Detection of tick-borne pathogens in Rhipicephalus sanguineus sensu lato and dogs from different districts of Portugal

Dogs are highly exposed to pathogens transmitted by ectoparasites. The Mediterranean climate of Southern Europe, together with the presence of stray and/or neglected pets in close proximity with humans, contribute for tick expansion and stand for increased risk to infections in humans due to the zoonotic potential of many of these agents.The aim of this study was to perform a molecular survey in dogs (suspected of tick-borne disease and/or infested with ticks), as well as in ticks collected from those animals, from 12 districts of Portugal to investigate the occurrence of Rickettsia spp. and other tick-borne pathogens (Babesia, Ehrlichia, Anaplasma and Hepatozoon). Additionally, a serological survey of spotted fever group Rickettsia in Portuguese dogs was performed using an in-house immunofluorescence assay (IFA).A total of 200 whole-blood samples and 221 Rhipicephalus sanguineus s. l. ticks were collected from dogs.A total of 14 (7 %) blood samples and 10 (4.5 %) ticks yielded presumptively positive 420-bp amplicons using the Rickettsia spp. partial ompB nested PCR. Screening of the ompB-positive samples using the gltA gene showed 8 positive ticks. All Rickettsia ompB and gltA sequences had the highest identity with R. massiliae. The Rickettsia-positive dogs were further tested for other tick-borne pathogens and were found to be infected with Babesia spp. (n = 5), but not with Ehrlichia, Anaplasma or Hepatozoon.Of the 149 dog serum specimens tested in the serological assay, 103 (69 %) were positive for IgG antibodies against spotted fever group Rickettsia. Antibodies were found in dogs from all the studied districts, in 55 (53 %) of the stray and in 48 (47 %) of the owned dogs.Our study detected and characterized for the first time R. massiliae in dogs from Portugal, broadening the geographical range of this canine pathogen and adding knowledge to the impact of this disease in dogs.     

Pathogens in ticks collected in Israel: I. Bacteria and protozoa in Hyalomma aegyptium and Hyalomma dromedarii collected from tortoises and camels

Ticks were collected from 30 Greek tortoise (Testudo graeca), and 10 Arabian camels (dromedary) (Camelus dromedarius) in Israel. All those collected from Greek tortoises belonged to Hyalomma aegyptium, while all specimens collected from the camels belonged to Hyalomma dromedarii. Out of 84 specimens of H. aegyptium, 31 pools were examined by PCR, while from 75 H. dromedarii specimens nine pools were studied. Out of 31 pools of H. aegyptium 26 were positive for pathogens or endosymbiont; 14 for one, 11 for two and one for three pathogens. Out of nine pools prepared from H. dromedarii, seven were positive for pathogens (two for C. burnetii and five for Leishmania infantum). In H. aegyptium, Rickettsia africae, Rickettsia aeschlimannii, Rickettsia endosymbiont, Coxiella burnetii, Hemolivia mauritanica, Babesia microti, Theileria sp., and Leishmania infantum was detected, while in H. dromedarii C. burnetii and L. infantum were found. None of the ticks were positive for Anaplasma/Ehrlichia, Listeria monocytogenes, Bartonella spp., Hepatozoon spp. and Toxoplasma gondii. H Rickettsia endosymbionts, C. burnetii, B. microti, Theileria sp. and L. infantum are reported for the first time in H. aegyptium, and C. burnetii and L. infantum for the first time in H. dromedarii.     

Spotted fever group rickettsiae in ticks, Morocco

A total of 370 ticks, encompassing 7 species from 4 genera, were collected during 2002-2006 from domestic animals and vegetation in the Taza region of northeastern Morocco. Rickettsial DNA was identified in 101 ticks (27%) by sequencing PCR products of fragments of the citrate synthase and outer membrane protein genes of Rickettsia spp. Seven rickettsiae of the spotted fever group were identified, including 4 pathogens: R. aeschlimannii in Hyalomma marginatum marginatum, R. massiliae in Rhipicephalus sanguineus, R. slovaca in Dermacentor marginatus, and R. monacensis in Ixodes ricinus. Two suspected pathogens were also detected (R. raoultii in D. marginatus and R. helvetica in I. ricinus). An incompletely described Rickettsia sp. was detected in Haemaphysalis spp. ticks.     

