Sarcocystis spp. of New and Old World Camelids: Ancient Origin, Present Challenges.

Sarcocystis spp. are coccidian protozoans belonging to the Apicomplexa phylum. As with other members of this phylum, they are obligate intracellular parasites with complex cellular machinery for the invasion of host cells. Sarcocystis spp. display dixenous life cycles, involving a predator and a prey as definitive and intermediate hosts, respectively. Specifically, these parasites develop sarcocysts in the tissues of their intermediate hosts, ranging in size from microscopic to visible to the naked eye, depending on the species. When definitive hosts consume sarcocysts, infective forms are produced in the digestive system and discharged into the environment via feces. Consumption of oocyst-contaminated water and pasture by the intermediate host completes the parasitic cycle. More than 200 Sarcocystis spp. have been described to infect wildlife, domestic animals, and humans, some of which are of economic or public health importance. Interestingly, Old World camelids (dromedary, domestic Bactrian camel, and wild Bactrian camel) and New World or South American camelids (llama, alpaca, guanaco, and vicuña) can each be infected by two different Sarcocystis spp: Old World camelids by S. cameli (producing micro- and macroscopic cysts) and S. ippeni (microscopic cysts); and South American camelids by S. aucheniae (macroscopic cysts) and S. masoni (microscopic cysts). Large numbers of Old and New World camelids are bred for meat production, but the finding of macroscopic sarcocysts in carcasses significantly hampers meat commercialization. This review tries to compile the information that is currently accessible regarding the biology, epidemiology, phylogeny, and diagnosis of Sarcocystis spp. that infect Old and New World camelids. In addition, knowledge gaps will be identified to encourage research that will lead to the control of these parasites.

Comparative Degradome Analysis of the Bovine Piroplasmid Pathogens Babesia bovis and Theileria annulata.

Babesia bovis and Theileria annulata are tick-borne hemoprotozoans that impact bovine health and are responsible for considerable fatalities in tropical and subtropical regions around the world. Both pathogens infect the same vertebrate host, are closely related, and contain similar-sized genomes; however, they differ in invertebrate host specificity, absence vs. presence of a schizont stage, erythrocyte invasion mechanism, and transovarial vs. transstadial transmission. Phylogenetic analysis and bidirectional best hit (BBH) identified a similar number of aspartic, metallo, and threonine proteinases and nonproteinase homologs. In contrast, a considerably increased number of S54 serine rhomboid proteinases and S9 nonproteinase homologs were identified in B. bovis, whereas C1A cysteine proteinases and A1 aspartic nonproteinase homologs were found to be expanded in T. annulata. Furthermore, a single proteinase of families S8 (subtilisin-like protein) and C12 (ubiquitin carboxyl-terminal hydrolase), as well as four nonproteinase homologs, one with dual domains M23-M23 and three with S9-S9, were exclusively present in B. bovis. Finally, a pronounced difference in species-specific ancillary domains was observed between both species. We hypothesize that the observed degradome differences represent functional correlates of the dissimilar life history features of B. bovis and T. annulata. The presented improved classification of piroplasmid proteinases will facilitate an informed choice for future in-depth functional studies.

Babesia microti Immunoreactive Rhoptry-Associated Protein-1 Paralogs Are Ancestral Members of the Piroplasmid-Confined RAP-1 Family.

Babesia, Cytauxzoon and Theileria are tick-borne apicomplexan parasites of the order Piroplasmida, responsible for diseases in humans and animals. Members of the piroplasmid rhoptry-associated protein-1 (pRAP-1) family have a signature cysteine-rich domain and are important for parasite development. We propose that the closely linked B. microti genes annotated as BMR1_03g00947 and BMR1_03g00960 encode two paralogue pRAP-1-like proteins named BmIPA48 and Bm960. The two genes are tandemly arranged head to tail, highly expressed in blood stage parasites, syntenic to rap-1 genes of other piroplasmids, and share large portions of an almost identical ~225 bp sequence located in their 5' putative regulatory regions. BmIPA48 and Bm960 proteins contain a N-terminal signal peptide, share very low sequence identity (<13%) with pRAP-1 from other species, and harbor one or more transmembrane domains. Diversification of the piroplasmid-confined prap-1 family is characterized by amplification of genes, protein domains, and a high sequence polymorphism. This suggests a functional involvement of pRAP-1 at the parasite-host interface, possibly in parasite adhesion, attachment, and/or evasion of the host immune defenses. Both BmIPA48 and Bm960 are recognized by antibodies in sera from humans infected with B. microti and might be promising candidates for developing novel serodiagnosis and vaccines.

