Anaplasma phagocytophilum in urban and peri-urban passerine birds in Ile-de-France.

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.

Italian peninsula as a hybridization zone of Ixodes inopinatus and I. ricinus and the prevalence of tick-borne pathogens in I. inopinatus, I. ricinus, and their hybrids.

BACKGROUND: Ixodes inopinatus was described from Spain on the basis of morphology and partial sequencing of 16S ribosomal DNA. However, several studies suggested that morphological differences between I. inopinatus and Ixodes ricinus are minimal and that 16S rDNA lacks the power to distinguish the two species. Furthermore, nuclear and mitochondrial markers indicated evidence of hybridization between I. inopinatus and I. ricinus. In this study, we tested our hypothesis on tick dispersal from North Africa to Southern Europe and determined the prevalence of selected tick-borne pathogens (TBPs) in I. inopinatus, I. ricinus, and their hybrids. METHODS: Ticks were collected in Italy and Algeria by flagging, identified by sequencing of partial TROSPA and COI genes, and screened for Borrelia burgdorferi s.l., B. miyamotoi, Rickettsia spp., and Anaplasma phagocytophilum by polymerase chain reaction and sequencing of specific markers. RESULTS: Out of the 380 ticks, in Italy, 92 were I. ricinus, 3 were I. inopinatus, and 136 were hybrids of the two species. All 149 ticks from Algeria were I. inopinatus. Overall, 60% of ticks were positive for at least one TBP. Borrelia burgdorferi s.l. was detected in 19.5% of ticks, and it was significantly more prevalent in Ixodes ticks from Algeria than in ticks from Italy. Prevalence of Rickettsia spotted fever group (SFG) was 51.1%, with significantly greater prevalence in ticks from Algeria than in ticks from Italy. Borrelia miyamotoi and A. phagocytophilum were detected in low prevalence (0.9% and 5.2%, respectively) and only in ticks from Italy. CONCLUSIONS: This study indicates that I. inopinatus is a dominant species in Algeria, while I. ricinus and hybrids were common in Italy. The higher prevalence of B. burgdorferi s.l. and Rickettsia SFG in I. inopinatus compared with that in I. ricinus might be due to geographical and ecological differences between these two tick species. The role of I. inopinatus in the epidemiology of TBPs needs further investigation in the Mediterranean Basin.

Multi-locus sequence analysis of Anaplasma bovis in goats and ticks from Thailand, with the initial identification of an uncultured Anaplasma species closely related to Anaplasma phagocytophilum-like 1.

Ticks and tick-borne pathogens (TTBP) pose a serious threat to animal and human health globally. Anaplasma bovis, an obligatory intracellular bacterium, is one of the more recent species of the Family Anaplasmaceae to be formally described. Owing to its diminutive size, microscopic detection presents a formidable challenge, leading to it being overlooked in laboratory settings lacking advanced equipment or resources, as observed in various regions, including Thailand. This study aimed to undertake a genetic analysis of A. bovis and determine its prevalence in goats and ticks utilizing three genetic markers (16S rRNA, gltA, groEL). A total of 601 goat blood and 118 tick samples were collected from 12 sampling sites throughout Thailand. Two tick species, Haemaphysalis bispinosa (n = 109), and Rhipicephalus microplus (n = 9) were identified. The results herein showed that 13.8 % (83/601) of goats at several farms and 5 % (1/20) of ticks were infected with A. bovis. Among infected ticks, A. bovis and an uncultured Anaplasma sp. which are closely related to A. phagocytophilum-like 1, were detected in each of H. bispinosa ticks. The remaining R. microplus ticks tested positive for the Anaplasma genus. A nucleotide sequence type network showed that A. bovis originated from Nan and Narathiwat were positioned within the same cluster and closely related to China isolates. This observation suggests the potential dispersal of A. bovis over considerable distances, likely facilitated by activities such as live animal trade or the transportation of infected ticks via migratory birds. The authors believe that the findings from this study will provide valuable information about TTBP in animals.

Prevalence of tick-borne pathogens in ticks collected from the wild mountain ungulates mouflon and chamois in 4 regions of France.

