Tick-borne Bacterial Pathogens Research Articles

Molecular Analysis of Tick-Borne Bacterial Pathogens from Ticks Infesting Animal Hosts in Kyrgyzstan, 2021

Molecular Analysis of Tick-Borne Bacterial Pathogens from Ticks Infesting Animal Hosts in Kyrgyzstan, 2021

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

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

Identification of Anaplasma marginale adhesins for entry into Dermacentor andersoni tick cells using phage display.

Anaplasma marginale is an obligate, intracellular, tick-borne bacterial pathogen that causes bovine anaplasmosis, an often severe, production-limiting disease of cattle found worldwide. Methods to control this disease are lacking, in large part due to major knowledge gaps in our understanding of the molecular underpinnings of basic host-pathogen interactions. For example, the surface proteins that serve as adhesins and, thus, likely play a role in pathogen entry into tick cells are largely unknown. To address this knowledge gap, we developed a phage display library and screened 66 A. marginale proteins for their ability to adhere to Dermacentor andersoni tick cells. From this screen, 17 candidate adhesins were identified, including OmpA and multiple members of the Msp1 family, including Msp1b, Mlp3, and Mlp4. We then measured the transcript of ompA and all members of the msp1 gene family through time, and determined that msp1b, mlp2, and mlp4 have increased transcript during tick cell infection, suggesting a possible role in host cell binding or entry. Finally, Msp1a, Msp1b, Mlp3, and OmpA were expressed as recombinant protein. When added to cultured tick cells prior to A. marginale infection, all proteins except the C-terminus of Msp1a reduced A. marginale entry by 2.2- to 4.7-fold. Except OmpA, these adhesins lack orthologs in related pathogens of humans and animals, including Anaplasma phagocytophilum and the Ehrlichia spp., thus limiting their utility in a universal tick transmission-blocking vaccine. However, this work greatly advances efforts toward developing methods to control bovine anaplasmosis and, thus, may help improve global food security.

Genotyping of Borrelia, Rickettsia and Anaplasma in Ixodes ricinus and Dermacentor reticulatus ticks in the Kaliningrad region

Background. Tick-borne bacterial and protozoal pathogens pose a significant public health problem. The aim of this study was to detect and genotype Borrelia, Rickettsia and Anaplasma in Ixodes ricinus and Dermacentor reticulatus ticks collected in the Kaliningrad region in 2021–2022. Materials and methods. The study included 862 I. ricinus and 803 D. reticulatus ticks (1665 in total) collected in 33 biotopes of the Kaliningrad region. Detection of the DNA of tick-borne pathogens was carried out in individual ticks by PCR using a set of specific primers, followed by sequencing and phylogenetic analysis. Results. The level of infection of I. ricinus ticks with Borrelia was 15.5%, and genotyping by the p66 gene sequence showed the presence of genetic material from four species: B. afzelii, B. garinii, B. valaisiana, and B. lusitaniae. In D. reticulatus ticks, no Borrelia genetic material was detected. The Rickettsia DNA has been found in both tick species. Moreover, the infection rate of I. ricinus ticks was 2.6%, and D. reticulatus — 21.2%. R. helvetica were found in I. ricinus ticks, and R. raoultii in meadow ticks when genotyping by gltA gene. Genetic markers of Anaplasma phagocytophilum have been found in I. ricinus and D. reticulatus ticks. Cases of co-infection of an individual tick have also been identified. Conclusion. Six different species of tick-borne pathogens were found in the I. ricinus and D. reticulatus ticks collected in the Kaliningrad region and R. helvetica, R. raoultii and A. phagocytophilum were identified for the first time.

Epidemiological Investigation of Tick-Borne Bacterial Pathogens in Domestic Animals from the Qinghai-Tibetan Plateau Area, China.

