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Background: Canine vector-borne diseases (CVBDs) are illnesses caused by pathogens transmitted by blood-feeding arthropods such as ticks and mosquitoes. Many CVBDs, including dirofilariosis, anaplasmosis, and ehrlichiosis, are globally distributed and may cause a variety of clinical signs in dogs. Several CVBD agents are zoonotic, making epidemiological surveillance a joint veterinary and public health effort. In this study, we determined the seropositivity of four pathogens from dogs on Saipan, Northern Mariana Islands, a US Commonwealth located in the western Pacific Ocean., Methods: Blood samples (n = 443) were collected from client-owned, owner surrendered, and shelter dogs that participated in an island-wide spay-and-neuter event in 2023. All samples were assessed using a commercial, point-of-care enzyme-linked immunosorbent assay (ELISA) test (SNAP ® 4Dx ® Plus, IDEXX Laboratory, Westbrook, Maine, USA) to detect the Dirofilaria immitis antigen and antibodies against Ehrlichia spp., Anaplasma spp., and Borrelia burgdorferi sensu lato. Risk factors were assessed for each pathogen through a univariate analysis, followed by a multivariable logistic regression., Results: Overall, 66.1% (n = 300/443) of the dogs tested positive for at least one pathogen, with the highest prevalence observed for Ehrlichia spp. (58.0%; n = 246/443), followed by Anaplasma spp. (43.1%; n = 184/443) and D. immitis (14.8%; n = 63/443). Among the dogs with a single pathogen detected (30.9%; n = 137/443), Ehrlichia spp. was most prevalent (64.9%; n = 89/137), followed by Anaplasma spp. (23.3%; n = 32/137) and D. immitis (11.6%; n = 16/137). For co-detection of two or more pathogens (36.7%; n = 163/443), Ehrlichia spp. + Anaplasma spp. presented the highest frequency (70.5%; n = 115/163), followed by Ehrlichia spp. + D. immitis (6.7%; n = 11/163), Anaplasma spp. + D. immitis (3.6%; n = 6/163), and Ehrlichia spp. + Anaplasma spp. + D. immitis (19.0%; n = 31/163). Age (P = < 0.001), residing district (P = 0.001), and ownership status (P = < 0.001) were significantly associated with D. immitis positive status in a univariable analysis. Age (P = < 0.001), residing district (P = 0.177), and ownership status (P = 0.014) were significant in a univariable analysis with Ehrlichia spp. as an outcome. Finally, Anaplasma spp. had a significant association with ownership status (P = < 0.001) as a risk factor in a univariable analysis., Conclusions: This study shows high seropositivity for CVBPs in a dog population living in a poorly studied area. The results of this study suggest that strategies for the prevention and control of these CVBDs should be reinforced on the Island of Saipan., Competing Interests: Declarations. Ethics approval and consent to participate: All canine samples were collected in adherence with animal use protocols approved by Texas A&M University’s Institutional Animal Care and Use Committee under the number 2022–0261. Verbal consent was received for each dog from either their owner or the shelter managers to participate in this study. The results of the IDEXX SNAP® 4Dx® Plus test were shared with either the owners of the dogs or the shelter managers. Consent for publication: All authors consent to publication. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Objectives: We sought to assess the performance of 3 laboratory tests on blood specimens for direct detection of Anaplasma phagocytophilum, the cause of human granulocytic anaplasmosis (HGA), in patients tested at a single medical institution in New York State., Methods: Direct tests included microscopic blood smear examination for intragranulocytic inclusions, polymerase chain reaction (PCR), and culture using the HL-60 cell line. The HGA cases testing positive by only 1 direct test were not included, unless HGA was confirmed by acute or convalescent serology using an indirect immunofluorescent assay., Results: From 1997 to 2009, 71 patients with HGA were diagnosed by at least 1 of the 3 direct test methods. For the subgroup of 55 patients who were tested using all 3 methods, culture was positive for 90.9% (50/55) vs 81.8% (45/55) for PCR vs 63.6% (35/55) for blood smear (P =.002). Most cultures (79.3%) were detected as positive within 1 week of incubation., Conclusions: Although using culture to detect A phagocytophilum is likely not amenable for implementation in most hospital laboratories, in our experience, culture had the highest yield among the direct tests evaluated., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society for Clinical Pathology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Human ehrlichiosis is a potentially fatal tickborne disease caused by 3 species: Ehrlichia chaffeensis, E. ewingii, and E. muris eauclairensis. In the United States, 234 confirmed cases of E. ewingii ehrlichiosis were reported to the Centers for Disease Control and Prevention through the National Notifiable Diseases Surveillance System during 2013-2021; average annual incidence was 0.08 cases/1 million population. E. ewingii ehrlichiosis was reported more commonly among older, White, non-Hispanic, and male patients. Incidence and case counts generally increased yearly, except for 2020 and 2021. The highest number of cases were reported from Missouri and Arkansas. We report the geographic expansion of E. ewingii ehrlichiosis and the continued public health challenge of clarifying clinical manifestations of this infection. Clinician education will be essential to implement molecular assays to properly diagnose E. ewingii infection in patients and gain a better understanding of the epidemiology of this emerging disease.

Canine monocytic ehrlichiosis (CME), induced by Ehrlichia canis, is an important infectious disease in dogs, characterized by various clinical signs and consequent immune dysfunction. This study aimed to characterize nuclear morphology, chromatin compaction, histone H3 acetylation, and DNA methylation in lymphocytes from dogs naturally infected with E. canis, compared with healthy controls. A total of 30 dogs were included in this study, comprising 15 healthy dogs and 15 dogs with confirmed E. canis infection, verified through polymerase chain reaction. Blood samples were collected from these dogs to isolate peripheral blood mononuclear cells. The isolated cells were prepared into smears and stained using the Feulgen reaction for subsequent analysis. These stained smears underwent video imaging analysis to assess nuclear morphology and chromatin parameters. Additionally, lymphocytes isolated from the PBMCs were analyzed to quantify global levels of histone H3 acetylation and DNA methylation. The results indicated significant increases in nuclear size and alterations in chromatin architecture in the lymphocytes of dogs with E. canis infection. A significant reduction in histone H3 acetylation was observed in this group, suggesting a potential mechanism of transcriptional repression. In contrast, no significant differences in DNA methylation were detected between the infected dogs and the healthy controls. In conclusion, our findings reveal distinct morphological and epigenetic alterations in lymphocytes associated with E. canis infection, thereby enhancing the understanding of the immune dysfunction observed in dogs with CME., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Ixodes ricinus is an important vector of infectious human and livestock diseases in Europe. Co-infections of pathogens in ticks and hosts have been reported. Tick cell lines offer a useful model system for study of co-infections. We present a review of the existing literature on co-infections in tick cell lines. Previous studies have demonstrated the usefulness of tick cell lines in studies on co-infection of different pathogens and their interaction with the tick microbiome. We also carried out a preliminary study to investigate the effects of co-culturing Borrelia burgdorferi and Anaplasma phagocytophilum on their growth and interactions with the Ixodes ricinus cell line IRE/CTVM19 over a 13-day period. Replication of both pathogens was quantified by real-time PCR. The presence of A. phagocytophilum appeared to have a slight inhibitory effect on the multiplication of B. burgdorferi , that were added subsequently. In contrast, the prior presence of B. burgdorferi appeared to have a stimulatory effect on A. phagocytophilum after 6 days in culture. We conclude that the IRE/CTVM19 tick cell line is suitable for simultaneous and continuous cultivation of both bacteria and can be applied in future research.

Ehrlichioses, caused by Ehrlichia species, are tick-borne diseases (TBDs) that affect animals and humans worldwide. This study aimed to investigate the molecular occurrence of Ehrlichia spp. in 530 animals (155 Dromedary camels, 199 goats, 131 cattle, and 45 sheep) in the Benadir and Lower Shabelle regions of Somalia. Blood DNA samples were tested for PCR targeting dsb and sodB genes of Ehrlichia spp. and PCS20 and map1 genes of E. ruminantium . The obtained sequences were submitted for phylogenetic analyses. Ehrlichia spp. were detected in 26.4% (140/530) of animals by dsb -PCR, with the highest prevalence in dromedary camels (54.8%), followed by cattle (29.8%), goats (7.0%), and sheep (4.4%). Dromedary camels, cattle, and goats had significantly higher infection odds compared to sheep ( p < 0.05). Among dsb -PCR-positive samples, 76.9% (30/39) of cattle tested sodB -positive, while other species were negative. E. ruminantium was detected in 13.7% (18/131) of cattle by pCS20 -PCR, but none were positive for the map1 gene. Phylogenetic analysis confirmed E. minasensis in camels, sheep, and goats and E. ruminantium in cattle, marking the first molecular evidence of E. minasensis in dromedary camels, sheep, and goats globally, and E. ruminantium in cattle from Somalia. These findings emphasize the need for further research on its economic and public health impact.

