Phenotypic and genotypic characterization of ticks and tick-borne pathogens from cattle in selected villages of Greater Letaba Municipality in Limpopo Province, South Africa.

Ticks are blood ectoparasites that feed on domestic, wild animals and humans. They spread a variety of infections such as protozoa, viruses, and bacteria. Moreover, cattle reared by smallholder farmers are susceptible to ticks and tick-borne pathogens. Therefore, accurate identification of ticks and detection of tick-borne pathogens is crucial. The main aim of this study was to identify and characterize ticks and tick-borne pathogens from selected villages in Greater Letaba Municipality, Limpopo Province, using morphological and molecular techniques. A total of 233 ticks were collected from cattle and identified morphologically using appropriate morphological keys. The following tick species were identified: Amblyomma hebraeum, Hyalomma rufipes, Hyalomma truncatum, Rhipicephalus appendiculatus, Rhipicephalus (Boophilus) decoloratus, Rhipicephalus (Boophilus) microplus, Rhipicephalus evertsi evertsi, and Rhipicephalus sanguineus. Rhipicephalus spp. was the most common species accounting to 73.8% of the identified ticks. The genomic DNA was extracted from the whole tick for tick identification and from midguts of the ticks for the detection of tick-borne pathogens, followed by amplification and sequencing. A total of 27 samples were positive for tick-borne pathogens: 23 samples tested positive for Theileria and four samples tested positive for Ehrlichia. Anaplasma and Rickettsial OmpB could not be detected from any of the samples. There was no obvious grouping of ticks and tick-borne pathogens on the bases of their locality. The findings of this study confirm previous reports that indicated that cattle reared by smallholder farmers harbor various ticks and tick-borne pathogens of veterinary, public health, and economic importance. Regular monitoring of tick infestations in villages around the study areas is recommended to avoid disease outbreaks.

Sample-to-answer direct real-time PCR detection of Anaplasma phagocytophilum, Ehrlichia spp., and Babesia spp. infections in whole-blood specimens.

Emerging tick-borne illnesses, such as anaplasmosis, babesiosis, or ehrlichiosis, are caused by obligate intracellular pathogens that have clinically comparable presentations. Diagnostics used in laboratories today are serologic assays and blood smear analyses, which have known diagnostic limits. This study evaluated the performance of a sample-to-answer direct real-time PCR laboratory-developed test for the multiplex qualitative detection of Anaplasma, Babesia, and Ehrlichia DNA in whole-blood specimens. Compared to two standard-of-care (SOC) methods, the DiaSorin tick-borne laboratory-developed test for Anaplasma detection demonstrated a positive percent agreement (PPA) and negative percent agreement (NPA) of 100% (95% CI, 0.80 to 1.0) and 89% (95% CI, 0.74 to 0.97), respectively with a discordant rate of 9.3% against microscopy. After discordant resolution, the NPA increased to 100%. For Babesia, the test demonstrated a PPA of 100% (95% CI, 0.90 to 1.0) and NPA of 100% (95% CI, 0.90 to 1.0). Compared to a SOC PCR method Anaplasma samples showed a PPA of 100% (95% CI, 0.66 to 1.0) and NPA of 100% (95% CI, 0.90 to 1.0). Ehrlichia results showed a PPA of 100% (95% CI, 0.69 to 1.0) and NPA of 100% (95% CI, 0.90 to 1.0). The total percent agreement was 98% (95% CI, 0.95 to 0.99) with a κ statistic of 0.95 (95% CI, 0.90 to 0.99) or almost perfect agreement compared to SOC methods. This laboratory-developed test for detecting Anaplasma, Babesia, and Ehrlichia DNA provides rapid and reliable detection of tick-borne infections without nucleic acid extraction. IMPORTANCE: This work demonstrates that detection of tick-borne illnesses, such as anaplasmosis, babesiosis, or ehrlichiosis, can be performed directly from whole blood with no extraction. The assay described here has a high positive and negative percent agreement with existing methods and is used as the standard of care. An increasing incidence of tick-borne illness combined with shortage of well-trained technologists to perform traditional manual testing, testing options that can be adapted to various lab settings, are of the utmost importance.

Prevalence and genetic diversity of Anaplasma and Ehrlichia in ticks and domesticated animals in Suizhou County, Hubei Province, China.