The Qinghai-Tibetan Plateau area (QTPA) features a unique environment that has witnessed the selective breeding of diverse breeds of domestic livestock exhibiting remarkable adaptability. Nevertheless, Anaplasma spp., Rickettsia spp., Coxiella spp., and Borrelia spp. represent tick-borne bacterial pathogens that pose a global threat and have substantial impacts on both human and animal health, as well as on the economy of animal husbandry within the Qinghai-Tibetan plateau area. In this study, a total of 428 samples were systematically collected from 20 distinct areas within the Qinghai Plateau. The samples included 62 ticks and 366 blood samples obtained from diverse animal species to detect the presence of Anaplasma spp., Rickettsia spp., Coxiella spp., and Borrelia spp. The prevalence of infection in this study was determined as follows: Anaplasma bovis accounted for 16.4% (70/428), A. capra for 4.7% (20/428), A. ovis for 5.8% (25/428), Borrelia burgdorferi sensu lato for 6.3% (27/428), Coxiella burnetii for 0.7% (3/428), and Rickettsia spp. for 0.5% (2/428). Notably, no cases of A. marginale and A. phagocytophilum infections were observed in this study. The findings revealed an elevated presence of these pathogens in Tibetan sheep and goats, with no infections detected in yaks, Bactrian camels, donkeys, and horses. To the best of our knowledge, this study represents the first investigation of tick-borne bacterial pathogens infecting goats, cattle, horses, and donkeys within the Qinghai Plateau of the Qinghai-Tibetan Plateau area. Consequently, our findings contribute valuable insights into the distribution and genetic diversity of Anaplasma spp., Rickettsia spp., Coxiella spp., and Borrelia spp. within China.

Nanopore-based metagenomics reveal a new Rickettsia in Europe

Accurate identification of tick-borne bacteria, including those associated with rickettsioses, pose significant challenges due to the polymicrobial and polyvectoral nature of the infections. We aimed to carry out a comparative evaluation of a non-targeted metagenomic approach by nanopore sequencing (NS) and commonly used PCR assays amplifying Rickettsia genes in field-collected ticks. The study included a total of 310 ticks, originating from Poland (44.2 %) and Bulgaria (55.8 %). Samples comprised 7 species, the majority of which were Ixodes ricinus (62.9 %), followed by Dermacentor reticulatus (21.2 %). Screening was carried out in 55 pools, using total nucleic acid extractions from individual ticks. NS and ompA/gltA PCRs identified Rickettsia species in 47.3 % and 54.5 % of the pools, respectively. The most frequently detected species were Rickettsia asiatica (27.2 %) and Rickettsia raoultii (21.8 %), followed by Rickettsia monacensis (3.6 %), Rickettsia helvetica (1.8 %), Rickettsia massiliae (1.8 %) and Rickettsia tillamookensis (1.8 %). Phylogeny construction on mutS, uvrD, argS and virB4 sequences and a follow-up deep sequencing further supported R. asiatica identification, documented in Europe for the first time. NS further enabled detection of Anaplasma phagocytophilum (9.1 %), Coxiella burnetii (5.4 %) and Neoehrlichia mikurensis (1.8 %), as well as various endosymbionts of Rickettsia and Coxiella. Co-detection of multiple rickettsial and non-rickettsial bacteria were observed in 16.4 % of the pools with chromosome and plasmid-based contigs. In conclusion, non-targeted metagenomic sequencing was documented as a robust strategy capable of providing a broader view of the tick-borne bacterial pathogen spectrum.

Molecular detection reveals diverse tick-borne bacterial and protozoan pathogens in two tick species from Yingshan County of Hubei Province, China in 2021–2022

Molecular detection reveals diverse tick-borne bacterial and protozoan pathogens in two tick species from Yingshan County of Hubei Province, China in 2021–2022

Molecular surveillance reveals a potential hotspot of tick-borne disease in Yakeshi City, Inner Mongolia

A molecular surveillance of tick-borne diseases was performed in Hulunbuir City, Inner Mongolia. A total of 149 ticks including three species (Ixodes persulcatus, Haemaphysalis concinna, and Dermacentor silvarum) were collected. As many as 11 tick-borne bacterial pathogens were identified in them. Some of them have high positive rates. For example, Candidatus Rickettsia tarasevichiae was detected with a high prevalence of 72.48%, while Candidatus Lariskella sp. was detected in 31.54% of ticks. For both Rickettsia raoultii and Anaplasma phagocytophilum, two distinct genotypes were identified based on their phylogenetic trees based on 16S rRNA, gltA, and groEL sequences. Remarkable genetic diversity was also observed for 16S and flaB genes of Borreliella garinii, an agent of Lyme disease. Rickettsia heilongjiangensis causing Far-Eastern spotted fever (2.68%, 4/149), Ehrlichia muris causing human ehrlichiosis (4.70%, 7/149), Borrelia miyamotoi causing relapsing fever (2.01%, 3/149), and Borreliella afzelii causing Lyme disease (2.01%, 3/149) were also detected. Additionally, a previously uncharacterized Anaplasma species closely related to Anaplasma ovis was identified. Herein we name it “Candidatus Anaplasma mongolica”. Based on these results, we propose that Yakeshi City might be a potential hotspot of tick-borne diseases.

