The detection of CD2+, CD4+, CD8+, and WC1+ T lymphocytes, B cells and macrophages in fixed and paraffin embedded bovine tissue using a range of antigen recovery and signal amplification techniques

In order to develop procedures to label the main bovine leucocyte populations in paraffin embedded sections, the immunoreactivity of 25 monoclonal antibodies (mAbs) to different leucocyte antigens was assessed with formal dichromate (FD5) and 10% formalin fixation, a battery of antigen retrieval (AR) methods, and the biotin-tyramide amplification system. All the leucocyte populations investigated (CD2+, CD4+, CD8+, WC1+ T lymphocytes, B cells and macrophages) were strongly and specifically detectable under an appropriate combination of mAb, AR method and signal amplification system. CD4 and CD8 required the most stringent conditions and could only be demonstrated in FD5 fixed sections. For detection of CD2, WC1+ T lymphocytes, B cells and macrophages, all the mAbs produced immunoreactivity in FD5 or formalin fixed tissues. The need to check a range of different AR methods is stressed, as the method of choice varied for each individual mAb. The incorporation of the signal amplification system was necessary to observe a strong signal and the complete distribution of CD4, CD8 and B cells. Fixation by FD5 proved to be better than formalin for the preservation of surface antigens but it was inferior for the detection of markers which were found to show cytoplasmic immunoreactivity, such as the macrophage marker MAC387 or the B cell markers BAQ155 or IL-A59.     

Serological and biochemical follow-up in cattle naturally infected with Fasciola hepatica, and comparison with a climate model for predicting risks of fasciolosis

Several biological parameters were measured in 31 heifers naturally infected with Fasciola hepatica during one grazing season in the Belgian Ardennes. A forecast model based on daily temperature used to assess the risk of fasciolosis was fitted to this assay. Cattle were turned out to two pastures. Each pasture was divided into two plots: one was treated with calcium cyanamide and the other was left untreated. The Lymnaea truncatula snails were counted on three different occasions. The results indicated a poor molluscicide efficiency. Body weight gains, anti-Fasciola antibody levels, faecal egg counts, levels of sorbitol dehydrogenase (SDH) and gamma-glutamyl transferase (gamma GT), packed cell volumes, white blood cells and differential leucocyte counts were determined monthly. No statistically significant difference was observed between animals from the two plots regardless of the recorded data. No correlation was found between body weight gains and other biological data. The sampling date had a significant effect on the antibody responses within a same group, and on the enzymatic levels for all groups combined. The forecast results were consistent with the recorded data. Temperature was a major bioclimatic constraint on the transmission of life cycle, and risk of infection occurred mainly in late spring (May/June) and in early September. Current results might be used to issue advice on the need for flukicide treatment of cattle. The indicators of the infection considered alone were useless and it is concluded that herd diagnosis of fasciolosis may rely on the rise of specific antibody levels, possibly associated with an increase in hepatic enzyme activities.     

REVERSIBLE MAGNETIC RESONANCE IMAGING ABNORMALITIES IN DOGS FOLLOWING SEIZURES

Reversible magnetic resonance (MR) imaging lesions have been described in humans following seizures. This condition has not yet been reported in animals. This paper describes reversible abnormalities identified in 3 dogs using MR imaging that was performed initially within 14 days of the last seizure and follow-up imaging that was performed after 10 to 16 weeks of anticonvulsant therapy. All three dogs had lesions in the piriform/temporal lobes, characterized by varying degrees of hyperintensity on T2-weighted images and hypointensity on T1-weighted images. In one dog, contrast enhancement was evident. On reevaluation, partial resolution occurred in all 3 dogs. In a fourth animal with an olfactory meningioma, similar appearing lesions in the temporal cortex and right and left piriform lobes were identified after seizure activity. A surgical biopsy of the temporal cortex and hippocampus was performed and edema, neovascularization, reactive astrocytosis, and acute neuronal necrosis were evident. These histologic findings are similar to those reported in humans with seizures. Recognizing the potential occurrence of reversible abnormalities in MR images is important in developing a diagnostic and therapeutic plan in canine patients with seizures. Repeat imaging after seizure control may help differentiate between seizure-induced changes and primary multifocal parenchymal abnormalities.     

Feeding-induced increases in insulin do not suppress secretion of growth hormone

Secretion of growth hormone (GH) is reduced for several hours after feeding when access to feed is restricted to a 2-hr period each day. We hypothesized that increased secretion of insulin after feeding inhibits release of GH from the anterior pituitary gland. Our objectives were to determine whether: 1) alloxan prevents concentrations of insulin from increasing after feeding steers; 2) concentrations of GH remain high after feeding alloxan-treated steers; and 3) GH-releasing hormone (GHRH) stimulates greater release of GH in alloxan-treated, than in control, steers after feeding. Steers were injected iv with either saline (control) or with alloxan (110 mg/kg) (n = 4 per group). Concentrations of insulin were not different (P = 0.61) between control and alloxan-treated steers before feeding (87.5 +/- 33.6 pmol/l). However, alloxan prevented insulin from increasing (P < 0.001) after feeding (131.8 pmol/1) compared with control steers (442.0 pmol/l) (pooled SEM = 47.5). Overall, GH was higher (P < 0.05) in alloxan-treated (6.4 ng/ml) than in control steers (3.7 ng/ml) (pooled SEM = 0.7), but GH decreased (P < 0.001) after feeding in both groups. Iv injection of GHRH stimulated release of GH 1 hr before, but not when injected 1 hr after feeding (P < 0.001). In addition, net areas under the GH curve were not significantly different between control and alloxan-treated groups. We conclude that increased concentrations of insulin after feeding do not mediate feeding-induced suppression of GH secretion in steers.     