Vector-borne pathogens, which are transmitted by blood-feeding arthropods to animals and people, are common in tropical regions where, combined with economic factors, can cause significant public health burden. A community-level study was undertaken in southwestern Guatemala to assess the presence of vector-borne pathogens in blood samples from humans (n = 98), their animals (n = 90), and ectoparasites (n = 83) over a period of 2 weeks. Human capillary blood was collected from participant's index finger, and animal venous blood (chickens, pigs, dogs, and cats) was collected from the jugular or cephalic veins at the enrollment period of a concurrent study. Ectoparasites (fleas, ticks, and lice) were collected from dogs at the time of the blood collection. Total DNA was extracted from the human blood, animal blood, and ectoparasites and assayed using published PCR assays for Anaplasma spp., Bartonella spp., and Ehrlichia spp. Ectoparasites were also tested for the presence of Rickettsia spp. DNA by PCR. Anaplasma spp. DNA was amplified from 1 of 39 (2.6 %) chickens and 1 of 6 (16.6 %) turkeys. All human and dog blood samples were negative for Bartonella spp. in the same community. Ehrlichia spp. DNA was amplified from 12 (60 %) of 20 dogs and sequencing documented Ehrlichia spp. in 2 dogs and the ticks and fleas collected from these dogs. All the Ehrlichia spp.-positive sequences showed 100 % homology to E. canis sequences and other uncultured Ehrlichia spp. strains isolated from animals. Rickettsia spp. DNA was not amplified from any of the ectoparasites assessed. Our findings suggest that Ehrlichia spp. are common in dogs and Anaplasma spp. are circulating in poultry in a rural community in southwest Guatemala. We expect these results to be used in awareness campaigns and public health interventions to reduce vector borne pathogens in the region., Competing Interests: Declaration of competing interest The authors declare that there is not any actual or potential conflict of interests regarding the publication of this study., (Copyright © 2024. Published by Elsevier B.V.)

Background: Ehrlichia spp. are obligate intracytoplasmic Gram-negative tickborne bacteria from the Anaplasmataceae family. Ehrlichiosis is considered an emerging disease in humans and animals. Studies on bovine ehrlichiosis are scarce in Northeastern Brazil. Therefore, the objectives of this work were to determine the frequency of antibodies anti-Ehrlichia sp. in dairy cattle herds in the Feira de Santana microregion, Bahia, and to confirm infection through PCR and nucleotide sequencing in selected seropositive animals., Results: Blood samples were collected from 256 clinically healthy adult cattle from four dairy farms. Plasma from each animal was tested for Ehrlichia sp. using an indirect immunofluorescence antibody test (IFAT) and a 1:40 cutoff. Of the 256 tested samples, 249 (97.26%) were seropositive for Ehrlichia sp. Forty blood samples (10 from each farm) from IFAT-positive cattle were examined by a PCR for Ehrlichia sp. and resulted in 13 infected animals; DNA samples from six PCR-positive cows were sequenced and matched 100% with Ehrlichia minasensis., Conclusions: The results represent the first serological and molecular evidence of E. minasensis in cattle from Bahia state and northeastern Brazil. Despite the limited number of tested cattle, the high frequency of seropositive animals in four farms suggests that Ehrlichia sp. is widely disseminated in the studied microregion. Considering that the infected cattle were asymptomatic, it is possible that they developed protective immunity against Ehrlichia sp., Competing Interests: Declarations. Ethical approval: The bovine samples used in this work came from a clinical screening of Trypanosoma vivax in dairy cattle. Due to this reason, ethical permission was not necessary to use the collected samples. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: Ehrlichia canis, a rickettsial organism, is responsible for causing ehrlichiosis, a tick-borne disease affecting dogs., Objectives: This study aimed to estimate ehrlichiosis prevalence and identify associated risk factors in pet dogs., Methods: A total of 246 peripheral blood samples were purposively collected from pet dogs in Dhaka, Mymensingh, and Rajshahi districts between December 2018 and December 2020. Risk factor data were obtained through face-to-face interviews with dog owners using a pre-structured questionnaire. Multivariable logistic regression analysis identified risk factors. Polymerase chain reaction targeting the 16S rRNA gene confirmed Ehrlichia spp. PCR results were further validated by sequencing., Results: The prevalence and case fatality of ehrlichiosis were 6.9% and 47.1%, respectively. Dogs in rural areas had 5.8 times higher odds of ehrlichiosis (odd ratio, OR: 5.84; 95% CI: 1.72-19.89) compared to urban areas. Dogs with access to other dogs had 5.14 times higher odds of ehrlichiosis (OR: 5.14; 95% CI: 1.63-16.27) than those without such access. Similarly, irregularly treated dogs with ectoparasitic drugs had 4.01 times higher odds of ehrlichiosis (OR: 4.01; 95% CI: 1.17-14.14) compared to regularly treated dogs. The presence of ticks on dogs increased ehrlichiosis odds nearly by 3 times (OR: 3.02; 95% CI: 1.02-8.97). Phylogenetic analysis, based on 17 commercially sequenced isolates, showed different clusters of aggregation, however, BAUMAH-13 (PP321265) perfectly settled with a China isolate (OK667945), similarly, BAUMAH-05 (PP321257) with Greece isolate (MN922610), BAUMAH-16 (PP321268) with Italian isolate (KX180945), and BAUMAH-07 (PP321259) with Thailand isolate (OP164610)., Conclusions: Pet owners and veterinarians in rural areas should be vigilant in monitoring dogs for ticks and ensuring proper preventive care. Limiting access to other dogs in high-risk areas can help mitigate disease spread. Tick prevention measures and regular treatment with ectoparasitic drugs will reduce the risk of ehrlichiosis in dogs. The observed genetic similarity of the Bangladeshi Ehrlichia canis strain highlights the need for ongoing surveillance and research to develop effective control and prevention strategies, both within Bangladesh and globally., Competing Interests: All authors declare that they have no conflicts of interest., (Copyright: © 2024 Kabir et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In August 2023, ehrlichiosis was confirmed in a patient in Italy with a Haemaphysalis punctata tick attached to his neck. Gene sequences of Ehrlichia canis from the tick and the patient were identical, indicating a potential risk for this uncommon infection for persons participating in outdoor activities.

Objectives: Crimean-Congo Hemorrhagic Fever (CCHF) is a tick-borne zoonotic viral disease that could be a public health concern. The overlapping of clinical symptoms of some acute bacterial febrile diseases with CCHF is of importance for clinical diagnosis. This study aimed to molecularly examination of Brucella, Coxiella burnetii, Borrelia, and Ehrlichia infections among individuals suspected of CCHF in Iran., Methods: In this study, 260 serum samples of suspected cases of CCHF with definitively negative laboratory test results for CCHF virus infection, were examined for Brucella spp., Coxiella burnetii, Borrelia spp., and Ehrlichia spp. by Real-time PCR., Results: According to the results, 3.46 % and 3.07 % of the patients were positive for brucellosis and Q fever, respectively. Notably, no cases of borreliosis or ehrlichiosis were detected. Among the positive cases for brucellosis (N = 9), three cases were identified as Brucella abortus infection. Individuals under the age of 43 displayed a significantly higher positivity rate for Q fever (p < 0.01). Furthermore, patients presenting with chills had a 5.81-fold increased likelihood of being infected with Q fever (95 % CI: 1.39-24.26) compared to those without chills. Notably, no other variables demonstrated a statistically significant association with Q fever infection., Discussion and Conclusions: The results of this study showed that bacterial infections such as Q fever and brucellosis should be considered as differential diagnoses of CCHF. It is recommended that other bacterial infections that can cause early clinical symptoms similar to CCHF should also be taken into consideration in future studies and serological and molecular investigations of these infections should be tested on a wide scale., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Anaplasma phagocytophilum is an emerging tick-borne zoonotic bacterium, which is considered a significant risk to the health and industry of cattle in tropical and sub-tropical regions worldwide. This research focuses on examining the worldwide occurrence of A. phagocytophilum in cattle. Several databases, including Scopus, PubMed, ProQuest, Web of Science, ScienceDirect, and Google Scholar, were searched for publications spanning October 2004 to November 2024. The pooled prevalence was calculated with a 95% confidence interval (CI) using a random-effects model based on the Freeman-Tukey double arcsine transformation. A total of 72 studies satisfied the inclusion criteria, revealing a global prevalence of A. phagocytophilum in cattle estimated at 8.5% (5.9%-11.5%). Mongolia (51.9%, 45.9%-56.2%) and Guatemala (51%, 41.2%-60.7%) were countries that accounted for the highest prevalence. Moreover, the infection was most prevalent in African region with prevalence of 11.3% (3.9%-21.5%). The highest prevalence rate was observed in hot-summer Mediterranean climate (13.7%, 4.7%-26.2%). The analysis indicated that immunological techniques were associated with the highest prevalence rate (14.2%, 6.5%-24.3%). The findings of the present research highlighted important geographical and environmental factors that affect the prevalence of disease. In the fields of veterinary medicine and public health, these findings enhance disease management plans and preventative initiatives., (© 2025 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