Identification and distribution of nine tick-borne spotted fever group Rickettsiae in the Country of Georgia

Rickettsial pathogens cause diseases that vary in severity and clinical presentation. Rickettsia species transmitted by ticks are mostly classified within the spotted fever group of rickettsiae (SFGR) and are often associated with febrile diseases. Preliminary studies have detected three human-pathogenic SFGR from ticks in Georgia: Rickettsia aeschlimannii, Rickettsia raoultii, and Rickettsia slovaca. To more broadly assess the presence of tick-borne rickettsiae from Georgia we examined 1594 ticks, representing 18 species from five genera (Ixodes, Hyalomma, Haemaphysalis, Dermacentor, and Rhipicephalus), collected from eight regions of Georgia. A total of 498 tick DNA samples extracted from single ticks or pooled ticks were assessed by molecular methods. Genus-specific Rick17b and species-specific qPCR assays were used to identify six rickettsiae: R. aeschlimannii, R. raoultii, R. slovaca, Rickettsia conorii subsp. conorii, Rickettsia massiliae, and Rickettsia monacensis. Tick samples that were positive for Rickettsia, but not identified by the species-specific assays, were further evaluated by multi-locus sequence typing (MLST) using sequences of four protein-coding genes (gltA, ompA,ompB, sca4). Three additional Rickettsia species were identified by MLST: Candidatus Rickettsia barbariae, Rickettsia helvetica, and Rickettsia hoogstraalii. Overall, nine species of Rickettsia (six human pathogens and three species with unknown pathogenicity) were detected from 12 tick species of five different genera. A distribution map for the tick-borne rickettsiae revealed six newly identified endemic regions in Georgia.     

Tick-borne pathogens in dogs,wild small mammals and their ectoparasites in the semi-arid Caatinga biome,northeastern Brazil

Caatinga is a biome exclusive to the semiarid zone of Brazil, where studies on ticks and tick-borne diseases are scarce. Herein, we investigated the occurrence of Rickettsia, Ehrlichia, and Coxiella in wild mammals, domestic dogs and their ectoparasites using molecular and serological techniques. During 2014–2016, blood samples and ectoparasites were collected from 70 small mammals (51 rodents, 18 marsupials, 1 wild canid) and 147 domestic dogs in three areas of the Caatinga. Through serological analyses of domestic dogs of the three areas, 8 to 11 % were seropositive for Rickettsia rickettsii, 9 to 37 % for Rickettsia amblyommatis, 61 to 75 % for Ehrlichia canis, and 0–5% for Coxiella burnetii. All wild mammals were seronegative for Rickettsia spp. and C. burnetii, except for one rodent (Wiedomys pyrrhorhinos) and one marsupial (Didelphis albiventris) that were seroreactive to C. burnetii, one wild canid (Cerdocyon thous) for R. amblyommatis, and two Rattus rattus for Rickettsia spp. Through PCR targeting DNA of Rickettsia, Ehrlichia or Coxiella, all blood samples were negative, except for the presence of Ehrlichia canis DNA in 8.8 % of the domestic dogs, and a recently reported novel agent, Ehrlichia sp. strain Natal, in one marsupial (Gracilinanus agilis). A total of 222 ticks, 84 fleas, and six lice were collected. Ticks were mostly Rhipicephalus sanguineus sensu lato, some Ixodes loricatus, Ornithodoros rietcorreai, Haemaphysalis sp., and Amblyomma spp.; fleas were Ctenocephalides felis felis, Pulex sp. and Polygenis (Polygenis) bohlsi jordani; and lice were Polyplax sp. and Gyropus sp. Through molecular detection of microorganisms, 9% of C. felis felis contained Rickettsia felis, 20 % of A. auricularium contained R. amblyommatis and 13 % of A. parvum contained ‘Candidatus Rickettsia andeanae’, whereas Ehrlichia canis DNA was detected in at least 6% of the R. sanguineus s.l. from one area. We report a variety of ectoparasites infesting small mammals and domestic dogs in the Caatinga biome, where these ectoparasites probably act as vectors of rickettsiae, ehrlichial agents (E. canis and Ehrlichia sp. strain Natal) and C. burnetii. Our results highlight to the potential risks of human infection by these tick-borne agents in the Caatinga biome.     