Comparison of clinical signs and hemodynamic variables used to monitor rabbits during halothane- and isoflurane-induced anesthesia

OBJECTIVE: To characterize variables used to monitor rabbits during inhalation anesthesia. ANIMALS: 8 male New Zealand White rabbits. PROCEDURE: Rabbits were similarly anesthetized with halothane (HAL) or isoflurane (ISO) in a crossover study; half received HAL followed by ISO, and the protocol was reversed for the remaining rabbits. After induction, minimum alveolar concentration (MAC) was determined for each agent, using the tail-clamp method, and variables were recorded at 0.8, 1.0, 1.5, and 2.0 MAC (order randomized). RESULTS: Mean +/- SEM MAC was 1.42 +/- 0.05 and 2.07 +/- 0.09% for HAL and ISO, respectively. Directly measured auricular mean arterial blood pressure was 52.8 +/- 5.6 and 54.8 +/- 6.1 mm Hg at 0.8 MAC for HAL and ISO, respectively, and decreased from these values in a parallel dose-dependent manner. Respiratory frequency remained constant (range, 69 to 78 breaths/min) over the range of HAL doses but incrementally decreased from a mean of 53 (at 0.8 MAC) to 32 breaths/min (at 2.0 MAC) for ISO. The PaCO2 was similar at 0.8 MAC for HAL and ISO and progressively increased with increasing doses of both agents; PaCO2 at 2.0 MAC for ISO was significantly greater than that at 2.0 MAC for HAL (79.8 +/- 13.7 vs 54.9 +/- 4.0 mm Hg, respectively). Eyelid aperture consistently increased in a dose-dependent manner for both anesthetics. CONCLUSIONS: Arterial blood pressure, PaCO2, and eyelid aperture consistently and predictably changed in rabbits in response to changes in anesthetic doses. The magnitude of respiratory depression was greater for ISO than for HAL.     

The use of chance-corrected agreement to diagnose canine compulsive disorder: an approach to behavioral diagnosis in the absence of a 'gold standard'.

This study assessed the diagnostic accuracy of formal diagnostic criteria for canine compulsive disorder (canine CD). Canine CD is a syndrome of abnormal behaviors that are believed to result from conflict or frustration. Differential diagnoses include normal conflict behavior and learned behavior. In studies of canine CD, confidence in the diagnosis comes with knowing the accuracy of the diagnostic method. This accuracy may be quantified as the chance-corrected agreement between the diagnostic method and a 'gold standard' diagnostic test. The present study examined the agreement between diagnoses of canine CD made by an expert (the 'gold standard') and by using formal diagnostic criteria. The owners of 84 dogs suspected of having CD received 2 telephone interviews. The first utilized a detailed, pre-tested questionnaire; a dog was then diagnosed with CD if the behavioral history met 7 diagnostic criteria. The second interview was given by a behavioral expert whose diagnosis was based on personal experience. The interviewers were blind to each other's diagnoses. The chance-corrected agreement between diagnoses was minimal (kappa = 0.02) and disagreement was associated with 3 of the formal criteria: a history of conflict or frustration, an increase in the number of contexts that elicit the behavior, and an increase in the daily frequency of the behavior. Reasons for the disagreement include the order of the interviews, response biases, the setting of the interviews, and, possibly, the diversity of the behaviors associated with canine CD. To the authors' knowledge, this type of study is the first in clinical ethology to address validation of the diagnostic method. The results indicate 3 developmental aspects of canine CD that should be examined in future work.     

Development of PCR-based tests for the identification of North American isolates of epizootic haemorrhagic disease virus.

A serogroup-specific polymerase chain reaction (PCR) assay and isolate identification strategies (restriction endonuclease analysis (REA) and nucleotide sequencing) were developed for the detection of North American isolates of epizootic haemorrhagic disease virus (EHDV). PCR primers (EHDV-pr4, EHDV-pr5) were designed to hybridize to the L3 gene of a North American isolate of EHDV serotype 1. Total nucleic acid was extracted from preparations of infected tissue culture and PCR was performed using a cDNA-PCR kit, according to the manufacturer's specifications. The PCR assay generated a 459 base pair product from North American isolates of EHDV serotypes 1 and 2, while bluetongue virus (BTV) serotypes 10, 11, 13, and 17, and cell controls, failed to demonstrate PCR products. Slight modifications allowed for the PCR detection of EHDV-1 and -2 in white-tailed deer blood (Odocoileus virginiatus); PCR fragments were not amplified from uninfected deer blood. A number of restriction endonucleases and sequencing primers were evaluated for their utility in isolate identification experiments. Specifically, REA employing HincII and cycle sequencing with an internal primer (EHDV-1-pr3) proved most successful for identifying isolate-specific genome markers. The techniques presented are expected to prove valuable for rapid and specific detection of possible future EHDV incursions in wild and domestic animal species.