Rhipicephalus Sanguineus Sensu Lato Research Articles

Genetic variability of Ehrlichia canis TRP36 in ticks, dogs, and red foxes from Eurasia

Ehrlichia canis is among the most prevalent tick-borne pathogens infecting dogs worldwide, being primarily vectored by brown dog ticks, Rhipicephalus sanguineus sensu lato (s.l.). The genetic variability of E. canis has been assessed by analysis of different genes (e.g., disulfide bond formation protein gene, glycoprotein 19, tandem repeat protein 36 - TRP36) in the Americas, Africa, Asia, and in a single dog sample from Europe (i.e., Spain). This study was aimed to assess the variations in the TRP36 gene of E. canis detected in naturally infected canids and R. sanguineus s.l. ticks from different countries in Asia and Europe. DNA samples from dogs (n = 644), foxes (n = 146), and R. sanguineus s.l. ticks (n = 658) from Austria, Italy, Iran, Pakistan, India, Indonesia, Malaysia, the Philippines, Singapore, Thailand, Vietnam, and Taiwan were included in this study. Ehrlichia canis 16S rRNA positive samples (n = 115 from the previous studies; n = 14 from Austria in this study) were selected for molecular examination by analyses of TRP36 gene. Out of 129 E. canis 16S rRNA positive samples from dogs (n = 88), foxes (n = 7), and R. sanguineus s.l. ticks (n = 34), the TRP36 gene was successfully amplified from 52. The phylogenetic analysis of the TRP36 pre-repeat, tandem repeat, and post repeat regions showed that most samples were genetically close to the United States genogroup, whereas two samples from Austria and one from Pakistan clustered within the Taiwan genogroup. TRP36 sequences from all samples presented a high conserved nucleotide sequence in the tandem repeat region (from 6 to 20 copies), encoding for nine amino acids (i.e., TEDSVSAPA). Our results confirm the US genogroup as the most frequent group in dogs and ticks tested herein, whereas the Taiwan genogroup was present in a lower frequency. Besides, this study described for the first time the US genogroup in red foxes, thus revealing that these canids share identical strains with domestic dogs and R. sanguineus s.l. ticks.

Spatial clustering of fourteen tick species across districts of Zimbabwe

BackgroundTicks transmit several diseases that result in high morbidity and mortality in livestock. Tick-borne diseases are an economic burden that negatively affect livestock production, cost countries billions of dollars through vaccine procurement and other disease management efforts. Thus, understanding the spatial distribution of tick hotspots is critical for identifying potential areas of high tick-borne disease transmission and setting up priority areas for targeted tick disease management. In this study, optimised hotspot analysis was applied to detect hotspots and coldspots of 14 common tick species in Zimbabwe. Data on the spatial distribution of tick species were obtained from the Epidemiology Unit of the Division of Veterinary Field Services of Zimbabwe.ResultsA total of 55,133 ticks were collected with Rhipicephalus decoloratus being the most common species (28.7%), followed by Amblyomma hebraeum (20.6%), and Rhipicephalus sanguineus sensu lato (0.06%) being the least common species. Results also showed that tick hotspots are species-specific with particular tick species occupying defined localities in the country. For instance, Amblyomma variegatum, Rhipicephalus appendiculatus, Rhipicephalus decoloratus, Rhipicephalus compostus, Rhipicephalus microplus, Rhipicephalus pravus, and Rhipicephalus simus were concentrated in the north and north eastern districts of the country. In contrast, Amblyomma hebraeum, Hyalomma rufipes, Hyalomma trancatum and Rhipicephalus evertsi evertsi were prevalent in the southern districts of Zimbabwe.ConclusionThe occurrence of broadly similar hotspots of several tick species in different districts suggests presence of spatial overlaps in the niche of the tick species. As ticks are vectors of several tick-borne diseases, there is high likelihood of multiple disease transmission in the same geographic region. This study is the first in Zimbabwe to demonstrate unique spatial patterns in the distribution of several tick species across the country. The results of this study provide an important opportunity for the development of spatially-targeted tick-borne disease management strategies.