In Silico Survey and Characterization of Babesia microti Functional and Non-Functional Proteases.

Human babesiosis caused by the intraerythrocytic apicomplexan Babesia microti is an expanding tick-borne zoonotic disease that may cause severe symptoms and death in elderly or immunocompromised individuals. In light of an increasing resistance of B. microti to drugs, there is a lack of therapeutic alternatives. Species-specific proteases are essential for parasite survival and possible chemotherapeutic targets. However, the repertoire of proteases in B. microti remains poorly investigated. Herein, we employed several combined bioinformatics tools and strategies to organize and identify genes encoding for the full repertoire of proteases in the B. microti genome. We identified 64 active proteases and 25 nonactive protease homologs. These proteases can be classified into cysteine (n = 28), serine (n = 21), threonine (n = 14), asparagine (n = 7), and metallopeptidases (n = 19), which, in turn, are assigned to a total of 38 peptidase families. Comparative studies between the repertoire of B. bovis and B. microti proteases revealed differences among sensu stricto and sensu lato Babesia parasites that reflect their distinct evolutionary history. Overall, this data may help direct future research towards our understanding of the biology and pathogenicity of Babesia parasites and to explore proteases as targets for developing novel therapeutic interventions.

Kinetic characterization of a novel cysteine peptidase from the protozoan Babesia bovis, a potential target for drug design.

C1A cysteine peptidases have been shown to play an important role during apicomplexan invasion and egress of host red blood cells (RBCs) and therefore have been exploited as targets for drug development, in which peptidase specificity is deterministic. Babesia bovis genome is currently available and from the 17 putative cysteine peptidases annotated four belong to the C1A subfamily. In this study, we describe the biochemical characterization of a C1A cysteine peptidase, named here BbCp (B. bovis cysteine peptidase) and evaluate its possible participation in the parasite asexual cycle in host RBCs. The recombinant protein was obtained in bacterial inclusion bodies and after a refolding process, presented typical kinetic features of the cysteine peptidase family, enhanced activity in the presence of a reducing agent, optimum pH between 6.5 and 7.0 and was inhibited by cystatins from R. microplus. Moreover, rBbCp substrate specificity evaluation using a peptide phage display library showed a preference for Val > Leu > Phe. Finally, antibodies anti-rBbCp were able to interfere with B. bovis growth in vitro, which highlights the BbCp as a potential target for drug design.

A novel type 1 cystatin involved in the regulation of Rhipicephalus microplus midgut cysteine proteases.

Rhipicephalus microplus is a cattle ectoparasite found in tropical and subtropical regions around the world with great impact on livestock production. R. microplus can also harbor pathogens, such as Babesia sp. and Anaplasma sp. which further compromise cattle production. Blood meal acquisition and digestion are key steps for tick development. In ticks, digestion takes place inside midgut cells and is mediated by aspartic and cysteine peptidases and, therefore, regulated by their inhibitors. Cystatins are a family of cysteine peptidases inhibitors found in several organisms and have been associated in ticks with blood acquisition, blood digestion, modulation of host immune response and tick immunity. In this work, we characterized a novel R. microplus type 1 cystatin, named Rmcystatin-1b. The inhibitor transcripts were found to be highly expressed in the midgut of partially and fully engorged females and they appear to be modulated at different days post-detachment. Purified recombinant Rmcystatin-1b displayed inhibitory activity towards typical cysteine peptidases with high affinity. Moreover, rRmcystatin-1b was able to inhibit native R. microplus cysteine peptidases and RNAi-mediated knockdown of the cystatin transcripts resulted in increased proteolytic activity. Moreover, rRmcystatin-1b was able to interfere with B. bovis growth in vitro. Taken together our data strongly suggest that Rmcystatin-1b is a regulator of blood digestion in R. microplus midgut.

