Molecular detection of Anaplasma bovis, Candidatus Anaplasma boleense and Rickettsia spp. in ticks infesting small ruminants.

Anaplasma spp. and Rickettsia spp. are intracellular vector-borne pathogens and harbored by a wide range of ticks and vertebrate hosts. Aim of this study was to molecularly characterize Anaplasma spp. and Rickettsia spp. in different ticks collected from livestock hosts in nine districts of Khyber Pakhtunkhwa (KP), Pakistan. In total, 862 ticks were collected from cattle, goats and sheep. Highest tick's infestation was observed on cattle 56.14% (32/57), followed by goats 45.45% (40/88), and sheep 42.05% (45/107). Rhipicephalus microplus (305/862, 35.38%) was predominant species, followed by Haemaphysalis sulcata (243/862, 28.19%), Hyalomma anatolicum (133/862, 15.42%), Haemaphysalis bispinosa (120/862, 13.92%), and Hyalomma kumari (61/862, 7.07%). A subset of 135 ticks were screened for Anaplasma spp. and Rickettsia spp. based on the amplification of partial 16 S rDNA and outer-membrane protein A (ompA) fragments, respectively. In total, 16 ticks (11.85%) were positive for Anaplasma spp. and Rickettsia spp. Obtained 16 S rDNA sequences for Anaplasma spp. detected in Ha. bispinosa and Ha. sulcata showed 99.98% identity with Anaplasma bovis, while other detected in Rh. microplus showed 99.84% identity with Candidatus Anaplasma boleense. Similarly, detected ompA sequence in Ha. sulcata showed 100% identity with Rickettsia sp. and 97.93% with Rickettsia slovaca, and another sequence detected in Rh. microplus showed 100% identity with Candidatus Rickettsia shennongii. In phylogenetic trees, these sequences clustered with corresponding species from Pakistan, China, Turkey, South Korea, South Africa, and Herzegovina. This is the first study reporting detection of A. bovis in Ha. bispinosa and Ha. sulcata, Ca. A. boleense in Rh. microplus collected from goats, and R. slovaca-like in Ha. sulcata. Our results enforce the need for regular surveillance of Rickettsiales in hard ticks infesting livestock in the region.

High serological and molecular prevalence of Ehrlichia canis and other vector-borne pathogens in dogs from Boa Vista Island, Cape Verde.

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.

Detection of tick-borne pathogens in the pangolin tick, Amblyomma javanense, from Vietnam and Laos, including a novel species of Trypanosoma.

Two species of Southeast Asian pangolins (the Chinese pangolin, Manis pentadactyla and the Malayan or Sunda pangolin, Manis javanica) are critically endangered species. Therefore, knowledge on their parasitic infections is very important, especially considering ticks that can transmit which pathogens. In this study, 32 pangolin ticks (Amblyomma javanense), that were collected in Vietnam and Laos, were analyzed with molecular methods for the presence of tick-borne pathogens. Two members of the family Anaplasmataceae were shown to be present in 14 pangolin ticks, i.e., Candidatus Anaplasma pangolinii and an Ehrlichia sp. In three ticks, a single Rickettsia genotype was also detected, and in seven ticks four 18S rRNA sequence variants of a Babesia sp. Most importantly, a novel protozoan agent, tentatively called here Trypanosoma sp. "PAT14" was detected in one A. javanense nymph. These results imply the first molecular finding of any species of Anaplasma, Ehrlichia and Babesia in pangolin ticks from Vietnam and Laos. On the other hand, detection of a new tick-associated Trypanosoma sp. in A. javanense from Southeast Asia is not only important from a taxonomic point of view, but it is also the first finding of any trypanosomes in the genus Amblyomma in Eurasia and adds pangolins to the potential placental mammalian hosts of any trypanosomes.

Prevalence of tick-borne bacterial pathogens in Germany-has the situation changed after a decade?

