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We report here one new, hospitalized case of Anaplasma phagocytophilum in Belgium. The clinical presentation of anaplasmosis, its treatment and the molecular and serological relevant laboratory methods are briefly developed.

Background: Equine granulocytic anaplasmosis (EGA) is a tick-borne disease caused by Anaplasma (A.) phagocytophilum. In Germany, this pathogen is transmitted primarily by Ixodes ricinus. There is limited knowledge about its prevalence in horses in Germany. The aim of this retrospective study was to analyze the results of serological and molecular testing for A. phagocytophilum in horses which were done in a commercial laboratory in Germany over fourteen years. Additionally, risk factors were evaluated, and hematological abnormalities were addressed in horses with positive PCR results., Methods: This retrospective study examined results of direct (Polymerase chain reaction [PCR]) and indirect (immunofluorescence antibody test [IFAT]) detection methods for A. phagocytophilum in horses on samples provided by German veterinarians and processed by the commercial laboratory LABOKLIN from 2008 to 2021. In horses with positive test results, a Complete Blood Count (CBC) and Serum Amyloid A (SAA) were also analyzed where possible., Results: In total, 1217/4834 horses tested positive (PCR: 190/1246 horses, 15.2%; IFAT: 1036/3849 horses, 26.9%). Seasonality and location, as classified by federal state, had a statistically significant impact on PCR results (P < 0.001 for both). In horses with positive PCR results, hematological abnormalities were detected in 112/118 horses (95%), with thrombocytopenia (86%) and anemia (52%) representing the most common findings. The remaining 6/118 horses (5%) showed no hematological abnormalities on CBC. SAA was measured in 35 horses with positive PCR results, which exclusively showed marked elevation., Conclusions: The seasonality of A. phagocytophilum infections confirmed by PCR testing was consistent with known peaks in vector activity in Germany. The high rate of horses with positive PCR results when compared to dogs and cats may be due to a lack of ectoparasite prophylaxis. Infections with A. phagocytophilum should be considered as a differential diagnosis in horses with cytopenia on CBC and SAA elevation, especially in the summer and after any possible tick exposure., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ingo Schaefer reports a relationship with Laboklin Laboratory for Clinical Diagnostics GmbH & Co that includes: employment. Elisabeth Mueller reports a relationship with Laboklin Laboratory for Clinical Diagnostics GmbH & Co that includes: employment. Declaration of interest Elisabeth Müller is the CEO of the commercial laboratory LABOKLIN (Bad Kissingen, Germany) and Ingo Schäfer an employee of the laboratory. This has not influenced the results of our study in any way., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Bovine parafilariosis is an emerging fly-borne disease in central Europe, characterized by seasonal occurrence of hemorrhagic exudations ('bleeding spots') from the end of winter to end of summer. In two cases from Germany reported here, one animal of a small herd in Bavaria and 20 animals on a farm in Baden-Württemberg presented bleeding spots from late March and late April 2020, respectively. Exudate samples from both cases were positive for larvated Parafilaria eggs. Examination of the skin and trimmed tissue after slaughter of the animal from Bavaria resulted in the collection of 11 nematodes (two males, eight females, one specimen in fragments). The animal's carcass presented typical yellow-greenish areas and bloody spots on the subcutaneous tissue of the flesh side of the skin. The nematodes were microscopically determined as Parafilaria bovicola. Basic morphometric measurements of two (one intact) male and six female nematodes are within the ranges of published data; length (male/female) 28.8/48.0-64.5 mm; width, 397.6 μm/430.7-527.6 μm; distance of cervical papillae to anterior end, 177.6/248.9-337.4; left spiculum/right spiculum (male), 365.3-379.4/149.5-180.3 μm; gubernaculum 45.0-48.1 μm; distance of vulva to anterior end (female), 37.3-66.0 mm. In order to gain information on P. bovicola in its vector, 91 cattle-visiting Musca autumnalis flies were collected from the affected animal in Bavaria (36 flies) and from co-pastured animals (55 flies) for PCR analysis and sequencing. A total of 14 flies were PCR-positive for filarial DNA, and sequencing of a fragment of the cox1 gene resulted in identification of P. bovicola (n = 10) and Thelazia gulosa (n = 5). This report presents further cases of bovine parafilariosis in Germany, provides morphometric data on male and female P. bovicola nematodes retrieved from cattle and identified DNA of P. bovicola and T.gulosa in M. autumnalis flies collected at a site of occurrence of bovine parafilariosis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

To address the lack of information on ticks infesting cattle in Egypt and the pathogens that they transmit, the current study aimed to (i) provide insight into tick species found on cattle in Egypt, (ii) identify the pathogens in ticks and their cattle hosts and (iii) detect pathogen associations in ticks and cattle. Tick samples and blood from their bovine hosts were collected from three different areas in Egypt (EL-Faiyum Oasis, Assiut Governorate and EL-Kharga Oasis). Tick species were identified by morphology and by sequence analysis of the cytochrome C oxidase subunit 1 (cox1) gene. Tick pools and blood samples from cattle were screened by the Reverse Line Blot hybridization (RLB) assay for the simultaneous detection of tick-borne pathogens, including Babesia, Theileria, Anaplasma, Ehrlichia, and Rickettsia spp., as well as the tick endosymbiont Midichloria mitochondrii. The RLB results were confirmed with specific conventional and semi-nested PCRs followed by sequencing. In total, 570 ticks (males, females and nymphs) were collected from 41 heads of cattle. Altogether 398 ticks belonged to the genus Hyalomma (397 Hyalomma excavatum and one Hyalomma scupense) while 172 ticks were identified as Rhipicephalus annulatus. Pooled H. excavatum ticks tested positive for several protozoa and bacteria with different minimum infection rates (MIRs): Theileria annulata (18.1 %), Babesia occultans (1.8 %), Anaplasma marginale (28.5 %), Anaplasma platys (0.25 %), Midichloria mitochondrii (11.6 %), Ehrlichia chaffeensis-like (1.8 %) and Ehrlichia minasensis (1 %). In R. annulatus, several agents were identified at different MIRs: T. annulata (2.3 %), B. bovis (0.6 %), A. marginale (18.0 %), A. platys (1.2 %), M. mitochondrii (2.9 %), E. minasensis (0.6 %). Pathogens co-detection in tick pools revealed A. marginale and T. annulata in 13.3 % samples followed by the co-detection of A. marginale and M. mitochondrii (8.4 %). In addition, triple