Absence of Borrelia spp., Candidatus Neoehrlichia mikurensis, and Anaplasma phagocytophilum in questing adult Dermacentor reticulatus ticks

To determine whether Dermacentor reticulatus ticks are infected by Borrelia spp., Candidatus Neoehrlichia mikurensis, or Anaplasma phagocytophilum, we collected questing adults in the outskirts of Berlin, Germany, examined them for the presence of DNA of these pathogens, and compared the infection rates to those of sympatric Ixodes ricinus ticks. Questing D. reticulatus adults appeared not to harbor the bacterial pathogens that are prevalent in I. ricinus ticks. Based on our sample size, the estimated prevalence of each of these pathogens in D. reticulatus ticks would be well below three tenth of a percent (<0.3 %). For pathogens which so rarely infect D. reticulatus ticks, this tick likely plays no epidemiologic vector role for either their enzootic transmission cycle in nature or their transmission to people.     

"Candidatus Neoehrlichia mikurensis" infection in a dog from Germany

"Candidatus Neoehrlichia mikurensis" is a new intracellular pathogen associated with human infection and death. "Candidatus Neoehrlichia mikurensis" infection in a chronically neutropenic dog from Germany was confirmed by DNA sequencing. The same organism was previously described from ticks and two sick human beings from Germany.     

Molecular characterization of Anaplasma phagocytophilum and Borrelia burgdorferi in Ixodes scapularis ticks from Pennsylvania

Ixodes scapularis ticks were collected in 2000 and 2001 from two areas in Pennsylvania and tested for the presence of Anaplasma phagocytophilum and Borrelia burgdorferi by PCR and DNA sequencing. Of the ticks collected from northwestern and southeastern Pennsylvania, 162 of 263 (61.6%) and 25 of 191 (13.1%), respectively, were found to be positive for B. burgdorferi. DNA sequencing showed >99% identity with B. burgdorferi strains B31 and JD1. PCR testing for A. phagocytophilum revealed that 5 of 263 (1.9%) from northwestern Pennsylvania and 76 of 191 (39.8%) from southeastern Pennsylvania were positive. DNA sequencing revealed two genotypes of A. phagocytophilum, the human granulocytic ehrlichiosis (HGE) agent and a variant (AP-Variant 1) that has not been associated with human infection. Although only the HGE agent was present in northwestern Pennsylvania, both genotypes were found in southeastern Pennsylvania. These data add to a growing body of evidence showing that AP-Variant 1 is the predominant agent in areas where both genotypes coexist.     

Molecular detection of Ehrlichia canis,Anaplasma bovis,Anaplasma platys,Candidatus Midichloria mitochondrii and Babesia canis vogeli in ticks from Israel

Ticks are vectors of important pathogens of human and animals. Therefore, their microbial carriage capacity is constantly being investigated. The aim of this study was to characterize the diversity of domestic animal pathogens in ticks collected from vegetation and the ground, from different parts of Israel. Non-engorged questing adult ticks were collected from 13 localities. A total of 1196 ticks in 131 pools—83 pools of Rhipicephalus turanicus and 48 of Rhipicephalus sanguineus (with two to ten ticks per pool)—were included in this study. In addition, 13 single free-roaming Hyalomma spp. ticks were collected. Screening by molecular techniques revealed the presence of Ehrlichia canis, Anaplasma platys, Anaplasma bovis and Babesia canis vogeli DNA in R. turanicus ticks. E. canis, A. bovis, B. canis vogeli and Candidatus Midichloria mitochondrii DNA sequences were detected in R. sanguineus ticks. Candidatus Midichloria mitochondrii DNA was also detected in Hyalomma spp. ticks. Neither Hepatozoon spp. nor Bartonella spp. DNA was detected in any of the ticks examined. This study describes the first detection of E. canis in the tick R. turanicus, which may serve as a vector of this canine pathogen; E. canis was the most common pathogen detected in the collected questing ticks. It also describes the first detection of A. bovis and Candidatus Midichloria mitochondrii in Israel. To the best of the author's knowledge, this is the first report describing the detection of DNA of the latter two pathogens in R. sanguineus, and of A. bovis in R. turanicus.     

