Comparative analysis of cellular immune responses and cytokine levels in sheep experimentally infected with bluetongue virus serotype 1 and 8.

Protective immunity in sheep with bluetongue virus (BTV) infection as well as the role of BTV-induced cytokines during immune response remains unclear. Understanding the basis immunological mechanisms in sheep experimentally infected with serotypes 1 and 8 (BTV-1 and -8) was the aim of this study. A time-course study was carried out in order to evaluate cell-mediated immune response and serum concentrations of cytokines (IL-1ß, TNFα, IL-12, IFNγ, IL-4 and IL-10) with inflammatory and immunological functions. Depletion of T cell subsets (mainly CD4(+), γδ and CD25(+)) together with the absence of cytokines (IFNγ and IL-12) involved in the regulation of cell-mediated antiviral immunity at the first stage of the disease suggested that both BTV-1 and BTV-8 might impair host's capability against primary infections which would favor viral replication and spreading. However, cellular immune response and cytokines elicited an immune response in sheep that efficiently reduced viremia in the final stage of the experiment. Recovery of T cell subsets (CD4(+) and CD25(+)) together with a significant increase of CD8(+) T lymphocytes in both infected groups were observed in parallel with the decrease of viremia. Additionally, the recovery of CD4(+) T lymphocytes together with the significant increase of IL-4 serum levels at the final stage of the experiment might contribute to humoral immune response activation and neutralizing antibodies production against BTV previously described in the course of this experiment. These results suggested that both cellular and humoral immune response may contribute to protective immunity against BTV-1 and BTV-8 in sheep. The possible role played by IL-10 and CD25(+) cells in controlling inflammatory and immune response in the final stage of the experiment has also been suggested.

The influence of acute and chronic hypercalcemia on the parathyroid hormone response to hypocalcemia in rabbits.

OBJECTIVE: To investigate the influence of acute and chronic hypercalcemia on the parathyroid hormone (PTH) response to hypocalcemia. DESIGN: The PTH response to hypocalcemia has been evaluated in three groups of rabbits: Group I, normal rabbits, Group II, normal rabbits subjected to an acute hypercalcemic clamp (induced by CaCl(2) infusion) and Group III, rabbits with chronic hypercalcemia (due to surgical reduction of renal mass). RESULTS: In Group I (baseline Ca(2+)=1.69+/-0.02 mM), hypocalcemia resulted in stimulation of PTH secretion which reached a maximum (PTHmax) of 91.7+/-6.4 pg/ml. In rabbits from Group II, which also had normal baseline Ca(2+) (1.70+/-0.02 mM), plasma Ca(2+) was maintained at an elevated level for 2 h, at around 2.05 mM. The PTH response to hypocalcemia in Group II was attenuated and the PTHmax in these rabbits was 45.6+/-7.4 pg/ml. In rabbits from Group III, baseline Ca(2+) was elevated (2.06+/-0.06 mM) for 1 month. The PTH response to hypocalcemia in Group III was esentially the same as in Group I and PTHmax reached levels of 94.8+/-9.9 pg/ml. CONCLUSIONS: A difference in PTH response to hypocalcemia has been found in rabbits after exposure to either acute or chronic hypercalcemia. After acute hypercalcemia, an attenuated PTH response to hypocalcemia has been identified. Chronic hypercalcemia, however, did not influence the PTH response to hypocalcemia.

Validation and clinical utility of a novel immunoradiometric assay exclusively for biologically active whole parathyroid hormone in the horse.

REASONS FOR PERFORMING STUDY: Parathyroid hormone (PTH) plays a critical role in the regulation of mineral metabolism in mammals. Until recently, the standard method for PTH measurement has been the 2nd generation intact-PTH (I-PTH) assay. Current evidence indicates that the I-PTH assay binds to the PTH molecule and to an inactive N-terminally truncated PTH fragment that tends to accumulate in the blood of uraemic patients. Therefore, a new 3rd generation PTH assay that detects only the whole PTH molecule (W-PTH; cyclase-activating PTH [CAP]) has been developed. OBJECTIVES: To validate this more specific W-PTH assay for measurement of equine PTH and evaluate its clinical utility. METHODS: W-PTH and I-PTH were measured in plasma samples from normal horses (adults and foals) and horses with nutritional secondary hyperparathyroidism (N2HPT) and with chronic renal failure (CRF). Replicate measurements and dilutional paralellism were used for assay validation. Changes in blood ionized calcium were induced by EDTA and CaCl2 administration. RESULTS: Performance of the W-PTH assay (accuracy, sensitivity, specificity and ability to detect changes in PTH in response to changes in calcium) was similar to that of the I-PTH assay. Surprisingly, the relative W-PTH concentration in normal horses and foals was higher than the relative I-PTH concentration. W-PTH values remained higher than I-PTH during acute hypo- and hypercalcaemia. An increase in both W-PTH and I-PTH concentrations was found in horses with N2HPT. In horses with CRF, W-PTH and I-PTH values were very low and no increase in I-PTH was observed. CONCLUSIONS: The W-PTH assay can be used for measurement of equine PTH. POTENTIAL RELEVANCE: The use of W-PTH assay is likely to improve the diagnosis of mineral metabolism in horses.