Ehrlichia chaffeensis is an obligatory intracellular bacterium that infects monocytes and macrophages and causes human monocytic ehrlichiosis. Ehrlichia translocated factor-3 (Etf-3) is a type IV secretion system effector that binds host-cell ferritin light chain and induces ferritinophagy, thus increasing cellular labile iron pool for Ehrlichia proliferation. To further characterize roles of Etf-3 in Ehrlichia infection, we produced immune libraries of Etf-3-specific nanobodies (Nbs). Based on distinct complementarity-determining region 3 sequences, we identified 16 and 15 families of anti-Etf-3 Nbs that could specifically bind the N- and C-terminal halves of Etf-3, respectively. Transfection with plasmids encoding the anti-Etf-3 Nbs N48 and N51, but not N59, significantly inhibited E. chaffeensis infection in HEK293 cells. All three Nbs colocalized with Etf-3-GFP in co-transfected RF/6A cells, but N48 and N51 had significantly higher binding affinities for recombinant Etf-3. Etf-3-GFP transfection-induced ferritinophagy and endogenous ferritin degradation was abrogated in HEK293 cells co-transfected with N48 or N51, but not with N59. To efficiently express Nbs in the infected host-cell cytoplasm, lipid nanoparticles-encapsulated mRNAs (LNP-mRNAs) encoding N48, N51, or N59 were created for delivery into cells or mice. Incubation of HEK293 cells or inoculation of mice with LNP-mRNA-N48 or LNP-mRNA-N51 significantly inhibited E. chaffeensis infection compared to those with LNP-mRNA-N59 or without LNP-mRNA. Our results demonstrate that Etf-3-specific Nbs delivered via LNP-mRNAs can inhibit Etf-3 functions and Ehrlichia infection., (Copyright © 2024. Published by Elsevier GmbH.)

Background: According to the worldwide growing number of examined tick-borne diseases, the aim of this study was to evaluate the changes in proteomic profile of human serum induced by the development of Erythema migrans (EM), human granulocytic anaplasmosis (AP) and co-infection before/after antibiotic therapy., Methods: A proteomics approach based on SDS-PAGE/LC-MS/MS analysis was used to determine the proteins expression and 15d-PGE2 adducts level with albumin isolated from the serum of patients and sex/age-matched healthy donors., Findings: In the serum proteome of the patients with EM or/and AP, significant changes occurred in the expression of the same top 15 modified proteins; however, each protein level was modified in the different way in terms of comparing B. burgdorferi and A. phagocytophilum infections, as well as before and after therapy. In the case of co-infection, the differences in protein expression before and after therapy were significantly lower than in the monoinfection. Moreover, both EM and AP infections/co-infection significantly increased the albumin ability to create adducts with 15d-PGE2. The therapy partially reversed this property only in the case of a single infection, but this effect was not observed for therapy of co-infections., Interpretation: The results demonstrate how challenging is the treatment of the tick-borne co-infections and how important is further analysis of this subject. Individual differences are also observed in each of examined in this study diseases, which makes it more difficult to develop a common biomarker for each of the tick-borne diseases., Competing Interests: Declaration of competing interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (Copyright © 2025 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Ehrlichiosis and anaplasmosis are vector-borne bacterial diseases produced by intracellular rickettsial species of the genus Ehrlichia and Anaplasma. Ehrlichia canis and Anaplasma spp. (A. platys and A. phagocytophilum) have reported cases of zoonotic transmision and are the main bacterial agents of canine ehrlichiosis and anaplasmosis. They normally present an asymptomatic or mild course in domestic and wild animals with some lethal cases reported. The main vector of these diseases in Europe are the castor bean tick (Ixodes ricinus) and the brown dog tick (Rhipicephalus sanguineus), although only in the latter, the main host is the domestic dogs. The aim of this work is to apply an integrative approach to convert ecological niche models (ENMs) into potential transmission risk models and understand the relative contribution of the two potential vectors (R. sanguineus and I. ricinus) to spread both diseases in the Iberian Peninsula and Balearic Islands., Results: Two ENMs meeting all criteria were successfully generated for R. sanguineus and I. ricinus with human footprint being the most relevant explanatory variable. The novelty of the study lies in the combination of independent ENMs for both species to estimate the disease transmission risk of specific bacteria (E. canis, A. platys and A. phagocytophilum). Only the transmission risk maps that had higher contribution of R. sanguineus than I. ricinus showed relevant and positive significant correlations between risk and seroprevalence in either of the two species of bacteria (R ≥ 0.4; p < 0.05). Regarding Anaplasma spp., the map having 10% contribution of I. ricinus (10I) and 90% of R. sanguineus (90R) inferred 47.4% of infected dogs in very high-risk areas. In the case of E. canis, the model showing a proportion of 25I-75R showed better validation power (53.4% of infected dogs in very high-risk areas)., Conclusion: The validation approach used in this study produced a good approximation to understand the relative contribution of the two tick species in bacterial disease transmission in dogs in the Iberian Peninsula and Balearic Islands. Rhipicephalus sanguineus appears as the main transmitter of both diseases in the study area (90% and 75% for anaplasmosis and ehrlichiosis respectively), in accordance with its higher abundance and host preference. This estimate may help veterinary staff, clinicians and owners to optimize the control of these diseases in certain vulnerable areas, and thus reduce the risk of infection in risk areas., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: None. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Red foxes (Vulpes vulpes) are found throughout the United Kingdom (UK), and can reach high population densities in urban areas. They are often infested with ticks which may carry tick-borne pathogens, leading to a risk of transmission to domestic animals and humans. This study investigated the prevalence of tick-borne pathogens in ticks sourced from red fox carcasses across Great Britain between 2018 and 2022. Tick species were identified using morphological keys and molecular barcoding, followed by specific pathogen testing using PCR. In total, 227 ticks were collected from 93 foxes. Pooling (n = 2) was undertaken for unengorged nymphs from the same tick species and fox host, with 203 homogenates tested in total (24 pools and 179 individual ticks). Ixodes hexagonus was the most abundant tick species sampled (73 %), of which 59 % were nymphs and 41 % were females. Less common were Ixodes ricinus (12 %) and Ixodes canisuga (15 %), the majority of which were females (73 % and 91 %, respectively). One Ixodes sp. larva was identified. Babesia DNA was identified in seven individual ticks and once in pooled ticks (n = 2); seven detections were in I. hexagonus and one in I. canisuga, with an overall detection rate of 7 % (95 % CI: 6 - 8 %). Sequence analysis confirmed that all Babesia detections in I. hexagonus were Babesia vulpes, with detection of Babesia Badger Type A in I. canisuga. Screening for Anaplasma phagocytophilum DNA through amplification of the msp2 gene yielded an overall detection rate of 4 % (detected in I. hexagonus only). Louping ill virus was not detected by qRT-PCR in any tick RNA tested. The majority of pathogen detections were in ticks from red foxes in rural areas of the UK, although a small number of Babesia detections were in ticks collected from semi-rural or urban red foxes. Additionally, B. vulpes was detected in GB red fox tissues, suggesting a potential role as a reservoir host. This study confirms the detection of tick-borne pathogens in ticks infesting UK red foxes and highlights the involvement of GB tick species in animal or human disease transmission., Competing Interests: Declaration of competing interests The authors declare that they have no competing interests., (Copyright © 2024. Published by Elsevier GmbH.)