Richness of hard ticks (Acari: Ixodidae) from Eastern Brazilian Amazonia, state of Pará, Brazil

ABSTRACT Ixodid or hard ticks (Acari: Ixodidae) occurs worldwide and are considered the most important vectors of pathogens to wild and domestic animals. Herein, we provide information on tick species infesting wildlife vertebrates, domestic animals (dog, donkey, goat, and horse), and humans as well as collected in the environment from 34 localities in the state of Pará, eastern Brazilian Amazonia. This is the first report to include a large geographical range of the tick fauna associated with their hosts in the eastern Amazonia. Overall, 3,396 tick specimens (600 larvae, 830 nymphs, 602 males, 1,364 females) were collected, comprising five genera and 23 species [Amblyomma cajennense sensu stricto (s.s.), Amblyomma calcaratum, Amblyomma coelebs, Amblyomma dissimile, Amblyomma geayi, Amblyomma goeldii, Amblyomma humerale, Amblyomma latepunctatum, Amblyomma longirostre, Amblyomma naponense, Amblyomma nodosum, Amblyomma oblongoguttatum, Amblyomma ovale, Amblyomma pacae, Amblyomma rotundatum, Amblyomma scalpturatum, Amblyomma varium, Dermacentor nitens, Haemaphysalis juxtakochi, Ixodes amarali, Ixodes luciae, Rhipicephalus microplus, and Rhipicephalus sanguineus sensu lato (s.l.)]. Additionally, we also provide new tick-host associations for A. cajennense s.s., A. calcaratum, A. coelebs, A. dissimile, A. humerale, A. latepunctatum, A. longirostre, A. naponense, A. nodosum, A. oblongoguttatum, A. rotundatum, A. scalpturatum, H. juxtakochi, I. luciae, and R. microplus.

Rhipicephalus sanguineus salivary gland extract as a source of immunomodulatory molecules.

Rhipicephalus sanguineus sensu lato (s.l.), popularly known as 'brown dog tick', is the primary vector of pathogens affecting dogs worldwide. To enter the host's organism, these pathogens utilise the anticoagulant, antiplatelet, anti-inflammatory and immunomodulatory actions of compounds present in the tick's saliva; such compounds are released by the ectoparasite in order to attach and feed on dogs. Nitric oxide (NO) is one of the regulatory factors in inflammation, apoptosis and immunomodulation. Here, we evaluated the in vitro activity of salivary gland extract of female dog ticks on the macrophage-derived J774 cell line, with and without lipopolysaccharide (LPS) stimulation. Cultures were evaluated for possible morphological alterations caused by exposure to the extract. There was no apparent in vitro cytotoxicity of the extract. Also, the NO secretory response in the non-LPS-stimulated cells was not inhibited. On the other hand, the extract presented modulatory action in the cultures of LPS-stimulated cells at a concentration of 0.1μg/mL, possibly through macrophage activation, and induced a significant decrease in NO secretion. These results confirm the modulatory potential of bioactive molecules in the salivary glands of R. sanguineus ticks.

Ectoparasites infesting animals living in close contact with human beings: a real trouble for One Health perspective?

ABSTRACT The number of domestic animals living with human beings is rapidly increasing in parallel with an enhanced risk of transmission of their parasites and the pathogens they might carry. The aim of this study was to assess the occurrence of hematophagous arthropods infesting domestic animals from Northeastern Brazil and to remark the implications of their occurrence on the epidemiology and control of selected veterinary and human diseases. From January 2017 to April 2019, ectoparasites infesting domestic cats, dogs and horses were collected for their respective hosts and identified. Overall, ectoparasites were sampled from 86 domestic animals, living in different anthropic settings. A total of 401 specimens (344 ticks and 57 fleas) were collected from different hosts [i.e., 10 (2.49%), 96 (23.94%) and 295 (73.57%) from cats, dogs and horses, respectively]. Two flea (i.e., Ctenocephalides canis and Ctenocephalides felis) and 5 tick species (i.e., Amblyomma ovale, Amblyomma sculptum, Dermacentor nitens, Rhipicephalus microplus and Rhipicephalus sanguineus sensu lato) were identified. This study provides data on the ectoparasite fauna infesting domestic animals from Northeastern Brazil. The diagnosis and treatment of these parasites should not be underestimated, considering the role that hematophagous arthropods display as vectors of pathogens of medical and veterinary concern.

