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.

First molecular detection of Sarcocystis suihominis in a domestic pig of Nigeria.

Sarcocystis are Apicomplexan protozoa with a dixenous life cycle that includes a predator and a prey as definitive and intermediate hosts, respectively. Domestic and wild pigs are intermediate hosts of S. suihominis, with formation of sarcocysts in their muscles, while humans and non-human primates act as final hosts. After ingesting raw or undercooked sarcocyst-infested pork, signs of gastroenteritis including inappetence, nausea, vomiting, and diarrhea may develop in humans. Moreover, excretion of infective forms with human feces leads to dissemination of the parasite in the environment. In this study, macroscopic sarcocysts of white color, oval shape, and a diameter of approximately 3-8 mm were found in the skeletal muscle of a slaughtered domestic pig (Sus scrofa domesticus) destined for human consumption in an abattoir of Makurdi, Benue State, Nigeria. Sarcocyst DNA was used as template to PCR amplify the near-complete length of the 18S rRNA gene and a fragment of the cytochrome c oxidase subunit 1 (cox-1) gene. Amplicons were sequenced and used to construct phylogenetic trees with selected available Sarcocystis spp. sequences. In both cases, the placement of the analyzed sequences with S. suihominis was strongly supported, confirming the species identity of this macroscopic sarcocyst-forming parasite. This constitutes the first molecular identification of S. suihominis in Nigeria and the African continent. Proximity between pigs and humans, and poor sanitary conditions frequently encountered in pig farms of Nigeria might favor the dissemination of this zoonotic parasite, posing a threat to public health.

Prevalence, risk factors and molecular epidemiology of neonatal cryptosporidiosis in calves: The Argentine perspective.

Cryptosporidium spp. are enteroparasitic protozoans that cause cryptosporidiosis in newborn calves. Clinical signs of the infection are diarrhoea and dehydration leading to decreased productivity and economic losses in cattle farms around the world. Additionally, cryptosporidiosis is a relevant zoonotic disease since the ingestion of oocysts can be fatal for children under five years of age, the elderly, and/or immunocompromised adults. This review aims to integrate existing knowledge on the epidemiological situation of calf cryptosporidiosis and associated risk factors in Argentina. In addition, the GP60 subtype diversity of the pathogen was analysed and related with the global distribution of corresponding GP60 subtypes. Depending on the study region and applied diagnostics, prevalence among calves up to 20 days of age varied between 25.2% and 42.5%, while a prevalence of 16.3-25.5% was observed at the age of 1-90 days. So far, molecular studies have determined exclusively Cryptosporidium parvum in preweaned calves. In addition, C. parvum infection was reported as the major cause of calf diarrhoea, followed by rotavirus A (RVA), while enteropathogens such as coronavirus, Escherichiacoli, and Salmonella sp. played a negligible role. Calf age of 20 days or less, incidence of diarrhoea, poorly drained soils, and large farm size were identified as risk factors for C. parvum-infection in Argentina. A total of nine GP60 subtypes (IIaAxxG1R1, xx = 16 to 24) were identified, showing a stepwise increase of the trinucleotide motif TCA, and including the zoonotic subtypes IIaA16G1R1, IIaA17G1R1, IIaA18G1R1, IIaA19G1R1, and IIaA20G1R1. We found that an increase in the A16→A24 trinucleotide repeat was accompanied by a gradual decrease in the global distribution of GP60 alleles, strongly suggesting that IIaA16G1R1 represents the primordial allelic variant of this group. Since identified GP60 alleles have a similar genetic background, we hypothesize that the continuous trinucleotide repeat array has been generated by stepwise repeat expansion of A16. The information gathered and integrated in this study contributes to an improved understanding of the epidemiological characteristics of bovine cryptosporidiosis in and beyond Argentina, which in turn can help to develop control strategies for this parasitosis of veterinary and medical relevance.

Degrade to survive: the intricate world of piroplasmid proteases.

