Tick-borne microorganisms in Amblyomma tigrinum (Acari: Ixodidae) from the Patagonian region of Argentina.

This study presents the results of the molecular detection of tick-borne microorganisms in Amblyomma tigrinum Koch collected near the city of Viedma, Río Negro, Argentina. Ticks were collected in their non-parasitic stage, on pet dogs and on Lycalopex gymnocercus (Pampa fox). Also, six tick samples from humans were analyzed. All ticks were morphologically identified to species level and genomic DNA was extracted. The DNA samples were examined by end point PCR assays to amplified DNA of Anaplasma sp., Babesia sp., Ehrlichia sp., Rickettsia sp. and Theileria sp. Although all tested DNA samples from the collected ticks resulted negative to the detection of Piroplasmida and Rickettsia spp., 16 samples (16.5%, including all hosts) were positive in the 16S rDNA gene PCR that detects bacteria from the Anaplasmataceae family. Phylogenetic analysis of seven obtained partial sequences resulted in the identification of three bacteria: two Ehrlichia spp. (related to Ehrlichia sp. strain Iberá and strain Viedma) and Candidatus Anaplasma boleense. The latter finding represents the first detection of this novel Candidatus species in A. tigrinum. Based on the results of this study, it must be assumed that the diversity of bacteria of the Anaplasmataceae family in Argentina is greater than previously thought, and that these bacteria can infect a wide range of domestic and wild animals.

Preliminary Study on Artificial versus Animal-Based Feeding Systems for Amblyomma Ticks (Acari: Ixodidae).

Hard ticks pose a threat to animal and human health. Active life stages need to feed on a vertebrate host in order to complete their life cycle. To study processes such as tick-pathogen interactions or drug efficacy and pharmacokinetics, it is necessary to maintain tick colonies under defined laboratory conditions, typically using laboratory animals. The aim of this study was to test a membrane-based artificial feeding system (AFS) applicable for Amblyomma ticks using Amblyomma tonelliae as a biological model. Adult ticks from a laboratory colony were fed in a membrane-based AFS. For comparison, other A. tonelliae adults were fed on calf and rabbit. The proportions of attached (AFS: 76%; calf/rabbit: 100%) and engorged females (AFS: 47.4%; calf/rabbit: 100%) in the AFS were significantly lower compared to animal-based feeding (p = 0.0265). The engorgement weight of in vitro fed ticks (x¯ = 658 mg; SD ± 259.80) did not significantly differ from that of ticks fed on animals (p = 0.3272, respectively 0.0947). The proportion of females that oviposited was 100% for all three feeding methods. However, the incubation period of eggs (x¯ = 54 days; SD ± 7) was longer in the AFS compared to conventional animal-based feeding (p = 0.0014); x¯ = 45 days; SD ± 2 in the rabbit and (p = 0.0144). x¯ = 48 days; SD ± 2 in the calf). Egg cluster hatching (x¯ = 41%; SD ± 44.82) was lower in the AFS than in the other feeding methods (rabbit: x¯ = 74%; SD ± 20; p = 0.0529; calf: x¯ = 81%; SD ± 22; p = 0.0256). Although the attachment, development, and the hatching of AFS ticks were below those from animal-based feeding, the method may be useful in future experiments. Nevertheless, further experiments with a higher number of tick specimens (including immature life stages) and different attractant stimuli are required to confirm the preliminary results of this study and to evaluate the applicability of AFS for Amblyomma ticks as an alternative to animal-based feeding methods.

Molecular detection and phylogenetic analysis of Anaplasma platys-like and Candidatus Anaplasma boleense strains from Argentina.