Solar tick trap with a pheromone lure – A stand-in approach for off-host control of Rhipicephalus sanguineus sensu lato ticks

Rhipicephalus sanguineus sensu lato (s.l.), the brown dog tick, transmits several important haemo-protozoan and rickettsial pathogens to dogs. Effective control of these ticks is entirely by the use of acaricidal preparations both on and off the host. Indiscriminate use of acaricides can cause environmental pollution and toxicity to humans as well as development of acaricidal resistance in ticks. As an alternative control strategy, a novel, eco-friendly and economical solar tick trap, which attracts ticks using pheromones and kills them by electrocution, was fabricated for use in kennels to control different stages of ticks. All stages of ticks were found to be lured to sex pheromone (SP), assembly pheromone (AP) and a combination of SP + AP impregnated vapour patches and electrocuted. There was a highly significant (p < 0.01) difference in the level of attraction between engorged and questing stages of ticks to the pheromones in the trap. AP lured a higher number of ticks, both engorged and questing, in comparison to other pheromones. The solar tick trap bearing vapour patch impregnated with AP was considered most suitable for use in integrated pest management (IPM) of ticks in heavily infested kennels. The solar trap remained effective for a month after which, replacement of pheromone impregnated vapour patch was necessary for continued and effective attraction of the ticks. This solar tick trap device, which uses pheromone without any acaricide, could prove to be an ideal green alternative for use in IPM of ticks in kennels.

Identification of Anaplasma marginale in long-eared hedgehogs (Hemiechinus auritus) and their Rhipicephalus turanicus ticks in Iran

Hedgehogs play a role in the eco-epidemiology of parasites, viruses and bacteria of veterinary and medical importance. In Europe, hedgehogs have been found infected with tick-transmitted Anaplasma phagocytophilum and A. marginale. In this study blood and ticks collected from 53 long-eared hedgehogs (Hemiechinus auritus) living in southeastern Iran close to Afghanistan and Pakistan borders were examined for Anaplasma spp. infection using microscopical and molecular biology methods. At microscopical examination Anaplasma-like inclusion bodies were found at the margin of erythrocytes in the blood smear of one infected hedgehog (prevalence 1.9 %). Each hedgehog was infested with three to eight adult ticks (average 4.7 ± 1.5). On the body of hedgehogs, the ears were the main site for attachment of ticks. Out of 248 collected adult ticks 81.4 % were Rhipicephalus sanguineus sensu lato and 18.5 % were R. turanicus. PCR and sequencing showed the presence of A. marginale infection in the blood of two hedgehogs (3.8 %) and R. turanicus ticks collected from them. This is the first report of A. marginale infection in hedgehogs in Iran. Epidemiological importance of this finding is discussed and current knowledge on the tick fauna of hedgehogs in the country is reviewed.

First survey of ticks, tick-borne pathogens (Theileria, Babesia, Anaplasma and Ehrlichia) and Trypanosoma evansi in protected areas for threatened wild ruminants in Tunisia.

