Molecular detection of Candidatus Ehrlichia pampeana (Rickettsiales: Anaplasmataceae) in Haemaphysalis juxtakochi (Acari: Ixodidae) from central Argentina.

The aim of this study was to report the first detection of Candidatus Ehrlichia pampeana in Haemaphysalis juxtakochi from Argentina. Free-living ticks were collected from vegetation by drag-flag method on five sample sites in Entre Ríos Province, central Argentina, belonging to the Espinal Phytogeographic Province. Molecular detection of order Rickettsiales agents was performed using gltA (Rickettsia spp.), 16 S rRNA and groEL (Anaplasmataceae) genes as targets. A total of 67 ticks of Amblyomma aureolatum (20 nymphs and 4 adults), Amblyomma dubitatum (15 nymphs) and Haemaphysalis juxtakochi (24 nymphs and 4 adults) were collected. While all tested ticks were negative for Rickettsia spp., and Anaplasmataceae agents could neither be detected in A. aureolatum nor in A. dubitatum, Candidatus Ehrlichia pampeana was detected in one male of H. juxtakochi. DNA sequences of this microorganism (16 S rDNA and groEL) are related to sequences of Ehrlichia ewingii. The findings of the current study represent the first report of this Ehrlichia strain for Argentina.

Novel Anaplasma (Rickettsiales: Anaplasmataceae) strain and Hepatozoon sp. cf. H. procyonis (Apicomplexa, Hepatozoidae) detected in Procyon cancrivorus (Carnivora, Procyonidae) from Argentina, with note of tick-host association.

The aim of this work is to report the first detection of Procyon cancrivorus naturally co-infected with Hepatozoon sp. cf. H. procyonis and a novel Anaplasma strain from South America and potential vector tick species associated. On August 30, 2016, a specimen of P. cancrivorus was found dead on the route in Chaco province, Argentina. A tick and a blood sample by cardiac puncture was collected from the specimen. DNA was extracted from blood sample and the tick was morphological identity as a female of Amblyomma ovale. Molecular detection of Anaplasmataceae family and Hepatozoon spp. agents was performed targeting two different loci: 16 S rRNA and 18 S rRNA gene. The phylogenetic analyses show that the Anaplasma sp. strain detected in P. cancrivorus in this study is similar to Anaplasma sp. strains previously detected in Nasua nasua and A. ovale from Brazil. Furthermore, Hepatozoon sp. of the H. procyonis group was amplified that is phylogenetically closely related to H. procyonis reported in N. nasua from Brazil. Since it was not exactly the same as the latter, it was decided to name at Hepatozoon sp. cf. H. procyonis. It is possible that, this potential new species of Anaplasma would be specific for Procyonidae family and there are two species of Hepatozoon linked to this family in South America. These results added to other published studies suggest that A. ovale could be a potential vector both for the new potential strain of Anaplasma and for the Hepatazoon sp. of the H. procyonis group.

Analysis of the tick communities associated to domestic mammals in rural areas of the Yungas montane forest from Argentina.

The aim of this work was to describe the tick community associated to domestic mammals in rural areas from the Yungas lower montane forest of Argentina. The circulation of tick-borne pathogens was also analyzed. Samples of ticks parasitizing cattle, horses, sheep and dogs were carried out in different seasons, and questing ticks were collected from vegetation and analyzed to detect the presence of Rickettsia, Ehrlichia, Borrelia and Babesia by a battery of different PCRs. The structure of the tick communities was analyzed through the Chao1 species richness estimator, the Shannon-Wiener index and the Horn index of community similarity. Eight tick species were collected in the study area: Amblyomma sculptum, Rhipicephalus microplus, Amblyomma hadanii, Dermacentor nitens, Amblyomma ovale, Haemaphysalis juxtakochi, Ixodes pararicinus and Rhipicephalus sanguineus sensu stricto. However, A. sculptum was by far the dominant species in the tick assemblages analyzed, and this was reflected in the low diversity values obtained. Dermacentor nitens, A. sculptum and R. microplus were the three species associated to horses. The predominance of A. sculptum was also observed in the tick samples obtained from dogs, even on two tick species, namely A. ovale and R. sanguineus s.s., which have dogs as the principal domestic host. Rhipicephalus microplus and A. sculptum were the most abundant ticks on cattle, while few specimens of I. pararicinus, A. hadanii and D. nitens were found on bovines. Dermacentor nitens ticks were found to be infected with B. caballi, which indicate the circulation of this pathogen of horses in the Yungas area. The detection of a strain of Borrelia sp. belonging to the B. burgdorferi s.l. complex in I. pararicinus is consistent with previous findings made in Argentina, but the public health relevance of this vector-microorganism association is far from being similar to that occurs in the northern hemisphere because there are practically no records of these tick species parasitizing humans in South America. The tick community of rural areas of the Yungas lower montane forest is composed by species which are potential vectors of pathogenic microorganism with veterinary and public health importance, circulating in a human-wildlife-livestock interface.

