The role of Ixodes trianguliceps tick larvae in circulation of Babesia microti in the Middle Urals

The nested PCR method with primers flanking a conserved fragment of the Babesia microti ss-rDNA gene was used to examine 834 larvae of Ixodes trianguliceps ticks engorged to a varying degree, taken off 237 hosts of 12 species (rodents and insectivores). The hosts were collected in southern taiga forests in the lowmountain area of the Middle Urals (Chusovoi District, Perm Province) in 2003–2010. Babesia DNA was detected in 89 (10.7%) larvae from 8 species of small mammals. According to the data obtained by PCR and microscopic methods, either B. microti DNA or the parasites themselves were found in the blood of 45.2% of the mammals. The nucleotide sequences of 15 amplicons of Babesia DNA obtained from larvae of I. trianguliceps ticks and their hosts were identical to those of B. microti available in GenBank. In 13 cases, they were similar to B. microti US-type (a human pathogen) and in two cases (those from I. trianguliceps and from the vole Clethrionomys rufocanus from which it was removed), to B. microti of the Munich strain which is not pathogenic to humans. The duration of feeding on small mammals seems to exert the main influence on the infection rate of I. trianguliceps larvae. The fully engorged larvae contained B. microti DNA more often and usually in greater amounts than those collected during the first days of blood-sucking. The latter usually revealed Babesia DNA in the minimum quantity (< 0.064 ng/μl). According to the data obtained, transovarial transmission of Babesia in I. trianguliceps is unlikely. The processes of horizontal and transstadial transmission appear to be of crucial importance for the functioning of the natural foci of babesiosis.     

Host distribution and pathogen infection of fleas (Siphonaptera) recovered from small mammals in Pennsylvania

The number of recognized flea‐borne pathogens has increased over the past decade. However, the true number of infections related to all flea‐borne pathogens remains unknown. To better understand the enzootic cycle of flea‐borne pathogens, fleas were sampled from small mammals trapped in central Pennsylvania. A total of 541 small mammals were trapped, with white‐footed mice (Peromyscus leucopus) and southern red‐backed voles (Myodes gapperi) accounting for over 94% of the captures. Only P. leucopus were positive for examined blood‐borne pathogens, with 47 (18.1%) and ten (4.8%) positive for Anaplasma phagocytophilum and Babesia microti, respectively. In addition, 61 fleas were collected from small mammals and tested for pathogens. Orchopeas leucopus was the most common flea and Bartonella vinsonii subspecies arupensis, B. microti, and a Rickettsia felis‐like bacterium were detected in various flea samples. To the best of our knowledge, this is the first report of B. microti DNA detected from a flea and the first report of a R. felis‐like bacterium from rodent fleas in eastern North America. This study provides evidence of emerging pathogens found in fleas, but further investigation is required to resolve the ecology of flea‐borne disease transmission cycles.     

Zoological prerequisites of human tick-borne infections in the Northern Urals

The results of studies in the pessimum of the taiga tick (Ixodes persulcatus Sch., 1930) range in the Urals have shown that this species occurs in the regions at latitudes of up to 60°50′ N. In Northern Ural mountains, the ticks show preference for secondary small-leaved forests growing on southern slopes, where their abundance along census routes reaches 7 ind./km. The abundance and distribution of taiga ticks hosts, birds and small mammals, have been estimated in the main types of their habitats. Larvae and nymphs of the taiga tick have been found on nine bird and seven small mammal species, with the main parasitic load falling on the northern red-backed vole (Clethrionomys rutilus P., 1779), which is widespread and abundant in the greater part of the study area. Molecular biological analysis of I. persulcatus ticks and blood samples from their hosts has revealed the presence of nucleic acids of pathogenic microorganisms A. phagocytophilum, E. muris, and A. burgdorferi sensu lato, the RNA of tick-borne encephalitis virus, and the DNA of Babesia microti.     

