Prevalence of Anaplasma phagocytophilum in questing Ixodes ricinus nymphs across twenty recreational areas in England and Wales

Human granulocytic anaplasmosis and tick-borne fever, affecting livestock, are diseases caused by an infection with the bacterium Anaplasma phagocytophilum. Its transmission dynamics between vertebrate hosts and ticks remain largely unknown and the potential impact on public health in the United Kingdom is unclear. This study aimed to assess the distribution and estimate the prevalence of A. phagocytophilum in questing Ixodes ricinus at recreational locations across England and Wales over six years. An additional objective was to investigate possible associations between prevalence, habitat and presence of ruminant hosts. Ixodes ricinus ticks were collected each spring at 20 recreational locations across England and Wales between 2014 and 2019. Nymphs were tested for infection with A. phagocytophilum by detection of bacterial genome in DNA extracts, targeting the msp2 gene locus. Positive samples were further investigated for the presence of different ecotypes based on the GroEL region. Of 3,919 nymphs tested, the mean infection prevalence was 3.6% [95%CI: 3.1-4.3] and ranged from 0 to 20.4%. Northern England had a higher overall prevalence (4.7% [95%CI: 3.4-6.4]) compared to Southern England (1.8% [95%CI: 1.3-2.5]) and the presence of sheep was associated with higher A. phagocytophilum prevalence (8.4% [95%CI: 6.9-10.1] vs 1.2% [95%CI: 0.8-1.7] when absent). There was also a negative correlation with the prevalence of Borrelia burgdorferi s.l. (causing Lyme borreliosis). When investigating the diversity of A. phagocytophilum, ecotype I accounted for 86.8% of samples and ecotype II for 13.2%. Our study presents an overview of A. phagocytophilum prevalence in questing I. ricinus in recreational areas across England and Wales and discusses the potential public and veterinary health relevance.     

Anaplasma phagocytophilum infection in Ixodes ricinus, Bavaria, Germany

Anaplasma phagocytophilum DNA was detected by real-time PCR, which targeted the msp2 gene, in 2.9% of questing Ixodes ricinus ticks (adults and nymphs; n = 2,862), collected systematically from selected locations in Bavaria, Germany, in 2006. Prevalence was significantly higher in urban public parks in Munich than in natural forests.     

The distribution and prevalence of B. burgdorferi genomospecies in Ixodes ricinus ticks in Ireland

Questing Ixodes ricinus ticks were collected from six locations throughout Ireland and 638 nymphs, 111 females and 118 males were investigated for infection with Borrelia burgdorferi sensu lato (s.l.). The total prevalence of B. burgdorferi s.l. in the ticks was determined as 14.9% by polymerase chain reaction (PCR) amplification of the spacer region of 5S-23S rRNA genes. Infection prevalence was significantly higher in adult (20.1%) Ixodes ricinus compared to nymphs (13.1%). The prevalence of infection in adult male and female ticks was similar (19.5% and 20.7% respectively). The genomospecies B. burgdorferi sensu stricto, B. afzelii, B. garinii and group VS116 were identified by reverse line blot (RLB) using genomospecies specific oligonucleotide probes. The most prevalent B. burgdorferi genomospecies identified were VS116 (34.6%), B. garinii (24.3%) and B. burgdorferi sensu stricto (18.4%). B. afzelii was uncommon (6.6%). Multiple infections were observed in 13.2% of the infected ticks. The distribution of the genomospecies showed geographical variation and also seemed to be influenced by the nature of the habitat. A broad range of genomospecies seemed to be associated with the presence of a wide spectrum of potential reservoir hosts in the habitat and also with a high overall prevalence of B. burgdorferi s.l.     