The aim of the study was to identify ticks present in the environment and wild Tunisian ruminants and to detect tick-borne pathogens and Trypanosoma evansi DNA in these specimens. Sampling was done throughout each season from the environment in three protected areas around Tunisia: El Feidja, Haddaj and Oued Dekouk. Ticks were collected also, from one fawn of Barbary red deer and eight naturally deceased wild ruminants (one Barbary red deer, five Scimitar-horned oryx, one Addax antelope and one Dorcas gazelle), all of which lived in various protected areas. PCR and nested PCRs were performed to detect the presence of Theileria spp., Babesia spp., Trypanosoma evansi, Ehrlichia spp., Anaplasma spp., Anaplasma bovis and Anaplasma phagocytophilum DNA in these tick specimens. A total of 352 ticks were collected, belonging to six different species: Hyalomma excavatum (80.6%), Hyalomma dromedarii (10.2%), Hyalomma marginatum (0.5%), Rhipicephalus bursa (0.5%), Rhipicephalus sanguineus sensu lato (5.1%) and Ixodes ricinus (2.8%). Pathogens have been detected in 25% of H. dromedarii, 9.1% of H. excavatum and 5% of R. sanguineus sensu lato. The percentage of detection of T. evansi was 0.2%. Ehrlichia spp.-Anaplasma spp. were detected in 10.1% of ticks. Anaplasma spp. and A. bovis were detected in 7.6%, and 0.8% of examined ticks, respectively. None of the Theileria spp., Babesia spp., or A. phagocytophilum DNA was detected in the tested ticks. To our knowledge, the present study represents the first identification of these six tick species and the first detection of rickettsial pathogens and T. evansi in North African wild ruminants' species. These results extend the knowledge about the diversity of ticks and tick-borne pathogens in wildlife and justify further investigations of the possible role of R. sanguineus sensu lato in the transmission of T. evansi.

Difference in tick infestations

The aim of this study was to identify the species and parasitic indices of ticks. A flock of 57 individuals (sheep, goats, cattle and dogs) in southern Algeria was studied from March 2019 to February 2020 during monthly visits. A total of 2544 ticks were collected by examining the entire body of the animal. Two tick species were identified: Hyalomma dromedarii (2430 ticks) and Rhipicephalus sanguineus sensu lato (114 ticks). The first species had annual activity and consisted only of adults, while the second species had spring and summer activity and contained both adults and nymphs. At the annual level, the prevalence of infestation, abundance, and intensity were 38.60%, 44.63 and 115.64 ticks per animal, respectively. At the monthly level there were species specific fluctuations, with a peak in activity in September.

Molecular Detection of Microorganisms Associated with Small Mammals and Their Ectoparasites in Mali.

Small mammals are the natural reservoirs for many zoonotic pathogens. Using molecular tools, we assessed the prevalence of bacteria and protozoans in small mammals and their ectoparasites in Faladjè, Bougouni, and Bamoko, Mali. A total of 130 small mammals belonging to 10 different species were captured, of which 74 (56.9%) were infested by ectoparasites, including Laelaps echidnina, Xenopsylla cheopis, Amblyomma variegatum, Rhipicephalus sanguineus sensu lato, and Haemaphysalis spp. nymphs. DNA of Bartonella was found in 14/75 (18.7%), 6/48 (12.5%), and 3/7 (42.8%) small mammals from Faladjè, Bougouni, and Bamako, respectively. In Faladjè, Bartonella DNA was detected in 31/68 (45.6%) of L. echidnina and 14/22 (63.6%) of X. cheopis. In Bougouni, it was found in 2/26 (7.7%) of L. echidnina and 10/42 (23.8%) of X. cheopis. The sequences of Bartonella obtained from small mammals were close to those of Bartonella mastomydis, Bartonella elizabethae, and uncultured Bartonella spp. In Faladjè, Coxiella burnetii DNA was detected in 64.4% (29/45) of Haemaphysalis spp. ticks, 4.5% (2/44) of Mastomys erythroleucus, 12.5% (1/8) of Praomys daltoni, and 1.5% (1/68) of L. echidnina. We found DNA of Wolbachia in X. cheopis from Faladjè and DNA of Rickettsia africae and Ehrlichia ruminantium in Am. variegatum from Bougouni. The results of our study show that several small mammal species harbor and may serve as potential reservoirs of Bartonella spp., likely to play a major role in the maintenance, circulation, and potential transmission of bacteria in Mali. The pathogenicity of these bacteria for humans or animals remains to be demonstrated.