Ticks are major vectors of various pathogens of health importance, such as bacteria, viruses and parasites. The problems associated with ticks and vector-borne pathogens are increasing in mountain areas, particularly in connection with global climate change. We collected ticks (n = 2,081) from chamois and mouflon in 4 mountainous areas of France. We identified 6 tick species: Ixodes ricinus, Rhipicephalus bursa, Rh. sanguineus s.l., Haemaphysalis sulcata, H. punctata and Dermacentor marginatus. We observed a strong variation in tick species composition among the study sites, linked in particular to the climate of the sites. We then analysed 791 ticks for DNA of vector-borne pathogens: Babesia/Theileria spp., Borrelia burgdorferi s.l., Anaplasma phagocytophilum, A. marginale, A. ovis, and Rickettsia of the spotted fever group (SFG). Theileria ovis was detected only in Corsica in Rh. bursa. Babesia venatorum (2 sites), Borrelia burgdorferi s.l. (B. afzelii and B. garinii; 2 sites) and Anaplasma phagocytophilum (3 sites) were detected in I. ricinus. Anaplasma ovis was detected at one site in I. ricinus and Rh. sanguineus s.l. SFG Rickettsia were detected at all the study sites: R. monacensis and R. helvetica in I. ricinus at the 3 sites where this tick is present; R. massiliae in Rh. sanguineus s.l. (1 site); and R. hoogstraalii and Candidatus R. barbariae in Rh. bursa in Corsica. These results show that there is a risk of tick-borne diseases for humans and domestic and wild animals frequenting these mountain areas.

Title: Prévalence d'agents pathogènes vectorisés chez des tiques collectées chez des ongulés sauvages (mouflons, chamois) dans 4 zones montagneuses en France. Abstract: Les tiques sont des vecteurs majeurs de différents agents pathogènes d'importance sanitaire, tels que des bactéries, des virus et des parasites. Les problématiques liées aux tiques et aux pathogènes vectorisés augmentent en zones de montagne, en lien notamment avec le réchauffement climatique. Nous avons collecté des tiques (n = 2 081) sur des chamois et des mouflons dans 4 zones montagneuses en France. Six espèces ont été identifiées : Ixodes ricinus, Rhipicephalus bursa, Rh. sanguineus s.l., Haemaphysalis sulcata, H. punctata et Dermacentor marginatus. Nous avons observé une forte variation de la composition en espèces de tiques entre les sites d'étude, en lien notamment avec le climat des sites. Nous avons ensuite recherché les ADN d'agents pathogènes vectorisés sur 791 tiques : Babesia/Theileria spp, Borrelia burgdorferi s.l., Anaplasma phagocytophilum, A. marginale, A. ovis, et de Rickettsia du groupe des fièvres boutonneuses (SFG). Theileria ovis a été détecté uniquement en Corse chez Rh. bursa. Babesia venatorum (2 sites), Borrelia burgdorferi s.l. (B. afzelii and B. garinii; 2 sites) et Anaplasma phagocytophilum (3 sites) ont été détectés chez I. ricinus. Anaplasma ovis a été détecté dans un site chez I. ricinus et Rh. sanguineus s.l.. Les Rickettsia SFG ont été détectées dans tous les sites d'étude : Rickettsia monacensis et R. helvetica chez I. ricinus dans les 3 sites où cette tique est présente; R. massiliae chez Rh. sanguineus s.l. (1 site); et R. hoogstraalii et Candidatus R. barbariae chez Rh. bursa en Corse. Ces résultats montrent un risque de transmission de maladies par les tiques pour les personnes et les animaux domestiques et sauvages fréquentant ces zones de montagne.

Antibody-blocking of a tick transporter impairs Anaplasma phagocytophilum colonization in Haemaphysalis longicornis ticks.