Detection of Rickettsia spp. and Anaplasma phagocytophilum in Ixodes ricinus ticks in a region of Middle Germany (Thuringia)

Rickettsia spp. and Anaplasma spp. are regarded as potentially emerging tick-borne pathogens, but so far data on prevalence rates in questing ticks and reports on human diseases in several parts of Europe are rarely available.In this study, 430 nymphs and 570 adult Ixodes (I.) ricinus ticks were collected from a frequently visited forest region of Thuringia (Zeitzgrund, near Stadtroda) in 2006 (n=506) and 2007 (n=494). Individual ticks were investigated for a part of the citrate synthase gene (gltA) of Rickettsia spp. and the 16S rRNA gene of Anaplasma phagocytophilum. Positive amplicons were identified with restriction fragment length polymorphism (RFLP) and/or sequencing. Overall, 14.7% (147/1000) of investigated ticks were infected with Rickettsia spp. After sequencing of 64/147 positive amplicons R. helvetica (29/64) was detected predominantly. Prevalence varied in different developmental stages between 9.3% (40/430) in nymphs and 18.8% (107/570) in adults. A. phagocytophilum-specific DNA was detected in 5.4% (54/1000) of ticks with an infection rate of 4.7% (20/430) in nymphs and 6.0% (34/570) in adults. In 1% (10/1000) of ticks coinfections with Rickettsia spp. and A. phagocytophilum were found. Our study provides interesting insights into the circulation and cocirculation of different rickettsial species and A. phagocytophilum in the same biotope.     

Prevalence of Rickettsia spp., Anaplasma phagocytophilum,and Coxiella burnetii in adult Ixodes ricinus ticks from 29 study areas in central and southern Sweden

A total of 887 adult Ixodes ricinus ticks (469 females and 418 males) from 29 different localities in Sweden were screened for Rickettsia, Anaplasma, and Coxiella DNA using PCR and then subjected to gene sequencing. Rickettsial DNA was detected in 9.5–9.6% of the ticks. Most of the positive ticks were infected with Rickettsia helvetica. One tick harbored another spotted fever rickettsia, closely related to or identical with R. sibirica not previously found in I. ricinus nor in Sweden. Six of the ticks (0.7%) were infected with an Anaplasma sp., presumably A. phagocytophilum. Coxiella burnetii DNA was not detected in any of the ticks. The detection of R. helvetica and A. phagocytophilum in several of the localities sampled suggests that these potentially human-pathogenic agents are common in Sweden.     

Detection of Rickettsia helvetica in Ixodes ricinus ticks collected from Pyrenean chamois in France

Seventy-one Ixodes ricinus ticks collected from Pyrenean chamois (Rupicapra pyrenaica) in the French Pyrenees were tested by real-time polymerase chain reaction to detect the presence of Rickettsia and Bartonella. Four ticks (6%) were positive for R. helvetica. The chamois carries infected ticks, and this enables the dissemination throughout the environment with this bacterium, a potential human pathogen.     

The role of different species of wild ungulates and Ixodes ricinus ticks in the circulation of genetic variants of Anaplasma phagocytophilum in a forest biotope in north-western Poland

The aim of this study was to reveal genetic variants of Anaplasma phagocytophilum strains occurring in different species of wild ungulates and in Ixodes ricinus ticks to check the role of the examined species in the circulation of the revealed variants in nature. The aim was also to determine if the detected variants of A. phagocytophilum are specific for particular game species as well as to examine their identity with other strains, including pathogenic ones. Sequences of the amplified groEL heat shock operon and msp2 gene fragments of A. phagocytophilum were obtained from samples collected between 2005 and 2007 from 14 roe deer (Capreolus capreolus), 13 red deer (Cervus elaphus), 1 fallow deer (Dama dama) and 4 wild boar (Sus scrofa) as well as 13 engorged and 11 questing I. ricinus ticks occurring in the area of Puszcza Wkrzańska Forest in north-western Poland. Analysis of the sequences showed the presence of five and four gene variants of groEL and msp2, respectively. The variants showed high identity with sequences derived from strains pathogenic to humans and/or domestic and companion animals. Cervids seem to play a more important role in the circulation of the detected variants in nature than wild boar. Some of the detected variants are not shared by roe and red deer. The results obtained on the basis of groEL and msp2 sequences are discrepant. Analysis of the groEL operon sequence provides more information on A. phagocytophilum strains than the msp2 gene sequence.     

