Morphological descriptions of the nymph and adults of Ornithodoros clarki, the larva and nymph of Ornithodoros rondoniensis, with notes on their phylogenetic relationships.

Based on tick specimens collected recently in Mexico, Nicaragua, Panama and Brazil, we provide morphological descriptions of the nymph and adults of Ornithodoros clarki Jones & Clifford, 1972 from the first three countries, and the larva and nymph of Ornithodoros rondoniensis (Labruna, Terassini, Camargo, Brandão, Ribeiro & Estrada-Peña, 2008) from Brazil. Also, an analysis of mitochondrial 16S rDNA sequences was performed to analyze the phylogenetic relationships of these tick species. Adults and nymphs of O. clarki and O. rondoniensis are unique among the Argasidae family by presenting exceptionally large spiracular plates with small goblets, and an integument with smooth polygonal mammillae. However, these two species are morphologically distinct based on specific patterns of coxal folds, idiosomal mammillae and pilosity, and female genital flap. In contrast, the larvae of O. clarki and O. rondoniensis are morphologically identical, except for a general larger size of the former species; this slight difference is corroborated by Principal Component Analysis (PCA) by using 40 morphometric variables. Phylogenetic analyses including 16S rDNA partial sequences of different Ornithodoros taxa from Central and South America indicate that O. rondoniensis from Brazil diverges from O. clarki from Mexico, Nicaragua and Panama. However, phylogenetic distance separating both alleged species is similar or slightly lower than the distances depicted for conspecific populations of a few other Ornithodoros species. Nonetheless, our primary criterion to maintain O. rondoniensis as a valid species is because its adult and nymphal stages do present distinct morphological traits that easily distinguish these postlarval stages from O. clarki.

Redescription of the larva, and description of the nymphal and adult stages of Ornithodoros peruvianus Kohls, Clifford & Jones, 1969 (Acari: Argasidae).

The soft tick Ornithodoros peruvianus Kohls, Clifford & Jones, 1969 was described as a parasite of bats in Peru upon the examination of engorged larvae only. Recently, larvae of this tick species were reported on bats from northern Chile. However, the adult and nymphal stages of O. peruvianus have remained undescribed. This study aimed to redescribe the larva of O. peruvianus based on unfed specimens, and to describe nymphs, the male and the female of this species. Ticks were collected on the walls inside three caves in northern Chile. Two females laid eggs in the laboratory. Part of the unfed larvae was separated for morphological and morphometrical analyses, and the remaining specimens were fed upon laboratory mice in order to obtain subsequent nymphal and adult stages. The first nymphal stage (N1) moulted either to male or to a second nymphal stage (N2) without feeding. Obtained N2 moulted either to male or female after one meal. PCR amplification of tick mitochondrial 16S rRNA of specimens from the three caves revealed almost identical sequences. The unfed larva of O. peruvianus has an elongated idiosoma, and fringed setae cover the ventral surfaces of coxae, palps and tarsi. Nymph 1 has a thin integument covered by incipient mammillae and barely noticeable dorsal disks; it lacks cheeks and possesses few short setae on the basis capitulum. Nymph 2 has a pair of small cheeks and resembles adult stages in its tegumentary traits and capitulum. Adult stages exhibit developed cheeks (larger in females) without the capacity to completely cover the capitulum. Very small and low mammillae cover the surface of the dorsal idiosoma in adults. As this feature also occurs in other bat-associated soft ticks, regardless of their phylogenetic relatedness, small mammillae in bat soft ticks are suggestive of convergent evolution.

Description of Ornithodoros montensis n. sp. (Acari, Ixodida: Argasidae), a parasite of the toad Rhinella arenarum (Amphibia, Anura: Bufonidae) in the Monte Desert of Argentina.

A new tick species of the genus Ornithodoros (Acari: Argasidae) was described from larvae collected on the toad Rhinella arenarum in a locality from Argentina belonging to the Monte Biogeographic Province. Ornithodoros montensis n. sp. was described based on morphological traits and sequences of the mitochondrial 16S rRNA gene. The diagnostic characters for this species are a combination of idiosoma oval, dorsal plate pyriform with posterior margin slightly concave, dorsal surface with 17 pairs of setae (7 anterolateral, 4 to 5 central and 5 to 6 posterolateral), ventral surface with 6 pairs of setae and 1 pair on anal valves, three pairs of sternal setae, postcoxal setae absent, and hypostome pointed apically with dental formula 3/3 in the anterior half and 2/2 posteriorly almost to base. Phylogenetic analysis of 16S rDNA sequences and a principal component analysis based on morphometric characters provided additional support to the description of O. montensis as an independent lineage within the genus Ornithodoros. Larvae of O. montensis are phylogenetically closely related to O. puertoricensis, O. rioplatensis, O. talaje s.s., O. guaporensis, O. hasei and O. atacamensis, all of them belonging to the "O. talaje group".

