The distribution of lymphocyte subsets in normal ovine skin.

The distribution of lymphocyte subsets in the skin of clinically normal sheep was studied using monoclonal antibodies to OvCD5, OvCD4, OvCD8, WC1, and CD45RA. Four different anatomical sites were examined in each of 38 sheep. Four different age groups ranging from 7 to 10-day-old lambs to 12 to 14-month-old adults were represented. The majority of lymphocytes in all age groups and at all sites were angiocentrically located within the superficial dermis. Total lymphocyte numbers at each site increased with age. The predominant cell type identified at all sites was WC1+ and the proportion of lymphocytes of this phenotype was significantly higher at wooled sites. Only occasional CD45RA +/- cells were present in any section.

A study of cattle-to-cattle transmission of Mycobacterium bovis infection.

Twenty steers, positive to the single intradermal comparative tuberculin test (SICTT), were selected from herds with a recent history of Mycobacterium bovis infection. Ten steers, negative to SICTT, were selected from herds with no history of M. bovis infection and served as in-contact animals. The animals were divided into 10 groups, each consisting of two SICTT-positive (reactor) animals and one in-contact animal. Each group was housed in an individual loose-box for a period of 1 year. Five of the groups were fed a restricted diet for part of the experiment. All cattle were slaughtered at the end of the study period and examined at post mortem. Transmission of infection to an in-contact animal occurred in four of the 10 groups. One of the four in-contact animals, which became infected, had a retropharyngeal lymph node tubercle and M. bovis was isolated from lymph nodes without visible lesions from the other three. Two of the infected in-contact animals without visible lesions did not show any detectable cell-mediated immune response. There was no evidence that dietary, restriction had any effect on transmission of disease.

Effects of suppressing cortisol following castration of bull calves on adrenocorticotropic hormone, in vitro interferon-gamma production, leukocytes, acute-phase proteins, growth, and feed intake.

The objective was to determine the effects of reducing the plasma cortisol rise in calves following castration on plasma ACTH concentrations, keyhole limpet hemocyanin (KLH)- and concanavalin A (Con A)-induced in vitro interferon (IFN)-gamma production, white blood cell (WBC) numbers, neutrophil:lymphocyte (N:L) ratio, plasma haptoglobin and fibrinogen concentrations, ADG, and ADFI. Forty 5-mo-old Friesian bull calves (169 +/- 1.7 kg) were assigned to four treatments: 1) control (CON); 2) oral metyrapone administration (MET); 3) surgical castration at 0 h on d 0 (SURG); and 4) oral metyrapone administration and surgical castration (MET+SURG). Cortisol, ACTH, IFN-gamma production, haptoglobin, fibrinogen, ADFI, and ADG were not different between CON and MET animals. The MET+SURG calves had lower (P < .001) peak and mean cortisol during .25 to 1.5 h than SURG animals, but area under the cortisol vs time curve from 0 to 12 h did not differ (P > .39) between SURG and MET+SURG calves. Peak ACTH concentrations and area under the ACTH vs time curve from 0 to 6 h were greater (P < .05) for MET+SURG than for SURG calves. There were no differences between MET+SURG and SURG animals in IFN-gamma production, WBC numbers, and ADFI. On d 1, MET+SURG and SURG animals had lower (P < .01) KLH- and Con A-induced IFN-gamma production and higher (P < .05) neutrophil numbers and N:L ratio compared with CON animals. Plasma haptoglobin on d 1 and 3 and fibrinogen concentrations on d 3 and 7 were elevated (P < .05) for MET+SURG and SURG compared with CON animals, whereas SURG animals had greater (P < .05) haptoglobin and fibrinogen concentrations than MET+SURG animals on d 7. The ADG of SURG calves was lower (P < .05) than that of MET+SURG calves during d 0 to 7. Metyrapone treatment partially suppressed cortisol and increased ACTH in castrated calves but did not alter the castration-induced suppression of IFN-gamma and increases in neutrophil numbers and the N:L ratio.

Detection of Mycobacterium bovis infection in cattle using an immunoassay for bovine soluble interleukin-2 receptor-alpha (sIL-2R-alpha) produced by peripheral blood T-lymphocytes following incubation with tuberculin PPD.