Molecular detection of tick-borne haemopathogens in shelter dogs and Rhipicephalus sanguineus (sensu lato) ticks from Peninsular Malaysia

Ticks are important vectors in transmitting various pathogens and they could jeopardize the health and welfare of humans and animals worldwide. The present study aimed to investigate the presence of important tick-borne haemopathogens (TBH) in dogs and ticks via polymerase chain reaction (PCR) assays. A total of 220 blood samples and 140 ticks were collected from 10 animal shelters in Peninsular Malaysia. Of 220 blood samples, 77 (35 %) were positive to TBH, of which 20 % were E. canis, 12 % were A. platys, 7 % were B. gibsoni and 7 % were B. vogeli. All ticks were identified as Rhipicephalus sanguineus with five samples (3.57 %) positive with TBH. Co-infections of TBH (0.45–9.55 %) in dogs were also observed in this study.

Identification of a non-host semiochemical from miniature pinscher, Canis lupus familiaris, that repels Rhipicephalus sanguineus sensu lato (Acari: Ixodidae)

It is already known that the beagle breed of domestic dogs produces semiochemicals capable of repelling the brown dog tick, Rhipicephalus sanguineus sensu lato (s.l.). With a view to discovering new non-host semiochemicals as tick repellents, we compared the semiochemicals produced by a putative tick-resistant breed of dog, miniature pinscher, with known tick-resistant (beagle) and tick-susceptible (English cocker spaniel) breeds. Two non-host compounds produced by beagles, i.e. 2-hexanone and benzaldehyde, were shown to be present in samples collected from all three breeds. Furthermore, two compounds, 6-methyl-5-hepten-2-one and 1,2,4-trimethylbenzene, were found in higher amounts in samples collected from miniature pinscher dogs. The mean amounts of benzaldehyde, 2-hexanone and 1,2,4-trimethylbenzene were similar for beagles and miniature pinschers (P > 0.05) and higher than the means observed for cocker spaniels (P < 0.05), whereas the mean amount of 6-methyl-5-hepten-2-one produced by miniature pinschers was significantly higher (P < 0.05) than for the other breeds of dogs. In Petri-dish assays with adult R. sanguineus s.l., 6-methyl-5-hepten-2-one was repellent for all observation periods evaluated for the two highest concentrations (0.100 and 0.200 mg.cm−2, P < 0.01). The obtained results support our hypothesis that miniature pinschers are a tick-resistant dog breed and agree with previous observations of miniature pinschers being the breed least parasitized by ticks. Furthermore, the non-host semiochemical 6-methyl-5-hepten-2-one has potential to be developed for use as a repellent for the protection of susceptible dogs from R. sanguineus s.l. ticks.

Molecular detection and characterization of Hepatozoon canis in stray dogs from Cuba.

Canine hepatozoonosis caused by Hepatozoon canis is a worldwide distributed tick-borne disease of domestic and wild canids that is transmitted by ingestion of Rhipicephalus sanguineus sensu lato (s.l.) ticks. The present study was aimed to determine the prevalence of Hepatozoon infections in 80 stray dogs from Havana Province in Cuba, and to confirm the species identity and phylogenetic relationships of the causative agent. Samples were screened by microscopical examination of thin blood smears for the presence of Hepatozoon spp. gamonts and by genus-specific SYBR green-based real-time PCR assay targeting the 18S rRNA gene. Direct microscopy examination revealed Hepatozoon gamonts in the peripheral blood of 8 dogs (10.0%; 95% CI: 4.80-18.0%), while 38 animals (47.5%; 95% CI: 36.8-58.4%) were PCR-positive, including all microscopically positive dogs. Hence, the agreement between the two detection methods was 'poor' (κ = 0.20). Hematological parameters did not differ significantly between PCR-positive and PCR-negative dogs (p > 0.05). The DNA sequences of the 18S rRNA gene of the Hepatozoon spp. from Cuban dogs showed a nucleotide identity >99% with those of 18S rRNA sequences of Hepatozoon canis isolates from Czech Republic, Brazil and Spain. Phylogenetic analysis revealed that obtained sequences clustered within the Hepatozoon canis clade, different from the Hepatozoon felis or Hepatozoon americanum clades. The present study represents the first molecular characterization of Hepatozoon canis in stray dogs within Cuba.

