Theileria annae is a tick-transmitted small piroplasmid that infects dogs and foxes in North America and Europe. Due to disagreement on its placement in the Theileria or Babesia genera, several synonyms have been used for this parasite, including Babesia Spanish dog isolate, Babesia microti-like, Babesia (Theileria) annae, and Babesia cf. microti. Infections by this parasite cause anemia, thrombocytopenia, and azotemia in dogs but are mostly subclinical in red foxes (Vulpes vulpes). Furthermore, high infection rates have been detected among red fox populations in distant regions strongly suggesting that these canines act as the parasite’s natural host. This study aims to reassess and harmonize the phylogenetic placement and binomen of T. annae within the order Piroplasmida. Four molecular phylogenetic trees were constructed using a maximum likelihood algorithm based on DNA alignments of: (i) near-complete 18S rRNA gene sequences (n = 76 and n = 93), (ii) near-complete and incomplete 18S rRNA gene sequences (n = 92), and (iii) tubulin-beta gene sequences (n = 32) from B. microti and B. microti-related parasites including those detected in dogs and foxes. All phylogenetic trees demonstrate that T. annae and its synonyms are not Theileria parasites but are most closely related with B. microti. The phylogenetic tree based on the 18S rRNA gene forms two separate branches with high bootstrap value, of which one branch corresponds to Babesia species infecting rodents, humans, and macaques, while the other corresponds to species exclusively infecting carnivores. Within the carnivore group, T. annae and its synonyms from distant regions segregate into a single clade with a highly significant bootstrap value corroborating their separate species identity. Phylogenetic analysis clearly shows that T. annae and its synonyms do not pertain to Theileria and can be clearly defined as a separate species. Based on the facts that T. annae and its synonyms have not been shown to have a leukocyte stage, as expected in Theileria, do not infect humans and rodents as B. microti, and cluster phylogenetically as a separate species, this study proposes to name this parasite Babesia vulpes sp. nov., after its natural host, the red fox V. vulpes.

SUMMARY: Bovine babesiosis caused by the tick-transmitted haemoprotozoansBabesia bovis, Babesia bigeminaandBabesia divergenscommonly results in substantial cattle morbidity and mortality in vast world areas. Although existing live vaccines confer protection, they have considerable disadvantages. Therefore, particularly in countries where large numbers of cattle are at risk, important research is directed towards improved vaccination strategies. Here a comprehensive overview of currently used live vaccines and of the status quo of experimental vaccine trials is presented. In addition, pertinent research fields potentially contributing to the development of novel non-live and/or live vaccines are discussed, including parasite antigens involved in host cell invasion and in pathogen-tick interactions, as well as the protective immunity against infection. The mining of available parasite genomes is continuously enlarging the array of potential vaccine candidates and, additionally, the recent development of a transfection tool forBabesiacan significantly contribute to vaccine design. However, the complication and high cost of vaccination trials hinderBabesiavaccine research, and have so far seriously limited the systematic examination of antigen candidates and prevented an in-depth testing of formulations using different immunomodulators and antigen delivery systems.

The tick-transmitted hemoparasiteBabesia boviscauses an acute infection that results in persistence and immunity against challenge infection in cattle that control the initial parasitemia. Resolution of acute infection with this protozoal pathogen is believed to be dependent on products of activated macrophages (Mφ), including inflammatory cytokines and nitric oxide (NO) and its derivatives.B. bovisstimulates inducible nitric oxide synthase (iNOS) and production of NO in bovine Mφ, and chemical donors of NO inhibit the growth ofB. bovisin vitro. However, the induction of inflammatory cytokines in Mφ by babesial parasites has not been described, and the antiparasitic activity of NO produced byB. bovis-stimulated Mφ has not been definitively demonstrated. We report that monocyte-derived Mφ activated byB. bovisexpressed enhanced levels of inflammatory cytokines interleukin-1β (IL-1β), IL-12, and tumor necrosis factor alpha that are important for stimulating innate and acquired immunity against protozoal pathogens. Furthermore, a lipid fraction ofB. bovis-infected erythrocytes stimulated iNOS expression and NO production by Mφ. Cocultures of Mφ andB. bovis-infected erythrocytes either in contact or physically separated resulted in reduced parasite viability. However, NO produced by bovine Mφ in response toB. bovis-infected erythrocytes was only partially responsible for parasite growth inhibition, suggesting that additional factors contribute to the inhibition ofB. bovisreplication. These findings demonstrate thatB. bovisinduces an innate immune response that is capable of controlling parasite replication and that could potentially result in host survival and parasite persistence.