Panola Mountain Ehrlichia (PME) is an emerging zoonotic pathogen, transmitted by Amblyomma americanum ticks in the southeastern United States. It is closely related to Ehrlichia ruminantium, the causative agent of heartwater disease. Heartwater disease is an often-fatal illness of ruminant livestock present in Africa and the Caribbean. The taxonomic relationship between PME and E. ruminantium has raised concerns about the pathogenicity of PME in livestock. To determine whether cattle could be naturally infected with PME in an endemic area, we conducted a one-year longitudinal study of Angus-breed beef calves in Fayetteville, Arkansas. One hundred seventy-seven calves born between September and October 2022 were sampled for blood and ticks in February, May, and September 2023. Blood and ticks from each animal were tested for bacteria in the family, Anaplasmataceae using quantitative and conventional PCR, and positive samples were sequenced for species identification. Panola Mountain Ehrlichia was detected in 2.34 % of male A. americanum collected in February, and 1.27 % of female, 0.95 % of male, and 0.43 % of nymphal A. americanum collected in May. No PME-positive ticks were collected in September. Active PME infections were detected in two calves: one which tested positive in May 2023 and one which tested positive in September 2023. Neither animal exhibited any signs of disease, and the animal PME-positive in May tested negative in September. Cattle are susceptible to PME, but the pathogen does not appear to cause obvious disease. However, all animals in this study were under one year old, and older animals may be more susceptible. Cattle are at risk of tick-borne illness in the winter as well as spring, and off-season acaricide applications may improve disease management., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Ehrlichiosis and anaplasmosis are rising tickborne infections posing significant risks to solid-organ transplant (SOT) patients. We present three cases highlighting clinical presentations, diagnostic challenges, and the benefits of microbial cell-free DNA (mcfDNA) sequencing. Emphasizing early diagnosis and preventive measures, we advocate for advanced diagnostic modalities to improve outcomes in this vulnerable population., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Ticks are prominent vectors of several zoonotic diseases. Tick-borne pathogens include the members of the genus Ehrlichia, which are obligate intracellular bacteria infecting immune and hematopoietic cells. Ehrlichia muris predominantly affects rodents, but was also reported to be a human pathogen. The known geographical distribution of this bacterium ranges from Asia, to the USA and eastern Europe. In the present work, we report the finding of E. muris in an Ixodes ricinus tick collected from a migratory bird (Turdus iliacus) in Italy, southern Europe. We sequenced the total DNA from this tick sample, and, thanks to a dedicated bioinformatic pipeline, selectively assembled the genome of the bacterium, which represents the first one for E. muris from Europe. Phylogenetic and comparative genomic analyses were then performed. Accounting for tick species distribution, bird migratory routes, and molecular phylogeny of the bacterium, it is likely that this bird transported the tick to Italy from an endemic area of E. muris, such as eastern Europe. In addition, comparative genomic analyses highlighted that E. muris and other Ehrlichia spp. display copy number variations in two families of membrane proteins, likely due to recent gene duplication, deletion and recombination events. These differences are probably a source of variability for surface antigens to evade host immunity, with a potential role in host adaptation and specificity. The present results underline the impact of migratory birds on the spread of tick-borne pathogens towards non-endemic areas, highlighting the need for further epidemiological surveillance at bird ringing stations in Italy, and advocating further investigations on possible local transmission of E. muris in competent mammalian hosts., Competing Interests: Declaration of competing interest We declare no conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Host-microbe interactions that facilitate entry into mammalian cells are essential for obligate intracellular bacterial survival and pathogenesis. Anaplasma phagocytophilum is an obligate intracellular bacterium that invades neutrophils to cause granulocytic anaplasmosis. The invasin-receptor pairs and signaling events that induce Anaplasma uptake are inadequately defined. A. phagocytophilum invasion protein A orchestrates entry via residues 9-21 (AipA 9-21 ) engaging an unknown receptor. Yeast two-hybrid screening suggested that AipA binds within C-terminal amino acids 851-967 of CD13 (aminopeptidase N), a multifunctional protein that, when crosslinked, initiates Src kinase and Syk signaling that culminates in endocytosis. Co-immunoprecipitation validated the interaction and confirmed that it requires the AipA N-terminus. CD13 ectopic expression on non-phagocytic cells increased susceptibility to A. phagocytophilum infection. Antibody blocking and enzymatic inhibition experiments found that the microbe exploits CD13 but not its ectopeptidase activity to infect myeloid cells. A. phagocytophilum induces Src and Syk phosphorylation during invasion. Inhibitor treatment established that Src is key for A. phagocytophilum infection, while Syk is dispensable and oriented the pathogen-invoked signaling pathway by showing that Src is activated before Syk. Disrupting the AipA-CD13 interaction with AipA 9-21 or CD13 781-967 antibody inhibited Src and Syk phosphorylation and also infection. CD13 crosslinking antibody that induces Src and Syk signaling restored infectivity of anti-AipA 9-21 -treated A. phagocytophilum . The bacterium poorly infected CD13 knockout mice, providing the first demonstration that CD13 is important for microbial infection in vivo . Overall, A. phagocytophilum AipA 9-21 binds CD13 to induce Src signaling that mediates uptake into host cells, and CD13 is critical for infection in vivo ., Importance: Diverse microbes engage CD13 to infect host cells. Yet invasin-CD13 interactions, the signaling they invoke for pathogen entry, and the relevance of CD13 to infection in vivo are underexplored. Dissecting these concepts would advance fundamental understanding of a convergently evolved infection strategy and could have translational benefits. Anaplasma phagocytophilum infects neutrophils to cause granulocytic anaplasmosis, an emerging disease for which there is no vaccine and few therapeutic options. We found that A. phagocytophilum uses its surface protein and recently identified protective immunogen, AipA, to bind CD13 to elicit Src kinase signaling, which is critical for infection. We elucidated the AipA CD13 binding domain, which CD13 region AipA engages, and established that CD13 is key for A. phagocytophilum infection in vivo. Disrupting the AipA-CD13 interaction could be utilized to prevent granulocytic anaplasmosis and offers a model that could be applied to protect against multiple infectious diseases., Competing Interests: The authors declare no conflict of interest.

Ehrlichiosis is a potentially life-threatening disease caused by infection with the obligatory intracellular bacteria Ehrlichia species. Ehrlichia japonica infection of mice provides an animal model of ehrlichiosis as it recapitulates full-spectrum and lethal ehrlichiosis in humans. The E. japonica transposon mutant of EHF0962 , which encodes a previously uncharacterized hypothetical protein, is attenuated in both infection and virulence in mice. EHF0962 was hence named here as resistance-inducing protein of Ehrlichia (RipE). Using this Δ ripE mutant, we studied how RipE protein contributes to Ehrlichia pathogenesis. Ehrlichia species have an intracellular developmental cycle and a brief extracellular stage to initiate a new cycle of infection. Majority of RipE proteins were expressed on the surface of the smaller infectious dense-core stage of bacteria. Extracellular Δ ripE E. japonica contained significantly less adenosine triphosphate (ATP) and lost infectivity more rapidly in culture compared with wild-type (WT) E. japonica . Genetic complementation in the Δ ripE mutant or overexpression of ripE in WT E. japonica significantly increased bacterial ATP levels, and RipE-overexpressing E. japonica was more virulent in mice than WT E. japonica . RipE is conserved among Ehrlichia species. Immunization of mice with recombinant RipE induced an in vitro infection-neutralizing antibody, significantly prolonged survival time after a lethal dose of E. japonica challenge, and cross-protected mice from infection by Ehrlichia chaffeensis , the agent of human monocytic ehrlichiosis. Our findings shed light on the extracellular stage of Ehrlichia , highlighting the importance of RipE and ATP levels in Ehrlichia for extracellular resistance and the next cycle of infection. Thus, RipE is a critical Ehrlichia protein for infection as such can be a potential vaccine target for ehrlichiosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Chien, Lin, Duan, Denton, Kawahara and Rikihisa.)

In recent years, vector-borne diseases have become widespread throughout the world and affect the health of humans and domestic animals. These diseases spread to areas where their primary vectors, fleas and ticks, thrive, particularly in tropical and subtropical climate regions, providing ideal conditions for their proliferation. The growing closeness between people and their pets increases the likelihood of bites from these ectoparasites, which represents a latent zoonotic risk. Therefore, the objective of the study was to determine the presence of Anaplasma spp., Ehrlichia spp., and Bartonella spp. in dogs treated at the Small Animal Clinic of the Faculty of Veterinary Medicine of the Universidad Nacional Mayor de San Marcos, located in Lima, Peru. Blood samples from 214 dogs were molecularly analyzed for hemopathogen detection. The results revealed prevalences of 2.6 % (6/214) for Anaplasma platys, 5.14 % (11/214) for Ehrlichia canis, and 0.46 % (1/214) for Bartonella rochalimae. No statistically significant relationship was found between the animal infection and the age, sex, breed, presence of fleas (Ctenocephalides felis) and ticks (Rhipicephalus sanguineus), and locality. This study reported molecularly for the first time the presence of A. platys, E. canis, and B. rochalimae in dogs from Lima city, and demonstrates the occurrence of zoonotic pathogens in pets treated at the veterinary clinic., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Equine Piroplasmosis (EP) and Equine Granulocytic Anaplasmosis (EGA) are diseases that affect horses, transmitted by ixodid ticks, causing a nonspecific febrile syndrome. Equine Piroplasmosis is endemic in Brazil, and most horses are in enzootic stability. Serological and molecular studies carried out on horses in Brazil have shown the presence of Anaplasma phagocytophilum, however, the clinical relevance of this infection has not yet been established. The present study aims to evaluate the importance of Babesia caballi, Theileria equi, and A. phagocytophilum as etiological agents in horses with clinical manifestations suggestive of these diseases in the metropolitan mesoregion of Rio de Janeiro. A total of 45 animals with clinical signs were submitted to DNA extraction followed by qPCR test. Anaplasma phagocytophilum, Neorickettsia risticii and Theileria haneyi were not found in any of the horses with clinical signs, however 62.2% were infected with at least one agent of EP. Theileria equi was the most frequent etiologic agent (35.5%), followed by coinfection (15.5%) and B. caballi (11.2%). These results suggest that A. phagocytophilum has minor clinical importance in the region, while EP is frequently found in symptomatic horses, representing an important differential diagnosis in suspected cases., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Despite the high global impacts of canine vector-borne diseases (CVBD) due to their wide distribution and zoonotic potential, the current epidemiological situation of CVBD in many tropical and subtropical regions remains unknown. This study examines the seroprevalence and molecular prevalence of Ehrlichia canis and other pathogens causing CVBDs (Leishmania infantum, Dirofilaria immitis, Babesia spp., Anaplasma spp. and Hepatozoon canis) in dogs living on the island of Boa Vista (Cape Verde Republic). Blood samples and infesting ticks were taken from 150 dogs across the island (stray, shelter, and pet dogs). Serum samples were tested using a rapid immunochromatographic test (Uranotest ® Quattro) that detects antibodies against E. canis, L. infantum, Anaplasma spp. and D. immitis antigen. Levels of serum antibodies against E. canis were measured using the immunofluorescence antibody test (IFAT). In addition, tick-borne pathogens in blood samples (Anaplasma spp., Babesia spp., Hepatozoon spp., and Ehrlichia canis) were detected by microscopy observation and/or PCR plus sequencing. The seroprevalence of E. canis was extremely high at 82% (123/150), as revealed by both immunochromatography and IFAT. Most dogs returning a seropositive test result (82.92%; 102/123) had antibody titres > 1:1280 but showed no clinical signs or notable laboratory abnormalities. Of the 123 animals testing seropositive for E. canis, 67 (54.47%) also presented antibodies against Anaplasma spp., and 13 (10.56%) showed the presence of Hepatozoon spp. gamonts in the blood smear. Ehrlichia canis infection was detected in 17.1% (25/146) of dogs tested by direct sequencing of polymerase chain reaction (PCR) products. Co-infections were detected in seven of these dogs: four dogs tested PCR-positive for both E. canis and A. platys, two dogs tested positive for E. canis and Hepatozoon spp., and one dog tested positive for E. canis, A. platys and Hepatozoon spp. Rhipicephalus sanguineus sensu lato was the only tick species found infesting the canine study population. The high prevalence of tick-borne pathogens detected in dogs from Boa Vista Island highlights a need for improved control measures designed to prevent the transmission of these pathogens., (© 2024. The Author(s).)