Anaplasma phagocytophilum infection in small mammal hosts of Ixodes ticks, western United States

A total of 2,121 small mammals in California were assessed for Anaplasma phagocytophilum from 2006 through 2008. Odds ratios were >1 for 4 sciurids species and dusky-footed woodrats. High seroprevalence was observed in northern sites. Ten tick species were identified. Heavily infested rodent species included meadow voles, woodrats, deer mice, and redwood chipmunks.     

High prevalence of Borrelia lusitaniae in Ixodes ricinus ticks in Tunisia

To investigate whether ticks of the genus Ixodes are infected by Borrelia burgdorferi complex, 490 unfed Ixodes ricinus ticks were collected by flagging in three different areas of Tunisia in 1998. DNAs extracted from 81 adults, 60 nymphs and 38 larvae were analysed after genic amplification of the noncoding spacer between the two copies of the rrl–rrf genes of B. burgdorferi sl. The prevalence of B. burgdorferi sl. in adults, nymphs and larvae was found to be 34, 33.3 and 2.6%, respectively. All DNAs (n = 61) but one were identified as belonging to different genotypes of B. lusitaniae by analysis of the restriction fragment length polymorphism of amplification products. In addition, 290 adults, 14 nymphs and 7 larvae were used to inoculate BSK-H medium to isolate spirochetes. Fifteen strains were isolated from adult ticks in the humid areas of Tunisia, whereas only one was obtained from larvae. Isolates were identified as B. lusitaniae (15/16) and B. garinii (1/16). These results provide new evidence for the existence of Lyme borreliosis in North Africa.     

Genetic diversity of Borrelia burgdorferi sensu lato isolates obtained from Ixodes ricinus ticks collected in Slovakia

In Europe, Borrelia burgdorferi sensu lato is diverse, including B.burgdorferi s.s., B.garinii, B.afzelii, B.valaisiana and B.lusitaniae. In this study, we focused on the distribution of the different B.burgdorferi species among Ixodes ricinus adult ticks collected in an endemic area within Slovakia. We compared results of prevalence of B.burgdorferi infection in ticks obtained by immunofluorescence (IF) and by isolation. Isolates were characterized by restriction fragment length polymorphism (RFLP) of the rrf-rrl intergenic spacer genes using MseI. Using immunofluorescence we observed that 56/114 (49%) ticks were infected by B.burgdorferi s.l. Males were found to be more often infected (32/57, 56%) than females (24/57, 42%) but the difference was not significant (p=0.1895). From the same 114 ticks a total of 37 isolates were obtained: 19 from males (33%) and 18 from females (32%). The RFLP identification revealed 25 B.afzelii (68%), 5 B.garinii (14%), 5 B.valaisiana (14%) and 2 B.lusitaniae (5%). The infection in ticks was more often detected by IF than by isolation (p=0.0153) and isolation success was higher when the infection degree in ticks was high (p=0.0397). The infection prevalence observed in this area is among the highest observed in Europe.     

Seasonal and habitat variation in the prevalence of Rickettsia helvetica in Ixodes ricinus ticks from Denmark

A total of 704 unfed ticks of the species Ixodes ricinus collected in Denmark were screened for Rickettsia DNA by a genus-specific real-time PCR. Of the nymphs, 4.7% (31/662) were positive for rickettsial DNA. Among the positive ticks, we observed a seasonal and habitat variation. The infection rate was highest in May as compared to July, August, and October. Ecotone (high tick density) showed an elevated prevalence as compared to spruce or beech forests. Sequencing revealed only DNA from R. helvetica.     

