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Migratory birds play a dual role as potential reservoirs of tick-borne pathogens, and potential dispersers of pathogen-containing ticks during their migratory journeys. Ixodes ricinus, a prevalent tick species in Northern and Western Europe, serves as a primary vector for Anaplasma phagocytophilum—a bacterium with implications for human and animal health. There is limited information available regarding A. phagocytophilum in birds. Our investigation focused on A. phagocytophilum prevalence in ticks collected from migratory birds in southeastern Sweden. The identification of ticks involved both molecular analyses for species determination and morphological classification to ascertain the developmental stage. The presence of A. phagocytophilum was determined using real-time PCR. Of the 1115 ticks analyzed from 4601 birds, 0.9% (n = 10), including I. ricinus and Ixodes frontalis, tested positive for A. phagocytophilum. Notably, common blackbirds (Turdus merula) yielded the highest number of A. phagocytophilum-infected ticks. The findings suggest that A. phagocytophilum is present in a small proportion of ticks infesting migratory birds in southeastern Sweden. Consequently, the role of birds as hosts for ticks infected with A. phagocytophilum appears to be low, suggesting that birds seem to play a minor indirect role in the geographic dispersal of A. phagocytophilum. [ABSTRACT FROM AUTHOR]

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.

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.

IntroductionTick-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.MethodsIxodes 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.ResultsBorreliella 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.DiscussionHere, 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.

Abstract Background Ticks are vectors of various pathogens, including tick-borne encephalitis virus causing TBE and bacteria such as Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum causing e.g. viral-bacterial co-infections (TBE + LB/HGA), which pose diagnostic and therapeutic problems. Since these infections are usually accompanied by inflammation and oxidative stress causing metabolic modifications, including phospholipids, the aim of the study was to assess the level of polyunsaturated fatty acids and their metabolism (ROS- and enzyme-dependent) products in the blood plasma of patients with TBE and TBE + LB/HGA before and after pharmacotherapy. Methods The total antioxidant status was determined using 2,20-azino-bis-3-ethylbenzothiazolin-6-sulfonic acid. The phospholipid and free fatty acids were analysed by gas chromatography. Lipid peroxidation was estimated by measuring small molecular weight reactive aldehyde, malondialdehyde and neuroprostanes. The reactive aldehyde was determined using gas chromatography coupled with mass spectrometry. The activity of enzymes was examined spectrophotometrically. An analysis of endocannabinoids and eicosanoids was performed using a Shimadzu UPLC system coupled with an electrospray ionization source to a Shimadzu 8060 Triple Quadrupole system. Receptor expression was measured using an enzyme-linked immunosorbent assay (ELISA). Results The reduced antioxidant status as a result of infection was accompanied by a decrease in the level of phospholipid arachidonic acid (AA) and docosahexaenoic acid (DHA) in TBE, an increase in DHA in co-infection and in free DHA in TBE with an increase in the level of lipid peroxidation products. The enhanced activity of enzymes metabolizing phospholipids and free PUFAs increased the level of endocannabinoids and eicosanoids, while decreased 15-PGJ2 and PGE2 was accompanied by activation of granulocyte receptors before pharmacotherapy and only tending to normalize after treatment. Conclusion Since classical pharmacotherapy does not prevent disorders of phospholipid metabolism, the need to support treatment with antioxidants may be suggested.

This study was targeted to evaluate the infection rate of Anaplasma phagocytophilum in cows in Mosul city-Iraq, using microscopic examination (ME) of the blood smears and nested polymerase chain reaction (N-PCR) technique to evaluate the efficiency of different diagnostic methods used in this study and to determine the parasitemia with the more common leucocytes infected with A. phagocytophilum. 50 blood samples were collected from cows in Mosul city. Blood smears were prepared for the primary detection of inclusion bodies (Morulae) inside leukocytes. The remaining blood was used to confirm the infection using the N-PCR technique. Results revealed that the infection rate of A. phagocytophilum in Mosul City was 58 and 72% using the ME method and N-PCR, respectively. Substantial compatibility was observed between the ME method and N-PCR technique based on the Kappa value 0.699 with a sensitivity of 80.55%, specificity of 100%, and accuracy of 86% of the ME method compared with the N-PCR technique. Infected cows with A. phagocytophilum were suffering from acute disease. Based on the ME of the blood smears, the total parasitemia of A. phagocytophilum inside the leukocytes ranged between 6-29%, with a mean of 17%. The highest significance of parasitemia in neutrophils was 40%, followed by lymphocytes was 25% compared with mononuclear cells, basophils, and eosinophils, which showed no significant change of parasitemia among them. In conclusion, A. phagocytophilum is widespread in cows in Mosul- Iraq; the ME method and N-PCR technique efficiently detect the A. phagocytophilum and neutrophils, which are mostly infected with bacteria.

Background. Infections transmitted to humans by the bites of ixodid ticks remain an urgent public health problem. In this work we explored the natural foci of tickborne infections located in the valley of the Chikoy River, which is a part of the buffer zone of the Baikal natural territory.The aim. To characterize the modern diversity and prevalence of tick-borne pathogens in the ecosystems of the valley of the Chikoy River (Trans-Baikal Territory, Russian Federation).Materials and methods. Thirteen sampling sites were located in typical biotopes throughout the Chikoy valley. In total 48 adult Ixodes persulcatus ticks, 1 female Haemaphysalis concinna tick and 38 specimens of small mammals were studied. All samples were tested for infection with seven tick-borne pathogens using multiplex real-time PCR.Results. No pathogens were detected in the H. concinna specimen. No R. sibirica and R. heilongjiangensis were detected both in ticks and in rodents. Among I. persulcatus, tick-borne encephalitis virus (TBEV), and the prevalence of Borrelia burgdorferi s. l. comprised 39.5 %, A. phagocytophilum – 16.7 %, B. miyamotoi – 8.3 % and Ehrlichia sp. – 2.1 %. Among infected ticks 6.2 % were co-infected with B. burgdorferi s. l. and A. phagocytophilum. Four rodent hosts of ticks and infections were identified: Myodes rufocanus (44.7 %), Apodemus peninsulae (39 %), Microtus oeconomus (13.2 %) and M. rutilus (2.6 %). Mean prevalence of B. burgdorferi s. l. in rodents comprised 39.5 %, B. miyamotoi – 28.9 %, Ehrlichia sp. – 21.1 % and A. phagocytophilum – 18.4 %. TBEV was detected in 5.3 % of rodents.Conclusion. At least five tick-borne pathogens circulate in the Chikoi River valley, i. e. TBEV, B. burgdorferi sensu lato, A. phagocytophilum, B. miyamotoi and Ehrlichia sp. The wide spread of I. persulcatus and abundance of competent rodent hosts of infections and ticks indicates that natural foci of tick-borne diseases are widely distributed in the Chikoi River valley.

Babesia microti is a protozoan that infects red blood cells, causing hemolytic anemia and flu-like symptoms in humans. Understanding co-infections is crucial for the better diagnosis, treatment, and management of tick-borne diseases. This study examined the prevalence of Babesia microti co-infection with other prevalent tick-borne pathogens in Pennsylvania. The dataset acquired from the Dr. Jane Huffman Wildlife Genetics Institute included passive surveillance data from Ixodes spp. from 2021 to 2023. Submitted ticks were screened for tick-borne pathogens using species-specific TaqMan qPCR. Of the 793 B. microti-positive ticks pulled for analysis, 65.0% were co-infected with other pathogens (n = 516). Notably, 60.9% of the B. microti-positive ticks were co-infected with Borrelia burgdorferi, 10.2% with Anaplasma phagocytophilum Ap-ha, and 7.5% carried a triple co-infection with B. burgdorferi and A. phagocytophilum Ap-ha. The rates of B. microti infection and its co-infections are on the rise, with patterns observed in Pennsylvania and other regions of the USA. While other studies have collected both nymphal and adult ticks to screen for co-infections in Pennsylvania, our study stood out as a unique contribution to the field by focusing exclusively on B. microti-positive ticks. The continued monitoring of tick-borne co-infections is vital to prevent misdiagnosis and ensure effective treatment regimens.

Migratory and local birds may disperse ticks and their associated pathogens. The aim of this study was to provide information regarding ticks infesting birds in Asturias, a region that accounts for most of the Lyme disease hospitalizations in Spain. From September 2021 to April 2023, trained and experienced bird-banders collected ticks from birds in two estuary and four forest locations. A total of 1698 birds (52 species, 38 genera, and 26 families) were captured. A total of 51 ticks (28 larvae, 20 nymphs, and 3 females) were collected from 43 birds, belonging to three species: Ixodes ricinus (31), Ixodes frontalis (18), and Haemaphysalis concinna (1). The average prevalence of tick infestation was 2.5% and the average tick burden was 1.2 ticks per infested host. The bird species Turdus merula, Parus major, Luscinia svecica, and Anthus pratensis were among the most infested. Anaplasma phagocytophilum was detected in one I. ricinus nymph collected from Phylloscopus collybita. We have not detected Borrelia burgdorferi s.l., Rickettsia spp., Coxiella burnetii, or piroplasmids in any of the 51 analyzed ticks. These results suggest low infestation rate in migratory/estuary birds and a higher rate in forest/sedentary ones. Despite this, the detection of pathogens, although with low prevalence, can pose a risk to public health.