Introduction: Tick-borne pathogens, such as Borreliella spp., Rickettsia spp., and Anaplasma spp., are frequently detected in Germany. They circulate between animals and tick vectors and can cause mild to severe diseases in humans. Knowledge about distribution and prevalence of these pathogens over time is important for risk assessment of human and animal health. Methods: Ixodes ricinus nymphs were collected at different locations in 2009/2010 and 2019 in Germany and analyzed for tick-borne pathogens by real-time PCR and sequencing. Results: Borreliella spp. were detected with a prevalence of 11.96% in 2009/2010 and 13.10% in 2019 with B. afzelii and B. garinii as dominant species. Borrelia miyamotoi was detected in seven ticks and in coinfection with B. afzelii or B. garinii. Rickettsia spp. showed a prevalence of 8.82% in 2009/2010 and 1.68% in 2019 with the exclusive detection of R. helvetica. The prevalence of Anaplasma spp. was 1.00% in 2009/2010 and 7.01% in 2019. A. phagocytophilum was detected in seven tick samples. None of the nymphs were positive for C. burnetii. Discussion: Here, observed changes in prevalence were not significant after a decade but require longitudinal observations including parameters like host species and density, climatic factors to improve our understanding of tick-borne diseases.

Molecular prevalence of Anaplasma marginale, Babesia bigemina, and Theileria orientalis and their associations with Mafriwal cattle's age groups.

Objective: This study was designed to determine the molecular prevalence of hemoparasites and their associations with Mafriwal cattle's age groups. Materials and Methods: Blood samples were taken from the coccygeal veins of calves (n = 92), yearlings (n = 95), lactating (n = 90), and dry (n = 94) cows, which were subjected to microscopic and molecular identification of hemoparasites. The prevalence rate was determined based on the proportion of infected samples in the observed samples. Associations between hemoparasitism and different age groups of Mafriwal cattle were determined by the odds ratio and Fisher's exact test. Results: Babesia bigemina was the most prevalent hemoparasite in monospecies infection (20.8%), while the co-infection of Anaplasma marginale and B. bigemina (36.4%) had the highest molecular prevalence. Highly significant associations of hemoparasitism were observed between calves and yearlings (p < 0.001, Odds ratio = 21.340, 95% CI = 3.200-907.871), lactating (p < 0.01, Odds ratio = 6.600, 95% CI = 1.808-36.516), and dry (p < 0.001, Odds ratio = 10.457, 95% CI = 2.363-96.242) cows. Nevertheless, calves and yearlings were 2-4 times more likely to be co-infected with multiple hemoparasite species in comparison to older age groups. Conclusion: Mafriwal cattle were more susceptible to hemoparasitism with advancing age, but the younger calves were more prone to be co-infected with multiple hemoparasite species.

High genetic diversity of Anaplasma ovis in sheep from Bosnia and Herzegovina.

BACKGROUND: Ovine anaplasmosis (sensu stricto) is a rickettsial blood disease caused by the tick-borne species Anaplasma ovis. The disease is characterized by mild anemia, fever, and icterus. A more severe clinical presentation is possible in non-endemic areas. There is no existing data on the presence of Anaplasma ovis in Bosnia and Herzegovina. However, given the country's location within the Mediterranean Basin and the recent molecular detection of Babesia ovis, it is plausible that sheep in the region could naturally be infected with this tick-borne pathogen. METHODS AND RESULTS: Blood samples from 81 sheep in the Podrinje and Herzegovina areas were examined by PCR. PCR positivity was found in 38 (46.9%) cases indicating a high number of infected sheep. Mixed infections with Babesia ovis and A.ovis were observed in 63.3% of cases. A higher number of positive sheep was recorded in the area of Herzegovina. Phylogenetic analysis of the gltA, groEL, and msp4 genes of A. ovis revealed numerous genotypes and significant genetic variability. This diversity was not related to geographic origin, tick-borne infection status, or sheep breeding practices in Podrinje and Herzegovina. CONCLUSIONS: The data obtained in this study suggest that the emergence of new genotypes and the high genetic variability of A. ovis are driven by specific local and micro-environmental factors.

Molecular Investigation of Small Ruminant Abortions Using a 10-Plex HRM-qPCR Technique: A Novel Approach in Routine Diagnostics.

