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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. [ABSTRACT FROM AUTHOR]

Coxiella burnetii, a highly adapted obligate intracellular bacterial pathogen and the cause of the zoonosis Q fever, is a reemerging public health threat. C. burnetii employs a Type IV secretion system (T4SS) to establish and maintain its intracellular niche and modulate host immune responses including the inhibition of apoptosis. Interactions between C. burnetii and caspase-1-mediated inflammasomes are not fully elucidated. This study confirms that C. burnetii does not activate caspase-1 during infection of mouse macrophages in vitro. C. burnetii –infected cells did not develop NLRP3 and ASC foci indicating its ability to avoid cytosolic detection. C. burnetii is unable to inhibit the pyroptosis and IL-1β secretion that is induced by potent inflammasome stimuli but rather enhances these caspase-1-mediated effects. We found that C. burnetii upregulates pro-IL-1β and robustly primes NLRP3 inflammasomes via TLR2 and MyD88 signaling. As for wildtype C. burnetii, T4SS-deficient mutants primed and potentiated NLRP3 inflammasomes. An in vivo model of pulmonary infection in C57BL/6 mice was developed. Mice deficient in NLRP3 or caspase-1 were like wildtype mice in the development and resolution of splenomegaly due to red pulp hyperplasia, and histologic lesions and macrophage kinetics, but had slightly higher pulmonary bacterial burdens at the greatest measured time point. Together these findings indicate that C. burnetii primes but avoids cytosolic detection by NLRP3 inflammasomes, which are not required for the clinical resistance of C57BL/6 mice. Determining mechanisms employed by C. burnetii to avoid cytosolic detection via NLRP3 inflammasomes will be beneficial to the development of preventative and interventional therapies for Q fever. [ABSTRACT FROM AUTHOR]

The apicomplexan hemoparasite, Theileria parva, causes East Coast fever (ECF), a frequently fatal disease of African cattle. Vaccine development has been impeded by incomplete understanding of protective immunity following natural exposure or the infection and treatment method (ITM) of immunization. This is attributable to a paucity of methods to characterize the memory T-cell repertoire following infection. To overcome this impediment, assays developed to study the immune response to other intracellular pathogens were adapted for use in studies with T. parva to enable definition of the phenotype and function of effector T cells in T. parva-immune cattle, facilitating vaccine development. As reported herein, stimulation of peripheral blood mononuclear cells (PBMC) from ITM-immunized steers with irradiated, autologous, T. parva-infected cell lines elicited a proliferative recall response comprised of CD45R0+/CCR7− CD4+ and CD8+ T cells. Subsequent co-incubation of stimulated cultures with infected cells demonstrated the presence of cytotoxic T cells (CTLs) with the ability to kill infected cells. Comparison of CTL activity in cultures depleted of CD4+ or CD8+ T cells demonstrated CTL activity was primarily attributed to CD8+ T cells. Importantly, stimulation of PBMC from vaccinated steers always elicited proliferation of CD4+ and CD8+ T cells. This was the first important observation obtained from the use of the assay described herein. [ABSTRACT FROM AUTHOR]

Theileria equi is a widely distributed apicomplexan parasite that causes severe hemolytic anemia in equid species. There is currently no effective vaccine for control of the parasite and understanding the mechanism that T. equi utilizes to invade host cells may be crucial for vaccine development. Unlike most apicomplexan species studied to date, the role of micronemes in T. equi invasion of host cells is unknown. We therefore assessed the role of the T. equi claudin-like apicomplexan microneme protein (CLAMP) in the invasion of equine erythrocytes as a first step towards understanding the role of this organelle in the parasite. Our findings show that CLAMP is expressed in the merozoite and intra-erythrocytic developmental stages of T. equi and in vitro neutralization experiments suggest that the protein is involved in erythrocyte invasion. Proteomic analyses indicate that CLAMP interacts with the equine erythrocyte α-and β- spectrin chains in the initial stages of T. equi invasion and maintains these interactions while also associating with the anion-exchange protein, tropomyosin 3, band 4.1 and cytoplasmic actin 1 after invasion. Additionally, serological analyses show that T. equi-infected horses mount robust antibody responses against CLAMP indicating that the protein is immunogenic and therefore represents a potential vaccine candidate. [ABSTRACT FROM AUTHOR]

Theileria parva is an economically important, intracellular, tick-transmitted parasite of cattle. A live vaccine against the parasite is effective against challenge from cattle-transmissible T. parva but not against genotypes originating from the African Cape buffalo, a major wildlife reservoir, prompting the need to characterize genome-wide variation within and between cattle- and buffalo-associated T. parva populations. Here, we describe a capture-based target enrichment approach that enables, for the first time, de novo assembly of nearly complete T. parva genomes derived from infected host cell lines. This approach has exceptionally high specificity and sensitivity and is successful for both cattle- and buffalo-derived T. parva parasites. De novo genome assemblies generated for cattle genotypes differ from the reference by ~54K single nucleotide polymorphisms (SNPs) throughout the 8.31 Mb genome, an average of 6.5 SNPs/kb. We report the first buffalo-derived T. parva genome, which is ~20 kb larger than the genome from the reference, cattle-derived, Muguga strain, and contains 25 new potential genes. The average non-synonymous nucleotide diversity (πN) per gene, between buffalo-derived T. parva and the Muguga strain, was 1.3%. This remarkably high level of genetic divergence is supported by an average Wright's fixation index (FST), genome-wide, of 0.44, reflecting a degree of genetic differentiation between cattle- and buffalo-derived T. parva parasites more commonly seen between, rather than within, species. These findings present clear implications for vaccine development, further demonstrated by the ability to assemble nearly all known antigens in the buffalo-derived strain, which will be critical in design of next generation vaccines. The DNA capture approach used provides a clear advantage in specificity over alternative T. parva DNA enrichment methods used previously, such as those that utilize schizont purification, is less labor intensive, and enables in-depth comparative genomics in this apicomplexan parasite. Author summary: An estimated 50 million cattle in sub-Saharan Africa are at risk of the deadly livestock disease East coast fever (ECF), caused by the parasite Theileria parva, which imposes tremendous economic hardship on smallholder farmers. An existing ECF vaccine protects against strains circulating among cattle, but not against T. parva derived from African Cape buffalo, its main wildlife carrier. Understanding this difference in protective efficacy requires characterization of the genetic diversity in T. parva strains associated with each mammalian host, a goal that has been hindered by the proliferation of T. parva in nucleated host cells, with much larger genomes. Here we adapted a sequence capture approach to target the whole parasite genome, enabling enrichment of parasite DNA over that of the host. Choices in protocol development resulted in nearly 100% parasite genome specificity and sensitivity, making this approach the most successful yet to generate T. parva genome sequence data in a high-throughput manner. The analyses uncovered a degree of genetic differentiation between cattle- and buffalo-derived genotypes that is akin to levels more commonly seen between species. This approach, which will enable an in-depth T. parva population genomics study from cattle and buffalo in the endemic regions, can easily be adapted to other intracellular pathogens. [ABSTRACT FROM AUTHOR]