Molecular identification of protozoal and bacterial organisms in domestic animals and their infesting ticks from north-eastern Algeria

A molecular survey was undertaken to determine the presence of protozoal and bacterial organisms in 120 ticks and 87 blood samples collected from mammals in north-eastern Algeria. Eight tick species were morphologically identified including 70 Rhipicephalus (Boophilus) annulatus, 23 Rhipicephalus bursa, five Rhipicephalus sanguineus sensu lato, 11 Hyalomma impeltatum, five Hyalomma scupense, two Hyalommma marginatum, one Hyalomma anatolicum and three Ixodes ricinus. Quantitative PCR screening of the ticks showed that Theileria annulata, “Candidatus Ehrlichia urmitei”, Theileria buffeli and Anaplasma platys were detected in Rh. annulatus. Rickettsia massiliae and Anaplasma ovis were detected in Rh. sanguineus s.l. and Rh. bursa. Rickettsia aeschlimannii was detected in Hy. marginatum, Hy. scupense and Hy. impeltatum. Finally, “Candidatus Rickettsia barbariae” was detected in Rh. bursa. In the screening blood samples, Theileria equi, T.annulata, T. buffeli, Babesia bovis, Anaplasma marginale, A. ovis and Borrelia spp. were detected in cattle. Theileria ovis, T. annulata, and A. ovis were detected in sheep. In addition, A. ovis and T. equi were detected in goats and equidea respectively. In this study, T. equi and “Candidatus Rickettsia barbariae” were identified for the first time in Algeria as well as potential new species of Ehrlichia and Anaplasma.Although molecular detection does not indicate vector/reservoir competence when investigating ticks removed from animals, this study expands the knowledge of the microorganisms detected in ticks in north-east of Algeria.     

Molecular detection of spotted fever group rickettsiae in ticks from Ethiopia and Chad

DNA extracted from 363 ticks collected in Ethiopia and 9 ticks collected in Chad, Africa were screened by PCR to detect DNA from spotted fever group rickettsiae. Fifteen ticks (4.1%) collected in Ethiopia and one tick (11%) collected in Chad tested positive when PCR targeting the gltA and ompA rickettsial genes was performed. PCR-positive products of the gltA and ompA genes were used for DNA sequencing. Rickettsia africae was detected in 12/118 Amblyomma lepidum and in 1/2 A. variegatum. Also, 2/12 Hyalomma marginatum rufipes collected in Ethiopia and one H. marginatum rufipes collected in Chad were positive for R. aeschlimannii. Our results confirm the previously reported presence of R. africae in Ethiopia and also show the first evidence of R. aeschlimannii in ticks collected in Ethiopia and Chad.     

Molecular detection of a novel Babesia sp. and pathogenic spotted fever group rickettsiae in ticks collected from hedgehogs in Turkey: Haemaphysalis erinacei,a novel candidate vector for the genus Babesia