The invasive Asian longhorned tick Haemaphysalis longicornis that vectors and transmits several animal pathogens is significantly expanding in the United States. Recent studies report that these ticks also harbor human pathogens including Borrelia burgdorferi sensu lato, Babesia microti, and Anaplasma phagocytophilum. Therefore, studies that address the interactions of these ticks with human pathogens are important. In this study, we report the characterization of H. longicornis organic anion-transporting polypeptides (OATPs) in interactions of these ticks with A. phagocytophilum. Using OATP-signature sequence, we identified six OATPs in the H. longicornis genome. Bioinformatic analysis revealed that H. longicornis OATPs are closer to other tick orthologs rather than to mammalian counterparts. Quantitative real-time PCR analysis revealed that OATPs are highly expressed in immature stages when compared to mature stages of these ticks. In addition, we noted that the presence of A. phagocytophilum upregulates a specific OATP in these ticks. We also noted that exogenous treatment of H. longicornis with xanthurenic acid, a tryptophan metabolite, influenced OATP expression in these ticks. Immunoblotting analysis revealed that antibody generated against Ixodes scapularis OATP cross-reacted with H. longicornis OATP. Furthermore, treatment of H. longicornis with OATP antibody impaired colonization of A. phagocytophilum in these ticks. These results not only provide evidence that the OATP-tryptophan pathway is important for A. phagocytophilum survival in H. longicornis ticks but also indicate OATP as a promising candidate for the development of a universal anti-tick vaccine to target this bacterium and perhaps other rickettsial pathogens of medical importance.

Tick-borne encephalitis virus transmitted singly and in duo with Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum bacteria by ticks as pathogens modifying lipid metabolism in human blood.

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.

Tick hemocytes have a pleiotropic role in microbial infection and arthropod fitness.

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.

A case of transfusion-transmission Anaplasma phagocytophilum from leukoreduced red blood cells.

BACKGROUND: Anaplasma phagocytophilum is a tick-borne bacterium and the cause of human granulocytic anaplasmosis (HGA). Here, we report a case of transfusion-transmitted (TT)-HGA involving a leukoreduced (LR) red blood cell (RBC) unit. CASE REPORT: A 64-year-old woman with gastric adenocarcinoma and multiple myeloma who received weekly blood transfusions developed persistent fevers, hypotension, and shortness of breath 1 week after receiving an RBC transfusion. Persistent fevers, new thrombocytopenia, and transaminitis suggested a tick-borne infection. RESULTS: The absence of blood parasites on thick and thin blood smears suggested that malaria and Babesia infection were not present, and the recipient tested negative for antibodies to Borrelia burgdorferi. Blood testing by polymerase chain reaction (PCR) for Ehrlichia and Anaplasma species identified A. phagocytophilum. Treatment with doxycycline resolved the infection; however, the recipient expired due to complications of her known malignancies. The recipient lived in a nursing home and did not have pets or spend time outdoors. The donor was a female in her 70s from Maine who was diagnosed with HGA 3 weeks after donating blood and whose LR-RBCs from the donation were transfused to the recipient 9 days following collection. CONCLUSION: This is a confirmed case of TT-HGA. Although rare, TT-HGA has been reported with LR-RBCs and platelets. In endemic areas, testing for tick-borne associated infections should be considered when investigating post-transfusion complications.

Molecular investigation of Anaplasma phagocytophilum and related strains among sheep flocks from different parts of Türkiye; with a note of phylogenetic analyses of Anaplasma phagocytophilum- like 1.

Anaplasma phagocytophilum is a vector-borne zoonotic pathogen and can infect various vertebrate hosts, especially cattle, sheep, goats, horses, and dogs. Molecular-based studies have revealed that the agent has a high genetic diversity and closely related strains circulate in hosts. In this study, 618 sheep blood samples obtained from different geographic regions of Türkiye were researched for A.phagocytophilum and related strains with PCR, RFLP, and DNA sequence analyses. The DNA of these pathogens was detected in 110 (17.79%) samples. RFLP assay showed that all positive samples were infected with A.phagocytophilum-like 1, whereas A.phagocytophilum-like 2 and A.phagocytophilum were not detected. Partial parts of 16 S rRNA gene of seven randomly selected positive samples were sequenced. The phylogenetic analyses of these isolates revealed that at least two A.phagocytophilum-like 1 isolates circulate among hosts in Türkiye and around the world. A.phagocytophilum-related strains have been reported in molecular-based studies over the last few years, but there is a lack of data on the vector competence, epidemiology, clinical symptoms, and genetic diversity of these pathogens. Therefore, large-scale molecular studies are still needed to obtain detailed data on the above-mentioned topics.

