Molecular identification of Babesia parasites isolated from Ixodes ricinus ticks collected in northwestern Poland

In the present study, PCR has been applied to detect and analyze DNA of Babesia spp. extracted from Ixodes ricinus ticks. Collection of I. ricinus was made in 6 forested areas of Zachodniopomorskie Voivodship, Poland, during 2 seasonal peaks of tick activity, i.e., spring and autumn, 2001. In total, 1,328 I. ricinus were collected and processed for PCR with F34 and R323 primers. Babesia spp. was detected in 28 (2% of 1,328 tested) ticks; 26 were identified as B. divergens. The other 2 were identified as B. microti. PCR was conducted with 18S rRNA specific primers and sequencing was processed to precisely identify and compare these isolates with B. microti and B. divergens sequences from Europe, North America, and Asia obtained from the GenBank. Analysis revealed that sequences of B. microti from northwestern Poland are almost identical (99.94%) with those referred to as "Munich strain"; both form a clade different from other European strains, as well as those from Asia and North America (called B. microti, sensu stricto). An investigation performed with B. divergens sequences showed that the sequence from northwestern Poland is 99.94% homologous to an isolate from Ireland ("Purnel"), and differs in just a few nucleotides from other European sequences. Phylogenetic analysis revealed that the sequence of B divergens isolated from Polish ticks form a group that comprise 4 European sequences from Great Britain and Ireland and is, therefore, closely related to other European and North American B. divergnens sequences.     

Splenomegaly and Reticulocytosis Caused by Babesia microti Infections in Natural Populations of the Montane Vole, Microtus montanus

ABSTRACT. A survey for Babesia microti in rodents was conducted at six sites within Grand Teton National Park, Wyoming. Blood and spleen smears, hematocrits, and reticulocyte counts were made on all of the animals to evaluate parameters for the diagnosis of babesiosis. Ticks were removed for identification. Of 257 Microtus montanus , 103 were infected with B. microti. In addition, five of 12 Microtus pennsylvanicus and one of three Arvicola richardsoni were parasitized by B. microti. Peromyscus maniculatus (n = 40) were not infected. Concurrent infections by Hepatozoon sp., Trypanosoma sp., and the bacterium, Grahamella sp., were noted in blood smears from a number of M. montanus. Splenomegaly and reticulocytosis were significant parameters associated with babesiosis while decreased hematocrit was not. Ticks removed from the voles were identified as Ixodes eastoni and were the probable vectors of the B. microti.     

Splenomegaly and reticulocytosis caused by Babesia microti infections in natural populations of the montane vole, Microtus montanus.

A survey for Babesia microti in rodents was conducted at six sites within Grand Teton National Park, Wyoming. Blood and spleen smears, hematocrits, and reticulocyte counts were made on all of the animals to evaluate parameters for the diagnosis of babesiosis. Ticks were removed for identification. Of 257 Microtus montanus, 103 were infected with B. microti. In addition, five of 12 Microtus pennsylvanicus and one of three Arvicola richardsoni were parasitized by B. microti. Peromyscus maniculatus (n = 40) were not infected. Concurrent infections by Hepatozoon sp., Trypanosoma sp., and the bacterium, Grahamella sp., were noted in blood smears from a number of M. montanus. Splenomegaly and reticulocytosis were significant parameters associated with babesiosis while decreased hematocrit was not. Ticks removed from the voles were identified as Ixodes eastoni and were the probable vectors of the B. microti.     

Immunity of dogs against Babesia canis, its vector tick Dermacentor reticulatus, and Ixodes ricinus in endemic area

Previous epidemiological studies allowed us to accurately define endemic areas of canine babesiosis and tick distribution in southeastern France (Martinod, 1983). Using a micro-ELISA test 100 dogs sera were tested with 3 antigens: Babesia canis, Dermacentor reticulatus and Ixodes ricinus. Antibodies against B. canis and its vector D. reticulatus were detected in an endemic area, sometimes with high levels (optical density 1.38 and 0.80 respectively). A correlation factor and regression lines were found between ELISA activity of B. canis and vector tick antigens, even for dogs which never showed any babesiosis symptoms. These results were compared with those of an area without any babesiosis. Furthermore I. ricinus antigens detected ELISA activity in sera of dogs; some cross reactions were observed between I. ricinus and D. reticulatus antigen.     