In this study, a total of 319 ticks were obtained from hedgehogs (Erinaceus concolor). All ticks were pooled into groups and screened by PCR for tick-borne pathogens (TBPs). PCR and sequence analyses identified the presence of a novel Babesia sp. in adult Haemaphysalis erinacei. In addition, the presence of natural transovarial transmission of this novel Babesia sp. was detected in Ha. erinacei. According to the 18S rRNA (nearly complete) and partial rRNA locus (ITS-1/5.8S/ITS-2) phylogeny, it was determined that this new species is located within the Babesia sensu stricto clade and is closely related to Babesia spp. found in carnivores. Furthermore, the presence of three pathogenic spotted fever group (SFG) rickettsiae was determined in 65.8% of the tick pools: Rickettsia sibirica subsp. mongolitimonae in Hyalomma aegyptium (adult), Hyalomma spp. (larvae), Rhipicephalus turanicus (adult), and Ha. erinacei (adult); Rickettsia aeschlimannii in H. aegyptium (adult); Rickettsia slovaca in Hyalomma spp. (larvae and nymphs) and H. aegyptium (adult). To our knowledge, this is the first report of R. sibirica mongolitimonae in H. aegyptium, Ha. erinacei, and Rh. turanicus, and the first report of R. slovaca in H. aegyptium. In addition, the presence of a single Hemolivia mauritanica haplotype was detected in H. aegyptium adults. Consequently, the presence of a novel Babesia sp. has been identified in a new candidate vector tick species in this study. Additionally, three SFG rickettsiae that cause infections in humans were identified in ticks collected from hedgehogs. Therefore, environmental wildlife monitoring for hedgehogs should be carried out for ticks and tick-borne pathogens in the region. Additionally, studies regarding the reservoir status of hedgehogs for the aforementioned pathogens must be carried out.     

Rickettsia spp. in Rhipicephalus sanguineus sensu lato ticks collected from stray dogs in Kerman city,Iran

Rickettsial diseases are recognized as one of the most important vector-borne infectious diseases for humans all over the world. Dogs and their ticks are considered the most important reservoirs for Rickettsia spp., especially in spotted fever group rickettsioses. The aim of the study was to investigate Rickettsia infections in ticks collected from stray dogs in southeastern Iran. In this study, 50 stray dogs in Kerman city were randomly selected, of 68% and 52% of which were above 8 months age and male, respectively. Ticks were collected from the dog skins. After identification of collected ticks, genomic DNA of all ticks was extracted. DNA samples were tested using real-time PCR for Rickettsia spp. infections. The species of Rickettsia in positive samples were determined using gltA gene amplification and sequencing. A total of 250 ticks were collected from 50 stray dogs and all of them belonged to Rhipicephalus sanguineus sensu lato. Totally, 10 pooled of 50 pooled ticks were positive for Rickettsia spp. in real-time PCR and the minimal Rickettsial infection rate was 4% in this study. The identified Rickettsia spp. included R. massiliae (n = 5), R. rhipicephali (n = 1), and R. sibirica (n = 1). In this study, molecular evidence of Rickettsia spp. infection was observed in collected ticks from stray dogs in southeast Iran. More sensitivity to human and animal health care systems in southeastern Iran is essential to the diagnosis of suspected clinical cases that are related to rickettsiosis.     

Prevalence of antibodies against Anaplasma spp., Borrelia burgdorferi sensu lato,Babesia gibsoni,and Ehrlichia spp. in dogs in the Republic of Korea

There is a lack of comprehensive studies on the seroprevalence of tick-borne pathogens in the Republic of Korea. Therefore, the aim of this study was to investigate the seroprevalences of Anaplasma spp. (A. phagocytophilum/A. platys), Borrelia burgdorferi sensu lato, Babesia gibsoni, Ehrlichia spp. (E. canis/E. ewingii), and Ehrlichia chaffeensis in dogs across the Republic of Korea in 2017 and 2018. A total of 2215 serum samples collected from 938 companion dogs, 969 shelter dogs, and 308 military working dogs were examined using commercial enzyme-linked immunosorbent assay (ELISA) and indirect fluorescence immunoassay (IFA) kits. Data collected for each animal, including breed, sex, age, region, season, and dog type, were used for statistical analysis. The overall seroprevalence was highest for Anaplasma spp. (15.1 %), followed by Ehrlichia spp. (10.3 %), B. burgdorferi sensu lato (6.4 %), E. chaffeensis (2.3 %), and B. gibsoni (1.7 %). One hundred and sixty-one dogs had antibodies against two or three different pathogens. The most common combinations were Anaplasma spp. - Ehrlichia spp. (2.1 %), Anaplasma spp. - E. chaffeensis (1.4 %), and Anaplasma spp. - B. burgdorferi sensu lato (1.2 %). Season was significantly associated with the seroprevalences of B. burgdorferi sensu lato and Ehrlichia spp., with dogs presenting the highest percentage of positive results during summer. Anaplasma spp. and B. gibsoni were significantly more prevalent in the northern and southern regions, respectively. The seroprevalences of Anaplasma spp., B. burgdorferi sensu lato, and Ehrlichia spp. were significantly higher in military working dogs, while the seroprevalence of E. chaffeensis was higher in companion dogs. The current findings are important for future surveillance of canine tick-borne pathogens and designing appropriate approaches for the diagnosis and control of these pathogens in the Republic of Korea.     