In silico identification of immunotherapeutic and diagnostic targets in the glycosylphosphatidylinositol metabolism of the coccidian Sarcocystis aucheniae.

Meat of the South American camelids (SACs) llama and alpaca is an important source of animal protein and income for rural families in the Andes, and a product with significant growth potential for local and international markets. However, infestation with macroscopic cysts of the coccidian protozoon Sarcocystis aucheniae, a parasitosis known as SAC sarcocystosis, significantly hampers its commercialization. There are no validated methods to diagnose the presence of S. aucheniae cysts other than carcass examination. Moreover, there are no available drugs or vaccines to cure or prevent SAC sarcocystosis. Identification of relevant molecules that act at the host-pathogen interface can significantly contribute to the control of this disease. It has been shown for other pathogenic protozoa that glycosylphosphatidylinositol (GPI) is a critical molecule implicated in parasite survival and pathogenicity. This study focused on the identification of the enzymes that participate in the S. aucheniae GPI biosynthetic pathway and the repertoire of the parasite GPI-anchored proteins (GPI-APs). To this aim, RNA was extracted from parasite cysts and the transcriptome was sequenced and translated into amino acid sequences. The generated database was mined using sequences of well-characterized GPI biosynthetic enzymes of Saccharomyces cerevisiae and Toxoplasma gondii. Eleven enzymes predicted to participate in the S. aucheniae GPI biosynthetic pathway were identified. On the other hand, the database was searched for proteins carrying an N-terminal signal peptide and a single C-terminal transmembrane region containing a GPI anchor signal. Twenty-four GPI-anchored peptides were identified, of which nine are likely S. aucheniae-specific, and 15 are homologous to membrane proteins of other coccidians. Among the latter, 13 belong to the SRS domain superfamily, an extensive group of coccidian GPI-anchored proteins that mediate parasite interaction with their host. Phylogenetic analysis showed a great degree of intra- and inter-specific divergence among SRS family proteins. In vitro and in vivo experiments are needed to validate S. aucheniae GPI biosynthetic enzymes and GPI-APs as drug targets and/or as vaccine or diagnostic antigens.

Cysteine Proteinase C1A Paralog Profiles Correspond with Phylogenetic Lineages of Pathogenic Piroplasmids.

Piroplasmid parasites comprising of Babesia, Theileria, and Cytauxzoon are transmitted by ticks to farm and pet animals and have a significant impact on livestock industries and animal health in tropical and subtropical regions worldwide. In addition, diverse Babesia spp. infect humans as opportunistic hosts. Molecular phylogeny has demonstrated at least six piroplasmid lineages exemplified by B. microti, B. duncani, C. felis, T. equi, Theileria sensu stricto (T. annulata, T. parva, and T. orientalis) and Babesia sensu stricto (B. bovis, B. bigemina, and B. ovis). C1A cysteine-proteinases (C1A-Cp) are papain-like enzymes implicated in pathogenic and vital steps of the parasite life cycle such as nutrition and host cell egress. An expansion of C1A-Cp of T. annulata and T. parva with respect to B. bovis and B. ovis was previously described. In the present work, C1A-Cp paralogs were identified in available genomes of species pertaining to each piroplasmid lineage. Phylogenetic analysis revealed eight C1A-Cp groups. The profile of C1A-Cp paralogs across these groups corroborates and defines the existence of six piroplasmid lineages. C. felis, T. equi and Theileria s.s. each showed characteristic expansions into extensive families of C1A-Cp paralogs in two of the eight groups. Underlying gene duplications have occurred as independent unique evolutionary events that allow distinguishing these three piroplasmid lineages. We hypothesize that C1A-Cp paralog families may be associated with the advent of the schizont stage. Differences in the invertebrate tick host specificity and/or mode of transmission in piroplasmid lineages might also be associated with the observed C1A-Cp paralog profiles.