After activation of T-lymphocytes with antigen there is an increase in the expression of interleukin-2 receptor-alpha (IL-2R-alpha) followed by the release of a soluble form of the molecule (sIL-2R-alpha) from the membrane of the stimulated cells. The present study investigates the novel use of the release of sIL-2R-alpha from activated T-lymphocytes as a marker of cell-mediated immunity (CMI) in cattle infected with Mycobacterium bovis. An enzyme immunoassay was used to detect sIL-2R-alpha produced following incubation of bovine peripheral blood mononuclear cells with mycobacterial antigens. Using this assay, 63/67 cattle naturally infected with M. bovis were identified whereas only 1/51 uninfected animals were considered to give a positive result. This assay is more convenient to use than lymphocyte proliferation assays which involve the use of radionucleosides. It should prove useful for monitoring the immunological activation of bovine T-lymphocytes in a variety of situations including the development of CMI responses in cattle to novel mycobacterial antigens or potential vaccines.

Effects of cortisol on in vitro interferon-gamma production, acute-phase proteins, growth, and feed intake in a calf castration model.

The objective of this study was to determine the effects of castration, with its presumed pain and inflammatory effects, including increased cortisol, and elevated cortisol per se on in vitro interferon-gamma (IFN-gamma) production, ADG, ADFI, and plasma haptoglobin and fibrinogen. Thirty Friesian bull calves (174 +/- 3.8 kg) were assigned to three treatments (given on d 0): 1) control (CON); 2) i.v. cortisol administration to mimic castration-induced increases in cortisol (CORT); and 3) surgical castration (SURG). Blood samples were collected for 12 h on d 0 and at 24 and 72 h after treatment for cortisol determination. Keyhole limpet hemocyanin (KLH)- and concanavalin A (Con A)-induced in vitro IFN-gamma production in blood, and plasma haptoglobin and fibrinogen were measured in blood samples taken before treatment on d 0 and on d 1 and 3. On d 0, CORT and SURG animals had higher peak cortisol (P < .001) and area under the cortisol curve (P < .001) than CON animals. There were no differences (P > .05) between CON, CORT, and SURG animals in cortisol at 24 and 72 h. There were no differences (P > .05) between CON and CORT animals in IFN-gamma production, haptoglobin, fibrinogen, ADG, and ADFI. Compared with CON animals, SURG animals had lower (P < .05) KLH-induced IFN-gamma on d 1 and CON A-induced IFN-gamma on d 1 and 3. Haptoglobin concentrations were greater (P < .05) for SURG than for CON animals on d 1 and 3. Fibrinogen concentrations were greater (P < .001) for SURG than for CON animals on d 3. The SURG animals had lower (P < .01) ADG and ADFI during d 0 to 7 than CON animals. In conclusion, castration decreased IFN-gamma production, ADG, and ADFI and increased haptoglobin and fibrinogen, and these effects seemed to be independent of plasma cortisol concentrations.

Development of a sandwich immunoassay for the detection of soluble bovine interleukin-2 receptor-alpha (sIL-2R-alpha) and its use to measure cell-mediated immunity in cattle.

Stimulation of T-lymphocytes with mitogens or antigens results in the production of the cytokine interleukin-2, which exerts its physiological effect by interacting with a specific IL-2 receptor on the cell surface. The alpha-chain of this receptor is induced and expressed on the cell surface after lymphocyte activation. Following continuous antigen stimulation, a smaller soluble form of this alpha-subunit (sIL-2R-alpha) is shed from the membrane of activated cells. This study describes a sandwich ELISA for bovine sIL-2R-alpha that was developed using monoclonal antibodies specific for bovine IL-2R-alpha (CD 25). The feasibility of using sIL-2R-alpha released by activated T-lymphocytes as an in vitro marker of cell-mediated immunity (CMI) in cattle is demonstrated. Calves were immunized with the foreign protein keyhole limpet haemocyanin (KLH) and the development of CMI was followed using sIL-2R-alpha release, IFN-gamma production and lymphocyte proliferation assay. The results showed that the release of sIL-2R-alpha by previously sensitized cells following stimulation with antigen is likely to be a useful marker of CMI in infectious diseases, and in the study of T cell antigens and/or novel vaccines. Using appropriate detection systems, the measurement of sIL-2R-alpha may also prove to be a useful marker of CMI in other species.

Cervico-thoracic vertebral osteomyelitis in 14 calves.

Fourteen cases of cervico-thoracic (C-T) vertebral osteomyelitis in calves were investigated over a 6 year period. The onset of clinical signs was between 2 and 9 weeks of age. There was no breed or sex predisposition. The clinical history prior to referral extended from 5 days to 8 weeks (mean 20 days). The most common clinical presentation was difficulty in rising with a tendency to knuckle or kneel on the forelimbs which displayed hypotonia and hyporeflexia. In over half the cases pain could be elicited on manipulation of the neck. The lesion in all cases involved one or more of the vertebrae from C6 to T1. The diagnosis was confirmed by radiology and/or at post mortem. Four animals were discharged after treatment, 10 animals were humanely destroyed. Salmonella dublin was isolated from the vertebral lesion in eight of the 10 calves at post mortem.

