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Background: Theileria orientalis is a tick-borne hemoparasite that causes anemia, ill thrift, and death in cattle globally. The Ikeda strain of T. orientalis is more virulent than other strains, leading to severe clinical signs and death of up to 5% of affected animals. Within the Asia-Pacific region, where it affects 25% of Australian cattle, T. orientalis Ikeda has a significant economic impact on the cattle industry. In 2017, T. orientalis Ikeda was detected in a cattle herd in Albermarle County, Virginia, United States. Months earlier, the U.S. was alerted to the invasion of the Asian longhorned tick, Haemaphysalis longicornis, throughout the eastern U.S. Abundant H. longicornis ticks were identified on cattle in the T. orientalis-affected herd in VA, and a subset of ticks from the environment were PCR-positive for T. orientalis Ikeda. A strain of T. orientalis from a previous U.S. outbreak was not transmissible by H. longicornis; however, H. longicornis is the primary tick vector of T. orientalis Ikeda in other regions of the world. Thus, the objective of this study was to determine whether invasive H. longicornis ticks in the U.S. are competent vectors of T. orientalis Ikeda., Methods: Nymphal H. longicornis ticks were fed on a splenectomized calf infected with the VA-U.S.-T. orientalis Ikeda strain. After molting, a subset of adult ticks from this cohort were dissected, and salivary glands assayed for T. orientalis Ikeda via qPCR. The remaining adult ticks from the group were allowed to feed on three calves. Calves were subsequently monitored for T. orientalis Ikeda infection via blood smear cytology and PCR., Results: After acquisition feeding on a VA-U.S.-T. orientalis Ikeda-infected calf as nymphs, a subset of molted adult tick salivary glands tested positive by qPCR for T. orientalis Ikeda. Adult ticks from the same cohort successfully transmitted T. orientalis Ikeda to 3/3 naïve calves, each of which developed parasitemia reaching 0.4-0.9%., Conclusions: Our findings demonstrate that U.S. H. longicornis ticks are competent vectors of the VA-U.S.-T. orientalis Ikeda strain. This data provides important information for the U.S. cattle industry regarding the potential spread of this parasite and the necessity of enhanced surveillance and control measures.

A novel apicomplexan parasite was serendipitously discovered in horses at the United States - Mexico border. Phylogenetic analysis based on 18S rDNA showed the erythrocyte-infective parasite to be related to, but distinct from, Theileria spp. in Africa, the most similar taxa being Theileria spp. from waterbuck and mountain zebra. The degree of sequence variability observed at the 18S rDNA locus also suggests the likely existence of additional cryptic species. Among described species, the genome of this novel equid Theileria parasite is most similar to that of Theileria equi, also a pathogen of horses. The estimated divergence time between the new Theileria sp. and T. equi, based on genomic sequence data, is greater than 33 million years. Average protein sequence divergence between them, at 23%, is greater than that of Theileria parva and Theileria annulata proteins, which is 18%. The latter two represent highly virulent Theileria spp. of domestic cattle, as well as of African and Asian wild buffalo, respectively, which differ markedly in pathology, host cell tropism, tick vector and geographical distribution. The extent of genome-wide sequence divergence, as well as significant morphological differences, relative to T. equi justify the classification of Theileria sp. as a new taxon. Despite the overall genomic divergence, the nine member equi merozoite antigen (EMA) superfamily, previously found as a multigene family only in T. equi, is also present in the novel parasite. Practically, significant sequence divergence in antigenic loci resulted in this undescribed Theileria sp. not being detectable using currently available diagnostic tests. Discovery of this novel species infective to equids highlights exceptional diversity within the genus Theileria, a finding with serious implications for apicomplexan parasite surveillance., (Published by Elsevier Ltd.)

Background: The apicomplexan hemoprotozoan parasite Theileria equi is one of the etiologic agents causing equine piroplasmosis, a disease of equines that is endemic throughout large parts of the world. Before 2009 the United States had been considered to be free of this parasite. Occasional cases had occurred but there was no evidence for endemic vector-borne transmission in the U.S. until a 2009 outbreak in Texas in which Dermacentor variabilis and Amblyomma cajennense were implicated as vectors. Although D. variabilis has previously been shown to be a competent laboratory vector, studies suggested A. cajennense was not a competent transstadial vector, even though the presence of this tick species on horses in South American is epidemiologicaly correlated with higher a prevalence of infection. In this study we tested the transstadial and intrastadial vector competence of D. variabilis and A. cajennense for T. equi., Methods: A tick passaged T. equi strain from the Texas outbreak and ticks colonized from engorged females collected off horses on the outbreak ranch in Texas were used for these studies. Nymph or adult ticks were fed on infected horses and transmission fed on naïve horses. Infections were tracked with PCR and serology, dissected tick tissues were tested with PCR., Results: A. cajennense transmitted T. equi intrastadially when adult ticks acquired infection by feeding on an infected horse, and transmitted to a naïve host on subsequent reattachment and feeding. D. variabilis failed to transmit in the same experiment. Transstadial transmission was not successful for either tick species. PCR on DNA isolated from eggs of females that had fed on an infected horse suggests that there is no transovarial passage of this parasite by either tick species., Conclusion: This work confirms that ticks from the Texas population of A. cajennense are competent intrastadial vectors of T. equi. We propose that the most likely natural mode of transmission for this parasite/vector combination in the Texas outbreak would have been biological transmission resulting from adult male ticks moving between infected and uninfected horses. The intrastadial mode of transmission should be considered as one equally possible scenario whenever implicating vectors of T. equi.