The ecology of Ixodes trianguliceps ticks and their role in the natural foci of ixodid tick-borne borrelioses in the Middle Urals

From 1994 to 2011, over 7000 individuals of small mammals were captured and examined for ticks in the natural foci of ixodid tick-borne borrelioses (ITBB) in the Middle Urals (Chusovskoy District of Perm Territory). Alongside with the taiga tick (Ixodes persulcatus), which is the main Borrelia vector, approximately 5700 feeding individuals of Ixodes trianguliceps have been identified. The latter species has been found to be about five times less abundant than the former. I. trianguliceps has been collected from small mammals belonging to 19 species. Its main hosts are common shrews (Sorex araneus), bank voles (Clethrionomys glareolus), and northern red-backed voles (C. rutilus). I. trianguliceps shows two seasonal peaks of abundance, spring-summer, and summer-autumn. Plating in BSK II medium yielded 72 isolates of Borrelia from a total of 1142 individuals of I. trianguliceps; 64 isolates have been identified with PCR and RFLP. The mean values of the Borrelia infestation rate in I. trianguliceps larvae, nymphs, and adults are 2.6, 10.2, and 8.1%, respectively, which is 5–10 times lower than in the taiga tick individuals collected from the same mammals. Borreliae obtained from I. trianguliceps (as well as those from I. persulcatus) have been identified as Borrelia garinii and B. afzelii, the former spirochete species being more frequent (about 90% of isolates from I. trianguliceps). Our results indicate that I. trianguliceps ticks participate in circulation of the ITBB causative agents in the forests of the Middle Urals. Rare occurrence of the tick and low rates of its infestation with borreliae suggest that the species is unlikely to play a significant role in the epizootic development in the natural foci of ITBB.     

PCR identification of the host DNA in the taiga tick (Ixodinae: <Emphasis Type="Italic">Ixodes persulcatus</Emphasis>) nymphs in St. Petersburg and its environs

Host DNA from hungry males and females of Ixodes persulcatus (a total of 143 specimens) collected in the wild in April–May 2008 and 2009 was identified by means of the PCR method. Amplification of each sample was performed using a primer species-specific by the 12S rRNA mitochondrial gene. Four species of small mammals (Apodemus uralensis, Clethrionomys glareolus, Microtus arvalis, and Sorex araneus) and two species of passerine birds (Fringilla coelebs and Parus major) were analyzed. Hosts were established for 44 samples (30.8% of the material); in five cases (3.5%), feeding on more than one host was revealed.     

Natural infections of small mammals with blood parasites on the borderland of boreal and temperate forest zones

Blood parasites of small mammals living in Białowieża Forest (eastern Poland) were investigated between 1996 and 2002. The following haemoparasite species were found:Trypanosoma (Herpetosoma) evotomys in bank voleClethrionomys glareolus; T. (H.) microti in root voleMicrotus oeconomus; Babesia microti in root vole;Hepatozoon erhardovae in bank vole andHepatozoon sp. in root vole. Some non-identifiedBartonella species were found in bank vole, root vole, field voleMicrotus agrestis, yellow-necked mouseApodemus flavicollis, common shrewSorex araneus, Eurasian water shrewNeomys fodiens, and Mediterranean water shrewN. anomalus. The prevalence and diversity of blood parasites were lower in shrews than small rodents. Totally, 52.0% of bank voles, 50.0% of root voles, 32.5% of common shrews, and 41.2% of Eurasian water shrews were infected with any of the blood parasites. Mixed infections were seldom observed in bank vole (17.3% of investigated individuals) and root vole (14.7%). No animals were infected with three or four parasites simultaneously. Infection of Białowieża small mammals with haemoparasites seemed to be similar to those described in other temperate forest regions rather than boreal ones. Infection rates of rodent species seem to be higher in their typical habitats: for bank vole it was the highest in mixed forest, whereas for root vole in sedge swamp. The results suggest that Arvicolidae play a greater role than Muridae or Soricidae in maintenance ofBabesia andHepatozoon foci in natural environments of central Europe.     