Effect of dietary restriction on cell-mediated immune responses in cattle infected with Mycobacterium bovis.

Nine M. bovis-infected cattle on a diet deficient in both protein and energy for 133 days lost approximately 17% of their original body weight. However, dietary restriction did not result in any significant reduction in skin sensitivity to PPD, in vitro production of IFN-gamma or lymphocyte blastogenesis. The number of circulating BoCD4+ cells and B cells were similar in both the malnourished and the control cattle. However, significantly lower numbers (P < 0.01) of circulating BoCD2+ cells, BoCD8+ cells, WC1+ gamma delta T cells and ACT2+ cells were found in the malnourished cattle. With the exception of inorganic phosphate, the changes in plasma biochemical parameters were unremarkable.

A sequential study of the bovine tuberculin reaction.

The sequential histopathological and immunocytochemical changes that characterize the tuberculin reaction were studied in 13 cattle experimentally sensitized to Mycobacterium bovis, and 14 cattle naturally infected with M. bovis. There were two distinct, temporally related patterns of morphological change that were similar for both groups of cattle. The first phase, between 6 hr and 24 hr after the intradermal injection of purified protein derivative (PPD), was characterized by a perivascular aggregation of WC1+ gamma delta T cells and neutrophils and the presence of leucocytoclastic vasculitis within the papillary dermis. The second phase of the reaction was characterized by increased numbers of infiltrating BoCD4+ cells, BoCD8+ cells and macrophages, as well as an increase in expression of the interleukin-2 (IL-2) receptor and the ACT2 antigen. Macrophages were the most numerous infiltrating leucocytes between 24 hr and 72 hr after the intradermal injection of PPD. At 72 hr, the reaction was characterized by intense perivascular cuffing with BoCD4+ cells, BoCD8+ cells and macrophages; gamma delta T cells and neutrophils were a minor component of the reaction and leucocytoclastic vasculitis was no longer observed. No B cells were detected in the dermis throughout the period of study. The increase in skin thickness was primarily because of inflammatory oedema that was contained within the area by a meshwork of fibrin deposited around the collagen bundles of the reticular dermis.

Effects of dexamethasone on cell-mediated immune responses in cattle sensitized to Mycobacterium bovis.

Systemic administration of dexamethasone led to a significant reduction in the size of the tuberculin reaction in response to intradermal injection of bovine purified protein derivative in 18 cattle experimentally sensitized to Mycobacterium bovis (P < 0.01) and 8 cattle naturally infected with M bovis (P < 0.001). The reaction in 6 of the 7 M bovis infected cattle that received dexamethasone was classified as negative for the standard interpretation of the single intradermal comparative tuberculin test. Significantly fewer BoCD2+ (P < 0.05) and BoCD4+ T cells (P < 0.001) were present at the reaction site and in blood of dexamethasone-treated cattle, compared with untreated control cattle. Significantly fewer cells expressing the interleukin-2 receptor and WC1+ gamma delta T cells (P < 0.001), and a significantly greater number of cells expressing the ACT2 antigen (P < 0.05) were found at the reaction site in dexamethasone treated cattle than in controls. The number of BoCD8+ T cells at the reaction site and in blood was not significantly affected by administration of dexamethasone. In vitro production of interferon-gamma by lymphocytes incubated with bovine purified protein derivative also was significantly lower (P < 0.01) in the dexamethasone treated cattle.

Effect of a recent injection of purified protein derivative on diagnostic tests for tuberculosis in cattle infected with Mycobacterium bovis.

The intradermal injection of avian and bovine purified protein derivative into 15 Mycobacterium bovis-infected cattle reduced the capacity of a second single intradermal comparative tuberculin test carried out seven days later to predict accurately the animals' infection status (P < 0.001). In vitro lymphyocyte blastogenesis was also reduced (P < 0.05) but the in vitro production of interferon-gamma was unaffected.

The tuberculin test.

Tuberculin tests in general use today rely on the response to intradermal injections of tuberculin with assessment of the injection site for swelling at 72 hours post injection. Estimates of the sensitivity of tuberculin tests range from 68-95% while specificity is estimated to be 96-99%. The sensitivity of the test is affected by the potency and dose of tuberculin administered, the interval post-infection, desensitisation, deliberate interference, post-partum immunosuppression and observer variation. Specificity is influenced by sensitisation as a result of exposure to M. avium, M. paratuberculosis and environmental mycobacteria and by skin tuberculosis.