co-detection with A. marginale, T. annulata and M. mitochondrii were found in 5.3 % of the tick pools. In cattle, the most common coinfection was with A. marginale and T. annulata (82.9 %) followed by the coinfection between A. marginale, T. annulata and B. bovis (4.9 %), A. marginale and B. bigemina (2.4 %) and finally the coinfection between T. annulata and B. occultans (2.4 %). Anaplasma platys, Babesia occultans, and E. minasensis were detected for the first time in Egypt in both cattle and ticks. These findings should be taken in consideration regarding human and animal wellbeing by the public health and veterinary authorities in Egypt., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Anaplasmosis and theileriosis are important diseases with great economic impact that affect the cattle industry worldwide. In this study, we describe the first molecularly confirmed clinical cases of anaplasmosis caused by Anaplasma marginale and of a concurrent infection with A. bovis and Theileria orientalis in Croatian cattle. Clinical signs of A. marginale-infected cows included fever, lethargy, dark urine, as well as icteric and reddish mucous membranes. Postmortem examination revealed icterus, urinary bladder filled with dark urine and splenomegaly. A marginale was observed within erythrocytes on Giemsa-stained spleen imprints. Three affected cows were successfully treated with oxytetracycline, after which no new deaths occurred in the herd. Three cows in a different herd died suddenly and were found to be concurrently infected with A. bovis and T. orientalis. Postmortem examination revealed generalized icterus and urinary bladder filled with dark urine. These cases of A. marginale, A. bovis and T. orientalis infection show that bovine anaplasmosis and theileriosis are present within Croatian cattle and should be included in differential diagnostic protocols., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

The species identification of tick vectors of Crimean-Congo hemorrhagic fever virus (CCHFV), especially Hyalomma (H.) species, is a prerequisite to understand the eco-epidemiology of this disease and to reveal vector and virus reservoir species. However, the morphologic species discrimination can be difficult for damaged or blood-fed ticks and in case of species intercrosses. Therefore, we used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and restriction fragment length polymorphism (RFLP) analysis to distinguish the most common Hyalomma species from sub-Saharan Africa (H. truncatum, H. rufipes and H. dromedarii). Within the last years, MALDI-TOF MS analysis based on tick leg proteins has been shown to be a reliable method to distinguish several tick species. For this purpose, a reference spectral library of several European, American and African tick species was established. In this study, six different Hyalomma species were tested, all of which were all clearly distinguishable by mass spectrometric analyses. Moreover, MALDI TOF- MS was able to confirm morphologic findings where sequencing provided ambiguous results. In addition, a polymerase chain reaction (PCR) based on the CO1 gene amplification of ticks has been developed for the unequivocal species identification by amplicon sequencing and specific restriction endonuclease cleavage pattern analysis. RFLP proved to be a feasible auxiliary discrimination tool for selected Hyalomma species when access to sequencing methods is not available, as for instance during field studies., (Copyright © 2020 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Anaplasma phagocytophilum is a Gram-negative obligate intracellular bacterium that replicates in neutrophil granulocytes. It is transmitted by ticks and causes tick-borne fever in domestic ruminants such as sheep, cattle and goats. However, in contrast to sheep and cattle little is known about the clinical course of infection in goats. We report here on three cases of symptomatic infection with A. phagocytophilum in two goats (Capra aegagrus hircus) and one water buffalo (Bubalus bubalis). The animals showed symptoms and laboratory findings similar to sheep and cattle. To our knowledge, this is the first report on the symptomatic infection of water buffalos with A. phagocytophilum. The infecting strains were genetically characterized by 16S rRNA gene, ankA gene and multilocus sequence typing (MLST). Four other strains from asymptomatically infected goats were also included. The ankA sequences from five goats were part of the formerly described ankA gene clusters I and IV that are known to contain A. phagocytophilum strains from sheep and cattle. However, the sequences from one goat and from the water buffalo belonged to ankA gene cluster II that was formerly described to be restricted to roe deer. A similar observation was made for MLST as three goats clustered with sequences from sheep and cattle, whereas three other goats and the water buffalo were found to be part of the roe deer cluster. However, since most of the strains from sheep and cattle were distinct from the roe deer strains, roe deer might not represent major reservoir hosts for tick-borne fever in domestic ruminants. When differing parts of the 16S rRNA gene were used for typing the results were conflicting. This shows that the use of a standardized typing method such as MLST is highly desirable to generate easily comparable results., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Wild vertebrates are involved in the transmission cycles of numerous pathogens. Additionally, they can affect the abundance of arthropod vectors. Urbanization, landscape and climate changes, and the adaptation of vectors and wildlife to human habitats represent complex and evolving scenarios, which affect the interface of vector, wildlife and human populations, frequently with a consequent increase in zoonotic risk. While considerable attention has focused on these interrelations with regard to certain major vector-borne pathogens such as Borrelia burgdorferi s.l. and tick-borne encephalitis virus, information regarding many other zoonotic pathogens is more dispersed. In this review, we discuss the possible role of wildlife in the maintenance and spread of some of these neglected zoonoses in Europe. We present case studies on the role of rodents in the cycles of Bartonella spp., of wild ungulates in the cycle of Babesia spp., and of various wildlife species in the life cycle of Leishmania infantum, Anaplasma phagocytophilum and Rickettsia spp. These examples highlight the usefulness of surveillance strategies focused on neglected zoonotic agents in wildlife as a source of valuable information for health professionals, nature managers and (local) decision-makers. These benefits could be further enhanced by increased collaboration between researchers and stakeholders across Europe and a more harmonised and coordinated approach for data collection., (Copyright © 2017 Elsevier B.V. All rights reserved.)