Ornithodoros savignyi, the Tick Vector of "Candidatus Borrelia kalaharica" in Nigeria.

Endemic tick-borne relapsing fever (TBRF) has not been documented in Nigeria, yet clinically compatible cases have been described, and soft tick species are endemic in surrounding countries. Consequently, our aim was to investigate if TBRF-associated Borrelia is present in Nigeria. To address this, we examined 49 soft tick pools to identify the tick species and to screen for Borrelia The tick species was revealed by 16S rRNA gene amplification and Sanger sequencing to be Ornithodoros savignyi, an aggressive, multihost, rapidly feeding species with significant veterinary impact. We detected a Borrelia organism in 3 of 49 pooled samples (6%). Molecular analysis of amplified 16S rRNA and flagellin genes and intragenic spacer fragments disclosed that this Borrelia organism was synonymous with the recently described organism "Candidatus Borrelia kalaharica," identified in a tourist returning to Germany from South Africa. Given the widespread endemic range of this tick vector, TBRF should be considered part of the differential diagnosis for patients with fever returning from arid areas of Africa and further afield.

A morphological and phylogenetic analysis of Ornithodoros marinkellei (Acari: Argasidae), with additional notes on habitat and host usage.

Ornithodoros marinkellei was described from larvae collected on Pteronotus spp. bats in Colombia and Panama. More recently, this tick was reported in the Brazilian Amazon. Because some morphometric differences were observed between O. marinkellei larvae from Colombia and Brazil, it was proposed that further investigations were needed to assess whether the differences could be attributed to intra- or inter-specific polymorphism. Herein, we collected O. marinkellei specimens in the type locality of Colombia, in Brazil, and in a new locality in Nicaragua, expanding the distribution of the species to Nicaragua. Morphometric analysis of larvae and adults, corroborated by a principal component analysis (PCA), indicated that the Brazilian specimens were larger than specimens from Colombia and Nicaragua. Phylogenetic analysis inferred from the mitochondrial 16S rRNA gene showed ticks from Colombia and Nicaragua more genetically related than any of them with ticks from Brazil, although ticks from the three countries grouped in a clade sister to a major clade containing sequences of various Neotropical Ornithodoros species. We concluded that ticks identified as O. marinkellei from Colombia, Nicaragua, and Brazil represent the same taxon, and that the genetic and morphological differences between them are likely to have a geographical bias. We redescribed the nymph of O. marinkellei, which has a vestigial hypostome, probably incompatible with blood feeding. We also report human infestation by O. marinkellei adults. As all reports of O. marinkellei adults have been from hot caves (temperature > 35 °C), this abiotic condition could be a limiting factor for the occurrence of this tick species.

Morphological and molecular description of immature stages of Ornithodoros savignyi (Acari: Argasidae).

This study was designed to provide more details about larva, first nymph, and second nymph of Ornithodoros savignyi using a combination of light microscopy (LM), scanning electron microscopy (SEM), and partial sequence of mitochondrial 16S ribosomal ribonucleic acid (rRNA). The main characteristics of larva are wrinkled integument with many grooves, gnathosoma without camerostome cheeks, hypostome with a pair of large teeth apically, and tarsus without humps. The comparisons between the first and the second nymphs are different shape and distribution of dorsal grooves; a few spots without mammilla on the dorsal surface of the second nymph; 27 and 63-65 pairs of setae on the dorsal surface of the first and second nymphs, respectively; small holes on mammillae that are more dense in the second nymph; basis capitulum with two pairs of small setae in the second nymph; and one pair of sate in the first nymph, hypostome with dental formula 2/2 in the first nymph, and 3/3 in the second nymph. The partial 16S rRNA sequence of the second nymph that was determined as O. savignyi (450 bp) was deposited in GenBank under the accession number KU163242.