Anaplasmataceae presence in Amblyomma calcaratum associated with anteaters (Tamandua tetradactyla) in the rainforest ecoregion,Argentina

Bacteria of the sister genera Ehrlichia and Anaplasma (Anaplasmataceae) are obligate intracellular Alphaproteobacteria that are transmitted mostly through arthropod vectors. These agents can infect different vertebrate cells, depending on the species involved, and can cause diseases in animals and humans. In this study, we evaluated the presence of Anaplasmataceae bacteria in Amblyomma calcaratum ticks collected from a road-killed Tamandua tetradactyla in the Rainforest ecoregion in Argentina. All samples were screened for Anaplasmataceae DNA using a real-time PCR assay targeting the 16S rRNA gene. Evidence of Anaplasmataceae DNA was detected in three out of thirty-nine Am. calcaratum ticks. Phylogenetic analysis of a portion of 16S rRNA gene positioned one sample (Ehrlichia sp. strain Ac124) with Ehrlichia sequences and the other two samples with Anaplasma sequences; Anaplasma sp. strain Ac145 close to Anaplasma odocoilei and Anaplasma sp. strain Ac152 in an ancestral position to most Anaplasma species. The groEL sequence obtained showed that Ehrlichia sp. strain Ac124 was phylogenetically related to Ehrlichia sp. strain Iberá reported infecting Amblyomma tigrinum from Iberá wetlands in Argentina. Phylogenetic analysis using the rpoB sequence positioned Anaplasma sp. strain Ac145 close to the canine pathogen Anaplasma platys, while Anaplasma sp. strain Ac152 was positioned close to the bovine pathogen Anaplasma marginale.In this study, three Anaplasmataceae agents were detected in adults of Am. calcaratum associated with a T. tetradactyla. These results suggest that the number of Anaplasmataceae species, as well as their distribution, is largely unknown.     

Diversity and geographic distribution of rickettsial agents identified in brown dog ticks from across the United States

Rhipicephalus sanguineus sensu lato, or brown dog ticks, transmit a variety of pathogens of veterinary and public health importance globally. Pathogens vectored by brown dog ticks and identified in the United States include Babesia vogeli, Ehrlichia canis, and several spotted fever group Rickettsia spp. (SFGR). Due to the challenge of collecting canine blood samples nationwide to screen for exposure to these pathogens, we took an indirect approach and tested brown dog ticks for molecular evidence of infection. Brown dog ticks (616 adults and 65 nymphs) collected from dogs and cats across the nation were tested by separate PCR assays detecting Babesia spp., E. canis, and SFGR. While no Babesia sp. was found, we identified rickettsial agents in 3.5% (24/681; 95% CI 2.4–5.2%) of the ticks. Pathogens and related organisms detected in ticks included E. canis (n = 1), Rickettsia amblyommatis (n = 3), Rickettsia massiliae (n = 11), Rickettsia monacensis (n = 3), Rickettsia montanensis (n = 5), and an undefined Rickettsia species (n = 1). These data demonstrate a wider geographic distribution of R. massiliae than previously known, and to the authors’ knowledge, reports R. monacensis in brown dog ticks for the first time. Due to the close association that brown dog ticks have with domestic dogs and humans, more research is needed to understand the full array of organisms, some of which are zoonotic, potentially transmitted by this widespread tick complex.     