Piroplasmids of the genera Babesia, Theileria, and Cytauxzoon are tick-transmitted parasites with a high impact on animals and humans. They have complex life cycles in their definitive arthropod and intermediate vertebrate hosts involving numerous processes, including invasion of, and egress from, host cells, parasite growth, transformation, and migration. Like other parasitic protozoa, piroplasmids are equipped with different types of protease to fulfill many of such essential processes. Blockade of some key proteases, using inhibitors or antibodies, hinders piroplasmid growth, highlighting their potential usefulness in drug therapies and vaccine development. A better understanding of the functional significance of these enzymes will contribute to the development of improved control measures for the devastating animal and human diseases caused by these pathogens.

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.

Molecular Detection and Differentiation of Arthropod, Fungal, Protozoan, Bacterial and Viral Pathogens of Honeybees.

The honeybee Apis mellifera is highly appreciated worldwide because of its products, but also as it is a pollinator of crops and wild plants. The beehive is vulnerable to infections due to arthropods, fungi, protozoa, bacteria and/or viruses that manage to by-pass the individual and social immune mechanisms of bees. Due to the close proximity of bees in the beehive and their foraging habits, infections easily spread within and between beehives. Moreover, international trade of bees has caused the global spread of infections, several of which result in significant losses for apiculture. Only in a few cases can infections be diagnosed with the naked eye, by direct observation of the pathogen in the case of some arthropods, or by pathogen-associated distinctive traits. Development of molecular methods based on the amplification and analysis of one or more genes or genomic segments has brought significant progress to the study of bee pathogens, allowing for: (i) the precise and sensitive identification of the infectious agent; (ii) the analysis of co-infections; (iii) the description of novel species; (iv) associations between geno- and pheno-types and (v) population structure studies. Sequencing of bee pathogen genomes has allowed for the identification of new molecular targets and the development of specific genotypification strategies.

Occurrence of sarcoptic mange in free-ranging vicuñas (Vicugna vicugna) of the Andean high plateau region of Argentina.

Free-ranging vicuñas (Vicugna vicugna) are handled in some areas of the Andean high plateau region following an ancestral practice known as chaku, which consists in their transient capture and shearing of their fiber for commercialization. In this study, 807 vicuñas captured during 12 chaku events that took place in 2019 in the province of Jujuy, Argentina, were examined for typical mange skin lesions. Twenty-eight of the examined vicuñas presented alopecia with erythema, exudation, hyperkeratosis, and/or bleeding scarred lesions, mostly in the chest, rear and front legs, and inguinal zone. Most of the cases (82%) appeared in Laguna Cucho at 4900 masl, where 23% of the animals presented these skin reactions. Microscopic evaluation of skin scrapings revealed the presence of a great number of 0.1- to 0.4-mm-long mites of different life cycle stages, morphologically compatible with the species Sarcoptes scabiei. This etiological agent was confirmed by PCR amplification and sequencing of a cox-1 species-specific segment. Histopathological examination of skin biopsies showed extensive infiltration of the dermis with lymphocytes, neutrophils and eosinophils, hyperplasia at different stages, epidermis degeneration, and hyperkeratosis. This is the first characterization of sarcoptic mange in free-ranging vicuñas by clinical examination, mite morphology, histopathological studies, and molecular confirmation in the region. Mange hampers the welfare of vicuñas and the economy of the local communities that organize chaku events since infested vicuñas cannot be sheared. Its long-term effects are unknown but it might affect the fitness and survival of this iconic South American camelid.

Unraveling the Complexity of the Rhomboid Serine Protease 4 Family of Babesia bovis Using Bioinformatics and Experimental Studies.

Babesia bovis, a tick-transmitted apicomplexan protozoon, infects cattle in tropical and subtropical regions around the world. In the apicomplexans Toxoplasma gondii and Plasmodium falciparum, rhomboid serine protease 4 (ROM4) fulfills an essential role in host cell invasion. We thus investigated B. bovis ROM4 coding genes; their genomic organization; their expression in in vitro cultured asexual (AS) and sexual stages (SS); and strain polymorphisms. B. bovis contains five rom4 paralogous genes in chromosome 2, which we have named rom4.1, 4.2, 4.3, 4.4 and 4.5. There are moderate degrees of sequence identity between them, except for rom4.3 and 4.4, which are almost identical. RT-qPCR analysis showed that rom4.1 and rom4.3/4.4, respectively, display 18-fold and 218-fold significantly higher (p < 0.01) levels of transcription in SS than in AS, suggesting a role in gametogenesis-related processes. In contrast, transcription of rom4.4 and 4.5 differed non-significantly between the stages. ROM4 polymorphisms among geographic isolates were essentially restricted to the number of tandem repeats of a 29-amino acid sequence in ROM4.5. This sequence repeat is highly conserved and predicted as antigenic. B. bovis ROMs likely participate in relevant host−pathogen interactions and are possibly useful targets for the development of new control strategies against this pathogen.