Comparative analysis of spermatids of Rhipicephalus sanguineus sensu lato (Ixodidae) and Ornithodoros rostratus ticks (Argasidae): morphophysiology aimed at systematics.

The phylogenetic relationships among tick species (Acari: Ixodida) have been revisited by several researchers over the last decades. Two subfamilies, Rhipicephalinae (Ixodidae) and Ornithodorinae (Argasidae), deserve special attention. The male reproductive system morphology, as well as the ultrastructure of the germ cells, may provide important information for phylogeny and systematics of metazoan groups, with spermatozoa exhibiting characters that can be used for this purpose. With that information in mind, this study aimed at evaluating, through a comparative analysis, the morphology of the male reproductive systems and germ cells of ticks species Rhipicephalus sanguineus and Ornithodoros rostratus. In order to do that, histology and scanning electron microscopy techniques were used. The results have shown that despite the similarities in the general morphology of the male reproductive system among studied Ixodida so far, there are morphological differences among the species studied herein, mainly the U-shaped testis (ancestral character) in O. rostratus and the pair testes (derived character) in R. sanguineus, and the general morphology of germ cells (spermatids V). Besides that, the morphological changes observed during the spermiogenesis appear to be different between the species studied here, probably characterizing the two families considered. The data generated in this study showed the importance of comparative internal morphology studies, mainly in regard to spermatology, despite the morphological data obtained herein not being enough to product a cladogram (sperm cladistics), it was already possible to observe clear differences among families Argasidae and Ixodidae in regard to the organization of their male reproductive systems and concerning the external morphology of spermatids. Data yet to be obtained through transmission electron microscopy techniques will allow the application of spermiocladistics and spermiotaxonomy as tools for tick systematics.

Ornithodoros sawaii (Ixodida: Argasidae) Larvae Collected from Hydrobates monorhis on Sogugul and Gaerin Islands, Jeollanam-do (Province), Republic of Korea.

The 65th Medical Brigade and Public Health Command District-Korea, in collaboration with the Migratory Bird Research Center, National Park Research Institute, conducted migratory bird tick surveillance at Sogugul and Gaerin Islands (small rocky bird nesting sites), Jeollanam-do (Province), Republic of Korea (ROK), on 30 July and 1 August 2009. Breeding seabirds captured by hands in their nesting burrows were banded, identified to species, and carefully examined for ticks during the nesting season. A total of 9 Ornithodoros sawaii larvae were removed from 4 adult Hydrobates monorhis (Swinhoe's storm petrel). The identification of the larvae of O. sawaii collected from migratory seabirds were molecularly confirmed using mitochondrial 16S rDNA primer sets.

Two new species of Ornithodoros (Ixodida; Argasidae) from the Southern Cone of South America.

Two new species of the genus Ornithodoros were described from larvae collected in Argentina and Chile. Ornithodoros xerophylus n. sp. was described from specimens collected on the small rodent Graomys centralis in Argentina. The diagnostic characters for this species are a combination of dorsal plate slightly oval with a length of approximately 250 µm, 16 pairs of dorsal setae, hypostome with apex rounded and dental formula 2/2 in most rows, 3/3 apically, and capsule of the Haller's organ oval in shape without reticulations. Larvae of Ornithodoros lahillei n. sp. were collected on the reptiles Philodryas chamissonis and Callopistes maculatus in Chile. The diagnostic characters for O. lahillei are a combination of dorsal plate subtriangular with margins corrugated and posterior margin convex, dorsal surface with 14 pairs of setae, absence of postcoxal setae, and hypostome with apex pointed and dental formula 3/3 in anterior third and 2/2 in the middle and basal portion. Phylogenetic analysis of 16S rDNA sequences and a Principal Component Analysis based on morphometric characters provided additional support to the description of O. lahillei and O. xerophylus as two independent lineages within the genus Ornithodoros.

Trans-oceanic host dispersal explains high seabird tick diversity on Cape Verde islands.

Parasites represent ideal models for unravelling biogeographic patterns and mechanisms of diversification on islands. Both host-mediated dispersal and within-island adaptation can shape parasite island assemblages. In this study, we examined patterns of genetic diversity and structure of Ornithodoros seabird ticks within the Cape Verde Archipelago in relation to their global phylogeography. Contrary to expectations, ticks from multiple, geographically distant clades mixed within the archipelago. Trans-oceanic colonization via host movements probably explains high local tick diversity, contrasting with previous research that suggests little large-scale dispersal in these birds. Although host specificity was not obvious at a global scale, host-associated genetic structure was found within Cape Verde colonies, indicating that post-colonization adaptation to specific hosts probably occurs. These results highlight the role of host metapopulation dynamics in the evolutionary ecology and epidemiology of avian parasites and pathogens.