Human anaplasmosis cases, caused by Anaplasma phagocytophilum, are increasing in the United States. This trend is explained, in part, by expansion in the geographic range of the primary vector, Ixodes scapularis. Multiple variants of A. phagocytophilum have been identified in field collected ticks, but only a single variant (human active, or "Ap-ha," variant) has been shown to be pathogenic in humans. Until recently, laboratory methods used to differentiate variants were cumbersome and seldomly used in large scale assessments of the pathogen's geographic distribution. As a result, many surveys reported A. phagocytophilum without segregating variants. Lack of discrimination among A. phagocytophilum variants could lead to overestimation of anaplasmosis risk to humans. Next Generation Sequencing (NGS) assays were recently developed to efficiently detect multiple Ixodes scapularis-borne human pathogens including Ap-ha. In this study, we utilized NGS to detect and differentiate A. phagocytophilum variants (Ap-ha vs. non ha) in host-seeking I. scapularis nymphs and adults collected across 23 states in the eastern United States from 2012 to 2023 as part of national tick surveillance efforts and research studies. Many of the included ticks were tested previously using a TaqMan PCR assay that could detect A. phagocytophilum but could not differentiate variants. We retested A. phagocytophilum infected ticks with NGS to differentiate variants. Anaplasma phagocytophilum (any variant) was identified in 165 (35 %) of 471 counties from which ticks were tested, whereas Ap-ha was detected in 70 (15 %) of 469 counties where variants were differentiated. Both variants were identified in 32 % (n = 40) of 126 counties with either variant detected. Among states where A. phagocytophilum (any variant) was detected, prevalence ranged from 2 % to 19 % in unfed adults and from 0.2 % to 7.8 % in unfed nymphs; prevalence of Ap-ha variant ranged from 0.0 % to 16 % in adults, and 0.0 % to 4.6 % in nymphs., (Copyright © 2024. Published by Elsevier GmbH.)

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., Competing Interests: Materials for this study were supplied by DiaSorin Molecular.

Human monocytotropic ehrlichiosis (HME) is a tick-borne bacterial infection caused by Ehrlichia chaffeensis. Most available data come from case reports, case series and retrospective studies, while prospective studies and clinical trials are largely lacking. To obtain a clearer picture of the currently known epidemiologic distribution, clinical and paraclinical presentation, diagnostic aspects, complications, therapeutic aspects, and outcomes of HME, we systematically reviewed the literature and analyzed and summarized the data. Cases of HME are almost exclusively reported from North America. Human infections due to other (non-chaffeensis) Ehrlichia spp. are rare. HME primarily presents as an unspecific febrile illness (95% of the cases), often accompanied by thrombocytopenia (79.1% of the cases), leukopenia (57.8% of the cases), and abnormal liver function tests (68.1% of the cases). Immunocompromized patients are overrepresented among reviewed HME cases (26.7%), which indicates the role of HME as an opportunistic infection. The incidence of complications is higher in immunocompromized compared to immunocompetent cases, with ARDS (34% vs 19.8%), acute renal failure (34% vs 15.8%), multi organ failure (26% vs 14.9%), and secondary hemophagocytic lymphohistiocytosis (26% vs 14.9%) being the most frequent reported. The overall case fatality is 11.6%, with a significant difference between immunocompetent (9.9%) and immunocompromized (16.3%) cases, and sequelae are rare (4.2% in immunocompetent cases, 2.5% in immunocompromised cases)., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Gygax et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Context: Pathogens can manipulate microbial interactions to ensure survival, potentially altering the functional patterns and microbiome assembly. The present study investigates how Anaplasma phagocytophilum infection affects the functional diversity, composition, and assembly of the Ixodes scapularis microbiome, with a focus on high central pathways-those characterized by elevated values in centrality metrics such as eigenvector, betweenness, and degree measures, in the microbial community., Methods: Using previously published data from nymphs' gut V4 region's amplicons of bacterial 16S rRNA, we predicted the functional diversity and composition in control and A. phagocytophilum-infected ticks and inferred co-occurrence networks of taxa and ubiquitous pathways in each condition to associate the high central pathways to the microbial community assembly., Results: Although no differences were observed concerning pathways richness and diversity, there was a significant impact on taxa and functional assembly when ubiquitous pathways in each condition were filtered. Moreover, a notable shift was observed in the microbiome's high central functions. Specifically, pathways related to the degradation of nucleosides and nucleotides emerged as the most central functions in response to A. phagocytophilum infection. This finding suggests a reconfiguration of functional relationships within the microbial community, potentially influenced by the pathogen's limited metabolic capacity. This limitation implies that the tick microbiome may provide additional metabolic resources to support the pathogen's functional needs., Conclusions: Understanding the metabolic interactions within the tick microbiome can enhance our knowledge of pathogen colonization mechanisms and uncover new disease control and prevention strategies. For example, certain pathways that were more abundant or highly central during infection may represent potential targets for microbiota-based vaccines., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background: Canine monocytic ehrlichiosis (CME) is considered a multisystemic, life-threatening, rickettsial, and tick-borne disease that affects canine species and is caused by Ehrlichia canis (E. canis) . Clinical signs of CME vary from asymptomatic to severe illness with three clinical phases. E. canis has the potential to infect humans., Aim: This study aimed to provide recent information as there is limited data about the disease in Egypt. Therefore, this work was conducted to study the molecular prevalence of E. canis and evaluate the corresponding risk factors, hematology, biochemistry, and molecular characterization of the genus Ehrlichia and E. canis species among Egyptian dogs., Methods: One hundred eighty dogs of both sexes from 3 months to 8 years from different breeds: stray and foreign breeds were examined for clinical signs in all seasons in two delta governorates: El-Dakahlia and El-Gharbia. Blood samples were collected from dogs for microscopic and haemato-biochemical analysis, and then molecular characterization of the genus Ehrlichia and species-specific E. canis was performed, followed by sequencing and phylogenetic analysis., Results: Out of 180 samples examined by polymerase chain reaction (PCR) assay, 42 (23.33%) were positive for the genus of Ehrlichia and the species-specific E. canis. Only twenty-four dogs (13.33%) were positive for PCR, infested with ticks, and showed fever, anemia, loss of body weight, pale mucous membrane of gum and conjunctiva, blindness, paralysis, hemoglobinuria, and Melena. The univariate logistic regression revealed that all variables, including age, season, tick infestation, hemorrhage from natural orifices, and ectoparasitic treatments per year, showed statistical significance ( p ≤ 0.05), except breed and sex, which also did not exhibit any relation between CME infection in multivariate logistic regression. The presence of morulae inside leukocytes in 66 dogs out of the total examined 180 (36.67%), only 39 (59.1%) were positive for morulae and PCR-positive for E. canis . Dogs positive for E. canis suffered from anemia, severe thrombocytopenia, the absolute value of WBCs and their fractions, alanine aminotransferas (ALT), AST, ALKP, γ-GT, total. P, T.BIL, urea, globulin, and creatinine were significantly increased in dogs infected with E. canis when compared to those with negative PCR results, while the levels of albumin and A: G ratios were significantly decreased., Conclusion: The current study proves the existence of E. canis in El-Dakahlia and El-Gharbia governorates, and this is the first large-scale study concerning the epidemiological, clinicopathological examination, molecular characterization, sequencing, and phylogenetic analysis of reported from the center of the Delta of the Nile in Egypt., Competing Interests: The authors of this paper have declared that no competing interests exist.