International interlaboratory validation of a nested PCR for molecular detection of Babesia bovis and Babesia bigemina, causative agents of bovine babesiosis.

Babesia bovis and B. bigemina are tick-transmitted parasites causing bovine babesiosis, characterized by significant morbidity and mortality leading to economic losses to the livestock industry in tropical and subtropical regions worldwide. Animals that recover from acute infection remain carriers with low parasitemia acting as a source of transmission, and often escape detection. An improved diagnosis of a B. bovis and/or B. bigemina infection of carrier animals is enabled by the availability of detection methods with high sensitivity. To this end, two nested PCR assays targeting the cytochrome b (cytb) genes of B. bovis and B. bigemina (cytb-nPCR), have been recently developed and an increased sensitivity with respect to reference protocols has been shown (Romero-Salas et al., 2016). In this study, the specificity against a panel of hemoparasites that potentially co-occur with B. bovis and B. bigemina was demonstrated to ensure applicability of the cytb-nPCR assays in a wide range of regions where bovine babesiosis is endemic. Furthermore, we compared both reported cytb-nPCR assays with reference nPCR and qPCR protocols for (i) their capability to detect carrier animals in the field, and (ii) their reproducibility when performed in different laboratories by independent operators. We show that, in a panel of bovine field samples (n = 100), the cytb-nPCR assays detected a considerably higher number of 25% B. bovis and 61% B. bigemina-positive animals compared to 7% and 20% B. bovis and 55% and 49% B. bigemina-positive animals when tested by reference nPCR and qPCR protocols, respectively. Cytb-nPCRs were also found superior in the detection of carrier animals when field samples from Africa were analyzed. In addition, both the B. bovis and B. bigemina cytb-nPCR assays were independently validated in a single blinded study in three laboratories. Importantly, no significant differences in the number/percentage of infected animals was observed using cytb-nPCR assays. In summary, the cytb-nPCR assays detected a considerably higher number of chronically infected B. bovis and B. bigemina carrier animals compared to reference nPCR and qPCR protocols, when applied in different epidemiological field situations. Furthermore, a high reproducibility between laboratories could be demonstrated.

Babesia bovis AMA-1, MSA-2c and RAP-1 contain conserved B and T-cell epitopes, which generate neutralizing antibodies and a long-lasting Th1 immune response in vaccinated cattle.

Vaccines against bovine babesiosis must, ideally, induce a humoral immune response characterized by neutralizing antibodies against conserved epitopes and a cellular Th1 immune response. In Babesia bovis, proteins such as AMA-1, MSA-2c, and RAP-1 have been characterized and antibodies against these proteins have shown a neutralizing effect, demonstrating the implication of B and T-cell epitopes in the immune response. There is evidence of the existence of B and T-cell epitopes in these proteins, however, it remains to be defined, the presence of conserved peptides in strains from around the world containing B and T-cell epitopes, and their role in the generation of a long-lasting immunity. The aim in this paper was to identify peptides of Babesia bovis AMA-1, MSA-2c, and RAP-1 that elicit a neutralizing and long-lasting Th1 immune response. Peptides containing B-cell epitopes of AMA-1, MSA-2c and RAP-1, were identified. The immune response generated by each peptide was characterized in cattle. All peptides tested induced antibodies that recognized intraerythrocytic parasites, however, only 5 peptides generated neutralizing antibodies in vitro: P2AMA-1 (6.28%), P3MSA-2c (10.27%), P4MSA-2c (10.42%), P1RAP-1 (32.45%), and P4RAP-1 (36.98%). When these neutralizing antibodies were evaluated as a pool, the inhibition percentage of invasion increased to 52.37%. When the T cellular response was evaluated, two peptides: P3MSA2c and P2AMA1 induced a higher percentage (>70%) of activated CD4 +/CD45RO+ T cells than unstimulated cells. Additionally, both peptides induced the production of gamma interferon (IFN-) in PBMCs from vaccinated cattle after one year proving the implication of a long-lasting Th1 immune response. In conclusion, we identified conserved peptides containing B and T-cell epitopes in antigens of B. bovis that elicit a Th1 immune response and showed evidence that peptides from the same protein elicit different immune responses, which has implication for vaccine development in bovine babesiosis.