Effects of exercise and EDTA administration on blood ionized calcium and parathyroid hormone in horses.

OBJECTIVE: To determine effects of exercise on blood ionized calcium (Ca2+) and plasma parathyroid hormone (PTH) concentrations in horses and to compare the effects of exercise-induced and EDTA-induced hypocalcemia on PTH secretion. ANIMALS: 17 horses entered in a show jumping competition and 5 horses given EDTA. PROCEDURE: Blood Ca2+ and plasma PTH concentrations were measured before and after exercise in the 17 horses entered in the jumping competition. In the other 5 horses, concentrations were measured during infusion of EDTA IV. RESULTS: Exercise resulted in a significant decrease in blood Ca2+ concentration and a significant increase in plasma PTH concentration, and blood Ca2+ concentration was correlated with plasma PTH concentration. Administration of EDTA resulted in hypocalcemia and an increase in PTH concentration. For the same decrease in Ca2+ concentration, magnitude of the exercise-induced increase in PTH concentration was similar to magnitude of the EDTA-induced increase. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the increase in plasma PTH concentration in horses after exercise is secondary to hypocalcemia and that the increase in PTH concentration seems to be commensurate with the decrease in Ca2+ concentration.

Plasma ionized calcium and parathyroid hormone concentrations in horses after endurance rides.

OBJECTIVE: To evaluate changes in plasma ionized calcium (Ca2+) and parathyroid hormone (PTH) concentrations in horses competing in endurance rides. DESIGN: Longitudinal clinical study. ANIMALS: 28 horses. PROCEDURE: Venous blood samples were obtained from horses before and after racing 80 km. Plasma pH and concentrations of Ca2+, PTH, inorganic phosphorus, albumin, lactate, and magnesium were measured. RESULTS: Overall, a significant decrease in mean (+/- SD) plasma Ca2+ concentration (from 6.44 +/- 0.42 to 5.64 +/- 0.42 mg/dl) and a significant increase in plasma PTH concentration (from 49.9 +/- 30.1 to 148.1 +/- 183.0 pg/ml) were found after exercise. Exercise also resulted in significant increases in plasma inorganic phosphorus, albumin, and lactate concentrations. No changes in plasma magnesium concentration or pH were detected after exercise. Plasma PTH concentration was not increased after exercise in 8 horses; in these horses, plasma PTH concentration decreased from 58.2 +/- 26.3 to 27.4 +/- 22.4 pg/ml, although plasma Ca2+ concentration was also decreased. CONCLUSIONS AND CLINICAL RELEVANCE: Plasma Ca2+ concentration was decreased after racing for 80 km, compared with values obtained before racing. In most horses, an increase in plasma PTH concentration that was commensurate with the decrease in plasma Ca2+ was detected; however, some horses had decreased plasma PTH concentrations.

Influence of feeding status, time of the day, and season on baseline adrenocorticotropic hormone and the response to thyrotropin releasing hormone-stimulation test in healthy horses.