Modulation of Macrophage Redox and Apoptotic Processes to Leishmania infantum during Coinfection with the Tick-Borne Bacteria Borrelia burgdorferi.

Canine leishmaniosis (CanL) is a zoonotic disease caused by protozoan Leishmania infantum. Dogs with CanL are often coinfected with tick-borne bacterial pathogens, including Borrelia burgdorferi in the United States. These coinfections have been causally associated with hastened disease progression and mortality. However, the specific cellular mechanisms of how coinfections affect microbicidal responses against L. infantum are unknown. We hypothesized that B. burgdorferi coinfection impacts host macrophage effector functions, prompting L. infantum intracellular survival. In vitro experiments demonstrated that exposure to B. burgdorferi spirochetes significantly increased L. infantum parasite burden and pro-inflammatory responses in DH82 canine macrophage cells. Induction of cell death and generation of mitochondrial ROS were significantly decreased in coinfected DH82 cells compared to uninfected and L. infantum-infected cells. Ex vivo stimulation of PBMCs from L. infantum-seronegative and -seropositive subclinical dogs with spirochetes and/or total Leishmania antigens promoted limited induction of IFNγ. Coexposure significantly induced expression of pro-inflammatory cytokines and chemokines associated with Th17 differentiation and neutrophilic and monocytic recruitment in PBMCs from L. infantum-seropositive dogs. Excessive pro-inflammatory responses have previously been shown to cause CanL pathology. This work supports effective tick prevention and risk management of coinfections as critical strategies to prevent and control L. infantum progression in dogs.

Molecular Detection of Anaplasma, Ehrlichia and Rickettsia Pathogens in Ticks Collected from Humans in the Republic of Korea, 2021.

Tick-borne pathogens (TBPs), transmitted by the bites of ticks, are of great medical and veterinary importance. They include bacteria, viruses, and protozoan parasites. To provide fundamental data on the risk of tick contact and public health strategies, we aimed to perform a molecular investigation on four tick-borne bacterial pathogens in ticks collected from humans across the Republic of Korea (ROK) in 2021. In total, 117 ticks were collected, including Haemaphysalis longicornis (56.4%), Amblyomma testudinarium (26.5%), Ixodes nipponensis (8.5%), H. flava (5.1%), and I. persulcatus (0.9%). Among the ticks, 20.5% (24/117) contained tick-borne bacterial pathogens, with infection rates of 17.9% for Rickettsia (Candidatus Rickettsia jingxinensis, R. tamurae, R. monacensis, and Candidatus Rickettsia tarasevichiae), 2.5% for Anaplasma (A. phagocytophilum, A. capra, and A. bovis), and 0.9% for Ehrlichia (Ehrlichia sp.). Additionally, the co-detection rate for R. monacensis and A. phagocytophilum was 0.9%. To our knowledge, this is the first report of A. capra and A. bovis detection in ticks collected from humans in the ROK. This study contributes to the understanding of the potential risk of tick contact and provides fundamental data for establishing a public health strategy for tick-borne disease management in the ROK.

Molecular Detection of Tick-Borne Bacterial and Protozoan Pathogens in Haemaphysalis longicornis (Acari: Ixodidae) Ticks from Free-Ranging Domestic Sheep in Hebei Province, China.