The objective of this study was to apply and preliminarily evaluate a High-Resolution Melting (HRM) analysis technique coupled with qPCR, that allows the simultaneous detection of 10 different ruminant abortogenic pathogens, for investigating abortions in sheep and goats throughout Greece. A total of 264 ovine and caprine vaginal swabs were obtained the week following the abortion from aborted females and analyzed using a commercially available kit (ID Gene™ Ruminant Abortion Multiplex HRM, Innovative Diagnostics). Results indicated a high prevalence of Coxiella burnetii and Chlamydophila spp., which were detected in 48.9% and 42.4% of the vaginal swabs, respectively. Results for these most commonly detected pathogens were compared with those of a well-established commercial qPCR kit, with near-perfect agreement. Toxoplasma gondii, Salmonella spp., Brucella spp., Anaplasma phagocytophilum, Campylobacter fetus, and Neospora caninum were also identified, the two latter reported for the first time in the country in small ruminants. Mixed infections occurred in 35.6% of the animals examined. This technique allows for the simultaneous detection of many abortogenic pathogens in an accurate and cost-effective assay. Detection of uncommon or not previously reported pathogens in various cases indicates that their role in ovine and caprine abortions may be underestimated.

Molecular Detection of Theileria ovis, Anaplasma ovis, and Rickettsia spp. in Rhipicephalus turanicus and Hyalomma anatolicum Collected from Sheep in Southern Xinjiang, China.

The Xinjiang Uygur Autonomous Region (Xinjiang) borders eight countries and has a complex geographic environment. There are almost 45.696 million herded sheep in Xinjiang, which occupies 13.80% of China's sheep farming industry. However, there is a scarcity of reports investigating the role of sheep or ticks in Xinjiang in transmitting tick-borne diseases (TBDs). A total of 894 ticks (298 tick pools) were collected from sheep in southern Xinjiang. Out of the 298 tick pools investigated in this study, Rhipicephalus turanicus (Rh. turanicus) and Hyalomma anatolicum (H. anatolicum) were identified through morphological and molecular sequencing. In the southern part of Xinjiang, 142 (47.65%), 86 (28.86%), and 60 (20.13%) tick pools were positive for Rickettsia spp., Theileria spp., and Anaplasma spp., respectively. Interestingly, the infection rate of Rickettsia spp. (73%, 35.10%, and 28.56-41.64%) was higher in Rh. turanicus pools than in H. anatolicum pools (4%, 4.44%, and 0.10-8.79%) in this study. Fifty-one tick pools were found to harbor two pathogens, while nineteen tick pools were detected to have the three pathogens. Our findings indicate the presence of Rickettsia spp., Theileria spp., and Anaplasma spp. potentially transmitted by H. anatolicum and Rh. turanicus in sheep in southern Xinjiang, China.

Sylvatic vector-borne pathogens including Cytauxzoon europaeus in the European wildcat (Felis silvestris) from southwestern Germany.

BACKGROUND: European wildcats (Felis silvestris) are widely distributed in Europe and a strictly protected species in Germany. Lately, anthropogenic protective efforts lead to increasing numbers of wildcats in southwestern Germany. Moreover, in recent years the numbers of domestic cats are increasing. Thus, the contact between domestic and wildcats may lead to the spread of zoonotic pathogens in both animal species. As data on vector-borne pathogens (VBPs) in wildcats from Germany are limited to date, the objective of this study was to investigate the presence and current distribution of VBPs in wildcats from southwestern Germany. METHODS: Skin and spleen samples from 117 European wildcats, originating from a regional carcass-monitoring program in southwestern Germany, were examined by real-time and conventional polymerase chain reaction (PCR) for the presence of Anaplasma phagocytophilum, Neoehrlichia mikurensis, Rickettsia spp., Bartonella spp., and Piroplasmida. RESULTS: In total, 6.8% (n = 8) of the wildcats were Rickettsia-positive, specified as R. helvetica. Three wildcats were positive for A. phagocytophilum (2.6%), one for Bartonella spp., namely B. taylorii (0.8%), and 84 for Cytauxzoon spp. (71.8%). Out of these 84 samples, 23 were further sequenced revealing very high identity levels (99.84-100%) to C. europaeus, which is considered to be pathogenic for domestic cats. All wildcats were negative for the presence of N. mikurensis DNA. CONCLUSIONS: European wildcats in southwestern Germany are hosting several VBPs. With the exception of Cytauxzoon spp., low prevalence rates of most examined pathogens suggest that wildcats are primarily incidental hosts for sylvatic pathogens associated with rodents, in contrast to domestic cats. However, the high prevalence of the cat-associated pathogen C. europaeus suggests that wildcats in southwestern Germany may serve as reservoirs for this pathogen.