The Piroplasmida Babesia, Cytauxzoon, and Theileria in farm and companion animals: species compilation, molecular phylogeny, and evolutionary insights.

The order Piroplasmida, including the genera Babesia, Cytauxzoon, and Theileria is often referred to as piroplasmids and comprises of dixenous hemoprotozoans transmitted by ticks to a mammalian or avian host. Although piroplasmid infections are usually asymptomatic in wild animals, in domestic animals, they cause serious or life-threatening consequences resulting in fatalities. Piroplasmids are particularly notorious for the enormous economic loss they cause worldwide in livestock production, the restrictions they pose on horse trade, and the negative health impact they have on dogs and cats. Furthermore, an increasing number of reported human babesiosis cases are of growing concern. Considerable international research and epidemiological studies are done to identify existing parasite species, reveal their phylogenetic relationships, and develop improved or new drugs and vaccines to mitigate their impact. In this review, we present a compilation of all piroplasmid species, isolates, and species complexes that infect domestic mammals and which have been well defined by molecular phylogenetic markers. Altogether, 57 taxonomic piroplasmid entities were compiled, comprising of 43 piroplasmid species, 12 well-defined isolates awaiting formal species description, and two species complexes that possibly mask additional species. The extrapolation of the finding of at least 57 piroplasmid species in only six domestic mammalian groups (cattle, sheep, goat, horse, dog, and cat) allows us to predict that a substantially higher number of piroplasmid parasites than vertebrate host species exist. Accordingly, the infection of a vertebrate host species by multiple piroplasmid species from the same and/or different phylogenetic lineages is commonly observed. Molecular phylogeny using 18S rRNA genes of piroplasmids infecting domestic mammals results in the formation of six clades, which emerge due to an anthropocentric research scope, but not due to a possibly assumed biological priority position. Scrutinizing the topology of inferred trees reveals stunning insights into some evolutionary patterns exhibited by this intriguing group of parasites. Contrary to expectations, diversification of parasite species appears to be dominated by host-parasite cospeciation (Fahrenholz's rule), and, except for piroplasmids that segregate into Clade VI, host switching is rarely observed. When only domestic mammalian hosts are taken into account, Babesia sensu lato (s.l.) parasites of Clades I and II infect only dogs and cats, respectively, Cytauxzoon spp. placed into Clade III only infect cats, Theileria placed into Clade IV exclusively infect horses, wheras Theileria sensu stricto (s.s.) of Clade V infects only cattle and small ruminants. In contrast, Babesia s.s. parasites of Clade VI infect all farm and companion animal species. We outline how the unique ability of transovarial transmission of Babesia s.s. piroplasmids of Clade VI facilitates species diversification by host switching to other host vertebrate species. Finally, a deterioration of sequence fidelity in databases is observed which will likely lead to an increased risk of artifactual research in this area. Possible measures to reverse and/or avoid this threat are discussed.

Molecular evidence of Leishmania spp. in spider monkeys (Ateles geoffroyi) from The Tuxtlas Biosphere Reserve, Veracruz, Mexico.

