Optimization of the suspension culture method for in vitro cultivation of Babesia bovis.

An evaluation of some system variables for the suspension-culture method of in vitro Babesia bovis cultivation has identified some variables as critical and suggested optimum values for others. Growth of Babesia was not affected by siliconizing the culture vessels or including HEPES buffer in the culture medium. THe culture pH, however, markedly influenced the growth rate. When supplemented with a large amount of serum, even Hanks's balanced salt solution was an adequate medium to support growth of B bovis, but growth improved with increasing medium complexity. A comparison of growth in the complex mammalian tissue culture mediums RPMI-1640, medium 199, and NCTC-135 showed no significant differences. Heparinized plasma (plasma with added heparin ) could not be substituted for serum. While serum could be stored by either freezing or refrigeration at 4 C for up to a week RBC rapidly lost their ability to support growth of B bovis when stored at 4 C. Preliminary experiments with the gaseous environment indicated that CO2 is required and that the optimum oxygen tension was near the normal atmospheric level of about 20%.

Continuous in vitro cultivation of Babesia bovis.

Babesia bovis was isolated from an experimentally infected calf (No. 1) and was maintained in vitro for 32 days by subculturing 14 times, using a total dilution of 192,000. A splenectomized calf was inoculated with subculture Babesia (isolate B). The agent (isolate C) was isolated and maintained in vitro for 17 days prior to inoculation of a third splenectomized calf. Babesia organisms (isolate D) were isolated and retained in vitro for 270 days. After 85 days in vitro, organisms from this third isolation (isolate D) were injected into a fourth splenectomized calf to test for infectivity. Responses of the three calves to cultural inoculation were similar to those responses that occurred from inoculation of infected bovine blood (calf 1). There was no observed change in the microscopic appearance of the Babesia in vitro with time.

Growth of Babesia bovis in bovine erythrocyte cultures.

Babesia bovis was cultured in a suspension of bovine erythrocytes incubated at 37 degrees C in Medium 199 with 50% bovine serum. The cells in culture were kept in suspension by slow stirring in spinner flasks and the medium was replaced at 24-hour intervals. Persistent multiplication of the parasite in a short series of subcultures suggests the feasibility of this approach for continuous culture.

Fluorescein diacetate staining of Anaplasma marginale as a possible measure of viability.

Fluorescein diacetate viability-staining technique was applied to Anaplasma marginale. Marginal bodies took up the stain and became fluorescent. When infected red blood cells were incubated at 55 C, the number of fluorescent Anaplasma decreased at a rate similar to the known loss of viability.

Changes in serum concentration of phagocytosis-stimulating factor in experimentally induced bovine anaplasmosis: preliminary findings.

To determine whether correlation exists between serum concentration of phagocytosis-stimulating factor (PSF) and in vivo phagocytic activity, 2 splenectomized steers were inoculated with Anaplasma marginale, and their serum PSF concentrations were monitored. At the time of the anemic crisis, serum PSF concentrations were elevated five to tenfold.