Ticks and tick-borne pathogens significantly threaten human and animal health worldwide. Haemaphysalis longicornis is one of the dominant tick species in East Asia, including China. In the present study, 646 Ha. longicornis ticks were collected from free-ranging domestic sheep in the southern region of Hebei Province, China. Tick-borne pathogens of zoonotic and veterinary importance (i.e., Rickettsia, Anaplasma, Ehrlichia, Borrelia, Theileria, and Hepatozoon spp.) were detected in the ticks using PCR assays and sequence analysis. The prevalence rates of these pathogens were 5.1% (33/646), 15.9% (103/646), 1.2% (8/646), 17.0% (110/646), 0.15% (1/646), and 0.15% (1/646), respectively. For Rickettsia spp., R. japonica (n = 13), R. raoultii (n = 6), and Candidatus R. jingxinensis (n = 14) were detected for the first time in the province, while several Anaplasma spp. were also detected in the ticks, including A. bovis (n = 52), A. ovis (n = 31), A. phagocytophilum (n = 10), and A. capra (n = 10). A putative novel Ehrlichia spp. was also found with a prevalence of 1.2% in the area. The present study provides important data for effectively controlling ticks and tick-borne diseases in the Hebei Province region of China.

Co-infection of tick-borne bacterial pathogens in ticks in Inner Mongolia, China.

Tick-borne infectious diseases pose a serious health threat in certain regions of the world. Emerging infectious diseases caused by novel tick-borne pathogens have been reported that are causing particular concern. Several tick-borne diseases often coexist in the same foci, and a single vector tick can transmit two or more pathogens at the same time, which greatly increases the probability of co-infection in host animals and humans and can lead to an epidemic of tick-borne disease. The lack of epidemiological data and information on the specific clinical symptoms related to co-infection with tick-borne pathogens means that it is not currently possible to accurately and rapidly distinguish between a single pathogen infection and co-infection with multiple pathogens, which can have serious consequences. Inner Mongolia in the north of China is endemic for tick-borne infectious diseases, especially in the eastern forest region. Previous studies have found that more than 10% of co-infections were in host-seeking ticks. However, the lack of data on the specific types of co-infection with pathogens makes clinical treatment difficult. In our study, we present data on the co-infection types and the differences in co-infection among different ecological regions through genetic analysis of tick samples collected throughout Inner Mongolia. Our findings may aid clinicians in the diagnosis of concomitant tick-borne infectious diseases.

The Use of the Antigenically Variable Major Surface Protein 2 in the Establishment of Superinfection during Natural Tick Transmission of Anaplasma marginale in Southern Ghana.

Many vector-borne pathogens, including Anaplasma spp., Borrelia spp., Trypanosoma spp., and Plasmodium spp., establish persistent infection in the mammalian host by using antigenic variation. These pathogens are also able to establish strain superinfection, defined as infection of an infected host with additional strains of the same pathogen despite an adaptive immune response. The ability to establish superinfection results in a population of susceptible hosts even with high pathogen prevalence. It is likely that antigenic variation, responsible for persistent infection, also plays a role in the establishment of superinfection. Anaplasma marginale, an antigenically variable, obligate intracellular, tickborne bacterial pathogen of cattle, is well suited for the study of the role of antigenically variant surface proteins in the establishment of superinfection. Anaplasma marginale establishes persistent infection by variation in major surface protein 2 (msp2), which is encoded by approximately six donor alleles that recombine into a single expression site to produce immune escape variants. Nearly all cattle in regions of high prevalence are superinfected. By tracking the acquisition of strains in calves through time, the complement of donor alleles, and how those donor alleles are expressed, we determined that simple variants derived from a single donor allele, rather than multiple donor alleles, were predominant. Additionally, superinfection is associated with the introduction of new donor alleles, but these new donor alleles are not predominantly used to establish superinfection. These findings highlight the potential for competition among multiple strains of a pathogen for resources within the host and the balance between pathogen fitness and antigenic variation.

Molecular detection of Rickettsia, Anaplasma, and Bartonella in ticks from free-ranging sheep in Gansu Province, China

