Seroprevalence of Anaplasma spp. and Ehrlichia spp. and molecular detection of Anaplasma phagocytophilum and Anaplasma platys in stray dogs in Bosnia and Herzegovina.

Stray dogs may be highly exposed to vector-borne pathogens (VBPs), including zoonotic agents, and therefore may pose a high risk of spreading infections to other animals and humans. Among the Anaplasmataceae, Anaplasma phagocytophilum, A. platys and Ehrlichia canis are commonly identified species in dogs in Europe; however, information on the occurrence of these pathogens in canine populations from Bosnia and Herzegovina (B&H) is still lacking. Thus, the aim of this study was to determine the seroprevalence of Anaplasma spp. and Ehrlichia spp. in stray dogs in the Sarajevo region of B&H and to identify A. phagocytophilum, A. platys, E. canis and E. ewingii by molecular techniques. A total of 903 blood samples of stray dogs were screened by SNAP 4Dx Plus Test for the presence of antibodies against A. phagocytophilum/A. platys and E. canis/E. ewingii. Real-time PCR assays were performed for the detection of Anaplasmataceae, A. phagocytophilum, A. platys, E. canis and E. ewingii in seropositive dogs. Antibodies to A. phagocytophilum/A. platys and/or E. canis/E. ewingii were detected in 187 (20.7%) samples. Seroprevalence was highest for A. phagocytophilum/A. platys (184/903, 20.4%). Two dogs had antibodies to E. canis/E. ewingii, while one dog was found to have antibodies to A. phagocytophilum/A. platys and to E. canis/E. ewingii. Forty-eight (25.7%) of the 187 seropositive dogs examined by Real-time PCR were positive for Anaplasmataceae. A. phagocytophilum was detected in 45 (24%) samples, while one sample was positive for A. phagocytophilum and A. platys. Two samples positive for Anaplasmataceae tested negative in the species-specific PCRs. E. canis or E. ewingii could not be detected in any of the Ehrlichia-seropositive dogs. These findings highlight the need for dog health monitoring, improving the health and welfare of stray dog population, and establishment of effective surveillance systems to combat VBDs.

Molecular prevalence and risk factors associated with tick-borne pathogens in cattle in western Kenya.

BACKGROUND: Tick-borne pathogens (TBPs) are of global importance, especially in sub-Saharan Africa where they represent a major constraint to livestock production. Their association with human disease is also increasingly recognized, signalling their zoonotic importance. It is therefore crucial to investigate TBPs prevalence in livestock populations and the factors associated with their presence. We set out to identify TBPs present in cattle and to determine associated risk factors in western Kenya, where smallholder livestock production is important for subsistence and market-driven income. RESULTS: Tick-borne pathogen infections in blood samples collected from cattle at livestock markets and slaughterhouses between May 2017 and January 2019 were identified by high-resolution melting analysis and sequencing of PCR products of genus-specific primers. Of the 422 cattle sampled, 30.1% (127/422) were infected with at least one TBP, while 8.8% (37/422) had dual infections. Anaplasma spp. (19.7%) were the most prevalent, followed by Theileria (12.3%), Ehrlichia (6.6%), and Babesia (0.2%) spp. Sequence analysis of the TBPs revealed them to be Anaplasma platys-like organisms (13.5%), Theileria velifera (7.4%), Anaplasma marginale (4.9%), Theileria mutans (3.1%), Theileria parva (1.6%), and Babesia bigemina (0.2%). Ehrlichia ruminantium, Rickettsia spp., and arboviruses were not detected. Exotic breeds of cattle were more likely to be infected with A. marginale compared to local breeds (OR: 7.99, 95% CI: 3.04-22.02, p <  0.001). Presence of ticks was a significant predictor for Anaplasma spp. (OR: 2.18, 95% CI: 1.32-3.69, p = 0.003) and Ehrlichia spp. (OR: 2.79, 95% CI: 1.22-7.23, p = 0.022) infection. Cattle sampled at slaughterhouses were more likely to be positive for Anaplasma spp. (OR: 1.64, 95% CI: 1.01-2.70, p = 0.048) and A. marginale (OR: 3.84, 95% CI: 1.43-12.21, p = 0.012), compared to those sampled at livestock markets. CONCLUSION: This study reports TBP prevalence and associated risk factors in western Kenya, factors which are key to informing surveillance and control measures.