Anaplasma phagocytophilum effector EgeA facilitates infection by hijacking TANGO1 and SCFD1 from ER-Golgi exit sites to pathogen-occupied inclusions.

The obligatory intracellular bacterium Anaplasma phagocytophilum causes human granulocytic anaplasmosis, an emerging zoonosis. Anaplasma has limited biosynthetic and metabolic capacities, yet it effectively replicates inside of inclusions/vacuoles of eukaryotic host cells. Here, we describe a unique Type IV secretion system (T4SS) effector, ER-Golgi exit site protein of Anaplasma (EgeA). In cells infected by Anaplasma, secreted native EgeA, EgeA-GFP, and the C-terminal half of EgeA (EgeA-C)-GFP localized to Anaplasma-containing inclusions. In uninfected cells, EgeA-C-GFP localized to cis-Golgi, whereas the N-terminal half of EgeA-GFP localized to the ER. Pull-down assays identified EgeA-GFP binding to a transmembrane protein in the ER, Transport and Golgi organization protein 1 (TANGO1). By yeast two-hybrid analysis, EgeA-C directly bound Sec1 family domain-containing protein 1 (SCFD1), a host protein of the cis-Golgi network that binds TANGO1 at ER-Golgi exit sites (ERES). Both TANGO1 and SCFD1 localized to the Anaplasma inclusion surface. Furthermore, knockdown of Anaplasma EgeA or either host TANGO1 or SCFD1 significantly reduced Anaplasma infection. TANGO1 and SCFD1 prevent ER congestion and stress by facilitating transport of bulky or unfolded proteins at ERES. A bulky cargo collagen and the ER-resident chaperon BiP were transported into Anaplasma inclusions, and several ER stress marker genes were not up-regulated in Anaplasma-infected cells. Furthermore, EgeA transfection reduced collagen overexpression-induced BiP upregulation. These results suggest that by binding to the two ERES proteins, EgeA redirects the cargo-adapted ERES to pathogen-occupied inclusions and reduces ERES congestion, which facilitates Anaplasma nutrient acquisition and reduces ER stress for Anaplasma survival and proliferation.

Impact of vaccination with the Anaplasma phagocytophilum MSP4 chimeric antigen on gene expression in the rabbit host.

There are currently no vaccines available to prevent and control of Anaplasma phagocytophilum, an intracellular bacterial pathogen transmitted by ticks that occurs in many regions of the world and causes disease in a wide range of domestic and wild hosts, including humans. Vaccines induce long-lasting immunity and could prevent or reduce transmission of this pathogen. Understanding how vaccines induce a protective response can be difficult due to the complexity of the immune system, which operates at many levels throughout the organism. New perspectives in vaccinology, based on systems biology approaches, integrate many scientific disciplines to fully understand the biological responses to vaccination, where a transcriptomic approach could reveal relevant information of the host immune system, allowing profiling for rational design of vaccine formulations, administration, and potential protection. In the present study we report the gene expression profiles by RNA-seq followed by functional analysis using whole blood samples from rabbits immunized with a recombinant chimeric protein containing peptides from the MSP4 protein of A. phagocytophilum, which showed satisfactory results in terms of potential protection. Transcriptomic analysis revealed differential expression of 720 genes, with 346 genes upregulated and 374 genes downregulated. Overrepresentation of biological and metabolic pathways correlated with immune response, protein signaling, cytoskeleton organization and protein synthesis were found. These changes in gene expression could provide a complete and unique picture of the biological response to the epitope candidate vaccine against A. phagocytophilum in the host.

