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• Iatrogenic inoculation with T. haneyi results in asymptomatic, persistent infection. • Initial infection with T. equiTX is associated with a marked neutropenia. • T. haneyi is undetectable with currently accepted diagnostics. • Superinfection regardless of the initial infecting species can be established. Theileria equi infection, exotic to the United States has reemerged through intravenous (iatrogenic) and tick-borne transmission. Surveillance at the US-Mexico border identified a new species, Theileria haneyi , (T. haneyi EP) (EP = Eagle Pass, Texas) which warranted additional investigation due to inability to detect by PCR targeting of T. equi ema-1 and EMA-1-cELISA validated for T. equi. Infection dynamics of T. haneyi EP were evaluated, including ability to superinfect in the presence of T. equi -Texas (T. equi TX), the isolate responsible for the reemergence of T. equi in the U S. Experimental infection with T. equiTX or T. haneyi EP revealed minimal clinical disease however, T. equiTX infection led to significantly greater neutropenia. Comparison of time to antibody detection following inoculation revealed significantly greater time to detectable anti- T. haneyi EP antibody (26.67 days post-inoculation (DPI)) than T. equi TX (11.67 DPI). Regardless of initial infection with either T. equi TX or T. haneyi EP, superinfection was established. Comparative analysis of antibody responses from a splenectomized horse infected with T. haneyi EP to that of a spleen intact horse infected with T. equi FL revealed a different antibody binding profile to T. haneyi EP, T. equi TX and T. equi FL merozoite antigen and limited shared antigen/cross-reactive antibody(s). Affinity purified T. equi EMA-1 and EMA-2 from T. equi FL were shown as targets for horse antibodies against T. haneyi. Data presented here show (1) T. haneyi EP can superinfect in the presence of T. equi TX infection and co-persists for minimally 25 months, (2) intravenous challenge with T. haneyi is subclinical, and (3) limited cross-reactive antibody between T. haneyi EP and T. equi includes reactivity to EMA-1 and EMA-2. [ABSTRACT FROM AUTHOR]

Introduction Theileria equi , an etiologic agent of equine piroplasmosis, is a tick-transmitted hemoprotozoan of the phylum Apicomplexa. Recent outbreaks of piroplasmosis in the United States have renewed interest in safe and effective treatment options. Although imidocarb dipropionate (IMD) is the drug of choice for clearance of T. equi , adverse reactions and recently documented resistance support the need for alternative therapeutic strategies. The recently described bumped kinase inhibitors (BKIs) are a new class of compounds that could potentially be used as safe and effective alternatives to IMD. In an initial effort to evaluate this potential, herein we determined the T. equi growth inhibitory activity of 11 BKIs relative to that of IMD and the previously tested BKI 1294. Because some BKIs have known human ether-à-go-go related gene (hERG) channel activity, we also assessed the hERG activity of each compound with the goal to identify those with the highest potency against T. equi coupled with the lowest potential for cardiotoxicity. Results Six BKIs inhibited T. equi growth in vitro , including the previously evaluated BKI 1294 which was used as a positive control. All six compounds were significantly less potent (higher 50% effective concentration (EC 50 )) than IMD. Two of those compounds were more potent than BKI 1294 control but had similar hERG activity. Although the remaining three compounds had similar to lower potency than BKI 1294, hERG EC 50 was higher for three of them (BKI 1735, BKI 1369 and BKI 1318). Conclusions The BKI compounds evaluated in this study inhibited T. equi in vitro and had diverse hERG activity. Based on these considerations, three compounds would be suitable for further evaluation. While these results provide a foundation for future work, in vivo pharmacokinetic, pharmacodynamics, and safety studies are needed before BKI compounds can be recommended for clinical use in T. equi infected horses. [ABSTRACT FROM AUTHOR]

Abstract: The Babesia bovis and B. bigemina apicomplexan protozoa in conjunction with the rickettsia Anaplasma marginale are intraerythrocytic pathogens that are responsible for the most prevalent and costly tick borne diseases (TBD''s) of cattle worldwide. These organisms are historically associated as they can cause clinically related hemolytic diseases in cattle, are all transmitted by Rhiphicephallus (Boophilus) ticks, and share an uncanny ability to evade the immune systems of the vertebrate hosts, causing persistent disease. In addition, acute babesiosis and anaplasmosis can be prevented quite effectively by combining tick control and vaccination with living attenuated organisms. However these methods of control have numerous limitations and improved approaches are needed. Importantly, immunizations of cattle with inactivated experimental Babesia and Anaplasma vaccines can elicit variable degrees of protection, indicating the feasibility for the development of inactivated or subunit vaccines. A new research toolbox that includes full genome sequencing combined with the improved ability to genetically modify the organisms is enhancing our understanding of their biology. An emerging paradigm is the use of recently developed Babesia and Anaplasma transfection methods for functional gene characterizations and for vaccine development. Promising recently identified subunit vaccine candidates are also emerging, including babesial proteases, putative rhoptry, microneme, and sexual stage antigens, as well as subdominant, conserved, A. marginale outer membrane major surface proteins. However, significant knowledge gaps on the role of key parasite molecules involved in cell invasion, adhesion, asexual and sexual reproduction, tick transmission, and evasion of the immune system, remain. A better understanding of the biology of these organisms and the protective immune responses will positively contribute toward the goal of developing improved immunological and pharmacological interventions against these elusive pathogens that are responsible for the most devastating TBD''s of cattle. Importantly, the currently available research toolbox provides basic research instruments for helping close current knowledge gaps which will aid the design and production of effective vaccines and alternative pharmacological interventions. [Copyright &y& Elsevier]