Molecular detection of zoonotic blood pathogens in ticks from illegally imported turtles in Italy.

International trade of animals may represent a gateway for the spreading of zoonotic pathogens and their vectors. Amongst animals, reptiles are commonly illegally imported worldwide, being Italy in the fifth position of importation of these animals. Thus, the current study analysed the pathogens associated with Hyalomma aegyptium ticks, which were collected from illegally imported tortoises from North Africa to Italy. All tick DNA samples were tested by conventional PCR for the presence of Anaplasma spp., Babesia spp., Borrelia spp., Coxiella burnetti, Ehrlichia spp., Hepatozoon spp., Rickettsia spp. and microfilariae of filarioids. Out of 22% (n=161) of ticks screened, 78.9% (n=127) were males and 21.1% (n=34) females. Among them, three male specimens collected from two different turtles (1.9%; 95% CI; 0.5-5.5) scored positive for Anaplasma spp./Ehrlichia spp., whereas all females were negative. BLAST analysis of the sequences obtained from positive samples revealed 99-99.3% nucleotide identity with the sequence of Ehrlichia ewingii available in GenBank. The finding of E. ewingii in ticks from imported reptiles warrants the need for imposing strict rules in the international trade of reptiles to effectively reduce the introduction of exotic pathogens and their vectors in new geographic areas.

Molecular detection of Rickettsia sp. cf. Rickettsia monacensis in Ixodes sp. cf. Ixodes affinis collected from white-tailed deer in Campeche, Mexico.

Deer encompass a group of large-sized vertebrates that serve as hosts for a wide variety of ectoparasites, mainly ticks. In Mexico, ticks have relevance as vectors of pathogenic microorganisms, and 20 species of hard ticks are associated with four species of deer, although only a single study has been conducted to detect bacterial agents associated with ticks from deer in the country. In February, 2019 three white-tailed deers (Odocoileus virginianus) were hunted from the locality of Chiná from the municipality of Campeche, Mexico. The sampled deers were parasitized by 26 ticks belonged to three species: Amblyomma mixtum (5♀, 1♂), Amblyomma ovale (2♀, 1♂), and Ixodes sp. cf. Ixodes affinis (15♀, 2♂). Specimens were screened individually for Anaplasma, Borrelia, Ehrlichia, and Rickettsia DNA by the amplification of several fragments of 16S rRNA, gltA, 17-kDa, and flaB genes. This study report for the first time the presence of Rickettsia sp. cf. Rickettsia monacensis in Ixodes sp. cf. Ixodes affinis in Mexico.

Rhipicephalus microplus and its vector-borne haemoparasites in Guinea: further species expansion in West Africa.

Rhipicephalus microplus is an ixodid tick with a pantropical distribution that represents a serious threat to livestock. West Africa was free of this tick until 2007, when its introduction into Benin was reported. Shortly thereafter, further invasion of this tick species into other West African countries was identified. In this paper, we describe the first detection of R. microplus in Guinea and list the vector-borne haemoparasites that were detected in the invading and indigenous Boophilus species. In 2018, we conducted a small-scale survey of ticks infesting cattle in three administrative regions of Guinea: N`Zerekore, Faranah, and Kankan. The tick species were identified by examining their morphological characteristics and by sequencing their COI gene and ITS-2 gene fragments. R. microplus was found in each studied region. In the ticks, we found the DNA of Babesia bigemina, Anaplasma marginale, Anaplasma platys, and Ehrlichia sp. The results of this study indicate that R. microplus was introduced into Guinea in association with cows from Mali and/or the Ivory Coast.

Molecular detection and characterisation of protozoan and rickettsial pathogens in ticks from cattle in the pastoral area of Karamoja, Uganda.

