghorn antelope (Antilocapra americana).

Five captive-raised pronghorn antelope (Antilocapra americana) were fed an alfalfa-grass hay diet containing 15 ppm total dietary selenium (Se) for 164 days. Four additional captive-raised pronghorns fed a similar diet containing approximately 0.3 ppm total dietary Se served as controls. None of the pronghorns had clinical signs attributable to the high Se hay. Plasma Se increased more rapidly than blood Se concentrations, from baseline concentrations (< 0.15 g/ml) to > 0.40 g/ml within the first 50 days on the high selenium diet, but thereafter declined to approximately 0.30 microgram/ml. Mean primary antibody response to hen egg albumin was less in pronghorn on Se hay. No significant gross or histological lesions attributable to selenosis were found, nor was there any evidence of dystrophic hoof growth. The greatest Se tissue concentrations were found in liver and kidney (5.67 to 10.4 micrograms/g and 2.36 to 3.14 micrograms/g, respectively) from experimental animals; liver and kidney from the controls contained considerably less (< or = 0.52 microgram/g and < or = 0.61 microgram/g, respectively). Exposure of pronghorns for more than 5 mo to a diet containing 15 ppm Se caused significant increases in plasma, liver and kidney Se concentrations, in the absence of clinical disease or pathologic lesions due to selenosis. Based on these results, we propose that pronghorns are less susceptible to selenosis than previously reported and that diagnostic criteria for the disease should be modified.

Immunization of mice against a synthetic N-terminal extracellular domain gonadotropin-releasing hormone receptor peptide: evidence for a direct uterine effect.

PROBLEM: Immature male and female mice were immunized with a synthetic peptide corresponding to amino acids 5-17 (ASLEQDPNHCSAI) of the mouse hypophyseal gonadotropin-releasing hormone (GnRH) receptor. METHOD: Effect of immunization (postpuberal) was restricted to the uterus. Pituitary-gonadal functions were not altered. RESULTS: The endometrial lining of immunized females was thin and lacked glandular development. These observations were corroborated in actively immunized and passively immunized adult females. CONCLUSIONS: Apparently endometrial cells express a unique surface antigen, though reactive with antipeptide antibodies, that differs from the prototype pituitary GnRH receptor. Antibodies that selectively inhibit endometrial maturation might be used to treat proliferative diseases of the uterus.

Naturally occurring selenosis in Wyoming.

A review of Wyoming State Veterinary Laboratory records for 1947-1987 revealed no substantiated cases of naturally occurring selenosis. However, older reports attributed thousands of animal deaths to selenium each year in this area. Beginning in August 1988, cases of suspected selenosis and selenium deficiency were solicited from veterinarians and producers by announcements in various statewide livestock publications. As of August 1991, 4 cases (all horses) of naturally occurring selenosis have been confirmed. Clinical signs were most often referable to epithelial damage, e.g., hoof lesions and loss of mane and tail. None involved neurologic signs. Sources of selenium included native range and grass hay.

Bovine respiratory syncytial virus-specific immune responses in cattle following immunization with modified-live and inactivated vaccines. Analysis of proliferation and secretion of lymphokines by leukocytes in vitro.

Cattle were immunized with vaccines containing modified-live or inactivated bovine respiratory syncytial virus (BRSV) and lymphocyte proliferative responses and cytokine secretion were monitored sequentially. Compared to pre-inoculated values, significant increases in proliferative responses to modified-live BRSV were detectable by Day 7 after the primary immunization with the vaccine containing inactivated BRSV, and by 7 days after the second immunization with modified-live virus. After a third immunization with the respective vaccines, proliferative responses to live BRSV were significantly higher in the group that received modified-live vaccine compared to the group that received inactivated vaccine. Proliferative responses to live BRSV corresponded with the presence of interleukin-2 (IL-2) in the supernatants from BRSV-stimulated leukocyte cultures and there were significantly higher levels of IL-2 in cultures from the group that received modified-live BRSV. An interferon species with the characteristics of interferon-alpha was also present in the supernatants from leukocyte cultures and there were no significant differences between the groups of vaccines. The predominant phenotype of proliferating cells in BRSV-stimulated leukocyte cultures derived from both groups of bovine vaccines was a BoCD4+ T-lymphocyte. These in vitro data suggest that both types of vaccines are capable of stimulating cell-mediated immune responses to BRSV in cattle.

Antigenic property of pediocin AcH produced by Pediococcus acidilactici H.

Pediocin AcH, a bacteriocin of Pediococcus acidilactici H, inhibits the growth of several food spoilage and pathogenic bacteria. The antigenic property of partially purified pediocin AcH was tested by immunizing mice and a rabbit. Pediocin AcH was not immunogenic in these animals as determined by immunoblotting even after conjugation to bovine serum albumin. The non-immunogenic nature of pediocin AcH, its non-toxicity to laboratory animals and its hydrolysis by gastric proteolytic enzymes may be considered favourably in its possible use as a food preservative.

Toward regulation of gonadal function by a synthetic hybrid molecule composed of gonadotropin and Fc fragment of immunoglobulin G.

