The effect of Johne's vaccination on tuberculin testing in farmed red deer (Cervus elaphus)

AIM: To assess the degree of interference with bovine tuberculin testing in farmed red deer that vaccination of young deer with an oil-adjuvanted vs aqueous formulation of live attenuated Mycobacterium paratuberculosis Strain 316F vaccines would be likely to cause, and to compare immunological responses between vaccine formulations.

METHODS: Five-month-old red deer (n=45) were randomly allocated to three treatment groups of 15 animals, which received either no vaccine, a single 2-ml dose of an oil-adjuvanted formulation or two 2-ml doses, 6 weeks apart, of an aqueous formulation of live attenuated M. paratuberculosis Strain 316F vaccine injected subcutaneously (S/C) in the neck (Control, Oil-adjuvant Ptb, and Aqueous Ptb groups, respectively). Injection-site reactions were described and measured on Weeks 3, 6 and 9. Animals were weighed and lymphocyte transformation tests (LTT) and antibody enzyme-linked immunosorbent assays (ELISA) using avian, bovine and Johnin tuberculin were conducted on blood samples collected at Weeks 0, 6, 12, 15, 24, 27, 36 and 39. A bovine mid-cervical skin test (MCT) was applied at Week 12, and comparative cervical skin tests (CCTs) at Weeks 24 and 36. At Week 42, the animals were slaughtered at a commercial deer slaughter premises and subjected to rigorous meat inspection.

RESULTS: Two animals were eliminated at the start of the trial due to a positive cross-reaction with bovine tuberculin in the initial LTT. Almost all animals reacted to the MCT at Week 12, with mean skin thicknesses of 3.9, 2.9 and 1.0 mm for the Oil- adjuvant Ptb, Aqueous Ptb and Control groups, respectively. When the CCT was conducted at Week 24, 2/15 Oil-adjuvant Ptb, 2/14 Aqueous Ptb and 1/14 Control animals were classified as CCT-positive to bovine tuberculin. By Week 36, all animals were CCT-negative. The Oil-adjuvant Ptb vaccination resulted in high persistent levels of antibody that reacted with bovine tuberculin, compared with negligible levels in the Aqueous Ptb group.

Overall, a single dose of the Oil-adjuvant Ptb vaccine in deer stimulated a vigorous, cross-reactive immune response, evidenced by high LTT, skin-test and antibody reactions to bovine tuberculin, with both cell-mediated and humoral characteristics. By comparison, two doses of the Aqueous Ptb vaccine produced less cross-reactivity and a bias towards a cell-mediated response. The Oil-adjuvant Ptb vaccine resulted in moderate injection- site lesions that were quite persistent, whereas the Aqueous Ptb vaccine resulted in smaller nodules that regressed more quickly.

CONCLUSIONS: Vaccination of farmed deer with an oil-adjuvanted Johne's vaccine has the potential to cause significant interference with routine tuberculin skin testing. The cross-reactivity should decline with time and the CCT should be able to clear MCT-positives, but there is a risk of false-positives to the blood test for tuberculosis (BTB), due to high persistent levels of antibody. The CCT could be used as a primary skin test in vaccinated deer on some farms.

The Aqueous Ptb caused fewer problems with skin testing and produced significantly less bovine antibody than the Oil-adjuvant Ptb, but stimulated persistent cell-mediated immune responses that may provide some protection against Johne's disease.     

Evidence from the use of isozyme markers of competition in swards between short-leaved and long-leaved perennial ryegrass

Intraspecific competition in Lolium perenne was studied using a replacement series of a short-leaved and a long-leaved genotype that was managed under different cutting frequencies and nitrogen supplies. Leaf length, tiller density and yield were recorded 6 months after sowing. Since the two competitors were labelled at the PG1/2 locus, it was possible to identify unambiguously the origin of tillers in the mixtures and to estimate the average length of lamina of each genotype. The difference in leaf length between the two competitors was maintained under particular types of management and generated contrasting competitive abilities leading to evolution of the genotypic composition of the mixtures. Thus, under infrequent cutting, competition for light resulted in the domination of the long-leaved genotype and in a concomitant intragenotypic selection for leaf length. Supplying nitrogen fertilizer emphasized the phenotypic plasticity of the genotypes, particularly of the dominated one. Frequent cutting reduced competition for light and the change towards the short-leaved genotype was far less rapid. The yielding and competitive abilities of ryegrass genotypes and the implications for breeding are briefly discussed.     

Acute myelogenous leukaemia in a mare

SUMMARY: A 5-year-old Thoroughbred mare presented with a 4 week history of weight loss, fever and leukopenia. Rectally, a large active foetus, thickened spleen and an abdominal mass were palpated. Leukopenia, mild anaemia, marked thrombocytopenia and hyperfibrinogenaemia were found. Cytology and cytochemical staining of a bone marrow aspirate supported a diagnosis of acute myelogenous leukaemia. The mare deteriorated despite medical therapy and was humanely euthanased.     

Regulation of uterine estrogen receptors (ER) by beta-adrenergic stimulation in immature rats

The effects of a 3-day intramuscular (i.m.) administration of clenbuterol (25 μ.g/ Kg), propranolol (12 mg/kg), clenbuterol (25 μg/kg) plus propranolol (12 mg/ Kg) and estradiol (0.5 μg) upon the female reproductive system were investigated in immature Sprague-Dawley rats. Clenbuterol and estradiol treatments induced a significant increase in uterus weight and in relative uterus weight, whereas in the groups treated with propranolol and clenbuterol plus propranolol no differences were detected versus controls. The uterine estrogen receptor levels were significantly increased by clenbuterol administration. In the rats dosed with propranolol and clenbuterol plus propranolol, no modifications occurred in estrogen receptor concentrations when compared with control values. Uterine progesterone receptors were never significantly affected by any of the considered treatments. Data obtained indicate that clenbuterol treatment induces an increase in uterus weight and in estrogen receptor levels and that these effects are regulated by acute beta-adrenergic stimulation, as the contemporaneous administration of high doses of a beta-blocker inhibit such effects.     