Ticks and tick-borne diseases (TBDs) significantly affect cattle production and the livelihoods of communities in pastoralist areas. Data on protozoan and rickettsial pathogens in ticks infesting cattle in Uganda is scanty; while it is an indicator of the likelihood of disease transmission and occurrence. A cross-sectional study was conducted amongst cattle in the Karamoja Region, northeastern Uganda, from July through September 2017, to determine the tick species diversity, identify protozoan and rickettsial pathogens in the ticks, and characterise pathogenic species by sequence and phylogenetic analyses. About 50 % of the ticks detected from each predilection site on each animal were collected from 100 purposively-selected cattle from 20 randomly-selected herds. Twelve tick species belonging to the genera Amblyomma, Rhipicephalus and Hyalomma were identified, the most abundant being Amblyomma lepidum (93.9 %), followed by Amblyomma variegatum (2.0 %) and Rhipicephalus evertsi evertsi (1.0 %). Tick species that have not been reported in recent studies amongst cattle in Uganda were found, namely Rhipicephalus pravus, Rhipicephalus praetextatus and Rhipicephalus turanicus. The ticks were grouped into 40 pools, by species and location, and the reverse line blot (RLB) hybridisation assay was used to detect pathogens from the ticks. The most frequently detected tick-borne parasites were Theileria mutans, Theileria velifera and Theileria parva, each observed in 25 % (10/40) of the tick pools. Tick-borne pathogens, namely Babesia rossi, Babesia microti and Theileria sp. (sable) that are not common to, or not known to infect, cattle were identified from ticks. The gene encoding Ehrlichia ruminantium pCS20 region, the Ehrlichia and Anaplasma 16S rRNA gene, and T. parva p67 sporozoite antigen gene were amplified, cloned and sequenced. Seven novel E. ruminantium pCS20 variants were identified, and these grouped into two separate clusters with sequences from other parts of Africa and Asia. The T. parva p67 sequences were of the allele type 1, and parasites possessing this allele type are commonly associated with East Coast fever in eastern Africa. Analysis of the Ehrlichia and Anaplasma 16S rRNA gene sequences showed that they were closely related to Rickettsia africae and to a new Ehrlichia species variant recently found in China. Our R. africae 16S rRNA sequences grouped with R. africae isolates from Nigeria, Egypt and Benin. The information on tick species diversity and pathogens in the various tick species provides an indicator of potential transmission amongst cattle populations, and to humans, and can be useful to estimate disease risk and in control strategies.

Detection of Ehrlichia sp. in Amblyomma sculptum parasitizing horses from Brazilian Pantanal wetland.

Bacteria of the genus Ehrlichia are transmitted by ticks and also are an important cause of infection in wild and domestic mammals. Infection with Ehrlichia spp. has been reported in horses, especially in the USA, Nicaragua and Brazil. In this study, we report the parasitism by Amblyomma sculptum, Rhipicephalus microplus and Dermacentor nitens ticks in horses from a ranch located in south Pantanal wetland. Molecular and serological analyzes to determine infection by Ehrlichia spp. in horses and their respective ticks were carried out. A total of 12 horses were submitted to blood collection to investigate antibodies by indirect immunofluorescence assay (IFA) using Ehrlichia canis crude antigens and to be tested by polymerase chain reaction (PCR) in order to amplify fragments of the 16S rRNA, dsb, groEL and sodB gene of Ehrlichia spp. A total of 164 tick specimens were removed from horses, stored in isopropanol and later identified as D. nitens, A. sculptum and R. microplus. DNA from ticks were extracted and subjected to the same PCR assays to detect Ehrlichia spp. Anti-Ehrlichia spp. antibodies were detected in five/12 (41.7 %) horses by IFA, with antibody titers ranging from 40 to 160. All horse DNA samples were negative for the 16S rRNA, dsb, groEL and sodB of Ehrlichia spp. One A. sculptum female was positive to all target genes of Ehrlichia. This tick was parasitizing an Ehrlichia-seropositive horse with antibody titer of 80. Nucleotide sequences of 16S rRNA, dsb, groEL and sodB genes showed close relationship with different strains of Ehrlichia detected in wild mammals, Amblyomma ticks and horses from Brazil and Argentina. Detection of anti-Ehrlichia sp. antibodies suggests that horses have been exposed to an ehrlichial agent in the Pantanal. Future studies on Ehrlichia infection should be carried out to better elucidate and to bring new information about equine ehrlichiosis, since these animals are important hosts of ticks in the Brazilian Pantanal wetlands.