Members of the variable merozoite surface antigen (vmsa) gene family ofBabesia bovisencode membrane proteins involved in erythrocyte invasion. In this study, we have identified and sequenced the complete 8.3-kb genomic locus containingmsa-2, a member of thevmsafamily, in the biologically cloned Mexico Mo7 strain. Four tandemly arranged copies ofmsa-2-related genes were found in the locus. The four genes, designatedmsa-2a₁(which corresponds to the originally describedmsa-2gene),msa-2a₂,msa-2b, andmsa-2c, were shown to be transcribed and expressed and encode proteins with open reading frames ranging in size from 266 (MSA-2c) to 317 (MSA-2a₁) amino acids. MSA-2a₁and -2a₂are the most closely related of the four proteins (90% identity), differing by (i) the number of 24-amino-acid repeats that comprise a surface-exposed B-cell epitope and (ii) the presence of a 32-amino-acid area of recombination between MSA-2a₂and -2b. In contrast,msa-2cis most closely related to the previously describedbabr 0.8gene in Australia strains ofB. bovis. Comparison of MSA-2 proteins in the Argentina R1A strain ofB. boviswith the Mexico Mo7 clone revealed a relatively high degree of conservation (83.6, 69.4, 79.1, and 88.7% amino acid identity for MSA-2a₁, -2a₂, -2b, and -2c, respectively), in contrast to the extensive MSA-1 sequence variation (52% identity) between the same two strains. Postinfection bovine immune serum contains antibodies that bound to each of the recombinant MSA-2 proteins. Blocking assays demonstrated the presence of unique B-cell epitopes in MSA-2a₁, -2b, and -2c. The results support the evolution of themsa-2locus through at least two gene duplications, with selection for multiple related but antigenically distinct merozoite surface proteins.

The Babesia bovis merozoite surface antigen 1 (MSA-1), a member of the variable merozoite surface antigen (VMSA) family, is an immunodominant glycoprotein which elicits antibodies that inhibit erythrocyte invasion. While antigenic polymorphism is a general feature of vmsa genes, the molecular basis and extent of msa-1 sequence polymorphism have not been well characterized. In this study we defined the msa-1 locus in the biologically cloned Mexico Mo7 strain of B. bovis and identified the sequence differences between MSA-1 antigenically dissimilar strains. We then determined whether sequences conserved between distinct msa-1 alleles would induce cross-reactive CD4 ⁺ T lymphocytes or inhibitory antibodies. The msa-1 locus in Mo7 contains a single msa-1 gene flanked by transcribed genes with no sequence homology to members of the VMSA gene family. Argentina B. bovis strains R1A and S2P have msa-1 genes with amino acid sequences that are 98.8% identical to each other, and antibodies against S2P MSA-1 cross-react with native R1A MSA-1. In contrast, identity between the Argentina and Mexico Mo7 msa-1 alleles is only 52%, with no continuous stretch of identity longer than 16 amino acids. Despite limited sequence conservation, antibodies against R1A MSA-1 were able to inhibit invasion of erythrocytes by Mo7 merozoites. The results indicate that inhibition-sensitive epitopes are conserved despite significant sequence divergence between Mexico and Argentina strain alleles and support a conserved functional role for polymorphic MSA-1 in erythrocyte invasion.