It is well known that dog shelters are a common source for parasitic infections in different countries worldwide. The present study was conducted in order to determine the prevalence of intestinal parasites and the frequency of polyparasitism in dogs living in two private shelters in Belgrade, Serbia. For this purpose, 134 faecal samples were examined for gastrointestinal parasites with the merthiolate-iodine-formalin concentration (MIFC)-method as well as for Giardia-coproantigen with an enzyme-linked-immunosorbent assay (ELISA). Taeniid eggs were identified by PCR and sequence analysis. Overall, at least one of nine different endoparasites was detected in 75.4% (101/134) of the dogs. Giardia duodenalis coproantigen was found most frequently (45.5%; 61/134), followed by eggs of Ancylostomatidae (41.0%; 55/134), oocysts of Hammondia/Neospora (11.2%; 15/134), eggs of Toxascaris leonina (9.7%; 13/134), oocysts of Isospora canis (8.2%; 11/134), eggs of Trichuris vulpis (6.7%; 9/134), cysts of Sarcocystis spp. (4.5%; 6/134), eggs of Toxocara canis (3.0%; 4/134) and eggs of Taenia spp. (1.5%; 2/134). The results of the study confirm a high parasitic burden in the investigated shelter dogs and call for an effective deworming program including an improved hygiene management in the affected facilities., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Tick-borne bovine anaplasmosis, caused by the obligate intracellular pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae), is a major constraint to cattle production in tropical and subtropical regions. From Madagascar, clinical cases were published but data based on molecular methods regarding the prevalence and genetic diversity of this pathogen on the island are lacking. The aims of this study were to investigate (1) the prevalence of A. marginale in Malagasy zebu cattle (Bos indicus) and their ticks with a species-specific real-time PCR, (2) the genetic diversity of A. marginale based on tandem repeats and microsatellites of the msp1α gene, and (3) the phylogenetic relationship between A. marginale isolates from Madagascar and strains found worldwide. Two hundred fourteen blood samples and 1822 ticks from 214 zebu cattle were collected. Rhipicephalus (R) microplus (40.2%) and Amblyomma (A) variegatum (59.8%) were identified on the cattle. A. marginale DNA was found in 89.7% of the examined zebu cattle and in 62.3% of the examined ticks. The tandem repeat and microsatellite analyses of the mspa1 gene showed high genetic diversity among the isolates between and within the different regions and high infection potential. Eighteen of the 25 tandem repeats identified have not been described before. Phylogenetic analysis revealed clustering of A. marginale strains from Madagascar with South Africa, America and Israel. A common ancestor may originate from South Africa and may have evolved due to phylogeographic characteristics or by a history of cattle movement. Its high prevalence in cattle and ticks, together with a low number of clinical manifestations and a high genetic heterogeneity among the investigated strains, confirms endemic stability of A. marginale in cattle from Madagascar., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Rickettsiae are emerging pathogens causing various types of spotted fever and typhus and are mostly transmitted by arthropods to humans and animals. In order to investigate the distribution of Rickettsiae of the spotted fever group (SFG) in small mammals as potential reservoirs and in fleas and ticks from these animals as potential vectors, a total of 91 small mammals (seven species) were captured and their ectoparasites were collected at seven sites around Leipzig, Saxony, Germany, in 2010 and 2011. Altogether, 91 skin samples, 125 fleas (five species) and 363 ticks (four species) were investigated for DNA of Rickettsia spp. with a real-time PCR targeting the gltA gene. A total of 26 (28.6%) rodents, 5 (3.9%) fleas and 151 (41.6%) ticks were positive for Rickettsia spp. by real-time PCR. Altogether 42 positive tick-, and all positive small mammal- and flea-samples were further determined to Rickettsia species level with a conventional PCR targeting the ompB gene followed by sequencing. Sequencing of 14 positive rodent samples revealed R. helvetica (n=12) and R. raoultii (n=2). Three Rickettsia spp. were detected in ticks: Rickettsia raoultii (59.6%), R. monacensis (4.8%) and R. helvetica (33.3%). In total 85.6% of Dermacentor reticulatus ticks and 20.4% of Ixodes ricinus ticks were positive. Rickettsia raoultii was found in 4 of the 5 positive fleas. To our knowledge this is the first detection of R. raoultii in Myodes glareolus and of R. helvetica in Apodemus agrarius from Germany. The high prevalence of R. helvetica in small mammals suggests that they may play an important role as potential natural reservoir hosts. The high prevalence in engorged I. ricinus for R. helvetica and in D. reticulatus ticks for R. raoultii, mostly deriving from uninfected mammals, leads to the conclusion that those tick species may serve as vectors for those Rickettsia spp. Detection of R. raoultii in fleas, parasitizing on their small mammal hosts, may indicate accidental uptake during feeding on hosts with bacteraemia rather than an active involvement of fleas in the transmission cycle of this Rickettsia species., (Copyright © 2016. Published by Elsevier B.V.)