Prevalence of Anaplasma phagocytophilum and Babesia divergens in Ixodes ricinus ticks from Lithuania and Norway

We detected Anaplasma phagocytophilum and Babesia divergens in Ixodes ricinus ticks collected from different locations in Lithuania and Norway by using the Taq Man based real-time PCR method. The msp2 gene of A. phagocytophilum and the 18S rRNA gene of B. divergens have been chosen as amplification targets. The overall infection rate of A. phagocytophilum in Norwegian ticks was 4.5% (10/224) and in Lithuanian ticks 3% (4/140). The prevalence varied in locations between 0% and 9% in Lithuania and in Norway. Three out of 140 (2%) ticks were infected with B. divergens in Lithuania and two out of 224 (0.9%) in Norway. The prevalence of B. divergens infection varied from 0% to 3% and from 0% to 4% in different sites in Lithuania and Norway, respectively.     

Detection and identification of Anaplasma phagocytophilum, Borrelia burgdorferi, and Rickettsia helvetica in Danish Ixodes ricinus ticks

Borreliosis is an endemic infection in Denmark. Recent serosurveys have indicated that human anaplasmosis may be equally common. The aim of this study was to look for Anaplasma phagocytophilum and related pathogens in Ixodes ricinus ticks and estimate their prevalence, compared to Borrelia, using PCR. Ticks were collected from three locations in Denmark: Jutland, Funen, and Bornholm. Ticks from Jutland and Funen were analysed individually, ticks from Bornholm were analysed in pools of 20. A. phagocytophilum was found in ticks from all areas. A. phagocytophilum was found in 23.6% of ticks from Jutland and Funen, while 11% were positive for Borrelia burgdorferi. The Borrelia genotype B. afzelii was most prevalent, followed by B. valaisiana, B. burgdorferi s.s. and B. garinii.A. phagocytophilum was found in 14.5% of nymphs and 40.5% of adult ticks, while Borrelia was found in 13% of nymphs and 8% of adult ticks. The difference in prevalence between Anaplasma and Borrelia in adult ticks supports the idea that their maintenance cycles in nature may be different. Ticks were also infected with Rickettsia helvetica. Our study indicates that A. phagocytophilum prevalence in ticks in Denmark is as high as Borrelia prevalence and that human anaplasmosis may be unrecognized.     

Detection of Anaplasma phagocytophilum and Ehrlichia sp. HF strains in Ixodes ricinus ticks in Brittany, France

DNA extracts from 156 tick pools, 18 blood specimens and 17 spleens from European woodmice (Apodemus sylvaticus) collected in Brittany, France were tested by PCR for the 16S rRNA gene of Anaplasmataceae. Positive amplicons were sequenced and confirmed, either by amplification and sequencing of a second gene, or by a second PCR specific for the P44 and gltA genes of Anaplasma phagocytophilum and the gltA gene of Ehrlichia sp. HF. In addition to A. phagocytophilum, the study detected Ehrlichia sp. HF for the first time in Ixodes ricinus ticks. This organism has only been detected previously in Ixodes ovatus ticks from Japan.     

Transmission of Anaplasma phagocytophilum to Ixodes ricinus ticks from sheep in the acute and post-acute phases of infection

A total of 60 sheep were exposed to Anaplasma phagocytophilum infection on an enclosed area of Ixodes ricinus-infested pasture in North Wales, United Kingdom, and rapidly acquired acute A. phagocytophilum infections detectable by PCR and blood smear examination. Of the ticks that had engorged in the previous instar on infected sheep, 52% of adult ticks and 28% of nymphs were PCR positive; a significant, 10-fold increase in prevalence compared to that of ticks that engorged on sheep preinfection was observed (P = 0.015). The likelihood that ticks were PCR positive, after feeding on the sheep and molting to the next instar, increased marginally with increasing numbers of infected neutrophils per milliliter of blood of their sheep host (P = 0.068) and increased significantly when they were collected from sheep carrying higher numbers of adult female ticks (P = 0.017), but increasing numbers of feeding nymphs had a significant negative effect on transmission (P = 0.049). The numbers of circulating neutrophils and of infected neutrophils also varied significantly with the numbers of ticks feeding on the sheep when the blood was collected. Our study suggests that ruminants are efficient reservoirs of A. phagocytophilum during the acute and post-acute phases of infection. The risk of ruminant-derived infections may, however, be strongly affected by variations in tick densities, which may influence transmission from acutely infected animals via effects on the numbers of infected cells in the blood and possibly by within-skin modulation of infection.     