A putative novel strain of Ehrlichia infecting Amblyomma tigrinum associated with Pampas fox (Lycalopex gymnocercus) in Esteros del Iberá ecoregion,Argentina

The current work evaluated road-killed Pampas foxes (Lycalopex gymnocercus) and their ticks for the presence of vector-borne agents in the ecoregion Esteros del Iberá in northeastern Argentina. Spleen, lung and blood samples and Amblyomma tigrinum adult ticks collected from the foxes were tested by polymerase chain reaction (PCR) assays targeting bacteria of the genera Ehrlichia, Anaplasma, and Rickettsia. All foxes tested were negative for the three genera, but evidence of Ehrlichia and Rickettsia infection was detected in the ticks. One A. tigrinum (out of 12 tested) was infected by an ehrlichial agent, here named Ehrlichia sp. strain Iberá, related to ehrlichial agents recently detected in platypuses in Tasmania (Ornithorhynchus anatinus) and in voles (Myodes rutilus and Myodes rufocanus) and shrews (Sorex araneus) in the Russian Far East. Regarding Rickettsia, all A. tigrinum ticks (100%) were infected by ´Candidatus Rickettsia andeanae´, a member of the spotted fever group rickettsia of unknown pathogenicity. Further research is necessary to unveil the ecology of Ehrlichia sp. strain Iberá as well as its zoonotic relevance. The species of the genus Ehrlichia are known to be pathogenic to mammals, including humans and domestic animals, thus the presence of this ehrlichial agent in A. tigrinum is a potential risk for veterinary and public health, as the adults of A. tigrinum are common parasites of dogs in rural and peri-urban environments, and humans are also frequently bitten by this tick species.     

Comparative analyses of mitochondrial and nuclear genetic markers for the molecular identification of Rhipicephalus spp.

The genus Rhipicephalus (Acari: Ixodidae) comprises a large number of vectors of pathogens of substantial medical and veterinary concern; however, species identification based solely on morphological features is often challenging. In the present study, genetic distance within selected Rhipicephalus species (i.e., Rhipicephalus bursa, Rhipicephalus guilhoni, Rhipicephalus muhsamae, Rhipicephalus sanguineus sensu lato and Rhipicephalus turanicus), were investigated based on molecular and phylogenetic analyses of fragments of the mitochondrial 16S, 12S and cytochrome c oxidase subunit 1 (cox1) genes, as well as of the whole sequences of the ribosomal internal transcribed spacer-2 (ITS-2) region. Mean values of inter-specific genetic distance (e.g., up to 12.6%, 11.1% and 15.2%), as well as of intra-specific genetic distance (e.g., 0.9%, 0.9% and 1%), calculated using the Kimura-2 parameter substitution model with uniform rates among sites for 16S, 12S and cox1 genes, respectively, confirmed the differentiation of the rhipicephaline species herein examined. The molecular identification was also supported by the distinct separation of species-specific clades inferred from the phylogenetic analyses of all mitochondrial sequences. Conversely, little interspecific divergence was detected amongst ribosomal ITS-2 sequences (i.e., up to 2.8%) for species belonging to the R. sanguineus complex, which resulted in the ambiguous placement of selected R. sanguineus s.l. and R. turanicus sequences in the corresponding phylogenetic tree. Results from this study confirm the suitability of mtDNA markers for the reliable identification of ticks within the Rhipicephalus genus and provide a framework for future studies of taxonomy, speciation history and evolution of this group of ticks.     

Genetic diversity of Anaplasma and Ehrlichia bacteria found in Dermacentor and Ixodes ticks in Mongolia

Anaplasma and Ehrlichia are tick-borne bacterial pathogens that cause human granulocytic anaplasmosis, human monocytic ehrlichiosis, and are severe threats to livestock economies like Mongolia. In this study, ticks were collected, identified, and pooled (n = 299) from three distinct environments across central Mongolia. Each pool was initially tested for Anaplasma/Ehrlichia using a 16S rRNA PCR assay that detects both genera, and specific PCR testing was done to identify those positive samples. Maximum likelihood estimation (MLE) of infection rates of ticks collected from the environment in Selenge aimag (province) found infection rates of Ixodes persulcatus ticks to be 2.0% (95% CI: 0.7, 4.3%) for A. phagocytophilum and 0.8% (95% CI: 0.1, 2.5%) for both nonspecific Ehrlichia and Anaplasma. Ehrlichia muris was only detected in I. persulcatus ticks collected from the Selenge aimag, where the MLE was 1.2% (95% CI: 0.1, 2.5%). The calculated MLE infection rate of Anaplasma spp. in questing Dermacentor nuttalli ticks ranged from 1.9% (95% CI: 1.1, 9.1%) in the Tov aimag to 2.3% (95% CI: 1.3, 10.8%) in the Selenge aimag. However, when examining MLE in ticks removed from livestock, estimates increase substantially, ranging from 7.8% (95% CI: 4.2, 13.3%) in Dornogovi to 22.5% (95% CI: 14.3, 34.3%) in Selenge, suggesting that livestock play a key role in disease maintenance. Considering the collective economic losses that can result from these pathogens and the potential for illness in nomadic herdsmen, these results highlight the need for enhanced TBD surveillance and prevention measures within Mongolia.     