Theileria equi has a biphasic life cycle in horses, with a period of intraleukocyte development followed by patent erythrocytic parasitemia that causes acute and sometimes fatal hemolytic disease. Unlike Theileria spp. that infect cattle (Theileria parva and Theileria annulata), the intraleukocyte stage (schizont) of Theileria equi does not cause uncontrolled host cell proliferation or other significant pathology. Nevertheless, schizont-infected leukocytes are of interest because of their potential to alter host cell function and because immune responses directed against this stage could halt infection and prevent disease. Based on cellular morphology, Theileria equi has been reported to infect lymphocytes in vivo and in vitro, but the specific phenotype of schizont-infected cells has yet to be defined. To resolve this knowledge gap in Theileria equi pathogenesis, peripheral blood mononuclear cells were infected in vitro and the phenotype of infected cells determined using flow cytometry and immunofluorescence microscopy. These experiments demonstrated that the host cell range of Theileria equi was broader than initially reported and included B lymphocytes, T lymphocytes and monocyte/macrophages. To determine if B and T lymphocytes were required to establish infection in vivo, horses affected with severe combined immunodeficiency (SCID), which lack functional B and T lymphocytes, were inoculated with Theileria equi sporozoites. SCID horses developed patent erythrocytic parasitemia, indicating that B and T lymphocytes are not necessary to complete the Theileria equi life cycle in vivo. These findings suggest that the factors mediating Theileria equi leukocyte invasion and intracytoplasmic differentiation are common to several leukocyte subsets and are less restricted than for Theileria annulata and Theileria parva. These data will greatly facilitate future investigation into the relationships between Theileria equi leukocyte tropism and pathogenesis, breed susceptibility, and strain virulence.

Background: Theileria equi is a tick-borne apicomplexan hemoparasite that causes equine piroplasmosis. This parasite has a worldwide distribution but the United States was considered to be free of this disease until recently., Methods: We used samples from 37 horses to determine genetic relationships among North American T. equi using the 18S rRNA gene and microsatellites. We developed a DNA fingerprinting panel of 18 microsatellite markers using the first complete genome sequence of T. equi., Results: A maximum parsimony analysis of 18S rRNA sequences grouped the samples into two major clades. The first clade (n = 36) revealed a high degree of nucleotide similarity in U.S. T. equi, with just 0-2 single nucleotide polymorphisms (SNPs) among samples. The remaining sample fell into a second clade that was genetically divergent (48 SNPs) from the other U.S. samples. This sample was collected at the Texas border, but may have originated in Mexico. We genotyped T. equi from the U.S. using microsatellite markers and found a moderate amount of genetic diversity (2-8 alleles per locus). The field samples were mostly from a 2009 Texas outbreak (n = 22) although samples from five other states were also included in this study. Using Weir and Cockerham's FST estimator (θ) we found strong population differentiation of the Texas and Georgia subpopulations (θ = 0.414), which was supported by a neighbor-joining tree created with predominant single haplotypes. Single-clone infections were found in 27 of the 37 samples (73%), allowing us to identify 15 unique genotypes., Conclusions: The placement of most T. equi into one monophyletic clade by 18S is suggestive of a limited source of introduction into the U.S. When applied to a broader cross section of worldwide samples, these molecular tools should improve source tracking of T. equi outbreaks and may help prevent the spread of this tick-borne parasite.

Theileria orientalis Ikeda, an emerging U.S. bovine hemoparasite, causes anemia, abortion, ill-thrift, and occasionally death. While Haemaphysalis longicornis is the primary vector, it is possible that other U.S. ticks are capable of parasite transmission and may contribute to disease spread. Dermacentor variabilis is highly prevalent in the U.S., exhibits a similar geographical distribution to T. orientalis, and is a competent vector of the related parasite, Theileria equi. Herein, we conducted controlled acquisition and transmission studies using splenectomized calves to assess whether D. variabilis can transstadially transmit T. orientalis. D. variabilis nymphs were applied to an infected, splenectomized calf for parasite acquisition and subsequently incubated to molt into adults. Freshly molted adults were applied to two splenectomized T. orientalis-naïve calves to investigate parasite transmission. Calves were monitored for 59 days, and no evidence of parasite transmission was detected using PCR for the T. orientalis Ikeda major piroplasm surface protein gene, blood smear cytology, complete blood counts, or physical examination. Salivary glands from a subset of D. variabilis adults were assessed for T. orientalis using PCR, and the parasite was not detected. These findings support the conclusion that D. variabilis is not capable of transstadial transmission of the U.S. T. orientalis Ikeda isolate. [ABSTRACT FROM AUTHOR]