Detection of Borrelia burgdorferi, Ehrlichia chaffeensis, and Anaplasma phagocytophilum in ticks (Acari: Ixodidae) from a coastal region of California

A study was conducted in Santa Cruz County to estimate the prevalence and distribution of the agents of Lyme disease, human granulocytic (HGE), and human monocytic (HME) ehrlichiosis in 1,187 adult ixodid ticks collected from eight public-use recreation areas over a 2-yr period. Borrelia burgdorferi, the causative agent of Lyme disease, was detected by a polymerase chain reaction (PCR) in 44 of 776 (5.67%) Ixodes pacificus ticks and in 3 of 58 (5.17%) Dermacentor variabilis ticks. Anaplasma phagocytophilum, the causative agent of HGE, was detected by PCR in 48 (6.19%) I. pacificus ticks and 5 (8.62%) D. variabilis ticks. Ehrlichia chaffeensis, the causative agent of HME, was detected by nested PCR in just five (0.64%) I. pacificus ticks and four (6.9%) D. variabilis ticks. Interestingly, eight (1.03%) I. pacificus ticks were co-infected with B. burgdorferi and A. phagocytophilum, and just one (0.12%) tick was co-infected with B. burgdorferi and E. chaffeensis. Less than 1% of 353 Dermacentor occidentalis ticks showed evidence of infection with any of the agents tested. To our knowledge, this is the first reported identification of A. phagocytophilum and E. chaffeensis in D. occidentalis ticks from California This study represents the first extensive survey of Lyme and the ehrlichial diseases across multiple areas of Santa Cruz County, and suggests that prevalence of B. burgdorferi in Santa Cruz County may be higher than other areas of the state.     

Distribution and molecular analysis of Lyme disease spirochetes, Borrelia burgdorferi, isolated from ticks throughout California.

Previous studies describing the occurrence and molecular characteristics of Lyme disease spirochetes, Borrelia burgdorferi, from California have been restricted primarily to isolates obtained from the north coastal region of this large and ecologically diverse state. Our objective was to look for and examine B. burdorferi organisms isolated from Ixodes pacificus ticks collected from numerous regions spanning most parts of California where this tick is found. Thirty-one isolates of B. burgdorferi were examined from individual or pooled I. pacificus ticks collected from 25 counties throughout the state. One isolate was obtained from ticks collected at Wawona Campground in Yosemite National Park, documenting the occurrence of the Lyme disease spirochete in an area of intensive human recreational use. One isolate from an Ixodes neotomae tick from an additional county was also examined. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot analysis, agarose gel electrophoresis, Southern blot analysis, and the polymerase chain reaction were used to examine the molecular and genetic determinants of these uncloned, low-passage-number isolates. All of the isolates were identified as B. burgdorferi by their protein profiles and reactivities with monoclonal and polyclonal antibodies, and all the isolates were typed by the polymerase chain reaction as North American-type spirochetes (B. burgdorferi sensu stricto). Although products of the ospAB locus were identified in protein analyses in all of the isolates, several isolates contained deleted forms of this locus that would result in the expression of chimeric OspA-OspB proteins. The analysis of OspC demonstrated that this protein was widely conserved among the isolates but was also quite variable in its molecular mass and the amount of it that was expressed.     

Prevalence of Lyme disease agents and several emerging pathogens in questing ticks from the German Baltic coast

In summer 2008, a total of 512 Ixodes ricinus (Acari: Ixodidae) ticks was collected from vegetation in four areas at the Baltic coast of Germany and tested for the presence of Lyme disease spirochetes. Among them, 293 ticks from three areas were screened for Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae), rickettsiae of the spotted fever group (Rickettsiales: Rickettsiaceae), and Babesia spp. (Piroplasmida: Babesiidae), respectively. Borrelia burgdorferi sensu lato genospecies (Spirochaetales: Spirochaetaceae) were detected in 3.1% of the tick samples. The prevalence ofA. phagocytophilum was 1.0%, rickettsiae were present in 8.5%, and pathogenic Babesia spp. in 8.9% of analyzed ticks. Coinfections occurred in five ticks. With this study we report first data on the coexistence of established and emerging pathogens in questing ticks from recreational areas of northeastern Germany, indicating the need of further studies for a reliable risk assessment.     