Anaplasma and Ehrlichia are tick-borne bacterial pathogens that cause anaplasmoses and ehrlichioses in humans and animals. In this study, we examined the prevalence of Anaplasma and Ehrlichia species in ticks and domesticated animals in Suizhou County, Hubei Province in the central China. We used PCR amplification and DNA sequencing of the 16S rRNA, groEL, and gltA genes to analyze. We collected 1900 ticks, including 1981 Haemaphysalis longicornis and 9 Rhipicephalus microplus, 159 blood samples of goats (n = 152), cattle (n = 4), and dogs (n = 3) from May to August of 2023. PCR products demonstrated that Anaplasma bovis, Anaplasma capra, and an Ehrlichia species were detected in the H. longicornis with the minimum infection rates (MIR) of 1.11%, 1.32%, and 0.05%, respectively; A. bovis, A. capra, and unnamed Anaplasma sp. were detected in goats with an infection rate of 26.31%, 1.31% and 1.97%, respectively. Anaplasma and Ehrlichia species were not detected from cattle, dogs and R. microplus ticks. The genetic differences in the groEL gene sequences of the Anaplasma in the current study were large, whereas the 16S rRNA and gltA gene sequences were less disparate. This study shows that ticks and goats in Suizhou County, Hubei Province carry multiple Anaplasma species and an Ehrlichia species, with relatively higher infection rate of A. bovis in goats. Our study indicates that multiple Anaplasma and Ehrlichia species exist in ticks and goats in the central China with potential to cause human infection.

Tissue-specific localization of tick-borne pathogens in ticks collected from camels in Kenya: insights into vector competence.

Background: Tick-borne pathogen (TBP) surveillance studies often use whole-tick homogenates when inferring tick-pathogen associations. However, localized TBP infections within tick tissues (saliva, hemolymph, salivary glands, and midgut) can inform pathogen transmission mechanisms and are key to disentangling pathogen detection from vector competence. Methods: We screened 278 camel blood samples and 504 tick tissue samples derived from 126 camel ticks sampled in two Kenyan counties (Laikipia and Marsabit) for Anaplasma, Ehrlichia, Coxiella, Rickettsia, Theileria, and Babesia by PCR-HRM analysis. Results: Candidatus Anaplasma camelii infections were common in camels (91%), but absent in all samples from Rhipicephalus pulchellus, Amblyomma gemma, Hyalomma dromedarii, and Hyalomma rufipes ticks. We detected Ehrlichia ruminantium in all tissues of the four tick species, but Rickettsia aeschlimannii was only found in Hy. rufipes (all tissues). Rickettsia africae was highest in Am. gemma (62.5%), mainly in the hemolymph (45%) and less frequently in the midgut (27.5%) and lowest in Rh. pulchellus (29.4%), where midgut and hemolymph detection rates were 17.6% and 11.8%, respectively. Similarly, in Hy. dromedarii, R. africae was mainly detected in the midgut (41.7%) but was absent in the hemolymph. Rickettsia africae was not detected in Hy. rufipes. No Coxiella, Theileria, or Babesia spp. were detected in this study. Conclusions: The tissue-specific localization of R. africae, found mainly in the hemolymph of Am. gemma, is congruent with the role of this tick species as its transmission vector. Thus, occurrence of TBPs in the hemolymph could serve as a predictor of vector competence of TBP transmission, especially in comparison to detection rates in the midgut, from which they must cross tissue barriers to effectively replicate and disseminate across tick tissues. Further studies should focus on exploring the distribution of TBPs within tick tissues to enhance knowledge of TBP epidemiology and to distinguish competent vectors from dead-end hosts.

Detection of Anaplasma and Ehrlichia bacteria in humans, wildlife, and ticks in the Amazon rainforest.

Tick-borne bacteria of the genera Ehrlichia and Anaplasma cause several emerging human infectious diseases worldwide. In this study, we conduct an extensive survey for Ehrlichia and Anaplasma infections in the rainforests of the Amazon biome of French Guiana. Through molecular genetics and metagenomics reconstruction, we observe a high indigenous biodiversity of infections circulating among humans, wildlife, and ticks inhabiting these ecosystems. Molecular typing identifies these infections as highly endemic, with a majority of new strains and putative species specific to French Guiana. They are detected in unusual rainforest wild animals, suggesting they have distinctive sylvatic transmission cycles. They also present potential health hazards, as revealed by the detection of Candidatus Anaplasma sparouinense in human red blood cells and that of a new close relative of the human pathogen Ehrlichia ewingii, Candidatus Ehrlichia cajennense, in the tick species that most frequently bite humans in South America. The genome assembly of three new putative species obtained from human, sloth, and tick metagenomes further reveals the presence of major homologs of Ehrlichia and Anaplasma virulence factors. These observations converge to classify health hazards associated with Ehrlichia and Anaplasma infections in the Amazon biome as distinct from those in the Northern Hemisphere.

