Amblyomma cajennense is an intrastadial biological vector of Theileria equi

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

Vaccination with an in vitro culture attenuated Babesia bovis strain safely protects highly susceptible adult cattle against acute bovine babesiosis

IntroductionLive in vivo attenuated Babesia bovis vaccines produced by sequential passages in splenectomized calves have historically been used to control acute bovine babesiosis in endemic areas worldwide. However, several constraints prevent the widespread use of these vaccines, including the need for several splenectomized calves to produce vaccine batches, and potential inconsistent parasite attenuation, which contraindicates their use for highly Babesia-susceptible adult cattle. Thus, the use of vaccines based on well-defined in vitro culture attenuated B. bovis strains emerges as a more sustainable and efficient alternative. Previous work demonstrated that the culture attenuated strain Att-S74-T3Bo is non-tick transmissible and able to safely protect calves against needle challenge with a B. bovis virulent strain.Methods and resultsHerein we evaluated safety and efficacy of Att-S74-T3Bo in preventing acute babesiosis in adult (>1.5 year of age) cattle. Results demonstrated that Att-S74-T3Bo vaccination of adult animals (n=5) induced self-limiting signs of acute infection and protected the vaccinated animals against challenge with the homologous virulent B. bovis strain Vir-S74-T3Bo. Att-S74-T3Bo-vaccinated adult cattle developed significant (P<0.05) monocytosis, with concomitant neutropenia and CD4+ leukopenia, in peripheral blood early after vaccination. Also, vaccinated animals developed a specific signature of pro- and anti-inflammatory cytokine expression in peripheral blood and significant levels of IgM, total IgG, IgG1, and IgG2 against the B. bovis immunodominant antigen RAP-1 CT. Strikingly, none of the vaccinated animals showed any signs of acute babesiosis after challenge with Vir-S74-T3Bo. In contrast, control adult cattle (n=5) showed pathognomonic symptoms of acute babesiosis, and significant decrease (P<0.05) in lymphocytes, monocytes, and neutrophils, starting on day 7 post-challenge. All control animals developed severe acute disease and were euthanized on days 10 through 12 days post-challenge.Discussion and conclusionEvidence from this study indicates that Att-S74-T3Bo safely protects highly susceptible adult cattle against challenge with a homologous virulent strain of B. bovis. In conclusion, Att-S74-T3Bo may be considered as a potential efficient and sustainable attenuated candidate vaccine strain to control acute bovine babesiosis in highly susceptible adult cattle. Future studies should focus on increasing the number of animals vaccinated, duration of immunity, and efficacy of this attenuated strain against heterologous virulent parasite strains

N-Glycosylation in piroplasmids : Diversity within simplicity

N-glycosylation has remained mostly unexplored in Piroplasmida, an order of tick-transmitted pathogens of veterinary and medical relevance. Analysis of 11 piroplasmid genomes revealed three distinct scenarios regarding N-glycosylation: Babesia sensu stricto (s.s.) species add one or two N-acetylglucosamine (NAcGlc) molecules to proteins; Theileria equi and Cytauxzoon felis add (NAcGlc)2-mannose, while B. microti and Theileria s.s. synthesize dolichol-P-P-NAcGlc and dolichol-P-P-(NAcGlc)2 without subsequent transfer to proteins. All piroplasmids possess the gene complement needed for the synthesis of the N-glycosylation substrates, dolichol-P and sugar nucleotides. The oligosaccharyl transferase of Babesia species, T. equi and C. felis, is predicted to be composed of only two subunits, STT3 and Ost1. Occurrence of short N-glycans in B. bovis merozoites was experimentally demonstrated by fluorescence microscopy using a NAcGlc-specific lectin. In vitro growth of B. bovis was significantly impaired by tunicamycin, an inhibitor of N-glycosylation, indicating a relevant role for N-glycosylation in this pathogen. Finally, genes coding for N-glycosylation enzymes and substrate biosynthesis are transcribed in B. bovis blood and tick stages, suggesting that this pathway is biologically relevant throughout the parasite life cycle. Elucidation of the role/s exerted by N-glycans will increase our understanding of these successful parasites, for which improved control measures are needed.Instituto de PatobiologíaFil: Florin-Christensen, Mónica. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: Florin-Christensen, Mónica. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rodriguez, Anabel Elisa. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: Rodriguez, Anabel Elisa. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Suarez, Carlos Esteban. Washington State University. College of Veterinary Medicine. Department of Veterinary Microbiology and Pathology; Estados UnidosFil: Suarez, Carlos Esteban. United States Department of Agricultural-Agricultural Research Service. Animal Disease Research Unit; Estados UnidosFil: Ueti, Massaro W. Washington State University. College of Veterinary Medicine. Department of Veterinary Microbiology and Pathology; Estados UnidosFil: Ueti, Massaro W. United States Department of Agricultural-Agricultural Research Service. Animal Disease Research Unit; Estados UnidosFil: Delgado, Fernando Oscar. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: Delgado, Fernando Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Echaide, Ignacio Eduardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Schnittger, Leonhard. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: Schnittger, Leonhard. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Assessment of Babesia bovis 6cys A and 6cys B as components of transmission blocking vaccines for babesiosis