Canine infection with Dirofilaria immitis, Borrelia burgdorferi, Anaplasma spp., and Ehrlichia spp. in the United States, 2013-2019.

BACKGROUND: Dogs in the US are commonly infected with vector-borne pathogens, including heartworm and tick-borne disease agents. The geographic distribution of both arthropod vectors and the pathogens they transmit continues to expand. METHODS: To describe the current geographic distribution and prevalence of antigen of Dirofilaria immitis and antibody to Borrelia burgdorferi, Ehrlichia spp., and Anaplasma spp. in dogs, we summarized over 144 million test results from 2013 to 2019, inclusive, by county, state, and region. Canine seroprevalence by state was compared to population-adjusted human reports of tick-borne diseases. RESULTS: Results varied regionally, with D. immitis antigen and Ehrlichia spp. antibodies more frequently detected in the Southeast (2.6% and 5.2%, respectively) and antibody to B. burgdorferi and Anaplasma spp. most common in the Northeast (12.1% and 7.3%, respectively). Overall, percent positive test results to D. immitis decreased in the Southeast by 33.3% when compared to earlier summaries using the same strategy (from 3.9 to 2.6%). Geographic expansion of areas where dogs commonly test positive for Ehrlichia spp. was evident, likely because of a change in the test made in 2012 to allow detection of antibodies to E. ewingii concomitant with expansion of vector tick populations. Percent positive test results to Ehrlichia spp. increased in every region; this shift was particularly pronounced in the Southeast, where percent positive test results increased fourfold (from 1.3 to 5.2%). Continued geographic expansion of B. burgdorferi and A. phagocytophilum was apparent in the Northeast, Midwest, and Upper South, although canine seroprevalence of antibody to B. burgdorferi was much lower than prior surveys in many Lyme-endemic areas. Annual reports of human cases of Lyme disease, ehrlichiosis, and anaplasmosis were associated with percent positive canine results by state for the three tick-borne disease agents (R2 = 0.812, 0.521, and 0.546, respectively). Within endemic areas, percent positive test results for all three tick-borne agents demonstrated evidence of geographic expansion. CONCLUSIONS: Large scale analysis of results from screening dogs in practice for evidence of vector-borne infections, including those with zoonotic importance, continues to be a valuable strategy for understanding geographic trends in infection risk over time.

Diversity and genotypic analysis of tick-borne pathogens carried by ticks infesting horses in Korea.

Ticks account for an extensive range of health and welfare issues in horses. In addition, tick-borne pathogens (TBPs) limit global animal trading and equine sporting events. Here, we assess the prevalence, co-infectivity and risk factors of TBPs in horse ticks in Korea. A total of 245 hard ticks, including 103 male and 142 female adults, were obtained from horses on Jeju Island during the spring to autumn seasons of 2013-2019. All collected ticks were identified as adult Haemaphysalis longicornis. We screened and analyzed each tick for the presence of several TBPs by polymerase chain reaction (PCR) analysis. Among the 245 ticks, we detected genes for three TBPs, Candidatus Rickettsia longicornii (22.9%), Ehrlichia canis (0.4%) and Theileria luwenshuni (0.4%), while Anaplasma spp. was not detected. TBPs were most prevalent in ticks harvested during the autumn season, and more abundant in the female than male adults. This is the first report of the genera Ehrlichia, Rickettsia and Theileria in horse ticks in Korea. TBPs in horse ticks are likely a reservoir for zoonotic transmission to other animals, including humans. Our findings demonstrate the need for further understanding of the prevalence and epidemiology of TBPs in wild and domestic animals.