Animal babesiosis: an emerging zoonosis also in Italy?

In Italy, babesiosis is widespread in several Central and Southern Regions, but few data are available on its presence in most Italian areas. In 2004 a project was financed by the MIUR to investigate on the babesiosis epidemiology in vertebrate and invertebrate hosts, and on the transmission risk for humans in Central and Northern Regions of the country. Microscopy and/or molecular tools were applied to blood samples of wild animals, livestock and pets, and to 1,677 ticks collected on animals or in the environment, with the aim of detect babesial parasites. Moreover, serological tests were used to evaluate the circulation of these protozoa among animals and people at risk. Microscopy identified as positive 5.0% of the animals, mostly living in Central Regions, but also in Northern areas considered Babesia-free. Serology evidenced the same general trend. PCR detected "piroplasm" DNA in 13.8% of the animals, and sequencing identified babesial parasites in 101/233 samples. The ticks were identified as belonging to 12 species, mostly represented by Ixodes ricinus, Rhipicephalus sanguineus and Dermacentor marginatus. Molecular analyses evidenced babesial parasites in 3.8% of them; in Rh. sanguineus was also demonstrated the vertical transmission of Babesia canis canis. To date 30 human sera have been analysed: 3 showed antibodies to B. microti. Animal babesiosis is largely present among pets, wild and farm animals, whereas goats seem refractory to the infection. In wild ungulates have been found the B. divergens-like, and the Babesia EU1 strains (reported in Italy in humans). Our findings evidenced the low reliability of microscopy in epidemiological studies, and the need of new/improved immunological tests to face diagnostic problems. The monitoring of infected areas and infection rates, joined to appropriate control programs, seems necessary to avoid the transmission of babesiosis to humans.     

Molecular evidence of coinfection of Borrelia burgdorferi sensu lato,human granulocytic ehrlichiosis agent,and Babesia microti in ticks from northwestern Poland

To assess the potential risk for tick-borne agents, Ixodes ricinus were collected from 2 sites in northwestern Poland. The ticks were tested by polymerase chain reaction for coinfection with Borrelia burgdorferi sensu lato (s. l.), human granulocytic ehrlichiosis (HGE) agent, and Babesia microti. Of the 533 processed ticks, 16.7% were positive for B. burgdorferi s. l., 13.3% for B. microti, and 4.5% for the HGE agent. Twenty ticks were coinfected with 2 or 3 of the pathogens.     

Detection of Kobe-type Babesia microti associated with Japanese human babesiosis in field rodents in central Taiwan and southeastern mainland China

Field rodent surveys for Babesia infection were performed from 2002 to 2005 in the vicinities of human babesiosis occurrences in Taiwan and mainland China. Babesia microti was identified by microscopical examination and/or PCR in 1 Rattus coxinga and 1 Crocidura horsfieldii in central Taiwan and in 13 Niviventer confucianus and 1 Apodemus agrarius in Zhejiang and Fujian Provinces of southeastern China. Of 15 B. microti samples detected by PCR, all except 1 were shown to be the Kobe-type, the aetiological small subunit rRNA gene-type of the first Japanese patient; the exception was also a Kobe-related type. The Kobe-type had been found in rodents only in a few places including the human infection occurrence place in Japan. The internal transcribed spacer 1 to 2 sequences of the Taiwanese and Chinese Kobe-types were very similar to each other but considerably different (approx. 94% pairwise identities) from that of the Japanese Kobe-type. A Taiwanese Kobe-type strain was serologically differentiated from the Kobe strain originating from the Japanese first patient. The distribution of the Kobe-type in the vicinities of human babesiosis occurrences in Taiwan and China as well as in Japan is suggestive of involvement of the Kobe-type in Asian human babesiosis.     