Prevalence of Rickettsiales (Anaplasma phagocytophilum and Rickettsia spp.) in hard ticks (Ixodes ricinus) in the city of Hamburg,Germany

To narrow the gap of missing knowledge on Rickettsia spp. and Anaplasma phagocytophilum infections in ticks in northwestern Germany and, at the same time, to provide first prevalence data on these pathogens in the city of Hamburg, a total of 1,400 questing Ixodes ricinus ticks were collected at ten different public green areas from April until October 2011. Ticks were examined using probe-based quantitative real-time PCR. A percentage of 3.6 % (51/1,400) ticks were tested positive for A. phagocytophilum infections divided into 2.1 % (3/141) adults [1.7 % (1/60) females and 2.5 % (2/81) males] and 3.8 % (48/1,259) nymphs. The percentage of infected ticks per sampling site varied statistically significantly from 0.7 % (1/140) to 12.1 % (17/140), whereas between sampling months, no statistically significant differences were observed (2.0–6.5 %, 4–13/140). The overall Rickettsia spp. infection rate was 52.5 % (735/1,400). In adult ticks, Rickettsia spp. infection rate was 56 % (79/141) divided into 61.7 % (37/60) infected females and 51.9 % (42/81) infected males. Nymphs showed an infection rate of 52.1 % (656/1,259). In contrast to A. phagocytophilum infections, no statistically significant differences in Rickettsia spp. infection rates among sampling sites (44.3–63.6 %, 62–89/140) were observed, whereas seasonal variations were obvious: the percentage of Rickettsia-positive ticks was significantly lower in April (36.5 %, 73/200) and May (29.5 %, 59/200) compared to the summer and fall months (55.0–64.5 %, 110–129/200). Rickettsia species differentiation via real-time pyrosequencing revealed Rickettsia helvetica as the only occurring species. Co-infections with both Rickettsia spp. and A. phagocytophilum were detected in 2.0 % (28/1,400) of the ticks. The present study revealed that in the city of Hamburg, the tick infection rate with A. phagocytophilum is comparable with other German data, whereas the Rickettsia spp. infection rate of 52.5 % is by far the highest prevalence detected in Germany so far. As the city of Hamburg has 1.8 million inhabitants and attracts millions of tourists every year, the potential health risk should not be underestimated.     

Prevalence of Theileria equi, Babesia caballi, and Anaplasma phagocytophilum in horses from the north of Portugal

Piroplasmid protozoa Theileria equi and Babesia caballi and zoonotic rickettsial bacterium Anaplasma phagocytophilum are important agents of equine vector-borne diseases (EVBD). This study aimed at investigating the prevalence of infections with or exposure to these pathogens in horses from the north of Portugal. Blood was randomly collected from 162 horses, living in 72 different stables, to prepare Giemsa-stained slide smears. Additionally, plasma samples were tested for antibodies to T. equi and B. caballi by two competitive enzyme-linked immunosorbent assays and to A. phagocytophilum by an indirect fluorescence antibody test. Five horses were positive to T. equi by microscopy (3.1 %), three to B. caballi (1.9 %), and none to A. phagocytophilum with no horse simultaneously positive for the two piroplasms. Clinically suspect animals had a significantly higher positivity to T. equi by microscopy in comparison with the nonsuspect ones (21.4 vs. 1.4 %). Twenty-nine horses were seropositive to T. equi (17.9 %), 18 to B. caballi (11.1 %), and 21 to A. phagocytophilum (13.0 %). Combined serology and microscopy positive results to T. equi and B. caballi were 19.1 and 11.7 %, respectively, with 33.3 % of the horses found positive to at least one agent. Forty horses were positive to single agents and 14 to more than one agent. An outdoor or mixed outdoor/indoor type of housing was found to be a risk factor for the combined positivity to T. equi. Infections with T. equi, B. caballi, and A. phagocytophilum are endemic in the north of Portugal. In addition to the treatment of positive horses, preventive measures should be put in practice to reduce exposure to and infection with agents of EVBD.     

Tick-borne encephalitis (TBE) virus prevalence and virus genome characterization in field-collected ticks (Ixodes ricinus) from risk,non-risk and former risk areas of TBE,and in ticks removed from humans in Germany

Tick-borne encephalitis (TBE) is recognized as the most important viral tick-borne zoonosis in 27 countries in Europe. In this study, ticks were collected in Germany from two non-risk areas in the states of Saxony-Anhalt and Mecklenburg-Western Pomerania, where several single human TBE cases have occurred in recent years. Ticks were also collected from a region in Thuringia, known to be a former risk area for TBE virus (TBEV), where numerous human cases were reported between 1960 and 1975. Detection of TBEV RNA was conducted by real-time RT-PCR. No TBEV was detected in any field-collected ticks. However, ticks were also collected from volunteers living in Bavaria. Three of 239 ticks from this collection were positive for TBEV genome and two genetically distinct TBEV strains were detected and characterized.     

