Equine piroplasmosis is a disease of Equidae, including horses, donkeys, mules, and zebras, caused by either of two protozoan parasites, Theileria equi or Babesia caballi. These parasites are biologically transmitted between hosts via tick vectors, and although they have inherent differences they are categorized together because they cause similar pathology and have similar morphologies, life cycles, and vector relationships. To complete their life cycle, these parasites must undergo a complex series of developmental events, including sexual-stage development in their tick vectors. Consequently, ticks are the definitive hosts as well as vectors for these parasites, and the vector relationship is restricted to a few competent tick species. Because the vector relationship is critical to the epidemiology of these parasites, we highlight current knowledge of the vector ecology of these tick-borne equine pathogens, emphasizing tick transmissibility and potential control strategies to prevent their spread.

Background: Transmission of arthropod-borne apicomplexan parasites that cause disease and result in death or persistent infection represents a major challenge to global human and animal health. First described in 1901 as Piroplasma equi, this re-emergent apicomplexan parasite was renamed Babesia equi and subsequently Theileria equi, reflecting an uncertain taxonomy. Understanding mechanisms by which apicomplexan parasites evade immune or chemotherapeutic elimination is required for development of effective vaccines or chemotherapeutics. The continued risk of transmission of T. equi from clinically silent, persistently infected equids impedes the goal of returning the U. S. to non-endemic status. Therefore comparative genomic analysis of T. equi was undertaken to: 1) identify genes contributing to immune evasion and persistence in equid hosts, 2) identify genes involved in PBMC infection biology and 3) define the phylogenetic position of T. equi relative to sequenced apicomplexan parasites. Results: The known immunodominant proteins, EMA1, 2 and 3 were discovered to belong to a ten member gene family with a mean amino acid identity, in pairwise comparisons, of 39%. Importantly, the amino acid diversity of EMAs is distributed throughout the length of the proteins. Eight of the EMA genes were simultaneously transcribed. As the agents that cause bovine theileriosis infect and transform host cell PBMCs, we confirmed that T. equi infects equine PBMCs, however, there is no evidence of host cell transformation. Indeed, a number of genes identified as potential manipulators of the host cell phenotype are absent from the T. equi genome. Comparative genomic analysis of T. equi revealed the phylogenetic positioning relative to seven apicomplexan parasites using deduced amino acid sequences from 150 genes placed it as a sister taxon to Theileria spp. Conclusions: The EMA family does not fit the paradigm for classical antigenic variation, and we propose a novel model describing the role of the EMA family in persistence. T. equi has lost the putative genes for host cell transformation, or the genes were acquired by T. parva and T. annulata after divergence from T. equi. Our analysis identified 50 genes that will be useful for definitive phylogenetic classification of T. equi and closely related organisms.

Tick-borne pathogens may be transmitted intrastadially and transstadially within a single vector generation as well as vertically between generations. Understanding the mode and relative efficiency of this transmission is required for infection control. In this study, we established that adult male Rhipicephalus microplus ticks efficiently acquire the protozoal pathogen Babesia equi during acute and persistent infections and transmit it intrastadially to naïve horses. Although the level of parasitemia during acquisition feeding affected the efficiency of the initial tick infection, infected ticks developed levels of ≥10 ⁴ organisms/pair of salivary glands independent of the level of parasitemia during acquisition feeding and successfully transmitted them, indicating that replication within the tick compensated for any initial differences in infectious dose and exceeded the threshold for transmission. During the development of B. equi parasites in the salivary gland granular acini, the parasites expressed levels of paralogous surface proteins significantly different from those expressed by intraerythrocytic parasites from the mammalian host. In contrast to the successful intrastadial transmission, adult female R. microplus ticks that fed on horses with high parasitemia passed the parasite vertically into the eggs with low efficiency, and the subsequent generation (larvae, nymphs, and adults) failed to transmit B. equi parasites to naïve horses. The data demonstrated that intrastadial but not transovarial transmission is an efficient mode for B. equi transmission and that persistently infected horses are an important reservoir for transmission. Consequently, R. microplus male ticks and persistently infected horses should be targeted for disease control.