Canine monocytic ehrlichiosis caused by Ehrlichia canis is an important rickettsial pathogen of dogs transmitted by Rhipicephalus sanguineus sensu lato ticks in India. Globally, molecular characterization of E. canis is done using different E. canis gene targets. This study aimed to characterize genetic diversity of uncultured Ehrlichia species from dogs by 16S rRNA and partial gp200 gene (termed as p43 region) sequences data. Phylogeny based on 16S rRNA gene did not reveal any region-specific lineages. The phylogeny based on 5' region of E. canis gp200 gene (termed as p43 region) revealed four major clusters (A, B, C and D) and the Indian isolates fall under clusters A and B. Cluster A is characterized by an insertion of unique 141 bp tandem repeat sequence. Similar tandem repeat sequence was found in one of the E canis isolates from east-Asia, suggesting a possible divergence within this species. The study shows evidence for divergence of a new lineage within E. canis. The location of this insertion at the 'ankyrin repeat domains' containing region is suggestive of its possible role in modulation of host responses., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Ehrlichia chaffeensis infects and proliferates inside monocytes or macrophages and causes human monocytic ehrlichiosis (HME), an emerging life-threatening tick-borne zoonosis. After internalization, E. chaffeensis resides in specialized membrane-bound inclusions, E. chaffeensis -containing vesicles (ECVs), to evade from host cell innate immune responses and obtain nutrients. However, mechanisms exploited by host cells to inhibit E. chaffeensis growth in ECVs are still largely unknown. Here we demonstrate that host cells recognize E. chaffeensis Ech&#95;1067, a penicillin-binding protein, and then upregulate the expression of PIT1, which is a phosphate transporter and transports phosphate from ECVs to the cytosol to inhibit bacterial growth. We found that host cells upregulate the PIT1 expression upon E. chaffeensis infection using transcriptome sequencing, qRT-PCR and Western blotting, and PIT1 is localized on the ECV membrane in infected THP-1 cells using confocal microscopy. Silence of PIT1 using shRNA enhances E. chaffeensis intracellular growth. Finally, we found that E. chaffeensis Ech&#95;1067 induces the upregulation of PIT1 expression through the MyD88-NF-κB pathway using recombinant protein for stimulation and siRNA for silence. Our findings deepen the understanding of the innate immune responses of host cells to inhibit bacterial intracellular growth and facilitate the development of new therapeutics for HME.

Background: MicroRNAs (miRNAs) represent a subset of small noncoding RNAs and carry tremendous potential for regulating gene expression at the post-transcriptional level. They play pivotal roles in distinct cellular mechanisms including inhibition of bacterial, parasitic, and viral infections via immune response pathways. Intriguingly, pathogens have developed strategies to manipulate the host's miRNA profile, fostering environments conducive to successful infection. Therefore, changes in an arthropod host's miRNA profile in response to pathogen invasion could be critical in understanding host-pathogen dynamics. Additionally, this area of study could provide insights into discovering new targets for disease control and prevention. The main objective of the present study is to investigate the functional role of differentially expressed miRNAs upon Ehrlichia chaf feensis, a tick-borne pathogen, infection in tick vector, Amblyomma americanum ., Methods: Small RNA libraries from uninfected and E. chaffeensis -infected Am. americanum midgut and salivary gland tissues were prepared using the Illumina Truseq kit. Small RNA sequencing data was analyzed using miRDeep2 and sRNAtoolbox to identify novel and known miRNAs. The differentially expressed miRNAs were validated using a quantitative PCR assay. Furthermore, a miRNA inhibitor approach was used to determine the functional role of selected miRNA candidates., Results: The sequencing of small RNA libraries generated >147 million raw reads in all four libraries and identified a total of >250 miRNAs across the four libraries. We identified 23 and 14 differentially expressed miRNAs in salivary glands, and midgut tissues infected with E. chaffeensis , respectively. Three differentially expressed miRNAs (miR-87, miR-750, and miR-275) were further characterized to determine their roles in pathogen infection. Inhibition of target miRNAs significantly decreased the E. chaffeensis load in tick tissues, which warrants more in-depth mechanistic studies., Conclusions: The current study identified known and novel miRNAs and suggests that interfering with these miRNAs may impact the vectorial capacity of ticks to harbor Ehrlichia . This study identified several new miRNAs for future analysis of their functions in tick biology and tick-pathogen interaction studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Kumar, Budachetri, Rikihisa and Karim.)

Ehrlichia chaffeensis is a bacterium belonging to the Anaplasmataceae family. In Mexico, only 2 species have been recorded in association with tick species and humans. The objective of the present study was to detect the presence of bacteria of the genus Ehrlichia in ticks collected from the Chamela-Cuixmala Biosphere Reserve, Jalisco, Mexico. The collected ticks were identified and analyzed individually by polymerase chain reaction to amplify a fragment of the Anaplasmataceae 16S rRNA gene and the Ehrlichia-specific dsb gene. A total of 204 ticks, corresponding to 5 species of Ixodidae and 1 of Argasidae, were collected from 147 mammals of 6 species and 4 orders; 57 ticks collected from vegetation were also included. Among the total ticks collected, 1.47% (3/204) was positive for Ehrlichia sp. DNA was obtained using the primers EHR 16SD and EHR 16SR for 16S rRNA and DSB-330 and DSB-728 for dsb. The positive samples corresponded to a larva (Amblyomma sp.) associated with Didelphis virginiana and 2 nymphs (Amblyomma cf. oblongoguttatum) infesting Nasua narica. None of the ticks collected from the vegetation tested positive for Ehrlichia sp. DNA on the basis of the 16S rRNA and dsb genes. The sequences from the larvae of Amblyomma sp. and the nymphs of A. cf. oblongoguttatum were similar to those of E. chaffeensis. The phylogenetic analysis inferred with maximum likelihood corroborated the identity as E. chaffeensis. Although the role of these tick species as vectors of E. chaffeensis is still undetermined, the presence of infected ticks in the area indicates a potential zoonotic risk., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America.)

Wild animals in general, birds in particular, play a key role in transporting ticks and propagating tick-borne pathogens. Several studies have confirmed the infection of birds with Anaplasma phagocytophilum, with overall prevalence varying widely from country to country and/or study to study. This zoonotic bacterium, transmitted mainly by ticks of the genus Ixodes, is responsible for granulocytic anaplasmosis in humans (HGA) and domestic animals (cats, dogs, horses). The disease is also called tick-borne fever (TBF) in ruminants. Extremely rare in the USA, TBF is very common in Europe, where it causes economic losses in livestock. Conversely, HGA is well established in the USA whereas only a few less severe cases have been observed in Europe. Current typing techniques support the existence of multiple variants with differences in virulence/pathogenicity and tropism for certain tick and host species. However, epidemiological cycles remain difficult to characterize in Europe. Several studies describe a cycle apparently involving only birds in Europe, but no such study has been conducted in mainland France. Our objectives were to search for A. phagocytophilum in passerine birds in the Ile-de-France region and to explore their diversity using groEL and ankA gene typing and multilocus sequence typing (MLST). Various tissues (spleen, liver, and skin) were collected from cadavers of 680 passerines between March and December 2021. The presence of A. phagocytophilum was detected by qPCR Taqman targeting the msp2 gene. Three blackbirds (Turdus merula) were found positive, representing detection rates of 0.4 % in all birds tested and 3.3 % in blackbirds. The higher frequency of detection in blackbirds could be at least partially explained by their lifestyle, as they feed on the ground. Analysis of the results of groEL and ankA typing and MLST from positive blackbirds support the hypothesis that the avian A. phagocytophilum strains in Ile-de-France are distinct from those found in mammals, and that they form their own cluster in Europe., Competing Interests: Declarations of competing interest The authors declare that they have no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Tick-borne diseases are important for animal and human health, because they can cause death if not diagnosed and treated early. Canine monocytic ehrlichiosis (CME) can cause high morbidity in dog populations. Rocky Mountain Spotted Fever (RMSF) is among the most virulent infectious in humans; dogs are also susceptible to infection. The aims of this study were to evaluate the presence of Ehrlichia canis and Rickettsia spp. infections in domestic dogs, and to identify tick species parasitizing dogs among urban areas of two municipalities (Sobral and Alcântaras) in the Ceará State, Northeastern Brazil. A total of 208 domiciled dogs was sampled. After clinical evaluation, blood samples and ticks were collected and submitted to Real-Time Polymerase Chain Reaction (RT-PCR) targeting E. canis DNA. Serum samples were screened by Indirect Immunofluorescence Assays (IFA) for antibodies against different strains of Rickettsia spp. previously recognized in Brazil. The results of this study indicate the molecular detection of E. canis in the state of Ceará, Brazil, where the proportion of canine infection in Sobral (9.9%) was higher than in Alcântaras (5.6%). Rhipicephalus sanguineus sensu lato was the prevalent tick species infesting the dogs in both municipalities (43.5 and 53.3%, respectively). Our serological results indicate that dogs of the study area were at low risk of exposure to these tick-borne Rickettsia spp. of the spotted fever group. Our study offers epidemiological data of these diseases to better understanding Rickettsiales epidemic and enzootic cycles in the Brazilian semiarid region, improving prevention and control measures., Competing Interests: Declaration of competing interest There were no conflicting interests that could have influenced the conduction of this study., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Bed bugs, common blood-feeding pests, have received limited attention regarding their potential involvement in emerging pathogen transmission. This study aimed to investigate the main vector-borne bacteria within bed bugs collected from Tunisian governorates and to genetically characterize the identified species. Molecular screening was conducted on field-collected bed bug samples, targeting zoonotic vector-borne bacteria from the Anaplasmataceae family, as well as the genera Rickettsia, Ehrlichia, Bartonella, and Borrelia. A total of 119 Cimex lectularius specimens were collected and grouped into 14 pools based on sampling Tunisian sites. Using genus-specific PCR assays, DNA of Rickettsia and Ehrlichia spp. was detected in a single pool. Sequencing and BLAST analysis of the obtained partial ompB and dsb sequences from positive samples revealed 100% similarity with those of Ehrlichia canis and Rickettsia felis available in GenBank. Obtained partial sequences showed phylogenetic similarity to R. felis and E. canis isolates found in dogs and ticks from American and European countries. To the best of our knowledge, this study is the first to investigate bed bugs in Tunisia and to report the worldwide identification of R. felis and E. canis DNA in the common bed bug, C. lectularius. These findings highlight the need for further research to explore the potential role of bed bugs in the epidemiology of these vector-borne bacteria., Competing Interests: Declaration of Competing Interest The authors declared that they have no potential conflicts of interest with respect to the research, authorship and publication of this article., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