Detection of Sarcocystis aucheniae in blood of llama using a duplex semi-nested PCR assay and its association with cyst infestation.

The protozoon Sarcocystis aucheniae is the causative agent of South American camelid (SAC) sarcocystosis. Infections are characterized by the presence of cysts in muscles which are in size and appearance similar to rice grains. As consumption of insufficiently cooked infected meat produces gastroenteritis, cyst-containing SAC meat is confiscated by sanitary authorities or depreciated with serious economic consequences for SAC breeders. In this work, a duplex semi-nested PCR was designed to simultaneously detect parasite and llama DNA in host blood samples. Species-specific regions of S. aucheniae 18S rRNA gene and Lama glama 16S mitochondrial gene were amplified, yielding bands of 583 and 257 bp, respectively, and separated by gel electrophoresis. The method proved to be highly sensitive, with a detection limit lower than one parasite per milliliter blood, and the inclusion of primers to detect llama-specific DNA resulted useful as a methodological control. Blood samples collected from llamas of Argentina and Bolivia (n = 225) were analyzed using this method, and 18.7 % resulted positive for S. aucheniae. No correlation was found between PCR results and llama age, sex or the finding of macroscopic cysts in meat after slaughter. Lack of molecular detection in the blood of some llamas harboring macrocysts suggests that parasite circulation in the bloodstream after encystment is under the detection threshold of the test or even absent, while PCR positive results in cyst-infested animals suggests that prior exposure to the parasite does not impede subsequent infections. The described method can be useful to detect active foci of infection, to assess the effectiveness of parasiticide treatments, and for the surveillance and tracing of definitive hosts.

Seropositivity to Sarcocystis infection of llamas correlates with breeding practices.

Production of llama (Lama glama) meat in rural communities of the Andean regions is largely affected by Sarcocystis spp. infection. Macroscopic cysts develop in muscles as a consequence of S. aucheniae parasitism, often resulting in meat downgrade or condemnation. Llama meat production is informal in Argentina but has broad perspectives for improvement, and would significantly benefit from the development of standardized control methodologies. This work analyzes whether the presence of anti-Sarcocystis spp. antibodies in llamas is influenced by factors such as geographic region and/or herd management practices. To this aim, an indirect ELISA was set up based on a ~23kDa soluble immunogenic protein fraction (Sa23), isolated from S. aucheniae macrocysts (Sa23-iELISA). Serum samples (n=507) were collected from llamas bred under three different conditions: (i) with no sanitation controls and in the presence of pastoral dogs by small producers of different localities of the Argentine Puna (Group I, n=237); (ii) with sanitation controls and no pastoral dogs, in fenced fields of an experimental agricultural station in the Argentine Puna (Group II, n=167); and (iii) with sanitation controls and no pastoral dogs in fenced fields of farms of the humid Pampas (Group III, n=103). Results of the Sa23-iELISA were expressed as percentages of positivity with respect to a reference Sarcocystis-positive serum. Notably, the percentage of sera that fell above the cut-off (31.5% positivity) in group (i) was significantly higher (p<0.001) than those of groups (ii) and (iii) (50% vs 23% and 26%, respectively). These results indicate that herd management practices constitute a critical risk factor for sarcocystiosis in llamas. Differences in these practices include feeding of dogs with raw Sarcocystis-infected llama meat, with the consequent maintenance of the parasite life cycle by the contamination of pastures and water with fecal-derived infective oocysts/sporocysts. Additionally, the itinerancy of llama herds in search for pastures and water sources possibly exposes animals to a higher number of infective foci. On the other hand, percentages of seropositive llamas kept under controlled conditions in the Puna or the humid Pampas were not significantly different, suggesting that climate, altitude, and/or pasture characteristics do not influence Sarcocystis-infection. Male gender and older age of llamas were found to be propensity factors for sarcocystiosis in llamas bred in La Puna under controlled conditions. Availability of diagnostic tools, as well as increased knowledge on the parasite and its epidemiology, will allow the design of control strategies for SAC sarcocystiosis.