Background: The tick-borne intra-erythrocytic apicomplexan Babesia caballi is one of the etiological agents of equine babesiosis, an economically important disease of equids in most tropical and subtropical areas of the world. Discovering candidate antigens for improved diagnostic tools and vaccines remains needed for controlling equine babesiosis. This study describes the B. caballi sbp4 (Bcsbp4) gene and protein (BcSBP4) and analyzes its antigenicity in infected equids. Methods: BLAST searches of an uncurated B. caballi assembly genome using the B. bovis SBP4 as a query were carried out, followed by PCR amplification and sequencing of a newly identified BcSBP4. Characterization of this novel gene and protein was performed by bioinformatics analysis, western blots, immunofluorescence (IFA) and an in vitro neutralization test using anti SBP4 peptide antibodies. Antigenicity of recombinant BcSBP4 (rBcSBP4) was tested with sera from field animals (n = 18) using an indirect ELISA (iELISA). Results: Babesia caballi genome searches using B. bovis SBP4 as a query allowed identification of a novel gene termed Bcsbp4. The Bcsbp4 gene encodes for a protein of 30.58 kDa, which is fully conserved among B. caballi isolates from USA and Egypt. Bioinformatics analysis indicates that BcSBP4 contains a signal peptide and lacks additional transmembrane domains. Expression of BcSBP4 in blood stages of B. caballi was confirmed by western blot and IFA using antibodies against synthetic peptides representing putative B-cell epitopes of BcSBP4 predicted by in silico analysis. In vitro neutralization tests using anti-BcSBP4 peptide antibodies showed a marginal, but statistically significant inhibitory effect on the infectivity of B. caballi merozoites in horse red blood cells. Sera from eight B. caballi-infected equids, but none out of ten negative equid control sera, gave a positive signal in an rBcSBP4 based iELISA. Conclusions: The Bcsbp4 gene is expressed in B. caballi blood stages. The BcSBP4 protein is a potential candidate for developing a novel serological test that could detect B. caballi infection in equids in tropical and subtropical countries worldwide. [ABSTRACT FROM AUTHOR]

Equine theileriosis, a tick-transmitted disease caused by the hemoprotozoan parasites Theileria equi and Theileria haneyi, affects equids throughout tropical and subtropical regions of the world. It is a significant regulatory concern in non-endemic countries, where testing for equine theileriosis is required prior to horse import to prevent parasite entry. Within endemic areas, infection causes significant morbidity and mortality, leading to economic losses. No vaccine for equine theileriosis is available, and current drug treatment protocols are inconsistent and associated with significant side effects. Recent work has revealed substantial genetic variability among equine theileriosis organisms, and analysis of ribosomal DNA from affected animals around the world indicates that the organisms can be grouped into five distinct clades. As these diverse parasites are capable of infecting a wide range of both tick and mammalian hosts, movement of different equine Theileria species between endemic countries, and eventually into non-endemic countries, is a significant concern. Furthermore, the substantial genetic variability of these organisms will likely render currently utilized importation diagnostic tests unable to detect all equine Theileria spp. To this end, more complete characterization of these diverse parasites is critical to the continued global control of equine theileriosis. This review discusses current knowledge of equine Theileria spp. in this context, and highlights new opportunities and challenges for workers in this field. [ABSTRACT FROM AUTHOR]

The infection and treatment (ITM) live vaccination method for control of Theileria parva infection in cattle is increasingly being adopted, particularly in Maasai pastoralist systems. Several studies indicate positive impacts on human livelihoods. Importantly, the first detailed protocol for live vaccine production at scale has recently been published. However, quality control and delivery issues constrain vaccination sustainability and deployment. There is evidence that the distribution of T. parva is spreading from endemic areas in East Africa, North into Southern Sudan and West into Cameroon, probably as a result of anthropogenic movement of cattle. It has also recently been demonstrated that in Kenya, T. parva derived from cape buffalo can 'breakthrough' the immunity induced by ITM. However, in Tanzania, breakthrough has not been reported in areas where cattle co‐graze with buffalo. It has been confirmed that buffalo in northern Uganda national parks are not infected with T. parva and R. appendiculatus appears to be absent, raising issues regarding vector distribution. Recently, there have been multiple field population genetic studies using variable number tandem repeat (VNTR) sequences and sequencing of antigen genes encoding targets of CD8+ T‐cell responses. The VNTR markers generally reveal high levels of diversity. The antigen gene sequences present within the trivalent Muguga cocktail are relatively conserved among cattle transmissible T. parva populations. By contrast, greater genetic diversity is present in antigen genes from T. parva of buffalo origin. There is also evidence from several studies for transmission of components of stocks present within the Muguga cocktail, into field ticks and cattle following induction of a carrier state by immunization. In the short term, this may increase live vaccine effectiveness, through a more homogeneous challenge, but the long‐term consequences are unknown. [ABSTRACT FROM AUTHOR]

East Coast Fever (ECF), caused by the tick-borne apicomplexan parasite Theileria parva , is a leading cause of morbidity and mortality in cattle of sub-Saharan Africa. The infection and treatment method (ITM) is currently the only vaccine available to control T. parva. Although ITM elicits levels of protection, its widespread adoption is limited by costs, laborious production process, and antibiotic co-treatment requirement, necessitating the development of a more sustainable vaccine. To this end, efforts have been concentrated in the identification of new T. parva vaccine antigens and in the development of suitable platforms for antigen expression. In this study, we investigated the molecular and antigenic properties of T. parva antigen Tp9 expressed by mammalian cells. Data indicate that Tp9 contains a signal peptide that is weakly functional in mammalian cells. Thus, Tp9 secretion from mammalian cells increased 10-fold after the native signal peptide was replaced with the human tissue plasminogen activator signal peptide (tPA). Sera from all T. parva -immune cattle recognized this recombinant, secreted Tp9. Additionally, PBMC from ITM-immunized cattle produced significant (p < 0.05) amounts of IFNγ following ex vivo exposure to Tp9, but this response varied between cattle of different MHC class I and class II genotypes. In addition, depletion experiments demonstrated that IFNγ to Tp9 was primarily produced by CD4+ T cells. Molecular analysis demonstrated that Tp9 presents a signal peptide that is weakly functional in mammalian cells, suggesting that it remains within lymphocytes during infection. Tp9 secretion from mammalian cells was substantially increased when the tPA secretion signal sequence was substituted for the native secretion signal sequence. Using full-length, recombinant Tp9 secreted from mammalian cells, we demonstrated that T. parva -immune cattle develop both humoral and cellular immune responses to this antigen. Collectively, these results provide rationale for further evaluation of Tp9 as a component of a T. parva subunit vaccine. [ABSTRACT FROM AUTHOR]