Prevalence and distribution of pathogen infection and permethrin resistance in tropical and temperate populations of Rhipicephalus sanguineus s.l. collected worldwide.

The brown dog tick, Rhipicephalus sanguineus sensu lato (s.l.) Latreille (Acari: Ixodidae), is a peridomestic, cosmopolitan parasite of dogs known to vector numerous pathogens of veterinary and medical importance. Recent phylogenetic analyses separate this tick into temperate and tropical lineages. Populations of Rh. sanguineus s.l. have been reported to exhibit sodium channel target site insensitivity to permethrin and etofenprox, which is likely due to the prolonged use of pyrethroids against many pests in and around the home. In this study, populations collected in the Caribbean, Africa, Asia, Europe and North America, were tested to identify the distribution of a known resistance mechanism, pathogen-vector interactions and phylogeny in relation to latitude. Using molecular assays, populations from 29 distinct locations were simultaneously geographically typed and screened for bacterial infection by Rickettsia, Ehrlichia, Babesia and Hepatozoon species, and for the presence of a sodium channel single nucleotide polymorphism known to confer permethrin resistance. Implications of these results on Rh. sanguineus s.l. management in association with geographical distribution will be discussed.

Sublethal concentrations of acetylcarvacrol affect reproduction and integument morphology in the brown dog tick Rhipicephalus sanguineus sensu lato (Acari: Ixodidae).

Rhipicephalus sanguineus sensu lato (s.l.), also known as the brown dog tick, is among the main tick species involved in the transmission of pathogens to humans and other animals and, therefore, the target of numerous control methods. However, due to the disadvantages of synthetic acaricides, the use of alternative products such as plant derivatives has been encouraged. This study aimed to evaluate the acaricidal potential of acetylcarvacrol and to determine its efficacy at sublethal doses for the control of R. sanguineus s.l. female ticks. In addition, as acetylcarvacrol was applied topically, morphological alterations in the integument were assessed. Acetylation of carvacrol was performed by reaction with acetic anhydride in a sodium hydroxide solution, being confirmed by infrared spectroscopy. The lethal concentration for 50 and 90% (LC50 and LC90) of unfed ticks and the efficacy of acetylcarvacrol in engorged females were determined after the Adult Immersion Test (AIT). For the evaluation of effects of acetylcarvacrol in the integument, routine histological techniques were employed after the AIT. The LC50 and LC90 in unfed females were 2.8 and 7.2 μL/mL, respectively. Regarding reproductive performance, after treatment with 8.0 μL/mL acetylcarvacrol 90.9% control was achieved, as ticks showed the lowest egg production index (EPI), hatching rate (HR), and fecundity rate (FR). In the integument, considerable morphological alterations were observed both in cuticle and epithelium. Thus, acetylcarvacrol affected R. sanguineus s.l. external coating and reproduction when applied at sublethal concentrations, probably contributing to a long-term control.

Morphophysiological analysis of the salivary glands of Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) exposed to ozonated water: A control strategy.

The tick Rhipicephalus sanguineus sensu lato has great medical and veterinary importance, mainly because the ability to transmit many diseases, causing harm to pets but also risks to public health. The blood spoliation and transmission of pathogens occur because of the immunosuppressive action of these ticks' saliva, a potent mixture of bioactive substances that is secreted by the salivary glands, one of the organs responsible for their biological success, and hence the target of studies for their control. Ozone has promise for use as an alternative acaricide, due to its proven efficiency in controlling agricultural and food pests, besides posing no risk of environmental contamination or to animal and human health. Therefore, this study evaluated the acaricidal potential of exposure of females of R. sanguineus s.l. to ozonated water at many concentrations and analysed the morphophysiological alterations of the salivary glands, employing histological and light microscopic techniques. The results demonstrated that the ozonated water at the concentrations investigated caused severe alterations in the salivary glands, bringing a new perspective for control of R. sanguineus s.l., through an ecologically correct method due to the absence of harm to non-target organisms and the environment.