Anaplasma phagocytophilum infection associated with strong inflammatory response in 3 cats.

Anaplasmosis is a vector-borne disease caused by Anaplasma (A.) spp. which currently is still rarely diagnosed in cats. This article describes 3 independent cases of anaplasmosis in cats from different regions of Germany presented to veterinarians in 2021. All cats showed unspecific clinical signs, such as fever, reduced general condition, and decreased appetite. One cat additionally had generalized limb pain, another showed reluctance to move as well as vomiting. On complete blood cell count, only 1 of 3 cats showed mild thrombocytopenia. A. phagocytophilum was detected in blood samples of all 3 cats by polymerase chain reaction. Additionally, in 2 cats (in which blood smears were evaluated) morulae could be detected within neutrophilic granulocytes. Initially, all 3 cats had highly elevated serum amyloid A (SAA) concentrations. Treatment with doxycycline caused a rapid improvement of clinical signs, followed by a decrease of SAA concentrations to normal levels as well as negative PCR results after a treatment duration of at least 28 days. In cats with fever, otherwise unspecific clinical signs with only mild or no hematological changes, elevated SAA concentrations, and previous exposure to ticks, attending veterinarians should consider anaplasmosis as differential diagnosis.

Genetic manipulation of an Ixodes scapularis cell line.

Although genetic manipulation is one of the hallmarks of model organisms, its applicability to non-model species has remained difficult due to our limited understanding of their fundamental biology. For instance, manipulation of a cell line originated from the black-legged tick Ixodes scapularis, an arthropod that serves as a vector for several human pathogens, has yet to be established. Here, we demonstrate the successful genetic modification of the commonly used tick ISE6 line through ectopic expression and clustered regularly interspaced palindromic repeats [(CRISPR)/CRISPR-associated protein 9 (Cas9)] genome editing. We performed ectopic expression using nucleofection and attained CRISPR-Cas9 editing via homology-dependent recombination. Targeting the E3 ubiquitin ligase x-linked inhibitor of apoptosis (xiap) and its substrate p47 led to an alteration in molecular signaling within the immune deficiency network and increased infection of the rickettsial agent Anaplasma phagocytophilum in I. scapularis ISE6 cells. Collectively, our findings complement techniques for the genetic engineering of I. scapularis ticks, which currently limit efficient and scalable molecular genetic screens in vivo.IMPORTANCEGenetic engineering in arachnids has lagged compared to insects, largely because of substantial differences in their biology. This study unveils the implementation of ectopic expression and CRISPR-Cas9 gene editing in a tick cell line. We introduced fluorescently tagged proteins in ISE6 cells and edited its genome via homology-dependent recombination. We ablated the expression of xiap and p47, two signaling molecules present in the immune deficiency (IMD) pathway of Ixodes scapularis. Impairment of the tick IMD pathway, an analogous network of the tumor necrosis factor receptor in mammals, led to enhanced infection of the rickettsial agent Anaplasma phagocytophilum. Altogether, our findings provide a critical technical resource to the scientific community to enable a deeper understanding of biological circuits in the black-legged tick I. scapularis.

Frequency of Anaplasma phagocytophilum, Borrelia spp., and coinfections in Ixodes ricinus ticks collected from dogs and cats in Germany.

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.

Molecular prevalence of vector borne bacterial pathogens in the blood samples of wild rodent species trapped from Saudi Arabia.