Anaplasma phagocytophilum, Babesia microti, and Borrelia burgdorferi in Ixodes scapularis, southern coastal Maine

Ixodes scapularis (deer ticks) from Maine were tested for multiple infections by polymerase chain reaction and immunofluorescence. In 1995, 29.5%, 9.5%, and 1.9% of deer ticks were infected with Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti, respectively. In 1996 and 1997, the number of A. phagocytophilum-infected ticks markedly declined. In 1995 through 1996, 4 (1.3%) of 301 were co-infected.     

Associations of Anaplasma phagocytophilum Bacteria Variants in Ixodes scapularis Ticks and Humans,New York,USA

Anaplasmosis, caused by the tickborne bacterium Anaplasma phagocytophilum, is an emerging public health threat in the United States. In the northeastern United States, the blacklegged tick (Ixodes scapularis) transmits the human pathogenic genetic variant of A. phagocytophilum (Ap-ha) and a nonpathogenic variant (Ap-V1). New York has recently experienced a rapid and geographically focused increase in cases of anaplasmosis. We analyzed A. phagocytophilum–infected I. scapularis ticks collected across New York during 2008–2020 to differentiate between variants and calculate an entomological risk index (ERI) for each. Ap-ha ERI varied between regions and increased in all regions during the final years of the study. Space-time scan analyses detected expanding clusters of Ap-ha located within documented anaplasmosis hotspots. Ap-ha ERI was more positively correlated with anaplasmosis incidence than non-genotyped A. phagocytophilum ERI. Our findings help elucidate the relationship between the spatial ecology of A. phagocytophilum variants and anaplasmosis.     

15-year Borrelia prevalence and species distribution monitoring in Ixodes ricinus/inopinatus populations in the city of Hanover,Germany

Lyme borreliosis, caused by Borrelia burgdorferi sensu lato (s.l.) spirochaetes, is the most common tick-borne disease (TBD) in the Northern Hemisphere. Rising incidences indicate that its epidemiology may be affected by global changes. Therefore, the current study aimed to assess changes in tick infection rates with Borrelia spp. over a 15-year monitoring period in the city of Hanover, Germany, as a follow-up to previous prevalence studies (years 2005, 2010 and 2015). To assess the epidemiological risk, ticks of the Ixodes ricinus/inopinatus-complex were sampled from April to October 2020 by the flagging method at 10 frequently visited recreation areas in Hanover. Analysis by quantitative real-time PCR of 2100 individual ticks revealed an overall Borrelia prevalence of 25.5% (535/2100). Regarding different tick developmental stages, nymphs showed a significantly lower Borrelia prevalence (18.4% [193/1050]) than adult ticks (32.6% [342/1050]). Comparison with previous years revealed a stable total Borrelia prevalence along with consistent infection rates in the different developmental stages over the 15-year monitoring period. Borrelia species differentiation by Reverse Line Blot was successful in 67.3% of positive ticks collected in 2020, with B. afzelii being the dominating species (59.2% of the differentiated infections), besides B. burgdorferi sensu stricto (s.s.), B. garinii, B. valaisiana, B. spielmanii, B. bavariensis and B. bissettiae and the relapsing fever spirochaete B. miyamotoi. Additionally, the proportion of infections attributed to B. afzelii showed a significant increase in 2020 compared to 2005 and 2015 (59.2% vs. 37.6% and 32.0% of successfully differentiated infections, respectively). Coinfections with Anaplasma phagocytophilum and Rickettsia spp. stayed stable comparing 2020 with previous years. Therefore, although changes in the Borrelia prevalence in questing ticks were not observed throughout the 15-year monitoring period, shifts in Borrelia species distribution may alter the epidemiological risk.     

