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: There have been ongoing efforts to identify anti-tick vaccine targets to protect cattle from infestation with cattle fever ticks Rhipicephalus (Boophilus) microplus. Two commercial vaccines based on the tick gut protein Bm86 have had variable effectiveness, which has led to poor acceptance, and numerous studies have attempted to identify vaccine antigens that will provide more consistently effective protection. Transcriptomic analysis of R. microplus led to identification of three aquaporin genes annotated to code for transmembrane proteins involved in the transport of water across cell membranes. Previous work showed that vaccination with full-length recombinant aquaporin 1 (RmAQP1) reduced tick burdens on cattle. Targeted silencing of aquaporin 2 (RmAQP2) expression suggested it might also be a good anti-tick vaccination target. Methods: Three synthetic peptides from the predicted extracellular domains of RmAQP2 were used to vaccinate cattle. Peptides were conjugated to keyhole limpet hemocyanin (KLH) as an antigenic carrier molecule. We monitored the antibody response with ELISA and challenged vaccinated cattle with R. microplus larvae. Results: There was a 25% reduction overall in the numbers of ticks feeding to repletion on the vaccinated cattle. Immune sera from vaccinated cattle recognized native tick proteins on a western blot and reacted to the three individual synthetic peptides in an ELISA. The vaccinated calf with the highest total IgG titer was not the most effective at controlling ticks; ratios of IgG isotypes 1 and 2 differed greatly among the three vaccinated cattle; the calf with the highest IgG1/IgG2 ratio had the fewest ticks. Ticks on vaccinated cattle had significantly greater replete weights compared to ticks on controls, mirroring results seen with RNA silencing of RmAQP2. However, protein data could not confirm that vaccination had any impact on the ability of the tick to concentrate its blood meal by removing water. Conclusions: A reduced number of ticks feed successfully on cattle vaccinated to produce antibodies against the extracellular domains of RmAQP2. However, our predicted mechanism, that antibody binding blocks the ability of RmAQP2 to move water out of the blood meal, could not be confirmed. Further study will be required to define the mechanism of action and to determine whether these vaccine targets will be useful components of an anti-tick vaccine cocktail. Graphical

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.

For most organisms, iron is an essential nutrient due to its role in fundamental cellular processes. Insufficient iron causes sub-optimal metabolism with potential effects on viability, while high levels of iron are toxic due to the formation of oxidative radicals, which damage cellular components. Many molecules and processes employed in iron uptake, storage, transport and metabolism are conserved, however significant knowledge gaps remain regarding these processes in ticks due to their unique physiology. In this study, we first identified and sequenced 13 genes likely to be involved in iron metabolism in Dermacentor andersoni cells. We then developed a method to reduce iron levels in D. andersoni cells using the iron chelator 2,2'-bipyridyl and measured the transcriptional response of these genes to iron reduction. The genes include a putative transferrin receptor, divalent metal transporter 1, duodenal cytochrome b, zinc/iron transporters zip7, zip13, zip14, mitoferrin, ferrochelatase, iron regulatory protein 1, ferritin1, ferritin2, transferrin and poly r(C)-binding protein. Overall, the transcriptional response of the target genes to iron reduction was modest. The most marked changes were a decrease in ferritin2, which transports iron through the tick hemolymph, the mitochondrial iron transporter mitoferrin, and the mitochondrial enzyme ferrochelatase. Iron regulatory protein1 was the only gene with an overall increase in transcript in response to reduced iron levels. This work lays the foundation for an improved understanding of iron metabolism in ticks which may provide molecular targets for the development of novel tick control methods and aid in the understanding of tick-pathogen interactions.

Background: Vector-borne diseases pose an increasing threat to global food security. Vaccines, diagnostic tests, and therapeutics are urgently needed for tick-borne diseases that affect livestock. However, the inability to obtain significant quantities of pathogen stages derived from ticks has hindered research. In vitro methods to isolate pathogens from infected tick vectors are paramount to advance transcriptomic, proteomic, and biochemical characterizations of tick-borne pathogens. Methods: Nymphs of Rhipicephalus appendiculatus were infected with Theileria parva by feeding on a calf during an acute infection. Isolation of sporozoites was accomplished by feeding infected adult ticks on an in vitro tick feeding system. Sporozoite viability was tested using in vitro bovine lymphocytes. Results: We isolated infectious T. parva sporozoites secreted into an in vitro tick feeding system. Infected adult R. appendiculatus ticks attached to and successfully fed on silicone membranes in the in vitro tick feeding system. Bovine blood in the receptacle was replaced with cell-free medium and the ticks were allowed to feed for 3 h to collect secreted T. parva sporozoites. Secreted sporozoites infected in vitro bovine lymphocytes, demonstrating that isolated sporozoites remained viable and infectious. Conclusions: This work is the first to report the isolation of mature infectious T. parva sporozoites using an in vitro tick feeding system, which represents a significant step towards the development of a more efficient control strategy for T. parva. Isolation of infectious tick-stage parasites will facilitate the examination of the vector-pathogen interface, thereby accelerating the development of next-generation vaccines and treatment interventions for tick-borne pathogens. Graphical