Molecular detection of Sarcocystis aucheniae in the blood of llamas from Argentina.

Sarcocystis aucheniae are apicomplexan protozoa that infect South American camelids (SACs), giving rise to macroscopic cysts similar to rice grains in skeletal muscles. Visual detection of macrocysts in slaughtered animals hampers commercialization of SAC meat, a highly relevant economic exploitation for Andean rural families. Importantly, the consumption of undercooked S. aucheniae-infested meat causes gastroenteritis. A carnivore definitive host, possibly the dog, acquires the parasite when feeding on infected SAC meat, and later eliminates infective oocysts in its feces. The parasite cycle is completed when SACs ingest contaminated water or pastures. We hypothesized that parasite DNA can be detected in SAC blood using molecular methods. In order to test this hypothesis, a seminested PCR format was specifically designed to target the hypervariable 18S rRNA gene region of S. aucheniae. PCR conditions were optimized using genomic DNA extracted from macrocyst bradyzoites. A detection limit of up to 1 parasite in 10µl of llama blood was established based on DNA samples extracted from aliquots of S. aucheniae bradyzoite-spiked non-infected llama blood. The seminested PCR allowed to detect natural infections of S. aucheniae in llama blood samples originating in the Andean flatlands of Argentina. Specific amplification of S. aucheniae DNA was corroborated by amplicon sequencing. This is the first report of S. aucheniae detection in llama blood, which provides a valuable diagnostic tool for epidemiological studies and for the evaluation of the efficacy of control measures for this parasitosis.

Detección molecular de Sarcocystis aucheniae en sangre de llamas de Argentina / Molecular detection of Sarcocystis aucheniae in the blood of llamas from Argentina

Sarcocystis aucheniae are apicomplexan protozoa that infect South American camelids (SACs), giving rise to macroscopic cysts similar to rice grains in skeletal muscles. Visual detection of macrocysts in slaughtered animals hampers commercialization of SAC meat, a highly relevant economic exploitation for Andean rural families. Importantly, the consumption of undercooked S. aucheniae-infested meat causes gastroenteritis. A carnivore definitive host, possibly the dog, acquires the parasite when feeding on infected SAC meat, and later eliminates infective oocysts in its feces. The parasite cycle is completed when SACs ingest contaminated water or pastures. We hypothesized that parasite DNA can be detected in SAC blood using molecular methods. In order to test this hypothesis, a seminested PCR format was specifically designed to target the hypervariable 18S rRNA gene region of S. aucheniae. PCR conditions were optimized using genomic DNA extracted from macrocyst bradyzoites. A detection limit of up to 1 parasite in 10 μl of llama blood was established based on DNA samples extracted from aliquots of S. aucheniae bradyzoite-spiked non-infected llama blood. The seminested PCR allowed to detect natural infections of S. aucheniae in llama blood samples originating in the Andean flatlands of Argentina. Specific amplification of S. aucheniae DNA was corroborated by amplicon sequencing. This is the first report of S. aucheniae detection in llama blood, which provides a valuable diagnostic tool for epidemiological studies and for the evaluation of the efficacy of control measures for this parasitosis.