Detection of Anaplasma phagocytophilum DNA in Ixodes ticks (Acari: Ixodidae) from Madeira Island and Setubal District, mainland Portugal

A total of 278 Ixodes ticks, collected from Madeira Island and Setubal District, mainland Portugal, were examined by polymerase chain reaction (PCR) for the presence of Anaplasma phagocytophilum. Six (4%) of 142 Ixodes ricinus nymphs collected in Madeira Island and 1 nymph and 1 male (2%) of 93 I. ventalloi collected in Setubal District tested positive for A. phagocytophilum msp2 genes or rrs. Infection was not detected among 43 I. ricinus on mainland Portugal. All PCR products were confirmed by nucleotide sequencing to be identical or to be most closely related to A. phagocytophilum. To our knowledge, this is the first evidence of A. phagocytophilum in ticks from Setubal District, mainland Portugal, and the first documentation of Anaplasma infection in I. ventalloi. Moreover, these findings confirm the persistence of A. phagocytophilum in Madeira Island's I. ricinus.     

Isolation of Arsenophonus nasoniae from Ixodes ricinus ticks in Slovakia

The tick Ixodes ricinus is the most prevalent and widely distributed tick species in Central Europe, commonly found in woodlands, heaths, and forests and particularly abundant in the Alpine region. This tick readily bites humans and transmits a number of bacterial and viral pathogens. We collected 10 live nymphs of I. ricinus ticks from vegetation in the Rovinka forest, Slovakia, and isolated a strain of Arsenophonus nasoniae from one tick using the BME/CTVM2 cell line. A new isolate was then subcultured on axenic media (Columbia agar supplemented with 5% sheep blood). To the best of our knowledge, this bacterium was never previously isolated from hard ticks or identified in ticks in Europe. We amplified and sequenced the 16S rRNA, rpoB, and ftsY genes. Limited genetic characterization showed that the isolated strain is almost identical to a strain from the parasitic wasp Nasonia vitripennis. Electron microscopy revealed a typical morphology of a Gram-negative bacterium, without pili or flagellae. Its role in human and animal pathology remains to be evaluated.     

Using next generation sequencing for molecular detection and differentiation of Anaplasma phagocytophilum variants from host seeking Ixodes scapularis ticks in the United States

Anaplasmosis is increasingly common in the United States, with cases being reported over an expanding geographic area. To monitor for changes in risk of human infection, the U.S. Centers for Disease Control and Prevention monitors the distribution and abundance of host-seeking vector ticks (Ixodes scapularis and Ixodes pacificus) and their infection with Anaplasma phagocytophilum. While several variants of A. phagocytophilum circulate in I. scapularis, only the human-active variant (Ap-ha) appears to be pathogenic in humans. Failure to differentiate between human and non-human variants may artificially inflate estimates of the risk of human infection. Efforts to differentiate the Ap-ha variant from the deer variant (Ap-V1) in ticks typically rely on traditional PCR assays coupled with sequencing of PCR products. However, laboratories are increasingly turning to Next Generation Sequencing (NGS) to increase testing efficiency, retain high sensitivity, and increase specificity compared with traditional PCR assays. We describe a new NGS assay with novel targets that accurately segregate the Ap-ha variant from other non-human variants and further identify unique clades within the human and non-human variants. Recognizing that not all investigators have access to NGS technology, we also developed a PCR assay based on one of the novel targets so that variants can be visualized using agarose gel electrophoresis without the need for subsequent sequencing. Such an assay may be used to improve estimates of human risk of developing anaplasmosis in North America.     

Occurrence of Borrelia burgdorferi in Ixodes ricinus in The Netherlands

In a 1989 surveillance to study the infection rate of Ixodes ricinus ticks with Borrelia burgdorferi, a total of 1838 nymphal and adult Ixodes ricinus ticks were collected from 20 locations in The Netherlands. By means of a culture method using modified Barbour-Kelly-Stoenner medium nymphs were examined in pools of 4 and adults were examined individually. With 63 out of 75 isolates propagation and serological identification were possible. All of these 63 isolates were identified as Borrelia burgdorferi by means of an indirect immunofluorescence assay. In nymphs the minimal infection rate was 2.4%. The rate of infection for adults was shown to be 14.3%. These results are in agreement with the percentages found by other investigators in West-Europe. In all locations examined Ixodes ricinus were found to be infected with Borrelia burgdorferi, although the infection rate varied. The results indicate that the infection is widespread among ticks in The Netherlands and the potential risk of acquiring Lyme borreliosis after a tick bite is serious.     