Morbidity and mortality in a large Irish feedlot; microbiological and serological findings in cattle with acute respiratory disease.

A survey of morbidity and mortality was undertaken in a slatted unit containing 6399 beef cattle over a 6 month period. The mortality rate was 0.78% and the morbidity rate was 12.7%. The interval from arrival to morbidity was 25.5 days +/- 0.9. Significantly more sick cattle were identified on either Mondays or Tuesdays than were seen on Saturdays or Sundays (P < 0.005). The mean interval to mortality (all diseases) was 48.5 days +/- 7.4. Respiratory disease was the most frequently recorded disease and was responsible for 62% of morbidity and 58% of mortality. Samples from 133 cattle, taken as they presented with acute onset respiratory disease, contained antibodies to Bovine Herpes Virus -1(BHV-1) (14.3%), Bovine Viral Diarrhoea Virus (BVDV) (78%), Bovine Respiratory Syncytial Virus (BRSV) (94%) and Parainfluenza type 1 Virus (P13V) (99%). When the same cattle were resampled 4 to 6 weeks later antibodies were found for BHV-1 (93.2%), BVDV (99.2%), BRSV (100%) and P13V (100%). Eleven of 27 bronchoalveolar lavage samples taken from the above group of cattle exhibited positive fluorescence for BHV-1 but all 27 samples were negative for BVDV, BRSV and P13V. Pasteurella multocida was isolated from eight of the 27 lavage samples and Pasteurella haemolytica from one sample.

Antagonism of antidiuretic hormone in domestic pigs (Sus scrofa).

1. Morphological and physiological aspects of renal function are shared by humans and swine. SK&F 101926 is a potent antagonist of vasopressin binding to V2 receptors and vasopressin stimulation of adenylate cyclase activity in renomedullary membranes from both species. 2. Unexpectedly, SK&F 101926 proved to be an antidiuretic agonist in humans. Hence, we evaluated SK&F 101926 for antidiuretic agonist and antagonist activities in conscious domestic pigs. 3. During water diuresis (Uosm < 230 mOsm/kg H2O), administration of SK&F 101926 (100 micrograms/kg, i.v.) produced a maximal Uosm of 192 +/- 18 mOsm/kg H2O, a concentration not significantly different from that in vehicle-treated pigs. 4. In hydropenia, SK&F 101926 produced a modest decrease in Uosm, from 945 to 629 mOsm/kg H2O (P < 0.05). 5. In in vitro studies subsequently performed using renomedullary tissue from the same pigs, SK&F 101926 displayed high affinity for V2 receptors (Kbind = 11.8 nM) and high potency to inhibit vasopressin-stimulation of adenylate cyclase (Ki = 3.9 nM). 6. No activity of SK&F 101926 to stimulate adenylate cyclase activity was detected. 7. We conclude that, in spite of its activity in in vitro assays, SK&F 101926 is a weak antidiuretic antagonist in domestic pigs. 8. These results underscore the limited utility of assessments of vasopressin receptor binding and vasopressin-stimulated adenylate cyclase activities in vitro to predict functional antidiuretic activities in vivo.

Identification and characterization of monoclonal antibodies reactive with bovine, caprine and ovine T-lymphocyte determinants by flow microfluorimetry.

Comparative flow microfluorimetric (FMF) analysis was used to identify and characterize 27 monoclonal antibodies (MoAbs) reactive with bovine T-lymphocytes. Determinants present on all circulating T-lymphocytes were recognized by 11 MoAbs, 8 of which blocked E rosette formation. Determinants present on only the BoCD4+ T-lymphocyte subset were detected by 9 MoAbs, while determinants restricted to the BoCD8+ T-lymphocyte subset were recognized by 7 MoAbs. Competitive labeling experiments demonstrated that determinants recognized by subset-specific MoAbs were present on BoCD4 or BoCD8 molecules. Comparative studies revealed that some determinants, both pan-T specific and subset-specific, were conserved on homologous (orthologous) molecules expressed on leukocytes from other species of ruminants. Polymorphism was evident with several determinants.

Ageing changes in the bovine kidney.

A study of macroscopically normal bovine kidneys from three age groups (neonatal calves, 2.5- to 3-year-old bullocks and cull cows), with no abnormalities on urine analysis, was carried out by light microscopy, immunofluorescence and electron microscopy. There was a slight increase in the proportion of involuted nephrons with increasing age but the proportion of nephrons affected was not greater than 10 per cent in any age group. In contrast to the findings of earlier workers, no evidence of diffuse proliferative glomerulonephritis was found in the material examined. It was concluded that the above techniques should be applied to the investigation of renal disease in cattle, as has already been done in man and small domestic animals.