Rickettsia spp. and Ehrlichia spp. in Amblyomma ticks parasitizing wild amphibious sea kraits and yellow-margined box turtles in Okinawa, Japan.

Recently, several tick-borne pathogens were detected in reptile-associated ticks. However, studies on the microorganisms in reptile-associated ticks in Japan are limited. This molecular survey thus aimed to identify and characterize tick-borne pathogens (Rickettsiaceae and Anaplasmataceae) in reptile-associated ticks in Japan. In total, 77 Amblyomma nitidum and 104 Amblyomma geoemydae were collected from wild amphibious sea kraits (Laticauda semifasciata, Laticauda colubrina, and Laticauda laticaudata) and from yellow-margined box turtles (Cuora flavomarginata evelynae), respectively. Conventional polymerase chain reaction was performed using the DNA extracted from the ticks to detect the selected pathogens. Sequencing analysis of four Rickettsia genes (gltA, ompA, ompB, and sca4) led to the identification of a putative novel Rickettsia sp. and Rickettsia aeschlimannii-like rickettsia in A. nitidum and A. geoemydae, respectively. Sequencing analysis of gltA and groEL of Anaplasmataceae revealed that the Ehrlichia spp. in these ticks were novel and related to Candidatus Ehrlichia occidentalis. This is the first study on the microorganisms in A. nitidium and the first record of Rickettsia and Ehrlichia in A. geoemydae. Further studies are required to understand their pathogenicity to humans and animals and their life cycle in the wild.

Molecular Detection and Identification of Candidatus Ehrlichia Hainanensis, A Novel Ehrlichia Species in Rodents from Hainan Province, China.

Ehrlichia (Anaplasmataceae family) are obligatory intracellular bacteria that infect humans and animals. They are hosted by mammals such as canines, bovines and wild rodents, and are vectored by ticks. In this study, we collected 121 rodent samples comprising 67 Niviventer fulvescens, 27 Rattus tanezumi, 24 Chiromyscus sp., 2 Rattus nitidus and 1 Leopoldamys edwardsi from Hainan province, which includes the second largest island in China. The presence and genetic diversity of Ehrlichia species was evaluated and characterized by amplification and sequencing of 16S rRNA, groEL and gltA genes. An Ehrlichia species was detected in 5 of the 67 Niviventer fulvescens samples (7.46%). The 16S rRNA, groEL and gltA genes showed the highest identity to known Ehrlichia sequences (99.20%, 89.87% and 83.86%, respectively). In the phylogenetic trees they formed a cluster distinct from all other species. We propose that this species is a putative novel Ehrlichia species, which we suggest be named Candidatus Ehrlichia hainanensis. Its pathogenicity to humans remains to be further researched, and molecular surveillance in local populations is needed.

The Prevalence of Different Human Pathogenic Microorganisms Transmitted by Ixodes Tick Vectors in Belarus.

Pathogens transmitted by ticks cause several important diseases in humans, including Lyme disease, the incidence of which has been increasing in Belarus. Between April and October 2017, a total of 504 questing Ixodid ticks (77% Ixodes ricinus and 23% Dermacentor reticulatus) were collected from six regions and city of Minsk, in Belarus. All ticks were analyzed by RT-PCR amplification for the presence of Borrelia burgdorferi sensu lato, tick-borne encephalitis virus (TBEV), Anaplasma phagocytophillum, Ehrlihia muris, and Borrelia miyamotoi. B. burgdorferi s.l. and Rickettsia spp. were the most commonly detected tick-borne pathogens, with prevalence rates of 31.08% and 33.7%, respectively. A. phagocytophillum was found in 104 (20.63%), and 107 (21.2%) ticks were positive for E. muris. TBEV was detected in 83 (16.47%). Circulation of Borrelia miyamotoi spirochete in I. ricinus ticks in Brest, Gomel, and Minsk region was detected for the first time. Our data provide a basis for further studies to determine the distribution and abundance of different tick species in Belarus and therefore a capacity to predict where cases of important tick-borne diseases may occur.