A conjugate of human chorionic gonadotropin (HCG) and Fc fragment of immunoglobulin G was prepared by covalent cross-linking using the heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio) propionate. Mouse Leydig tumor cells expressing receptors for luteinizing hormone were specifically lysed in vitro as a consequence of complement fixation via the Fc component of the hybrid molecule. Furthermore, administration of HCG-Fc to rams caused an acute depression in circulatory testosterone. This novel concept of targeted inhibition of gonadal function could prove to have future applications in control of reproductive processes.

Enzyme-linked immunosorbent assay for efficient detection of antibody to bluetongue virus in pronghorn (Antilocapra americana).

An indirect enzyme-linked immunosorbent assay (ELISA), using cell-associated viral antigen, was developed for detection of antibody to bluetongue virus (BTV) in field-collected pronghorn (Antilocapra americana) sera. To test the applicability of the ELISA to seroepizootiologic studies, pronghorn serum samples from three Wyoming counties (USA) were tested. Bluetongue virus ELISA results were compared to those of the bluetongue immunodiffusion assay. Discrepant serum samples were retested for reaction to either BTV or epizootic hemorrhagic disease virus. The pronghorn BTV ELISA gave rapid, quantitative, objective results and should facilitate testing large numbers of sera for BT diagnostic and seroepizootiologic studies.

Effects of cyclopiazonic acid and aflatoxin singly and in combination on selected clinical, pathological and immunological responses of guinea pigs.

Cyclopiazonic acid (CPA) and aflatoxin are known sometimes to coexist in nature but little is known of possible biological interaction in mammals that consume mixtures of these two mycotoxins. Guinea pigs were dosed orally with CPA (2.2 mg/kg) or aflatoxin (0.045 mg B1/kg) singly or in combination. Effects of toxin consumption were determined on clinical health, body weight gain, pathological change, and several immunologically related parameters including delayed cutaneous hypersensitivity, antibody response, complement hemolytic titer, intracutaneous mitogen (PHA) and in vitro lymphocyte blastogenesis. In contrast to an earlier study by others, significant synergy between these two toxins was demonstrated in reduced rate of body weight gain, lethality and histologic changes (vacuolization) in hepatocytes. Reductions in complement titer, intradermal PHA, delayed cutaneous hypersensitivity response and in vitro lymphocyte blastogenesis were related to aflatin activity. No effects on antibody formation to Brucella abortus were observed with either toxin or the combination of toxins. Cyclopiazonic acid appeared to restore the suppressive effects of aflatoxin in delayed cutaneous hypersensitivity response and in vitro lymphocyte blastogenesis.

Decline in sialic acid composition of cellular membranes isolated from ovine corpora lutea during prostaglandin-induced luteolysis: apparent independence of autoimmune recognition.

Sialic acid was quantified in plasma membranes of corpora lutea isolated during prostaglandin (PG) F2 alpha-induced luteolysis in sheep. Concentrations of sialic acid within membranes decreased after injection of PGF2 alpha, and before signs of luteal regression (i.e., a decline in tissue concentrations of progesterone) were manifested. Removal of residues of sialic acid from luteal membranes was not associated with cellular binding of gamma globulin, as monitored by indirect immunofluorescence microscopy. We suggest that desialylation of luteal membranes could be an important aspect of the mechanism of luteolysis. Such a process does not appear to involve participation of autoantibody.

Immunoregulation of luteolysis.

It is proposed that the immune/inflammatory system plays a yet unrecognized role in the mechanics of prostaglandin (PG) F2 alpha-induced luteal regression. Eosinophils are specifically attracted into luteal tissues and activated to degranulate (i.e. secrete cytotoxins) before symptoms of luteolysis are manifested (sheep). Further, because eosinophils are often associated with tissue reactions involving antigen-antibody binding, it is hypothesized that a luteal cell antigen could be expressed/unmasked as a result of the action of PGF2 alpha. Identification of the antigen by an appropriate autoantibody (e.g. complement-fixing) is an alternative mode by which cellular destruction can be mediated. Sialic acid residues that coat the surface of luteal membranes might act as a protective agent to autoimmune recognition. The hypothesis that luteolysis comprises an autoimmune reaction is extended to indicate that rescue of the corpus luteum from regression during early pregnancy involves a local immunosuppressive mechanism.

Bovine interleukin 2: production by an E-rosette-defined lymphocyte subpopulation.

E-rosette-separated bovine peripheral blood lymphocyte subpopulations were examined for ability to produce interleukin 2 (IL 2). Sequential E-rosetting techniques resulted in three T-cell subpopulations and a non-T population. Separated cells were stimulated with Con A and the resulting culture supernatants were assayed for IL 2 activity on IL 2-dependent cells. The bovine T-cell subpopulation which rosetted with both neuraminidase-treated and 2-aminoethylisothiouronium bromide (AET)-treated erythrocytes was found to produce significantly more IL 2 than the other T-cell subpopulations or the non-T population. These results suggest that this population may have a T-helper cell function. IL 2-dependent cells were found to be predominately T-cells by E-rosetting, were lymphoblastoid in appearance and surface immunoglobulin negative. Conditioned media containing IL 2 were used to demonstrate cytotoxic T-cell activity against allogeneic lymphocytes in peripheral blood lymphocytes.