Recombinant herpesvirus vaccine vectors offer distinct advantages in next-generation vaccine development, primarily due to the ability to establish persistent infections to provide sustainable antigen responses in the host. Recombinant bovine herpesvirus-4 (BoHV-4) has been previously shown to elicit protective immunity in model laboratory animal species against a variety of pathogens. For the first time, we describe the induction of antigen-specific immune responses to two delivered antigens in the host species after intranasal nebulization of recombinant BoHV-4 expressing the chimeric peptide containing the bovine viral diarrhea virus (BVDV) glycoprotein E2 and the bovine herpesvirus 1 (BoHV-1) glycoprotein D (BoHV-4-A-CMV-IgK-gE2gD-TM). In this study, four cattle were immunized via intranasal nebulization with the recombinant BoHV-4 construct. Two of the cattle were previously infected with wild-type BoHV-4, and both developed detectable serologic responses to BVDV and BoHV-1. All four immunized cattle developed detectable viral neutralizing antibody responses to BVDV, and one steer developed a transient viral neutralizing response to BoHV-1. Approximately one year after immunization, immunosuppressive doses of the glucocorticoid dexamethasone were administered intravenously to all four cattle. Within two weeks of immunosuppression, all animals developed viral neutralizing antibody responses to BoHV-1, and all animals maintained BVDV viral neutralizing capacity. Overall, nebulization of BoHV-4-A-CMV-IgK-gE2gD-TM persistently infects cattle, is capable of eliciting antigen-specific immunity following immunization, including in the presence of pre-existing BoHV-4 immunity, and recrudescence of the virus boosts the immune response to BoHV-4-vectored antigens. These results indicate that BoHV-4 is a viable and attractive vaccine delivery platform for use in cattle. [ABSTRACT FROM AUTHOR]

Gammaherpesviruses in the genus Macavirus establish clinically unapparent persistent infections in reservoir species. Transmission of some of these viruses, including alcelaphine herpesvirus 1 (AlHV-1) and ovine herpesvirus 2 (OvHV-2), to clinically susceptible species in the order Artiodactyla can result in malignant catarrhal fever (MCF), a usually fatal lymphoproliferative disease. Serology can be used to identify MCF virus (MCFV)-infected carrier animals. However, all current serological assays utilize AlHV-1 antigens, thus none is specific for OvHV-2. In situations where sheep and other MCFV carriers are present, such as in zoos and game farms, an OvHV-2-specific assay would determine if OvHV-2 is present in the population. In this study, a recombinant protein containing a truncated OvHV-2 Ov8 glycoprotein was expressed and evaluated as a suitable target antigen to specifically detect OvHV-2 infection using an enzyme linked immunosorbent assay (ELISA). A competitive inhibition (CI)-ELISA that detects an epitope conserved among all MCFVs was used to categorize, as positive or negative, sera from 205 domestic sheep. The Ov8 assay showed 100% diagnostic sensitivity, 98.97% diagnostic specificity, 99.07% positive predictive value, and 100% negative predictive value and very high agreement (kappa = 0.990 and 95% CI = 0.971–1.000) with the CI-ELISA. Sera from animals infected with MCFVs other than OvHV-2 did not cross-react with Ov8 (100% negative predictive value). These data support the use of the Ov8 ELISA as an OvHV-2-specific diagnostic assay. [ABSTRACT FROM AUTHOR]

Background: Exercise‐induced pulmonary hemorrhage (EIPH) refers to bleeding from the lungs in association with strenuous exercise. It has been documented in race horses but little information exists on EIPH in barrel racing horses. Hypothesis/Objectives: Our goals were to evaluate the presence of EIPH in barrel racing horses and estimate its prevalence in the Pacific Northwest. Animals: 149 barrel racing horses enrolled at events in WA (11), ID (3), and MT (33). Methods: Observational cross‐sectional study. Data collected included signalment, history of illness, respiratory disease, race division, and pre‐race medications. Endoscopy was performed and tracheobronchoscopic (TBE) EIPH score was assigned based on quantity of blood in the trachea (0 = no blood to 4 = abundance of blood within the trachea). After TBE, bronchoalveolar lavage (BAL) was performed. Erythrocyte (red blood cell, RBC) counts were obtained from bronchoalveolar lavage fluid (BALF). Statistical analysis included linear and logistic regression, Fisher's exact t test, and calculation of correlation coefficient. Significance was set at P < .05. Results: The prevalence of EIPH based on TBE EIPH score was 54%. When based on BALF RBC count >1,000 cells, EIPH prevalence was 66%. Race time did not significantly affect the presence of EIPH. A significant (P < .0001) positive linear relationship between the TBE and BAL erythrocyte count was identified, but its strength was poor (r2 = .15). Conclusions and Clinical Importance: EIPH occurs in over 50% of barrel racing horses in the Pacific Northwest. Precise determination of the impact of EIPH on health of barrel racers requires further study. [ABSTRACT FROM AUTHOR]

Coxiella burnetii is a globally distributed zoonotic bacterial pathogen that causes abortions in ruminant livestock. In humans, an influenza-like illness results with the potential for hospitalization, chronic infection, abortion, and fatal endocarditis. Ruminant livestock, particularly small ruminants, are hypothesized to be the primary transmission source to humans. A recent Netherlands outbreak from 2007–2010 traced to dairy goats resulted in over 4,100 human cases with estimated costs of more than 300 million euros. Smaller human Q fever outbreaks of small ruminant origin have occurred in the United States, and characterizing shedding is important to understand the risk of future outbreaks. In this study, we assessed bacterial shedding and seroprevalence in 100 sheep from an Idaho location associated with a 1984 human Q fever outbreak. We observed 5% seropositivity, which was not significantly different from the national average of 2.7% for the U.S. (P>0.05). Furthermore, C. burnetii was not detected by quantitative PCR from placentas, vaginal swabs, or fecal samples. Specifically, a three-target quantitative PCR of placenta identified 0.0% shedding (exact 95% confidence interval: 0.0%-2.9%). While presence of seropositive individuals demonstrates some historical C. burnetii exposure, the placental sample confidence interval suggests 2016 shedding events were rare or absent. The location maintained the flock with little or no depopulation in 1984 and without C. burnetii vaccination during or since 1984. It is not clear how a zero-shedding rate was achieved in these sheep beyond natural immunity, and more work is required to discover and assess possible factors that may contribute towards achieving zero-shedding status. We provide the first U.S. sheep placental C. burnetii shedding update in over 60 years and demonstrate potential for C. burnetii shedding to reach undetectable levels after an outbreak event even in the absence of targeted interventions, such as vaccination. [ABSTRACT FROM AUTHOR]

Delivery of various forms of recombinant Theileria parva sporozoite antigen (p67) has been shown to elicit antibody responses in cattle capable of providing protection against East Coast fever, the clinical disease caused by T. parva. Previous formulations of full-length and shorter recombinant versions of p67 derived from bacteria, insect, and mammalian cell systems are expressed in non-native and highly unstable forms. The stable expression of full-length recombinant p67 in mammalian cells has never been described and has remained especially elusive. In this study, p67 was expressed in human-derived cells as a full-length, membrane-linked protein and as a secreted form by omission of the putative transmembrane domain. The recombinant protein expressed in this system yielded primarily two products based on Western immunoblot analysis, including one at the expected size of 67 kDa, and one with a higher than expected molecular weight. Through treatment with PNGase F, our data indicate that the larger product of this mammalian cell-expressed recombinant p67 cannot be attributed to glycosylation. By increasing the denaturing conditions, we determined that the larger sized mammalian cell-expressed recombinant p67 product is likely a dimeric aggregate of the protein. Both forms of this recombinant p67 reacted with a monoclonal antibody to the p67 molecule, which reacts with the native sporozoite. Additionally, through this work we developed multiple mammalian cell lines, including both human and bovine-derived cell lines, transduced by a lentiviral vector, that are constitutively able to express a stable, secreted form of p67 for use in immunization, diagnostics, or in vitro assays. The recombinant p67 developed in this system is immunogenic in goats and cattle based on ELISA and flow cytometric analysis. The development of a mammalian cell system that expresses full-length p67 in a stable form as described here is expected to optimize p67-based immunization. [ABSTRACT FROM AUTHOR]

