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.

Identificación de Rickettsias sp. y hemoparásitos en garrapatas de la especie Rhipicephalus sanguineus en perros de la ciudad de San Juan Bautista, departamento de Misiones, Paraguay / Identification of Rickettsias sp.and hemoparasites in ticks of the genus Rhipicephalus sanguineusin dogs from the city of San Juan Bautista, department of Misiones, Paraguay

El objetivo principal de esta investigación fue identificar la presencia de Rickettsias sp. y hemoparásitos en garrapatas de la especie Rhipicephalus sanguineus en perros. Se analizaron un total de 62 garrapatas adultas cargadas recolectadas de 62 perros del barrio Concepción, zona urbana de la ciudad de San Juan Bautista, en el departamento de Misiones, Paraguay, durante el año 2017. La muestra incluyó perros de diversas edades, razas y sexos. Para el análisis, se realizó un frotis directo de la hemolinfa de las garrapatas extraídas de los caninos, y posterior tinción Wright. Los resultados revelaron que el 13% de las garrapatas resultaron positivas para Rickettsias sp. y hemoparásitos. En detalle, el 6,5% mostró la presencia de cuerpos elementales de Ehrlichia spp., mientras que otro 6,5% presentó cuerpos vermiformes de Babesia spp. El 87% restante de las muestras resultaron negativas para Rickettsias sp. y hemoparásitos. En cuanto a los perros cuyas garrapatas dieron positivo para Rickettsias sp. y hemoparásitos, el 50% eran machos, y el otro 50% eran hembras. En cuanto a la edad de los perros muestreados, el 25% tenían menos de 1 año, el 50% tenían entre 1 y 5 años, y el 25% restante tenían más de 5 años. Con relación a la raza de los perros cuyas garrapatas resultaron positivas para Rickettsias sp. y hemoparásitos, el 37,5% eran de raza pura y el 62,5% eran de raza mestiza.

Ehrlichia spp. infection worsens cardiac damage in dogs with canine visceral leishmaniasis.

In endemic areas for canine visceral leishmaniasis (CVL), the occurrence of coinfection with other pathogens, such as Ehrlichia spp., has been associated with worsening of the clinical condition. The study aimed to evaluate the occurrence of histological changes in the myocardia of dogs naturally infected with Leishmania chagasi with or without coinfection with Ehrlichia spp.. We evaluated paraffin-embedded myocardial sections from 31 dogs, affected by either L. chagasi alone or coinfected with L. chagasi and Ehrlichia spp., to compare the extent and degree of cardiac damage. The blocks were divided into two groups. G1 (dogs infected only by L. chagasi) and G2 (dogs coinfected with L. chagasi and Ehrlichia spp.). The right atrium free wall, right ventricle free wall, left ventricle, and interventricular septum of all groups were evaluated. Cardiac alterations were observed in 41.93% (52/124) of the fragments evaluated and inflammatory infiltrate was the most common pattern found. The G2 group showed a higher incidence of myocarditis, with 61.53% (32/52), compared to the G1 group, in which 20 out of 72 cases (27.7%) exhibited histopathological changes (p <0.05). These findings confirmed that coinfection can potentiate cardiac damage in dogs.

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.

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.

Hemopathogens in naturally infected bovine fetuses in Brazil.

The transplacental transmission of parasites and hemoparasites is crucial for understanding the epidemiology of diseases. This study aimed to assess the prevalence of hemopathogens in bovine fetuses at various gestational periods. Samples were obtained from a slaughterhouse in the state of Minas Gerais, Brazil, and a total of 236 fetuses were collected. DNA extracted from blood samples (145) and organ samples (a pool of brain and spleen) (236) underwent a nested PCR (nPCR) assay to detect Babesia spp., Theileria spp., Trypanosoma vivax, Anaplasma marginale, Anaplasma bovis, Anaplasma phagocytophilum, Ehrlichia minasensis, and hemotropic Mycoplasma spp. Additionally, serological analysis of 145 plasma samples was conducted using the indirect fluorescent antibody test-IFAT to detect IgG against Babesia bovis, Babesia bigemina, A. marginale, and Trypanosoma vivax. The observed prevalence of transplacental transmission was 19.3 %, 6.2 %, 42.7 % and 2.7 %, for A. marginale, B. bigemina, 'Candidatus M. haemobos', and Mycoplasma wenyonii, respectively. The prevalence of A. marginale by gestational trimester was 16 % (13/81) in the second trimester and 23 % (14/60) in the third trimester, with no positive samples in the first trimester. Regarding the species B. bovis and B. bigemina, all evaluated animals tested negative by nPCR, and no serological evidence for B. bovis was found by the IFAT. Babesia bigemina demonstrated an overall seroprevalence of 6.2 % (9/145), with 4.8 % (7/145) in the last trimester and 1.3 % (2/145) in the second trimester of pregnancy. In total, 42.7 % (62/145) of blood samples were positive for 'Candidatus M. haemobos', with 42 % (34/81) in the middle trimester, and 43 % (26/60) in the final trimester of pregnancy. Mycoplasma wenyonni was detected in 2.7 % (4/145) blood samples, all in coinfection with 'C. M. haemobos'. The prevalence by pregnancy trimester was 25 % (1/4) in the first trimester; 1.2 % (1/81) in the second trimester and 3.3 % (2/60) in the third trimester of pregnancy. Hemopathogen DNA was detected in fetus blood samples but not the brain or spleen samples. All the samples were negative for T. vivax, Theileria spp., Anaplasma spp. and Ehrlichia spp. Overall, in this study, approximately 70 % of fetuses were positive for one or more of the studied parasites. No significant associations were observed between pairs of pathogens, except 'C. M. haemobos' and A. marginale.