Detection of tick-borne pathogens in Rhipicephalus bursa ticks collected from the autochthonous Garrano breed of horses in Portugal.

The Garrano is a semi-feral horse breed native to several mountains in the northern Iberian Peninsula. Despite being endangered, this unique breed of pony has managed to survive in the wild and continues to be selectively bred, highlighting their remarkable resilience and adaptability to harsh environments. Wildlife plays a critical role in the survival of tick vectors in their natural habitats and the transfer of tick-borne pathogens, as they can serve as reservoir hosts for many agents and amplifiers for these vectors. The semi-feral lifestyle of the Garrano horses makes them particularly vulnerable to exposure to numerous tick species throughout the year. Therefore, the aim of this study was to investigate the occurrence of Anaplasma, Ehrlichia, Babesia, Theileria, and spotted fever rickettsiae in the Garrano horse ticks to obtain a knowledge of circulating agents in this host population. The collected ticks (n = 455) were identified as Rhipicephalus bursa. DNA specimens were organized in pools of 5 ticks, for molecular screening. Pools PCR results confirmed the presence of Candidatus Rickettsia barbariae (n = 12 for the ompB gene, n = 11 for the ompA gene and n = 6 for the gltA gene), Babesia bigemina (n = 1), Babesia caballi (n = 3), Theileria equi (n = 15) and Theileria haneyi (n = 1).These results confirm the circulation of an emerging rickettsial spotted fever group member, Candidatus R. barbariae, in R. bursa ticks. Our findings demonstrated that Candidatus R. barbariae co-circulates with B. bigemina and T. equi, which are vectored by R. bursa. We are reporting for the first time, the detection of T. haneyi among R. bursa ticks feeding in the Garrano horses in Portugal. Surveillance studies for tick-borne infections are essential to provide information that can facilitate the implementation of preventive and control strategies.

Distribution and pathogen prevalence of field-collected ticks from south-western Korea: a study from 2019 to 2022.

Hard ticks are known vectors of various pathogens, including the severe fever with thrombocytopenia syndrome virus, Rickettsia spp., Coxiella burnetii, Borrelia spp., Anaplasma phagocytophilum, and Ehrlichia spp. This study aims to investigate the distribution and prevalence of tick-borne pathogens in southwestern Korea from 2019 to 2022. A total of 13,280 ticks were collected during the study period, with H. longicornis accounting for 86.1% of the collected ticks. H. flava, I. nipponensis and A. testudinarium comprised 9.4%, 3.6%, and 0.8% of the ticks, respectively. Among 983 pools tested, Rickettsia spp. (216 pools, 1.6% MIR) were the most prevalent pathogens across all tick species, with R. japonica and R. monacensis frequently detected in I. nipponensis and Haemaphysalis spp., respectively. Borrelia spp. (28 pools, 0.2% MIR) were predominantly detected in I. nipponensis (27 pools, 13.8% MIR, P < 0.001). Co-infections, mainly involving Rickettsia monacensis and Borrelia afzelii, were detected in I. nipponensis. Notably, this study identified R. monacensis for the first time in A. testudinarium in South Korea. These findings offer valuable insights into the tick population and associated pathogens in the region, underscoring the importance of tick-borne disease surveillance and prevention measures.

First molecular survey of tick-borne protozoan and bacterial pathogens in the questing tick population in Bangladesh.