Molecular detection and phylogeny of Anaplasma spp. closely related to Anaplasma phagocytophilum in small ruminants from China

The genus Anaplasma comprises eight bacterial species that are obligate intracellular pathogens that affect human and animal health. The zoonotic species A. phagocytophilum is the causative agent of tick-borne fever in ruminants, and of granulocytic anaplasmosis in horses, dogs, and humans. Recently, novel strains related to A. phagocytophilum (A. phagocytophilum-like 1/Japanese variant and A. phagocytophilum-like 2/Chinese variant) have been identified. The aim of this study was to reveal the prevalence and phylogeny of A. phagocytophilum and related stains in small ruminants and ticks in China based on sequences of the 16S rRNA combined restriction fragment length polymorphism (RFLP) and groEL genes. PCR–RFLP and phylogenetic analyses based on the 16S rRNA gene showed the presence of A. phagocytophilum-like 1 and 2 variants in sampled animals from China, with prevalence rates of 22.6% (303/1338) and 0.7% (10/1338), respectively. Only A. phagocytophilum-like 1 DNA was found in Haemaphysalis longicornis. The phylogeny based on the groEL gene showed inclusion of A. phagocytophilum-like 1 and some A. phagocytophilum-like 2 strains in two unique clades distinct from, but related to, Japanese and Chinese strains of related A. phagocytophilum, respectively. One noteworthy result was that the SSAP2f/SSAP2r primers detected Ehrlichia spp. strains. Moreover, the A. phagocytophilum-like 1 and 2 strains should be considered in the differential diagnosis of caprine and ovine anaplasmosis. Further investigations should be conducted to provide additional epidemiological information about A. phagocytophilum and A. phagocytophilum-like variants in animals and ticks.     

Inter-annual variation in prevalence of Borrelia burgdorferi sensu stricto and Anaplasma phagocytophilum in host-seeking Ixodes scapularis (Acari: Ixodidae) at long-term surveillance sites in the upper midwestern United States: Implications for public health practice

The geographic range of the blacklegged tick, Ixodes scapularis, and its associated human pathogens have expanded substantially over the past 20 years putting an increasing number of persons at risk for tick-borne diseases, particularly in the upper midwestern and northeastern United States. Prevention and diagnosis of tick-borne diseases rely on an accurate understanding by the public and health care providers of when and where persons may be exposed to infected ticks. While tracking changes in the distribution of ticks and tick-borne pathogens provides fundamental information on risk for tick-borne diseases, metrics that incorporate prevalence of infection in ticks better characterize acarological risk. However, assessments of infection prevalence are more labor intensive and costly than simple measurements of tick or pathogen presence. Our objective was to examine whether data derived from repeated sampling at longitudinal sites substantially influences public health recommendations for Lyme disease and anaplasmosis prevention, or if more constrained sampling is sufficient. Here, we summarize inter-annual variability in prevalence of the agents of Lyme disease (Borrelia burgdorferi s.s.) and anaplasmosis (Anaplasma phagocytophilum) in host-seeking I. scapularis nymphs and adults at 28 longitudinal sampling sites in the Upper Midwestern US (Michigan, Minnesota, and Wisconsin). Infection prevalence was highly variable among sites and among years within sites. We conclude that monitoring infection prevalence in ticks aids in describing coarse acarological risk trends, but setting a fixed prevalence threshold for prevention or diagnostic decisions is not feasible given the observed variability and lack of temporal trends. Reducing repeated sampling of the same sites had minimal impact on regional (Upper Midwest) estimates of average infection prevalence; this information should be useful in allocating scarce public health resources for tick and tick-borne pathogen surveillance, prevention, and control activities.