Abstract Background Anaplasma phagocytophilum is a Gram-negative obligate intracellular bacterium that replicates in neutrophil granulocytes. It is transmitted by ticks of the Ixodes ricinus complex and causes febrile illness called granulocytic anaplasmosis primarily in humans, horses, dogs, sheep, cattle and goats. In comparison, clinically apparent disease has been described rarely in cats especially compared to dogs and horses. It is currently unknown whether cats are less susceptible to A. phagocytophilum or whether granulocytic anaplasmosis might be underdiagnosed in cats. Methods To address this question, we examined clinical signs and laboratory findings in seven A. phagocytophilum infected cats from Germany and Switzerland. We then genetically characterized feline A. phagocytophilum strains and compared them to those from other hosts showing clinically apparent disease. For this purpose, ankA-based, groEL-based and multilocus sequence typing (MLST) were applied. Furthermore, the concordance between these typing methods was assessed. Results Fever, lethargy and anorexia were the most common clinical signs in cats suffering from granulocytic anaplasmosis. The most frequent laboratory finding was thrombocytopenia. All three typing methods consistently indicated that the A. phagocytophilum strains found infecting cats are the same as those that cause disease in humans, dogs and horses. In general, the three typing methods applied exhibited high concordance. Conclusions The genetic characterization of the feline A. phagocytophilum strains indicates that strain divergence is not the explanation for the fact that granulocytic anaplasmosis is much less frequently diagnosed in cats than in dogs and horses. Otherwise, it may be possible that cats are less susceptible to the same strains than dogs and horse are. However, due to the unspecific clinical signs, it should be considered that granulocytic anaplasmosis may be under-diagnosed in cats. Graphical Abstract

Abstract Background Anaplasma phagocytophilum is characterized by a worldwide distribution and distinguished from other Anaplasmataceae by the broadest range of mammalian hosts and high genetic diversity. The role carnivores play in the life cycle of A. phagocytophilum in Europe is uncertain. Currently, only the red fox is considered a suitable reservoir host. In this study, we focused on native and invasive medium-sized carnivore species that live in sympatry and represent the most abundant species of wild carnivores in Poland. Methods A total of 275 individual spleen samples from six carnivore species (Vulpes vulpes, Meles meles, Procyon lotor, Nyctereutes procyonoides and Martes spp.) were screened combining nested PCR and sequencing for A. phagocytophilum targeting a partial groEL gene with subsequent phylogenetic analysis inferred by the maximum likelihood method. Results The DNA of A. phagocytophilum was detected in 16 of 275 individuals (5.8%). Eight unique genetic variants of A. phagocytophilum were obtained. All detected haplotypes clustered in the clade representing European ecotype I. Three variants belonged to the subclade with European human cases together with strains from dogs, foxes, cats, and wild boars. Conclusions While carnivores might have a restricted role in the dissemination of A. phagocytophilum due to their relatively low to moderate infection rates, they hold significance as hosts for ticks. Consequently, they could contribute to the transmission of tick-borne infections to humans indirectly, primarily through tick infection. This underscores the potential risk of urbanization for the A. phagocytophilum life cycle, further emphasizing the need for comprehensive understanding of its ecological dynamics . Graphical Abstract

Vector-borne diseases (VBDs) are considered as (re-)emerging, but information on the transmission cycles and wildlife reservoirs is often incomplete, particularly with regard to urban areas. The present study investigated blood samples from European hedgehogs (Erinaceus europaeus) presented at wildlife rehabilitation centres in the region of Hanover. Past exposure to B. burgdorferi sensu lato (s.l.) and tick-borne encephalitis virus (TBEV) was assessed by serological detection of antibodies, while current infections with Borrelia spp., Anaplasma phagocytophilum, Rickettsia spp., Neoehrlichia mikurensis, Bartonella spp., Babesia spp. and Spiroplasma ixodetis were investigated by (q)PCR. Of 539 hedgehogs tested for anti-Borrelia antibodies, 84.8% (457/539) were seropositive, with a higher seropositivity rate in adult than subadult animals, while anti-TBEV antibodies were detected in one animal only (0.2%; 1/526). By qPCR, 31.2% (168/539) of hedgehog blood samples were positive for Borrelia spp., 49.7% (261/525) for A. phagocytophilum, 13.0% (68/525) for Bartonella spp., 8.2% for S. ixodetis (43/525), 8.0% (42/525) for Rickettsia spp. and 1.3% (7/525) for Babesia spp., while N. mikurensis was not detected. While further differentiation of Borrelia spp. infections was not successful, 63.2% of the A. phagocytophilum infections were assigned to the zoonotic ecotype I and among Rickettsia spp. infections, 50.0% to R. helvetica by ecotype- or species-specific qPCR, respectively. Sequencing revealed the presence of a Rickettsia sp. closely related to Rickettsia felis in addition to a Bartonella sp. previously described from hedgehogs, as well as Babesia microti and Babesia venatorum. These findings show that hedgehogs from rehabilitation centres are valuable sources to identify One Health pathogens in urban areas. The hedgehogs are not only exposed to pathogens from fleas and ticks in urban areas, but they also act as potent amplifiers for these vectors and their pathogens, relevant for citizens and their pets.

Multiple ticks (Acari: Ixodoidea) carrying Rickettsials bacteria have significant importance for both human and animal health. Thus, the purpose of this work was to genetically analyze tick species and their associated Rickettsiales bacteria in animal hosts. In order to achieve these objectives, various animals (including camels, cattle, goats, sheep, dogs, and mice) were inspected in four districts (Mardan, Peshawar, Kohat, and Karak) of Khyber Pakhtunkhwa to collect ticks, while blood samples were collected from all the symptomatic and asymptomatic cattle in all four districts. A total of 234 ticks were obtained from 86 out of 143 (60.14%) host animals, which were morphologically identified as Rhipicephalus turanicus, Rhipicephalus microplus, Haemaphysalis cornupunctata, and Hyalomma asiaticum. Among these, their representative ticks (126/234, 53.85%) were processed for molecular confirmation using cytochrome c oxidase (cox1) gene. Obtained cox1 sequences of four different tick species showed 99.72%-100% maximum identity with their corresponding species reported from Pakistan, China, India, and Kazakhstan and clustered phylogenetically. This study presented the first genetic report of Hy. asiaticum ticks in Pakistan. Moreover, genetically confirmed tick species were molecularly analyzed by PCR for detection of Rickettsiales DNA using partial fragments of 16S rDNA, 190-kDa outer membrane protein A (ompA), and 120-kDa outer membrane protein B (ompB) genes. In addition, blood samples were analyzed to identify Rickettsiales bacteria using the aforementioned genes. Rickettsiales bacteria were found in 24/126 (19.05%) ticks and 4/16 (25.00%) in symptomatic cattle's blood. The obtained ompA and ompB sequences from Hy. asiaticum ticks showed 99.73%-99.87% with Candidatus Rickettsia shennongii and unidentified Rickettsia sp., whereas the obtained 16S rDNA sequences from cattle's blood and ticks (Hae. cornupunctata) showed 99.67% highest identity with Anaplasma phagocytophilum. The 16S rDNA sequence of Rickettsiales DNA from Rh. turanicus ticks showed 100% identity with Ehrlichia canis and unidentified Ehrlichia sp. Obtained sequences of Rickettsiales bacteria were grouped along with their respective species in phylogenetic trees, which were previously reported in Greece, Cuba, Iraq, Turkey, Pakistan, South Korea, and China (mainland and Taiwan). This extensive study explores the wide range of damaging ticks and their corresponding tick-borne bacteria in the area, suggesting a possible danger to both livestock and human communities. [ABSTRACT FROM AUTHOR]

Uncovering the complexity of systems in non-model organisms is critical for understanding arthropod immunology. Prior efforts have mostly focused on Dipteran insects, which only account for a subset of existing arthropod species in nature. Here we use and develop advanced techniques to describe immune cells (hemocytes) from the clinically relevant tick Ixodes scapularis at a single-cell resolution. We observe molecular alterations in hemocytes upon feeding and infection with either the Lyme disease spirochete Borrelia burgdorferi or the rickettsial agent Anaplasma phagocytophilum. We reveal hemocyte clusters exhibiting defined signatures related to immunity, metabolism, and proliferation. Depletion of phagocytic hemocytes affects hemocytin and astakine levels, two I. scapularis hemocyte markers, impacting blood-feeding, molting behavior, and bacterial acquisition. Mechanistically, astakine alters hemocyte proliferation, whereas hemocytin affects the c-Jun N-terminal kinase (JNK) signaling pathway in I. scapularis. Altogether, we discover a role for tick hemocytes in immunophysiology and provide a valuable resource for comparative biology in arthropods. Rolandelli et al. define how immune cells from the tick Ixodes scapularis are affected by infection and show that this cell population have distinct signatures and functions in immunity, metabolism and proliferation. [ABSTRACT FROM AUTHOR]