Little is known about the role of endemic ticks as vectors for bacterial and protozoan pathogens for animals and humans in Madagascar and their interaction in anthropogenic habitats where humans, their livestock and native Malagasy species (vectors and hosts) come into more frequent contact than in natural forest ecosystems. The aims of the study were (1) to test whether habitat degradation is associated with increased infestation of tortoises by ticks and (2) to investigate whether ticks carried Babesia, Borrelia or Rickettsia species that might be pathogenic for humans and livestock. We studied hard ticks of two endemic Malagasy tortoises, Astrochelys radiata and Pyxis arachnoides in March and April 2013 in southwest Madagascar. Two tortoise habitats were compared, the National Park of Tsimanampetsotsa and the adjacent degraded pasture and agricultural land at the end of the wet season. Ticks were screened for protozoan and bacterial pathogens via PCR on DNA isolated from ticks using genus-specific primers. Only one out of 42 A. radiata collected from both habitats had ticks. The low prevalence did not allow further analyses of the effect of habitat degradation. Forty-two P. arachnoides were found in the anthropogenic habitat and 36 individuals in the national park. Tick infestation rates of P. arachnoides differed significantly between the two study sites. Tortoises inside the park had lower tick prevalence than outside (8 of 36 (22%) versus 32 of 42 individuals (76%)) and infected animals tended to have fewer ticks inside than outside the park. All ticks collected in both habitats were adults of the ixodid tick Amblyomma chabaudi, which is supposed to be a host-specific tick of P. arachnoides. Screening for Borrelia sp. and Babesia sp. was negative in all ticks. But all A. chabaudi ticks were infected with Rickettsia africae, known to cause spotted fever in humans. Thus, habitat degradation seems to be linked to higher infestation of tortoises with ticks with possible consequences for humans and their livestock., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Tick-borne spotted fever group (SFG) rickettsioses are emerging infectious diseases in Sub-Saharan Africa. In Madagascar, the endemicity of tick-borne rickettsiae and their vectors has been incompletely studied. The first part of the present study was conducted in 2011 and 2012 to identify potential anthropophilic tick vectors for SFG rickettsiae on cattle from seven Malagasy regions, and to detect and characterize rickettsiae in these ticks. Amblyomma variegatum was the only anthropophilic tick species found on 262 cattle. Using a novel ompB-specific qPCR, screening for rickettsial DNA was performed on 111 A. variegatum ticks. Rickettsial DNA was detected in 96 of 111 ticks studied (86.5%). Rickettsia africae was identified as the only infecting rickettsia using phylogenetic analysis of ompA and ompB gene sequences and three variable intergenic spacers from 11 ticks. The second part of the study was a cross-sectional survey for antibodies against SFG rickettsiae in plasma samples taken from healthy, pregnant women at six locations in Madagascar, two at sea level and four between 450 and 1300m altitude. An indirect fluorescent antibody test with Rickettsia conorii as surrogate SFG rickettsial antigen was used. We found R. conorii-seropositives at all altitudes with prevalences between 0.5% and 3.1%. Our results suggest that A. variegatum ticks highly infected with R. africae are the most prevalent cattle-associated tick vectors for SFG rickettsiosis in Madagascar. Transmission of SFG rickettsiosis to humans occurs at different altitudes in Madagascar and should be considered as a relevant cause of febrile diseases., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

The diverse tick fauna as well as the abundance of tick populations in Romania represent potential risks for both human and animal health. Spotted fever group (SFG) rickettsiae are recognized as important agents of emerging human tick-borne diseases worldwide. However, the epidemiology of rickettsial diseases has been poorly investigated in Romania. In urban habitats, companion animals which are frequently exposed to tick infestation, play a role in maintenance of tick populations and as reservoirs of tick-borne pathogens. Therefore, the aim of the present study was to investigate the occurrence of SFG rickettsiae in ticks infesting dogs in a greater urban area in South-eastern Romania. Adult ixodid ticks (n=205), including Rhipicephalus sanguineus sensu lato (n=120), Dermacentor reticulatus (n=76) and Ixodes ricinus (n=9) were collected from naturally infested dogs and were screened for SFG rickettsiae using conventional PCR followed by sequencing. Additionally, ticks were screened for DNA of Babesia spp., Hepatozoon spp., Ehrlichia canis, and Anaplasma platys. Four zoonotic SFG rickettsiae were identified: Rickettsia raoultii (16%) and Rickettsia slovaca (3%) in D. reticulatus, Rickettsia monacensis (11%) in I. ricinus, and Rickettsia conorii (0.8%) in Rh. sanguineus s.l. Moreover, pathogens of veterinary importance, such as B. canis (21%) in D. reticulatus and E. canis (7.5%) in Rh. sanguineus s.l. were identified. The findings expand the knowledge on distribution of SFG rickettsiae as well as canine pathogens in Romania. Additionally, this is the first report describing the molecular detection of R. conorii in ticks from Romania., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Two cases of acute babesiosis in captive reindeer (Rangifer tarandus) in two