The Rhipicephalus microplus tick is a notorious blood-feeding ectoparasite of livestock, especially cattle, responsible for massive losses in animal production. It is the main vector for transmission of pathogenic bacteria and parasites, including Babesia bovis, an intraerythrocytic apicomplexan protozoan parasite responsible for bovine Babesiosis. This study describes the development and testing of a live B. bovis vaccine expressing the protective tick antigen glutathione-S-transferase from Haemaphysalis longicornis (HlGST). The B. bovis S74-T3B parasites were electroporated with a plasmid containing the bidirectional Ef-1α (elongation factor 1 alpha) promoter of B. bovis controlling expression of two independent genes, the selectable marker GFP-BSD (green fluorescent protein–blasticidin deaminase), and HlGST fused to the MSA-1 (merozoite surface antigen 1) signal peptide from B. bovis. Electroporation followed by blasticidin selection resulted in the emergence of a mixed B. bovis transfected line (termed HlGST) in in vitro cultures, containing parasites with distinct patterns of insertion of both exogenous genes, either in or outside the Ef-1α locus. A B. bovis clonal line termed HlGST-Cln expressing intracellular GFP and HlGST in the surface of merozoites was then derived from the mixed parasite line HlGST using a fluorescent activated cell sorter. Two independent calf immunization trials were performed via intravenous inoculation of the HlGST-Cln and a previously described control consisting of an irrelevant transfected clonal line of B. bovis designated GFP-Cln. The control GFP-Cln line contains a copy of the GFP-BSD gene inserted into the Ef-1α locus of B. bovis in an identical fashion as the HIGST-Cln parasites. All animals inoculated with the HlGST-Cln and GFP-Cln transfected parasites developed mild babesiosis. Tick egg fertility and fully engorged female tick weight was reduced significantly in R. microplus feeding on HlGST-Cln-immunized calves. Collectively, these data show the efficacy of a transfected HlGST-Cln B. bovis parasite to induce detectable anti-glutathione-S-transferase antibodies and a reduction in tick size and fecundity of R. microplus feeding in experimentally inoculated animals. [ABSTRACT FROM AUTHOR]

Background: Ebola virus (EBOV) is a Category A pathogen that is a member of Filoviridae family that causes hemorrhagic fever in humans and non-human primates. Unpredictable and devastating outbreaks of disease have recently occurred in Africa and current immunoprophylaxis and therapies are limited. The main limitation of working with pathogens like EBOV is the need for costly containment. To potentiate further and wider opportunity for EBOV prophylactics and therapies development, innovative approaches are necessary. Methods: In the present study, an antigen delivery platform based on a recombinant bovine herpesvirus 4 (BoHV-4), delivering a synthetic EBOV glycoprotein (GP) gene sequence, BoHV-4-syEBOVgD106ΔTK, was generated. Results: EBOV GP was abundantly expressed by BoHV-4-syEBOVgD106ΔTK transduced cells without decreasing viral replication. BoHV-4-syEBOVgD106ΔTK immunized goats produced high titers of anti-EBOV GP antibodies and conferred a long lasting (up to 6 months), detectable antibody response. Furthermore, no evidence of BoHV-4- syEBOVgD106ΔTK viremia and secondary localization was detected in any of the immunized animals. Conclusions: The BoHV-4-based vector approach described here, represents: an alternative antigen delivery system for vaccination and a proof of principle study for anti-EBOV antibodies generation in goats for potential immunotherapy applications. [ABSTRACT FROM AUTHOR]

Background: Tick-borne Babesia bigemina is responsible for acute and potentially lethal hemolytic disease in cattle. The development of genetic manipulation tools necessary to the better understanding of parasite biology is currently limited by the lack of a complete parasite genome and experimental tools such as transfection. Effective promoters, required to regulate expression of transgenes, such as the elongation factor-1 alpha (ef-1α), have been identified in other apicomplexans such as Babesia bovis and Plasmodium falciparum. Methods: The B. bigemina ef-1a locus was defined by searching a partial genome library of B. bigemina (Sanger Institute). Presence of an intron in the 5' untranslated region was determined by 5' Rapid Amplification of cDNA Ends (RACE) analysis. Promoter activity was determined by measurement of luciferase expression at several time points after electroporation, efficiency of transfections and normalization of data was determined by quantitative PCR and by the percentage of parasitized erythrocytes. Results: The ef-1α locus contains two identical head to head ef-1α genes separated by a 1.425 kb intergenic (IG) region. Significant sequence divergence in the regions upstream of the inverted repeats on each side of the B. bigemina IG region suggest independent regulation mechanisms for controlling expression of each of the two ef-1α genes. Plasmid constructs containing the 5' and 3' halves of the IG regions controlling the expression of the luciferase gene containing a 3' region of a B. bigemina rap-1a gene, were generated for the testing of luciferase activity in transiently transfected parasites. Both halves of the ef-1α IG region tested showed the ability to promote high level production of luciferase. Moreover, both B. bigemina ef-1α promoters are also active in transiently transfected B. bovis and conversely, a B. bovis ef-1α promoter is active in transiently transfected B. bigemina. Conclusions: Collectively these data demonstrate the existence of two distinct promoters with homologous and heterologous promoter function in B. bigemina and B. bovis which is described for the first time in Babesia species. This study is of significance for development of interspecies stable transfection systems for B. bigemina and for B. bovis. [ABSTRACT FROM AUTHOR]

Apicomplexa tick-borne hemoparasites, including Babesia bovis, Babesia microti, and Theileria equi are responsible for bovine and human babesiosis and equine theileriosis, respectively. These parasites of vast medical, epidemiological, and economic impact have complex life cycles in their vertebrate and tick hosts. Large gaps in knowledge concerning the mechanisms used by these parasites for gene regulation remain. Regulatory genes coding for DNA binding proteins such as members of the Api-AP2, HMG, and Myb families are known to play crucial roles as transcription factors. Although the repertoire of Api-AP2 has been defined and a HMG gene was previously identified in the B. bovis genome, these regulatory genes have not been described in detail in B. microti and T. equi. In this study, comparative bioinformatics was used to: (i) identify and map genes encoding for these transcription factors among three parasites’ genomes; (ii) identify a previously unreported HMG gene in B. microti; (iii) define a repertoire of eight conserved Myb genes; and (iv) identify AP2 correlates among B. bovis and the better-studied Plasmodium parasites. Searching the available transcriptome of B. bovis defined patterns of transcription of these three gene families in B. bovis erythrocyte stage parasites. Sequence comparisons show conservation of functional domains and general architecture in the AP2, Myb, and HMG proteins, which may be significant for the regulation of common critical parasite life cycle transitions in B. bovis, B. microti, and T. equi. A detailed understanding of the role of gene families encoding DNA binding proteins will provide new tools for unraveling regulatory mechanisms involved in B. bovis, B. microti, and T. equi life cycles and environmental adaptive responses and potentially contributes to the development of novel convergent strategies for improved control of babesiosis and equine piroplasmosis. [ABSTRACT FROM AUTHOR]