Order Rodentia is the most speciose among mammals and the members of this order are known to host more than 60 zoonotic diseases and rodents are a potential health threat to humans. This study was designed to report the molecular prevalence and phylogenetic evaluation of various blood borne bacterial pathogens (Anaplasma ovis, Anaplasma phagocytophilum, Anaplasma marginale and Bartonella spp.) in the blood samples of four wild rodent species [Meriones rex (N = 27), Acomys dimidiatus (N = 18), Myomys yemeni (N = 6) and Rattus rattus (N = 3)] that were trapped during August till October 2020 from Al Makhwah governorate in Saudi Arabia. Results revealed by 9/54 (16.6%) rodents amplified Msp4 gene and 2/54 (3.7%) rodents amplified rpoB gene of Anaplasma ovis and Bartonella spp. respectively. Anaplasma phagocytophilum and Anaplasma marginale were not detected among enrolled rodent species. Meriones rex was the most highly infected rodent species. DNA sequencing and BLAST analysis confirmed the presence of Anaplasma ovis and the Bartonella koehlerae in rodent blood samples. Phylogenetic analysis of both pathogens showed that Saudi isolates were clustered together and were closely related to isolates that were reported from worldwide countries. Risk factor analysis revealed that prevalence of both bacterial pathogens was not restricted to a particular rodent species or a rodent sex (P > 0.05). In conclusion, we are reporting for the very first time that Saudi rodents are infected with Anaplasma ovis and rodents can be infected with Bartonella koehlerae. Similar studies at large scale are recommended in all those areas of Saudi Arabia that are unexplored for the incidence and prevalence of bacterial pathogens among the rodents that are living near human dwellings in order to prevent bacterial infections in local people as well as in livestock.

Serological investigation of seven zoonotic pathogens in companion dogs in South Korea, 2018-2021.

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.

Epidemiological, clinical, and laboratory characteristics of human granulocytic anaplasmosis in North India.

Human granulocytic anaplasmosis (HGA) is an emerging, rickettsial tick-borne disease caused by Anaplasma phagocytophilum. Sero-epidemiological data demonstrate that this pathogen has a worldwide distribution. The diagnosis of HGA requires a high index of clinical suspicion, even in endemic areas. In recent years, HGA has increasingly been reported from Asia and described in China, Japan, and Korea. We serologically and molecularly screened 467 patients with clinical suspicion of Anaplasmosis. The present study describes the epidemiology, clinical, and laboratory details of 6 confirmed and 43 probable cases of human granulocytic anaplasmosis. One of the HGA patients developed secondary invasive opportunistic Aspergillus fumigatus and Acinetobacter baumanii infection during the illness, which resulted in a fatal infection. The HGA patients without severe complications had excellent treatment responses to doxycycline. The emergence of this newly recognized tick-borne zoonotic HGA in North India is a significant concern for public health and is likely underdiagnosed, underreported, and untreated. Hence, it is also essential to establish a well-coordinated system for actively conducting tick surveillance, especially in the forested areas of the country.IMPORTANCEThe results of the present study show the clinical and laboratory evidence of autochthonous cases of Anaplasma phagocytophilum in North India. The results suggest the possibility of underdiagnosis of HGA in this geographical area. One of the HGA patients developed secondary invasive opportunistic Aspergillus fumigatus and Acinetobacter baumanii infection during the illness, which resulted in a fatal infection.

The role of Rab27 in tick extracellular vesicle biogenesis and pathogen infection.

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.

Ceramide-1-phosphate is a regulator of Golgi structure and is co-opted by the obligate intracellular bacterial pathogen Anaplasma phagocytophilum.

Many intracellular pathogens structurally disrupt the Golgi apparatus as an evolutionarily conserved promicrobial strategy. Yet, the host factors and signaling processes involved are often poorly understood, particularly for Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis. We found that A. phagocytophilum elevated cellular levels of the bioactive sphingolipid, ceramide-1-phosphate (C1P), to promote Golgi fragmentation that enables bacterial proliferation, conversion from its non-infectious to infectious form, and productive infection. A. phagocytophilum poorly infected mice deficient in ceramide kinase, the Golgi-localized enzyme responsible for C1P biosynthesis. C1P regulated Golgi morphology via activation of a PKCα/Cdc42/JNK signaling axis that culminates in phosphorylation of Golgi structural proteins, GRASP55 and GRASP65. siRNA-mediated depletion of Cdc42 blocked A. phagocytophilum from altering Golgi morphology, which impaired anterograde trafficking of trans-Golgi vesicles into and maturation of the pathogen-occupied vacuole. Cells overexpressing phosphorylation-resistant versions of GRASP55 and GRASP65 presented with suppressed C1P- and A. phagocytophilum-induced Golgi fragmentation and poorly supported infection by the bacterium. By studying A. phagocytophilum, we identify C1P as a regulator of Golgi structure and a host factor that is relevant to disease progression associated with Golgi fragmentation.IMPORTANCECeramide-1-phosphate (C1P), a bioactive sphingolipid that regulates diverse processes vital to mammalian physiology, is linked to disease states such as cancer, inflammation, and wound healing. By studying the obligate intracellular bacterium Anaplasma phagocytophilum, we discovered that C1P is a major regulator of Golgi morphology. A. phagocytophilum elevated C1P levels to induce signaling events that promote Golgi fragmentation and increase vesicular traffic into the pathogen-occupied vacuole that the bacterium parasitizes. As several intracellular microbial pathogens destabilize the Golgi to drive their infection cycles and changes in Golgi morphology is also linked to cancer and neurodegenerative disorder progression, this study identifies C1P as a potential broad-spectrum therapeutic target for infectious and non-infectious diseases.