Questing ticks carry various tick-borne pathogens (TBPs) that are responsible for causing tick-borne diseases (TBDs) in humans and animals around the globe, especially in the tropics and sub-tropics. Information on the distribution of ticks and TBPs in a specific geography is crucial for the formulation of mitigation measures against TBDs. Therefore, this study aimed to survey the TBPs in the questing tick population in Bangladesh. A total of 2748 questing hard ticks were collected from the pastures in Sylhet, Bandarban, Sirajganj, Dhaka, and Mymensingh districts through the flagging method. After morphological identification, the ticks were grouped into 142 pools based on their species, sexes, life stages, and collection sites. The genomic DNA extracted from tick specimens was screened for 14 pathogens, namely Babesia bigemina (AMA-1), Babesia bovis (RAP-1), Babesia naoakii (AMA-1), Babesia ovis (18S rRNA), Theileria luwenshuni (18S rRNA), Theileria annulata (Tams-1), Theileria orientalis (MPSP), Anaplasma marginale (groEL), Anaplasma phagocytophilum (16S rRNA), Anaplasma bovis (16S rRNA), Anaplasma platys (16S rRNA), Ehrlichia spp. (16S rRNA), Rickettsia spp. (gltA), and Borrelia (Bo.) spp. (flagellin B) using genus and species-specific polymerase chain reaction (PCR) assays. The prevalence of the detected pathogens was calculated using the maximum likelihood method (MLE) with 95 % confidence interval (CI). Among 2748 ixodid ticks, 2332 (84.86 %) and 416 (15.14 %) were identified as Haemaphysalis bispinosa and Rhipicephalus microplus, respectively. Haemaphysalis bispinosa was found to carry all the seven detected pathogens, while larvae of R. microplus were found to carry only Bo. theileri. Among the TBPs, the highest detection rate was observed in A. bovis (20/142 pools, 0.81 %, CI: 0.51-1.20), followed by T. orientalis (19/142 pools, 0.72 %, CI: 0.44-1.09), T. luwenshuni (9/142 pools, 0.34 %, CI: 0.16-0.62), B. ovis (4/142 pools, 0.15 %, CI: 0.05 - 0.34) and Bo. theileri (4/142 pools, 0.15 %, CI: 0.05-0.34), Ehrlichia ewingii (3/142 pools, 0.11 %, CI: 0.03-0.29), and Babesia bigemina (1/142, 0.04 %, CI: 0.00 - 0.16). This study reports the existence of T. luwenshuni, E. ewingii, and Bo. theileri in Bangladesh for the first time. The novel findings of this study are the foremost documentation of transovarian transmission of B. bigemina and E. ewingii in H. bispinosa and also provide primary molecular evidence on the presence of E. ewingii and Bo. theileri in H. bispinosa. Therefore, this study may shed light on the circulating TBPs in ticks in the natural environment and thereby advocate awareness among physicians and veterinarians to control and prevent TBDs in Bangladesh.

Cervids as Sentinels for Rickettsia spp. in Portugal.

Cervids are highly exposed to ticks, however, their role in the life cycle of these rickettsiae has not been fully elucidated. Given the expanding distribution and growing population of deer species in Portugal, coupled with their direct and indirect interactions with humans during hunting, it becomes crucial to explore their role as sentinels and potential reservoirs of Rickettsia. The present investigation aimed to detect and evaluate exposure to Rickettsia in free-living deer from Portugal. Blood samples (n = 77) were collected from hunted game animals (red deer and fallow deer) from different areas throughout Portugal (Idanha-a-Nova, Monte Fidalgo, Montalvão and Arraiolos) and sera were tested by immunofluorescence assay, to detect antibodies. Additionally, blood DNA samples were screened for SFGR by nested-polymerase chain reaction targeting a fragment of the outer membrane protein B (ompB) gene, as well as for Anaplasma and Ehrlichia spp. targeting the 16S rRNA gene. Thirty-five per cent (25 deer and two fallow deer) tested positive (sera with a titer ≥1:64) for IgG antibodies against Rickettsia conorii. No rickettsial DNA was detected by PCR for the ompB gene, and all DNA samples tested negative for Anaplasma and Ehrlichia. As far as we know, this study is the first screening of cervid species in Portugal for Rickettsia antibodies. The findings suggest that these animals serve as useful sentinel indicators for the circulation of rickettsiae, offering a complementary perspective to studies focused on ticks. The increasing numbers of hunted deer in Portugal and the potential zoonotic features of Rickettsia spp. highlight the importance of continued surveillance directed at tick-borne diseases, especially those involving wild animals.

Ehrlichia effector TRP120 manipulates bacteremia to facilitate tick acquisition.