Background: Members of the Anaplasmataceae family, such as the Anaplasma and Ehrlichia species, cause economic losses and public health risks. However, the exact economic impact has not been comprehensively assessed in Mozambique due to limited data available on its basic epidemiology. Therefore, we investigated the molecular occurrence and identity of Anaplasma and Ehrlichia spp. infecting beef cattle in Maputo province, Mozambique., Methods: A total of 200 whole blood samples were collected from apparently healthy beef cattle. Whole blood DNA was extracted and tested for presence of Anaplasma spp. and Ehrlichia ruminantium DNA through amplification of the 16S rRNA and map1 genes. Positive samples to Anaplasma spp. were subject to PCR assay targeting the A. marginale-msp5 gene. Amplicons obtained were purified, sequenced and subject to phylogenetic analyses., Results: Anaplasma spp., A. marginale and E. ruminantium were detected in 153 (76.5%), 142 (71%) and 19 (9.5%) of all the samples analyzed, respectively. On this same sample group, 19 (9.5%) were co-infected with A. marginale and E. ruminantium. The 16S rRNA sequences of Anaplasma spp. obtained were phylogenetically related to A. marginale, A. centrale and A. platys. Phylogenetic analysis revealed that A. marginale-msp5 nucleotide sequences were grouped with sequences from Asia, Africa and Latin America, whereas E. ruminantium-map1 DNA nucleotide sequences were positioned in multiple clusters., Conclusion: Cattle in Maputo Province are reservoirs for multiple Anaplasma species. A high positivity rate of infection by A. marginale was observed, as well as high genetic diversity of E. ruminantium. Furthermore, five new genotypes of E. ruminantium-map1 were identified., (© 2024. The Author(s).)

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., Competing Interests: The authors declare no conflict of interest.

Tick-borne pathogens (TBPs) in dogs are a major global health concern, with their zoonotic importance often being neglected in developing countries due to a lack of surveillance. This study aimed to highlight the incidence of six important TBPs belonging to the genera Babesia, Theileria, Hepatozoon, Anaplasma, Ehrlichia, and Rickettsia in a total of 230 community dogs from two sites: Lumbini and the Kathmandu Valley, of Nepal. A total of 75 (32.6 %) dogs were found to be infected with at least one TBP, with 11 (4.7 %) being co-infected with more than one TBP. The detection rates of TBPs were 13.9 % (n = 32) for Ehrlichia canis, 9.1 % (n = 21) for Anaplasma platys, 8.6 % (n = 20) for Babesia vogeli, and 6.5 % (n = 15) for Babesia gibsoni. None of the samples were positive for Theileria, Hepatozoon, or Rickettsia. There was a significant association between A. platys and E. canis infections, respectively, with the locations from which the samples were collected. Infections of TBPs in community dogs might be the source of infection for pet dogs or even humans in shared habitats. Further studies are needed to determine the prevalence and diversity of TBPs in dogs in other regions of Nepal. As some of these parasites are zoonotic, concerted efforts are required to raise awareness of, and control efforts for, these tick-borne pathogens., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Introduction: Canine monocytic ehrlichiosis (CME) is a disease caused by the Gram-negative bacteria Ehrlichia canis, a bacterium that affects domestic dogs but can also infect humans. The diagnosis implies a challenge due to its diversity in clinical manifestations., Methodology: The frequency of E. canis infection, risk factors, and clinical-pathological parameters associated with seropositivity were calculated with the PROC FREQ TABLES and PROC LOGISTIC procedures of the SAS statistical software., Results: The study showed a seroprevalence of 26.62% (156/586). Association between seropositivity and risk factors was found. The age and the presence of ticks including clinical signs such as anorexia, seizures, cough, petechiae, epistaxis, and hematochezia, as well as multiple blood and biochemical alterations were analyzed. The logistic regression analysis showed a high predictive power (c = 0.98) for CME for thrombocytopenia, leukopenia, and anemia., Conclusions: The high prevalence of E. canis in endemic areas makes its diagnosis difficult. Thus, clinical signs must be considered, along with blood and biochemical alterations, as a possible predictor of the disease., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2023 Gerardo Pavel Espino-Solís, Erika Astrid Flores-Lira, Alberto Barreras-Serrano, Issa Carolina García-Reynoso, Antonio de la Mora Covarrubias, Florinda Jiménez Vega, Angélica Escárcega-Ávila.)

The aim of this study was to characterize the gene expression of host immune- and cellular responses to a Norwegian virulent strain of Anaplasma phagocytophilum, the cause of tick-borne fever in sheep. Ten sheep were intravenously inoculated with a live virulent strain of A. phagocytophilum. Clinical-, observational-, hematological data as well as bacterial load, flow cytometric cell count data from peripheral blood mononuclear cells and host's gene expression post infection was analysed. The transcriptomic data were assessed for pre-set time points over the course of 22 days following the inoculation. Briefly, all inoculated sheep responded with clinical signs of infection 3 days post inoculation and onwards with maximum bacterial load observed on day 6, consistent with tick-borne fever. On days, 3-8, the innate immune responses and effector processes such as IFN1 signaling pathways and cytokine mediated signaling pathways were observed. Several pathways associated with the adaptive immune responses, namely T-cell activation, humoral immune responses, B-cell activation, and T- and B-cell differentiation dominated on the days of 8, 10 and 14. Flow-cytometric analysis of the PBMCs showed a reduction in CD4 + CD25 + cells on day 10 and 14 post-inoculation and a skewed CD4:CD8 ratio indicating a reduced activation and proliferation of CD4-T-cells. The genes of important co-stimulatory molecules such as CD28 and CD40LG, important in T- and B-cell activation and proliferation, did not significantly change or experienced downregulation throughout the study. The absence of upregulation of several co-stimulatory molecules might be one possible explanation for the low activation and proliferation of CD4-T-cells during A. phagocytophilum infection, indicating a suboptimal CD4-T-cell response. The upregulation of T-BET, EOMES and IFN-γ on days 8-14 post inoculation, indicates a favoured CD4 Th1- and CD8-response. The dynamics and interaction between CD4 + CD25 + and co-stimulatory molecules such as CD28, CD80, CD40 and CD40LG during infection with A. phagocytophilum in sheep needs further investigation in the future., (© 2023. The Author(s).)