The brown dog tick Rhipicephalus sanguineus sensu Roberts, 1965 across Australia: Morphological and molecular identification of R. sanguineus s.l. tropical lineage

The brown dog tick Rhipicephalus sanguineus (Latreille, 1806) is the most widely distributed tick species globally. Throughout the world there are at least two divergent lineages on dogs that are traditionally grouped into what was known as R. sanguineus. The species R. sanguineus was recently redescribed using a neotype reported from countries with a temperate climate. The second lineage distributed in countries with primarily tropical climates is currently designated R. sanguineus s.l. tropical lineage. Here, we present a comprehensive genetic evaluation of Australian brown dog ticks from across the continent that complements the morphological study of R. sanguineus sensu Roberts (1965). A total of 294 ticks were collected from dogs around Australia ― including New South Wales, Queensland, the Northern Territory and Western Australia ― for morphological identification. All ticks were morphologically identified as R. sanguineus sensu Roberts (1965). DNA was isolated from a single leg from morphologically characterised individuals from New South Wales (n = 14), Queensland (n = 18), Northern Territory (n = 7) and Western Australia (n = 13), together with ticks from Fiji (n = 1) and the Seychelles (n = 1) for comparison with Australian ticks. The study revealed three cox1 haplotypes clustered only with R. sanguineus s.l. tropical lineage’. An updated distribution of R. sanguineus sensu Roberts (1965) is compared to the 1965 distribution. In the Australian context, R. sanguineus s.l. has appeared in north-western New South Wales but remains absent from coastal New South Wales. Despite both temperate and tropical climates being present in Australia, only R. sanguineus s.l. tropical lineage was found. The evidence does not support the presence of the strictly defined brown dog tick, R. sanguineus by Nava et al. (2018) in Australia, because the examined ticks are genetically and morphologically distinct. We recommend using the term brown dog tick, R. sanguineus sensu Roberts (1965) for specimens from Australia.     

Vector competence of Rhipicephalus sanguineus sensu stricto for Anaplasma platys

Anaplasma platys is a Gram-negative, obligate intracellular bacteria that causes canine infectious cyclic thrombocytopenia in dogs. The brown dog tick Rhipicephalus sanguineus sensu lato is presumed to be the vector of A. platys based on the overlap in distribution of R. sanguineus and A. platys infections, detection of A. platys DNA in both flat and engorged field-collected R. sanguineus, and the fact that dogs are primary hosts of both brown dog ticks and A. platys. However, the only study evaluating the vector competence of R. sanguineus for A. platys under controlled laboratory conditions reported an apparent inability of ticks to acquire or maintain the pathogen. In 2016, engorged female Rhipicephalus sanguineus sensu stricto ticks were collected off dogs to start a laboratory tick colony. After one generation of colony maintenance on tick-naïve and pathogen-free New Zealand White rabbits, a rabbit used to feed F1 adults seroconverted to Anaplasma phagocytophilum antigen. PCR and subsequent DNA sequencing identified the presence of A. platys in both the adult ticks fed on this rabbit and their resulting F2 progenies. Retrospective testing of all previous (P and F1) life stages of this colony demonstrated that the infection originated from one field-collected A. platys-infected female whose progeny was propagated in the laboratory and produced the PCR-positive F1 adults. Over the following (F2-F4) generations, the prevalence of A. platys in this colony reached 90–100 % indicating highly efficient transovarial and horizontal transmission, as well as transstadial maintenance, of this pathogen by R. sanguineus s.s.     