Equine pituitary pars intermedia function can be assessed by the measurement of baseline and thyrotropin releasing hormone (TRH)-induced concentrations of adrenocorticotropic hormone (ACTH); however, these measurements may be affected by the environment. Therefore, a prospective observational study evaluated the influence of feeding, time of the day, and season on baseline and TRH-induced concentrations of ACTH in healthy horses. Baseline ACTH was measured in 50 horses before and 2 h after feeding. Six research horses were subjected to a crossover study in which 6 TRH tests were performed in 2 different seasons, March-April (MA) and July-September (JS), at 2 different times of the day, 8 AM and 8 PM, and, under 2 different conditions relative to feeding status, fasted and 2 h after feeding. Differences between fasted and fed horses were found in baseline ACTH, 17.1 ± 1.8 versus 46.1 ± 7.6 pg/mL (P = 0.003) and TRH-stimulated ACTH: 124.1 ± 21.3 versus 192.6 ± 33.1 pg/mL (P = 0.029) at 10 min, and 40.1 ± 4.9 versus 73.2 ± 13.4 pg/mL (P = 0.018) at 30 min post TRH injection. No differences were found between tests performed at different times of the day. Basal ACTH concentrations were greater in JS than in MA, 17.1 ± 1.8 versus 11.9 ± 0.6 pg/mL (P = 0.006). A seasonal influence was also found in stimulated ACTH values, which were much greater in JS 122.7 ± 36.7 versus 31.2 ± 7.4 pg/mL, at 10 min (P = 0.03) and 39.0 ± 7.2 versus 19.8 ± 3.1 pg/mL, at 30 min (P = 0.03). In addition to season, feeding is a potential confounding factor when measuring baseline or stimulated ACTH in horses. In conclusion, feeding status should be standardized for the diagnosis of equine pituitary pars intermedia dysfunction.

Cell-mediated immune response during experimental acute infection with bovine viral diarrhoea virus: evaluation of blood parameters.

Acute infections with bovine viral diarrhoea virus (BVDV), a major pathogen of cattle, are often asymptomatic or produce only mild clinical symptoms. However, they may play an important role in the bovine respiratory disease complex by exerting a marked immunosuppressive effect, as a result of the death of the immunocompetent cell populations involved in controlling innate and adaptive immune responses, together with a marked reduction of both cytokine expression and co-stimulatory molecule synthesis. Although experimental research and field studies have shown that acute BVDV infection enhances susceptibility to secondary infection, the precise mechanism involved in BVDV-induced immunosuppression remains unclear. The present study is aimed at measuring a range of blood parameters in a single group of fourteen calves infected with non-cytopathic BVDV-1. Focus has been put on those related to the cell-mediated immune response just as leucocyte populations and lymphocyte subpopulations, serum concentrations of cytokines (IL-1ß, TNF-α, IFN-γ, IL-12, IL-4 and IL-10) and acute phase proteins [haptoglobin, serum amyloid A (SAA), fibrinogen and albumin], as well as BVDV-specific antibodies and viremia. After non-cytopathic BVDV-1 infection, clinical signs intensity was never more than moderate coinciding with the presence of viremia and leucocyte and lymphocyte depletion. An early increase in TNF-α, IFN-γ and IL-12 levels in contrast to IL-1ß was observed in line with a raise in haptoglobin and SAA levels on the latest days of the study. As regards IL-4 levels, no evidence was found of any changes. However, a slight increase in IL-10 was observed, matching up the TNF-α decline during the acute phase response. These findings would help to increase our knowledge of the immune mechanisms involved in acute infection with non-cytopathic BVDV-1 strains, suggesting the existence of a clear tendency towards a type 1 immune response, thereby enhancing resistance against viral infections.

Comparative study of clinical courses, gross lesions, acute phase response and coagulation disorders in sheep inoculated with bluetongue virus serotype 1 and 8.

Bluetongue virus serotypes 1 (BTV-1) and 8 (BTV-8) have been described as the most prevalent in Europe during recent outbreaks displaying intense virulence, sheep being among the most severely affected livestock species. However, BTV pathogenesis is still unclear. This study sought to elucidate differences in the pathogenetic mechanisms of BTV-1 and -8 in sheep. For this purpose, a time-course study was carried out, with sequential sacrifices in order to relate pathological lesions to changes in a range of virological and serological parameters. A greater virulence of BTV-1 was probed. BTV-1 infected sheep showed a longer clinical course, with a significant increase of clinical signs and more severe gross lesions than BTV-8 infected sheep. These differences appear not to be attributable to greater virus replication, suggesting viral loads did not influence in the pathogenicity of these serotypes. While both groups displayed an early, intense antibody response, they still developed clinical signs and lesions characteristic of bluetongue, indicating a lack of correlation between antibody levels and protection against the disease. Both acute phase response (APR) and thrombocytopenia induced by BTV-1 in sheep were more intense. Furthermore, an association between acute phase proteins (APPs) concentrations and the evolution of clinical signs and gross lesions was also observed, suggesting the existence of a direct link between the pathogenicity of BTV serotypes, the severity of vascular lesions and the serum concentrations of APPs. To our knowledge, this is the first verification of a measurable APR in sheep with both experimental and naturally occurring bluetongue.