Background: Babesia bovis reproduces sexually in the gut of its tick vector Rhipicephalus microplus, which involves expression of 6cys A and 6cys B proteins. Members of the widely conserved 6cys superfamily are candidates for transmission blocking vaccines (TBV), but intricacies in the immunogenicity of the 6cys proteins in the related Plasmodium parasites required the identification of transmission blocking domains in these molecules for vaccine design. Hereby, the immunogenic efficacy of recombinant (r) B. bovis 6cys A and B proteins as a TBV formulation was studied. Methods: The immunogenicity of r6cys A and 6cys B proteins expressed in a eukaryotic system was evaluated in a cattle immunization trial (3 immunized and 3 control calves). A B. bovis sexual stage induction in vitro inhibition assay to assess the ability of antibodies to block the production of sexual forms by the parasite was developed. Results: Immunized cattle generated antibodies against r6cys A and r6cys B that were unable to block sexual reproduction of the parasite in ticks. Additionally, these antibodies also failed in recognizing native 6cys A and 6cys B and peptides representing 6cys A and 6cys B functional domains and in inhibiting the development of sexual forms in an in vitro induction system. In contrast, rabbit antibodies generated against synthetic peptides representing predicted B-cell epitopes of 6cys A and 6cys B recognized recombinant and native forms of both 6cys proteins as well as peptides representing 6cys A and 6cys B functional domains and were able to neutralize development of sexual forms of the parasite in vitro. Conclusions: These data, combined with similar work performed on Plasmodium 6cys proteins, indicate that an effective 6cys protein-based TBV against B. bovis will require identifying and targeting selected regions of proteins containing epitopes able to reduce transmission

Genetic characterization of Theileria equi infecting horses in North America: evidence for a limited source of U.S. introductions

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

Comparative efficacy of buparvaquone and imidocarb in inhibiting the in vitro growth of Babesia bovis

Introduction:B. bovis is an apicomplexan parasite responsible for bovine babesiosis, a tick-borne disease with a worldwide impact. The disease remains inefficiently controlled, and few effective drugs, including imidocarb dipropionate (ID), are currently available in endemic areas. The objective of this study was to evaluate whether buparvaquone (BPQ), a drug currently used to treat cattle infected with the Babesia-related Theileria spp. parasites, could be active against Babesia parasites. Herein, we compared the effect of ID and BPQ on B. bovis growth in vitro erythrocyte culture.Methods:We compared the effect of ID and BPQ on the culture-adapted Texas T2Bo strain of B. bovis. In vitro cultured parasites were incubated with ID and BPQ at two starting parasitemia levels (PPE), 0.2% and 1%. In vitro cultured parasites were treated with ID or BPQ at concentrations ranging from 10 to 300 nM, during 4 consecutive days. Parasitemia levels were daily evaluated using microscopic examination. Data was compared using the independent Student’s t-test.Results and discussion:Both ID and BPQ significantly inhibited (p < 0.05) the growth of B. bovis, regardless of the initial parasitemia used. At 1% parasitemia, BPQ had lower calculated inhibitory concentration 50 (IC50: 50.01) values than ID (IC50: 117.3). No parasites were found in wells with 0.2% starting parasitemia, treated previously with 50 nM of BPQ or ID, after 2 days of culture without drugs. At 1% parasitemia, no parasite survival was detected at 150 nM of BPQ or 300 nM of ID, suggesting that both drugs acted as babesiacidals.Conclusion:Overall, the data suggests that BPQ is effective against B. bovis and shows a residual effect that seems superior to ID, which is currently the first-line drug for treating bovine babesiosis globally

Differential paired stage-specific expression of Babesia bovis cysteine-rich GCC2/GCC3 domain family proteins (BboGDP) during development within Rhipicephalus microplus

Abstract Background Babesia bovis, an intra-erythrocytic apicomplexan parasite, is one of the causative agents of bovine babesiosis, the most important tick-borne disease of cattle in tropical and subtropical regions. Babesia bovis has a complex life-cycle that includes sexual development within the tick vector. The development of a transmission blocking vaccine to control bovine babesiosis requires the identification of antigens displayed on the surface of the parasite during its development within tick vectors. Four B. bovis cysteine-rich GCC2/GCC3 domain protein (BboGDP) family members were previously identified and are differentially expressed as discrete pairs by either blood stages or kinetes. In this study we focused on two family members, BboGDP1 and -3, that are expressed by Babesia parasites during tick infection. Methods and results Transcription analysis using quantitative PCR demonstrated that BboGDP1 and -3 were upregulated in in vitro-induced sexual stage parasites and during parasite development in the tick midgut. Moreover, protein expression analysis of BboGDP1 and -3 during the development of sexual stages in in vitro culture was consistent with their transcription profile. Live immunofluorescence analysis using polyclonal antibodies confirmed surface expression of BboGDP1 and -3 on in vitro-induced sexual stage parasites. In addition, fixed immunofluorescence analysis showed reactivity of anti-BboGDP1 and -3 polyclonal antibodies to kinetes. Conclusions The collective data indicate that BboGDP1 and -3 are expressed by kinetes and on the surface of sexual stages of the parasites. The identified parasite surface membrane proteins BboGDP1 and -3 are potential candidates for the development of a B. bovis transmission blocking vaccine. Graphical Abstrac