A new species of soft tick from dry tropical forests of Brazilian Caatinga.

Ornithodoros tabajara n. sp. is described from laboratory-reared larvae and adult specimens collected in the Brazilian Caatinga. This new species shares the ecological niche with Ornithodoros rietcorreai and is likely associated with colonial rodents of genus Kerodon. However, O. tabajara n. sp. is morphologically easy to distinguish from O. rietcorreai and other Neotropical Ornithodoros by a unique combination of characters: larva with 17 pairs of dorsal setae (seven anterolateral, three central and seven posterolateral), sub-oval dorsal plate, hypostome blunt apically with dentition formula 2/2 along its extension, only one pair of posthypostomal setae, six pairs of sternal setae, posteromedian setae absent, and leave-shaped anal valves; alive adults with whitish islands of mammillae symmetrically distributed on dorsum (not visible in ethanol-preserved specimens), and median disk merging with posteromedian file. A phylogenetic analysis performed with mitochondrial 16S rDNA sequences points O. tabajara n. sp. as O. rietcorreai's sister taxon, which rises the hypothesis of sympatric speciation.

Nuclear (18S-28S rRNA) and mitochondrial genome markers of Carios (Carios) vespertilionis (Argasidae) support Carios Latreille, 1796 as a lineage embedded in the Ornithodorinae: re-classification of the Carios sensu Klompen and Oliver (1993) clade into its respective subgenera.

Argasid systematics remains controversial with widespread adherence to the Hoogstraal (1985) classification scheme, even though it does not reflect evolutionary relationships and results in paraphyly for the main genera of soft ticks (Argasidae), namely Argas and Ornithodoros. The alternative classification scheme, proposed by Klompen and Oliver (1993), has problems of its own: most notably paraphyly of the subgenus Pavlovskyella and the controversial grouping together of the subgenera Alectorobius, Antricola, Carios, Chiropterargas, Nothoaspis, Parantricola, Reticulinasus and Subparmatus into the genus Carios. Recent phylogenetic analyses of 18S/28S rRNA sequences and mitochondrial genomes agree with the scheme of Klompen and Oliver (1993), with regard to the paraphyly of Pavlovskyella, placement of Alveonasus, Ogadenus, Proknekalia and Secretargas in the Argasinae and placement of Carios and Chiropterargas in the Ornithodorinae (Mans et al., 2019). The Carios clade and its constituent subgenera remain controversial, since the phylogenetic position of its type species Carios (Carios) vespertilionis Latreille, 1796 (formerly Argas vespertilionis) has not been determined with confidence. The current study aimed to resolve Carios sensu lato Klompen and Oliver, 1993, and Carios sensu stricto Hoogstraal, 1985, by determining and analysing phylogenetic nuclear and mitochondrial markers for C. (C.) vespertilionis. Both the nuclear and mitochondrial markers support placement of Carios s.s. within the subfamily Ornithodorinae, but to the exclusion of the clade that includes the 6 other subgenera that are part of Carios s.l. Klompen and Oliver (1993), namely Alectorobius, Antricola, Nothoaspis, Parantricola, Reticulinasus and Subparmatus. These 6 subgenera form a monophyletic clade that might be placed as new subgenera within the genus Alectorobius, or elevated to genera. Given the substantial differences in biology among these subgenera, we propose that these 6 subgenera be elevated to genera. Thus, we propose to modify the classification scheme of Mans et al. (2019) so that the subfamily Argasinae now has six genera, Alveonasus, Argas (subgenera Argas and Persicargas), Navis, Ogadenus, Proknekalia and Secretargas, and the subfamily Ornithodorinae has nine genera, Alectorobius, Antricola (subgenera Antricola and Parantricola), Carios, Chiropterargas, Nothoaspis, Ornithodoros (subgenera Microargas, Ornamentum, Ornithodoros, Pavlovskyella and Theriodoros), Otobius, Reticulinasus and Subparmatus (genera indicated in bold).

First records and molecular-phylogenetic analyses of three tick species (Ixodes kaiseri, Hyalomma lusitanicum and Ornithodoros coniceps) from Malta.