Ticks pose a serious threat to public health as carriers and often vectors of zoonotic pathogens. There are few systematic studies on the prevalence and genetic diversity of tick-borne bacterial pathogens in Western China. In this study, 465 ticks were collected from free-ranging sheep in Gansu Province in China. Ticks were divided into 113 pools and tick DNA was extracted from these ticks. PCR assays were performed using specific primers to screen for tick-borne pathogens as well as sequence analysis based on the 16S rRNA (rrs), ompB, gltA, ompA genes for Rickettsia, rrs, groEL genes for Anaplasma, and ssrA and rpoB genes for Bartonella. The PCR results showed that the minimum infection rates with Rickettsia, Anaplasma, and Bartonella were 16.8% (78/465), 18.9% (88/465), and 0.9% (4/465), respectively. Sequence analysis based on the concatenated sequences of rrs-ompB-gltA-ompA indicated that the Rickettsia species identified in the ticks belonged to Rickettsia raoultii, Rickettsia slovaca, and Rickettsia sibirica, respectively; phylogenetic analysis based on the groEL gene showed that all Anaplasma strains identified were Anaplasma ovis; and phylogenetic analysis based on the ssrA and rpoB genes indicated that all Bartonella strains in the ticks belonged to Bartonella melophagi. The results of this study showed that ticks in Gansu Province harbored multiple pathogens that may cause rickettsial diseases and bartonellosis. These diseases were neglected in the area and physicians and public health workers need to pay attention to their diagnoses to prevent human infection.

Epidemiology of tick-borne pathogens of cattle and tick control practices in coastal Kenya

Tick-borne diseases (TBD) are a major constraint to livestock health and productivity in sub-Saharan Africa. Nonetheless, there are relatively few robust epidemiologic studies documenting TBD and its management in different endemic settings in Kenya. Therefore, a cross-sectional study using multi-stage cluster sampling was undertaken to characterize the epidemiology of TBD and management factors among zebu cattle reared under an extensive system in coastal Kenya. Blood samples from 1486 cattle from 160 herds in 14 villages were screened for the presence of tick-borne bacterial and protozoan pathogens using PCR with high-resolution melting analysis and sequencing. Standardized questionnaires were used to collect data on herd structure and herd management practices, and a mixed-effect logistic regression model to identify risk factors for tick-borne pathogens (TBPs). The application of chemical acaricide was the primary method for tick control (96.3%, 154/160), with the amidine group (mainly Triatix®, amitraz) being the most frequently used acaricides. Respondents identified East Coast fever as the most important disease and Butalex® (buparvaquone) was the most commonly administered drug in response to perceived TBD in cattle. The overall animal- and herd-level prevalence for TBPs were 24.2% (95% confidence interval (CI): 22.0–26.4%) and 75.6% (95% CI: 68.2–82.1%), respectively. Cattle were infected with Anaplasma marginale (10.9%, 95% CI: 9.4–12.6), Theileria parva (9.0%, 95% CI: 7.5–10.5), Anaplasma platys (2.6%, 95% CI: 1.9–3.6), Theileria velifera (1.1%, 95% CI: 0.7–1.8), Babesia bigemina (0.5%, 95% CI: 0.2–1.0), and Anaplasma sp. (0.1%, 95% CI: 0.0–0.4). Moreover, 21 cattle (1.4%) were co-infected with two TBPs. None of the assessed potential risk factors for the occurrence of either A. marginale or T. parva in cattle were statistically significant. The intra-herd correlation coefficients (lCCs) computed in this study were 0.29 (A. marginale) and 0.14 (T. parva). This study provides updated molecular-based information on the epidemiological status of TBPs of cattle and herd management practices in coastal Kenya. This information can be used in designing cost-effective control strategies for combating these TBD in the region.

Detection and phylogenetic analysis of tick-borne bacterial and protozoan pathogens in a forest province of eastern China

Ticks, as obligate blood-sucking ectoparasites, feed on a broad range of vertebrates and transmit a great diversity of pathogenic microorganisms. Some tick-borne pathogens (TBPs) are endemic in China, whereas epidemiological studies are limited in Jiangxi, a forest province located in eastern China. Here, we have determined the positivity rates of TBPs in humans, rodents, dogs, goats and ticks, and performed the molecular characterization of TBPs in Jiangxi province. We found a high positivity rate of TBPs in the collected samples, demonstrating 23 (12.92%) samples positive for more than one TBPs. Of those, 11 (6.18%) samples were positive for Rickettsia spp., six (3.37%) Ehrlichia spp./Anaplasma spp., one (0.56%) Bartonella spp., two (1.12%) Borrelia spp., and five (2.81%) Babesia spp. The positivity rates of TBPs varied among ticks, animals, and humans as follow: goats (14/37, 37.84%), ticks (8/35, 22.86%), and dogs (1/11, 9.09%). Humans and rodents were negative for TBP presence. Phylogenetic analyses of these TBP sequences revealed the presence of Rickettsia japonica, Ehrlichia minasensis, and an unclassified Babesia spp. in goats, and Anaplasma phagocytophilum, Borrelia valaisiana, and an unclassified Bartonella spp. in ticks. Furthermore, R. japonica infection was exclusively found in goats with the positivity rate of 29.73%. Our study is the first report of R. japonica in goats around the world. These findings suggest high TBP positivity rates among goats, ticks, and dogs, and diverse TBPs in goats and ticks in the studied sites. Therefore, our results underscore the urgent need to assess TBP-tick-vertebrate-environment interactions and the risk of tick borne disease exposure in humans in the future.