Babesia bovis, an intra-erythrocytic tick-borne apicomplexan protozoan, is one of the causative agents of bovine babesiosis. Its life cycle includes sexual reproduction within cattle fever ticks, Rhipicephalus spp. Six B. bovis 6-Cys gene superfamily members were previously identified (A, B, C, D, E, F) where their orthologues in Plasmodium parasite have been shown to encode for proteins required for the development of sexual stages. The current study identified four additional 6-Cys genes (G, H, I, J) in the B. bovis genome. These four genes are described in the context of the complete ten 6-Cys gene superfamily. The proteins expressed by this gene family are predicted to be secreted or surface membrane directed. Genetic analysis comparing the 6-Cys superfamily among five distinct B. bovis strains shows limited sequence variation. Additionally, A, B, E, H, I and J genes were transcribed in B. bovis infected tick midgut while genes A, B and E were also transcribed in the subsequent B. bovis kinete stage. Transcription of gene C was found exclusively in the kinete. In contrast, transcription of genes D, F and G in either B. bovis infected midguts or kinetes was not detected. None of the 6-Cys transcripts were detected in B. bovis blood stages. Subsequent protein analysis of 6-Cys A and B is concordant with their transcript profile. The collective data indicate as in Plasmodium parasite, certain B. bovis 6-Cys family members are uniquely expressed during sexual stages and therefore, they are likely required for parasite reproduction. Within B. bovis specifically, proteins encoded by 6-Cys genes A and B are markers for sexual stages and candidate antigens for developing novel vaccines able to interfere with the development of B. bovis within the tick vector. [ABSTRACT FROM AUTHOR]

Respiratory failure and death in East Coast Fever (ECF), a clinical syndrome of African cattle caused by the apicomplexan parasite Theileria parva, has historically been attributed to pulmonary infiltration by infected lymphocytes. However, immunohistochemical staining of tissue from T. parva infected cattle revealed large numbers of CD3- and CD20-negative intralesional mononuclear cells. Due to this finding, we hypothesized that macrophages play an important role in Theileria parva disease pathogenesis. Data presented here demonstrates that terminal ECF in both Holstein and Boran cattle is largely due to multisystemic histiocytic responses and resultant tissue damage. Furthermore, the combination of these histologic changes with the clinical findings, including lymphadenopathy, prolonged pyrexia, multi-lineage leukopenia, and thrombocytopenia is consistent with macrophage activation syndrome. All animals that succumbed to infection exhibited lymphohistiocytic vasculitis of small to medium caliber blood and lymphatic vessels. In pulmonary, lymphoid, splenic and hepatic tissues from Holstein cattle, the majority of intralesional macrophages were positive for CD163, and often expressed large amounts of IL-17. These data define a terminal ECF pathogenesis in which parasite-driven lymphoproliferation leads to secondary systemic macrophage activation syndrome, mononuclear vasculitis, pulmonary edema, respiratory failure and death. The accompanying macrophage phenotype defined by CD163 and IL-17 is presented in the context of this pathogenesis. [ABSTRACT FROM AUTHOR]

Theileria parva is a tick-transmitted intracellular apicomplexan pathogen of cattle in sub-Saharan Africa that causes East Coast fever (ECF). ECF is an acute fatal disease that kills over one million cattle annually, imposing a tremendous burden on African small-holder cattle farmers. The pathology and level of T. parva infections in its wildlife host, African buffalo (Syncerus caffer), and in cattle are distinct. We have developed an absolute quantification method based on quantitative PCR (qPCR) in which recombinant plasmids containing single copy genes specific to the parasite (apical membrane antigen 1 gene, ama1) or the host (hypoxanthine phosphoribosyltransferase 1, hprt1) are used as the quantification reference standards. Our study shows that T. parva and bovine cells are present in similar numbers in T. parva-infected lymphocyte cell lines and that consequently, due to its much smaller genome size, T. parva DNA comprises between 0.9% and 3% of the total DNA samples extracted from these lines. This absolute quantification assay of parasite and host genome copy number in a sample provides a simple and reliable method of assessing T. parva load in infected bovine lymphocytes, and is accurate over a wide range of host-to-parasite DNA ratios. Knowledge of the proportion of target DNA in a sample, as enabled by this method, is essential for efficient high-throughput genome sequencing applications for a variety of intracellular pathogens. This assay will also be very useful in future studies of interactions of distinct host-T. parva stocks and to fully characterize the dynamics of ECF infection in the field. [ABSTRACT FROM AUTHOR]

Background The apicomplexan hemoparasite Theileria equi is a causative agent of equine piroplasmosis, eradicated from the United States in 1988. However, recent outbreaks have sparked renewed interest in treatment options for infected horses. Imidocarb dipropionate is the current drug of choice, however variation in clinical response to therapy has been observed. Methods We quantified the in vitro susceptibility of two T. equi isolates and a lab generated variant to both imidocarb dipropionate and a bumped kinase inhibitor compound 1294. We also evaluated the capacity of in vitro imidocarb dipropionate exposure to decrease susceptibility to that drug. The efficacy of imidocarb dipropionate for clearing infection in four T. equi infected ponies was also assessed. Results We observed an almost four-fold difference in imidocarb dipropionate susceptibility between two distinct isolates of T. equi. Four ponies infected with the less susceptible USDA Florida strain failed to clear the parasite despite two rounds of treatment. Importantly, a further 15- fold decrease in susceptibility was produced in this strain by continuous in vitro imidocarb dipropionate exposure. Despite a demonstrated difference in imidocarb dipropionate susceptibility, there was no difference in the susceptibility of two T. equi isolates to bumped kinase inhibitor 1294. Conclusions The observed variation in imidocarb dipropionate susceptibility, further reduction in susceptibility caused by drug exposure in vitro, and failure to clear T. equi infection in vivo, raises concern for the emergence of drug resistance in clinical cases undergoing treatment. Bumped kinase inhibitors may be effective as alternative drugs for the treatment of resistant T. equi parasites. [ABSTRACT FROM AUTHOR]

Babesia bovis is a tick-borne intraerythocytic protozoan responsible for acute disease in cattle which can be controlled by vaccination with attenuated B. bovis strains. Emerging B. bovis transfection technologies may increase the usefulness of these live vaccines. One use of transfected B. bovis parasites may be as a vaccine delivery platform. Previous transfection methods for B. bovis were limited by single expression sites and intracellular expression of transfected antigens. This study describes a novel transfection system in which two exogenous genes are expressed: one for selection and the other for a selected antigen designed to be delivered to the surface of the parasites. The strategy for duplicating the number of transfected genes was based on the use of the putative bidirectional promoter of the B. bovis 1.4 Kb ef-1α intergenic region. The ability of this region to regulate two independent expression sites was demonstrated using a luciferase assay on transiently transfected B. bovis parasites and then incorporated into a stable transfection plasmid to control independent expression of the selectable marker GFP-BSD and another gene of interest. A chimeric gene was synthetized using sequences from the protective B-cell epitopes of Rhipicephalus microplus tick antigen Bm86 along with sequences from the surface exposed B. bovis major surface antigen-1. This chimeric gene was then cloned into the additional expression site of the transfection plasmid. Transfection of the B. bovis Mo7 strain with this plasmid resulted in stable insertion into the ef-1α locus and simultaneous expression of both exogenous genes. Expression of the Bm86 epitopes on the surface of transfected merozoites was demonstrated using immunofluorescence analyses. The ability to independently express multiple genes by the inclusion of a bidirectional promoter and the achievement of surface expression of foreign epitopes advances the potential of transfected B. bovis as a future vaccine delivery platform. [ABSTRACT FROM AUTHOR]

