E. Lee Belden

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.

Subpopulations of bovine lymphocytes separated by rosetting techniques

Abstract A combination of E-rosetting techniques with both neuraminidase- and AET-treated sheep erythrocytes (RBC) was used to enumerate subsets of bovine T lymphocytes. Direct (anti-Ig) rosetting procedures were used to enumerate B lymphocytes bearing surface immunoglobulin (Ig). Approximately 10% of bovine peripheral blood lymphocytes formed rosettes only with neuraminidase-treated RBC; 20% formed rosettes with either neuraminidase- or AET-treated RBC; 30% formed rosettes only with AET-treated RBC; 25% possessed surface Ig, as shown by rosette formation with anti-Ig-coupled RBC; and the remaining 15% lacked both E receptors and surface Ig. These five populations were physically separated by centrifugation on Ficoll-Diatrizoate and recovered for functional analysis. The procedures reported here should be useful for the identification of lymphocyte populations responsible for recognition, effector, and cooperative functions in the bovine immune system.

VALIDATION OF A BRUCELLA ABORTUS COMPETITIVE ENZYME-LINKED IMMUNOSORBENT ASSAY FOR USE IN ROCKY MOUNTAIN ELK (CERVUS ELAPHUS NELSONI)

Brucellosis caused by infection with Brucella abortus is present in some elk (Cervus elaphus nelsoni) of the Greater Yellowstone Area (parts of Wyoming, Montana, and Idaho, USA). Since 1985, the Wyoming Game and Fish Department has vaccinated elk on elk feedgrounds in northwestern Wyoming during the winter months using B. abortus strain 19 (strain 19). Analysis of this vaccination program is hampered by the inability of standard serologic tests to differentiate between strain 19 vaccinated elk and those exposed to field strain B. abortus. In 1993, a competitive enzyme-linked immunosorbent assay (cELISA) was licensed to serologically differentiate between strain 19 vaccinated cattle and cattle exposed to field strain B. abortus. Seven groups of elk sera representing various B. abortus exposure histories were used to validate the cELISA test for elk. The cELISA test differentiated strain 19 vaccinated elk from elk that were challenged with B. abortus strain 2308, a pathogenic laboratory strain. The specificity of the cELISA was 96.8% for elk vaccinated with strain 19 only and sampled between 6 mo and 2 yr post vaccination, or with no B. abortus exposure. The sensitivity of the cELISA was 100%. The cELISA test will be useful in evaluating sera collected from elk in vaccinated, brucellosis endemic herds in the Greater Yellowstone Area.

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.

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.

INFECTION OF C57BL/6 MICE BY TRYPANOSOMA MUSCULI MODULATES HOST IMMUNE RESPONSES DURING BRUCELLA ABORTUS COCOLONIZATION

Abstract Brucellosis, which results in fetal abortions in domestic and wildlife animal populations, is of major concern in the US and throughout much of the world. The disease, caused by Brucella abortus, poses an economic threat to agriculture-based communities. A moderately efficacious live attenuated vaccine (B. abortus strain RB51) exists. However, even with vaccine use, outbreaks occur. Evidence suggests that elk (Cervus canadensis), a wild host reservoir, are the source of recent outbreaks in domestic cattle herds in Wyoming, USA. Brucella abortus establishes a chronic, persistent infection in elk. The molecular mechanisms allowing the establishment of this persistent infective state are currently unknown. A potential mechanism could be that concurrent pathogen burdens contribute to persistence. In Wyoming, elk are chronically infected with Trypanosoma cervi, which may modulate host responses in a similar manner to that documented for other trypanosomes. To identify any synergistic relationship between the two pathogens, we simulated coinfection in the well-established murine brucellosis model using Trypanosoma musculi and B. abortus S19. Groups of C57BL/6 mice (Mus musculus) were infected with either B. abortus strain 19 (S19) or T. musculi or both. Sera were collected weekly; spleens from euthanized mice were tested to determine bacterial load near the end of normal brucellosis infection. Although changes in bacterial load were observed during the later stages of brucellosis in those mice coinfected with T. musculi, the most significant finding was the suppression of gamma interferon early during the infection along with an increase in interleukin-10 secretion compared with mice infected with either pathogen alone. These results suggest that immune modulatory events occur in the mouse during coinfection and that further experiments are warranted to determine if T. cervi impacts Brucella infection in elk.

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.

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.