The Maltese islands are situated south of mainland Europe and north of Africa, therefore are expected to share tick species and tick-borne pathogens with both continents. This situation highlights the importance of studying ticks in this country. Nevertheless, the tick fauna of Malta appears to be a seldom investigated issue, with hitherto only five tick species reported in the country. Here, as part of a tick collection campaign continuing since 2016 in Malta, three tick species new to the country are reported and analyzed in comparison with GenBank data. Ixodes kaiseri (collected from North African hedgehog in Malta) had unique cytochrome c oxidase subunit I (cox1) and 16S rRNA gene haplotypes (with 98.1-99.3 % sequence identity to I. kaiseri from Europe and China). Phylogenetically, these haplotypes from Malta clustered separately from other, mainland-associated haplotypes, with high support. On the other hand, Ornithodoros coniceps (collected from domestic chicken in Malta) had identical or nearly identical cox1 and 16S rRNA gene haplotypes with soft ticks reported from France, northern Africa and western African islands, similarly to Hyalomma lusitanicum (collected from rabbit and cat in Malta) in comparison with conspecific ticks in Spain and Portugal. These results are most likely related to differences in host associations and corresponding translocality of these three tick species. Taken together, results of the present study add three new tick species to those five already known to be present in Malta.

First report of Newly Identified Ornithodoros Species in the Republic of Korea.

Ticks and tick-borne diseases are important issues worldwide because of their effects on animal and human health. The genus Ornithodoros, which is included in the family Argasidae, is typically associated with wild animals, including seabirds. In this study, samples from the nests of seabirds and surrounding soil were collected to investigate Ornithodoros spp. from 9 uninhabited islands in the western, eastern, and southern parts of Korea from April 2017 to October 2018. The islands are known as the breeding places of migratory and resident birds. Ticks were collected from soil and nest material of seabirds using a Tullgren funnel and identified using 16S rRNA and the cytochrome c oxidase 1 gene (COI), and host animals of soft ticks were identified using the mitochondrial DNA cytochrome b gene by a polymerase chain reaction. In the sequence identity of the 16S rRNA gene fragment of Ornithodoros sp., Ornithodoros sawaii was identified as the closest homologous sequence, and the new Ornithodoros sp. was newly identified. We found that the newly identified Ornithodoros sp. in the Republic of Korea was located in uninhabited islands used as breeding places by the black-tailed gull, Larus crassirostris.

Ornithodoros cerradoensis n. sp. (Acari: Argasidae), a member of the Ornithodoros talaje (Guérin-Méneville, 1849) group, parasite of rodents in the Brazilian Savannah.

Ornithodoros cerradoensis n. sp. is described from field-collected and laboratory reared nymphs, males, females, and larvae parasitizing the rodents Cavia aperea and Thrichomys sp. in the Brazilian Savannah. This new species is morphologically and genetically related with the Ornithodoros talaje group and can be separated from other Neotropical species using the following combination of characters: larva with 18 pairs of setae on dorsum (seven anterolateral, four central and seven posterolateral), hypostome with median dentition 2/2; adults provided with large mammillae; dorsal disks surrounded by bulked marginal ridges delimiting barely pebbled areas; three disks in the anterolateral file, and median disk not merging with the posteromedian file. Feeding assays in the laboratory demonstrated that (1) larvae of O. cerradoensis are slow-feeders (∼6 days), (2) first nymphal instar (N1) molts to second instar (N2) without feeding, and (3) N2 and third nymphal instar (N3) engorge rapidly (minutes). With the exception of Ornithodoros hasei nymphs that depict flattened bodies, O. cerradoensis N1, N2, and N3 highly resemble homologous instars of other species in O. talaje sensu lato, therefore are not suitable for morphological comparisons within the group. In addition to morphological signature of larvae and adults that separate this new species; results of cross-mating attempts between O. cerradoensis and Ornithodoros guaporensis a morphologically and phylogenetically closely related species that also parasitizes rodents in the Brazilian Savannah; a Principal Component Analysis using larval characters; and a phylogenetic analysis using mitochondrial markers, support O. cerradoensis as an independent lineage within the Ornithodorinae.

New records and phylogenetic position of Ornithodoros knoxjonesi (Ixodida: Argasidae).