The current study tested the hypothesis that removal of maltose binding protein (MBP) from recombinant antigen used for plate coating would improve the specificity of a commercial Anaplasma antibody competitive enzyme-linked immunosorbent assay (cELISA). The number of 358 sera with significant MBP antibody binding (≥30%I) in Anaplasma-negative herds was 139 (38.8%) when tested using the recombinant major surface protein 5 (rMSP5)-MBP cELISA without MBP adsorption. All but 8 of the MBP binders were rendered negative (<30%I) using the commercial rMSP5-MBP cELISA with MBP adsorption, resulting in 97.8% specificity. This specificity was higher than some previous reports, so to improve the specificity of the commercial cELISA, a new recombinant antigen designated rMSP5–glutathione S-transferase (GST) was developed, eliminating MBP from the antigen and obviating the need for MBP adsorption. Using the rMSP5-GST cELISA, only 1 of 358 Anaplasma-negative sera, which included the 139 sera with significant (≥30%I) MBP binding in the rMSP5-MBP cELISA without MBP adsorption, was positive. This resulted in an improved diagnostic specificity of 99.7%. The rMSP5-GST cELISA without MBP adsorption had comparable analytical sensitivity to the rMSP5-MBP cELISA with MBP adsorption and had 100% diagnostic sensitivity when tested with 135 positive sera defined by nested polymerase chain reaction. Further, the rMSP5-GST cELISA resolved 103 false-positive reactions from selected sera with possible false-positive reactions obtained using the rMSP5-MBP cELISA with MBP adsorption and improved the resolution of 29 of 31 other sera. In summary, the rMSP5-GST cELISA was a faster and simpler assay with higher specificity, comparable sensitivity, and improved resolution in comparison with the rMSP5-MBP cELISA with MBP adsorption. [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]

The global importance of the hemoparasite Theileria haneyi to equine health was recently shown by its resistance to imidocarb dipropionate (ID) and its interference with T. equi clearance by ID in some co-infected horses. Genetic characterization of T. haneyi revealed marked genomic reduction compared to T. equi, and initial experiments demonstrated reduced clinical severity in spleen-intact horses. Furthermore, in early experiments, splenectomized horses survived T. haneyi infection and progressed to an asymptomatic carrier state, in stark contrast to the high fatality rate of T. equi in splenectomized horses. Thus, we hypothesized that T. haneyi is less virulent than T. equi. To objectively assess virulence, clinical data from nine splenectomized, T. haneyi-infected horses were evaluated and compared to published data on T. equi-infected, splenectomized horses. Seven of eight splenectomized, T. haneyi-infected horses survived. Further, in six horses co-infected with T. equi and T. haneyi, only horses cleared of T. equi by ID survived splenectomy and became asymptomatic carriers. The reduced virulence of T. haneyi in splenectomized horses instructs why T. haneyi was, until recently, undetected. This naturally occurring comparative reduction in virulence in a natural host provides a foundation for defining virulence mechanisms of theileriosis and Apicomplexa in general. [ABSTRACT FROM AUTHOR]

Members of the CCp protein family have been previously described to be expressed on gametocytes of apicomplexan Plasmodium parasites. Knocking out Plasmodium CCp genes blocks the development of the parasite in the mosquito vector, making the CCp proteins potential targets for the development of a transmission-blocking vaccine. Apicomplexans Babesia bovis and Babesia bigemina are the causative agents of bovine babesiosis, and apicomplexan Theileria equi causes equine piroplasmosis. Bovine babesiosis and equine piroplasmosis are the most economically important parasite diseases that affect worldwide cattle and equine industries, respectively. The recent sequencing of the B. bovis and T. equi genomes has provided the opportunity to identify novel genes involved in parasite biology. Here we characterize three members of the CCp family, named CCp1, CCp2 and CCp3, in B. bigemina, B. bovis and T. equi. Using B. bigemina as an in vitro model, expression of all three CCp genes and proteins was demonstrated in temperature-induced sexual stages. Transcripts for all three CCp genes were found in vivo in blood stages of T. equi, and transcripts for CCp3 were detected in vivo in blood stages of B. bovis. However, no protein expression was detected in T. equi blood stages or B. bovis blood stages or B. bovis tick stages. Collectively, the data demonstrated a differential pattern of expression of three orthologous genes of the multidomain adhesion CCp family by B. bigemina, B. bovis and T. equi. The novel CCp members represent potential targets for innovative approaches to control bovine babesiosis and equine piroplasmosis. [ABSTRACT FROM AUTHOR]

Small ruminant lentiviruses include members that infect sheep (ovine lentivirus [OvLV]; also known as ovine progressive pneumonia virus/maedi-visna virus) and goats (caprine arthritis encephalitis virus [CAEV]). Breed differences in seroprevalence and proviral concentration of OvLV had suggested a strong genetic component in susceptibility to infection by OvLV in sheep. A genetic marker test for susceptibility to OvLV has been developed recently based on the TMEM154 gene with validation data from over 2,800 sheep representing nine cohorts. While no single genotype has been shown to have complete resistance to OvLV, consistent association in thousands of sheep from multiple breeds and management conditions highlight a new strategy for intervention by selective breeding. This genetic marker-assisted selection (MAS) has the potential to be a useful addition to existing viral control measures. Further, the discovery of multiple additional genomic regions associated with susceptibility to or control of OvLV suggests that additional genetic marker tests may be developed to extend the reach of MAS in the future. This review will cover the strengths and limitations of existing data from host genetics as an intervention and outline additional questions for future genetic research in sheep, goats, small ruminant lentiviruses, and their host-pathogen interactions. [ABSTRACT FROM AUTHOR]

Background: Like human immunodeficiency virus (HIV), ovine lentivirus (OvLV) is macrophage-tropic and causes lifelong infection. OvLV infects one quarter of U.S. sheep and induces pneumonia and body condition wasting. There is no vaccine to prevent OvLV infection and no cost-effective treatment for infected animals. However, breed differences in prevalence and proviral concentration have indicated a genetic basis for susceptibility to OvLV. A recent study identified TMEM154 variants in OvLV susceptibility. The objective here was to identify additional loci associated with odds and/or control of OvLV infection. Methodology/Principal Findings: This genome-wide association study (GWAS) included 964 sheep from Rambouillet, Polypay, and Columbia breeds with serological status and proviral concentration phenotypes. Analytic models accounted for breed and age, as well as genotype. This approach identified TMEM154 (nominal P = 9.2 x 10-7; empirical P = 0.13), provided 12 additional genomic regions associated with odds of infection, and provided 13 regions associated with control of infection (all nominal P<1 x 10-5). Rapid decline of linkage disequilibrium with distance suggested many regions included few genes each. Genes in regions associated with odds of infection included DPPA2/DPPA4 (empirical P = 0.006), and SYTL3 (P = 0.051). Genes in regions associated with control of infection included a zinc finger cluster (ZNF192, ZSCAN16, ZNF389, and ZNF165; P = 0.001), C19orf42/TMEM38A (P = 0.047), and DLGAP1 (P = 0.092). Conclusions/Significance: These associations provide targets for mutation discovery in sheep susceptibility to OvLV. Aside from TMEM154, these genes have not been associated previously with lentiviral infection in any species, to our knowledge. Further, data from other species suggest functional hypotheses for future testing of these genes in OvLV and other lentiviral infections. Specifically, SYTL3 binds and may regulate RAB27A, which is required for enveloped virus assembly of human cytomegalovirus. Zinc finger transcription factors have been associated with positive selection for repression of retroviral replication. DLGAP1 binds and may regulate DLG1, a known regulator of HIV infectivity. [ABSTRACT FROM AUTHOR]