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.

Molecular identification and characterization of Rickettsia spp. and other tick-borne pathogens in cattle and their ticks from Huambo, Angola.

Ticks are one of the most common vectors of a broad variety of pathogenic agents that significantly affects cattle production causing reduced productivity and important economic losses, while simultaneously having an impact on human health due to the zoonotic risk. In much of the territory of Angola urban population has grown rapidly in recent decades, sharing today close contact with large farms that are generally owned by city residents, providing the ideal conditions for vector-borne pathogens (VBP) transmission between animals and humans. Here we studied the occurrence of Rickettsia, Anaplasma, Ehrlichia, Babesia and Theileria in domestic cattle (n = 98) from Huambo, Angola, and their ticks (n = 116) to obtain a more detailed knowledge into the spectrum of tick-borne agents circulating in this population. We morphologically identified Amblyomma variegatum, Rhipicephalus decoloratus and R. evertsi mimeticus ticks, further confirmed by molecular analysis of the 12S rDNA and 16S rDNA genes. Although none of the bovine blood showed to be positive for R. africae by the ompB, ompA and gltA assays, five ticks showed to be positive for R. africae by the ompB, ompA and the gltA PCRs. Two 18S rRNA sequences were retrieved from bovine blood and one sequence from A. variegatum tick, showing 100% identity with Theileria mutans. By using a PCR targeting the 16S rRNA gene of Anaplama spp. we have also obtained six bovine blood samples showing 99-100 % nucleotide sequence identity with A. capra, two showing 98 % nucleotide sequence identity with A. phagocytophilum and three showing 98-100 % nucleotide sequence identity with A. platys. None of the ticks were positive. The present study shows the presence of a wide range of vector-borne diseases in domestic cattle and their ticks in Huambo province, Angola. Given the lack of both animal and human health infrastructures in this rural region, swift diagnosis and treatment is hampered which could produce a more severe impact on health.

Serological prevalence of six vector-borne pathogens in dogs presented for elective ovariohysterectomy or castration in the South central region of Texas.

BACKGROUND: Most vector-borne pathogens cause zoonotic diseases. These zoonoses often have wild animal reservoirs that play a significant role in disease epidemiology. However, pet animals have also been implicated in transmission of zoonotic agents to humans. To exemplify, dogs are competent reservoir hosts for several zoonotic vector-borne bacteria and protozoa. Despite that vector-borne diseases can be life-threatening for both pets and humans, studies on pathogen seroprevalence are very limited. Therefore, the objective of this study was to determine the serological prevalence of six zoonotic vector-borne agents in dogs from the South Central region of Texas (US). Electronic medical records of dogs, presenting over 2014-2019 for elective ovariohysterectomy or castration at a high volume spay and neuter clinic, were reviewed for serological testing. Sera from 418 dogs were tested for the Dirofilaria immitis antigen, and antibodies to Anaplasma phagocytophilum, Anaplasma platys, Borrelia burgdorferi, Ehrlichia canis, and Ehrlichia ewingi, using a commonly available commercial test kit. Descriptive statistics were computed to characterize the respective seroprevalence rates of the dog population. The study involved 192 (46%) male and 226 (54%) female dogs. RESULTS: Overall, 85 (20%) dogs tested positive for at least one of the 6 pathogens investigated. The highest seroprevalence rate averaged over the 6-year period was 11.7% for D. immitis followed by 8.4% for E. canis and/or E. ewingii, 4.3% for A. phagocytophilum and/or A. platys, and 0.2% for B. burgdorferi. The co-exposure or co-infection was only detected in 3.8% of the dog population. CONCLUSIONS: Together, opportunistic testing of dogs presenting for elective surgical procedures may provide an effective way of assessing seroprevalence and/or risk factors for common vector-borne diseases within a geographic region of concern.

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.

Molecular prevalence of emerging Anaplasma and Ehrlichia pathogens in apparently healthy dairy cattle in peri-urban Nairobi, Kenya.