Larvae of Ornithodoros knoxjonesi collected at five localities in three countries were studied using morphological and molecular methods to confirm this species' taxonomic validity. The larva of O. knoxjonesi is characterized as having 14 pairs of dorsal setae, eight pairs of ventral setae, plus a posteromedian seta; an elongate dorsal plate, tapered anteriorly; and a hypostome that is narrower near its midlength, with posteriorly projecting denticles. Although the larvae of O. knoxjonesi and Ornithodoros peropteryx are morphologically quite similar, the larva of O. knoxjonesi is characterized as having dorsal setae that are wider at the tip than at the base, while in O. peropteryx these setae are narrower at the tip than at the base; moreover, the dorsal setae are shorter in O. knoxjonesi (Al 0.037-0.065; Pl 0.035-0.059) than in O. peropteryx (Al 0.120-0.132; Pl 0.080-0.096). These species also differ in that O. knoxjonesi possesses only the Al seta on tarsus I, whereas O. peropteryx has both Al and Pl setae. And while both species have two setae on coxae I-III, in O. knoxjonesi the anterior seta is tapering and smooth and the posterior is fringed, while both setae are fringed in O. peropteryx. At the molecular level, based on a maximum likelihood analysis using approximately 400 bp of the mitochondrial 16S rDNA gene, O. knoxjonesi appears as an independent lineage, separated from O. peropteryx by a genetic distance of 16.28 %. Balantiopteryx plicata is a common host of O. knoxjonesi; however, in this work we report Pteronotus personatus and Pteronotus gymnonotus as new hosts of this tick species.

Description of a new soft tick species (Acari: Argasidae: Ornithodoros) parasite of Octodon degus (Rodentia: Octodontidae) in northern Chile.

A new argasid (Argasidae) tick is herein described based on morphology and molecular data obtained from larvae parasitizing Octodon degus and from ticks collected inside burrows in northern Chile. Unfed laboratory-reared larvae were mounted in slides for morphometrical and morphological analyses. Larvae of Ornithodoros octodontus n. sp. share morphological traits with Ornithodoros quilinensis and Ornithodoros xerophylus, two species associated with rodents in the Argentinean Chaco. However, a longer hypostome with two rows of 21 and 22 denticles each one, and conspicuous leaf-shaped anal plates separate O. octodontus. While nymphal stages of O. octodontus lack cheeks and possess a micromammillated dorsal integument, adults have cheeks and exhibit markedly irregular mammillae along their dorsal surface. Phylogenetic analyses of neotropical Argasidae based on mitochondrial 16S rDNA sequences point that O. octodontus forms a monophyletic group with O. xerophylus and an unidentified Ornithodoros sp. from Bolivia, all of them associated with burrow-dweller rodents. Ornithodoros aragaoi and Ornithodoros davisi, two rare species collected once only in the Peruvian Andean Plateau during 1955 are morphologically closely related with adults and nymphs of O. octodontus. Biological observations of O. octodontus revealed autogenic females. For the moment, subgeneric classification of this new species depends on further biological studies. The fauna of ticks occurring in Chile is now represented by 22 species, 11 belonging to the Argasidae family.

Ornithodoros savignyi: soft tick apyrase belongs to the 5'-nucleotidase family.

Salivary apyrases are nucleotide-metabolising enzymes that blood-feeding parasites utilise for modulation of extracellular nucleotides to prevent platelet activation and aggregation. In this study a 5'-nucleotidase specific degenerate primer was used to identify homologous transcripts from Ornithodoros savignyi salivary gland cDNA. Two 5'-nucleotidase isoforms that share significant sequence identity to putative apyrases from Rhipicephalus appendiculatus and Ixodes scapularis were identified. Structure prediction showed a tertiary structure similar to periplasmic ecto-5'-nucleotidase from Escherichia coli, with high conservation of functional residues. The O. savignyi 5'-nucleotidase isoform I was recombinantly expressed in Pichia pastoris. Cross-reactivity was demonstrated with polyclonal anti-apyrase antisera produced against O. savignyi apyrase. Subsequent Edman sequencing and MS/MS analysis of purified O. savignyi apyrase identified peptide sequence fragments that shared sequence identity with both newly identified 5'-nucleotidase isoforms. It was concluded that wild-type apyrase is a mixture of the isoforms identified from the salivary glands of O. savignyi. These results represent the first confirmation of a soft (argasid) tick apyrase that belongs to the 5'-nucleotidase family of enzymes.