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 104.9 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Rhipicephalus (Boophilus) microplus is an economically important tick of cattle involved in the transmission of Babesia bovis, the etiological agent of bovine babesiosis. Commercial anti-tick vaccines based on the R. microplus Bm86 glycoprotein have shown some effect in controlling tick infestation; however their efficacy as a stand-alone solution for tick control has been questioned. Understanding the role of the Bm86 gene product in tick biology is critical to identifying additional methods to utilize Bm86 to reduce R. microplus infestation and babesia transmission. Additionally, the role played by Bm86 in R. microplus fitness during B. bovis infection is unknown. Results: Here we describe in two independent experiments that RNA interference-mediated silencing of Bm86 decreased the fitness of R. microplus females fed on cattle during acute B. bovis infection. Notably, Bm86 silencing decreased the number and survival of engorged females, and decreased the weight of egg masses. However, gene silencing had no significant effect on the efficiency of transovarial transmission of B. bovis from surviving female ticks to their larval offspring. The results also show that Bm86 is expressed, in addition to gut cells, in larvae, nymphs, adult males and ovaries of partially engorged adult R. microplus females, and its expression was significantly down-regulated in ovaries of ticks fed on B. bovis-infected cattle. Conclusion: The R. microplus Bm86 gene plays a critical role during tick feeding and after repletion during blood digestion in ticks fed on cattle during acute B. bovis infection. Therefore, the data indirectly support the rationale for using Bm86-based vaccines, perhaps in combination with acaricides, to control tick infestation particularly in B. bovis endemic areas. [ABSTRACT FROM AUTHOR]

Background: Babesia are emerging health threats to humans and animals in the United States. A collaborative effort of multiple disciplines to attain optimal health for people, animals and our environment, otherwise known as the One Health concept, was taken during a research workshop held in April 2009 to identify gaps in scientific knowledge regarding babesioses. The impetus for this analysis was the increased risk for outbreaks of bovine babesiosis, also known as Texas cattle fever, associated with the re-infestation of the U.S. by cattle fever ticks. Results: The involvement of wildlife in the ecology of cattle fever ticks jeopardizes the ability of state and federal agencies to keep the national herd free of Texas cattle fever. Similarly, there has been a progressive increase in the number of cases of human babesiosis over the past 25 years due to an increase in the white-tailed deer population. Human babesiosis due to cattle-associated Babesia divergens and Babesia divergens-like organisms have begun to appear in residents of the United States. Research needs for human and bovine babesioses were identified and are presented herein. Conclusions: The translation of this research is expected to provide veterinary and public health systems with the tools to mitigate the impact of bovine and human babesioses. However, economic, political, and social commitments are urgently required, including increased national funding for animal and human Babesia research, to prevent the reestablishment of cattle fever ticks and the increasing problem of human babesiosis in the United States. [ABSTRACT FROM AUTHOR]

Previous studies initiated defining the role of host genetics in influencing the outcome of exposure to ovine progressive pneumonia virus. However, specific genes influencing host control of virus replication and disease progression have not been identified. This study, using 383 ewes of the Columbia, Polypay, and Rambouillet breeds, tested the hypothesis that host control of OPPV as measured by provirus levels in the peripheral blood associates with certain breeds and MHC class II Ovis aries ( Ovar) -DRB1 expressed alleles. Rambouillet ewes were less likely to have measurable provirus levels as compared to Columbia ewes at ages 5 and 6 ( P value < 0.02), and they exhibited lower provirus levels when compared to both Columbia and Polypay ewes of the same ages ( P value < 0.05). The presence of DRB1*0403- or DRB1*07012-expressed alleles were significantly associated ( P value = 0.019 and 0.0002, respectively) with lower OPP provirus levels but only were only found in 11% of the ewe flock. Analysis of each segregating amino acid in the β1 domain of DR β-chain revealed that amino acids Y31, T32, N37, T51, Q60, or N74 significantly associated ( P value range = 0.0003–0.018) with lower OPP provirus levels, whereas amino acids H32, A38, or I67 associated ( P value range = 0.013–0.043) with higher OPP provirus levels. These results suggest that Ovar-DRB1 contributes as one host genetic factor that controls OPP provirus levels, but does not fully account for the breed-specific OPP proviral differences. [ABSTRACT FROM AUTHOR]

Determining the genomic organization of the Ovis aries ( Ovar) major histocompatibility complex class IIa region is essential for future functional studies related to antigen presentation. In this study, a bacterial artificial chromosome (BAC) library of genomic DNA from peripheral blood leukocytes (PBL) of a Rambouillet ram was constructed, and BAC clone consisting of the major histocompatibility complex (MHC) class II DQB2, DQA2, DQB1, DQA1, and DRB1 loci was identified and completely sequenced. The BAC clone consists of 160,889 bp of finished sequence with the loci arranged in the following order: DQB2, DQA2, DQB1, DQA1, and DRB1 with 14.3, 25, 6.6, and 40.9 Kb spanning between the loci, respectively. All five of these loci were transcribed in the animal used to generate the MHC class II BAC clone. Repeat or retrotransposable elements along with MHC class II cis promoter elements consisting of S, X, and Y boxes were identified in the sequence. In addition, 16 non-coding conserved sequences amongst primates, carnivores, and ruminants were identified ( p < 0.001). These conserved sequences include binding sites for transcription factors with known roles in immune cells, and they provide a basis for further functional investigation of the genes in this region. This is the first ruminant finished sequence of the DQB2-DRB1 region, and this sequence information will aid in whole genome and transcriptome analyses of MHC class II. [ABSTRACT FROM AUTHOR]

Persistence of Anaplasma spp. in the animal reservoir host is required for efficient tick-borne transmission of these pathogens to animals and humans. Using A. marginale infection of its natural reservoir host as a model, persistent infection has been shown to reflect sequential cycles in which antigenic variants emerge, replicate, and are controlled by the immune system. Variation in the immunodominant outer-membrane protein MSP2 is generated by a process of gene conversion, in which unique hypervariable region sequences (HVRs) located in pseudogenes are recombined into a single operon-linked msp2 expression site. Although organisms expressing whole HVRs derived from pseudogenes emerge early in infection, long-term persistent infection is dependent on the generation of complex mosaics in which segments from different HVRs recombine into the expression site. The resulting combinatorial diversity generates the number of variants both predicted and shown to emerge during persistence. [ABSTRACT FROM AUTHOR]