Molecular Detection and Seroprevalence of Babesia microti among Stock Farmers in Khutul City,Selenge Province,Mongolia

Babesiosis is an emerging tick-borne disease in humans worldwide; however, little is known about the frequency of infection or prevalence of this disease in other parts of the world, excluding North America. In this study, we aimed to investigate Babesia microti infection frequency in a human population in Mongolia. One hundred blood samples were collected from stock farmers living in Khutul city of Selenge province, Mongolia. The sera and DNA from blood samples were evaluated for the presence of B. microti infection by using indirect fluorescent antibody (IFA) tests and PCR. The positive detection rates obtained using the IFA tests and PCR assays were 7% and 3%, respectively. This study is the first to detect of B. microti infections based on antibody seroprevalence or PCR assays for the presence of B. microti DNA in a Mongolian population.     

Genetic Variability in the Yellow-Necked Field Mouse (<Emphasis Type="Italic">Sylvaemus flavicollis</Emphasis> Melch., 1834, Muridae,Rodentia) at the Eastern Border of the Range

The first characterization of the genetic variability in populations of S. flavicollis on the eastern border of the range is presented. Seven individuals from the most northeastern habitat of the species (Middle Urals) were karotyped. No deviations from the standard chromosome set, either by the chromosome number or morphology, were revealed. Analysis of the mtDNA cytochrome b gene sequences (1133 bp) in 44 individuals from five populations on the eastern border of the species range (Middle and Southern Urals) resulted in identification of 17 haplotypes. All haplotypes were new and not found earlier in other parts of the species range. The genetic diversity indices and analysis of the demographic and genetic structure indicate a relatively recent origin of the populations under study as a result of rapid expansion. Phylogenetic analysis (97 haplotypes, including the GenBank data) showed that all haplotypes described at the eastern border of the range belonged to the same phylogroup distributed in the Balkan region, Northern and Eastern Europe, and Russia (Samara oblast). Close relationships between the examined populations and the populations from the northern part of the species range in Eastern Europe was demonstrated.     

Phenotypic variability of the pearly heath, Coenonympha arcania L. (Lepidoptera, Satyridae) in natural and anthropogenically transformed habitats in the Middle and South Urals

Analysis of variation of the wing length and eyespots in Coenonympha arcania (Linnaeus, 1761) (Lepidoptera, Satyridae) in the Middle and South Urals showed greater differences between populations from the forest and steppe zones than between populations from natural habitats and those subjected to chemical and radionuclide pollution. Based on small values of the fluctuating asymmetry (FA) index in all the habitats examined, it may be concluded that C. arcania finds favorable environment in both natural and anthropogenically transformed habitats in the Middle and South Urals.     

Molecular differentiation and taxonomy of the sunwatcher toad-headed agama species complex <Emphasis Type="Italic">Phrynocephalus</Emphasis> superspecies <Emphasis Type="Italic">helioscopus</Emphasis> (Pallas 1771) (Reptilia: Agamidae)

Lizards of the sunwatcher toad-headed agama species complex Phrynocephalus superspecies helioscopus, mostly distributed in Central Asia and Middle East, were examined using analysis of variation at the mitochondrial cytochrome oxidase c subunit I gene fragment and fingerprint analysis of nuclear DNA (inter-SINE PCR technique). A total of 86 individual tissue samples from 53 populations, to the full extent representing different parts of the species complex range, were subjected to molecular genetic examination, and surprisingly deep differentiation was revealed. The populations analyzed split into 12 isolated phylogroups, many of which were characterized by a narrow range and genetic isolation. Monophyly of sunwatcher (Ph. helioscopus) and Persian (Ph. persicus) toad-headed agamas was confirmed. However, both of these species probably represent the species complexes. Zoogeography of Central Asiais discussed.     