General review of tick species present in Portugal

At present, 24 species are known to occur in Portugal: Argas vespertilionis, Ornithodoros maritimus and Ornithodoros erraticus in Argasidae; Ixodes acuminatus, Ixodes bivari, Ixodes canisuga, Ixodes frontalis, Ixodes hexagonus, Ixodes ricinus, Ixodes simplex simplex, Ixodes ventalloi, Ixodes vespertilionis, Dermacentor marginatus, Dermacentor pictus', Haemaphysalis hispanica, Haemaphysalis inermis, Haemaphysalis punctata, Rhipicephalus bursa, Rhipicephalus pusillus, Rhipicephalus sanguineus, Rhipicephalus turanicus, Hyalomma lusitanicum, Hyalomma marginatum marginatum and Boophilus annulatus in Ixodidae. The more relevant diseases transmitted to cattle by ticks, particulary in Ribatejo and Alentejo regions, are the babesiosis due to Babesia bigemina and Babesia bovis, the theileriosis by Theileria annulata and the anaplasmosis due to Anaplasma marginale; the theileriosis by Theileria mutans2 may not be considered significant. The sheep and goats parasitoses transmitted by ticks are of less importance than the cattle diseases. However the babesiosis due to Babesia motasi and Babesia ovis and also theileriosis by Theileria hirci3 are present in some districts of the country.     

Molecular characterization of human pathogen Babesia EU1 in Ixodes ricinus ticks from Slovenia

New cases of human babesiosis were recently reported in Europe. The etiological agent was identified as Babesia EU1, a zoonotic pathogen with previously unreported molecular characteristics. On the basis of a comparison of the complete babesial 18S rRNA gene, we have generated strong molecular evidence that Ixodes ricinus ticks from Slovenia are infected with EU1.     

A molecular marker for the identification of the zoonotic reservoirs of Lyme borreliosis by analysis of the blood meal in its European vector Ixodes ricinus.

The efficacy of the mitochondrially encoded cytochrome b gene as a molecular marker for the discrimination of the reservoir host species of the Lyme borreliosis spirochete, Borrelia burgdorferi sensu lato (s.l.), in its European vector Ixodes ricinus (Acari: Ixodidae) was determined. Degenerate PCR primers were designed which amplified orthologous regions of the cytochrome b gene in several animal species which act as B. burgdorferi s.l. reservoirs and hosts for I. ricinus. PCR products were amplified and characterized by hybridization and restriction fragment length polymorphism analysis. Restriction fragment length polymorphism analysis of a 638-bp PCR product with HaeIII and DdeI revealed unique restriction fragment profiles, which allowed the taxonomic identification of animals to the genus level. A system was devised for the detection of the larval host blood meal from the remnants in unfed nymphal I. ricinus ticks by nested PCR amplification. An inverse correlation was demonstrated between amplicon size and successful PCR amplification of host DNA from the nymphal stage of the tick. The stability of the cytochrome b product as a marker for the identification of the larval host species in the nymphal instar was demonstrated up to 200 days after larval ingestion (approximately 165 days after molting) by reverse line blotting with a host-specific probe. This assay has the potential for the determination of the reservoir hosts of B. burgdorferi s.l. by using extracts from the same individual ticks for both the identification of the host species and the detection of the Lyme borreliosis spirochete.     