German zoos in 2009 and 2012 triggered this study to investigate the occurrence and species diversity of Babesia parasites infecting reindeer in different zoos and deer parks in Germany. Between June and December 2013, blood samples were taken from 123 clinically inapparent reindeer from 16 different facilities. Samples were tested for the presence of Babesia species DNA by conventional PCR and sequence analysis of part of the 18S rRNA gene. Also, Giemsa-stained smears of reindeer blood samples were examined for parasitaemia by light microscopy. The overall PCR-prevalence in blood samples was 23.6% (n=29). Comparison of sequenced amplicons with GenBank entries possibly revealed up to five different Babesia species: B. venatorum (n=19), B. capreoli (n=2) and B. capreoli-like (n=4), B. odocoilei-like (n=2) and B. divergens (n=1), while one sample turned out to be a Theileria sp. Out of the 16 facilities in the study, 12 housed at least one positive animal. In Giemsa-stained blood smears, intra-erythrocytic Babesia parasites were detected in samples of three reindeer from three locations. The high prevalence of Babesia infections implicates babesiosis to be a relevant infectious disease threat for captive reindeer in Germany. Consequently, reindeer with clinical signs compatible to those of acute babesiosis should either be tested for the presence of Babesia spp. DNA or blood smears should be examined for parasitaemia., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Small mammals serve as reservoir hosts for tick-borne pathogens, especially for those which are not transmitted transovarially in ticks - such as Babesia microti. Molecular investigations on the prevalence of B. microti in wild small mammals and on attached ticks from differently structured areas may provide information on the circulation of B. microti in different ecological niches. In 2012 and 2013, 622 rodents (396 Myodes glareolus, 178 Apodemus flavicollis, 36 Apodemus sylvaticus, 4 Apodemus agrarius, 7 Microtus arvalis, 1 Microtus agrestis) were captured from three differently structured habitats (urban, sylvatic, recultivated) in Germany. Attached ticks were collected from 449 small mammals (3250 Ixodes ricinus, 7 Ixodes trianguliceps, 133 Dermacentor reticulatus). A representative selection of a maximum of 5 ticks per developmental stage and species per 30 rodents of each species, location and year resulting in 965 ticks was further investigated. DNA was extracted from tick, blood and spleen samples, and tested by PCR for the partial 18S rRNA gene of B. microti with subsequent sequencing. The prevalence was significantly higher in rodents from the sylvatic site (4.6%) than in rodents captured at both other sites (-0.6%) (χ(2)=11.95; p=0.00125). Body and spleen weight of infected M. glareolus from the sylvatic site were significantly higher compared to those from non-infected individuals from that site (p=0.00288 and p=0.00017, respectively). Babesia microti DNA was detected in 3 out of 965 attached ticks (0.3%; 95%CI: 0-1) from all sites, but they derived exclusively from rodents captured at the sylvatic site. At the same site, I. ricinus nymphs (7.7%; 95%CI: 1-25.3) were significantly more often infected than I. ricinus larvae (0%; 95%CI: 0-1.3)(χ(2)=26.72; p<0.0001). The majority of positive rodents was also found at that site. I. trianguliceps occurred exclusively and the majority of M. glareolus at that site. Thus, it may be assumed that the circulation of B. microti is more efficient where this tick species and voles exist sympatrically than in areas with a predominant occurrence of Apodemus species., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

In this preliminary study, a novel DNA microarray system was tested for the diagnosis of bovine piroplasmosis and anaplasmosis in comparison with microscopy and PCR assay results. In the Dakahlia Governorate, Egypt, 164 cattle were investigated for the presence of piroplasms and Anaplasma species. All investigated cattle were clinically examined. Blood samples were screened for the presence of blood parasites using microscopy and PCR assays. Seventy-one animals were acutely ill, whereas 93 were apparently healthy. In acutely ill cattle, Babesia/Theileria species (n=11) and Anaplasma marginale (n=10) were detected. Mixed infections with Babesia/Theileria spp. and A. marginale were present in two further cases. A. marginale infections were also detected in apparently healthy subjects (n=23). The results of PCR assays were confirmed by DNA sequencing. All samples that were positive by PCR for Babesia/Theileria spp. gave also positive results in the microarray analysis. The microarray chips identified Babesia bovis (n=12) and Babesia bigemina (n=2). Cattle with babesiosis were likely to have hemoglobinuria and nervous signs when compared to those with anaplasmosis that frequently had bloody feces. We conclude that clinical examination in combination with microscopy are still very useful in diagnosing acute cases of babesiosis and anaplasmosis, but a combination of molecular biological diagnostic assays will detect even asymptomatic carriers. In perspective, parallel detection of Babesia/Theileria spp. and A. marginale infections using a single microarray system will be a valuable improvement., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

We report here one new, hospitalized case of Anaplasma phagocytophilum in Belgium. The clinical presentation of anaplasmosis, its treatment and the molecular and serological relevant laboratory methods are briefly developed.