Sarcocystis aucheniae es un protozoo apicomplexa que infecta a camélidos sudamericanos (CS), dando lugar a la formación de quistes macroscópicos similares a granos de arroz en los músculos esqueléticos. La detección visual de macroquistes en animales faenados dificulta la comercialización de la carne de CS, una explotación de gran relevancia para la economía de las familias rurales andinas. Es importante destacar que el consumo de carne infectada con S. aucheniae no suficientemente cocida causa gastroenteritis. Un hospedador definitivo carnívoro, posiblemente el perro, adquiere el parásito cuando se alimenta de carne de CS infectada y luego elimina ooquistes infectivos en las heces. El ciclo del parásito se completa cuando un CS ingiere agua o pasturas contaminadas. Hemos hipotetizado que es posible detectar ADN del parásito en la sangre de CS usando métodos moleculares. Para poner a prueba esta hipótesis, se diseñó una PCR semianidada que utiliza como blanco una región del gen 18S ARNr específica para S. aucheniae. Se optimizaron las condiciones de la PCR usando ADN genómico extraído de bradizoítos presentes en macroquistes. Se estableció un límite de detección de un parásito en 10 μl de sangre de llama, basado en muestras de ADN extraído de alícuotas de sangre de llama no infectada a las que se agregaron cantidades conocidas de bradizoítos de S. aucheniae. Más aún, la PCR semianidada permitió la detección de infecciones naturales por este parásito en muestras de sangre de llama de la Puna argentina. La amplificación específica de ADN de S. aucheniae fue corroborada por secuenciación de los productos de amplificación. Este es el primer reporte de la detección de S. aucheniae en sangre de llama. Además, este estudio contribuye una herramienta diagnóstica valiosa para estudios epidemiológicos y para la evaluación de la efectividad de medidas de control para esta parasitosis.

Characterization of a papain-like cysteine protease essential for the survival of Babesia ovis merozoites.

Babesia ovis, a tick-transmitted intraerythrocytic protozoan parasite, causes severe infections in small ruminants from Southern Europe, Middle East, and Northern Africa. With the aim of finding potential targets for the development of control methods against this parasite, sequence analysis of its genome led to the identification of four putative cysteine proteases of the C1A family. Orthology between B. ovis, B. bovis, T. annulata, and T. parva sequences showed that each B. ovis C1A peptidase sequence clustered within one of the four ortholog groups previously reported for these piroplasmids. The ortholog of bovipain-2 of B. bovis and falcipain-2 of Plasmodium falciparum, respectively, was designated "ovipain-2" and further characterized. In silico analysis showed that ovipain-2 has the typical topology of papain-like cysteine peptidases and a highly similar predicted three dimensional structure to bovipain-2 and falcipain-2, suggesting susceptibility to similar inhibitors. Immunoblotting using antibodies raised against a recombinant form of ovipain-2 (r-ovipain-2) demonstrated expression of ovipain-2 in in vitro cultured B. ovis merozoites. By immunofluorescence, these antibodies reacted with merozoites and stained the cytoplasm of infected erythrocytes. This suggests that ovipain-2 is secreted by the parasite and could be involved in intra- and extracellular digestion of hemoglobin and/or cleavage of erythrocyte proteins facilitating parasite egress. A significant reduction in the percentage of parasitized erythrocytes was obtained upon incubation of B. ovis in vitro cultures with anti-r-ovipain-2 antibodies, indicating an important functional role for ovipain-2 in the intra erythrocytic development cycle of this parasite. Finally, studies of the reactivity of sera from B. ovis-positive and negative sheep against r-ovipain-2 showed that this protease is expressed in vivo, and can be recognized by host antibodies. The results of this study suggest that ovipain-2 constitutes a potential target for immunotherapies and drug development against ovine babesiosis.

Molecular identification of Sarcocystis aucheniae as the macrocyst-forming parasite of llamas.