Abstract: With the recently sequenced Babesia bovis genome, a large pool of genes with unknown function was identified. The ability to complement and knock-out both unknown and previously identified genes would be a valuable tool to better understand gene function in B. bovis parasites. This review describes recent advances in the development of transient and stable transfection systems for B. bovis. Transient transfection constructs were initially generated using the promoter and the 3′ region of the rap-1 genes of B. bovis controlling expression of luciferase as a reporter. Successful expression of luciferase in B. bovis parasites using this plasmid introduced by classic electroporation of B. bovis infected erythrocytes was followed by the identification and characterization of stronger promoters, such as the ef-1α promoter, using transient transfection techniques. Further refinement of the transient transfection technique included development of the ability to transfect free merozoites using nucleofection, an alternative method to electroporation that results in higher transfection yields and improved viability of transfected parasites. Availability of the transient transfection system was critical for the further development of a stable transfection technique using a plasmid designed to target integration of a gfp-bsd gene into the B. bovis ef-1α locus. Several parasite lines resistant to the anti-babesial drug blasticidin (bsd) and constitutively expressing the gfp-bsd gene were generated after transfection. Integration of the gfp-bsd cassette into the genome was demonstrated by Southern blot and sequence analysis. Taken together these experiments demonstrated the feasibility to introduce, integrate and express exogenous genes in B. bovis. The stable transfection protocol was reproducible and used to transfect at least two distinct B. bovis strains. Further development of these transfection systems will facilitate functional analysis of B. bovis genes and will improve our understanding of the biology of and immunological response to this parasite. [Copyright &y& Elsevier]

• This is the first RNA-seq data study of the SZ and UO stages of E. necatrix. • Our data reveal new features of the E. necatrix transcriptional landscape. • It provides resources for future studies on E. necatrix. • It offers insights into the mechanisms of parasite invasion of host cells. Eimeria necatrix is one of the most pathogenic chicken coccidia and causes avian coccidiosis, an enteric disease of major economic importance worldwide. Eimeria parasites have complex developmental life cycles, with an exogenous phase in the environment and an endogenous phase in the chicken intestine. Oocysts excreted by chickens rapidly undergo meiosis and cell division to form eight haploid sporozoites (SZ). SZ liberated from sporocysts in the chicken intestine migrate to their preferred site of development to initiate cellular invasion. To date, almost nothing is known about the proteins that mediate parasite invasion in E. necatrix. In order to discover genes with functions involved in cellular invasion, the transcriptome profiles of E. necatrix unsporulated oocysts (UO) and SZ were analyzed using a combination of third-generation single-molecule real-time sequencing (TGS) and second-generation sequencing (SGS) followed by qRT-PCR validation. Correction of TGS long reads by SGS short reads resulted in 34,932 (UO) and 23,040 (SZ) consensus isoforms. After subsequent assembly, a total of 4949 and 4254 genes were identified from UO and SZ libraries, respectively. A total of 8376 genes were identified as differentially expressed genes (DEGs) between SZ and UO. Compared to UO, 4057 genes were upregulated and 4319 genes were downregulated in SZ. Approximately 1399 and 1758 genes were defined as stage-specific genes in SZ and UO, respectively. Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that 2978 upregulated SZ genes were clustered into 29 GO terms, and 857 upregulated SZ genes were associated with 26 KEGG pathways. We also predicted a further 50 upregulated SZ genes and 73 upregulated UO genes encoding microneme proteins, apical membrane antigens, rhoptry neck proteins, rhoptry proteins, dense granule proteins, heat shock proteins, calcium-dependent protein kinases, cyclin-dependent kinases, cGMP-dependent protein kinase, and glycosylphosphatidylinositol-anchored surface antigens. Our data reveal new features of the E. necatrix transcriptional landscape and provide resources for the development of novel vaccine candidates against E. necatrix infection. [ABSTRACT FROM AUTHOR]

• C. felis probe-based ddPCR can detect cytauxzoonosis 1 day prior to clinical signs. • Provides absolute quantification of parasite load and requires small sample volume. • Allows for monitoring of parasite load over time and in response to therapy. • Probe-based design can be utilized in traditional real-time PCR systems. Cytauxzoonosis is a tick-borne disease of domestic cats with high mortality and narrow therapeutic window, particularly in the southcentral and southeastern United States. The causative agent is the apicomplexan protozoal parasite Cytauxzoon felis and is primarily transmitted by Amblyomma americanum, the lone star tick. Currently there is no vaccine available to prevent cytauxzoonosis and treatment is often ineffective if not initiated early enough in the course of disease. Early diagnosis and therapeutic intervention are therefore crucial for the survival of infected cats. Several methods are available for diagnosis of cytauxzoonosis, with PCR being the most sensitive. However, current PCR assays, which employ double-stranded DNA intercalating dyes to detect C. felis infection, have inherent limitations such as the potential for false positive detection of non-specific amplification products and inability to provide absolute quantification of parasite load. The objective of this study was to develop a probe-based droplet digital PCR (ddPCR) assay capable of detection and quantification of C. felis load over time and during treatment. The C. felis ddPCR assay was able to (i) reliably detect and quantify C. felis DNA in clinical blood samples from cats with acute cytauxzoonosis and (ii) monitor clinical parasite load in response to anti-protozoal treatment through absolute quantification of C. felis DNA over time. When tested on blood samples from cats with experimental C. felis infection, the assay was able to detect infection in cats as early as 24 h prior to the development of clinical signs. In addition, we demonstrate that this probe-based design can be utilized in traditional real-time PCR systems, with similar detection capabilities as compared to ddPCR. The C. felis probe-based ddPCR was also able to detect infection in samples with lower parasite loads when compared to existing nested PCR assays, although these results were not significant due to small sample size. To the author's knowledge, this is the first reported probe-based ddPCR assay to detect Cytauxzoon felis infection in domestic cats. [ABSTRACT FROM AUTHOR]