The present study aimed at the molecular detection of Anaplasma spp. in different samples obtained from cattle, goats and free-living Rhipicephalus microplus ticks from Argentina. DNA of members of the Anaplasmataceae family was detected by different PCR assays. The phylogenetic analyses of the obtained partial DNA sequences of the 16 S rDNA gene resulted in the identification of two different Anaplasma spp.: (I) Anaplasma platys-like bacteria (in blood sample from cattle and pools of R. microplus larvae and (II) Candidatus Anaplasma boleense (in blood samples from goats and one pool of R. microplus larvae of R. microplus). Candidatus A. boleense was found in two provinces that belong to different biogeographic regions, which leads to the conclusion that this bacterium may be widely distributed in Argentina. Interestingly, both Anaplasma spp. were found in the same R. microplus population in Chaco province, indicating that these two strains of Anaplasma are circulating in the same tick population. The results of this work represent the first report of the circulation of A. platys-like bacteria and Ca. A. boleense in domestic ruminants and free-living R. microplus ticks in Argentina. Further studies to determine the prevalence of infection, dispersion, clinical impact, transmission routes and cross-reactivity in serological tests of both Anaplasma species are needed.

Molecular Detection of Candidatus Rickettsia andeanae and Ehrlichia sp. in Amblyomma pseudoconcolor Aragão, 1908 (Acari: Ixodidae) from the Argentinian Patagonia.

This study presents the molecular detection of Candidatus Rickettsia andeanae and Ehrlichia sp. in Amblyomma pseudoconcolor Aragão, 1908 (Acari: Ixodidae) collected on a large hairy armadillo (Chaetophractus villosus (Desmarest, 1804)). On 12 October 2020, a specimen of C. villosus was found dead on the road in Río Negro province, Argentina. Molecular detection of Rickettsia and Ehrlichia agents was performed amplifying the gltA and 16S rRNA gene, respectively. One tick, determined morphologically and genetically as A. pseudoconcolor, was collected on C. villosus. The rickettsial agent detected in A. pseudoconcolor was identified as Candidatus Rickettsia andeanae. The Ehrlichia sp. strain showed high sequence similarity to different uncultured Ehrlichia sp. detected in horses, capybaras and Ixodes ornithorhynchi from Nicaragua, Brazil and Australia, respectively. The results of this study and previous findings suggest that A. pseudoconcolor may be a potential vector of some Rickettsia and Ehrlichia bacteria of unknown pathogenicity.

Assays to evaluate the transovarial transmission of Anaplasma marginale by Rhipicephalus microplus.

The aim of this study was to evaluate the vectorial competence of Rhipicephalus microplus to transmit Anaplasma marginale transovarially, by analyzing the results of three different but complementary assays. First, larvae of R. microplus were fed on a calf infected with the isolate S1P of A. marginale. The engorged females obtained were analyzed by PCR and incubated for oviposition. After hatching, larvae were analyzed by PCR and fed on susceptible splenectomized cattle. Although A. marginale was detected in the females, no A. marginale DNA was amplified from the larvae and transmission of A. marginale to cattle was not recorded. In the second experiment, R. microplus larvae were fed on cattle naturally infected with field isolates of A. marginale and experimentally infected with the isolate S1P of A. marginale. After detachment, engorged females were incubated for oviposition. The offspring were analyzed by PCR, with negative results. Finally, free-living larvae of R. microplus collected from pasture on farms with cattle infected with A. marginale were analyzed by PCR for Anaplasma infection. All samples analyzed were negative for A. marginale. The results of this work indicate that transovarial transmission of A. marginale by R. microplus is unlikely to occur.

Phylogenetic divergence between Rickettsia amblyommatis strains from Argentina.

The aim of this study was to investigate if there is a phylogenetic divergence of Rickettsia amblyommatis strains from different phytogeographic provinces of Argentina and South America. Therefore, ompA and ompB gene sequences of R. amblyommatis DNA detected in Amblyomma hadanii and Amblyomma neumanni ticks from the Yungas and Chaco phytogeographic provinces, respectively, were obtained. These sequences were compared with GenBank sequences from R. amblyommatis from different countries of the American continent. The analyses shown that ompA haplotypes of R. amblyommatis form two clusters which differ phylogenetically. Regarding to ompB sequences, three groups of R. amblyommatis strains were detected. It could be shown that strains of R. amblyommatis detected in A. hadanii from the phytogeographic province Yungas are phylogenetic different to them detected in A. neumanni from the phytogeographic province Chaco. Furthermore, it could be observed that strains detected in different tick species associated with different phytogeographic provinces of the American continent differ phylogenetically.