Flow cytometric analysis of intracellular complexity and CD45 expression for use in rapid differentiation of leukocytes in bovine blood samples.

OBJECTIVE: To develop an efficient and reliable method that accurately differentiates bovine lymphocytes from monocytes in leukograms. SAMPLE POPULATION: Blood samples from 30 healthy cows and 1 calf with bovine leukocyte adhesion deficiency. PROCEDURE: Flow cytometric analysis of intracellular complexity and CD45 expression on bovine leukocytes was compared with results for conventional light microscopy methods. Verification of leukocyte subpopulations determined by intracellular complexity and CD45 expression was conducted, using 2-color phenotypic analysis with selected monoclonal antibodies. RESULTS: The CD45 and side-scatter properties of bovine leukocytes clearly differentiated cell types, including neutrophils, eosinophils, monocytes, and lymphocytes. CONCLUSIONS AND CLINICAL RELEVANCE: This is a rapid assay that is simple to use. More importantly, it is more accurate than the conventional method that involves the use of blood slides and light microscopy, because of the ability of the assay to readily distinguish bovine monocytes and lymphocytes. Rapid preparation of samples and short analysis times allow for efficient and reliable examination of a large number of samples, and the task of viewing slides by light microscopy is eliminated. The labor-savings benefit of this procedure is most apparent in research environments that require frequent processing of batches of blood samples.     

Long-term variation in brown trout, Salmo trutta L., stocking success in a large lake: interplay between availability of suitable prey and size at release

Abstract –  Factors affecting long-term variation in brown trout, Salmo trutta L., stocking success were examined in a large lake, Lake Oulujärvi, in central Finland. Brown trout were stocked in spring (late May to early June) in 1974–1991 and in summer (late June to early July) in 1992–2001. The biomass of the vendace, Coregonus albula (L.), population (prey) at release time had the largest positive effect on stocking success within both periods: biomass of adult vendace in spring and both 0+ and adult vendace in summer. Increasing the size of stocked fish had a positive effect if the vendace available at release were only adults. The increasing trend of predator-catch-per-unit-effort (CPUE) [combined CPUE of northern pike Esox lucius L., burbot Lota lota (L.), and pike-perch Stizostedion lucioperca (L.)] through the study period and its negative effect on trout stocking success suggested an increasing effect of predation within the entire time series.     

Fate of Escherichia coli and Escherichia coli O157 in soils and drainage water following cattle slurry application at 3 sites in southern Scotland

Abstract. Slurry from farm animals may contaminate water supplies, rivers and bathing waters with faecal coliforms, such as Escherichia coli . Where animals harbour the O157 strain the hazard to human health is particularly high, but both the hazard level, and the low incidence and sporadic nature of the excretion of E. coli O157 make it difficult to study this strain under field conditions. The survival of total E. coli and of E. coli O157 were compared in the laboratory for two soils under controlled temperature and moisture. E. coli O157 die-off rate was the same as or quicker than for total E. coli . This result meant that field experiments studying the fate of total E. coli should give a satisfactory evaluation of the risk of water contamination by the O157 strain. In four field experiments at three sites, slurry containing total E. coli numbers of 2.2 × 10⁴ to 5.7 × 10⁵ colony forming units per mL (c.f.u. mL^–1) was applied to drained field plots. Field die-off was faster than expected from laboratory experiments, especially in one experiment where two weeks dry weather followed application. In all but this experiment, the first drain flow events after slurry application led to very high E. coli concentrations in the drains (10³ to 10⁴ c.f.u. mL^–1). E. coli O157 was present in the slurry used for two of the experiments (33 c.f.u. per 100 mL in each case). However the proportion of E.coli O157 was very low (about 1 in 10⁵) and it was not detected in the drainage water. After the first week E. coli drainage water numbers decreased rapidly but they were 1–10 c.f.u. mL^–1 for much of the sampling period after slurry application (1–3 months).     

Bovine sire effects on daughters' in vitro blood neutrophil functions, lymphocyte blastogenesis, serum complement and conglutinin levels

Blood neutrophil functions, lymphocyte blastogenic responses, serum complement, and serum conglutinin activity of 98 lactating Holstein cows from two genetic lines were evaluated. The genetic lines were produced in a selection experiment that created and perpetuated genetic differences in milk production for up to seven generations. No significant differences between the two genetic lines of cows were found for neutrophil function, lymphocyte blastogenic responses, serum complement levels, or serum conglutinin levels. Significant differences between sire progeny groups within lines were found for unstimulated and mitogen-stimulated lymphocyte blastogenesis (P less than 0.0001), and almost all neutrophil functions (antibody independent neutrophil cytotoxicity, antibody dependent neutrophil cytotoxicity, ingestion of bacteria, iodination, chemiluminescence, chemokinesis, and chemotaxis (P less than or equal to 0.05)). Sire progeny group differences (P less than or equal to 0.0001) within lines for serum complement and conglutinin activity were also found. Neutrophil chemiluminescence activity (positive relationship; P less than or equal to 0.001), concanavalin A-stimulated lymphocyte blastogenesis (positive relationship; P less than or equal to 0.004), and serum conglutinin activity levels (negative relationship; P less than or equal to 0.01) each had small but significant associations with the total milk somatic cell count. Cows seropositive for bovine leukosis virus had increased resting and mitogen-stimulated lymphocyte blastogenic activity and were associated with increased in vitro neutrophil random migration and production of superoxide anion. Estimates of genetic parameters of various immune cell functions, of serum complement and of conglutinin levels for daughters of 11 sires with 4-6 daughters in the data set were determined. In this report, genetic variation was demonstrated for nonspecific humoral and cellular immunity.     