Flow cytofluorimetric analysis of lymphocyte subset alterations in cattle infected with bovine viral diarrhea virus.

Clinically normal, bovine viral diarrhea virus (BVDV)-seronegative, 7 to 9-month-old steers were inoculated intranasally with NY-1, a noncytopathic strain of BVDV, or exposed intramuscularly to killed BVDV. Indirect immunofluorescence staining with monoclonal antibodies specific for bovine leukocyte subsets followed by flow cytometric analysis was used to monitor subsequent hematologic alterations. Infection with BVDV resulted in a transient leukopenia which was characterized by decreases in the absolute numbers of circulating T lymphocytes, including BoT4+ ("helper") and BoT8+ ("cytotoxic/suppressor") subsets, B lymphocytes, and neutrophils. There was no significant variation in numbers of non-T, non-B ("null") lymphocytes or monocytes. Exposure to inactivated BVDV in a combination vaccine did not cause significant alteration in the circulating numbers of any major leukocyte subset; however, significant variation was seen in the BoT4/BoT8 ratios and in the numbers of cells expressing major histocompatibility complex (MHC) class II antigens.

Bovine interleukin 2: production and characterization.

The production of bovine IL 2 was studied and IL 2 was partially characterized. PMA at 5 ng/ml + Concanavalin A at 5 micrograms/ml treatment of peripheral blood mononuclear cells gave a greater yield of IL 2 activity in the supernatants than Con A, PMA or sodium periodate treatments alone. Macrophage depletion increased yields as did the addition of indomethacin, a prostaglandin E2 inhibitor. Bovine IL 2 was sensitive to trypsin, relatively stable at pH 2-9, 2-ME resistant and sensitive to increasing molar concentrations of urea. The activity of bovine IL 2 was reduced by over 45% at 70 degrees C for 30 min and 95% at 90 degrees C for 30 min. Bovine IL 2 was more stable at 4 degrees C than at room temperature and the stability at room temperature could be improved by inclusion of 1% BSA. Bovine IL 2 eluted from DEAE-Sephadex as a broad peak with 0.1-0.2 M NaCl. Peak activity corresponded to a molecular weight of approximately 16,000 daltons on Sephadex G-100.

Bovine antibody dependent cell-mediated cytotoxicity (ADCC) effector cells.

ADCC effector cells from bovine blood were separated by centrifugation, adherence and rosetting techniques. Each enriched cell population, peripheral blood mononuclear cells (PBM), null lymphocytes, monocytes and neutrophils, was then examined for its capacity to mediate ADCC. Utilizing heterologous sensitizing antisera it was found that monocytes had approximately twice the ADCC activity of null lymphocytes and that neutrophils had essentially no activity. However, when homologous sensitizing antisera were used it was found that neutrophils possessed the greatest activity followed by monocytes and null cells. Results confirm the existence of an ADCC active null lymphocyte in the bovine.

Lymphokine production in T-lymphocyte subsets defined by active E-rosette formation.

Human T-cell subsets, defined by active E rosette formation, were examined for their ability to produce leucocyte migration inhibitory factor (LIF) in response to mitogen or specific antigen. It was determined that T cells enriched for active rosette-forming (A+) cells produced LIF in response to concanavalin A, whereas T cells depleted of active rosette-forming (A-) cells did not. Similarly, A+ cells from a tuberculin-sensitive donor produced LIF in response to tuberculin purified protein derivative, whereas A- cells from the same donor failed to produce the mediator. Thus, T-cell production of LIF in humans appears to be restricted to those T cells capable of active rosette formation.

Mitogen and mixed lymphocyte culture responses of isolated bovine lymphocyte subpopulations.

Examinations were made of the mitogen and mixed lymphocyte culture (MLC) responses of subpopulations of bovine lymphocytes isolated by density sedimentation following sequential E-rosetting with aminoethylisothiouronium bromide- and neuraminidase-treated sheep erythrocytes (EAET, EN). These procedures consistently separated 3 populations of E-rosetting T cells from a 4th population of non-E-rosetting cells (B and null cells). The isolated B and null cell population did not respond to phytohemagglutinin, concanavalin A, or pokeweed mitogen and responded minimally in mixed lymphocyte culture. Conversely, isolated T cells responded well to each of these stimuli. Moreover, differences in responsiveness were found among the 3 T-cell populations isolate by differential rosetting. T cells with receptors for both EAET and EN rosette formation were the most responsive to the mitogens used and demonstrated maximum activity in MLC. T cells with only EAET receptors had equivalent activity in MLC and less activity in response to mitogens. Cells with only EN receptors were the least active T-cell population in these assays. The different reactivities of these T-cell populations in lymphoproliferative assays indicated that they represent distinct subsets of bovine T cells.