In silico selection of functionally important proteins from the mialome of Ornithodoros erraticus ticks and assessment of their protective efficacy as vaccine targets.

BACKGROUND: New candidate protective antigens for tick vaccine development may be identified by selecting and testing antigen candidates that play key biological functions. After blood-feeding, tick midgut overexpresses proteins that play essential functions in tick survival and disease transmission. Herein, Ornithodoros erraticus midgut transcriptomic and proteomic data were examined in order to select functionally significant antigens upregulated after feeding to be tested as vaccine candidate antigens. METHODS: Transcripts annotated as chitinases, tetraspanins, ribosomal protein P0 and secreted proteins/peptides were mined from the recently published O. erraticus midgut transcriptome and filtered in a second selection step using criteria based on upregulation after feeding, predicted antigenicity and expression in the midgut proteome. Five theoretical candidate antigens were selected, obtained as recombinant proteins and used to immunise rabbits: one chitinase (CHI), two tetraspanins (TSPs), the ribosomal protein P0 (RPP0) and one secreted protein PK-4 (PK4). RESULTS: Rabbit vaccination with individual recombinant candidates induced strong humoral responses that mainly reduced nymph moulting and female reproduction, providing 30.2% (CHI), 56% (TSPs), 57.5% (RPP0) and 57.8% (PK4) protection to O. erraticus infestations and 19.6% (CHI), 11.1% (TSPs), 0% (RPP0) and 8.1% (PK4) cross-protection to infestations by the African tick Ornithodoros moubata. The joint vaccine efficacy of the candidates was assessed in a second vaccine trial reaching 66.3% protection to O. erraticus and 25.6% cross-protection to O. moubata. CONCLUSIONS: These results (i) indicate that argasid chitinases and RPP0 are promising protective antigens, as has already been demonstrated for ixodid chitinases and RPP0, and could be included in vaccines targeting multiple tick species; (ii) reveal novel protective antigens tetraspanins and secreted protein PK-4, never tested before as protective antigens in ticks; and (iii) demonstrate that multi-antigenic vaccines increased vaccine efficacy compared with individual antigens. Lastly, our data emphasize the value of the tick midgut as a source of protective candidate antigens in argasids for tick control.

Comparative vector competence of the Afrotropical soft tick Ornithodoros moubata and Palearctic species, O. erraticus and O. verrucosus, for African swine fever virus strains circulating in Eurasia.

African swine fever (ASF) is a lethal hemorrhagic disease in domestic pigs and wild suids caused by African swine fever virus (ASFV), which threatens the swine industry globally. In its native African enzootic foci, ASFV is naturally circulating between soft ticks of the genus Ornithodoros, especially in the O. moubata group, and wild reservoir suids, such as warthogs (Phacochoerus spp.) that are bitten by infected soft ticks inhabiting their burrows. While the ability of some Afrotropical soft ticks to transmit and maintain ASFV is well established, the vector status of Palearctic soft tick species for ASFV strains currently circulating in Eurasia remains largely unknown. For example, the Iberian soft tick O. erraticus is a known vector and reservoir of ASFV, but its ability to transmit different ASFV strains has not been assessed since ASF re-emerged in Europe in 2007. Little is known about vector competence for ASFV in other species, such as O. verrucosus, which occurs in southern parts of Eastern Europe, including Ukraine and parts of Russia, and in the Caucasus. Therefore, we conducted transmission trials with two Palearctic soft tick species, O. erraticus and O. verrucosus, and the Afrotropical species O. moubata. We tested the ability of ticks to transmit virulent ASFV strains, including one of direct African origin (Liv13/33), and three from Eurasia that had been involved in previous (OurT88/1), and the current epizooties (Georgia2007/1 and Ukr12/Zapo). Our experimental results showed that O. moubata was able to transmit the African and Eurasian ASFV strains, whereas O. erraticus and O. verrucosus failed to transmit the Eurasian ASFV strains. However, naïve pigs showed clinical signs of ASF when inoculated with homogenates of crushed O. erraticus and O. verrucosus ticks that fed on viraemic pigs, which proved the infectiousness of ASFV contained in the ticks. These results documented that O. erraticus and O. verrucosus are unlikely to be capable vectors of ASFV strains currently circulating in Eurasia. Additionally, the persistence of infection in soft ticks for several months reaffirms that the infectious status of a given tick species is only part of the data required to assess its vector competence for ASFV.