Detection of a rickettsia closely related to Rickettsia aeschlimannii, "Rickettsia heilongjiangensis," Rickettsia sp. strain RpA4, and Ehrlichia muris in ticks collected in Russia and Kazakhstan

Using PCR, we screened 411 ticks from four genera collected in Russia and Kazakhstan for the presence of rickettsiae and ehrlichiae. In Russia, we detected "Rickettsia heilongjiangensis," Rickettsia sp. strain RpA4, and Ehrlichia muris. In Kazakhstan, we detected Rickettsia sp. strain RpA4 and a rickettsia closely related to Rickettsia aeschlimannii. These agents should be considered in a differential diagnosis of tick-borne infections in these areas.     

Infection and co-infection rates of Anaplasma phagocytophilum variants, Babesia spp., Borrelia burgdorferi, and the rickettsial endosymbiont in Ixodes scapularis (Acari: Ixodidae) from sites in Indiana, Maine, Pennsylvania, and Wisconsin

In total, 394 questing adult blacklegged ticks, Ixodes scapularis Say (Acari: Ixodidae), collected at four sites were analyzed by polymerase chain reaction (PCR) for five microbial species: Anaplasma phagocytophilum, Babesia microti, Babesia odocoilei, Borrelia burgdorferi, and the rickettsial I. scapularis endosymbiont. Identities of genetic variants of A. phagocytophilum were determined by sequencing a portion of the 16S DNA. In 55% of infected ticks (193/351), a single agent was detected. In 45% (158/351), two or more agents were detected; 37% harbored two agents and 8% harbored three agents. One male tick, collected from Ft. McCoy, WI, harbored all four microbial genera The highest rates of co-infection were by the Ixodes endosymbiont and B. burgdorferi (95/351). Two species of Babesia co-occurred within a single tick population in Wells National Estuarine Research Reserve, Wells, ME, whereas only B. odocoilei was found in other tick populations. Only A. phagocytophilum human anaplasmosis variant was detected in questing ticks from Tippecanoe River State Park, IN; from Wells; and Ft. McCoy, whereas a single infected tick from Presque Isle, PA, was infected by AP-Variant 1. Partially engorged ticks from deer in Tippecanoe River State Park were all infected with AP-Variant 1. Frequency of infections with each agent varied among populations. Rates and types of co-infections were not significantly different from random except for the Ixodes endosymbiont and B. burgdorferi in male ticks, which co-occurred less frequently than expected. Thus, I. scapularis hosts an array of pathogenic and symbiotic agents and potential evidence of interactions among microbial species was observed.     

Relative encounter frequencies and prevalence of selected Borrelia, Ehrlichia, and Anaplasma infections in Amblyomma americanum and Ixodes scapularis (Acari: Ixodidae) ticks from central New Jersey

To evaluate their relative importance in tick-borne disease transmission in New Jersey, host-seeking Amblyomma americanum (L.) and Ixodes scapularis Say adults and nymphs were collected during spring activity periods in 2003 and 2004 to determine relative frequencies at which these ticks were encountered from an area known to be hyperendemic for Lyme disease. Although similar numbers of the two species were encountered during early spring of both years, A. americanum were encountered more often later in the season and exhibited a longer activity period than I. scapularis. A. americanum nymphs were collected at frequencies between 2.6 and 7.3 times higher than I. scapularis nymphs. Polymerase chain reaction (PCR) analysis of 121 A. americanum adults yielded infection prevalences of 9.1% for Borrelia lonestari, 12.3% for Ehrlichia chaffeenensis, and 8.2% for E. ewingii, and coinfection prevalences of 4.1% for E. chaffeensis/E. ewingii and 0.8% for E. chaffeensis/B. lonestari. Infection prevalences in 147 I. scapularis adults were 50.3% for B. burgdorferi, 6.1% for Anaplasma (Ehrlichia) phagocytophilum, and 1.4% for a recently described novel Borrelia species, whereas the coinfection prevalences were 2.7% for B. burgdorferi/A. phagocytophilum, 0.7% for B. burgdorferi/novel Borrelia, and 0.7% for A. phagocytophilum/novel Borrelia. The B. burgdorferi infection prevalence in I. scapularis was considerably higher than that in A. americanum. However, the higher A. americanum encounter frequencies compared with I. scapularis may result in increased risk of acquiring exposure to A. americanum-transmitted pathogens. The potential public health implications of these results are discussed.