RAPHIDOPHYCEAE [CHADEFAUD EX SILVA] SYSTEMATICS AND RAPID IDENTIFICATION: SEQUENCE ANALYSES AND REAL‐TIME PCR ASSAYS1

Species within the class Raphidophyceae were associated with fish kill events in Japanese, European, Canadian, and U.S. coastal waters. Fish mortality was attributable to gill damage with exposure to reactive oxygen species (peroxide, superoxide, and hydroxide radicals), neurotoxins, physical clogging, and hemolytic substances. Morphological identification of these organisms in environmental water samples is difficult, particularly when fixatives are used. Because of this difficulty and the continued global emergence of these species in coastal estuarine waters, we initiated the development and validation of a suite of real‐time polymerase chain reaction (PCR) assays. Sequencing was used to generate complete data sets for nuclear encoded small‐subunit ribosomal RNA (SSU rRNA; 18S); internal transcribed spacers 1 and 2, 5.8S; and plastid encoded SSU rRNA (16S) for confirmed raphidophyte cultures from various geographic locations. Sequences for several Chattonella species (C. antiqua, C. marina, C. ovata, C. subsalsa, and C. verruculosa), Heterosigma akashiwo, and Fibrocapsa japonica were generated and used to design rapid and specific PCR assays for several species including C. verruculosa Hara et Chihara, C. subsalsa Biecheler, the complex comprised of C. marina Hara et Chihara, C. antiqua Ono and C. ovata, H. akashiwo Ono, and F. japonica Toriumi et Takano using appropriate loci. With this comprehensive data set, we were also able to perform phylogenetic analyses to determine the relationship between these species.     

Recombinase polymerase amplification with lateral flow strip for detecting Babesia microti infections

Babesia microti is one of the most important pathogens causing humans and rodents babesiosis—an emerging tick-borne disease that occurs worldwide. At present, the gold standard for the detection of Babesia is the microscopic examination of blood smears, but this diagnostic test has several limitations. The recombinase polymerase amplification with lateral flow (LF-RPA) assay targeting the mitochondrial cytochrome oxidase subunit I (cox I) gene of B. microti was developed in this study. The LF-RPA can be performed within 10–30 min, at a wide range of temperatures between 25 and 45 °C, which is much faster and easier to perform than conventional PCR. The results showed that the LF-RAP can detect 0.25 parasites/μl blood, which is 40 times more sensitive than the conventional PCR based on the V4 variable region of 18S rRNA. Specificity assay showed no cross-reactions with DNAs of related apicomplexan parasites and their host. The applicability of the LF-RPA method was further evaluated using two clinical human samples and six experimental mice samples, with seven samples were positively detected, while only three of them were defined as positive by conventional PCR. These results present the developed LF-RPA as a new simple, specific, sensitive, rapid and convenient method for diagnosing infection with B. microti. This novel assay was the potential to be used in field applications and large-scale sample screening.     

Distribution of gamasid mites on small mammals in Yunnan Province, China

An investigation of gamasid mites on the body surface of small mammals was carried out in Yunnan Province of China from 1990 to 2004. The small mammal hosts were captured from 25 counties which represent five geographical subregions, namely Middle Subregion of Hengduan Mountains, Southern Subregion of Hengduan Mountains, Eastern Plateau Subregion of Yunnan, Western Plateau Subregion of Yunnan and Southern Moun- tainous Subregion of Yunnan. The captured 10 803 small mammal hosts belong to nine families, 29 genera and 52 species in four orders （Rodentia, Insectivora, Scandentia and Lagomorpha）. A total of 68 571 gamasid mites were collected from the body surface of the captured small mammal hosts and all the gamasid mites were identified to 10 families, 33 genera and 112 species. This paper lists all the mite species, together with their taxonomic position （genera and families） and their corresponding hosts. Much more mite species were found in the Middle Subregion of Hengduan Mountains than in other geographical subregions. The total individuals of mites and small mammals in the Middle Subregion of Hengduan Mountains are also the most plentiful in the five geographical subregions. Three dominant mite species and three dominant small mammal hosts were determined as the dominant species in the investigated areas of Yunnan Province. The dominant hosts are Rattus flavipectus （which accounts for 34.85% of the total individuals）, Apodemus chevrieri （13.43%） and Rattus norvegicus （10.40%） while the dominant gamasid mite species are Laelaps nuttalli （Hirst, 1915） （27.84%）, Laelaps echidninus （Berlese, 1887） （18.38%） and Laelaps guizhouensis （Gu et Wang, 1981） （14.79%）. The results showed the high species diversity of gamasid mites in Yunnan Province and the uneven distribution feature in different subregions.     

