Ronald D. Schultz

Evaluation of Five Antibody Detection Tests for Diagnosis of Bovine Paratuberculosis

ABSTRACT Five diagnostic tests based on enzyme-linked immunosorbent assay (ELISA) technology for bovine paratuberculosis were evaluated by using individual serum or milk samples from 359 dairy cattle in seven paratuberculosis-free herds and 2,094 dairy cattle in seven Mycobacterium paratuberculosis-infected dairy herds. Three independent laboratories using three different culture procedures completed fecal cultures for M. paratuberculosis on these cattle and found 417 cows to be shedding M. paratuberculosis in their feces. An animal that was fecal culture positive for M. paratuberculosis by any of the three laboratories was considered a confirmed case of infection. The specificity of three ELISAs (two on serum and one on milk) was ≥99.8%. The specificity of the remaining two ELISAs, both done on serum, was 94.9 and 84.7%. Four of the five ELISAs evaluated produced similar sensitivity in detecting fecal culture-positive cattle (27.8 to 28.9%). Serum ELISA “D” had the lowest specificity (84.7%) and the highest sensitivity (44.5%), but if the cutoff value defining a positive test was changed from 125 to 250% (of the positive control) the sensitivity and specificity, 31.8 and 97.5%, respectively, were comparable to those of the other four assays. If the case definition for M. paratuberculosis infection was based on the culture results of a single laboratory instead of the combined results of three laboratories, ELISA sensitivity estimates were 45.7 to 50.0%. With the exception of ELISA D, assay agreement was high (kappa 0.66 to 0.85) for categorical assay interpretations (positive or negative), but linear regression of quantitative results showed low correlation coefficients (r2 = 0.40 to 0.68) due to the fact that ELISA results for some cows were high in one assay but low in another assay. Likelihood ratio analysis showed a direct relationship between the magnitude of ELISA result and the odds of a cow shedding M. paratuberculosis in its feces. If used judiciously and interpreted quantitatively, these ELISAs are useful tools in support of paratuberculosis control programs in dairy herds.

ADHERENCE TO BOVINE NEUTROPHILS AND SUPPRESSION OF NEUTROPHIL CHEMILUMINESCENCE BY MYCOPLASMA BOVIS

The adherence of viable and heat-treated Mycoplasma bovis to bovine peripheral blood neutrophils was studied by specific immunofluorescence staining and flow cytometry. Viable and heat-treated M. bovis cells, adhered to bovine neutrophils in dose-dependent fashion within a 30 min incubation. Fluorescence quenching using crystal violet indicated that unopsonized M. bovis cells remained on the surface of bovine neutrophils without experiencing significant ingestion. The effect of M. bovis adherence on neutrophil microbicidal function was examined by measuring luminol enhanced chemiluminescence (CL). Adherent M. bovis cells did not elicit a bovine neutrophil CL response over a 75 min incubation period. M. bovis inhibited the capacity of bovine neutrophils to mount a CL response. Inhibition occurred whether viable or heat-treated M. bovis cells were used and it occurred when neutrophils were stimulated with opsonized zymosan (OZ) or phorbol myristate acetate (PMA). Inhibition of the PMA stimulated neutrophil CL response required cytadherence by M. bovis cells. These findings suggest that activation of the bovine neutrophil respiratory burst was inhibited at or distal in the pathway to the activation of protein kinase C (PKC), the site of PMA stimulation, and that it was mediated by a direct interaction between the adhering M. bovis cells and the bovine neutrophil membrane.

Age and Long-term Protective Immunity in Dogs and Cats

Vaccination can provide an immune response that is similar in duration to that following a natural infection. In general, adaptive immunity to viruses develops earliest and is highly effective. Such anti-viral immune responses often result in the development of sterile immunity and the duration of immunity (DOI) is often lifelong. In contrast, adaptive immunity to bacteria, fungi or parasites develops more slowly and the DOI is generally short compared with most systemic viral infections. Sterile immunity to these infectious agents is less commonly engendered. Old dogs and cats rarely die from vaccine-preventable infectious disease, especially when they have been vaccinated and immunized as young adults (i.e. between 16 weeks and 1 year of age). However, young animals do die, often because vaccines were either not given or not given at an appropriate age (e.g. too early in life in the presence of maternally derived antibody [MDA]). More animals need to be vaccinated to increase herd (population) immunity. The present study examines the DOI for core viral vaccines in dogs that had not been revaccinated for as long as 9 years. These animals had serum antibody to canine distemper virus (CDV), canine parvovirus type 2 (CPV-2) and canine adenovirus type-1 (CAV-1) at levels considered protective and when challenged with these viruses, the dogs resisted infection and/or disease. Thus, even a single dose of modified live virus (MLV) canine core vaccines (against CDV, cav-2 and cpv-2) or MLV feline core vaccines (against feline parvovirus [FPV], feline calicivirus [FCV] and feline herpesvirus [FHV]), when administered at 16 weeks or older, could provide long-term immunity in a very high percentage of animals, while also increasing herd immunity.