Background: Ticks are vectors of various pathogens, including tick-borne encephalitis virus causing TBE and bacteria such as Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum causing e.g. viral-bacterial co-infections (TBE + LB/HGA), which pose diagnostic and therapeutic problems. Since these infections are usually accompanied by inflammation and oxidative stress causing metabolic modifications, including phospholipids, the aim of the study was to assess the level of polyunsaturated fatty acids and their metabolism (ROS- and enzyme-dependent) products in the blood plasma of patients with TBE and TBE + LB/HGA before and after pharmacotherapy. Methods: The total antioxidant status was determined using 2,20-azino-bis-3-ethylbenzothiazolin-6-sulfonic acid. The phospholipid and free fatty acids were analysed by gas chromatography. Lipid peroxidation was estimated by measuring small molecular weight reactive aldehyde, malondialdehyde and neuroprostanes. The reactive aldehyde was determined using gas chromatography coupled with mass spectrometry. The activity of enzymes was examined spectrophotometrically. An analysis of endocannabinoids and eicosanoids was performed using a Shimadzu UPLC system coupled with an electrospray ionization source to a Shimadzu 8060 Triple Quadrupole system. Receptor expression was measured using an enzyme-linked immunosorbent assay (ELISA). Results: The reduced antioxidant status as a result of infection was accompanied by a decrease in the level of phospholipid arachidonic acid (AA) and docosahexaenoic acid (DHA) in TBE, an increase in DHA in co-infection and in free DHA in TBE with an increase in the level of lipid peroxidation products. The enhanced activity of enzymes metabolizing phospholipids and free PUFAs increased the level of endocannabinoids and eicosanoids, while decreased 15-PGJ2 and PGE2 was accompanied by activation of granulocyte receptors before pharmacotherapy and only tending to normalize after treatment. Conclusion: Since classical pharmacotherapy does not prevent disorders of phospholipid metabolism, the need to support treatment with antioxidants may be suggested. [ABSTRACT FROM AUTHOR]

Based on the current situation of Korean culture and society, the population of companion animals in South Korea is growing rapidly along with zoonotic risks. The current data regarding zoonotic infections in companion dogs reported in Korea is sparse. This study aims to investigate the seroprevalence of seven potential zoonotic pathogens in companion dogs in South Korea: Anaplasma phagocytophilum, Borrelia burgdoferi, Ehrlichia canis, Coxiella burnetii, Brucella canis, Leptospira spp. and canine influenza A virus. A total of 284 serum samples were collected from 2018 to 2021, and the immunoglobulin G (IgG) antibodies against 7 zoonotic pathogens were detected using enzyme‐linked immunosorbent assays. Samples were divided into five groups and analysed based on age. IgG antibodies against six of the seven pathogens were detected. The highest seropositivity rate was detected for canine influenza A virus exposure (59.1%) for which the rates were the highest in dogs under 1 year old and declined with age. Positivity rates of the other pathogens were relatively low: 1.76% for Leptospira spp., 1.40% for A. phagocytophilum and E. canis, 1.06% for B. canis and 0.35% for B. burgdoferi. No antibodies against C. burnetii were detected in this study. The exposure of dogs in South Korea to six zoonotic pathogens was serologically confirmed, highlighting a potential risk for human infection. The zoonotic risk of companion dogs cannot be neglected, and implementation of One Health approach should be advocated to establish effective preventive measures. [ABSTRACT FROM AUTHOR]

A 11-month-old intact male Cavalier King Charles Spaniel (CKCS) dog was presented at the Teaching Hospital of National Chung Hsin University (NCHU-VMTH) due to thrombocytopenia without relevant clinical signs and no significant improvement after medication treatment. As per the complete blood profile, there was significant thrombocytopenia (platelet count 1– 3 1 × 1 0 3 / μ L) with mean platelet volume (MPV) ranging from 20.3 to 25.7 fl. Tests for Babesia canis, B. gibsoni, Ehrlichia canis and Anaplasma phagocytophilum yielded negative results. Despite treatment with antibiotics and immunosuppressive drugs, the dog's condition did not improve. The dog's platelet morphology and absence of symptoms raised the suspicion of inherited macrothrombocytopenia. Genomic DNA sequencing analysis of the β 1-tubulin gene revealed a single-point mutation, specifically c.745 G>A. This genetic analysis confirmed the presence of a β 1-tubulin mutation, marking the first reported case of such a mutation in a CKCS in Taiwan. Despite the diagnosis of this condition, both partial thromboplastin time (PTT) and active partial thromboplastin time (aPTT) were within normal ranges before neutering. When diagnosing the causes of thrombocytopenia in young patients, especially when there are no related clinical signs, it is crucial to consider prioritizing inherited macrothrombocytopenia, then thrombocytopenia secondary to parasite infection, or immune-mediated thrombocytopenia (IMT) to minimize unnecessary medication. [ABSTRACT FROM AUTHOR]

Background: A newly discovered zoonotic infection carried by ixodid ticks, Anaplasma capra, affects a wide variety of hosts, including numerous mammals. A. capra most likely infects erythrocytes or endothelial cells in mammals. This study aimed to investigate the A. capra pathogen in goats in Türkiye's Van province. Methods: A total of 200 goat blood samples were examined. Goat samples were subjected to partial amplification of the gltA gene fragment using a nested polymerase chain reaction. Results: A. capra DNA was detected in 0.5% of goat blood samples. Phylogenetic analysis of a partial gltA gene fragment showed that the Eastern Türkiye isolate, closely grouped with A. capra isolates reported from wild and domestic ruminants in France, Türkiye, and Kyrgyzstan, formed a distinct clade. Conclusions: This is the first report of A. capra in goats in Van province, Eastern Türkiye. [ABSTRACT FROM AUTHOR]

Anaplasmosis, a tick-borne disease with worldwide distribution, impacts ruminants, equines, carnivores, and humans. This study aimed to investigate Anaplasma phagocytophilum in horses from Ardabil province and Anaplasma ovis in small ruminants from East Azerbaijan province using the Nested Polymerase Chain Reaction (PCR) method. Blood samples were taken from the jugular vein of 100 healthy horses in the Ardabil province and 156 healthy sheep and goats (116 sheep and 40 goats) in the East Azerbaijan province during the spring and summer seasons of 2016 in northwest Iran. The collected blood samples were stored at -20°C until the molecular experiments were conducted. Nested PCR was employed to detect A. phagocytophilum in horses and A. ovis in small ruminants using extracted DNA and amplifying 16S rRNA and msp4 genes. The Chi-square test of independence was used to determine the relationship between Anaplasma spp., infection, and independent variables, including age, gender, animal species, and sampling location. None of the 100 samples collected from horses in the Ardabil province were positive for A. phagocytophilum. In the East Azerbaijan province, 11 out of the 156 (7.05%) blood samples collected from sheep and goats tested positive for A. ovis. In addition, A. ovis infection was not significantly related to the independent variables. Phylogenetic analysis showed that the sequence obtained in this study (MH790273) had 100% homology with the sequence obtained from sheep infected with Anaplasma in Ahvaz province (JQ621903.1). The findings of this study can contribute to the prevention and control of anaplasmosis in farm animals in northwestern Iran. [ABSTRACT FROM AUTHOR]

Objective: The objectives of this study were to determine the richness, abundance, and diversity of bacteria in stray dogs (Canis lupus familiaris) infested by ticks in Comarca Lagunera, northern Mexico, and to establish their pathogenic and or/zoonotic potential. Materials and Methods: Blood samples from 12 dogs were collected, and their deoxyribonucleic acid was extracted. The V3-V4 region of the 16S ribosomal ribunocleic acid gene was amplified by polymerase chain reaction. Next-generation sequencing (NGS) was performed on a MiSeq Illumina platform, and the data were analyzed using quantitative insights into microbial ecology. Results: The operational taxonomic units resulted in 23 phyla, 54 classes, 89 orders, 189 families, 586 genera, and 620 bacterial species; among them, 64 species and/or bacterial genera with pathogenic or zoonotic potential were identified, some of which have been reported in the literature as relevant to public health (Anaplasma phagocytophilum, Brucella spp., Clostridium spp., Corynebacterium affermentants, Cutibacterium spp., Dietzia spp., Ehrlichia canis, Fusobacterium necrophorum, Leptotrichia spp., Mycobacterium spp., Paracoccus spp., and Roseomonas gilardii). Conclusion: This research offers relevant information on the prevalence of tick-borne diseases as well as other potential zoonotic diseases in the blood of stray dogs parasitized by ticks in northern Mexico. New molecular biology and massive NGS techniques may play an important role in the study and documentation of bacterial profiles from animals in close proximity to humans. [ABSTRACT FROM AUTHOR]