BACKGROUND: Anaplasma and Ehrlichia species are tick-borne pathogens of both veterinary and public health importance. The current status of these pathogens, including emerging species such as Ehrlichia minasensis and Anaplasma platys, infecting cattle in Kenya, remain unclear, mainly because of limitation in the diagnostic techniques. Therefore, we investigated the Anaplasma and Ehrlichia species infecting dairy cattle in Nairobi, Kenya using molecular methods. RESULTS: A total of 306 whole blood samples were collected from apparently healthy dairy cattle. Whole blood DNA was extracted and tested for presence of Anaplasma and Ehrlichia DNA through amplification and sequencing of the 16S rDNA gene. Sequence identity was confirmed using BLASTn analysis while phylogenetic reconstruction was performed to determine the genetic relationship between the Kenyan isolates and other annotated genotypes available in GenBank. Anaplasma and Ehrlichia species were detected in 19.9 and 3.3% of all the samples analyzed, respectively. BLASTn analysis of the sequences against non-redundant GenBank nucleotide database revealed infections with A. platys (44.8%), A. marginale (31%) and A. bovis (13.8%). All four sequenced Ehrlichia spp. were similar to Ehrlichia minasensis. Nucleotide polymorphism was observed for A. platys, A. bovis and E. minasensis. The Anaplasma species clustered in four distinct phylogenetic clades including A. marginale, A. platys, A. bovis and some unidentified Anaplasma spp. The Kenyan Ehrlichia minasensis clustered in the same clade with isolates from America and Australia but distant from E. ruminantium. CONCLUSION: This study provides the first report of infection of dairy cattle in Kenya with A. platys and E. minasensis, which are emerging pathogens. We conclude that cattle in peri-urban Nairobi are infected with various species of Anaplasma and E. minasensis. To understand the extent of these infections in other parts of the country, large-scale screening studies as well as vector identification is necessary to inform strategic control.

Molecular detection of pathogens in ticks and fleas collected from companion dogs and cats in East and Southeast Asia.

BACKGROUND: Ticks and fleas are considered amongst the most important arthropod vectors of medical and veterinary concern due to their ability to transmit pathogens to a range of animal species including dogs, cats and humans. By sharing a common environment with humans, companion animal-associated parasitic arthropods may potentially transmit zoonotic vector-borne pathogens (VBPs). This study aimed to molecularly detect pathogens from ticks and fleas from companion dogs and cats in East and Southeast Asia. METHODS: A total of 392 ticks and 248 fleas were collected from 401 infested animals (i.e. 271 dogs and 130 cats) from China, Taiwan, Indonesia, Malaysia, Singapore, Thailand, the Philippines and Vietnam, and molecularly screened for the presence of pathogens. Ticks were tested for Rickettsia spp., Anaplasma spp., Ehrlichia spp., Babesia spp. and Hepatozoon spp. while fleas were screened for the presence of Rickettsia spp. and Bartonella spp. RESULT: Of the 392 ticks tested, 37 (9.4%) scored positive for at least one pathogen with Hepatozoon canis being the most prevalent (5.4%), followed by Ehrlichia canis (1.8%), Babesia vogeli (1%), Anaplasma platys (0.8%) and Rickettsia spp. (1%) [including Rickettsia sp. (0.5%), Rickettsia asembonensis (0.3%) and Rickettsia felis (0.3%)]. Out of 248 fleas tested, 106 (42.7%) were harboring at least one pathogen with R. felis being the most common (19.4%), followed by Bartonella spp. (16.5%), Rickettsia asembonensis (10.9%) and "Candidatus Rickettsia senegalensis" (0.4%). Furthermore, 35 Rhipicephalus sanguineus ticks were subjected to phylogenetic analysis, of which 34 ticks belonged to the tropical and only one belonged to the temperate lineage (Rh. sanguineus (sensu stricto)). CONCLUSION: Our data reveals the circulation of different VBPs in ticks and fleas of dogs and cats from Asia, including zoonotic agents, which may represent a potential risk to animal and human health.

