Claudia Thiel

Genetic Variants of Anaplasma phagocytophilum in Wild Caprine and Cervid Ungulates from the Alps in Tyrol, Austria

The occurrence of genetic variants of Anaplasma phagocytophilum was studied in wild ungulates from the northern and central eastern Alps in Tyrol, Austria. For this purpose, spleen samples collected from 53 game animals during the hunting season 2008/2009 (16 roe deer [Capreolus capreolus], 10 red deer [Cervus elaphus], 16 Alpine chamois [Rupicapra r. rupicapra], 7 Alpine ibex [Capra i. ibex], and 4 European mouflons [Ovis orientalis musimon]) were analyzed. Thirty-five animals originated from the Karwendel mountains, 12 from the Kaunertal area (Ötztal Alps), and the remaining from other mountainous areas in Tyrol. DNA extracts were screened with a real-time polymerase chain reaction targeting the msp2 gene of A. phagocytophilum. A total of 23 (43.4%) samples, from all ungulate species studied, were A. phagocytophilum positive. As of the date of this article, A. phagocytophilum has not been reported in the Alpine ibex. The positive samples were investigated further with polymerase chain reactions for amplification of the partial 16S rRNA, groEL, and msp4 genes. Sequence analysis using forward and reverse primers revealed seven different 16S rRNA gene variants. No variant could be attributed to any particular ungulate species. The groEL gene revealed 11 different variants, which grouped in the phylogenetic analysis into two distinct clusters: one cluster contained the sequences from roe deer, whereas the sequences of the other species formed the second cluster. The msp4 gene showed a high degree of variability in the amplified part with a total of 10 different sequence types. The results show that the wild mountain ungulates were infected to a considerable extent with various variants of A. phagocytophilum. The pathogenicity of the variants and the reservoir competence of the species investigated in this study deserve further attention in future studies.

Spotted Fever Group Rickettsiae in Ticks, Germany

To explore increased risk for human Rickettsia spp. infection in Germany, we investigated recreational areas and renatured brown coal surface-mining sites (also used for recreation) for the presence of spotted fever group rickettsiae in ticks. R. raoultii (56.7%), R. slovaca (13.3%), and R. helvetica (>13.4%) were detected in the respective tick species.

Prevalence of Anaplasma phagocytophilum in Ixodes ricinus in Bavarian public parks, Germany.

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.

Candidatus Neoehrlichia mikurensis and Anaplasma phagocytophilum : Prevalences and investigations on a new transmission path in small mammals and ixodid ticks

BackgroundSmall mammals are crucial for the life history of ixodid ticks, but their role and importance in the transmission cycle of tick-borne pathogens is mostly unknown. Candidatus Neoehrlichia mikurensis (CNM) and Anaplasma phagocytophilum are both tick-borne pathogens, and rodents are discussed to serve as main reservoir hosts for CNM but not for the latter especially in Germany. Analysing the prevalence of both pathogens in small mammals and their ticks in endemic regions may help to elucidate possible transmission paths in small mammal populations and between small mammals and ticks.MethodsIn 2012 and 2013, small mammals were trapped at three different sites in Germany. DNA was extracted from different small mammal tissues, from rodent neonates, foetuses and from questing and attached ticks. DNA samples were tested for CNM and A. phagocytophilum by real-time PCR. Samples positive for A. phagocytophilum were further characterized at the 16S rRNA gene locus.ResultsCNM was detected in 28.6% of small mammals and in 2.2% of questing and 3.8% of attached ticks. Altogether 33 positive ticks were attached to 17 different hosts, while positive ticks per host ranged between one and seven. The prevalences for this pathogen differed significantly within small mammal populations comparing sites (χ2: 13.3987; p: 0.0004) and between sexes. Male rodents had an approximately two times higher chance of infection than females (OR: 1.9652; 95% CI: 1.32-2.92). The prevalence for CNM was 31.8% (95% CI: 22-44) in rodent foetuses and neonates (23 of 67) from positive dams, and 60% (95% CI: 35.7-80.25) of positive gravid or recently parturient rodents (9 out of 15) had at least one positive foetus or neonate. Anaplasma phagocytophilum was detected at a low percentage in rodents (0-5.6%) and host-attached ticks (0.5-2.9%) with no significant differences between rodent species. However, attached nymphs were significantly more often infected than attached larvae (χ2: 25.091; p: <0.0001).ConclusionThis study suggests that CNM is mainly a rodent-associated pathogen and provides evidence for a potential transplacental transmission in rodents. In contrast, most of the rodent species captured likely represent only accidental hosts for A. phagocytophilum at the investigated sites.