The domestic South American camelids (SACs), llama (Lama glama) and alpaca (Lama paco), are frequently found to be infected with Sarcocystis parasites. Infections give rise in skeletal muscle to macroscopic cysts (1-5mm long) that resemble rice seeds, each containing several million living bradyzoites. The finding of cysts prevents commercialization of SAC meat, an important source of income for rural families in the Andean flatlands. Thus, development of diagnostic methods to facilitate the control of these infections is highly desirable, and the first step to this end is the unequivocal species identification of the causative agent. Based on the cyst form and size, the infecting parasite has been described as Sarcocystis aucheniae; however, this traditional approach is not reliable as similar cysts may contain different species. To date, molecular identification has been done for a single isolate of S. aucheniae from an alpaca in Australia. In order to verify the identity of the species present in SACs of South America, the complete 18S rRNA gene was PCR-amplified and sequenced from macrocyst DNA obtained from three llamas of the Andean flatlands. A phylogenetic Bayesian analysis was carried out using the analyzed and available 18S rRNA sequences of Sarcocystis spp. In the constructed tree, all of the new 18S rRNA gene sequences segregated in a single clade together with the 18S rRNA gene sequence reported from an alpaca in Australia, demonstrating that the isolated parasite is S. aucheniae, and that this parasite indiscriminately infects both domestic SACs. This work represents the first molecular identification of the causative agent of SAC sarcocystiosis in South America, and can contribute to the development of control methods for this neglected parasitosis.

Babesia bovis expresses Bbo-6cys-E, a member of a novel gene family that is homologous to the 6-cys family of Plasmodium.

A novel Babesia bovis gene family encoding proteins with similarities to the Plasmodium 6cys protein family was identified by TBLASTN searches of the B. bovis genome using the sequence of the P. falciparum PFS230 protein as query, and was termed Bbo-6cys gene family. The Bbo-cys6 gene family contains six genes termed Bbo-6cys-A, B, C, D, E and F encoding for proteins containing an arrangement of 6 cysteine residues. The Bbo-6cys genes A, B, C, D, and E are tandemly arranged as a cluster of Chromosome 2 in the B. bovis genome, whereas gene F is located in a distal region in the same chromosome. The Bbo-6cys-E gene, with higher homology to PFS230, was selected for further examination. Immunoblot analysis using recombinant Bbo-6cys-E protein and B. bovis-positive bovine serum demonstrated expression by the parasite and immunogenicity during B. bovis infection. Immunofluorescence analysis using anti-Bbo-6cys-E antibodies confirmed expression of Bbo-6cys-E in in vitro blood stages of B. bovis. In addition, polyclonal antisera against both recombinant Bbo-6cys-E and specific synthetic peptides containing predicted B-cell epitopes of Bbo-6cys-E, significantly inhibited erythrocyte invasion by B. bovis in in vitro neutralization assays, suggesting an important functional role for this protein. Identification of this new gene family in B. bovis and further investigation on its biological significance may aid our understanding of the bovine, tick and parasite relationships and the development of improved control methods against B. bovis infection in cattle.

Bovipain-2, the falcipain-2 ortholog, is expressed in intraerythrocytic stages of the tick-transmitted hemoparasite Babesia bovis.

BACKGROUND: Cysteine proteases have been shown to be highly relevant for Apicomplexan parasites. In the case of Babesia bovis, a tick-transmitted hemoparasite of cattle, inhibitors of these enzymes were shown to hamper intraerythrocytic replication of the parasite, underscoring their importance for survival. RESULTS: Four papain-like cysteine proteases were found to be encoded by the B. bovis genome using the MEROPS database. One of them, the ortholog of Plasmodium falciparum falcipain-2, here named bovipain-2, was further characterized. Bovipain-2 is encoded in B. bovis chromosome 4 by an ORF of 1.3 kb, has a predicted molecular weight of 42 kDa, and is hydrophilic with the exception of a transmembrane region. It has orthologs in several other apicomplexans, and its predicted amino acid sequence shows a high degree of conservation among several B. bovis isolates from North and South America. Synteny studies demonstrated that the bovipain-2 gene has expanded in the genomes of two related piroplasmids, Theileria parva and T. annulata, into families of 6 and 7 clustered genes respectively. The bovipain-2 gene is transcribed in in vitro cultured intra-erythrocyte forms of a virulent and an attenuated B. bovis strain from Argentina, and has no introns, as shown by RT-PCR followed by sequencing. Antibodies against a recombinant form of bovipain-2 recognized two parasite protein bands of 34 and 26 kDa, which coincide with the predicted sizes of the pro-peptidase and mature peptidase, respectively. Immunofluorescence studies showed an intracellular localization of bovipain-2 in the middle-rear region of in vitro cultured merozoites, as well as diffused in the cytoplasm of infected erythrocytes. Anti-bovipain-2 antibodies also reacted with B. bigemina-infected erythrocytes giving a similar pattern, which suggests cross-reactivity among these species. Antibodies in sera of two out of six B. bovis-experimentally infected bovines tested, reacted specifically with recombinant bovipain-2 in immunoblots, thus demonstrating expression and immunogenicity during bovine-infecting stages. CONCLUSIONS: Overall, we present the characterization of bovipain-2 and demonstrate its in vitro and in vivo expression in virulent and attenuated strains. Given the involvement of apicomplexan cysteine proteases in essential parasite functions, bovipain-2 constitutes a new vaccine candidate and potential drug target for bovine babesiosis.