Ticks (Acari: Ixodidae, Argasidae) associated with wild birds in Argentina.

The aim of this study was to report tick infestations on wild birds from four Phytogeographic Provinces of Argentina. A total of 1085 birds was captured (124 species, 97 genera, 29 families and 13 orders), and ticks were collected from 265 birds (48 species, 40 genera and five orders). A total of 1469 ticks (1102 larvae, 363 nymphs and 4 females) belonging to 15 tick species (Amblyomma calcaratum, Amblyomma dubitatum, Amblyomma nodosum, Amblyomma ovale, Amblyomma parvum, Amblyomma sculptum, Amblyomma tigrinum, Amblyomma triste, Haemaphysalis juxtakochi, Haemaphysalis leporispalustris, Ixodes auritulus sensu lato, Ixodes pararicinus, Ixodes silvanus, Ixodes sp. cf. I. affinis and Ornithodoros sp. cf. O. mimon). Eighty-one new associations between bird species and stages of tick species are detected. The families Thamnophilidae, Turdidae, Thraupidae, Passerellidae, Furnariidae and Troglodytidae were the most prevalent. According to the Phytogeographic Provinces involved in this study, the prevalence of infection for each of them in birds was: (1) Chaco: 28.2% (11 tick species); (2) Yungas: 22.0% (8 tick species); (3) Espinal: 11.1% (2 tick species); and (4) Pampa: 3.9% (1 tick species). This study provided information on the diversity of tick species that parasitize wild birds, the variability of the specific tick-bird associations between the different Phytogeographic Provinces and the relevance of some families of birds as hosts of different tick species.

Two novel Ehrlichia (Rickettsiales: Anaplasmataceae) strains detected in ticks (Ixodida, Ixodidae) and opossums (Didelphimorphia: Didelphidae) in Argentina.

The aim of this study is to determine if there is circulation of microorganisms of the genus Ehrlichia in opossums Didelphis albiventris and their ticks from the Humid Chaco in Argentina. Blood samples of 15 specimens of the opossum D. albiventris were analysed. Immature stages of the ticks Amblyomma ovale (Larvae=26; Nymphs=10), Amblyomma sculptum (Larvae=86; Nymphs=6) and Ornithodoros sp. cf. O. mimon (Larvae=90) were also analyzed. DNA was extracted individually from blood samples and ticks. Molecular detection of Ehrlichia agents was performed targeting two different loci: 16S rRNA and dsb gen. The phylogenetic analyses showed that the Ehrlichia sp. detected in D. albiventris in this study is identical to Ehrlichia sp. strain Natal previously detected in two marsupials from Brazil. Furthermore, a new Ehrlichia strain was amplified from an A. ovale nymph (named as Ehrlichia sp. strain El Bagual) which is phylogenetically closely related to a strain of Ehrlichia sp. detected in Bradypus tridactylus in Brazil. The findings of the current study represent the first report of these two strains of Ehrlichia for Argentina, showing that the diversity of Ehrlichia spp. is greater than previously assumed. Further studies should determine the epidemiological relevance of these findings.

Reestablishment of Rhipicephalus secundus Feldman-Muhsam, 1952 (Acari: Ixodidae).

Rhipicephalus secundus is reestablished as a valid tick name within the Rhipicephalus sanguineus group and removed from the synonymy list of Rhipicephalus turanicus. Morphological re-description of both male and female of R. secundus and the analysis of its phylogenetic position based on mitochondrial DNA sequences are presented. The morphological re-description was made with tick specimens collected on goat in Israel. The phylogenetic analyses showed that R. secundus belong to a different clade from those formed by R. turanicus sensu stricto (s.s.) and R sanguineus s.s., and by other taxa from the R. sanguineus group. Rhipicephalus secundus is morphologically related to R. turanicus, but the scutal punctation pattern of both male and female allows the morphological differentiation between R. secundus and R. turanicus, punctations being clearly more numerous and larger in the latter. Both male and female of R. secundus can be differentiated from those of R. sanguineus s.s. by the shape of the spiracular plate. In males, the dorsal prolongation of the spiracular plate is equal to the breadth of the adjacent festoon in R. secundus, while it is narrower than the breadth of the adjacent festoon in R. sanguineus s.s. The dorsal prolongation of the spiracular plate in the female of R. secundus is wider than in the female of R. sanguineus s.s. The genital apertures of the females of R. secundus and R. sanguineus are both U-shaped, but in R. sanguineus s.s. it is broader than in R. secundus. Considering the results obtained in this study, it can be stated that R. secundus is present at least in Israel, Palestinian Territories, Turkey, Albania and southern Italy, but it is necessary to carry out additional studies to determine the geographical range and host usage of this species.