Ixodes ricinus ticks are an important vector and reservoir of pathogenic microorganisms causing dangerous infectious diseases in humans and animals. The presence of ticks in urban greenery is a particularly important public health concern due to the potential for humans and companion animals to be exposed to tick-borne diseases there. The study assessed the prevalence of Borrelia burgdorferi and Anaplasma phagocytophilum infection in I. ricinus ticks feeding on dogs. The study consisted in analyses of I. ricinus ticks collected in 2018–2020 from owned and stray dogs in the north-eastern part of Lubelskie province (eastern Poland). An AmpliSens PCR kit was used for qualitative detection and differentiation of tick-borne infections. Infections of B. burgdorferi and A. phagocytophilum were detected in 10.9% and 12.9% of the examined ticks, respectively. One tick (0.7%) was co-infected by both pathogens. Infection with B. burgdorferi was significantly more highly prevalent in ticks collected from the owned dogs than from the strays (18.7% and 2.8%, respectively), whereas the prevalence of A. phagocytophilum was similar in both groups (12.0% and 13.9%, respectively). The co-infection observed in the study suggests the possibility of simultaneous infection by both pathogens from a single tick bite. The presence of pathogens in ticks collected from dogs is a factor in assessing infection risk not only to companion animals but also to their owners, who are in close contact with their dogs and visit the same green areas recreationally. [ABSTRACT FROM AUTHOR]

Background: Changing geographical and seasonal activity patterns of ticks may increase the risk of tick infestation and tick-borne pathogen (TBP) transmission for both humans and animals. Methods: To estimate TBP exposure of dogs and cats, 3000 female I. ricinus from these hosts were investigated for Anaplasma phagocytophilum and Borrelia species. Results: qPCR inhibition, which was observed for ticks of all engorgement stages but not questing ticks, was eliminated at a template volume of 2 µl. In ticks from dogs, A. phagocytophilum and Borrelia spp. prevalence amounted to 19.0% (285/1500) and 28.5% (427/1500), respectively, while ticks from cats showed significantly higher values of 30.9% (464/1500) and 55.1% (827/1500). Accordingly, the coinfection rate with both A. phagocytophilum and Borrelia spp. was significantly higher in ticks from cats (17.5%, 262/1500) than dogs (6.9%, 104/1500). Borrelia prevalence significantly decreased with increasing engorgement duration in ticks from both host species, whereas A. phagocytophilum prevalence decreased only in ticks from dogs. While A. phagocytophilum copy numbers in positive ticks did not change significantly over the time of engorgement, those of Borrelia decreased initially in dog ticks. In ticks from cats, copy numbers of neither A. phagocytophilum nor Borrelia spp. were affected by engorgement. Borrelia species differentiation was successful in 29.1% (365/1254) of qPCR-positive ticks. The most frequently detected species in ticks from dogs were B. afzelii (39.3% of successfully differentiated infections; 70/178), B. miyamotoi (16.3%; 29/178), and B. valaisiana (15.7%; 28/178), while B. afzelii (40.1%; 91/227), B. spielmanii (21.6%; 49/227), and B. miyamotoi (14.1%; 32/227) occurred most frequently in ticks from cats. Conclusions: The differences in pathogen prevalence and Borrelia species distribution between ticks collected from dogs and cats may result from differences in habitat overlap with TBP reservoir hosts. The declining prevalence of A. phagocytophilum with increasing engorgement duration, without a decrease in copy numbers, could indicate transmission to dogs over the time of attachment. The fact that this was not observed in ticks from cats may indicate less efficient transmission. In conclusion, the high prevalence of A. phagocytophilum and Borrelia spp. in ticks collected from dogs and cats underlines the need for effective acaricide tick control to protect both animals and humans from associated health risks. [ABSTRACT FROM AUTHOR]

Background: Haemaphysalis longicornis is drawing attentions for its geographic invasion, extending population, and emerging disease threat. However, there are still substantial gaps in our knowledge of viral composition in relation to genetic diversity of H. longicornis and ecological factors, which are important for us to understand interactions between virus and vector, as well as between vector and ecological elements. Results: We conducted the meta-transcriptomic sequencing of 136 pools of H. longicornis and identified 508 RNA viruses of 48 viral species, 22 of which have never been reported. Phylogenetic analysis of mitochondrion sequences divided the ticks into two genetic clades, each of which was geographically clustered and significantly associated with ecological factors, including altitude, precipitation, and normalized difference vegetation index. The two clades showed significant difference in virome diversity and shared about one fifth number of viral species that might have evolved to "generalists." Notably, Bandavirus dabieense, the pathogen of severe fever with thrombocytopenia syndrome was only detected in ticks of clade 1, and half number of clade 2-specific viruses were aquatic-animal-associated. Conclusions: These findings highlight that the virome diversity is shaped by internal genetic evolution and external ecological landscape of H. longicornis and provide the new foundation for promoting the studies on virus-vector-ecology interaction and eventually for evaluating the risk of H. longicornis for transmitting the viruses to humans and animals. 4querEumuJMtVvn4JLJ-5M Video Abstract [ABSTRACT FROM AUTHOR]

Expanding geographic distribution and increased populations of ticks has resulted in an upsurge of human-tick encounters in the United States (US), leading to an increase in tickborne disease reporting. Limited knowledge of the broadscale spatial range of tick species is heightened by a rapidly changing environment. Therefore, we partnered with the Forest Inventory and Analysis (FIA) program of the Forest Service, U.S. Department of Agriculture and used passive tick surveillance to better understand spatiotemporal variables associated with foresters encountering three tick species (Amblyomma americanum L., Dermacentor variabilis Say, and Ixodes scapularis L.) in the southeastern US. Eight years (2014–2021) of tick encounter data were used to fit environmental niche and generalized linear models to predict where and when ticks are likely to be encountered. Our results indicate temporal and environmental partitioning of the three species. Ixodes scapularis were more likely to be encountered in the autumn and winter seasons and associated with soil organic matter, vegetation indices, evapotranspiration, temperature, and gross primary productivity. By contrast, A. americanum and D. variabilis were more likely to be encountered in spring and summer seasons and associated with elevation, landcover, temperature, dead belowground biomass, vapor pressure, and precipitation. Regions in the southeast least suitable for encountering ticks included the Blue Ridge, Mississippi Alluvial Plain, and the Southern Florida Coastal Plain, whereas suitable regions included the Interior Plateau, Central Appalachians, Ozark Highlands, Boston Mountains, and the Ouachita Mountains. Spatial and temporal patterns of different tick species can inform outdoorsmen and the public on tick avoidance measures, reduce tick populations by managing suitable tick habitats, and monitoring areas with unsuitable tick habitat for potential missed encounters. Author summary: The study highlights the significance of passive tick surveillance data collected by Forest Inventory and Analysis crews in providing valuable information into the presence and distribution of ticks in the southeastern United States. We used ecological niche modeling and generalized linear models to assess the geographic regions and temporal periods associated with where and when ticks are likely to be encountered. In the region, ticks remain active throughout the year with their distribution influenced by climatic and topographical factors. Of interest, maximum temperature was a significant environmental variable for all three species suggesting that distributions may be altered as the climate warms. Elevation and landcover were important variables for both Amblyomma americanum and Dermacentor variabilis, whereas Ixodes scapularis populations were correlated with evapotranspiration, vegetation indices, and soil organic matter. The research also identified new tick occurrence records providing data in a region with minimal infrastructure for tick surveillance, but with many ticks and tick-borne diseases. Continued long-term passive surveillance with collaborations with academic and government partnerships will help monitor tick distribution changes resulting from landscape and temperature changes which affect public health risks. [ABSTRACT FROM AUTHOR]

Simple Summary: Bacteria continuously circulate in the natural environment and frequently cross into the human environment. While some of these bacteria can have a positive effect on human health, they are often accompanied by pathogenic forms. The aim of the present study was to more closely examine the zoonotic, tick-borne bacterium Anaplasma phagocytophilum, the causative agent of human and animal anaplasmosis. The study examined tissue samples from roe deer and red deer. Anaplasma phagocytophilum samples were genotyped based on DNA amplification, sequencing and phylogenetic analysis. Our results showed that detected bacteria from red deer belong to ecotype I and cluster I, which is why those bacteria are potentially pathogenic to humans. Detected bacteria from roe deer were mainly in ecotype II and clusters II and III, which means those bacteria are not pathogenic for humans. Background: The Gram-negative bacterium Anaplasma phagocytophilum is an intracellular pathogen and an etiological agent of human and animal anaplasmosis. Its natural reservoir comprises free-ranging ungulates, including roe deer (Capreolus capreolus) and red deer (Cervus elaphus). These two species of deer also constitute the largest group of game animals in Poland. The aim of the study was to genotype and perform a phylogenetic analysis of A. phagocytophilum strains from roe deer and red deer. Methods: Samples were subjected to PCR amplification, sequencing, and phylogenetic analysis of strain-specific genetic markers (groEL, ankA). Results: Five haplotypes of the groEL gene from A. phagocytophilum and seven haplotypes of ankA were obtained. The phylogenetic analysis classified the groEL into ecotypes I and II. Sequences of the ankA gene were classified into clusters I, II, and III. Conclusions: Strains of A. phagocytophilum from red deer were in the same ecotype and cluster as strains isolated from humans. Strains of A. phagocytophilum from roe deer represented ecotypes (I, II) and clusters (II, III) that were different from those isolated from red deer, and these strains did not show similarity to bacteria from humans. However, roe deer can harbor nonspecific strains of A. phagocytophilum more characteristic to red deer. It appears that the genetic variants from red deer can be pathogenic to humans, but the significance of the variants from roe deer requires more study. [ABSTRACT FROM AUTHOR]