Aptamer selection against cell extracts containing the zoonotic obligate intracellular bacterium, Anaplasma phagocytophilum.

A. phagocytophilum is a zoonotic and tick-borne bacterium, threatening human and animal health. Many questions persist concerning the variability of strains and the mechanisms governing the interactions with its different hosts. These gaps can be explained by the difficulty to cultivate and study A. phagocytophilum because of its strict intracellular location and the lack of specific tools, in particular monoclonal antibodies, currently unavailable. The objective of our study was to develop DNA aptamers against A. phagocytophilum, or molecules expressed during the infection, as new study and/or capture tools. Selecting aptamers was a major challenge due to the strict intracellular location of the bacterium. To meet this challenge, we set up a customized selection protocol against an enriched suspension of A. phagocytophilum NY18 strain, cultivated in HL-60 cells. The implementation of SELEX allowed the selection of three aptamers, characterized by a high affinity for HL-60 cells infected with A. phagocytophilum NY18 strain. Interestingly, the targets of these three aptamers are most likely proteins expressed at different times of infection. The selected aptamers could contribute to increase our understanding of the interactions between A. phagocytophilum and its hosts, as well as permit the development of new diagnostic, therapeutic or drug delivery appliances.

Tick-borne pathogens in questing adults Dermacentor reticulatus from the Eastern European population (north-eastern Poland).

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.

Historical associations and spatiotemporal changes of pathogen presence in ticks in Canada: A systematic review.

BACKGROUND: Starting in the early 20th century, ticks and their pathogens have been detected during surveillance efforts in Canada. Since then, the geographic spread of tick vectors and tick-borne pathogens has steadily increased in Canada with the establishment of tick and host populations. Sentinel surveillance in Canada primarily focuses on Ixodes scapularis, which is the main vector of Borrelia burgdorferi, the bacterium causing Lyme disease. Other tick-borne pathogens, such as Anaplasma, Babesia, and Rickettsia species, have lower prevalence in Canada, but they are emerging or re-emerging in tick and host populations. AIMS/MATERIALS & METHODS: Here, we assessed the historical associations between tick vectors, hosts and pathogens and identified spatiotemporal clusters of pathogen presence in ticks in Canada using data extracted from the literature. RESULTS: Approximately one-third of ticks were infected with a pathogen, and these ticks were feeding primarily on bird and mammal hosts. B. burgdorferi was the most detected pathogen and I. scapularis harboured the greatest number of pathogens. We identified several spatial outliers of high pathogen presence in ticks in addition to five spatiotemporal clusters in southern Canada, all of which have long-established tick populations. Six spatiotemporal clusters of high pathogen presence in ticks were also identified based on surveillance method, with four clusters associated with passive surveillance and two clusters associated with active surveillance. DISCUSSION: Our review represents the first systematic assessment of the literature that identifies historical associations and spatiotemporal changes in tick-host-pathogen disease systems in Canada over broad spatial and temporal scales. CONCLUSION: As distinct spatiotemporal clusters were identified based on surveillance method, it is imperative that surveillance efforts employ standardized methods and data reporting to comprehensively assess the presence, spread and risk of tick-borne pathogens in tick and host populations.