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.

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.

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.

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.

Assessment of the effect of long-term serum storage for retrospective serologic diagnosis of canine monocytic ehrlichiosis (Ehrlichia canis).

There is currently sparse information on the possible effect of long-term storage of serum specimens for the retrospective serodiagnosis of canine monocytic ehrlichiosis (CME). The aim of this study was to assess the agreement between the original serologic outcome and the results of a repeat indirect fluorescent antibody (IFA) assay for the detection of IgG antibodies against E. canis. A secondary aim was to compare the diagnostic performance of two commercially available point-of-care (POC) immunochromatographic (IC) assays. Archived serum samples originally tested as positive (n=66) or negative (n=19) for E. canis IgG antibodies and kept frozen at -20°C for a median of 22 years, were retrospectively examined by IFA and by two POC IC assays. Cohen's Kappa coefficient (0.748, p < 0.0001), indicated a substantial agreement between the original and repeat serologic testing results. An almost identical high sensitivity and moderate specificity were established for the two POC IC assays. Canine serum specimens on long-term storage may still be of value for seroepidemiologic surveys investigating the exposure to E. canis.

Tick-borne bacterial agents in Hyalomma asiaticum ticks from Xinjiang Uygur Autonomous Region, Northwest China.

BACKGROUND: Hyalomma ticks are widely distributed in semi-arid zones in Northwest China. They have been reported to harbor a large number of zoonotic pathogens. METHODS: In this study, a total of 334 Hyalomma asiaticum ticks infesting domestic animals were collected from four locations in Xinjiang, Northwest China, and the bacterial agents in them were investigated. RESULTS: A putative novel Borrelia species was identified in ticks from all four locations, with an overall positive rate of 6.59%. Rickettsia sibirica subsp. mongolitimonae, a human pathogen frequently reported in Europe, was detected for the second time in China. Two Ehrlichia species (Ehrlichia minasensis and Ehrlichia sp.) were identified. Furthermore, two Anaplasma species were characterized in this study: Candidatus Anaplasma camelii and Anaplasma sp. closely related to Candidatus Anaplasma boleense. It is the first report of Candidatus Anaplasma camelii in China. CONCLUSIONS: Six bacterial agents were reported in this study, many of which are possible or validated pathogens for humans and animals. The presence of these bacterial agents may suggest a potential risk for One Health in this area.

Ticks (Acari: Ixodidae) on resident and migratory wild birds in Orinoquia region, Colombia.

Several species of hard ticks, including those of the genera Ixodes, Haemaphysalis, Amblyomma, and Rhipicephalus, are of medical and veterinary importance and have been reported in association with Neotropical wild birds. Colombia, known for its great bird diversity, has 57 confirmed tick species. However, there are few studies on the association between wild birds and ticks in Colombia. The Orinoquia region, a migratory center in Colombia, provides a unique opportunity to study wild bird-tick associations and their implications for tick-borne disease dynamics. Our study, conducted between October and December 2021, aimed to identify hard ticks infesting resident and migratory wild birds in the department of Arauca and to assess the presence of bacteria from the genera Anaplasma, Borrelia, Ehrlichia, Rickettsia, and piroplasms. A total of 383 birds were examined, of which 21 were infested. We collected 147 ticks, including Amblyomma dissimile (larvae), Amblyomma longirostre (nymphs), Amblyomma mixtum (adults), and Amblyomma nodosum (larvae and nymphs). We did not detect bacterial DNA in the tested ticks; however, piroplasm DNA was detected in ticks from three of the infested birds. Of the 21 bird-tick associations, six are new to the Americas, and interesting documentation of piroplasm DNA in A. longirostre, A. nodosum, and A. dissimile ticks from wild birds in the region. This study provides valuable insights into the ticks associated with wild birds and their role in the dispersal of ticks and pathogens in Colombia, enhancing our understanding of tick life cycles and tick-borne disease dynamics.

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.

Molecular detection and diversity of tick-borne rickettsial pathogens in ticks collected from camel (Camelus dromedarius) in Upper Egypt.

Tick-borne rickettsial pathogens pose significant threats to public and animal health. In Upper Egypt, limited information exists regarding the prevalence and diversity of such tick-borne pathogens. Therefore, this study aimed to conduct a comprehensive investigation to elucidate the presence and variety of tick-borne rickettsial pathogens in Upper Egyptian camels. Our results revealed a prevalence of 2.96 % for Anaplasma marginale and 0.34 % for Candidatus Anaplasma camelii among Hyalomma ticks. However, Ehrlichia spp. weren't detected in our study. The identification of Ca. A. camelii in H. dromedari ticks was documented for the first time, suggesting a potential mode of transmission in camels. Notably, this study marks the first documentation of Rickettsia aeschlimannii with a prevalence of 6.06 % in the study area. Furthermore, we detected Coxiella burnetii in a prevalence of 8.08 % in Hyalomma ticks, indicating a potential risk of Q fever transmission. Molecular techniques results were confirmed by sequencing and phylogenetic analysis and provided valuable insights into the epidemiology of these pathogens, revealing their diversity. This study is vital in understanding tick-borne rickettsial pathogens' prevalence, distribution, and transmission dynamics in Upper Egypt. In conclusion, our findings emphasize the importance of continued research to enhance our understanding of the epidemiology and impact of these pathogens on both animal and human populations.

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.

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.

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.