The Prevalence of Rickettsial and Rickettsial-Like Diseases in Patients with Undifferentiated Febrile Illness - Hainan Province, China, 2018-2021.

Introduction: Rickettsial and Rickettsial-like diseases, resulting from obligate intracellular Gram-negative bacteria, pose a growing public health threat in China. To assess the current prevalence of these diseases on Hainan Island, a study was conducted on 9 bacterial pathogens found in patients with undifferentiated febrile illness (UFI) treated in Haikou between 2018 and 2021 using a TaqMan Polymerase Chain Reaction (TaqMan PCR) array. Methods: Blood samples (n=503) were collected from patients with UFI between 2018 and 2021. The samples were screened for Rickettsia spp., Orientia tsutsugamushi (O. tsutsugamushi), Anaplasma. phagocytophilum (A. phagocytophilum), Ehrlichia chaffeensis, Coxiella burnetii, Chlamydia psittaci, Brucella spp., Burkholderia pseudomallei, and Borrelia burgdorferi using a TaqMan PCR array. Positive samples (Ct<35) underwent confirmation through nested PCR, sequencing, and phylogenetic analysis. Results: O. tsutsugamushi and A. phagocytophilum were detected in the patients at positive rates of 14.51% (73/503) and 5.57% (28/503), respectively. Co-infection of O. tsutsugamushi and A. phagocytophilum was identified in scrub typhus (ST) positive populations from Hainan (10.96%, 8/73), Guangxi (61.54%, 8/13), and Yunnan (5.36%, 3/56) provincial-level administrative divisions (PLADs) of China. Conclusion: An increased prevalence rate of ST and a decreased prevalence of rickettsioses were observed in patients with UFI in Hainan compared to a decade ago. The co-infection of O. tsutsugamushi and A. phagocytophilum poses a current public health threat in China.

Investigation of Tick-Borne Pathogens in Ixodes Ticks from Bosnia and Herzegovina.

Limited information is available regarding the presence of tick-borne pathogens and their distribution within Ixodes species in Bosnia and Herzegovina. This study aimed to identify Rickettsia spp., Babesia spp., Anaplasma phagocytophilum, and Borrelia burgdorferi sensu lato (s.l.) in Ixodes ticks collected from domestic and wild animals and vegetation in different regions across Bosnia and Herzegovina. A total of 7438 adult ticks, including 4526 Ixodes ricinus, Ixodes canisuga, and Ixodes hexagonus, were collected. Real-time PCR screening of 450 pooled I. ricinus samples revealed a 22.1% infection rate with at least one pathogen. Rickettsia spp. (6.3%) were found in ticks from dogs, cats, and goats, Babesia spp. (3.1%) in ticks from dogs and cattle, A. phagocytophilum (8.8%) in ticks from dogs, goats, and cattle, and B. burgdorferi s.l. (3.4%) in ticks from dogs and cats. Mixed infections with B. burgdorferi s.l. and A. phagocytophilum, as well as B. burgdorferi s.l. and Rickettsia spp., were found in two pools of I. ricinus from dogs and cats, respectively. Additionally, co-infection with Rickettsia spp. and A. phagocytophilum was confirmed in three tick pools from dogs and goats. Each tick from these pooled samples was individually retested to confirm the presence of pathogens. In the examined pooled samples of I. canisuga (1) and I. hexagonus (6), none of the tested pathogens were detected. Our findings represent the first detection of Rickettsia spp., Babesia spp., A. phagocytophilum, and B. burgdorferi s.l. in I. ricinus collected from domestic animals and vegetation in Bosnia and Herzegovina. Considering the established infection rates, the detection of tick-borne pathogens in adult ticks collected from domestic animals and vegetation enriches the current knowledge of the presence of tick-borne pathogens at the local, regional, national, and broader levels.