Theileria orientalis Ikeda has caused an epidemic of bovine anemia and abortion across several U.S. states. This apicomplexan hemoparasite is transmitted by Haemaphysalis longicornis ticks; however, it is unknown if other North American ticks are competent vectors. Since the disease movement is largely determined by the host tick range(s), the prediction of the T. orientalis spread among U.S. cattle populations requires determination of additional competent tick vectors. Although Rhipicephalus microplus has mostly been eradicated from the U.S., outbreaks in populations occur frequently, and the U.S. remains at risk for reintroduction. Since R. microplus is a vector of Theileria equi and T. orientalis DNA has been detected in R. microplus, the goal of this study was to determine whether R. microplus is a competent vector of T. orientalis. Larval R. microplus were applied to a splenectomized, T. orientalis Ikeda-infected calf for parasite acquisition, removed as molted adults, and applied to two T. orientalis naïve, splenectomized calves for transmission. After 60 days, the naïve calves remained negative for T. orientalis by PCR and cytology. Additionally, T. orientalis was not detected in the salivary glands or larval progeny of acquisition-fed adults. These data suggest that R. microplus is not a competent vector of the U.S. T. orientalis Ikeda isolate. [ABSTRACT FROM AUTHOR]

Theileria equi has a biphasic life cycle in horses, with a period of intraleukocyte development followed by patent erythrocytic parasitemia that causes acute and sometimes fatal hemolytic disease. Unlike Theileria spp. that infect cattle (Theileria parva and Theileria annulata), the intraleukocyte stage (schizont) of Theileria equi does not cause uncontrolled host cell proliferation or other significant pathology. Nevertheless, schizont-infected leukocytes are of interest because of their potential to alter host cell function and because immune responses directed against this stage could halt infection and prevent disease. Based on cellular morphology, Theileria equi has been reported to infect lymphocytes in vivo and in vitro, but the specific phenotype of schizont-infected cells has yet to be defined. To resolve this knowledge gap in Theileria equi pathogenesis, peripheral blood mononuclear cells were infected in vitro and the phenotype of infected cells determined using flow cytometry and immunofluorescence microscopy. These experiments demonstrated that the host cell range of Theileria equi was broader than initially reported and included B lymphocytes, T lymphocytes and monocyte/macrophages. To determine if B and T lymphocytes were required to establish infection in vivo, horses affected with severe combined immunodeficiency (SCID), which lack functional B and T lymphocytes, were inoculated with Theileria equi sporozoites. SCID horses developed patent erythrocytic parasitemia, indicating that B and T lymphocytes are not necessary to complete the Theileria equi life cycle in vivo. These findings suggest that the factors mediating Theileria equi leukocyte invasion and intracytoplasmic differentiation are common to several leukocyte subsets and are less restricted than for Theileria annulata and Theileria parva. These data will greatly facilitate future investigation into the relationships between Theileria equi leukocyte tropism and pathogenesis, breed susceptibility, and strain virulence. [ABSTRACT FROM AUTHOR]

We report an outbreak of equine piroplasmosis in southern Texas, USA, in 2009. Infection prevalence reached 100% in some areas (292 infected horses). Amblyomma cajennense was the predominant tick and experimentally transmitted Theileria equi to an uninfected horse. We suggest that transmission by this tick species played a role in this outbreak. [ABSTRACT FROM AUTHOR]

Theileria equi has a biphasic life cycle in horses, with a period of intraleukocyte development followed by patent erythrocytic parasitemia that causes acute and sometimes fatal hemolytic disease. Unlike Theileria spp. that infect cattle (Theileria parva and Theileria annulata), the intraleukocyte stage (schizont) of Theileria equi does not cause uncontrolled host cell proliferation or other significant pathology. Nevertheless, schizont-infected leukocytes are of interest because of their potential to alter host cell function and because immune responses directed against this stage could halt infection and prevent disease. Based on cellular morphology, Theileria equi has been reported to infect lymphocytes in vivo and in vitro, but the specific phenotype of schizont-infected cells has yet to be defined. To resolve this knowledge gap in Theileria equi pathogenesis, peripheral blood mononuclear cells were infected in vitro and the phenotype of infected cells determined using flow cytometry and immunofluorescence microscopy. These experiments demonstrated that the host cell range of Theileria equi was broader than initially reported and included B lymphocytes, T lymphocytes and monocyte/macrophages. To determine if B and T lymphocytes were required to establish infection in vivo, horses affected with severe combined immunodeficiency (SCID), which lack functional B and T lymphocytes, were inoculated with Theileria equi sporozoites. SCID horses developed patent erythrocytic parasitemia, indicating that B and T lymphocytes are not necessary to complete the Theileria equi life cycle in vivo. These findings suggest that the factors mediating Theileria equi leukocyte invasion and intracytoplasmic differentiation are common to several leukocyte subsets and are less restricted than for Theileria annulata and Theileria parva. These data will greatly facilitate future investigation into the relationships between Theileria equi leukocyte tropism and pathogenesis, breed susceptibility, and strain virulence. [ABSTRACT FROM AUTHOR]