Molecular detection and phylogenetic characterization of Theileria equi in horses (Equus caballus) from a peri-urban area of Argentina.

To investigate the presence of Theileria equi in an endemic area of equine piroplasmosis 42 horses (Equus caballus) from Corrientes City, Argentina were sampled. Eighty-one percent (34 blood samples) of the analyzed horses were tested positive to the presence of piroplasmid 18S rDNA. All these samples could be identified as T. equi by amplifying the specific EMA-1 (merozoite antigen 1) gene of this species. Phylogenetic analysis of an obtained 18S rDNA complete sequence from one strain resulted in the identification of this sample as T. equi sensu stricto (genotype A). This study presents the first molecular detection and characterization of T. equi by the complete 18S rDNA sequence in Argentina. Based on these results further studies should be carried out to investigate the distribution and heterogeneity of presented genotypes of T. equi in Argentina, which is essential for the diagnosis, prevention and treatment of equine piroplasmosis.

Exotic tick detected in Argentina on a tourist returning from South Africa.

The aim of this study was to report the finding of a nymph attached to an Argentinean tourist returning from South Africa. The nymph specimen was morphologically analysed, submitted to DNA extraction and amplifying the 16S rRNA mitochondrial gene. Additionally, the nymph DNA was screened for Rickettsia, Ehrlichia and Anaplasma infection. The nymph was determined to belong to Amblyomma marmoreum species complex. No specific diagnosis was achieved because the comparative descriptions of species in this complex contain important discordances, and the DNA sequence obtained in the present study is positioned within the same clade with sequences of A. marmoreum see above, but the genetic divergence with them (4.96 and 5.76%) indicate that they belong to different species. No DNA of the Rickettsiales order bacterial was detected in the A. marmoreum species complex nymph.

Epidemiological link between canine monocytic ehrlichiosis caused by Ehrlichia canis and the presence of Rhipicephalus sanguineus sensu stricto in Argentina.

In this work, we analyze data that support an epidemiological link between cases of canine monocytic ehrlichiosis (CME) by Ehrlichia canis and the presence of Rhipicephalus sanguineus sensu stricto as vector in an endemic area for this tick in Argentina. In a blood sample of a 1-year-old toy poodle with CME compatible clinical signs, which showed CME typical morulae in monocytes in Giemsa-stained blood smear, DNA of E. canis was detected by PCR. Further, DNA of E. canis was also detected in a female of R. sanguineus s.s. collected on the infected dog. Rhipicephalus sanguineus s.s. is the only member of the R. sanguineus group that prevails in the study area. The results of this study suggest that R. sanguineus s.s. may play a more important role in the transmission of E. canis than it was assumed so far. The epidemiological link between CME cases and R. sanguineus s.s. as vector in temperate areas of Argentina described in this work contrast previous studies which found that R. sanguineus sensu lato "tropical lineage" (which is absent in the study area) is competent to transmit E. canis but not R. sanguineus s.s.

Borrelia genospecies in Ixodes sp. cf. Ixodes affinis (Acari: Ixodidae) from Argentina.

The aim of this work was to evaluate the presence of Borrelia infection in Ixodes sp. cf. Ixodes affinis ticks from Argentina. Specimens of Ixodes sp. cf. I. affinis were collected on vegetation and birds in five locations belonging the most humid part of the Chaco Biogeographic Province. Specimens were tested for Borrelia infection by nested-PCR targeting the flaB gene and the rrfA-rrlB intergenic spacer region (IGS), sequenced and phylogenetically analyzed. A total of 48 Ixodes sp. cf. I. affinis (12 questing adults from vegetation and 20 nymphs and 16 larvae on nine bird species: Arremon flavirostris, Basileuterus culicivorus, Campylorhamphus trochilirostris, Myiothlypis leucoblephara, Tachyphonus rufus, Thlypopsis sordida, Turdus amaurochalinus, Turdus rufiventris and Troglodytes aedon) were collected. Twelve adults, 14 nymphs and 11 larvae (3 individually and 8 in 3 pools) were analyzed. Partial sequences were detected in 6 adults, 11 nymphs and 4 larvae (2 individual and 2 pools). Phylogenetically, the Borrelia found in Ixodes sp. cf. I. affinis belongs to the B. burgdorferi sensu lato (s. l.) complex. The partial sequences obtained from the borrelian gene flaB and IGS were associated to two groups formed by sequences previously detected in Ixodes fuscipes, Ixodes longiscutatus and Ixodes pararicinus from the Southern Cone of America in northern Argentina, southern Brazil and Uruguay. The results of this work suggest that the haplotypes of B. burgdorferi s. l. complex detected in the three species of the I. ricinus complex distributed in the Southern Cone of America are related and widely distributed.