Ehrlichia species are obligatory intracellular bacteria that cause a potentially fatal disease, human ehrlichiosis. The biomolecular mechanisms of tick acquisition of Ehrlichia and transmission between ticks and mammals are poorly understood. Ehrlichia japonica infection of mice recapitulates the full spectrum of human ehrlichiosis. We compared the pathogenicity and host acquisition of wild-type E. japonica with an isogenic transposon mutant of E. japonica that lacks tandem repeat protein 120 (TRP120) (ΔTRP120). Both wild-type and ΔTRP120 E. japonica proliferated similarly in cultures of mammalian and tick cells. Upon inoculation into mice, both wild-type and ΔTRP120 E. japonica multiplied to high levels in various tissues, with similar clinical chemistry and hematologic changes, proinflammatory cytokine induction, and fatal disease. However, the blood levels of ΔTRP120 E. japonica were almost undetectable within 24 h, whereas the levels of the wild type increased exponentially. Greater than 90% of TRP120 was released from infected cells into the culture medium. Mouse blood monocytes exposed to native TRP120 from culture supernatants showed significantly reduced cell surface expression of the transmigration-related markers Ly6C and CD11b. Larval ticks attached to mice infected with either wild-type or ΔTRP120 E. japonica imbibed similar amounts of blood and subsequently molted to nymphs at similar rates. However, unlike wild-type E. japonica, the ΔTRP120 mutant was minimally acquired by larval ticks and subsequent molted nymphs and, thus, failed to transmit to naïve mice. Thus, TRP120 is required for bacteremia but not disease. These findings suggest a novel mechanism whereby an obligatory intracellular bacterium manipulates infected blood monocytes to sustain the tick-mammal transmission cycle. IMPORTANCE: Effective prevention of tick-borne diseases such as human ehrlichiosis requires an understanding of how disease-causing organisms are acquired. Ehrlichia species are intracellular bacteria that require infection of both mammals and ticks, involving cycles of transmission between them. Mouse models of ehrlichiosis and tick-mouse transmission can advance our fundamental understanding of the pathogenesis and prevention of ehrlichiosis. Herein, a mutant of Ehrlichia japonica was used to investigate the role of a single Ehrlichia factor, named tandem repeat protein 120 (TRP120), in infection of mammalian and tick cells in culture, infection and disease progression in mice, and tick acquisition of E. japonica from infected mice. Our results suggest that TRP120 is necessary only for Ehrlichia proliferation in circulating mouse blood and ongoing bacteremia to permit Ehrlichia acquisition by ticks. This study provides new insights into the importance of bacterial factors in regulating bacteremia, which may facilitate tick acquisition of pathogens.

Detection of Anaplasma spp. and Ehrlichia spp. in dogs from a veterinary teaching hospital in Italy: a retrospective study 2012-2020.

Anaplasma phagocytophilum, Anaplasma platys and Ehrlichia canis, responsible of diseases in dogs, are tick-borne pathogens with a proven or potential zoonotic role that have shown increasing prevalence worldwide. The aims of this retrospective study were to assess the frequency of Anaplasma spp. and Ehrlichia spp. exposure in dogs tested in a veterinary teaching hospital in Italy over a 9-year period, to compare the performance of the diagnostic tests used, to evaluate correlations with clinical data, and to genetically analyse the identified bacteria. During the study period, 1322 dogs tested by at least one of the rapid immunoenzymatic test, indirect immunofluorescent antibody test or end-point PCR assay for Anaplasmataceae detection were included. Dogs were tested if they had clinical signs or clinicopathological alteration or risk factors related to infection, and if they were potential blood-donor animals. Ninety-four of 1322 (7.1%) dogs tested positive for at least one pathogen: 53 (4.3%) for A. phagocytophilum, one (0.1%) for A. platys and 63 (4.6%) for E. canis. The number of dogs tested increased and the positivity rate progressively declined over the years. Comparison of tests showed a near-perfect agreement between serological tests and a poor agreement between PCR and indirect assays. A breed predisposition has been highlighted for A. phagocytophilum infection in hunting breed dogs and for E. canis infection in mixed breed dogs. Phylogeny confirmed potential zoonotic implications for A. phagocytophilum and showed no correlation of the identified bacteria with the geographical origin. Our study provides new insights into possible risk factors in dogs and evidenced discordant results between different tests, suggesting that a combination of serological and molecular assays is preferable for a correct diagnosis.

First report of Anaplasma spp., Ehrlichia spp., and Rickettsia spp. in Amblyomma gervaisi ticks infesting monitor lizards (Varanus begalensis) of Pakistan.