Wild canines which are closely related to dogs constitute a potential reservoir for haemoparasites by both hosting tick species that infest dogs and harbouring tick-transmitted canine haemoparasites. In this study, the prevalence of Babesia spp. and Theileria spp. was investigated in German red foxes (Vulpes vulpes) and their ticks. DNA extracts of 261 spleen samples and 1953 ticks included 4 tick species: Ixodes ricinus (n=870), I. canisuga (n=585), I. hexagonus (n=485), and Dermacentor reticulatus (n=13) were examined for the presence of Babesia/Theileria spp. by a conventional PCR targeting the 18S rRNA gene. One hundred twenty-one out of 261 foxes (46.4%) were PCR-positive. Out of them, 44 samples were sequenced, and all sequences had 100% similarity to Theileria annae. Similarly, sequencing was carried out for 65 out of 118 PCR-positive ticks. Theileria annae DNA was detected in 61.5% of the sequenced samples, Babesia microti DNA was found in 9.2%, and Babesia venatorum in 7.6% of the sequenced samples. The foxes were most positive in June and October, whereas the peak of tick positivity was in October. Furthermore, the positivity of the ticks was higher for I. canisuga in comparison to the other tick species and for nymphs in comparison to adults. The high prevalence of T. annae DNA in red foxes in this study suggests a reservoir function of those animals for T. annae. To our knowledge, this is the first report of T. annae in foxes from Germany as well as the first detection of T. annae and B. microti in the fox tick I. canisuga. Detection of DNA of T. annae and B. microti in three tick species collected from foxes adds new potential vectors for these two pathogens and suggests a potential role of the red fox in their natural endemic cycles., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Canine granulocytic anaplasmosis (CGA) is caused by the rickettsial microorganism Anaplasma phagocytophilum. CGA is typically characterized by fever, thrombocytopenia, lethargy, anorexia, arthropy, and other nonspecific clinical signs. Skin lesions have been described in naturally infected lambs and humans. The pathophysiology of CGA is not entirely clear, and the persistence of the organism after the resolution of clinical signs has been described. The aim of the study was to investigate if A. phagocytophilum can be detected in canine lesional skin biopsies from A. phagocytophilum-seropositive dogs with etiologically unclear skin lesions that improved after the treatment with doxycycline. Paraffin-embedded lesional skin biopsies were allocated into separate groups: biopsies from A. phagocytophilum-seropositive dogs responsive to treatment with doxycycline (n=12), biopsies from A. phagocytophilum-seronegative dogs (n=2), and biopsies in which skin lesions histopathologically resembled a tick bite (n=10). The serological status of the latter group was unknown. Histology of the seropositive and seronegative dog skin lesions did not indicate an etiology. DNA was extracted, and a conventional PCR for partial 16S rRNA gene was performed. Anaplasma phagocytophilum DNA was amplified from 4/12 seropositive dogs' skin biopsies. All sequences were 100% identical to the prototype A. phagocytophilum human strain (GenBank accession number U02521). Anaplasma phagocytophilum was not amplified from the 2 seronegative and 10 suspected tick bite dogs. Serum antibody titers of the PCR-positive dogs ranged from 1:200 to 1:2048. Histopathologically, a mild-to-moderate perivascular to interstitial dermatitis composed of a mixed cellular infiltrate and mild-to-moderate edema was seen in all seropositive dogs. In 8/12 seropositive dogs, vascular changes as vasculopathy, fibrinoid necrosis of the vessel walls, and leukocytoclastic changes were observed. In summary, our results support the hypothesis that the persistence of A. phagocytophilum in the skin may be causative for otherwise unexplained skin lesions in seropositive dogs., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

The aim of the present study was to provide a preliminary insight into the diversity of tick-borne pathogens circulating at the domestic host-tick interface in Romania. For this, feeding and questing ticks were analyzed by real-time polymerase chain reaction (PCR) for the presence of Anaplasma phagocytophilum, Anaplasma platys, Ehrlichia canis, Borrelia burgdorferi sensu latu, and by PCR and subsequent sequencing for Rickettsia spp., Babesia spp. and Theileria spp. A total of 382 ticks, encompassing 5 species from 4 genera, were collected in April-July 2010 from different areas of Romania; of them, 40 were questing ticks and the remainder was collected from naturally infested cattle, sheep, goats, horses or dogs. Tick species analyzed included Ixodes ricinus, Dermacentor marginatus, Hyalomma marginatum, Rhipicephalus bursa, and Rhipicephalus sanguineus. Four rickettsiae of the spotted fever group of zoonotic concern were identified for the first time in Romania: Rickettsia monacensis and Rickettsia helvetica in I. ricinus, and Rickettsia slovaca and Rickettsia raoultii in D. marginatus. Other zoonotic pathogens such as A. phagocytophilum, Borrelia afzelii, and Babesia microti were found in I. ricinus. Pathogens of veterinary