The black-handed spider monkey (Ateles geoffroyi) is a platyrrhine primate distributed in southern Mexico, Central America, and part of South America. Two subspecies inhabit Mexico: Ateles geoffroyi vellerosus and Ateles geoffroyi yucatanensis, both threatened with extinction. Serological evidence of exposure of spider monkeys to various groups of parasites such as Trypanosoma cruzi in México and Leishmania spp. in Brazil has been reported. The genus Leishmania encompasses about 23 species of flagellate protozoa that are transmitted by the bite of females of Phlebotominae sand flies. These parasites cause a zoonotic disease called leishmaniasis, which generates skin, mucocutaneous and/or visceral manifestations. The aim of the present study was to demonstrate the presence of Leishmania sp. in spider monkeys from the Tuxtlas Biosphere Reserve, Veracruz, Mexico. Blood samples from 10 free- ranging specimens of A. geoffroyi yucatanensis and 11 specimens in captivity of A. geoffroyi vellerosus were collected and used. The samples were subjected to a conventional Polymerase Chain Reaction test for the identification of a 116 bp fragment of a region from the kinetoplast minicircle of the parasite. Our analyzes showed that 71.4% of the sampled animals had fragment sizes compatible with Leishmania spp. The implications involve the survival of the specimens and the possibility that these primates act as sentinels of the disease. Furthermore, it is the first report suggesting the presence of Leishmania spp. in A. geoffroyi vellerosus and A. geoffroyi yucatanensis in Veracruz, Mexico.

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.

Use of the Leptospira sp. ligB C-terminus coding region as a diagnostic tool of animal leptospirosis.

Leptospirosis is the most widespread zoonosis worldwide, and it can cause reproductive failures in livestock, while in humans may vary from a mild fever to multi-organ failure and death. Due to this, in this study, we evaluated the usefulness of the segment encoding LigB C-terminus region, only present in pathogenic as target for a diagnostic PCR. This new PCR yielded a 100 % positivity for pathogenic Leptospira species and no cross-reactivity was found with intermediate or non-pathogenic species, or with other microorganisms, demostrating its high analytical specificity. The estimated analytical sensitivity was higher in serum samples than in blood or urine samples (6-9 × 102 lept/mL and 6-9 × 105 and 6-9 × 106 lept/mL, respectively). Multiple sequence alignment of the target region from different pathogenic Leptospira species confirmed that this gene region is highly conserved among these species, with few single nucleotide polymorphisms. The ligb-ct PCR here developed appears as a useful tool for the molecular diagnosis of leptospirosis.

The repertoire of serine rhomboid proteases of piroplasmids of importance to animal and human health.

Babesia, Theileria and Cytauxzoon are tick-borne apicomplexan protozoans of the order Piroplasmida, notorious for the diseases they cause in livestock, pets and humans. Host cell invasion is their Achilles heel, allowing for the development of drug or vaccine-based therapies. In other apicomplexans, cleavage of the transmembrane domain of adhesins by the serine rhomboid proteinase ROM4 is required for successful completion of invasion. In this study, we record and classify the rhomboid repertoire encoded in the genomes of 10 piroplasmid species pertaining to the lineages Babesia sensu stricto (s.s., Clade VI), Theileria sensu stricto (Clade IV), Theileria equi (Clade IV), Cytauxzoon felis (Clade IIIb) and Babesia microti (Clade I), as defined by Schnittger et al. (2012). Fifty-six piroplasmid rhomboid-like proteins were assigned by phylogenetic analysis and bidirectional best hit to the ROM4, ROM6, ROM7 or ROM8 groups, and their crucial motifs for conformation and function were identified. Forty-four of these rhomboids had either been incorrectly classified or misannotated. Babesia s.s. encode five or three ROM4 proteinase paralogs, whereas the remaining piroplasmids encode two ROM4 paralogs. All piroplasmids encode a single ROM6, ROM7 and ROM8. Thus, an increased paralog number of ROM4 is the only feature distinguishing Babesia s.s. from other piroplasmid lineages. Piroplasmid ROM6 is related to the mammalian mitochondrial rhomboid and, accordingly, N-terminal mitochondrial targeting signal sequences was found in some cases. ROM6 is the only rhomboid encoded by piroplasmids that is ubiquitous in other organisms. ROM8 represents a pseudoproteinase that is highly conserved between studied piroplasmids, suggesting that it is important in regulatory functions. ROM4, ROM6, ROM7 and ROM8 are exclusively present in Aconoidasida, which comprises piroplasmids and Plasmodium, suggesting a relevant functional role in erythrocyte invasion. The correct classification and designation of piroplasmid rhomboids presented in this study facilitates an informed choice for future in-depth study of their functions.