Immunologie Methods for the Detection of Humoral and Cellular Immunity

: A variety of immunologic techniques have been introduced during the past few years. Many of these techniques are being applied to clinical specimens in an attempt to help the practicing veterinarian make a diagnosis. The introduction of new techniques requires extensive testing with clinically normal and diseased patients. It is essential for the practicing veterinarian to understand that the techniques available for the detection of immunologic disorders in the dog and cat are not routine diagnostic procedures and that adequate information has not been developed for any of the techniques described to assure the clinical significance of either positive or negative results (Table 3). This should not discourage the practitioner from submitting samples, but should encourage him or her to question the significance of those results and to attempt to correlate them with history and clinical signs before arriving at a final diagnosis.

Feline leukemia virus immunity induced by whole inactivated virus vaccination.

A fraction of cats exposed to feline leukemia virus (FeLV) effectively contain virus and resist persistent antigenemia/viremia. Using real-time PCR (qPCR) to quantitate circulating viral DNA levels, previously we detected persistent FeLV DNA in blood cells of non-antigenemic cats considered to have resisted FeLV challenge. In addition, previously we used RNA qPCR to quantitate circulating viral RNA levels and determined that the vast majority of viral DNA is transcriptionally active, even in the absence of antigenemia. A single comparison of all USDA-licensed commercially available FeLV vaccines using these modern sensitive methods has not been reported. To determine whether FeLV vaccination would prevent nucleic acid persistence, we assayed circulating viral DNA, RNA, antigen, infectious virus, and virus neutralizing (VN) antibody in vaccinated and unvaccinated cats challenged with infectious FeLV. We identified challenged vaccinates with undetectable antigenemia and viremia concomitant with persistent FeLV DNA and/or RNA. Moreover, these studies demonstrated that two whole inactivated virus (WIV) adjuvanted FeLV vaccines (Fort Dodge Animal Healths Fel-O-Vax Lv-K) and Schering-Plough Animal Healths FEVAXYN FeLV) provided effective protection against FeLV challenge. In nearly every recipient of these vaccines, neither viral DNA, RNA, antigen, nor infectious virus could be detected in blood after FeLV challenge. Interestingly, this effective viral containment occurred despite a weak to undetectable VN antibody response. The above findings reinforce the precept of FeLV infection as a unique model of effective retroviral immunity elicited by WIV vaccination, and as such holds valuable insights into retroviral immunoprevention and therapy.

Effect of infection by bovine viral diarrhea virus (BVDV) in vitro on interleukin-1 activity of bovine monocytes☆

The effect of bovine viral diarrhea virus (BVDV) infection in vitro on the interleukin-1 (IL-1) activity of bovine monocytes was studied. Supernatants from BVDV-infected monocytes suppressed IL-1-stimulated proliferation of mouse thymocytes and masked lipopolysaccharide-stimulated IL-1 activity of bovine monocytes in the mouse comitogen thymocyte assay. Suppression of mouse thymocyte proliferation was restored by the addition of IL-1. IL-1 inhibitory activity was induced both by the prototype variants BVDV/NADL cytopathic and BVDV/NY-1 noncytopathic and by BVDV variants isolated from persistently infected cattle. Suppressed IL-1 activity was also found in supernatants from monocytes from persistently infected cattle following infection with BVDV in vitro. No differences in levels of IL-1 mRNA synthesis were detected between BVDV-infected and uninfected monocytes by RNA-cDNA hybridization. These results suggest that infection of bovine monocytes with BVDV results in the production and/or activation of a soluble inhibitor of IL-1 activity.

Mycoplasma bovis suppression of bovine lymphocyte response to phytohemagglutinin.

The effect of viable Mycoplasma bovis on the in vitro bovine peripheral blood lymphocyte response to phytohemagglutinin (PHA) was studied. Results showed that M. bovis did not act as a mitogen for bovine lymphocytes. Viable M. bovis produced a dose and time dependent suppression of the PHA stimulated lymphocyte response. Suppression was not a result of differences in the viability of infected or control lymphocyte cultures. The suppressive effect of M. bovis was found to be independent of the concentration of PHA used in the test and the lymphocyte response could not be restored by supplementation of the culture medium with arginine. Delay for 48 h after PHA stimulation before adding M. bovis to the lymphocyte cultures diminished, but did not prevent, the suppression of the lymphocyte response. These results show that suppression of the lymphocyte response does not require the presence of M. bovis during the period of PHA stimulation, and that M. bovis was capable of interrupting [3H]-thymidine incorporation in lymphocytes which were actively synthesizing DNA.