Zoonotic Tick-Borne Pathogens in Ticks from Vegetation and Alpine Ibex (Capra ibex) in the Maritime Alps, Italy.

In the Maritime Alps (northwestern Italy), we collected ticks from vegetation and Alpine ibex (Capra ibex). Ixodes ricinus was the most abundant species in the study area, questing up to 1824 m a.s.l. and infesting 28 out of 72 ibexes. Haemaphysalis punctata, H. sulcata and Dermacentor marginatus were also collected. The abundance of questing ticks significantly decreased with altitude, with beechwoods being the preferred habitat. By PCR, we identified Borrelia burgdorferi s.l. in questing I. ricinus (28.3%; 95%CI: 19.4-38.6) but not in specimens collected from animals. Rickettsia spp. infected both questing (20.6%; 95%CI: 12.9-30.3) and on-host (30.2%; 95%CI: 21.2-40.4) I. ricinus. Anaplasma phagocytophilum was detected in 4.3% (95%CI: 1.2-10.8) of questing I. ricinus and in 45.3% (95%CI: 34.6-56.4) of I. ricinus collected from ibex. Female I. ricinus collected on animals were significantly more infected with A. phagocytophilum than females collected from vegetation (OR = 11.7; 95%CI: 3.8-48.1). By amplifying and sequencing a fragment of the groEL gene, we identified 13 groEL haplotypes, clustering with ecotypes I and II; ecotype I, prevalent in our sample, is considered zoonotic. Our study demonstrates the presence of different tick-borne zoonotic agents in the study area, encompassing a wide altitudinal range, as confirmed by the ticks found on ibex, a typical mountain-dwelling mammal. The results also confirm the altitudinal range expansion of ticks and associated pathogens in the Alps and suggest that Alpine ibex may act as a reservoir for A. phagocytophilum, as do other wild ungulate species.

Host-pathogen associations inferred from bloodmeal analyses of Ixodes scapularis ticks in a low biodiversity setting.

Tick-borne pathogen emergence is dependent on the abundance and distribution of competent hosts in the environment. Ixodes scapularis ticks are generalist feeders, and their pathogen infection prevalence depends on their relative feeding on local competent and non-competent hosts. The ability to determine what host a larval life stage tick fed on can help predict infection prevalence, emergence, and spread of certain tick-borne pathogens and the risks posed to public health. Here, we use a newly developed genomic target-based technique to detect the source of larval bloodmeals by sampling questing nymphs from Block Island, RI, a small island with a depauperate mammalian community. We used previously designed specific assays to target all known hosts on this island and analyzed ticks for four human pathogenic tick-borne pathogens. We determined the highest proportion of larvae fed on avian species (42.34%), white-footed mice (36.94%), and white-tailed deer (20.72%) and occasionally fed on feral cats, rats, and voles, which are in low abundance on Block Island. Additionally, larvae that had fed on white-footed mice were significantly more likely to be infected with Borrelia burgdorferi and Babesia microti, while larvae that had fed on white-footed mice or white-tailed deer were significantly more likely to be infected with, respectively, mouse- and deer-associated genotypes of Anaplasma phagocytophilum. The ability to detect a nymph's larval host allows for a better understanding of tick feeding behavior, host distribution, pathogen prevalence, and zoonotic risks to humans, which can contribute to better tick management strategies. IMPORTANCE: Tick-borne diseases, such as Lyme disease, babesiosis, and anaplasmosis, pose significant public health burdens. Tick bloodmeal analysis provides a noninvasive sampling method to evaluate tick-host associations and combined with a zoonotic pathogen assay, can generate crucial insights into the epidemiology and transmission of tick-borne diseases by identifying potential key maintenance hosts. We investigated the bloodmeals of questing Ixodes scapularis nymphs. We found that avian hosts, white-footed mice, and white-tailed deer fed the majority of larval ticks and differentially contributed to the prevalence of multiple tick-borne pathogens and pathogen genotypes in a low biodiversity island setting. Unraveling the intricate network of host-vector-pathogen interactions will contribute to improving wildlife management and conservation efforts, to developing targeted surveillance, and vector and host control efforts, ultimately reducing the incidence of tick-borne diseases and improving public health.

