Individual heterogeneity in erythrocyte susceptibility to Babesia divergens is a critical factor for the outcome of experimental spleen-intact sheep infections

Abstract

Susceptibility of sheep erythrocytes to Babesia divergens was investigated in vitro and a high inter-individual variability in their ability to support parasite population development was demonstrated, with some individuals having refractory red blood cells (RBC). As neither changes in growth conditions nor the use of different B. divergens strains influenced the level of susceptibility, the main factor postulated for this variability is the erythrocyte itself. Sheep therefore represent an excellent in vitro model to study the parasite-erythrocyte interaction. In addition, the existence of refractory RBC should help in the identification of the erythrocyte components required for B. divergens development. Experimental infections were carried out on spleen-intact sheep characterized by refractory or fully susceptible erythrocyte types. These differences translated into the successful infection of only those animals with susceptible erythrocytes: infected animals showed no clinical signs, but maintained an asymptomatic persistent infection, as usually observed in the natural bovine host. Sheep therefore represent model organisms that can allow us to study interactions between B. divergens and its vertebrate host at different levels of biological organisation, from the target cell to the intact animal, and represent an experimental infection model of concomitant immunity. Only a low percentage (13%) of the sheep population tested possessed susceptible erythrocytes and the potential role of sheep as a natural host or reservoir of B. divergens is discussed.

Figure 1.

Variability in the susceptibility of erythrocytes prepared from 19 sheep. A₄₀₅ of the culture supernatants and counts of parasitemia were measured 49 and 66 h after culture inoculation with B. divergens clone 2802C D5 (2.10⁶ iRBC/mL) in 2 mL-well (RPMI 1640, sheep serum at 10%, 1.10⁹ RBC/mL) for each sheep. Each A₄₀₅ data point represents the average value of the triplicate culture pellets, and the error bars indicate the standard deviation. Parasitemia was calculated from the RBC obtained by mixing the triplicate culture pellets. In the insert, the regression analysis performed on the data from each method at 66 h post-inoculation is shown, as well as the regression coefficient. This experiment was also performed with the clone Rouen F5 with similar results (see Tab. I; Exp. 3).

Figure 2.

Influence of medium composition on the observed variability of erythrocyte susceptibility. B. divergens clone 2802C D5 was inoculated into media prepared with RBC of varying degrees of susceptibility and sera from different origins (5 different FCS – and 2 different sheep sera – SS) used at 10% (A) or at different concentrations (10, 20, 30 and 40%) (B and C). Each A₄₀₅ data point represents the average value of the triplicate wells, and the error bars indicate the standard deviation. This experiment was also conducted with the clone Rouen F5 with similar results.

Figure 3.

Influence of B. divergens clone on the observed variability of erythrocyte susceptibility. The susceptibility of the erythrocytes from 8 sheep to clones obtained from different B. divergens isolates was tested. Each A₄₀₅ data point represents the average value of the triplicate wells, and the error bar indicates the standard deviation. This experiment was performed three times: once with the inoculation of the clones at the same concentration and twice with inoculation concentrations adapted to the growth rate of each clone (only results from the last run are presented here). Similar results were obtained from these experiments.

Figure 4.

Evaluation of the prevalence of susceptible sheep in a random population. The susceptibility of the erythrocytes of 30 sheep (“Vendéen” breed) was tested in vitro with B. divergens clone Rouen F5. Each A₄₀₅ data point represents the average value of triplicate measures, and the error bar indicates the standard deviation. This experiment was performed twice with similar results.