Pharmacokinetics of intravenous ceftiofur sodium and concentration in body fluids of foals

The objectives of this study were to determine pharmacokinetics of intravenous (i.v.) ceftiofur in foals, to compare ultra-high performance liquid chromatography tandem mass spectometry (UPLC-MS/MS) and microbiologic assay for the measurement of ceftiofur concentrations, and to determine the minimum inhibitory concentration ( MIC ) of ceftiofur against common equine bacterial pathogens. In a cross-over design, ceftiofur sodium was administered i.v. to six foals (1–2 days-of-age and 4–5 weeks-of-age) at dosages of 5 and 10 mg/kg. Subsequently, five doses of ceftiofur were administered i.v. to six additional foals between 1 and 5 days of age at a dose of 5 mg/kg q 12 h. Concentrations of desfuroylceftiofur acetamide (DCA), the acetamide derivative of ceftiofur and desfuroylceftiofur-related metabolites were measured in plasma, synovial fluid, urine, and CSF by use of UPLC-MS/MS. A microbiologic assay was used to measure ceftiofur activity for a subset of plasma samples. Following i.v. administration of ceftiofur at a dose of 5 mg/kg to 1–2 day-old foals, DCA had a t _½ of 7.8 ± 0.1 h, a body clearance of 74.4 ± 8.4 mL/h/kg, and an apparent volume of distribution of 0.83 ± 0.09 L/kg. After multiple i.v. doses at 5 mg/kg, DCA concentrations in CSF were significantly lower than concurrent plasma concentrations. Ceftiofur activity using a microbiologic assay significantly underestimated plasma concentrations of DCA. The MIC of ceftiofur required to inhibit growth of 90% of isolates of Escherichia coli , Pasteurella spp, Klebsiella spp, and β-hemolytic streptococci was <0.5 μg/mL. Intravenous administration of ceftiofur sodium at the rate of 5 mg/kg every 12 h would provide sufficient coverage for the treatment of susceptible bacterial isolates.     

Ultrasound during mid‐gestation: Agreement with physical foetal and placental measurements and use in predicting gestational age in sheep

To determine the effects of poor maternal nutrition and litter size on foetal growth during mid‐gestation, pregnant ewes (n = 82) were fed 100%, 60% or 140% of NRC TDN beginning at day 30.2 ± 0.2 of gestation. Transabdominal ultrasound was performed weekly between day 46.0 ± 0.4 and 86.0 ± 0.7 to monitor foetal heart width (HW), umbilical diameter (UMB), rib width (RW) and placentome outer (OD) and inner diameter (ID). Data were analysed with repeated‐measures using the mixed procedure for effects of maternal diet, litter size and gestation, and equations predictive of gestational age were generated using the regression procedure. To determine the agreement of ultrasound measurement and actual size, ewes (n = 20–21) were euthanized at day 45 or 90 to obtain corresponding postmortem measurements for Bland–Altman analysis. The HW, UMB and placentome OD and ID increased with gestation (p < .0001) but were unaffected by maternal diet or litter size (p ≥ .12). Ultrasound underestimated postmortem measurements of HW (14.8%), UMB (7.3%), placentome OD (4.5%) and ID (37.3%) at day 90 of gestation. Ultrasound underestimated RW at day 45 (7.7%) but overestimated RW (23.8%) at day 90, indicating inconsistent bias when reporting RW by ultrasound. Combining the HW, UMB, RW and placentome OD generated the strongest equation predictive of gestational age (R² = .91). These findings indicate that during mid‐gestation, maternal diet or litter size did not affect HW, UMB or placentome diameters and these factors can be used to estimate gestational age.     

Progesterone and Caspase-3 Activation in Equine Cyclic Corpora Lutea

Soon after ovulation, the newly formed corpus luteum (CL) starts secreting progesterone (P(4)), necessary for implantation. The CL, an ovarian transient endocrine organ, undergoes growth and regression throughout its life span. The objective of this study was to evaluate if caspase-3 mediates cell death in the equine cyclic luteal structures and relate it to luteal endocrine function. Blood and luteal tissue were collected during the breeding season after slaughter from 38 randomly assigned cycling mares. Luteal tissues were classified as corpora haemorrhagica (CH; n = 7); mid luteal phase corpora lutea (Mid-CL; n = 17); late or regressing corpora lutea (Late-CL; n = 9) and corpora albicans (CA; n = 5). Plasma P(4) concentration, determined by radioimmunoassay, showed a significant increase from CH to Mid-CL (p < 0.001), followed by a decrease to Late-CL (p < 0.001) and CA (p < 0.001). Caspase-3 processing and poly (ADP) ribose polymerase (PARP) degradation were assessed by western blotting. Active caspase-3 was twofold increased in Mid-CL, Late-CL and CA as compared with CH (p < 0.05). Immunocytochemistry also showed a significant increase in caspase-3 expression in large luteal cells in all structures when compared with CH (p < 0.05). Consistently, the endogenous caspase-3 substrate, PARP, was markedly degraded from CH to CA (p < 0.05). In fact, the ratio of full-length to degraded PARP showed a significant decrease from CH to Mid-CL, Late-CL and CA (p < 0.05). Finally, the decrease in P(4) from Mid- to Late-CL coincided with no further increases in apoptosis. In conclusion, these results suggest that the effector caspase-3 of apoptosis, might play an important role during luteal tissue involution in the mare, even though its relationship with P(4) remains to be elucidated.