Ticks pose significant health risks to both wildlife and humans due to their role as vectors for various pathogens. In this study, we investigated tick infestation patterns, tick-associated pathogens, and genetic relationships within the tick species Amblyomma gervaisi, focusing on its prevalence in monitor lizards (Varanus bengalensis) across different districts in Pakistan. We examined 85 monitor lizards and identified an overall mean intensity of 19.59 ticks per infested lizard and an overall mean abundance of 11.98 ticks per examined lizard. All collected ticks (n = 1019) were morphologically identified as A. gervaisi, including 387 males, 258 females, 353 nymphs, and 21 larvae. The highest tick prevalence was observed in the Buner district, followed by Torghar and Shangla, with the lowest prevalence in Chitral. Lizard captures primarily occurred from May to October, correlating with the period of higher tick infestations. Molecular analysis was conducted on tick DNA, revealing genetic similarities among A. gervaisi ticks based on 16S rDNA and ITS2 sequences. Notably, we found the absence of A. gervaisi ITS2 sequences in the NCBI GenBank, highlighting a gap in existing genetic data. Moreover, our study identified the presence of pathogenic microorganisms, including Ehrlichia sp., Candidatus Ehrlichia dumleri, Anaplasma sp., Francisella sp., Rickettsia sp., and Coxiella sp., in these ticks. BLAST analysis revealed significant similarities between these pathogenic sequences and known strains, emphasizing the potential role of these ticks as vectors for zoonotic diseases. Phylogenetic analyses based on nuclear ITS2 and mitochondrial 16S rDNA genes illustrated the genetic relationships of A. gervaisi ticks from Pakistan with other Amblyomma species, providing insights into their evolutionary history. These findings contribute to our understanding of tick infestation patterns, and tick-borne pathogens in monitor lizards, which has implications for wildlife health, zoonotic disease transmission, and future conservation efforts. Further research in this area is crucial for a comprehensive assessment of the risks associated with tick-borne diseases in both wildlife and humans.

Molecular and next-generation sequencing analysis of tick-borne pathogens of Rhipicephalus ticks (Acari: Ixodidae) in cattle and dogs.

Ticks are small and adaptable arachnid ectoparasites and global carriers of various pathogens that threaten both human and animal health. They are present in many parts of China. A total of 858 ticks were collected from various regions and hosts, then subjected to species identification based on morphological and molecular characteristics, as described in the authors' previous study. Eighty-three individual tick samples were selected for screening pathogens based on metagenomic next-generation sequencing (mNGS) and polymerase chain reaction (PCR) assays. The genomic DNA of tick species was extracted, and amplification of the bacterial 16S rRNA gene was carried out from DNA of individual ticks using V3-V4 hypervariable regions, before subjecting to metagenomic analysis. Each tick underwent specific PCR tests for identifying the bacterial species present, including Anaplasma, Ehrlichia, Coxiella, and Rickettsia, and also protozoans such as Babesia, Theileria, and Hepatozoon. Illumina NovaSeq sequencing results revealed that the dominant phylum and family in Rhipicephalus spp. were Bacteroidota and Muribaculaceae, respectively. Alpha diversity patterns varied depending on tick sex (R. linnaei only), species and location, but not on host. Furthermore, bacterial pathogens, including A. marginale (58 %, 29/50), A. platys (6 %, 3/50), E. minasensis (2 %, 1/50), Ehrlichia sp. (10 %, 5/50), T. sinensis (24 %, 12/50), T. orientalis (54 %, 27/50) and Coxiella-like bacteria (CLB) (80 %, 40/50) were detected in R. microplus, while E. canis (33.33 %, 10/30), H. canis (20 %, 6/30) and CLB (100 %, 30/30) were detected in R. linnaei. Also, Anaplasma sp. (33.33 %, 1/3), A. marginale (33.33 %, 1/3), R. felis (33.33 %, 1/3) and CLB (100 %, 3/3) were detected in R. haemaphysaloides. Dual and triple co-infections involving pathogens or CLB were detected in 84.00 % of R. microplus, 66.66 % of R. haemaphysaloides, and 33.00 % of R. linnaei. The report on microbial communities and pathogens, which found from Rhipicephalus spp. in Hainan Island, is an important step towards a better understanding of tick-borne disease transmission. This is the first report in the area on the presence of Anaplasma sp., A. marginale, R. felis and Coxiella, in R. haemaphysaloides.