Background: The blacklegged tick, Ixodes scapularis, transmits most vector-borne diseases in the US. It vectors seven pathogens of public health relevance, including the emerging human pathogen Anaplasma phagocytophilum. Nevertheless, it remains critically understudied compared to other arthropod vectors. Ixodes scapularis releases a variety of molecules that assist in the modulation of host responses. Recently, it was found that extracellular vesicles (EVs) carry several of these molecules and may impact microbial transmission to the mammalian host. EV biogenesis has been studied in mammalian systems and is relatively well understood, but the molecular players important for the formation and secretion of EVs in arthropods of public health relevance remain elusive. RabGTPases are among the major molecular players in mammalian EV biogenesis. They influence membrane identity and vesicle budding, uncoating, and motility. Methods: Using BLAST, an in silico pathway for EV biogenesis in ticks was re-constructed. We identified Rab27 for further study. EVs were collected from ISE6 tick cells after knocking down rab27 to examine its role in tick EV biogenesis. Ixodes scapularis nymphs were injected with small interfering RNAs to knock down rab27 and then fed on naïve and A. phagocytophilum-infected mice to explore the importance of rab27 in tick feeding and bacterial acquisition. Results: Our BLAST analysis identified several of the proteins involved in EV biogenesis in ticks, including Rab27. We show that silencing rab27 in I. scapularis impacts tick fitness. Additionally, ticks acquire less A. phagocytophilum after rab27 silencing. Experiments in the tick ISE6 cell line show that silencing of rab27 causes a distinct range profile of tick EVs, indicating that Rab27 is needed to regulate EV biogenesis. Conclusions: Rab27 is needed for successful tick feeding and may be important for acquiring A. phagocytophilum during a blood meal. Additionally, silencing rab27 in tick cells results in a shift of extracellular vesicle size. Overall, we have observed that Rab27 plays a key role in tick EV biogenesis and the tripartite interactions among the vector, the mammalian host, and a microbe it encounters. [ABSTRACT FROM AUTHOR]

Powassan virus is an emerging tick-borne pathogen capable of causing severe neuroinvasive disease. As the incidence of human Powassan virus grows both in magnitude and geographical range, the development of sensitive detection methods for diagnostics and surveillance is critical. In this study, a Taqman-based triplex real-time PCR assay was developed for the simultaneous and quantitative detection of Powassan virus and Powassan virus lineage II (deer tick virus) in Ixodes scapularis ticks. An exon–exon junction internal control was built-in to allow for accurate detection of RNA quality and the failure of RNA extraction. The newly developed assay was also applied to survey deer tick virus in tick populations at 13 sites on Cape Cod and Martha's Vineyard Island in Massachusetts. The assay's performance was compared with the Luminex xMAP MultiFLEX Vector-borne Panel 2. The results suggested that the real-time PCR method was more sensitive. Powassan virus infection rates among ticks collected from these highly endemic tick areas ranged from 0.0 to 10.4%, highlighting the fine-scale geographic variations in deer tick virus presence in this region. Looking forward, our PCR assay could be adopted in other Powassan virus surveillance systems. [ABSTRACT FROM AUTHOR]

Ticks are vectors for transmitting tick-borne pathogens (TBPs) in animals and humans. Therefore, tick identification is necessary to understand the distribution of tick species and the pathogens they carry. Unfortunately, data on dog ticks and the TBPs they harbor in Malawi are incomplete. This study aimed to identify dog ticks and the TBPs they transmit in Malawi. One hundred thirty-two ticks were collected from 87 apparently healthy but infested domestic dogs in four districts of Malawi, which were pooled into 128 tick samples. The ticks were morphologically identified under a stereomicroscope using identification keys, and species identification was authenticated by polymerase chain reaction (PCR) through the amplification and sequencing of 12S rRNA and cytochrome c oxidase subunit I (CO1) genes. The tick species identified were Rhipicephalus sanguineus sensu lato (58.3%), Haemaphysalis elliptica (32.6%), and Hyalomma truncatum (9.1%). Screening for TBPs using species-specific PCR assays revealed that 48.4% of the ticks were infected with at least one TBP. The TBP detection rates were 13.3% for Anaplasma platys, 10.2% for Babesia rossi, 8.6% for B. vogeli, 6.3% for Ehrlichia canis, 3.9% for A. phagocytophilum, 3.1% for B. gibsoni, 2.3% for B. canis and 0.8% for Hepatozoon canis. Co-infections of up to three pathogens were observed in 48.4% of the positive samples. This is the first study to identify dog ticks and the TBPs they harbor in Malawi. These findings provide the basis for understanding dog tick distribution and pathogens they carry in Malawi. This study necessitates the examination of ticks from more study locations to have a better picture of tick challenge, and the development of ticks and tick-borne disease control methods in Malawi. [ABSTRACT FROM AUTHOR]

Simple Summary: Tick-borne diseases are increasingly spreading due to global warming. Although the migratory abilities of ticks are limited, the spread of ticks between companion animals and humans has increased with the recent increase in the number of companion animals. For this reason, it is important to know the species of ticks that are parasitizing companion animals and whether they carry disease-causing organisms. Our research team collected ticks from pet trails and pet hospitals in the Daejeon area over a period of three years and confirmed the presence of pathogens through polymerase chain reaction (PCR) analysis. As a result of examining approximately 29,200 ticks, the dominant species was found to be H. longicornis, and the presence of four types of pathogens was confirmed. Through this study, we confirmed the possibility of tick-borne disease transmission between companion animals and humans and the emergence of a new tick (A. testudinarium) due to global warming in the Daejeon area. With the ongoing global warming-induced climate change, there has been a surge in vector-borne diseases, particularly tick-borne diseases (TBDs). As the population of companion animals grows, there is growing concern from a One Health perspective about the potential for these animals to spread TBDs. In this study, ticks were collected from companion animals and the surrounding environment in Daejeon Metropolitan City, Korea, using flagging and dragging, and CO2 trap methods. These ticks were then subjected to conventional (nested) PCR for severe fever with thrombocytopenia syndrome virus (SFTSV), Anaplasma spp., Ehrlichia spp., and Borrelia spp. We identified a total of 29,176 ticks, consisting of three genera and four species: H. longicornis, H. flava, I. nipponensis, and A. testudinarium. Notably, H. longicornis was the predominant species. The presence of A. testudinarium suggested that the species traditionally found in southern regions are migrating northward, likely as a result of climate change. Our PCR results confirmed the presence of all four pathogens in both companion animals and the surrounding environment, underscoring the potential for the indirect transmission of tick-borne pathogens to humans through companion animals. These findings emphasize the importance of the ongoing surveillance of companion animals in the management and control of TBDs. [ABSTRACT FROM AUTHOR]

Ixodid ticks are distributed across all countries of the Western Balkans, with a high diversity of species. Many of these species serve as vectors of pathogens of veterinary and medical importance. Given the scattered data from Western Balkan countries, we have conducted a comprehensive review of available literature, including some historical data, with the aim to compile information about all recorded tick species and associated zoonotic pathogens in this region. Based on the collected data, the tick fauna of the Western Balkans encompasses 32 tick species belonging to five genera: Ixodes, Haemaphysalis, Dermacentor, Rhipicephalus and Hyalomma. A range of pathogens responsible for human diseases has also been documented, including viruses, bacteria and parasites. In this review, we emphasize the necessity for integrated surveillance and reporting, urging authorities to foster research by providing financial support. Additionally, international and interdisciplinary collaborations should be encouraged that include the exchange of expertise, experiences and resources. The present collaborative effort can effectively address gaps in our knowledge of ticks and tick-borne diseases. [ABSTRACT FROM AUTHOR]

Alectorobius species are soft ticks primarily infesting birds, such as swallows, while Dermacentor species are hard ticks mainly infesting mammals, such as small ruminants. This study for the first time reported on the morphological and molecular bases of two tick species, namely A. coniceps and a Dermacentor sp. in Pakistan. The former species was examined in swallows' nests in Khyber Pakhtunkhwa province, while the latter species was examined in small ruminants in Balochistan province. In total, 25 ticks were collected, with 14 ticks morphologically identified as A. coniceps (males = 9 and females = 5) and 11 ticks identified as Dermacentor sp. (males = 7 and females = 4). Following morphological identification, molecular identification was gained by obtaining 16S rDNA and cox1 sequences for these ticks. The BLAST results for the 16S rDNA and cox1 sequences from A. coniceps shared a maximum identity of 97.46% and 96.49% with the same species from Malta. The BLAST analysis of the 16S rDNA and cox1 sequences from Dermacentor sp. showed maximum identities of 98.42% and 97.45% with Dermacentor pavlovskyi from China. The phylogenetic analysis based on 16S rDNA and cox1 of A. coniceps showed a close evolutionary relationship with the same species. The case of Dermacentor sp., based on 16S DNA and cox1, indicated a close evolutionary relationship with Dermacentor pavlovskyi from China. [ABSTRACT FROM AUTHOR]