Ovar-DRB1 is part of the major histocompatibility complex (MHC) class II of sheep and functions by presenting extracellular-derived peptides to the immune system. Although there are over 100 different Ovar-DRB1 DNA sequences reported in GenBank, only two Ovar-DRB1 mRNA sequences have been reported. As a first step in understanding MHC Class II function as it relates to disease progression in sheep, Ovar-DRB1 transcripts encoding the peptide-binding site or the first domain (β1) of Ovar-DRB1 in a 32-ewe–lamb flock were identified and characterized by using reverse transcriptase–polymerase chain reaction, cloning, sequencing, and phylogenetic analysis. Fourteen new Ovar-DRB1 β1 cDNA sequences out of a total of 15 Ovar-DRB1 β1 cDNA sequences in a ewe–lamb flock of 32 sheep were identified. One Ovar-DRB1 β1 cDNA sequence was 100% identical to M93432, one of the two Ovar-DRB1 mRNA sequences reported in GenBank. Twelve out of 15 Ovar-DRB1 β1 cDNA sequences were 100% identical to the corresponding previously reported Ovar-DRB1 genomic DNA sequences, indicating that these Ovar-DRB1 genomic DNA sequences are also transcribed. One of three of the remaining Ovar-DRB1 β1 cDNA sequences, DRB1*07012, had a synonymous substitution resulting in an identical deduced amino acid sequence to DRB1*0701. Two of the remaining three Ovar-DRB1 β1 cDNA sequences had nucleotide differences and subsequent deduced amino acid sequence differences when compared to known Ovar-DRB1 β1 genomic DNA sequences, and therefore, DRB1*0206 and DRB1*0353 represent new Ovar-DRB1 β1 expressed alleles. Phylogenetic analysis of the 15 Ovar-DRB1 β1 cDNA sequences revealed that DRB1*0206 had a strong phylogenetic relationship to DRB1*0203, and DRB1*0353 had a strong phylogenetic relationship to DRB1*0303. [ABSTRACT FROM AUTHOR]

Bacterial pathogens in the genusAnaplasmagenerate surface coat variants by gene conversion of chromosomal pseudogenes into single-expression sites. These pseudogenes encode unique surface-exposed hypervariable regions flanked by conserved domains, which are identical to the expression site flanking domains. In addition,Anaplasma marginalegenerates variants by recombination of oligonucleotide segments derived from the pseudogenes into the existing expression site copy, resulting in a combinatorial increase in variant diversity. Using theA. marginalegenome sequence to track the origin of sequences recombined into themsp2expression site, we demonstrated that the complexity of the expressedmsp2increases during infection, reflecting a shift from recombination of the complete hypervariable region of a given pseudogene to complex mosaics with segments derived from hypervariable regions of different pseudogenes. Examination of the complete set of 1183 variants with segmental changes revealed that 99% could be explained by one of the recombination sites occurring in the conserved flanking domains and the other within the hypervariable region. Consequently, we propose an‘anchoring’ model for segmental gene conversion whereby the conserved flanking sequences tightly align and anchor the expression site sequence to the pseudogene. Associated with the recombination sites were deletions, insertions and substitutions; however, these are a relatively minor contribution to variant generation as these occurred in less than 2% of the variants. Importantly, the anchoring model, which can account for more variants than a strict segmental sequence identity mechanism, is consistent with the number ofmsp2variants predicted and empirically identified during persistent infection. [ABSTRACT FROM AUTHOR]

Identifying the reservoirs of a pathogen is vital for control of sporadic disease and epidemics. Listeria monocytogenes is a zoonotic foodborne pathogen that is responsible for 28% of food-related deaths in the United States annually, as well as a major cause of massive product recalls worldwide. To examine the role of the dairy farm as a potential source or reservoir for L. monocytogenes subtypes shown to cause human listeriosis, we compared the pulsed-field gel electrophoresis (PFGE) restriction enzyme digestion profiles of L. monocytogenes dairy farm-associated strains (milk, environmental, and bovine) to human sporadic and epidemic disease strains. Twenty-three percent of human sporadic strains had PFGE patterns identical to that of farm isolate(s). Additionally, three farm environmental strains and one human sporadic strain had a PFGE pattern identical to a strain of L. monocytogenes responsible for the 1985 California epidemic. These data indicate that this epidemic strain continues to cause sporadic human illness and has a potential dairy farm as a reservoir. [ABSTRACT FROM AUTHOR]

Explores the role of adaptive and innate immune responses in control of parasites. Elimination of parasites; Results of treatment with anti-interferon-ϒ antibody; Depletion of natural killer cells.

Examines the importance of pregnancy status and fetal prion genetics in the placentomas of scrapie infected sheep. Genotypes of the placentoma components by breeding; Importance of immunohistochemistry; Correlation between stage of pregnancy and spatial distribution.

Focuses on the efficient use of a small genome to generate antigenic diversity in tick-borne ehrlichial pathogens. Antigenic variation in major surface proteins of tick-borne bacterial pathogens; Presence of a single genomic expression site for major surface protein 2; Schematic representation of genomic pseudogenes.

Fifteen newborn Saanen goats were orally infected with a biologically cloned isolate of caprine arthritis-encephalitis virus (CAEV-63). At 33.5 months after infection, 8 of 12 seropositive goats had CAEV-63-specific neutralizing antibody. Five neutralization-positive goats developed clinically apparent arthritis of carpal joints, three of which had periarticular swelling indicating severe synovitis. Arthritis was not evident in age-matched controls or infected goats without neutralizing antibody. Multiple viral isolates were obtained from synovial fluid or synovial fluid cells ofarthritic joints between 36.4 and 44.9 months after infection, and successive isolates from individual joints were defined as antigenic variants expressing type-specific neutralization epi-topes. Thus, the evolution of neutralizing antibody does not arrest viral replication or development ofprogressive inflammatory lesions. Rather, some antigenic variants produced in the presence of neutralizing antibody are clonally expanded within carpal joints, and severe joint inflammation is specifically associated with these variants. [ABSTRACT FROM PUBLISHER]

The apicomplexan parasite Theileria haneyi is one of two known causative agents of equine theileriosis. It causes milder clinical disease than its more virulent counterpart, Theileria equi, in experimentally infected horses, and can superinfect T. equi-positive horses. The current equi merozoite antigen 1 (EMA1)-based competitive enzyme-linked immunosorbent assay (ELISA)used in the U.S. to detect equine theileriosis detects T. equi but not T. haneyi, and the complexity of molecular assays precludes widespread use for epidemiologic studies. In order to facilitate urgently needed studies on the prevalence of T. haneyi, the goal of this study was to develop a sensitive and specific serologic assay for the diagnosis of T. haneyi based on the equi merozoite antigen 11 (ThEMA11). To achieve this objective, ThEMA11 was recombinantly expressed in eukaryotic cells and its antigenicity assessed using sera from T. haneyi-experimentally infected horses. Confirmation of sera reactivity enabled design and optimization of an indirect ELISA. Specificity of the ELISA for T. haneyi was assessed using a cohort of sera from horses experimentally infected and confirmed PCR-positive for either T. equi or T. haneyi. Data from field samples further demonstrate that the ThEMA11 ELISA is capable of identifying T. haneyi antibodies in horses from multiple continents around the world. [ABSTRACT FROM AUTHOR]