Molecular screening of tick-borne microorganisms in ticks from rural areas of Panama, with the first record of Ehrlichia minasensis in Rhipicephalus microplus from Central America.

During a molecular screening of rickettsial, borrelial and protozoal agents in ticks from rural areas of Panama, we detected DNA of Rickettsia amblyommatis in four Amblyomma mixtum collected from horses in a locality in Los Santos province. Ehrlichia minasensis and Anaplasma marginale were identified from three and two Rhipicephalus microplus, respectively, collected on cows from Chiriqui province. No DNA from other tick-borne agents was found. These results increase the information of R. amblyommatis and A. marginale in Panama, and also mark the first record of E. minasensis in Central America.

A molecular toolbox for fast and convenient diagnosis of emerging and reemerging bacterial pathogens causing fever of intermediate duration.

PURPOSE: Fever of intermediate duration (FID) is defined as a fever in the community without a specific origin or focus, with a duration between 7 and 28 days. FID is often caused by pathogens associated with animal contact or their arthropods parasites, such as ticks, fleas, or lice. The purpose of this work is to design a collection of molecular tools to promptly and accurately detect common bacterial pathogens causing FID, including bacteria belonging to genera Rickettsia, Bartonella, Anaplasma, and Ehrlichia, as well as Coxiella burnetii. METHODS: Reference DNA sequences from a collection of Rickettsia, Bartonella, Anaplasma, and Ehrlichia species were used to design genus-specific primers and FRET probes targeted to conserved genomic regions. For C. burnetii, primers previously described were used, in combination with a newly designed specific probe. Real-time PCR assays were optimized using reference bacterial genomic DNA in a background of human genomic DNA. RESULTS: The four real-time PCR assays can detect as few as ten copies of target DNA from those five genera of FDI-causing bacteria in a background of 300 ng of human genomic DNA, mimicking the low microbial load generally found in patient's blood. CONCLUSION: These assays constitute a fast and convenient "toolbox" that can be easily implemented in diagnostic laboratories to provide timely and accurate detection of bacterial pathogens that are typical etiological causes of febrile syndromes such as FID in humans.

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

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

Molecular Evidence of Ehrlichia canis (Rickettsiales: Anaplasmataceae) in Ticks (Ixodida: Ixodidae) Associated with Dogs (Carnivora: Canidae) from Nuevo Leon, Mexico.

Background: Ehrlichia canis is transmitted by ticks causing Canine monocytic ehrlichiosis, which is considered one of the most critical tickborne pathogens. Materials and Methods: This study aimed to identify by PCR technique E. canis in ticks associated with dogs from urban and rural homes in Nuevo Leon, Mexico. The study was conducted at 13 localities in eight municipalities from 2012 to 2021. Results: A total of 1873 ticks of three species were captured: Amblyomma tenellum, Dermacentor variabilis, and Rhipicephalus sanguineus s.l. The overall infection rate of E. canis in ticks was 59.12% (149/252). Of the 15 sequences, three haplotypes were identified. Conclusion: The urban transmission cycle of canine ehrlichiosis is demonstrated, where the potential vector is the tick R. sanguineus s.l.

Investigation of tick-borne bacterial microorganisms in Haemaphysalis ticks from Hebei, Shandong, and Qinghai provinces, China.

Tick-borne microorganisms in many tick species and many areas of China are still not thoroughly investigated. In this study, 224 ticks including two species (Haemaphysalis longicornis and Haemaphysalis qinghaiensis) were collected from four cities in Hebei, Shandong, and Qinghai provinces, China. Ticks were screened for the presence of tick-borne bacterial microorganisms including Rickettsia, Anaplasmataceae (Anaplasma, Ehrlichia, Neoehrlichia, etc.), Coxiella, Borrelia, and Bartonella. Two Anaplasma species (Anaplasma ovis and Anaplasma capra) were detected in H. longicornis from Xingtai City of Hebei Province, with a positive rate of 3 % and 8 %, respectively. A Coxiella species was detected in H. longicornis ticks from all three locations in Hebei and Shandong provinces, with the positive rate ranging from 30 to 75 %. All the 16S and rpoB sequences were very similar (99.77-100 % identity) to Coxiella endosymbiont of Haemaphysalis ticks. An Ehrlichia species was detected in H. qinghaiensis (6/66, 9 %) from Xining City, Qinghai Province. The 16S and groEL sequences had 100 % and 97.40-97.85 % nucleotide identities to "Candidatus Ehrlichia pampeana" strains, respectively, suggesting that it may be a variant of "Candidatus Ehrlichia pampeana". All the ticks were negative for Rickettsia, Borrelia, and Bartonella. Because all the ticks were removed from goats or humans and were partially or fully engorged, it is possible that the microorganisms were from the blood meal but not vectored by the ticks. Our results may provide some information on the diversity and distribution of tick-borne pathogens in China.