Spotted Fever Group Rickettsia spp. Diversity in Ticks and the First Report of Rickettsia hoogstraalii in Romania.

Simple SummaryTicks are important parasites that feed on the blood of various host species, representing the most important arthropods transmitting diseases in Europe. Continuous changes in both tick distribution and abundance are related to multiple factors, including climate change. These changes have strong implications for both animal and human health; therefore, continuous surveillance of tickborne diseases is required for an appropriate evaluation of the potential risks faced by animals and humans in a given area. The spotted fever group Rickettsia comprises a large number of zoonotic agents with an increasing importance recognized in the last 30 years. The aim of this study was to evaluate these bacteria in ticks in Romania. Five Rickettsia species were identified in different tick species, with new pathogen–tick associations reported. Rickettsia hoogstraalii, one member of this group, was detected for the first time in Romania and in Rhipicephalus rossicus ticks. This species was first described in 2006 in Croatia, and its pathogenicity is not well known. In addition, the detection of R. raoultii and R. monacensis in unfed larvae of Haemaphysalis punctata reinforce the hypothesis of transmission of Rickettsia from female ticks to larvae; therefore the bite of larvae could pose a health risk.Tickborne bacterial pathogens have been described worldwide as risk factors for both animal and human health. Spotted fevers caused by Rickettsiae may cause non-specific symptoms, which make clinical diagnosis difficult. The aim of the current study was to evaluate and review the diversity of SFG Rickettsiae in ticks collected in 41 counties in Romania. A total of 2028 questing and engorged ticks collected in Romania belonging to five species were tested by PCR amplification of Rickettsia spp. gltA and 17-D gene fragments: Ixodes ricinus (n = 1128), Dermacentor marginatus (n = 507), D. reticulatus (n = 165), Rhipicephalus rossicus (n = 128) and Haemaphysalis punctata (n = 100). Five Rickettsia species were identified following DNA sequence analysis: R. helvetica, R. monacensis, R. slovaca, R. raoultii, and R. hoogstraalii. The most common species detected was R. monacensis. Moreover, R. hoogstraalii was detected for the first time in Romania and in R. rossicus ticks. The detection of R. raoultii and R. monacensis in questing larvae of Hae. punctata suggests the possible transovarial transmission of these Rickettsia species in ticks. The detection of R. hoogstraalii for the first time in Romania increases the reported SFG Rickettsia diversity in the country.

Detection of Tick-Borne Bacterial and Protozoan Pathogens in Ticks from the Zambia-Angola Border.

Tick-borne diseases (TBDs), including emerging and re-emerging zoonoses, are of public health importance worldwide; however, TBDs tend to be overlooked, especially in countries with fewer resources, such as Zambia and Angola. Here, we investigated Rickettsia, Anaplasmataceae, and Apicomplexan pathogens in 59 and 96 adult ticks collected from dogs and cattle, respectively, in Shangombo, a town at the Zambia–Angola border. We detected Richkettsia africae and Rickettsia aeschilimannii in 15.6% of Amblyomma variegatum and 41.7% of Hyalomma truncatum ticks, respectively. Ehrlichia minasensis was detected in 18.8% of H. truncatum, and Candidatus Midichloria mitochondrii was determined in Hyalomma marginatum. We also detected Babesia caballi and Theileria velifera in A. variegatum ticks with a 4.4% and 6.7% prevalence, respectively. In addition, Hepatozoon canis was detected in 6.5% of Rhipicephalus lunulatus and 4.3% of Rhipicephalus sanguineus. Coinfection of R. aeshilimannii and E. minasensis were observed in 4.2% of H. truncatum. This is the first report of Ca. M. mitochondrii and E. minasensis, and the second report of B. caballi, in the country. Rickettsia africae and R. aeschlimannii are pathogenic to humans, and E. minasensis, B. caballi, T. velifera, and H. canis are pathogenic to animals. Therefore, individuals, clinicians, veterinarians, and pet owners should be aware of the distribution of these pathogens in the area.