Molecular detection of rickettsial bacteria in ticks of the genus Ixodes from the Southern Cone of America.

To obtain information about rickettsial bacteria in ticks of the genus Ixodes from Argentina and Uruguay, specimens of I. fuscipes (previously named as I. aragaoi), Ixodes pararicinus, Ixodes sp. cf. I. affinis, and Ixodes sp. were tested targeting the rickettsial gltA and ompA genes. Rickettsial bacteria was detected in all of these species. Rickettsia found in Ixodes sp. is closely related to Ca. Rickettsia mendelii, which was previously detected in I. ricinus of the Czech Republic and later in I. brunneus from the USA. Phylogenetic analyses of the Rickettsia strains found in I. fuscipes, I. pararicinus, and Ixodes sp. cf. I. affinis showed that these strains form a clade together with R. buchneri detected in I. scapularis from the USA, which is closely related to R. monacensis from Europe. Rickettsia buchneri, R. cooleyi and the Rickettsia detected in I. fuscipes, I. pararicinus, and Ixodes sp. cf. I. affinis clustered together in a clade well supported, which suggest that they are different strains of R. buchneri. The phylogenetic analysis shows that Ixodes ticks that are closely related in evolutionary terms (i.e. Ixodes species from the I. ricinus complex, I. brunneus-Ixodes sp.) share closely related rickettsial strains. The results of this study show that rickettsial bacteria are present in Ixodes ticks from Argentina and describe the first detection of Ca. R. mendelii in South America.

Seasonal dynamics, geographical range size, hosts, genetic diversity and phylogeography of Amblyomma sculptum in Argentina.

The aim of this work was to generate knowledge on ecological aspects of Amblyomma sculptum in Argentina, such as seasonal dynamics, geographical range size, hosts, genetic diversity and phylogeography. Adult and immature A. sculptum ticks were collected in different localities of Argentina to know the geographical range size and hosts. The genetic diversity of this tick was studied through analyses of 16S rDNA sequences. To describe the seasonal dynamics, free-living ticks were monthly collected from October 2013 to October 2015. A. sculptum shows a marked ecological preference for Chaco Húmedo eco-region and "Albardones" forest of the great rivers in the wetlands in the Chaco Biogeographical Province, and for Selvas Pedemontanas and Selva Montana in the Yungas Biogeographical Province. This species has low host specificity, and it has large wild and domestic mammals as principal hosts to both immature and adult stages. Amblyomma sculptum is characterized by a one-year life cycle. Larvae peak in early winter, nymphs peaked during mid-spring, and adults during late summer and mid-summer. The genetic divergence was low and the total genetic variability was attributable to differences among populations. This fact could be associated to stochastics process linked to micro-habitat variations that could produce a partial restriction to gene flow among populations. The geographic regions do not contribute much to explain the A. sculptum population genetic structure, with an ancestral haplotype present in most populations, which gives rise to the rest of the haplotypes denoting a rapid population expansion.

Ticks infesting cattle and humans in the Yungas Biogeographic Province of Argentina, with notes on the presence of tick-borne bacteria.

This study was performed to determine the tick species that infest cattle and humans throughout an altitudinal gradient in the Yungas Biogeographic Province of Argentina. The presence of tick-borne bacteria of the genera Rickettsia, Ehrlichia and Borrelia in the collected ticks was also evaluated. Samples of ticks parasitizing cattle and humans were carried out in different seasons. Questing ticks (adults and nymphs) were collected from vegetation and analyzed to detect the presence of Rickettsia, Ehrlichia and Borrelia by a battery of different PCRs. Five species of hard ticks were found parasitizing cattle: Amblyomma sculptum, Amblyomma tonelliae, Amblyomma hadanii, Haemaphysalis juxtakochi and Ixodes pararicinus. Amblyomma sculptum (immature and adults), A. tonelliae (immature and adults), A. hadanii (larvae) and one nymph of I. pararicinus were found attached to humans. Rickettsia amblyommatis was detected in one nymph of A. hadanii. DNA of a Borrelia genospecies belonging to the B. burgdorferi s.l. complex (phylogenetically related to haplotypes previously reported in Ixodes aragaoi from Uruguay and I. pararicinus from Argentina) was detected in adults of I. pararicinus. Amblyomma sculptum and I. pararicinus appear to be the tick species more frequent on cattle in the YBP from Argentina, and A. sculptum and A. tonelliae, were the main ticks found attached to humans. The medical importance of the bacteria of the genus Rickettsia and Borrelia detected in this work remains unknown.

