A Culture-Adapted Strain of Babesia bovis Has Reduced Subpopulation Complexity and Is Unable to Complete Its Natural Life Cycle in Ticks

Abstract

Babesia bovis natural field strains are composed of several geno-phenotypically distinct subpopulations. This feature, together with possible epigenetic modifications, may facilitate adaptation to variable environmental conditions. In this study we compare geno-phenotypical features among long-term (more than 12 years) (LTCP) and short-term cultured B. bovis parasites (STCP) derived from the B. bovis S74-T3Bo strain. LTCPs intraerythrocytic forms are smaller in size than STCPs and have faster in vitro growth rate. In contrast to its parental strain, the LTCP lack expression of the sexual stage specific 6cysA and 6cysB proteins and are unable to develop sexual forms upon in vitro sexual stage induction. Consistently, in contrast to its parental strain, LTCPs have reduced virulence and are not transmissible to cattle by vector competent Rhipicephalus microplus (R. microplus). Similar to previous comparisons among attenuated and virulent B. bovis strains, the LTCP line has decreased genomic diversity compared to the STCP line. Thus, LTCP may contribute to our understanding of adaptive mechanisms used by the parasites in response to environmental changes, protective immunity, virulence, and transmission by ticks. In addition, LTCPs may be considered as candidates for a non-tick transmissible vaccine against bovine babesiosis.

Keywords: Babesia bovis; attenuation; in vitro culture; sexual stages; tick; transmission.

Figure 1

Phenotypic differences among B. bovis LTCP and STCP. (A) morphological differences of LTCP vs STCP detected by light microscopy. Bar: 5 µm. (B) The average length and width were calculated as pixels from the images taken by light microscopy and is represented in the “y” axis. (*) Indicates significant differences in average parasite length and width between LTCP and STCP. (C) In vitro growth curve comparison of LTCP and STCP B. bovis strains. (*) Indicates significant differences in the growth rate in the 2nd and 3rd day of the experiment.

Figure 2

Comparative analysis of LTCP vs STCP ability to develop sexual forms in an in vitro sexual Xa-induction system. (A) Parasite morphologies detected by light microscopy upon in vitro Xa-induction of sexual forms. Non induced cultures were used as a control for both strains. Bar 5 µm. (B) Comparative RT-PCR analysis for the detection of transcripts of sexual stage maker genes, 6cys A and 6cys B between LTCP and STCP. Transcripts derived from the rap-1 gene were used as a positive control. RNA extracts were derived from (NIC) non-induced and (IC) induced in vitro cultures. M represents the sizes of molecular markers in base pairs. PC: Genomic positive control. (C) Western blot analysis for detection of 6cys A protein expression as a sexual stage marker using non-induced (NIC) and induced (IC) in vitro cultures of LTCP and STCP. The monoclonal antibody BABB75, reactive with B. bovis RAP-1, was used as a positive control. M represents the sizes of molecular markers in kiloDaltons (kD). The amplifications were performed without the addition of reverse transcriptase (-) and on cDNA generated from total RNA extracted from B. bovis infected RBCs (+).

Figure 3

(A) Illustration of the location of the gene BBOV_III10230 used for the detection of DNA polymorphisms, gene diversity, and phylogenetic analysis among different B. bovis strains. (B) Nucleotide sequence representations of PCR amplicons derived from STCP and LTCP. (C) Phylogenetic comparison between the STCP (2 distinct genes amplified by PCR: subP1 and subP2), LTCP, MO7 and EKO clonal lines. (D) The Table shows identity percent comparison for the amplified genomic area among the different investigated parasite strains.

Figure 4

Schematic illustration and clinical parameters derived from the tick transmission experiments. The upper panel illustrate the acquisition feeding phase of the experiment, which was performed using splenectomized animals. Changes in the PCV and rectal temperature are illustrated on the right side of the panel. The lower panel illustrate the transmission phase of the experiment, performed on spleen intact animals. Changes in the PCV and rectal temperature are illustrated on the right side of the panel. Calf C-1482 infected with STC parasites via infected larvae was euthanized due to acute clinical signs at day 14 post application of control B. bovis infected larvae.

Figure 5

Analysis of animal and tick parameters for B. bovis infections in the tick transmission experiments. Giemsa-stained slides of a representative hemolymph sample to detect kinetes collected from ticks feeding on either C-1457 or C-1458 animals using 43x power objective of the light microscope. The upper panel represent samples collected from ticks fed on animal C-1458 infected with S74-T3Bo STCP stabilate. The lower panel represent samples collected from ticks fed on animal C-1457 infected with LTCP strain “lack of kinetes”.