The Diversity of Spotted Fever Group Rickettsia Found in Ixodidae Hard Ticks Removed from Humans in Karachay-Cherkessia, North Caucasus, Russia.

Karachay-Cherkessia is the region in the Russian Federation that has been overlooked in terms of research on the human bacterial pathogens transmitted by ticks. In this study, we investigated the infection status of ticks with Rickettsia, Borrelia, Coxiella burmetii, Anaplasma phagocytophilum, and Ehrlichia chaffeensis/Ehrlichia muris associated with natural focal infections in Karachay-Cherkessia. A total amount of 159 ticks from three species across three genera (Ixodes ricinus, Dermacentor marginatus, Hyalomma scupense) removed from humans were collected for analysis. Additionally, we used 53 individual ticks and 40 tick pools from the vegetation of three species (I. ricinus, D. marginatus, and Rhipicephalus bursa). Screening of the studied pathogens was performed by using commercial qPCR kits. Sanger sequencing utilizing partial sequences of gltA and ompA genes was employed to identify the Rickettsia species. Our findings revealed the presence of DNA from five species within the spotted fever group Rickettsia, namely Rickettsia raoultii, R. slovaca, R. helvetica, R. monacensis, and R. aeschlimannii. Moreover, two distinct genotypes were identified within R. aeschlimannii, R. helvetica, and R. monacensis. Additionally, DNA from Borrelia burgdoferi s.l., B. miyamotoi, and A. phagocytophilum was detected in the tested ticks. This study provides valuable insights into the prevalence and the diversity of bacterial pathogens transmitted by ticks in the Karachay-Cherkessia region.

The Impact of Altitude on Tick-Borne Pathogens at Two Mountain Ranges in Central Slovakia.

Ticks are ectoparasites of a wide range of animals and are important vectors of numerous pathogens affecting humans, livestock, and pets. This study investigates possible correlations between selected factors, altitude, soil pH, and a factor called 'amount' (number of ticks examined in pooled samples) on the occurrence of I. ricinus ticks positive for selected tick-borne microorganisms. Questing I. ricinus ticks were collected in 2016 and 2017 across various altitudes, at two mountain ranges in central Slovakia. Tick pools were screened for the presence of Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato (Bbsl), Babesia/Theileria spp., Rickettsia spp., and tick-borne encephalitis virus (TBEV) using molecular methods. Regression analysis was employed to evaluate relationships between selected factors and the occurrence of vector-borne microorganisms. This study revealed a statistically significant influence of altitude on the occurrence of A. phagocytophilum; increasing altitude of the sampling site was associated with increased probability of pathogen occurrence. For Babesia/Theileria spp., neither altitude nor soil pH significantly affected pathogen occurrence. The occurrence of Bbsl was notably impacted by both altitude and soil pH; higher altitudes were associated with a decreased probability of pathogen presence, whereas higher soil pH increased the likelihood of pathogen occurrence. The presence of Rickettsia in a pooled sample was not affected by altitude and soil pH, but the 'amount' factor was a significant predictor, increasing the probability of pathogen detection. Neither altitude nor soil pH had a significant impact on TBEV occurrence. The regression models showed moderate goodness-of-fit levels to the data, underscoring their utility in examining the role of altitude and soil pH on pathogen occurrence. However, they explained only a small portion of the overall variance in pathogen occurrence, indicating the presence of other significant factors not covered in this study.

Molecular Survey on the Occurrence of Tick-Borne Bacteria in Wild Birds from Central Italy.