A double antibody sandwich enzyme-linked immunosorbent assay for detection of secreted antigen 1 of Babesia microti using hamster model

A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) targeting secreted antigen 1 of Babesia microti (BmSA1) was developed for detection of B. microti infection. The optimized DAS-ELISA was sensitive enough to detect circulating BmSA1 by day 2 post-infection, in sequential sera of a hamster infected with B. microti. This detection was 4 days earlier than antibody detection by indirect ELISA. The kinetics of circulating BmSA1 coincided with the profile of parasitemia. The specificity of this assay was evaluated using sera from animals experimentally infected with different species of Babesia. The DAS-ELISA had a higher sensitivity than the microscopic examination of Giemsa-stained blood smears for detection of the infection in hamsters. Taken together, these results indicated that BmSA1 could be a potential marker for surveillance of human babesiosis.     

Borrelia burgdorferi and Babesia microti: efficiency of transmission from reservoirs to vector ticks (Ixodes dammini)

In endemic regions, Peromyscus leucopus, the mouse reservoir of the Lyme disease spirochete (Borrelia burgdorferi) and the piroplasm causing human babesiosis (Babesia microti), is nearly universally infected with both agents. Paradoxically, spirochetal infection is nearly twice as prevalent as is babesial infection in populations of field-collected nymphal Ixodes dammini, the tick vector. In the laboratory, a similarly disproportionate rate of infection was observed among nymphal ticks, feeding as larvae, on either B. burgdorferi- or B. microti-infected mice. Ticks which fed on mice with concurrent spirochetal and babesial infections also exhibited twice the incidence of spirochetal infection over that of the piroplasm. These data suggest that the efficiency of acquisition and transstadial passage of B. burgdorferi and B. microti infection differ by a factor of two. This discrepancy may explain differences observed both in the prevalence of infection in ticks collected in the field, as well as the apparently greater risk of spirochetal infection to humans in endemic areas.     

Babesia divergens in France: descriptive and analytical epidemiology

Babesia divergens cause of bovine babesiosis transmitted by Ixodes ricinus, is widely spread especially in West, Central and South-West parts of France. It occurs with two annual peaks, in spring and autumn. The study was carried out during a period of two years (1991-1993) in four farms in the Sarthe area, in order to know the distribution and the ecology of I. ricinus, and to show the presence of B. divergens. Cattle are parasitised as early as March essentially by adult ticks, according to a seasonal distribution (spring, autumn). The flag method allows to catch essentially the larvae and the nymphs on the pastures; nymphs appear as early as March, and larvae one to two months later. Ectoparasite collection on trapped micromammals (Apodemus sylvaticus, Clethrionomys glareolus, etc.) in pastures hedges, is used to detect small I. ricinus populations, mostly larvae. A new ELISA method has been used for the study of the kinetics of anti-B. divergens antibodies in 236 cattle during two years. Most of the animals (60%) show a high antibody level, essentially at the end of the pasture season; the percentage of positive animals decreases during winter and increases again during the pasture season. Calves become seropositive since their first months on pastures. Adults show asymptomatic infections several times along the year, mostly during spring and autumn; only three clinical babesiosis cases have been observed during the whole study, in animals exhibiting nevertheless a high specific antibody level.     

Factors driving the abundance of ixodes ricinus ticks and the prevalence of zoonotic I. ricinus-borne pathogens in natural foci

Environmental factors may drive tick ecology and therefore tick-borne pathogen (TBP) epidemiology, which determines the risk to animals and humans of becoming infected by TBPs. For this reason, the aim of this study was to analyze the influence of environmental factors on the abundance of immature-stage Ixodes ricinus ticks and on the prevalence of two zoonotic I. ricinus-borne pathogens in natural foci of endemicity. I. ricinus abundance was measured at nine sites in the northern Iberian Peninsula by dragging the vegetation with a cotton flannelette, and ungulate abundance was measured by means of dung counts. In addition to ungulate abundance, data on variables related to spatial location, climate, and soil were gathered from the study sites. I. ricinus adults, nymphs, and larvae were collected from the vegetation, and a representative subsample of I. ricinus nymphs from each study site was analyzed by PCR for the detection of Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum DNA. Mean prevalences of these pathogens were 4.0% ± 1.8% and 20.5% ± 3.7%, respectively. Statistical analyses confirmed the influence of spatial factors, climate, and ungulate abundance on I. ricinus larva abundance, while nymph abundance was related only to climate. Interestingly, cattle abundance rather than deer abundance was the main driver of B. burgdorferi sensu lato and A. phagocytophilum prevalence in I. ricinus nymphs in the study sites, where both domestic and wild ungulates coexist. The increasing abundance of cattle seems to increase the risk of other hosts becoming infected by A. phagocytophilum, while reducing the risk of being infected by B. burgdorferi sensu lato. Controlling ticks in cattle in areas where they coexist with wild ungulates would be more effective for TBP control than reducing ungulate abundance.     