importance were also identified, including Theileria equi in H. marginatum, Babesia occultans in D. marginatus and H. marginatum, Theileria orientalis/sergenti/buffeli-group in I. ricinus and in H. marginatum and E. canis in R. sanguineus. These findings show a wide distribution of very diverse bacterial and protozoan pathogens at the domestic host-tick interface in Romania, with the potential of causing both animal and human diseases., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The hard tick Ixodes ricinus is the most common tick in Central Europe and plays an important role as a vector of several pathogens. In the complex life cycles of these pathogens, the role of wild animals as natural reservoirs has been discussed. The aims of this study were to investigate the role of roe deer (Capreolus capreolus) as a potential reservoir host for Babesia spp., Anaplasma phagocytophilum, and Rickettsia spp. Therefore, we explored the differences in the infection rates of roe deer and engorged and questing ticks with these pathogens from a single forest site with special attention to coinfection. Blood, spleen, and skin samples of a total of 95 roe deer individuals were screened by molecular methods for these pathogens from September 2010 to January 2012 in the 'Angelberger Forst' (Bavaria, Germany). Moreover, 331 engorged ticks from 44 roe deer individuals and 199 host-seeking ticks from the same area were screened. Altogether, the following prevalence rates and a high diversity of species were detected for the respective pathogens in individual animals and ticks: (i) Babesia spp.: roe deer, 89.5%; engorged ticks, 7.3%; questing ticks: adults, 2.5%, nymphs, 3.3%. Sequencing revealed B. venatorum, B. capreoli, and B. microti. (ii) A. phagocytophilum: roe deer 98.9%; engorged ticks, 86.1%; questing ticks: adults, 8.9%, nymphs, 0.8%. (iii) Rickettsia spp.: roe deer, 0%; engorged ticks, 16.6%; questing ticks: adults, 13.9%, nymphs, 17.5%. Sequencing revealed R. helvetica. Furthermore, several coinfections were detected in both roe deer and ticks. The high prevalence rates of B. capreoli and A. phagocytophilum in roe deer support their role as reservoir hosts for these pathogens, but no evidence for a role of roe deer in the life cycle of R. helvetica could be provided., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Due to the availability of adequate habitats in urban environments, e.g. city parks and recreational green areas, ticks from such settings may also carry pathogens of veterinary and public health concern. Thus, tick-borne infections may readily be identified in companion animals residing in urbanised areas. To investigate the presence of vector-borne pathogens in Kiev, Ukraine, 52 engorged adult ticks, 33 Dermacentor reticulatus and 19 Ixodes ricinus, were collected from 15 dogs in the spring of 2010, and further 23 canine EDTA-blood samples were obtained in the spring of 2011 from client-owned patients presented in a veterinary clinic in Kiev. DNA of 9 pathogens was detected by PCR in ticks and canine EDTA-blood samples: Babesia canis canis, Anaplasma phagocytophilum, Rickettsia helvetica, Ri. monacensis, Ri. raoultii, and Dirofilaria repens (by proxy) were identified in engorged ticks and B. c. canis, Hepatozoon canis, Di. immitis, Di. repens, and Mycoplasma haemocanis in canine EDTA-blood samples. This is the first description of Ri. raoultii in the Ukraine. This study adds information on the occurrence of vector-borne pathogens of veterinary and public health importance in Kiev, Ukraine., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

The cattle tick Rhipicephalus (Boophilus) microplus causes expressive damage to livestock in Brazil and other countries. Its control is becoming more difficult due to the development of resistance in populations. Early detection of resistance can help in developing effective control strategies. This study evaluated the susceptibility of R. microplus to cypermethrin and chlorpyriphos and was the first attempt to identify the mechanism of resistance (target site insensitivity) in cattle tick populations from Minas Gerais state (Southeastern Brazil). Engorged female ticks were collected from 10 ranches within the state of Minas Gerais, and susceptibility was evaluated with the larval packet test (LPT) using technical grade cypermethrin and chlorpyriphos. It was possible to analyze LPT results of seven populations. Target site insensitivity was investigated in all 10 isolates by using molecular approaches for detection of the T2134A substitution within the domain III S6 segment and the C190A in the domain II S4-5 linker from the para-type sodium channel gene. LPT showed that all seven populations were resistant to cypermethrin with resistance ratio (RR) ranging from 16.0 to 25.0 and 85.7% were resistant to chlorpyriphos (RR=2.2-15.6). Although the T2134A mutation was not detected, the C190A mutation was highly prevalent, being present in 82-100% of the alleles sampled in field populations. A significant correlation was found between the LC50 values for cypermethrin and the frequency of the C190A mutation suggesting that it might be responsible for the phenotypic resistance detected., (Copyright © 2012 Elsevier B.V. All rights reserved.)