Possible Association between Selected Tick-Borne Pathogen Prevalence and Rhipicephalus sanguineus sensu lato Infestation in Dogs from Juarez City (Chihuahua), Northwest Mexico-US Border.

Tick-borne bacterial pathogens (TBBPs) show a worldwide distribution and represent a great impact on public health. The brown dog tick (Rhipicephalus sanguineus) is a vector of several pathogens that affect dogs and sometimes humans as well. In addition, TBBPs represent a diagnostic challenge and imply financial resources and medical treatment for long periods of time. In the present study, R. sanguineus s. l. was identified as the main tick species naturally parasitizing dogs that inhabit. Juárez City, Chihuahua, in the Paso del Norte region, Mexico–US Border, representing 99.8% of the cases. Additionally, an end-point PCR was performed to search for whether pathogens in R. sanguineus s. l. can transmit in DNA extracted from ticks and dog blood samples. This is the first molecular detection of Rickettsia rickettsi infecting domestic dogs in Mexico; however, other pathogens were also identified, such as Ehrlichia canis and Anaplasma platys in both ticks and dog blood samples, while Anaplasma phagocytophilum was identified only in dog blood samples. Moreover, co-detection in tick pools and co-infection in the analyzed dog blood samples could be found. Similarly, this research showed that dogs were found mostly parasitized by adult female ticks, increasing the possibility of transmission of E. canis.

Phylogenetic Studies of Coxiella-Like Bacteria and Spotted Fever Group Rickettsiae in Ticks Collected From Vegetation in Chaiyaphum Province, Thailand.

Ticks can transmit a wide variety of pathogens, including bacteria. Here, we report the detection of tick-associated bacteria in Chaiyaphum Province, northeastern Thailand. There have been few reports of tick-borne bacterial pathogens in the study areas, which are evergreen forests dominated by plateaus at elevations of approximately 1,000 m. In total, 94 ticks were collected from vegetation. They were screened for the presence of Coxiella, Francisella, Rickettsia, and Borrelia bacteria using PCR assays. In this study, we found ticks from two genera, Haemaphysalis and Amblyomma, that were positive for Coxiella-like bacteria (CLB) and Rickettsia. Francisella and Borrelia spp. were not detected in these two tick genera. The results revealed the evolutionary relationships of CLB in Amblyomma testudinarium, Haemaphysalis lagrangei, and Haemaphysalis obesa ticks using the 16S rRNA and rpoB markers, which clustered together with known isolates of ticks from the same genera. In contrast, the groEL marker showed different results. On the basis of the groEL phylogenetic analysis and BLAST results, three groups of CLB were found: (1) CLB from A. testudinarium grouped as a sister clade to CLB from Ixodes ricinus; (2) CLB from Haemaphysalis lagrangei was distantly related to CLB from Haemaphysalis wellingtoni; and (3) CLB from A. testudinarium grouped as sister clade to CLB from Amblyomma from French Guiana and Brazil. For Rickettsia studies, phylogenetic trees of the gltA, ompB, and sca4 genes revealed two groups of Spotted Fever Group (SFG) Rickettsiae: (1) SFG Rickettsiae that formed a sister clade with Rickettsia tamurae AT-1 (belong to the Rickettsia helvetica subgroup) in A. testudinarium and (2) SFG Rickettsiae that formed a distantly related group to Rickettsia rhipicephali 3-7-female6-CWPP (belong to the Rickettsia massiliae subgroup) in A. testudinarium. This study expanded our knowledge of the diversity of tick-borne Coxiella and Rickettsia bacteria. The pathogenic roles of these bacteria also need to be investigated further.