Molecular detection of pathogens in ticks associated with domestic animals from the Colombian Caribbean region.

Tick-borne diseases constitute a problem for livestock and public health. Given the socio-economic and environmental conditions of the Colombian Caribbean, ticks are particularly abundant, in turn exposing domestic animals and people in contact with them to such diseases. This study evaluates the presence of Babesia spp., Anaplasma spp., Coxiella spp. and Borrelia spp. in domestic animal ticks (Amblyomma mixtum, A. dissimile, Dermacentor nitens, Rhipicephalus sanguineus and R. microplus) by conventional PCR. Findings show a prevalence of 12.5% of Babesia, 0% of Borrelia, 39.4% of Anaplasma and 52.9% of Coxiella, whereas 6.2% of a total sample of 104 tick pools presented coinfections between Babesia and Anaplasma. Among the molecularly identified species are Ba. vogeli, Ba. bigemina and A. marginale, in addition to two Coxiella species-one being C. mudrowiae and the other similar to an undescribed endosymbiont of Rhipicephalus sp. It is necessary to evaluate the vector capacity of ticks such as A. mixtum, D. nitens and R. sanguineus in the transmission of A. marginale. Moreover, it is necessary to explore the role that bacteria of the genus Coxiella might have both in the health of humans and animals, and in the metabolism and reproduction of ticks. This is the first report on Babesia vogeli and B. bigemina in ticks from the Colombian Caribbean, representing a risk to animal and human health.

Molecular detection of Rickettsia spp., Anaplasma platys and Theileria equi in ticks collected from horses in Tayrona National Park, Colombia.

Horses are among the domestic animals that closely interact with humans and are highly parasitized by ticks, which are the primary vectors of zoonoses. As horses in Tayrona National Natural Park (PNNT) are used as a means of transporting goods, luggage and people, they are in constant contact with wild animals, workers and tourists from different countries. These factors increase the transmission risk of hemoparasites. The purpose of this study was to determine the presence of Rickettsia sp., Anaplasma sp., and Theileria sp., in horse ticks in this protected area using conventional PCR. We collected 343 ticks of genera Amblyomma, Rhipicephalus and Dermacentor. Of the 61 samples analyzed by PCR, 18 (29.5%) individuals were positive for Rickettsia sp., 15 (24.5%) for Anaplasma sp. and 4 (6.6%) for Theileria sp. This is the first report of these hemoparasite genera in ticks associated with horses in this preserved natural area, demonstrating the importance of additional studies on the presence and epidemiology of hemoparasites and their vectors in domestic and wild animals in conserved areas with a high flow of tourists.

New insights into the systematics of the afrotropical Amblyomma marmoreum complex (Acari: Ixodidae) and the genome of a novel Rickettsia africae strain using morphological and metagenomic approaches.

The Amblyomma marmoreum complex includes afrotropical species, such as Amblyomma sparsum, a three-host tick that parasitizes reptiles, birds, and mammals, and is a recognized vector of Ehrlichia ruminantium. However, the lack of morphological, genetic and ecological data on A. sparsum has caused considerable confusion in its identification. In this study, we used microscopy and metagenomic approaches to analyze A. sparsum ticks collected from a puff adder snake (Bitis arietans) in southwest Senegal (an endemic rickettsioses area) in order to supplement previous morphological descriptions, provide novel genomic data for the A. marmoreum complex, and describe the genome of a novel spotted fever group Rickettsia strain. Based on stereoscope and scanning electron microscopy (SEM) morphological evaluations, we provide high-quality images and new insights about punctation and enameling in the adult male of A. sparsum to facilitate identification for future studies. The metagenomic approach allowed us assembly the complete mitochondrial genome of A. sparsum, as well as the nearly entire chromosome and complete plasmid sequences of a novel Rickettsia africae strain. Phylogenomic analyses demonstrated a close relationship between A. sparsum and Amblyomma nuttalli for the first time and confirmed the position of A. sparsum within the A. marmoreum complex. Our results provide new insights into the systematics of A. sparsum and A. marmoreum complex, as well as the genetic diversity of R. africae in the Afrotropical region. Future studies should consider the possibility that A. sparsum may be a vector for R. africae.

New insights into the molecular phylogeny, biogeographical history, and diversification of Amblyomma ticks (Acari: Ixodidae) based on mitogenomes and nuclear sequences.

BACKGROUND: Amblyomma is the third most diversified genus of Ixodidae that is distributed across the Indomalayan, Afrotropical, Australasian (IAA), Nearctic and Neotropical biogeographic ecoregions, reaching in the Neotropic its highest diversity. There have been hints in previously published phylogenetic trees from mitochondrial genome, nuclear rRNA, from combinations of both and morphology that the Australasian Amblyomma or the Australasian Amblyomma plus the Amblyomma species from the southern cone of South America, might be sister-group to the Amblyomma of the rest of the world. However, a stable phylogenetic framework of Amblyomma for a better understanding of the biogeographic patterns underpinning its diversification is lacking. METHODS: We used genomic techniques to sequence complete and nearly complete mitochondrial genomes -ca. 15 kbp- as well as the nuclear ribosomal cluster -ca. 8 kbp- for 17 Amblyomma ticks in order to study the phylogeny and biogeographic pattern of the genus Amblyomma, with particular emphasis on the Neotropical region. The new genomic information generated here together with genomic information available on 43 ticks (22 other Amblyomma species and 21 other hard ticks-as outgroup-) were used to perform probabilistic methods of phylogenetic and biogeographic inferences and time-tree estimation using biogeographic dates. RESULTS: In the present paper, we present the strongest evidence yet that Australasian Amblyomma may indeed be the sister-group to the Amblyomma of the rest of the world (species that occur mainly in the Neotropical and Afrotropical zoogeographic regions). Our results showed that all Amblyomma subgenera (Cernyomma, Anastosiella, Xiphiastor, Adenopleura, Aponomma and Dermiomma) are not monophyletic, except for Walkeriana and Amblyomma. Likewise, our best biogeographic scenario supports the origin of Amblyomma and its posterior diversification in the southern hemisphere at 47.8 and 36.8 Mya, respectively. This diversification could be associated with the end of the connection of Australasia and Neotropical ecoregions by the Antarctic land bridge. Also, the biogeographic analyses let us see the colonization patterns of some neotropical Amblyomma species to the Nearctic. CONCLUSIONS: We found strong evidence that the main theater of diversification of Amblyomma was the southern hemisphere, potentially driven by the Antarctic Bridge's intermittent connection in the late Eocene. In addition, the subgeneric classification of Amblyomma lacks evolutionary support. Future studies using denser taxonomic sampling may lead to new findings on the phylogenetic relationships and biogeographic history of Amblyomma genus.