Dermacentor reticulatus is tick species with an expanding geographical range in Europe, which creates the possibility of spreading microorganisms of significant veterinary and medical importance. The study aimed to investigate the prevalence and genetic diversity of Rickettsia spp., Babesia spp., Borrelia spp. and Anaplasma phagocytophilum in adult D. reticulatus ticks from the Eastern European population in the urban and the natural biotopes of north-eastern Poland. Microorganisms were detected by PCR and identified by DNA sequencing. The overall infection rate of at least one of the pathogens was 29.6%. The predominantly was Rickettsia spp. (27.1%) (with R. raoultii—9.1%) followed by Babesia spp. (2.4%) with B. canis (1.5%) as the most frequent. Based on 18S rRNA gene sequence, three B. canis genotypes were revealed. The prevalence of R. raoultii and B. canis was significantly higher in ticks from natural biotopes. The infection rates of B. afzelii and A. phagocytophilum were determined at 0.9% and 0.3%, respectively. Co-infections were detected in 3.8% of infected ticks. In diagnosing tick-borne diseases in humans, tick-borne lymphadenopathy should not be excluded. The prevalence of different genotypes of B. canis suggests differences in the clinical picture of canine babesiosis in the area. [ABSTRACT FROM AUTHOR]

The ability of ticks to adapt to different ecological zones, coupled with the spread of infectious pathogens negatively affects livestock production and thus, there is a need for better control strategies. However, control measures within a geographical region can only be effective if there is available information on tick population dynamics and ecology. This study focused on ticks infesting livestock in the Kassena-Nankana Districts of the Upper East Region of Ghana. The ticks were morphologically identified, variables such as season, animal host, and predilection sites were recorded, and the data were analyzed using STATA version 13. Out of 448 livestock examined, tick infestation in cattle was (78.60%), followed by sheep (25%) and goats (5.88%). A total of 1,550 ticks including nymphs (303) and adults (1,247) were collected. Adult ticks were found to be significantly associated with season (p < 0.001), with a high burden in the wet season. The nymph burden and body parts of livestock hosts were significantly associated with more nymphs collected from male animals than females (p < 0.001). Three genera of ticks, Amblyomma (62.97%), Hyalomma (18.71%), and Rhipicephalus (18.32%) were morphologically identified with the most predominant tick species recorded as Amblyomma variegatum (62.97%). Matured A. variegatum was sampled primarily in the wet season with their predilection site as the udder/scrotum (p < 0.001). However, adult Hyalomma truncatum was observed to have a significant association with the anal region (p < 0.001). Findings from this study are essential for formulating tick control measures to prevent the spread of infectious pathogens. [ABSTRACT FROM AUTHOR]

Background: Ixodid ticks, particularly Rhipicephalus sanguineus s.l., are important vectors of various disease-causing agents in dogs and humans in Cuba. However, our understading of interactions among tick-borne pathogens (TBPs) in infected dogs or the vector R. sanguineus s.l. remains limited. This study integrates microfluidic-based high-throughput real-time PCR data, Yule's Q statistic, and network analysis to elucidate pathogen-pathogen interactions in dogs and ticks in tropical western Cuba. Methods: A cross-sectional study involving 46 client-owned dogs was conducted. Blood samples were collected from these dogs, and ticks infesting the same dogs were morphologically and molecularly identified. Nucleic acids were extracted from both canine blood and tick samples. Microfluidic-based high-throughput real-time PCR was employed to detect 25 bacterial species, 10 parasite species, 6 bacterial genera, and 4 parasite taxa, as well as to confirm the identity of the collected ticks. Validation was performed through end-point PCR assays and DNA sequencing analysis. Yule's Q statistic and network analysis were used to analyse the associations between different TBP species based on binary presence-absence data. Results: The study revealed a high prevalence of TBPs in both dogs and R. sanguineus s.l., the only tick species found on the dogs. Hepatozoon canis and Ehrlichia canis were among the most common pathogens detected. Co-infections were observed, notably between E. canis and H. canis. Significant correlations were found between the presence of Anaplasma platys and H. canis in both dogs and ticks. A complex co-occurrence network among haemoparasite species was identified, highlighting potential facilitative and inhibitory roles. Notably, H. canis was found as a highly interconnected node, exhibiting significant positive associations with various taxa, including A. platys, and E. canis, suggesting facilitative interactions among these pathogens. Phylogenetic analysis showed genetic diversity in the detected TBPs. Conclusions: Overall, this research enhances our understanding of TBPs in Cuba, providing insights into their prevalence, associations, and genetic diversity, with implications for disease surveillance and management. [ABSTRACT FROM AUTHOR]

Background: Acanthamoeba is an environmental host for various microorganisms. Acanthamoeba is also becoming an increasingly important pathogen as a cause of keratitis. In Acanthamoeba keratitis (AK), coinfections involving pathogenic bacteria have been reported, potentially attributed to the carriage of microbes by Acanthamoeba. This study assessed the presence of intracellular bacteria in Acanthamoeba species recovered from domestic tap water and corneas of two different AK patients and examined the impact of naturally occurring intracellular bacteria within Acanthamoeba on the severity of corneal infections in rats. Methodology/Principal findings: Household water and corneal swabs were collected from AK patients. Acanthamoeba strains and genotypes were confirmed by sequencing. Acanthamoeba isolates were assessed for the presence of intracellular bacteria using sequencing, fluorescence in situ hybridization (FISH), and electron microscopy. The viability of the bacteria in Acanthamoeba was assessed by labelling with alkyne–functionalized D–alanine (alkDala). Primary human macrophages were used to compare the intracellular survival and replication of the endosymbiotic Pseudomonas aeruginosa and a wild type strain. Eyes of rats were challenged intrastromally with Acanthamoeba containing or devoid of P. aeruginosa and evaluated for the clinical response. Domestic water and corneal swabs were positive for Acanthamoeba. Both strains belonged to genotype T4F. One of the Acanthamoeba isolates harboured P. aeruginosa which was seen throughout the Acanthamoeba's cytoplasm. It was metabolically active and could be seen undergoing binary fission. This motile strain was able to replicate in macrophage to a greater degree than strain PAO1 (p<0.05). Inoculation of Acanthamoeba containing the intracellular P. aeruginosa in rats eyes resulted in a severe keratitis with increased neutrophil response. Acanthamoeba alone induced milder keratitis. Conclusions/Significance: Our findings indicate the presence of live intracellular bacteria in Acanthamoeba can increase the severity of acute keratitis in vivo. As P. aeruginosa is a common cause of keratitis, this may indicate the potential for these intracellular bacteria in Acanthamoeba to lead to severe polymicrobial keratitis. Author summary: In recent years, Acanthamoeba spp. have become an increasingly important human pathogen, causing serious, debilitating, and sometimes deadly infections. It can cause a rare but severe and potentially blinding corneal infection known as Acanthamoeba keratitis, which is extremely painful, difficult to diagnose, and treat. In a remarkable dual role, Acanthamoeba species act as phagocytic predator, consuming other microbes, but also as environmental hosts for a wide range of bacteria. With the increasing prevalence of coinfections among AK patients, which often lead to the severe outcomes, it is important to identify the intracellular bacteria hosted by Acanthamoeba spp. Using a series of experiments we confirmed the presence of intracellular P. aeruginosa in an Acanthamoeba strain isolated from AK patient domestic tap water. Our study findings showed that amoeba adapted P. aeruginosa exhibited enhanced intracellular survival and replication in human monocyte derived macrophages (hMDMs) compared to the wild–type strain PAO1. Here, for the first time, we investigated the role of naturally acquired viable intracellular P. aeruginosa in the development of Acanthamoeba keratitis in rats. The presence of intracellular bacteria in Acanthamoeba resulted into acute keratitis, highlighting the importance of identifying endosymbionts harbored by Acanthamoeba for accurate differential diagnostics and prognostic evaluations of Acanthamoeba keratitis. [ABSTRACT FROM AUTHOR]