Ehrlichia spp. and Anaplasma spp. in Xenarthra mammals from Brazil, with evidence of novel 'Candidatus Anaplasma spp.'

Anaplasmataceae agents are obligatory intracellular Gram-negative α-proteobacteria that are transmitted mostly by arthropod vectors. Although mammals of the Superorder Xenarthra (sloths, anteaters, and armadillos) have been implicated as reservoirs for several zoonotic agents, only few studies have sought to detect Anaplasmataceae agents in this group of mammals. This study aimed to investigate the occurrence and genetic diversity of Anaplasma spp. and Ehrlichia spp. in blood and spleen samples of free-living Xenarthra from four different states in Brazil (São Paulo, Mato Grosso do Sul, Rondônia, and Pará). Nested and conventional PCR screening assays were performed to detect the rrs and dsb genes of Anaplasma spp. and Ehrlichia spp., respectively. The assays were positive in 27.57% (91/330) of the Anaplasma spp. and 24.54% (81/330) of the Ehrlichia spp. Of the 91 positive Anaplasma spp. samples, 56.04% were positive in a conventional PCR assay targeting the 23S-5S intergenic region. Phylogenetic and distance analyses based on the rrs gene allocated Anaplasma sequences from sloths captured in Rondônia and Pará states in a single clade, which was closely related to the A. marginale, A. ovis, and A. capra clades. The sequences detected in southern anteaters from São Paulo were allocated in a clade closely related to sequences of Anaplasma spp. detected in Nasua nasua, Leopardus pardalis, and Cerdocyon thous in Brazil. These sequences were positioned close to A. odocoilei sequences. Genotype analysis corroborated previous findings and demonstrated the circulation of two distinct Anaplasma genotypes in animals from north and southeast Brazil. The first genotype was new. The second was previously detected in N. nasua in Mato Grosso do Sul state. The intergenic region analyses also demonstrated two distinct genotypes of Anaplasma. The sequences detected in Xenarthra from Pará and Rondônia states were closely related to those in A. marginale, A. ovis, and A. capra. Anaplasma spp. sequences detected in Xenarthra from São Paulo and were allocated close to those in A. phagocytophilum. The analyses based on the dsb gene grouped the Ehrlichia spp. sequences with sequences of E. canis (São Paulo, Mato Grosso do Sul, and Pará) and E. minasensis (Rondônia and Pará). The data indicate the occurrence of E. canis and E. minasensis and two possible new Candidatus species of Anaplasma spp. in free-living mammals of the Superorder Xenarthra in Brazil.

Molecular screening of Anaplasmataceae in ticks collected from cattle in Corsica, France.

Bacteria belonging to the family Anaplasmataceae cause infections in humans and domestic animals. The consequences of infection can be significant economic losses for farmers. To better understand the epidemiology of tick-borne Anaplasmataceae in Corsica, we used molecular methods to detect and characterize Anaplasmataceae in ixodid ticks collected from cattle. Anaplasmataceae were detected by using a real-time polymerase chain reaction (PCR) targeting the 23S rRNA gene. Partial sequencing of rpoB and groEL allowed identifying species and conducting phylogenetic analyses. Infection rates were calculated using maximum likelihood estimation (MLE) with 95% confidence intervals (CIs). In total, 597 Rhipicephalus bursa, 216 Hyalomma marginatum, and seven Ixodes ricinus were collected from cattle during July-August 2017 and July-December 2018. Overall, Anaplasmataceae DNA was detected in 15 of 255 tick pools (MLE = 1.7%; 95% CI 0.9-2.7%). The molecular analysis revealed two species within the genus Anaplasma: A. marginale and A. phagocytophilum. We also detected bacteria within the genus Ehrlichia: we confirmed the detection of E. minasensis DNA in H. marginatum and R. bursa tick pools collected from cattle in Corsica and detected, for the first time to our knowledge, Candidatus E. urmitei in Corsican R. bursa ticks and a potential new species, Candidatus E. corsicanum. Further studies are needed to ascertain the pathogenesis and zoonotic potential of the strains and their importance for animals and public health.