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.

Characterization of GASA-1, a new vaccine candidate antigen of Babesia bovis.

Surface proteins bound to the cell membrane by glycosylphosphatidylinositol (GPI) anchors are considered essential for the survival of pathogenic protozoans. In the case of the tick-transmitted hemoparasite Babesia bovis, the most virulent causative agent of bovine babesiosis, the GPI-anchored proteome was recently unraveled by an in silico approach. In this work, one of the identified proteins, GASA-1 (GPI-Anchored Surface Antigen-1), was thoroughly characterized. GASA-1 is 179 aa long and has the characteristic features of a GPI-anchored protein, including a signal peptide, a hydrophilic core and a hydrophobic tail that harbors a GPI anchor signal. Transcriptomic analysis shows that it is expressed in pathogenic and attenuated B. bovis strains. Notably, the gasa-1 gene has syntenic counterparts in B. bigemina and B. ovata, which also encode GPI-anchored proteins. This is highly unusual since all piroplasmid GPI-anchored proteins described so far have been found to be species-specific. Sequencing of gasa-1 alleles from B. bovis geographical isolates originating from Argentina, USA, Brazil, Mexico and Australia showed over 98 % identity in both nucleotide and amino acid sequences. A recombinant form of GASA-1 (rGASA-1) was generated in E. coli and anti-rGASA-1 antibodies were raised in mice. Fixed and live immunofluorescence assays showed that GASA-1 is expressed in in vitro cultured B. bovis merozoites and surface-exposed. Moreover, incubation of B. bovis in vitro cultures with anti-GASA-1 antibodies partially, but significantly, reduced erythrocyte invasion, indicating that this protein bears neutralization-sensitive antibody epitopes. Splenocytes of rGASA-1-inoculated mice showed a specific proliferative response when exposed to the recombinant protein, indicating that GASA-1 bears T-cell epitopes. Finally, sera from a group of B. bovis-infected cattle reacted with the recombinant protein, demonstrating that GASA-1 is expressed during natural infection of bovines with B. bovis, and suggesting that it is immunodominant. The high degree of conservation among B. bovis isolates and the presence of syntenic genes in other Babesia species suggest a relevant role of GASA-1 and GASA-1-like proteins for parasite survival, especially considering that, due to their surface location, they are exposed to the selection pressure of the host immune system. The highlighted features of GASA-1 make it an interesting candidate for the development of vaccines against bovine babesiosis.

Development of a loop-mediated isothermal amplification (LAMP) and a direct LAMP for the specific detection of Nosema ceranae, a parasite of honey bees.

Nosema ceranae is a ubiquitous microsporidian pathogen infecting the midgut of honey bees. The infection causes bee nosemosis, a disease associated with malnutrition, dysentery, and lethargic behavior, and results in considerable economic losses in apiculture. The use of a rapid, sensitive, and inexpensive DNA-based molecular detection method assists in the surveillance and eventual control of this pathogen. To this end, a loop-mediated isothermal amplification (LAMP) assay targeting the single-copy gene encoding the polar tube protein 3 (PTP3) has been developed. Genomic DNA of N. ceranae-infected forager bees sampled from distant geographic regions could be reliably amplified using the established LAMP assay. The N. ceranae-LAMP showed higher sensitivity than a classical reference PCR (98.6 vs 95.7%), when both approaches were applied to the detection of N. ceranae. LAMP detected a ten-fold lower infection rate than the reference PCR (1 pg vs 10 pg genomic DNA, respectively). In addition, we show highly specific and sensitive detection of N. ceranae from spore preparations in a direct LAMP format. No cross-reactions with genomic DNA and/or spores from N. apis, often co-infecting A. mellifera, or from N. bombi, infecting bumble bees, were observed. This low-cost and time-saving molecular detection method can be easily applied in simple laboratory settings, facilitating a rapid detection of N. ceranae in honey bees in epidemiological studies, surveillance and control, as well as evaluation of therapeutic measures against nosemosis.