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.     

Vaccination of the brushtail possum (Trichosurus vulpecula) against Mycobacterium bovis infection with bacille Calmette-Guérin: the response to multiple doses.

In New Zealand, the brushtail possum (Trichosurus vulpecula) is the principal wildlife vector of bovine tuberculosis. Control of infected possum populations contributes to the control of tuberculosis in domestic livestock. Vaccination is potentially a complementary strategy to population control, but to be cost-effective, administration of the vaccine to possums would need to be from an appropriately designed automatic vaccinator. Possums themselves would activate the vaccinator so that it would deliver an aerosol spray of vaccine. There would be no direct way to prevent possums receiving multiple doses of vaccine. This study examined the effect on protective immunity of repeated vaccination. Captive possums were vaccinated with BCG strain pasteur 1173P2 either 12 times at weekly intervals, twice at 6-weekly intervals, or once. Vaccination was by a combination of intranasal aerosol and conjunctival instillation. Eight weeks after the last dose of vaccine, all possums were challenged intratracheally with Mycobacterium bovis strain 83/6235. Vaccination induced a significant immune response as measured by the lymphocyte proliferation assay (LPA). A significant level of protection, as measured by the response to challenge, developed in all the vaccinated possum groups, but protection was greatest in the group vaccinated 12 times. It was concluded that protection would be enhanced if vaccinations were repeated at short intervals (weekly), but no benefit or detriment resulted from revaccination after longer intervals (1-2 months).     

Norsalsolinol uptake into secretory vesicles via vesicular monoamine transporter and its secretion by membrane depolarization or purinoceptor stimulation in PC12 cells.

The intracellular dynamics of norsalsolinol, a neurotoxin candidate causing parkinsonism-like symptoms, in PC12 cells was studied. We found that dopamine and norsalsolinol are co-localized to secretory granule layer by sucrose density gradient in norsalsolinol-treated PC12 cells. The norsalsolinol was actively taken up into isolated secretory vesicle fraction from PC12 cells with a Km value of 41.5+/-6.8 microM. The uptake of 10 microM of norsalsolinol was sensitive to reserpine (1 microM), an inhibitor of vesicular dopamine transporter, and dopamine, an endogenous substrate, but insensitive to GBR-12909, an inhibitor of dopamine transporter on plasma membrane. In norsalsolinol-treated PC12 cells, exposure to high K+ or ATP resulted in simultaneous release of norsalsolinol and dopamine. Time course of a release of dopamine and that of norsalsolinol evoked by 50 mM KCl or 100 microM ATP corresponded to each other. These releases were dependent on the concentrations of secretagogues. These data suggest that norsalsolinol is taken up with dopamine into secretory vesicle via vesicular catecholamine transporter.     

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.