Presence of Borrelia in different populations of Ixodes pararicinus from northwestern Argentina.

This work was performed to evaluate the presence of Borrelia in different populations of Ixodes pararicinus from northwestern Argentina (Jujuy, Salta and Tucumán provinces). Questing adults and nymphs of I. pararicinus were collected from vegetation, and I. pararicinus nymphs were also collected on birds. Eighty-two ticks were tested for Borrelia presence by PCR targeting the gene flagellin and the rrfA-rrlB intergenic spacer region. Pools of ticks positive to Borrelia were formed by two nymphs collected on Turdus rufiventris in Tucumán, one nymph collected on Syndactyla rufosuperciliata in Jujuy, one nymph collected on Turdus nigriceps in Tucumán, three nymphs collected on T. nigriceps in Tucumán, and two females collected from vegetation in Salta. Two haplotypes of Borrelia sp. belonging to the Borrelia burgdorferi sensu lato complex were found. One of them is closely related to the haplotypes of Borrelia genospecies previously reported in I. aragaoi from Uruguay (haplotypes D and E) and in I. pararicinus from Jujuy Province in Argentina. The second haplotype (detected in the sample of Salta) is closely related to the haplotypes A, B and C associated with I. aragaoi from Uruguay. All these results suggest that the presence of B. burgdorferi s.l. genospecies in I. pararicinus ticks is widespread along the entire distribution of this tick species in northwestern Argentina. However, the Borrelia presence in I. pararicinus cannot be directly assumed as a phenomenon of medical relevance, because Ixodes ticks are not relevant as human parasites in South America, and none of the two Borrelia genospecies detected in this work is related to any of the Borrelia genospecies currently known to be pathogenic to humans.

Bacteria of the genera Ehrlichia and Rickettsia in ticks of the family Ixodidae with medical importance in Argentina.

The aim of this study was to get an overview about the occurrence of bacteria from the genus Ehrlichia and Rickettsia in ixodid ticks with medical importance in Argentina. Therefore, in 2013 and 2014, free-living ticks were collected in different provinces of northern Argentina. These ticks were determined as Amblyomma sculptum, Amblyomma neumanni, Amblyomma parvum, Amblyomma triste, Amblyomma ovale, Amblyomma tonelliae and Haemaphysalis juxtakochi. All samples were tested to determine the infection with Ehrlichia spp. and Rickettsia spp. by PCR assays. Rickettsial DNA was detected in all tested tick species, with the exception of A. tonelliae. 'Candidatus Rickettsia amblyommii', 'Candidatus Rickettsia andeanae', and Rickettsia parkeri were found in A. neumanni, A. parvum, and A. triste, respectively. Another rickettsial species, Rickettsia bellii, was found in A. sculptum, A. ovale and H. juxtakochi. None of the tested ticks showed infection with Ehrlichia. The results of the study demonstrate that Rickettsia species belonging to the spotted fever group are associated with various species of Amblyomma throughout a wide area of northern Argentina, where cases of Amblyomma ticks biting humans are common.

Borrelia burgdorferi sensu lato in Ixodes cf. neuquenensis and Ixodes sigelos ticks from the Patagonian region of Argentina.

This study was conducted to detect Borrelia burgdorferi sensu lato infection in ixodid ticks from the Patagonia region in the south of Argentina. Therefore, ticks were collected on rodents in the provinces of Chubut, Río Negro and Santa Cruz. These ticks were identified as nymphs of Ixodes cf. neuquenensis and Ixodes sigelos. The B. burgdorferi s.l. infection was tested by a battery of PCR methods targeting the gene flagellin (fla) and the rrfA-rrlB intergenic spacer region (IGS). Three pools of I. sigelos nymphs from Chubut and Santa Cruz provinces as well as one pool of I. cf. neuquenensis nymphs from Río Negro province were tested positive in the fla-PCR. The samples of I. sigelos were also positive for the IGS-PCR. Phylogenetically, the haplotypes found in the positive ticks belong to the B. burgdorferi s.l. complex, and they were closely related to Borrelia chilensis, a genospecies isolated from Ixodes stilesi in Chile. The pathogenic relevance of the Borrelia genospecies detected in both I. neuquenensis and I. sigelos is unknown.