Understanding the determinants of efficient tick-borne microbial transmission is needed to better predict the emergence of highly transmissible pathogen strains and disease outbreaks. Although the basic developmental cycle ofAnaplasmaandEhrlichiaspp. within the tick has been delineated, there are marked differences in the ability of specific strains to be efficiently tick transmitted. Using the highly transmissible St. Maries strain ofAnaplasma marginaleinDermacentor andersonias a positive control and two unrelated nontransmissible strains, we identified distinct barriers to efficient transmission within the tick. The Mississippi strain was unable to establish infection at the level of the midgut epithelium despite successful ingestion of infected blood following acquisition feeding on a bacteremic animal host. This inability to colonize the midgut epithelium prevented subsequent development within the salivary glands and transmission. In contrast,A. marginalesubsp.centralecolonized the midgut and then the salivary glands, replicating to a titer indistinguishable from that of the highly transmissible St. Maries strain and at least 100 times greater than that previously associated with successful transmission. Nonetheless,A. marginalesubsp.centralewas not transmitted, even when a large number of infected ticks was used for transmission feeding. These results establish that there are at least two specific barriers to efficient tick-borne transmission, the midgut and salivary glands, and highlight the complexity of the pathogen-tick interaction.

The protozoan parasite Babesia equi replicates within erythrocytes. During the acute phase of infection, B. equi can reach high levels of parasitemia, resulting in a hemolytic crisis. Horses that recover from the acute phase of the disease remain chronically infected. Subsequent transmission is dependent upon the ability of vector ticks to acquire B. equi and, following development and replication, establishment of B. equi in the salivary glands. Although restriction of the movement of chronically infected horses with B. equi is based on the presumption that ticks can acquire and transmit the parasite at low levels of long-term infection, parasitemia levels during the chronic phase of infection have never been quantified, nor has transmission been demonstrated. To address these epidemiologically significant questions, we established long-term B. equi infections (>1 year), measured parasitemia levels over time, and tested whether nymphal Boophilus microplus ticks could acquire and, after molting to the adult stage, transmit B. equi to naive horses. B. equi levels during the chronic phase of infection ranged from 10 ^3.3 to 10 ^6.0 /ml of blood, with fluctuation over time within individual horses. B. microplus ticks fed on chronically infected horses with mean parasite levels of 10 ^5.5 ± 10 ^0.48 /ml of blood acquired B. equi , with detection of B. equi in the salivary glands of 7 to 50% of fed ticks, a range encompassing the percentage of positive ticks that had been identically fed on a horse in the acute phase of infection with high parasitemia levels. Ticks that acquired B. equi from chronically infected horses, as well as those fed during the acute phase of infection, successfully transmitted the parasite to naive horses. The results unequivocally demonstrated that chronically infected horses with low-level parasitemia are competent mammalian reservoirs for tick transmission of B. equi .

Arthropod-borne apicomplexan pathogens that cause asymptomatic persistent infections present a significant challenge due to their life-long transmission potential. Although anti-microbials have been used to ameliorate acute disease in animals and humans, chemotherapeutic efficacy for apicomplexan pathogen elimination from a persistently infected host and removal of transmission risk is largely unconfirmed. The recent re-emergence of the apicomplexan Theileria equi in U.S. horses prompted testing whether imidocarb dipropionate was able to eliminate T. equi from naturally infected horses and remove transmission risk. Following imidocarb treatment, levels of T. equi declined from a mean of 10^4.9 organisms/ml of blood to undetectable by nested PCR in 24 of 25 naturally infected horses. Further, blood transfer from treated horses that became nested PCR negative failed to transmit to naïve splenectomized horses. Although these results were consistent with elimination of infection in 24 of 25 horses, T. equi-specific antibodies persisted in the majority of imidocarb treated horses. Imidocarb treatment was unsuccessful in one horse which remained infected as measured by nested PCR and retained the ability to infect a naïve recipient via intravenous blood transfer. However, a second round of treatment eliminated T. equi infection. These results support the utility of imidocarb chemotherapy for assistance in the control and eradication of this tick-borne pathogen. Successful imidocarb dipropionate treatment of persistently infected horses provides a tool to aid the global equine industry by removing transmission risk associated with infection and facilitating international movement of equids between endemic and non-endemic regions.