Ehrlichia is Gram negative obligate intracellular bacterium that cause human monocytotropic ehrlichiosis (HME). HME is characterized by acute liver damage and inflammation that may progress to fatal toxic shock. We previously showed that fatal ehrlichiosis is due to deleterious activation of inflammasome pathways, which causes excessive inflammation and liver injury. Mammalian cells have developed mechanisms to control oxidative stress via regulation of nuclear factor erythroid 2 related 2 (NRF2) signaling. However, the contribution of NRF2 signaling to Ehrlichia-induced inflammasome activation and liver damage remains elusive. In this study, we investigated the contribution of NRF2 signaling in hepatocytes (HCs) to the pathogenesis of Ehrlichia-induced liver injury following infection with virulent Ixodes ovatus Ehrlichia (IOE, AKA E. japonica). Employing murine model of fatal ehrlichiosis, we found that virulent IOE inhibited NRF2 signaling in liver tissue of infected mice and in HCs as evidenced by downregulation of NRF2 expression, and downstream target GPX4, as well as decreased NRF2 nuclear translocation, a key step in NRF2 activation. This was associated with activation of non-canonical inflammasomes pathway marked by activation of caspase 11, accumulation of reactive oxygen species (ROS), mitochondrial dysfunction, and endoplasmic reticulum (ER) stress. Mechanistically, treatment of IOE-infected HCs with the antioxidant 3H-1,2-Dithiole-3-Thione (D3T), that induces NRF2 activation, attenuated oxidative stress and caspase 11 activation, as well as restored cell viability. Importantly, treatment of IOE-infected mice with D3T resulted in attenuated liver pathology, decreased inflammation, enhanced bacterial clearance, prolonged survival, and resistance to fatal ehrlichiosis. Our study reveals, for the first time, that targeting anti-oxidative signaling pathway is a key approach in the treatment of severe and potential Ehrlichia-induced acute liver injury and sepsis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Sharma et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Hepatocytes play a crucial role in host response to infection. Ehrlichia is an obligate intracellular bacterium that causes potentially life-threatening human monocytic ehrlichiosis (HME) characterized by an initial liver injury followed by sepsis and multi-organ failure. We previously showed that infection with highly virulent Ehrlichia japonica ( E. japonica ) induces liver damage and fatal ehrlichiosis in mice via deleterious MyD88-dependent activation of CASP11 and inhibition of autophagy in macrophage. While macrophages are major target cells for Ehrlichia , the role of hepatocytes (HCs) in ehrlichiosis remains unclear. We investigated here the role of MyD88 signaling in HCs during infection with E. japonica using primary cells from wild-type (WT) and MyD88 -/- mice, along with pharmacologic inhibitors of MyD88 in a murine HC cell line. Similar to macrophages, MyD88 signaling in infected HCs led to deleterious CASP11 activation, cleavage of Gasdermin D, secretion of high mobility group box 1, IL-6 production, and inflammatory cell death, while controlling bacterial replication. Unlike macrophages, MyD88 signaling in Ehrlichia -infected HCs attenuated CASP1 activation but activated CASP3. Mechanistically, active CASP1/canonical inflammasome pathway negatively regulated the activation of CASP3 in infected MyD88 -/- HCs. Further, MyD88 promoted autophagy induction in HCs, which was surprisingly associated with the activation of the mammalian target of rapamycin complex 1 (mTORC1), a known negative regulator of autophagy. Pharmacologic blocking mTORC1 activation in E. japonica -infected WT, but not infected MyD88 -/- HCs, resulted in significant induction of autophagy, suggesting that MyD88 promotes autophagy during Ehrlichia infection not only in an mTORC1-indpenedent manner, but also abrogates mTORC1-mediated inhibition of autophagy in HCs. In conclusion, this study demonstrates that hepatocyte-specific regulation of autophagy and inflammasome pathway via MyD88 is distinct than MyD88 signaling in macrophages during fatal ehrlichiosis. Understanding hepatocyte-specific signaling is critical for the development of new therapeutics against liver-targeting pathogens such as Ehrlichia ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Teymournejad, Sharma, Abdelwahed, Kader, Ahmed, Elkafas and Ismail.)

Background: Anaplasma phagocytophilum is a tick-borne zoonotic bacterium that is the aetiologic pathogen of tick-borne fever (TBF) in ruminants. In clinical bovine cases of TBF, abortion and stillbirth may be observed. However, in this regard, the pathophysiology of TBF has not yet been completely elucidated, and no clear guidelines to diagnose A. phagocytophilum-related abortions and perinatal mortalities (APM) are available., Methods: This exploratory study aimed to investigate the presence of A. phagocytophilum in bovine cases of APM and determine whether placental or fetal spleen tissue has the greatest sensitivity for A. phagocytophilum identification. The placenta and fetal spleen of 150 late-term bovine APM cases were analysed using real-time PCR to detect A. phagocytophilum., Results: A total of 2.7% of sampled placentas were positive for A. phagocytophilum, while none of the fetal spleen samples was., Limitations: No histopathology to detect associated lesions was performed. Consequently, no evidence of causality between the detection of A. phagocytophilum and APM events could be achieved., Conclusion: The detection of A. phagocytophilum suggests a potential role of this pathogen in bovine APM, and placental tissue seems to be the most suitable tissue for its identification., (© 2023 British Veterinary Association.)

Canine ehrlichiosis is an important tick-borne disease caused by bacteria in the Ehrlichia genus with species such as E. canis, E. ewingii and E. chaffeensis resulting in a severe dog illness. This study determined the occurrence of canine ehrlichiosis antibodies and its associated factors in Kenya and Tanzania. This was a retrospective study that evaluated laboratory records of 400 samples from Kenya and Tanzania submitted to Pathologists Lancet Kenya for the IDEXX SNAP 4Dx™ Plus test between the years 2016 and 2021. Records of all samples submitted to the Pathologists Lancet Kenya veterinary laboratory for the diagnostic tests were retrieved, examined, and compiled. Descriptive statistics and univariable and multivariable logistic regression were considered during analysis. The overall proportion of samples that tested positive for canine ehrlichiosis was 23% (92/400). Samples from Kenya accounted for 61% (245/400) of samples, and the percent positive was 31% (29/245). The samples from Tanzania accounted for 39% (155/400), and the percent positive was 69% (63/155). In the final model, the odds of a sample testing positive was 1.7 times for those submitted from July to December compared with those submitted from January to June. Blood samples of dogs from Tanzania had 5.31 times the odds of testing positive on the SNAP test when compared with those from Kenya. This study reports high percent positive in samples originating from Tanzania and those received during the year's second half., (© 2023. The Author(s).)

The coinfections by some microorganisms have been related to severe diseases in humans and animals, where immunosuppressive agents favor opportunistic behavior of other pathogens. A 4-month-old, female mixed-breed dog with a two-week history of inappetence, prostration, emaciation, and respiratory distress was admitted at a veterinary hospital in Brazil. Tachycardia, pale mucous membranes, severe respiratory distress, and a large number of ticks (Rhipicephalus sanguineus s.l.) in different body regions were observed at clinical examination. Hematological examination of dog showed leukocytosis, neutrophilia, mild anemia, and thrombocytopenia, whereas unremarkable values in biochemical tests. Thoracic radiography revealed a pleural effusion image. Blood and the pleural fluid (purulent aspect) samples were subjected to qPCR (16S rRNA and dsb genes) and sequencing, which identified Ehrlichia canis and Anaplasma platys coinfection. An aggregate of coccoid-to-branching or long filamentous microorganisms, surrounded by pyogranulomatous inflammatory reaction was seen at the cytology of the pleural fluid. Bacteriological culture of pleural effusion showed colonies compatible with the genus Nocardia, which revealed gram-positive filamentous organisms with a tendency of fragmentation and were identified as Nocardia otitidiscaviarum in mass spectrometry (MALDI-TOF MS). Therapy of N. otitidiscaviarum isolate using levofloxacin (supported by a previous in vitro susceptibility testing) and doxycycline for E. canis and A. platys resulted in complete resolution of the clinical picture. Here, we report for the first time a triple coinfection by Nocardia otitidiscaviarum, A. platys, and E. canis in a dog with pleural effusion, where debilitating or immunosuppressive conditions induced by A. platys and E. canis coinfection probably contributed to the opportunistic behavior of N. otitidiscaviarum., (© 2023. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Ehrlichia chaffeensis is a tick-transmitted monocytic ehrlichiosis agent primarily causing the disease in people and dogs. We recently described the development and characterization of 55 random mutations in E. chaffeensis , which aided in defining the critical nature of many bacterial genes for its growth in a physiologically relevant canine infection model. In the current study, we tested 45 of the mutants for their infectivity ability to the pathogen's tick vector; Amblyomma americanum . Four mutations resulted in the pathogen's replication deficiency in the tick, similar to the vertebrate host. Mutations causing growth defects in both vertebrate and tick hosts included in genes coding for a predicted alpha/beta hydrolase, a putative dicarboxylate amino acid:cation symporter, a T4SS protein, and predicted membrane-bound proteins. Three mutations caused the bacterial defective growth only in the tick vector, which represented putative membrane proteins. Ten mutations causing no growth defect in the canine host similarly grew well in the tick vector. Mutations in 28 genes/genomic locations causing E. chaffeensis growth attenuation in the canine host were recognized as non-essential for its growth in the tick vector. The tick non-essential genes included genes coding for many metabolic pathway- and outer membrane-associated proteins. This study documents novel vector- and host-specific differences in E. chaffeensis for its functional gene requirements., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Author RG is on the editorial board as a specialty Chief Editor for the Veterinary and Zoonotic Infection section of the Frontiers of Cellular and Infection Microbiology., (Copyright © 2023 Jaworski, Wang, Nair, Liu and Ganta.)