Enzootic Babesia microti in Maine

Human babesiosis in the northeastern United States caused by Babesia microti (Apicomplexa: Piroplasmida) is mainly reported from coastal New England sites, where deer ticks (Ixodes dammini) are common. However, the piroplasm has been detected in microtine rodents elsewhere in association with I. angustus or other nidicolous ticks, suggesting that the agent is widely distributed but zoonotically significant only where a human-biting "bridge" vector is present. To determine whether this piroplasm may be enzootic in areas where I. dammini is absent, we surveyed small mammals collected from 2 sites in Maine, where I. angustus or I. muris is common but I. dammini is not. Of 43 chipmunks, voles, deer mice, and shrews examined, 3 (6.9, 95% confidence interval 0 to 14.5) were parasitemic, as determined by blood smear or polymerase chain reaction targeting a piroplasm-specific portion of the 18S ribosomal DNA gene. Phylogenetic analysis of the sequenced amplification products demonstrates the presence of 2 forms of B. microti. We conclude that B. microti may be enzootic in the absence of I. dammini but that human risk relates to dense infestations of this human-biting tick.     

Prevalence of Borrelia burgdorferi sensu lato genospecies in Ixodes ricinus ticks in Europe: a metaanalysis

In Europe, Borrelia burgdorferi genospecies causing Lyme borreliosis are mainly transmitted by the tick Ixodes ricinus. Since its discovery, B. burgdorferi has been the subject of many epidemiological studies to determine its prevalence and the distribution of the different genospecies in ticks. In the current study we systematically reviewed the literature on epidemiological studies of I. ricinus ticks infected with B. burgdorferi sensu lato. A total of 1,186 abstracts in English published from 1984 to 2003 were identified by a PubMed keyword search and from the compiled article references. A multistep filter process was used to select relevant articles; 110 articles from 24 countries contained data on the rates of infection of I. ricinus with Borrelia in Europe (112,579 ticks), and 44 articles from 21 countries included species-specific analyses (3,273 positive ticks). These data were used to evaluate the overall rate of infection of I. ricinus with Borrelia genospecies, regional distributions within Europe, and changes over time, as well as the influence of different detection methods on the infection rate. While the infection rate was significantly higher in adults (18.6%) than in nymphs (10.1%), no effect of detection method, tick gender, or collection period (1986 to 1993 versus 1994 to 2002) was found. The highest rates of infection of I. ricinus were found in countries in central Europe. B. afzelii and B. garinii are the most common Borrelia species, but the distribution of genospecies seems to vary in different regions in Europe. The most frequent coinfection by Borrelia species was found for B. garinii and B. valaisiana.     