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.

Identical 18S rRNA haplotypes of Hepatozoon canis in dogs and foxes in Brandenburg, Germany

Hepatozoon canis is a blood parasite of the suborder Adeleorina infecting wild and domestic canids. Transmission occurs by oral uptake of Rhipicephalus sanguineus sensu lato vector ticks infected with H. canis, but vertical transmission is also assumed to be possible. In German foxes, a high prevalence of H. canis has previously been reported despite the fact that R. sanguineus s.l. is not endemic. In the absence of knowledge about local transmission pathways, foxes should be considered to be possible reservoirs of H. canis and contribute to infection of domestic dogs. The present study aimed to determine how often foxes and dogs are infected in Brandenburg (Germany) and if identical or different H. canis 18S rRNA haplotypes are found in these host species. Hepatozoon spp. were detected by PCR in 46/1050 (4.4 %) of dog blood and 176/201 (77.6 %) of fox spleen samples from Brandenburg. Sequencing of 19 dog and 56 fox samples identified all as H. canis. For nine positive dogs, owners stated that they had never left Germany suggesting that autochthonous transmission occurs not only in foxes but also in dogs. Sequences for seven of these possible autochthonous cases were obtained and six were identical to the predominant haplotype found in the foxes. Haplotype network analysis confirmed that many dogs, including some without travel history, carried the same or very similar 18S rRNA haplotypes as the foxes suggesting that both hosts participate in the same epidemiological cycle.

Molecular detection of a Borrelia sp. in nymphs of Amblyomma brasiliense ticks (Acari: Ixodidae) from Iguaçu National Park, Brazil, genetically related to Borrelia from Ethiopia and Côte d’Ivoire

Gram-negative spirochetes of the genus Borrelia are transmitted to vertebrate hosts through the tick bite during blood intake. Pathogenic Borrelia species may cause relapsing fever or Lyme borreliosis in humans. Our study aimed to molecularly detect and characterize bacteria of this genus in ticks collected in the Iguaçu National Park, located in southern Brazil. Ticks were collected from the environment (free living) and from hosts from May 2015 to July 2017. In total, 829 ticks were tested, being 741 from the environment and 88 from hosts; 128 ticks were larvae, 523 were nymphs, and 178 were adults (80 males and 98 females). The species identified were: Amblyomma brasiliense (42.9 %), Amblyomma coelebs (16.8 %), Amblyomma sp. (15.0 %), Amblyomma incisum (10.3 %), Amblyomma ovale (8.7 %), Haemaphysalis juxtakochi (5.5 %), Rhipicephalus microplus (0.5 %), Amblyomma longirostre (0.2 %), and Rhipicephalus sanguineus sensu lato (0.1 %). DNA extraction was performed with pools of larvae or nymphs, or individually with adults, for a total of 394 samples. The PCR technique used to detect Borrelia DNA was performed with two rounds of amplification reactions targeting a segment of the flagellin B gene (flaB). Amplification occurred in only one DNA sample, which was obtained from nymphs of A. brasiliense collected from the vegetation of a trail. The nucleotide sequence analysis revealed 90.8 % identity to a sequence of Borrelia sp. from Côte d’Ivoire, and 89.1 % identity to a sequence from Ethiopia. Phylogenetic analysis revealed that the Borrelia sequence from A. brasiliense was distinct from the Borrelia species of the groups belonging to Lyme borreliosis, relapsing fever and the one associated with reptile and echidna as hosts. The sequence is likely from a putative new species of Borrelia and was detected for the first time in A. brasiliense ticks.

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.