Phylogenetic relationships of the Amblyomma cajennense complex (Acari: Ixodidae) at mitogenomic resolution.

The genus Amblyomma is the third most diverse in the number of species within the Ixodidae, with practically half of its species distributed in the Americas, though there are also species occurring in Africa, Asia, and Australia. Within the genus, there are several species complexes with veterinary and public health importance. The Amblyomma cajennense complex, in the Americas, is represented by six species with a wide distribution, from Texas to northern Argentina. We combined two sequencing techniques to generate complete mitogenomes of species belonging to the Amblyomma cajennense complex: genome skimming and long-range PCRs sequencing methods. Thus, we generated seven new mitochondrial genomes for all species of the Amblyomma cajennense complex, except for Amblyomma interandinum. Genetic distances between the mitogenomes corroborate the clear differentiation between the five species of the Amblyomma cajennense complex. The phylogenetic relationships of these species had previously been evaluated by combining partial nuclear and mitochondrial genes and here these relationships are corroborated with a more robust framework of data, which demonstrates that the conjunction of mitochondrial and nuclear partial genes can resolve close relationships when entire genes or genomes are unavailable. The gene order, structure, composition, and length are stable across these mitogenomes, and they share the general characteristics of Metastriata. Future studies should increase the number of available mitogenomes for this genus, especially for those species from the Indo-Pacific region and Africa, by means of a better understanding of their relationships and evolutionary process.

Molecular detection of Candidatus Rickettsia colombianensi in ticks (Acari, Ixodidae) collected from herpetofauna in San Juan de Carare, Colombia.

Knowledge about ticks and Rickettsiae in Colombia is still limited and the areas of the country where studies have been conducted are scarce. In this study, ticks (Acari, Ixodidae) associated with reptiles and amphibians in San Juan de Carare, Santander department, were morphologically and molecularly identified and tested for the presence of Rickettsia. For the molecular characterization of ticks, CO1, 12S and 16S sequences were generated and compared with other sequences available in genbank. Our analyses confirmed that the collected ticks were Amblyomma dissimile, and we provide the first report of this species parasitizing the snake Leptodeira septentrionalis. Of the samples analyzed, 69% were positive for Rickettsia sp. using the gltA, ompA and sca1 genes. Rickettsia sequences generated in this study were also compared to sequences downloaded from genbank by Bayesian inference (BI) and maximum likelihood (ML) phylogenetic analyzes. The presence of a single Rickettsia species, Candidatus Rickettsia colombianensi, was identified. This study expands the knowledge regarding the distribution of A. dissimile ticks and Rickettsiae in Colombia.

Hemogregarine and Rickettsial infection in ticks of toads from northeastern Colombia.

The toads Rhinella spp. are in constant contact with humans and domestic animals and are commonly parasitized by ticks, which are also potential vectors of pathogenic microorganisms, such as apicomplexans and rickettsia. However, little is known about microorganisms associated with toad ticks. In this work, we molecularly evaluated the presence of Rickettsia spp. and hemogregarines in ticks of Rhinella horribilis and R. humboldti in the Colombian Caribbean, finding two different species of Rickettsia: the colombianensi strain and one close to R. bellii. In the case of hemogregarines, since only 18S gene sequences are available, it is difficult to define species and place them correctly in a phylogeny, but most of our samples show a 99% identity with Hemolivia stellata, while others identical to each other seem to form another clade within this genre. All collected ticks were identified as Amblyomma dissimile, representing the first time that H. stellata was recorded in this tick. The prevalence of both microorganisms was very high, which makes it necessary to generate robust phylogenies to clarify their taxonomic diversity and to correctly define their ecological role and pathogenicity, which should be taken into account in amphibian conservation plans and veterinary medicine.

Rickettsial infection in ticks (Acari: Ixodidae) from reptiles in the Colombian Caribbean.

Although more reptiles are illegally traded in Colombia than any other group of animals, for both local and international markets, little is known about ticks associated with reptiles or pathogens associated with these ticks. In this study, ticks were collected from reptiles in Magdalena, Cesar and La Guajira regions in northern Colombia, and identified morphologically using taxonomic keys and molecularly by sequencing of the COI gene. In addition, Rickettsia spp. were detected by PCR amplifying the gltA, 16S rRNA, and sca1 genes. Ticks were identified as Amblyomma dissimile, representing the first record for this species in 17 of the 26 species of reptiles sampled. Additionally, our data expand the distribution range for this tick in Colombia. Rickettsia spp. DNA was detected in 18 ticks (3.6%) from 16 reptiles. Sequence analyses indicated that most of the Rickettsia were members of the R. monacensis clade, most closely related to Rickettsia sp. strain Colombianensi. We detected one tick infected with a Rickettsia with 99% identity to R. bellii.