This study aimed to provide up-to-date information on the dynamics of tick infestations on foxes in Thuringia, as the most recent information available was published in 1997. Fox carcasses that had been sent to the Thuringian State Authority for Food Safety and Consumer Protection (Thüringer Landesamt für Lebensmittelsicherheit und Verbraucherschutz - TLLV), between January 1st and December 31st, 2009, were examined for the presence of ticks. All ticks collected were stored at -20 °C before being identified and classified according to their developmental stage and sex. Out of a total of 1286 foxes examined, 989 (76.9%) were infested with ticks. A total of 13,227 ticks were collected from the foxes. The stage most frequently found was the larva (48.1%), followed by the adult (34.1%), and the nymphal stage (17.8%). Regarding the adult stage, Ixodes ricinus was the most frequent tick species detected (82.2%), followed by I. canisuga (10.8%) and I. hexagonus (6.7%). Dermacentor reticulatus ticks were very rare (0.3%). With regard to nymphs, I. canisuga and I. hexagonus were the most frequent tick species found, and this was also assumed for the larval stage. The results indicate the occurrence of tick infestations in foxes throughout the year, mainly by I. ricinus, I. canisuga, and I. hexagonus, with seasonal variations. Foxes were infested by I. ricinus ticks significantly more frequently from April to September. This applied to all tick developmental stages, but especially to adults. In contrast to I. ricinus, the infestation of foxes with I. canisuga and I. hexagonus was significantly higher from January to March and from October to December, especially with the immature developmental stages., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

The European hedgehog (Erinaceus europaeus) is a common insectivore in most parts of Europe and is frequently infested by the ticks Ixodes ricinus and I. hexagonus. I. ricinus ticks have been found infected with Anaplasma phagocytophilum, an obligate intracellular bacterium, but little is known about the potential of the hedgehog as a reservoir host. In this study, the infection with A. phagocytophilum and the genetic variants involved were investigated in a captive hedgehog population which was kept in a fenced, natural grass and bush garden habitat, and also in its ticks. Additionally hedgehogs from hedgehog caretaking stations were investigated. EDTA blood and ticks were collected from the captive hedgehog population once a month from March to October 2007 and in March and April 2008. All 3 developmental stages of I. ricinus and I. hexagonus occurred on the hedgehogs. After DNA extraction, the samples were screened for A. phagocytophilum with a real-time PCR, and selected samples were further investigated with a nested PCR targeting the partial 16S rRNA gene, followed by sequencing. One hundred thirty-six out of 220 hedgehog blood samples (61.8%) from altogether 48 individuals, 413 out of 563 I. ricinus samples and 90 out of 338 I. hexagonus samples were PCR-positive. Thirty-two hedgehogs were positive more than once, most frequently twice or 3 times, but also up to 9 times. Sequencing of the partial 16S rRNA gene resulted in 6 variants, but one variant ('A') was the most frequent which appeared in 93.8% of the positive hedgehogs. This variant (equaling Frankonia II, GenBank AF136712) has recently been reported from human, equine, and canine granulocytic anaplasmosis cases and thus, its specific association with hedgehogs is an important finding in the epidemiology of A. phagocytophilum in Europe. The high infection rate of both hedgehogs and ticks with A. phagocytophilum and the simultaneous infestation with 2 tick species of all developmental stages suggest that the hedgehog may be a suitable reservoir for at least some variants of A. phagocytophilum., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Previous studies have shown that public parks provide extraordinary habitats for ticks and tick-borne pathogens. Therefore, the aim of this study was to investigate the tick abundance (ticks/100 m(2)) in urban areas and the prevalence of Anaplasma phagocytophilum. Collection of ticks was performed by the flagging method in selected Bavarian public parks in a 2-year survey. DNA from 30 ticks of each developmental stage, sampling site and month was extracted and screened by specific real-time PCR for A. phagocytophilum. Selected positive samples were further genetically differentiated by nested PCR, targeting the 16S rRNA gene. A total of 13,403 Ixodes ricinus were collected with an average tick abundance of 15-53 ticks/100 m(2) in 2009 and 15-35 ticks/100 m(2) in 2010, depending on the sampling site. DNA of A. phagocytophilum was detected in 11.6% (n=146) and 8.5% (n=50) of adult females, 13.3% (n=164) and 9.2% (n=51) of adult males as well as 5% (n=60) and 3.9% (n=29) of nymphs for 2009 (n=3685) and 2010 (n=1884), respectively. 16S rRNA gene analysis revealed 6 different genetic variants, 2 of which were 100% identical to known sequences in humans. The results give strong evidence that the occurrence of A. phagocytophilum in I. ricinus is highly variable in different habitat types due to geographical, climatic, and biological factors as well as different genetic variants of A. phagocytophilum circulated in ticks with a noticeable habitat and host tropism., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Anaplasma phagocytophilum is an obligate intracellular bacterium causing granulocytic anaplasmosis in dogs, horses, and humans and tick-borne fever of ruminants. The bacterium has been detected in a variety of other mammals including wild ruminants without overt clinical signs of disease. Isolates in cell culture have been obtained from humans, dogs, horses, sheep, and ticks, but no strain from wild ruminants exists in cell culture in Europe. From September to November 2010, EDTA blood samples were collected from the jugular vein of 19 shot roe deer from a forest in southern Germany. The presence of specific A. phagocytophilum DNA was demonstrated with a real-time PCR targeting the msp2 gene in all 19 animals. Subsequently, blood cells were used to inoculate the tick cell line IDE8. The first infected IDE8 cells were detected in Giemsa-stained smears 57 days post inoculation. Only one roe deer yielded a positive culture which has been propagated for 9 consecutive passages thus far representing 228 days in culture. Further analysis of the A. phagocytophilum strain was performed by PCR followed by sequencing for the partial 16S rRNA, groEL, msp2, and msp4 genes. Phylogenetic topology of groEL and msp4 sequences placed the roe deer isolate in close proximity to sequences available from roe deer and goats from the neighbouring Alpine regions of Austria and Switzerland, and of msp2 with other ruminant species. This represents the first isolation of A. phagocytophilum in a tick cell line directly from an infected wild ruminant reservoir host, Capreolus capreolus, in Europe. The availability of a cultured A. phagocytophilum strain isolated from roe deer will allow us to study the biological characteristics and the pathogenic potential of this strain as well as to compare its host tropism and its genetic and antigenetic properties with those of other A. phagocytophilum strains from other animal species., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

For the first time, a female specimen of Ixodes frontalis was collected using the flagging method in a public park in the Bavarian city of Ingolstadt, Germany. Previous reports were from migratory birds only. Therefore, this is the first evidence that I. frontalis may be part of the tick fauna of Germany., (Copyright © 2011 Elsevier GmbH. All rights reserved.)