Simply Summary: Anaplasmosis is a widely spread emerging tick-borne disease, generally transmitted by the Ixodes tick species. Two members of the Anaplasma genus—A. phagocytophilum (infecting neutrophils) and A. platys (infecting platelets)—cause granulocytic anaplasmosis and thrombocytopenia in some animals and humans. Our case report describes granulocytic anaplasmosis in two dogs routinely tested for blood donation. The blood smear was negative for tick-borne pathogens, and the haematology findings indicated thrombocytopenia in both dogs. The dogs were mildly positive in the ELISA tests for the detection of antibodies for Anaplasma spp. The qRT-PCR result was negative for A. platys but positive for A. phagocytophilum. In conclusion, the molecular test is mandatory for confirmation of Anaplasma spp. infection, and we suggest it should be implemented in all blood donors. This study describes granulocytic anaplasmosis in two dogs naturally infected with Anaplasma phagocytophilum. The 3-year-old dogs (male and female) came from the same household and were referred to the Faculty of Veterinary Medicine in Iasi for blood donation. They were subject to standard routine tests: haematology blood test, blood smear, and serological tests (VETSCAN® FLEX4 and IDEXX SNAP 4Dx Plus). The female dog had no medical problems, while the male dog experienced joint pain. The blood smear was negative for tick-borne pathogens, and the haematology findings indicated thrombocytopenia in both dogs, with the male dog also displaying eosinophilia. The two dogs were mildly positive in the ELISA tests for the detection of Anaplasma spp. antibodies; therefore, the blood samples were tested using the qRT-PCR method for Anaplasma platys and Anaplasma phagocytophilum. The qRT-PCR result was negative for A. platys, but it was positive for A. phagocytophilum. The treatment consisted of the administration of doxycycline for 28 days. In conclusion, the high number of cases with non-specific clinical signs, the different sensitivity and specificity of the immunochromatographic serological tests, as well as the possibility of confusing the morula during the cytological examination, make the molecular test mandatory for precise diagnosis. [ABSTRACT FROM AUTHOR]

Ticks carry and transmit disease pathogens that cause many human illnesses. Many of these diseases in humans are accompanied by a wide variety of symptoms making tick-borne illnesses often difficult to diagnose. The rate of reported tick-borne illnesses in humans has increased during the past decades in the United States, alongside a rise in concern for public health and safety. In northern Nebraska, the American dog tick (Dermacentor variabilis) is the primary tick encountered by humans and domestic animals in the region. This species is a known vector for spotted fever group (SFG) rickettsioses and tularemia (Francisella tularensis). Both bacteria are pathogenic in humans, and prevalence rates in tick populations can provide important information on possible public health implications for an area. Our study surveyed ticks in Brown County, Nebraska, where we collected ticks from May through June in 2022. Multiplex and singleplex PCR analyses were conducted on DNA samples from individuals to screen for the six most common tick-borne pathogens in the central United States: SFG rickettsiae, Borrelia burgdorferi, F. tularensis, Ehrlichia chaffeensis, Ehrlichia ewingii, and Anaplasma phagocytophilum. We collected 291 American dog ticks during collections in Brown County, consisting of 129 males and 162 females. Five individuals tested positive for SFG rickettsiae, and 15 tested positive for tularemia. This research brings attention to possible concerns of tick-borne illness in north-central Nebraska, particularly transmission of F. tularensis from tick bites. Tularemia can be fatal, but early detection and treatment with antibiotics improves patient prognosis. Information on rates of pathogen prevalence in ticks can lead to more accurate and rapid diagnoses by healthcare providers and better educate citizens about risks of contracting tick-borne diseases while conducting outdoor activities. [ABSTRACT FROM AUTHOR]

Tick-borne viral diseases have become an increasingly important public health concern. Tamdy virus (TAMV) is a tick-borne virus of the genus Orthonairovirus in the family Nairoviridae. While some studies have suggested that TAMV is a pathogen associated with human febrile illness, its epidemiology and the risk of TAMV spill-over remain poorly understood. Ticks were collected in Xinjiang, China, and grouped into pools. RT-PCR assays were used to detect TAMV RNA in these pools. The seroprevalence of TAMV was investigated using Immunofluorescence assays, Western blotting, and Luciferase immunoprecipitation system (LIPS) assays. TAMV RNA was detected in 17 out of 363 tick pools, resulting in a minimum infection rate (MIR) of 4.7%. Hyalomma asiaticum and Dermacentor nuttalli were identified as major tick vectors of TAMV. Phylogenetic analysis demonstrated that TAMV strains from Xinjiang are closely related to strains from other countries. Seroprevalence studies showed that TAMV exposure has been occurring in Xinjiang since at least 2006. Antibody responses to TAMV were detected in 1.1% (26/2296) of animals, including domestic animals and wild rodents. The seropositivity rates were as follows: sheep (1.7%), dog (2.3%), Marmota monax (0.8%), Meriones meridianus (3.5%). The research findings reveal that TAMV can be transmitted by ticks to various animal species, posing a significant public health risk. The wide distribution of TAMV and its tick vectors emphasise the importance of early preparedness and control measures. This study highlights the necessity for maintaining vigilance in addressing emerging zoonotic diseases transmitted by ticks. [ABSTRACT FROM AUTHOR]

Tick‐associated viruses remain a substantial zoonotic risk worldwide, so knowledge of the diversity of tick viruses has potential health consequences. Despite their importance, large amounts of sequences in public data sets from tick meta‐genomic and ‐transcriptomic projects remain unannotated, sequence data that could contain undocumented viruses. Through data mining and bioinformatic analysis of more than 37,800 public meta‐genomic and ‐transcriptomic data sets, we found 83 unannotated contigs exhibiting high identity with known tick viruses. These putative viral contigs were classified into three RNA viral families (Alphatetraviridae, Orthomyxoviridae and Chuviridae) and one DNA viral family (Asfarviridae). After manual checking of quality and dissimilarity towards other sequences in the data set, these 83 contigs were reduced to five contigs in the Alphatetraviridae from four putative viruses, four in the Orthomyxoviridae from two putative viruses and one in the Chuviridae which clustered with known tick‐associated viruses, forming a separate clade within the viral families. We further attempted to assess which previously known tick viruses likely represent zoonotic risks and thus deserve further investigation. We ranked the human infection potential of 133 known tick‐associated viruses using a genome composition‐based machine learning model. We found five high‐risk tick‐associated viruses (Langat virus, Lonestar tick chuvirus 1, Grotenhout virus, Taggert virus and Johnston Atoll virus) that have not been known to infect human and two viral families (Nairoviridae and Phenuiviridae) that contain a large proportion of potential zoonotic tick‐associated viruses. This adds to the knowledge of tick virus diversity and highlights the importance of surveillance of newly emerging tick‐associated diseases. [ABSTRACT FROM AUTHOR]

Background: Ticks are parasitic organisms that can transmit a wide range of pathogenic microorganisms. They are the second most common vectors of diseases affecting humans and animals. The occurrence and transmission of tick-borne diseases has recently shown increasing or fluctuating trends. Methods: DNA was extracted after the collection of tick samples. A library targeting the V4 hypervariable region of the 16S rRNA gene was constructed. After quality control, next-generation sequencing was performed with the Illumina NextSeq platform to analyze microbial diversity within the ticks. Results: Samples were gathered between May 2023 and July 2023. A total of 77 ticks from five distinct regions were selected for next-generation sequencing. Molecular identification confirmed that all sequenced samples belonged to Haemaphysalis longicornis. The most abundant bacteria belonged to the phylum Proteobacteria, which was present in all samples. Variations in sample richness and evenness were observed among sampling sites (Shannon index, P = 0.019). The bacterial diversity in LT exhibited the highest value, with an average of 2.449. Rickettsia and Coxiella were the predominant bacterial species, both of which are classified as tick-borne pathogens. The linear discriminant analysis effect size revealed significant differences in microbial composition among groups, except for the PC and LY groups, and identified distinct biomarkers for each group. Conclusions: Our findings indicated the high relative abundance of both pathogenic bacteria and non-pathogenic endosymbionts in H. longicornis and the potential for pathogen transmission to residents. However, further validation through human case studies is necessary. Health care providers should be aware of the possibility of the occurrence of these diseases. [ABSTRACT FROM AUTHOR]

Objectives: "Q fever," which is caused by Coxiella burnetii, is endemic in India. In addition to livestock, rodents have also been reported to be associated with enzootic maintenance, favoring pathogen transmission. Currently, however, no data are available on the role of rodents in "Q fever" transmission in India. Methods: A cross-sectional study was undertaken in 39 Puducherry villages to screen Coxiella burnetii in synanthropic rodents (rats and shrews) and their ectoparasites (ticks, mites, and fleas) by real-time and conventional PCR protocols targeting the pathogen specific IS 1111 and com 1 genes. Results: One hundred forty animals were trapped (107 shrews and 33 rats). The ticks, mites, and fleas infesting the rodents were identified as Rhipicephalus sanguineus, Leptotrombidium deliense and Schoengastiella spp., and Xenopsylla cheopis, respectively. PCR screening of the DNA extracted from the rodent/shrew blood samples and their ectoparasites tested negative for C. burnetii. Conclusions: Synanthropic rodents, such as rodents/shrews and their ectoparasites do not have a pivotal part in the enzootic maintenance and spread of Q fever to humans and livestock in Puducherry. [ABSTRACT FROM AUTHOR]