Comprehensive screening of tick-borne microorganisms indicates that a great variety of pathogens are circulating between hard ticks (Ixodoidea: Ixodidae) and domestic ruminants in natural foci of Anatolia

Grazing domestic ruminants serve as important reservoirs and/or amplificatory hosts in the ecology of tick-borne pathogens (TBPs) and tick vectors in the natural foci; however, many enzootic life cycles including ruminants and ticks are still unknown. This study investigated a wide range of TBPs circulating among ticks and grazing ruminants in the natural foci of Anatolia, Turkey. Tick specimens (n = 1815) were collected from cattle, sheep, and goats in three ecologically distinct areas (wooded, transitional, and semi-arid zones) of Anatolia and identified by species: Dermacentor marginatus, Dermacentor reticulatus, Hyalomma anatolicum, Hyalomma excavatum, Hyalomma marginatum, Hyalomma scupense, Haemaphysalis inermis, Haemaphysalis parva, Haemaphysalis punctata, Haemaphysalis sulcata, Ixodes ricinus, Rhipicephalus bursa, and Rhipicephalus turanicus. PCR-sequencing analyses revealed TBPs of great diversity, with 32 different agents identified in the ticks: six Babesia spp. (Babesia occultans, Babesia crassa, Babesia microti, Babesia rossi, Babesia sp. tavsan1, and Babesia sp. Ucbas); four Theileria spp., including one putative novel species (Theileria annulata, Theileria orientalis, Theileria ovis, and Theileria sp.); one Hepatozoon sp.; four Anaplasma spp., including one novel genotype (Anaplasma phagocytophilum, Anaplasma marginale, Anaplasma ovis, and Anaplasma sp.); six unnamed Ehrlichia spp. genotypes; Neoehrlichia mikurensis; nine spotted fever group rickettsiae, including one putative novel species (Rickettsia aeschlimannii, Rickettsia slovaca, Rickettsia hoogstraalii, Rickettsia monacensis with strain IRS3, Rickettsia mongolitimonae, Rickettsia raoultii, Candidatus Rickettsia goldwasserii, Candidatus Rickettsia barbariae, and Rickettsia sp.); and Borrelia valaisiana. Detailed phylogenetic analyses showed that some of the detected pathogens represent more than one haplotype, potentially relating to the tick species or the host. Additionally, the presence of Neoehrlichia mikurensis, an emerging pathogen for humans, was reported for the first time in Turkey, expanding its geographical distribution. Consequently, this study describes some previously unknown tick-borne protozoan and bacterial species/genotypes and provides informative epidemiological data on TBPs, which are related to animal and human health, serving the one health concept.     

Detection of Leishmania infantum,Babesia canis,and rickettsiae in ticks removed from dogs living in Italy

The aims of this study were to determine natural infections by Anaplasma phagocytophilum/Anaplasma platys, Bartonella henselae, Ehrlichia canis, Leishmania infantum, Rickettsia spp., Babesia spp., and Hepatozoon spp. by molecular methods in ticks (n = 91) removed from dogs with clinical signs and laboratory abnormalities compatible with tick-borne diseases (n = 22) living in Italy and to assess the distribution and species of ticks encountered. Ticks from dogs living in southern Italy were all identified as Rhipicephalus sanguineus (n = 25), ticks from central Italy included Rh. sanguineus (n = 8) and Ixodes ricinus (n = 9), ticks from northern Italy included Rh. sanguineus (n = 45), Dermacentor marginatus (n = 3), and one I. ricinus. Leishmania infantum, Rickettsia spp., and Babesia canis were the only pathogens detected in 7 (8%), 4 (4%), and 2 (2%) out of 91 ticks, respectively. L. infantum was detected in I. ricinus from central Italy and in Rh. sanguineus from northern and central Italy. Rickettsia conorii and Ri. massiliae were detected in Rh. sanguineus ticks from central and southern Italy (Sicily), respectively. Bab. canis was detected in D. marginatus ticks from northern Italy.