Wide bovine tick-borne pathogen spectrum: Predominancy of Theileria annulata and the first molecular detection of Ehrlichia minasensis in Turkey.

Vector-borne diseases indulge in severe economic losses in the livestock industry by adversely affecting cattle breeding in tropical and subtropical zone countries, including Turkey, encompassing a wide land area representing diverse climatic conditions. This study aimed to investigate significant bovine tick-borne piroplasm, rickettsia, and some other bacterial agents by genus- or species-specific PCR and nested PCR techniques in Turkey. A total of 210 cattle blood samples were collected from sixteen provinces in different geographical regions of Turkey. PCR analyses were performed targeting the detection of Babesia/Theileria/Hepatozoon sp. 18S rRNA, Babesia/Theileria sp. 18S rRNA (V4), B. bigemina RAP-1a, B. bovis SBP-4, B. ovata AMA-1, B. naoaki AMA-1, T. annulata Tams-1, T. orientalis MPSP, T. mutans 18S rRNA, Anaplasma/Ehrlichia sp. 16S rRNA, A. marginale MSP4, A. bovis 16S rRNA, A. phagocytophilum 16S rRNA, A. capra 16S rRNA, E. ruminantium pSC20, Mycoplasma sp. 16S rRNA, and Coxiella burnetii 16S rRNA genes. Overall, 133 (63.3%) cattle were found to be infected with at least one of the following protozoan or bacterial pathogens; B. bovis, B. bigemina, B. occultans, T. annulata, T. orientalis, A. marginale, A. phagocytophilum, and Mycoplasma sp. The total prevalence of pathogens was determined as follows; 0.5% B. bovis, 0.5% B. bigemina, 1.4% B. occultans, 41.0% T. annulata, 1.4% T. orientalis, 10.5% A. marginale, 13.8% A. phagocytophilum, 0.5% A. bovis, 2.9% Uncultured Anaplasma sp., 0.5% E. minasensis, 0.5% Uncultured Ehrlichia sp., and 23.3% Mycoplasma sp. Moreover, large part of the total infection (n:133) was composed of single infections (63.9%); however, double (24.8%), triple (7.5%), quadruple (2.3%), and quintuple (1.5%) co-infections were also encountered. In addition to some bovine pathogens such as B. occultans, T. orientalis, A. bovis, M. wenyonii, and Candidatus Mycoplasma haemobos, which were rarely reported in Turkey, sequencing and phylogenetic analysis revealed the first detection of Uncultured Ehrlichia sp. (0.5%), and E. minasensis (0.5%) with 100% nucleotide sequence identities. The study also indicates that the spectrum of pathogens harbored by Turkish cattle is quite wide, and these pathogens cause multiple co-infections with various combinations, and T. annulata stands out as the primary bovine pathogen among them.

Phylogenetic analysis of Ehrlichia species isolated from horses and dogs in Iran.

This study aimed to determine the prevalence and phylogenetic analysis of Ehrlichia spp. in horses and dogs in Iran. Blood samples were collected from 400 animals, including 200 horses and 200 dogs, from five different provinces in Iran. Polymerase chain reaction (PCR) was used to detect Ehrlichia spp. based on amplification of the 16S rRNA gene. The semi-nested PCR method was used to amplify the dsb, TRP36, and gltA genes. The results showed that 4.5 % of the samples (3 % horses and 6 % dogs) were positive for Ehrlichia sp. The highest prevalence was observed in Kerman and Khuzestan, while the lowest was found in West Azerbaijan, Golestan, and Mazandaran. The study suggests that the populations of dogs and horses in the country should be considered important factors in the epidemiology of ehrlichiosis. Phylogenetic analysis based on the dsb and TRP36 genes revealed that the prevalent species were E. canis and E. ruminantium.