Objective: Distribution of the bacterium flora in ticks has an essential role in mapping and preventing local tick-borne diseases. The purpose of the current study was to map the bacterium flora and identify the main pathogens in ticks in grazing areas of Yunnan province. Methods: The V3-V4 region of 16S rRNA amplifier sequencing was used to analyze the tick-borne pathogens in Rh. Microplus. A prevalence survey on B. burgdorferi s.l., B. miyamotoi, E. chaffeensis, A. phagocytophilum, Coxiella burnetiid, and the spotted fever group rickettsiae was carried out using PCR. Phylogenetic analysis was used to identify and cluster the main prevalent microbe amplicons. Results: One hundred five genera and 117 species were detected in 50 ticks. Rickettsia, Anaplasma, Borrelia, Coxiella, and Ehrlichia were identified in tick samples by high-throughput sequencing at the genus level. Pathogen prevalence testing showed that the ticks were positive for B. burgdorferi s.l. (9/50 [18%]), A. phagocytophilum (1/50 [2%]), and the spotted fever group rickettsiae (35/50 [70%]), which were closely related to Candidatus R. jiangxinensis. Conclusion: The spotted fever group rickettsiae was the dominant pathogens in Xishuangbanna. Dual co-infection (1/50 [14%]) with Candidatus R. jiangxinensis and Borrelia garinii was demonstrated. V3-V4 high-throughput sequencing of the 16S rRNA gene was not sensitive to identify species for some bacteria, so more accurate and comprehensive analysis is necessary. [ABSTRACT FROM AUTHOR]

Ticks are among the most important group of vectors for pathogens causing vector-borne diseases in Europe. Over the last two decades, numerous reports strongly emphasize on the spreading and increased abundance of ticks in Romania. Moreover, molecular reports have also documented on the diverse tick-borne pathogens circulating at the domestic and wild host-tick interface. Among them, (re)-emerging tick-borne pathogens, including causative agents of zoonotic diseases, with high impact on the animal and human health are reported. Anaplasma phagocytophilum, the causative agent of granulocytic anaplasmosis in humans and animals is also regarded as an emerging tick-borne pathogen with a growing concern. Little is known about its occurrence in domestic animals in Romania. Therefore, the present study aimed on molecular testing for A. phagocytophilum infection in cattle and horses, Romania. For this, blood samples were tested by using a Real-Time PCR technique. The results were interpreted based on the cut-off cycle threshold of Ct<45. Following the amplification protocol, the methods was validated (the positive controls showed the average Ct=27.4285); of the tested samples, for two cattle amplification curves with the Ct<45 were registered, while one equine sample was inconclusive. These preliminary molecular data document the presence of the investigated pathogen. Further studies are planned to asses the prevalence and genetic characterization of A. phagocytophilum variants circulating in domestic animals, in Romania. [ABSTRACT FROM AUTHOR]

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Abstract Background The zoonotic intracellular alpha-proteobacterium Anaplasma phagocytophilum is a tick-transmitted pathogen. The associations between vertebrate reservoirs and vectors are described as wide-ranging, and it was previously shown that the pathogenicity of A. phagocytophilum differs depending on the combination of pathogen variant and infected host species. This leads to the question of whether there are variations in particular gene loci associated with different virulence. Therefore, this study aims at clarifying existing host-variant combinations and detecting possible reservoir hosts. To understand these interactions, a complex toolset for molecular epidemiology, phylogeny and network theory was applied. Methods Sequences of up to four gene loci (msp4, msp2, groEL and 16S rRNA) were evaluated for different isolates from variable host species, including, for example, dogs, cattle and deer. Variant typing was conducted for each gene locus individually, and combinations of different gene loci were analysed to gain more detailed information about the genetic plasticity of A. phagocytophilum. Results were displayed as minimum spanning nets and correlation nets. Results The highest diversity of variants for all gene loci was observed in roe deer. In cattle, a reduced number of variants for 16S rRNA [only 16S-20(W) and 16S-22(Y)] but multiple variants of msp4 and groEL were found. For dogs, two msp4 variants [m4-20 and m4-2(B/C)] were found to be linked to different variants of the other three gene loci, creating two main combinations of gene loci variants. Cattle are placed centrally in the minimum spanning net analyses, indicating a crucial role in the transmission cycles by possibly bridging the vector-wildlife cycle to infections of humans and domestic animals. The minimum spanning nets confirmed previously described epidemiological cycles of the bacterium in Europe, showing separation of variants originating from wildlife animals only and a set of variants shared by wild and domestic animals. Conclusions In this comprehensive study of 1280 sequences, we found a high number of gene variants only occurring in specific hosts. Additionally, different hosts show unique but also shared variant combinations. The use of our four gene loci expand the knowledge of host–pathogen interactions and may be a starting point to predict future spread and infection risks of A. phagocytophilum in Europe. Graphical abstract

Abstract Background Vector-borne pathogens (VBPs) are a major threat to humans, livestock and companion animals worldwide. The combined effect of climatic, socioeconomic and host composition changes favours the spread of the vectors, together with the expansion of invasive carnivores contributing to the spread of the pathogens. In Europe, the most widespread invasive species of carnivores are raccoons (Procyon lotor) and raccoon dogs (Nyctereutes procyonoides). This study focused on the detection of four major groups of VBPs namely Babesia, Hepatozoon, Anaplasma phagocytophilum and Bartonella in invasive and native carnivores in the Czech Republic, with the emphasis on the role of invasive carnivores in the eco-epidemiology of said VBPs. Methods Spleen samples of 84 carnivores of eight species (Canis aureus, Canis lupus, Lynx lynx, P. lotor, Martes foina, Lutra lutra, Mustela erminea and N. procyonoides) were screened by combined nested PCR and sequencing for the above-mentioned VBPs targeting 18S rRNA and cytB in hemoprotozoa, groEL in A. phagocytophilum, and using multilocus genotyping in Bartonella spp. The species determination is supported by phylogenetic analysis inferred by the maximum likelihood method. Results Out of 84 samples, 44% tested positive for at least one pathogen. Five different species of VBPs were detected in P. lotor, namely Bartonella canis, Hepatozoon canis, Hepatozoon martis, A. phagocytophilum and Bartonella sp. related to Bartonella washoensis. All C. lupus tested positive for H. canis and one for B. canis. Three VBPs (Hepatozoon silvestris, A. phagocytophilum and Bartonella taylorii) were detected in L. lynx for the first time. Babesia vulpes and yet undescribed species of Babesia, not previously detected in Europe, were found in N. procyonoides. Conclusions Wild carnivores in the Czech Republic are hosts of several VBPs with potential veterinary and public health risks. Among the studied carnivore species, the invasive raccoon is the most competent host. Raccoons are the only species in our study where all the major groups of studied pathogens were detected. None of the detected pathogen species were previously detected in these carnivores in North America, suggesting that raccoons adapted to local VBPs rather than introduced new ones. Babesia vulpes and one new, probably imported species of Babesia, were found in raccoon dogs. Graphical abstract

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.

Multiple ticks (Acari: Ixodoidea) carrying Rickettsiales bacteria have significant importance for both human and animal health. Thus, the purpose of this work was to genetically analyze tick species and their associated Rickettsiales bacteria in animal hosts. In order to achieve these objectives, various animals (including camels, cattle, goats, sheep, dogs, and mice) were inspected in four districts (Mardan, Peshawar, Kohat, and Karak) of Khyber Pakhtunkhwa to collect ticks, while blood samples were collected from all the symptomatic and asymptomatic cattle in all four districts. A total of 234 ticks were obtained from 86 out of 143 (60.14%) host animals, which were morphologically identified as Rhipicephalus turanicus, Rhipicephalus microplus, Haemaphysalis cornupunctata, and Hyalomma asiaticum. Among these, their representative ticks (126/234, 53.85%) were processed for molecular confirmation using cytochrome c oxidase (cox1) gene. Obtained cox1 sequences of four different tick species showed 99.72%–100% maximum identity with their corresponding species reported from Pakistan, China, India, and Kazakhstan and clustered phylogenetically. This study presented the first genetic report of Hy. asiaticum ticks in Pakistan. Moreover, genetically confirmed tick species were molecularly analyzed by PCR for detection of Rickettsiales DNA using partial fragments of 16S rDNA, 190-kDa outer membrane protein A (ompA), and 120-kDa outer membrane protein B (ompB) genes. In addition, blood samples were analyzed to identify Rickettsiales bacteria using the aforementioned genes. Rickettsiales bacteria were found in 24/126 (19.05%) ticks and 4/16 (25.00%) in symptomatic cattle’s blood. The obtained ompA and ompB sequences from Hy. asiaticum ticks showed 99.73%–99.87% with Candidatus Rickettsia shennongii and unidentified Rickettsia sp., whereas the obtained 16S rDNA sequences from cattle’s blood and ticks (Hae. cornupunctata) showed 99.67% highest identity with Anaplasma phagocytophilum. The 16S rDNA sequence of Rickettsiales DNA from Rh. turanicus ticks showed 100% identity with Ehrlichia canis and unidentified Ehrlichia sp. Obtained sequences of Rickettsiales bacteria were grouped along with their respective species in phylogenetic trees, which were previously reported in Greece, Cuba, Iraq, Turkey, Pakistan, South Korea, and China (mainland and Taiwan). This extensive study explores the wide range of damaging ticks and their corresponding tick-borne bacteria in the area, suggesting a possible danger to both livestock and human communities.