Birds are known to be carriers of ticks infected by tick-borne pathogens, including bacteria. However, not many studies have been carried out on avian tissues to detect these agents. The aim of the present survey was to investigate, using PCR, the presence of Anaplasma phagocytophilum, Bartonella spp., Borrelia burgdorferi sensu lato, Chlamydia psittaci, Coxiella burnetii, Ehrlichia canis, Francisella tularensis, and Rickettsia spp. in the spleens collected from 300 wild birds of different orders and species from Central Italy. A total of 53 (17.67%) samples were PCR positive for at least one investigated pathogen. One (0.33%) bird was positive for Bartonella spp., five (1.67%) birds were positive for C. burnetii, eleven (3.67%) for B. burgdorferi s.l., and thirty-six (12%) for C. psittaci. No coinfection was detected. All samples were negative for A. phagocytophilum, E. canis, F. tularensis, and Rickettsia spp. The findings showed that wild birds may harbor different zoonotic tick-borne bacteria; therefore, they can contribute to the diffusion of these agents.

Molecular detection and characterization of Anaplasma marginale and Babesia canis vogeli infecting dogs in Luxor, Egypt.

Tick-borne diseases in animals are increasing rapidly worldwide, but there is insufficient information about tick-borne diseases infecting dogs in southern Egypt. Thus, in the current study, we detected the presence of Anaplasma marginale (A. marginale) and Babesia canis vogeli (B. canis vogeli) in the blood of dogs. The results revealed that 4/100 (4%) were positive, and a higher infection rate was found in males (75%), than females (25%). The phylogenetic analysis for the major surface protein 4 (msp4) gene in this study was compared with amplicons separate from other reported isolates with alignment by identity 100% with cattle and camels from Egypt, and the phylogenetic analysis for the B. canis vogeli small subunit ribosomal RNA (SSU rRNA) gene in this study identified identity by 99.89% with dogs from Egypt. This report is considered the first report in southern Egypt about A. marginale in dogs based on the sequence analysis of the msp4 gene, providing new data for the classification and identification of A. marginale in dogs compared to A. marginale isolated from other animals in southern Egypt.

Occurrence and molecular prevalence of Anaplasmataceae, Rickettsiaceae and Coxiellaceae in African wildlife: A systematic review and meta-analysis.

INTRODUCTION: Tick-borne pathogens (TBPs) constitute an emerging threat to public and animal health especially in the African continent, where land-use change, and wildlife loss are creating new opportunities for disease transmission. A review of TBPs with a focus on ticks determined the epidemiology of Rhipicephalus ticks in heartwater and the affinity of each Rickettsia species for different tick genera. We conducted a systematic review and meta-analysis to collect, map and estimate the molecular prevalence of Anaplasmataceae, Rickettsiaceae and Coxiellaceae in African wildlife. MATERIALS AND METHODS: Relevant scientific articles were retrieved from five databases: PubMed, ScienceDirect, Scopus, Ovid and OAIster. Publications were selected according to pre-determined exclusion criteria and evaluated for risk of bias using the appraisal tool for cross-sectional studies (AXIS). We conducted an initial descriptive analysis followed by a meta-analysis to estimate the molecular prevalence of each pathogen. Subgroup analysis and meta-regression models were employed to unravel associations with disease determinants. Finally, the quality of evidence of every estimate was finally assessed. RESULTS: Out of 577 retrieved papers, a total of 41 papers were included in the qualitative analysis and 27 in the meta-analysis. We retrieved 21 Anaplasmataceae species, six Rickettsiaceae species and Coxiella burnetii. Meta-analysis was performed for a total of 11 target pathogens. Anaplasma marginale, Ehrlichia ruminantium and Anaplasma centrale were the most prevalent in African bovids (13.9 %, CI: 0-52.4 %; 20.9 %, CI: 4.1-46.2 %; 13.9 %, CI: 0-68.7 %, respectively). Estimated TBPs prevalences were further stratified per animal order, family, species and sampling country. DISCUSSION: We discussed the presence of a sylvatic cycle for A. marginale and E. ruminantium in wild African bovids, the need to investigate A. phagocytophilum in African rodents and non-human primates as well as E. canis in the tissues of wild carnivores, and a lack of data and characterization of Rickettsia species and C. burnetii. CONCLUSION: Given the lack of epidemiological data on wildlife diseases, the current work can serve as a starting point for future epidemiological and/or experimental studies.