PCR detection of Anaplasma phagocytophilum in goat flocks in an area endemic for tick-borne fever in Switzerland

Central Switzerland is a highly endemic region for tick-borne fever (TBF) in cattle, however, little is known about A. phagocytophilum in goats. In the present study, 72 animals from six goat flocks (373 EDTA blood-samples) in Central Switzerland were analysed for A. phagocytophilum DNA. A real-time PCR targeting the msp2 gene of A. phagocytophilum was performed and in positive samples the partial 16S rRNA, groEL and msp4 gene were amplified for sequence analysis. Four DNA extracts were positive. Different sequence types on basis of the amplified genes were found. For comparison, sequences of A. phagocytophilum from 12 cattle (originating from Switzerland and Southern Germany) were analysed. The 16S rRNA gene sequences from cattle were all identical amongst each other, but the groEL and msp4 gene differed depending on the origin of the cattle samples and differed from the variants from goats. This study clearly provides molecular evidence for the presence of different types of A. phagocytophilum in goat flocks in Switzerland, a fact which deserves more thorough attention in clinical studies.

Diversity of Babesia and Rickettsia Species in Questing Ixodes ricinus: A Longitudinal Study in Urban, Pasture, and Natural Habitats

In a previous study, our group investigated the Babesia spp. prevalence in questing Ixodes ricinus ticks from nine city parks in South Germany in the years 2009 and 2010. We showed predominant prevalence of B. venatorum (in previous literature also known as Babesia sp. EU1), especially in those parks in a more natural condition and with occurrence of large wild animals, such as roe deer. To obtain longitudinal data and to broaden the knowledge about this pathogen, further investigations were carried out in 2011 and 2012 in four of those city parks. Two additional habitat types were chosen for comparison of prevalence data and species analysis focusing on occurrence of potential reservoir hosts. A total of 10,303 questing I. ricinus were collected in four city parks, a pasture, and a natural area in Bavaria, and a representative number of samples were investigated for prevalence of DNA of Babesia spp. (n=4381) and Rickettsia spp. (n=2186) by PCR. In the natural and pasture area, a significantly higher Babesia spp. prevalence compared to the urban area was detected. The natural area revealed sequences of B. microti, B. venatorum, and B. capreoli. In the pasture and urban habitat, predominantly B. venatorum was found, whereas B. capreoli was less frequent and only one B. microti-infected tick was found. All B. microti sequences were 100% identical to the zoonotic Jena/Germany strain. For Rickettsia spp., the significantly highest prevalence was also detected in the natural and pasture areas, whereas lower prevalence was found in the urban area. Sequence analysis revealed R. helvetica (98%) and R. monacensis (2%). Prevalence rates and occurrence of Babesia spp. and Rickettsia spp. differed in urban, pasture and natural sites, most likely depending on the habitat structure (natural or cultivated) and therefore on the appearance and availability of reservoir hosts like roe deer or small mammals.

Anaplasma phagocytophilum in Questing Ixodes ricinus Ticks: Comparison of Prevalences and Partial 16S rRNA Gene Variants in Urban, Pasture, and Natural Habitats

ABSTRACT Urban, natural, and pasture areas were investigated for prevalences and 16S rRNA gene variants of Anaplasma phagocytophilum in questing Ixodes ricinus ticks. The prevalences differed significantly between habitat types, and year-to-year variations in prevalence and habitat-dependent occurrence of 16S rRNA gene variants were detected.

Relationship of Molecular and Clinical Findings on Anaplasma phagocytophilum Involved in Natural Infections of Dogs

Clinical manifestations of canine granulocytic anaplasmosis (CGA), caused by Anaplasma phagocytophilum—an obligate intracellular tick-transmitted bacterium infecting neutrophilic granulocytes—range from self-limiting to severe. Commonly, lethargy, inappetence, and fever and, further, lameness,

Prevalence and genetic characterization of Anaplasma marginale in zebu cattle (Bos indicus) and their ticks (Amblyomma variegatum, Rhipicephalus microplus) from Madagascar

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.