The Effect of Exogenous Hormone Treatment on Spermiation in Rhynchocypris oxycephalus (Sauvage and Dabry)

For the evaluation of hormonal control of spermiation in fish, a method to quanify the spermiation response of mature Rhynchocypris oxycephalus (Sauvage and Dabry) to hormonal therapy is described. Spermatocrit was determined after 7 min centrifugation at 18,000 ± g and sperm density was estimated by a standard hemocytomer method. Sperm density can be predicted from spermatocrit since their relationship is linear as described by the regression equation, Y = 3.68X - 27.18 ( R ² = 0.82, N = 50). where Y is spermatocrit and × is sperm density. Milt production by mature R. oxycephalus was highest at 24 h after injection of 1,000 IU human chorionic gonadotropin (HCG) and 50 μg luteinizing hormone-releasing hormone analogue (LHRHa) per kg body weight. Increased milt production coincided with low spermatocrit and sperm density levels. These results demonstrate that spermiation in mature R. oxycephalus can be reliably evaluated by a spermatocrit method and that HCG and LHRHa are effective in stimulating of spermiation in this species.     

0–7–21 Radiation Therapy for the Treatment of Canine Oral Melanoma

Eighteen dogs with malignant melanoma of the oral cavity were treated with high-dose per fraction (0–7–21) radiation therapy. Eight hundred cGy was administered on days 0, 7, and 21 for a total dose of 2,400 cGy in 3 weeks. Of 17 dogs evaluated, 9 (53%) had a complete remission and 5 (30%) achieved a partial remission with an overall response rate of 83%. Local failure occurred in 2 of the 9 dogs where a complete response was initially observed. One dog died of intercurrent disease, and one died of metastatic disease without evidence of local recurrence. Five dogs are alive and free of disease 9 to nineteen months from the initiation of therapy. The 0–7–21 protocol was well-tolerated, and acute radiation reactions were low-grade and limited to the skin. The results of this study demonstrate that oral melanomas in dogs are responsive to radiation. 0–7–21 radiation therapy offers a viable alternative to radical excision, especially when tumor volume or location would require cosmetically or functionally debilitating surgery.     

Effect of Split Nitrogen Application on Growth and Yield of Wheat (T. aestivum L.) Genotypes with Different N-Assimilation Potential

A field experiment was conducted with different nitrogen regimes to assess the growth and yield performance of wheat genotypes which differ in nitrate assimilation potential. Genotypic differences in biomass accumulation were observed at different growth stages. The nitrogen treatment had little effect on biomass accumulation at early stages of growth, while at later stages of growth there was enhanced biomass accumulation when N was applied in more than two splits. On an average, genotypes with high nitrate reductase activity (the 'HNR' genotypes) accumulated 14.2 % more biomass than the genotypes with low nitrate reductase activity ('LNR' genotypes) when an extra dose (40 kg N ha⁻¹) of nitrogen was given at the time of anthesis. The application of nitrogen in more than two splits increased grain yield of both 'HNR' and 'LNR' genotypes mainly by increasing grain weight per ear. The application of an extra dose of nitrogen (40 kg N ha⁻¹) at the time of anthesis increased grain yield of 'HNR' genotypes by 38.5 % as compared to 'LNR' genotypes.     

Hyperlipemia and Ketonuria in an Alpaca and a Llama

An alpaca and a llama in late stages of gestation were evaluated for lethargy, anorexia, and recumbency. Both camelids had cloudy, white, turbid serum, elevated serum triglyceride (1564, 5658 mg/dL, respectively) and cholesterol (158, 297 mg/dL, respectively) concentrations, and ketonuria. Signs of fetal stress were evident ultrasonographically in the alpaca, and a live cria was delivered by Cesarean section performed under general anesthesia. The alpaca developed severe metabolic acidosis, hepatic lipidosis, and acute renal failure secondary to renal lipidosis and died 36 hours after admission despite medical therapy. Histopathology revealed renal and hepatic lipidosis and neutrophilic pancreatitis. The cria died 72 hours after birth. The llama responded to IV electrolyte, dextrose, and regular crystalline insulin therapy. The pregnancy was maintained, and the llama was discharged from the hospital 20 days after admission. Two months after discharge, the llama gave birth to a live, 5 kg cria. Findings of hypertriglyceridemia, hypercholesterolemia, elevated sorbitol dehydrogenase activity, metabolic acidosis, azotemia, and ketonuria occurred in these two camelids. Based on this report, camelids appear to be similar to both horses and cattle in their response to severe energy imbalances in late gestation.