Bovine natural cell mediated cytotoxicity (NCMC): activation by cytokines.

Incubation of bovine peripheral blood mononuclear leukocytes (PBML) with the cytokines (CK) IL-2, alpha-IFN, gamma-IFN or IL-4 resulted in significant increases in natural cell mediated cytotoxicity (NCMC) over endogenous levels, as determined in an 18 h 51Cr-release assay using the human K562 or mouse Yac-1 target cell lines. Endogenous cytotoxic activity of bovine natural effector cells (NEC) using K562 or Yac-1 target cells was minimal (killing less than 8%). After 18 h of incubation with the CK hurIL-2, alpha-bovrIFN, gamma-bovrIFN or hurIL-4, NEC had significant increases in cytotoxic activity for both K562 and Yac-1 target cells. Significant increases in cytotoxic activity were not found after incubation of NEC with IL-1 or beta-IFN. Specific killing varied with CK concentration in a dose dependent manner and was proportional to effector:target cell ratio. Activation of the bovine NEC by CK was rapid, occurring within 6-12 h of incubation with alpha-IFN or gamma-IFN and within 12-18 h of incubation with IL-2. Incubation of bovine PBML with IL-2 and alpha- or gamma-IFN or with alpha-IFN and gamma-IFN showed that these CK do not act in a synergistic manner to increase NCMC in the bovine NEC.

Effect of experimental infection of cattle with bovine herpesvirus-1 (BHV-1) on the ex vivo interaction of bovine leukocytes with Mannheimia (Pasteurella) haemolytica leukotoxin.

Abstract Mannheimia (Pasteurella) haemolytica A1 produces an extracellular leukotoxin (LKT) that is reported to bind the β2-integrin CD11a/CD18 (LEA-1) on ruminant leukocytes. LKT binding induces activation, and subsequent cytolysis, of these cells. It is well known that active viral infection greatly increases the susceptibility of cattle to pasteurellosis. To better understand the mechanism by which this occurs, we investigated the effects of experimental in vivo infection of cattle with bovine herpes virus-1 (BHV-1) on the ex vivo interaction of bovine leukocytes with the M. haemolytica LKT. In this study, we demonstrated that active BHV-1 infection increased the expression of the β2-integrin CD11a/CD18 (as defined by the mAb BAT75) on bovine peripheral blood neutrophils, enhanced the binding of LKT to bronchoalveolar lavage (BAL) leukocytes and peripheral blood neutrophils, and increased the killing of BAL leukocytes and peripheral blood leukocytes by LKT. In addition, BHV-1 greatly increased the number of BAL, resulting in many more LKT-responsive cells being present in the lungs. These findings might explain in part the increased susceptibility of BHV-1 infected cattle to pneumonic pasteurellosis.

Growth hormone receptor and regulation of gene expression in fetal lymphoid cells.

The role of growth hormone (GH) in modulating the adult immune response is receiving increased attention; however, its role in the development of immune competence in the fetus has not been defined. In order to begin to address the role of GH in the ontogeny of the immune response. cells from bovine fetal spleen and thymus were examined for GH receptor and responsiveness to GH. Northern analysis and ligand binding studies showed that growth hormone receptor (GHR) was readily detected in early- and mid-gestational fetal thymocytes, but it was less readily detected in thymocytes from older fetuses. In contrast, GHR was easily detected in splenocytes at all fetal ages. Thymocytes and splenocytes from mid-gestational fetuses expressed low levels of cell surface GHR by flow cytofluorometric analysis, and CD4+ and CD8 (single positive) thymocyte subsets were positive. Northern analyses were employed to determine the effects of in vitro GH treatment on expression of several proto-oncogenes, cytokines, and GHR in thymocytes from fetuses at approximately mid-gestation. GH treatment for 30 min down-regulated c-jun and c-fos mRNA approximately 2- and 2.8-fold, respectively. After 6 h treatment, GH increased transcript levels for interleukin (IL)-1alpha, IL-1beta, IL-6, and GM-CSF about 2.5-, 2.2-, 3-, and 2-fold, respectively. GH also down-regulated the expression of its own receptor about 3.2-fold after 8 h of incubation. The presence of GHR in fetal lymphoid cells and its temporal and spatial regulation suggest a potential role for GH in the development and or function of the fetal bovine immune system. Although the mechanism(s) is unclear, our results suggest that GH is intimately involved in lymphocyte function and expression of certain cytokines during a critical period of fetal immune development.