Identification and characterization of a novel secreted antigen 1 of Babesia microti and evaluation of its potential use in enzyme-linked immunosorbent assay and immunochromatographic test

Here, we identified a novel secreted antigen designated as Babesia microti secreted antigen 1 (BmSA1) by immunoscreening a B. microti cDNA expression library using the sera from hamsters immunized with plasma, putatively containing secreted antigens, from B. microti-infected hamsters. Antibodies raised in mice immunized with recombinant BmSA1 (rBmSA1) recognized a native 33-kDa parasite protein. An enzyme-linked immunosorbent assay (ELISA) of rBmSA1 detected specific antibodies as early as 6 and 4 days post-infection in sera from a hamster experimentally infected with B. microti Gray strain (US type) and a mouse experimentally infected with B. microti Munich strain (rodent isolate), respectively. Moreover, a rapid immunochromatographic test (ICT) using rBmSA1 detected specific antibodies in a hamster experimentally infected with B. microti from day 6 to at least day 270 post-infection, which was quite consistent with the results of the ELISA. In addition, analysis of the sera involved in the first case of human babesiosis in Japan (Kobe type) showed that specific antibodies were detectable in the patient and the positive donor by ELISA using rBmSA1, and the ICT result was identical to the ELISA data. Taken together, these results indicated that BmSA1 could be a promising and universal target for developing both ELISA and ICT for the serodiagnosis of human babesiosis and for an epidemiological survey of its rodent reservoir.     

Human babesiosis, an emerging zoonosis

Data of human babesiosis are shortly reviewed with particular emphasis to Europe. In Europe, most cases of human babesiosis are caused by Babesia divergens. Although both phenotypic and genotypic features suggest that zoonotic B. microti may occur in Europe, convincing medical evidence is lacking. Recently a non-Babesia divergens organism causing zoonotic infection has been found in Italy and Austria. Overall, the seroprevalence against both B. microti and B. divergens microrganisms in human ranges 1.5%-11.5% in Europe.     

Distinct combinations of Borrelia burgdorferi sensu lato genospecies found in individual questing ticks from Europe

The genetic diversity of Borrelia burgdorferi sensu lato was assessed in individual adult Ixodes ricinus ticks from Europe by direct PCR amplification of spirochetal DNA followed by genospecies-specific hybridization. Analysis of mixed infections in the ticks showed that B. garinii and B. valaisiana segregate from B. afzelii. This and previous findings suggest that host complement interacts with spirochetes in the tick, thereby playing an important role in the ecology of Lyme borreliosis.     

Detection of Babesia spp. DNA in small mammals and ixodic ticks on the territory of north Ural, west Siberia and far east of Russia

Totally, 932 small mammals and 458 questing adult Ixodes persulcatus from Sverdlovsk and Novosibirsk regions and Khabarovsk Territory, as well as 128 Haemaphysalis japonica, 34 H. concinna and 29 Dermacentor silvarum from Khabarovsk Territory were examined for the presence of Babesia by nested PCR based on the 18S rRNA gene. Babesia microti DNA was found in samples of small mammals from all the studied regions--in 36.2% of samples from Sverdlovsk region, 5.3% of samples from Novosibirsk region, and 6.7% of samples from Khabarovsk Territory. The determined B. microti 18S rRNA gene sequences from Novosibirsk region (6 sequences) and from Khabarovsk Territory (10 sequences) were identical to each other and to the sequences of pathogenic for human B. microti US-type, while the determined B. microti 18S rRNA gene sequences from Sverdlovsk region (12 sequences) were identical to those of B. microti strain Munich. B. microti were found most frequently in samples of Myodes spp., they were found also in Microtus spp., Apodemus spp., Sorer spp., and Sicista betulinav. It was shown that one of 347 analyzed I. persulcatus from Novosibirsk region and one of 77 I. persulcatus from Khabarovsk Territory contained B. microti US-type DNA. One I. persulcatus from Novosibirsk region contained B. divergens DNA. In this work B. divergens was for the first time determined in I. persulcatus and B. microti in I. persulcatus in Asian part of Russia. Three different genetic variants of Babesia sensu stricto were found in three H. japonica from Khabarovsk Territory. The first genetic variant was closely related to Babesia sp. revealed in a feral raccoon in Japan (99.9% similarity on the basis of 18S rRNA gene sequences). Two others Babesia genetic variants were most similar to the ovine pathogen Babesia crassa (97.1-97.6% similarity on the basis of 18S rRNA gene sequences).