Free fatty acids induce cell differentiation to infective forms in Trypanosoma cruzi.

Intestinal extracts of Triatoma infestans induce cell differentiation of Trypanosoma cruzi epimastigotes into the infective metacyclic form. Part of this effect can be explained by the presence of haemoglobin fragments, which stimulate trypanosomal adenylate cyclase. In this work we examined the metacyclogenic activity of lipids present in this intestinal extract. We found that lipid extracts of the intestinal extract have significant stimulatory effects that reside with the free-fatty-acid fraction, especially oleic acid. These compounds stimulate de novo diacylglycerol formation and protein kinase C activity in the parasite. Moreover, metacyclogenesis is stimulated by phorbol esters and cell-permeant diacylglycerol, while protein kinase C down-regulation or incubation with inhibitors of this kinase abrogates this effect. These results indicate that free fatty acids are a novel signal, inducing metacyclogenesis, acting through a pathway involving diacylglycerol biosynthesis and protein kinase C activation.

Immunodominant epitopes in Babesia bovis rhoptry-associated protein 1 that elicit memory CD4(+)-T-lymphocyte responses in B. bovis-immune individuals are located in the amino-terminal domain.

Babesia bovis rhoptry-associated protein 1 (RAP-1), which confers partial protection against B. bovis challenge, is recognized by antibodies and T lymphocytes from cattle that have recovered from infection and are immune to subsequent challenge. RAP-1 is a 60-kDa protein with an N-terminal (NT) region that contains four cysteine residues conserved among all Babesia RAP-1 family members and a C-terminal (CT) region that contains multiple, degenerate, tandem 23-amino-acid (aa) repeats. To define the location of CD4(+)-T-cell epitopes for vaccine development using a recombinant protein or minigene construct, a series of truncated recombinant RAP-1 proteins and peptides were tested for stimulation of T-cell lines derived from B. bovis-immune cattle. CD4(+)-T-cell lines from three B. bovis-immune cattle with different DRB3 haplotypes responded to the NT region of RAP-1, whereas T cells from only one animal responded weakly to the CT region. T-cell lines from the three individuals recognized two to six NT-region peptides spanning aa 134 to 316 and representing at least four dominant epitopes. Using RAP-1-specific CD4(+)-T-cell clones, two NT-region epitopes, EYLVNKVLYMATMNYKT (aa 187 to 203) and EAPWYKRWIKKFR (aa 295 to 307), and one CT-region repeat epitope, FREAPQATKHFL, which is present twice at aa positions 391 to 402 and 414 to 425, were identified. Several peptides representing degenerate repeats of the agonist CT-region peptide FREAPQATKHFL neither stimulated responses of T-cell clones specific for this peptide nor inhibited responses to the agonist peptide. Upon stimulation with specific antigen, T-cell clones specific for NT or CT epitopes produced gamma interferon. The presence of T-helper-cell epitopes in the NT domain of RAP-1, which is highly conserved among otherwise antigenically different strains of B. bovis, supports the inclusion of this region in vaccine constructs to be tested in cattle.