Giardia microti in pet Microtus guentheri: Evidence of a parasite never detected in Italy

The genus Giardia includes several species distinguished by morphological, biological and molecular features. Currently, eight species within the genus are retained as valid. In Italy no identification of Giardia species other than Giardia duodenalis has been so far reported. Fecal samples were collected from two Günther's Voles (Microtus guentheri) positive to Giardia cysts by microscopic investigation and immunofluorescence. The voles were born in Milan (Northern Italy) from two gravid females imported from the Netherlands and kept for sale in a pet shop in Varese (Northern Italy). Positive feces were subjected to a nested PCR to amplify a 18S rRNA fragment for molecular characterization. A phylogenetic analysis was conducted to compare the obtained sequence with those of all other Giardia species available in GenBank for the 18S locus, using the Maximum Likelihood (ML) method by R software. Sequence analyses unambiguously identified the isolates as belonging to G. microti, showing 99% of identity with those of its isolates available in GenBank. A well-defined cluster, supported by significant bootstrap values and corresponding to the G. microti cluster, including sequences obtained from M. guentheri, was evidenced in the ML tree, confirming species assignment. The present finding represents the first report of G. microti from pet animals in Italy.     

Evidence of <Emphasis Type="Italic">Babesia microti</Emphasis> infection in multi-infected <Emphasis Type="Italic">Ixodes persulcatus</Emphasis> ticks in Russia

To detect Babesia-infected Ixodes persulcatus Shulze in a suburb of St. Petersburg, Russia, 738 adult ticks were studied using Babesia specific primers and PCR techniques. The entire sample (more than 1,200 individuals) was screened for the presence of Borrelia spp., Ehrlichia spp. and tick-borne encephalitis virus (TBEV). All 7 ticks infected with Babesia microti, were also infected with other pathogens (all 7 among 417 infected ticks, zero amongst the remaining 321 naive ones (χ² = 5.25, p < 0.05). Babesia microti occurred twice with Borrelia afzelii, 3 times with Borrelia garinii, once with both, and once with both B. garinii and TBEV. The prevalence of infection with Borrelia spp. was 34.0%, with Ehrlichia spp. 6.2%, with TBEV 1.5%, and with Ba microti 0.9%. Babesia microti infection was not found in combination with Ehrlichia sp. or Borrelia burgdorferi sensu stricto. The latter pathogen (prevalence 2.6%), just like Ba. microti, was not encountered as a monoinfection. The data suggest that Ba. microti infection can only survive in I. persulcatus in combination with Borrelia spp. (7 of 7 infections). The disease in humans is more severe and longer-lasting when more than one pathogen is involved. Our observations show that the well known St. Petersburg focus of tick-borne encephalitis and Lyme disease is also a focus of ehrlichiosis and babesiosis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ancient DNA sequences point to a large loss of mitochondrial genetic diversity in the saiga antelope (Saiga tatarica) since the Pleistocene

Prior to the Holocene, the range of the saiga antelope (Saiga tatarica) spanned from France to the Northwest Territories of Canada. Although its distribution subsequently contracted to the steppes of Central Asia, historical records indicate that it remained extremely abundant until the end of the Soviet Union, after which its populations were reduced by over 95%. We have analysed the mitochondrial control region sequence variation of 27 ancient and 38 modern specimens, to assay how the species’ genetic diversity has changed since the Pleistocene. Phylogenetic analyses reveal the existence of two well‐supported, and clearly distinct, clades of saiga. The first, spanning a time range from >49 500 ¹⁴C ybp to the present, comprises all the modern specimens and ancient samples from the Northern Urals, Middle Urals and Northeast Yakutia. The second clade is exclusive to the Northern Urals and includes samples dating from between 40 400 to 10 250 ¹⁴C ybp. Current genetic diversity is much lower than that present during the Pleistocene, an observation that data modelling using serial coalescent indicates cannot be explained by genetic drift in a population of constant size. Approximate Bayesian Computation analyses show the observed data is more compatible with a drastic population size reduction (c. 66–77%) following either a demographic bottleneck in the course of the Holocene or late Pleistocene, or a geographic fragmentation (followed by local extinction of one subpopulation) at the Holocene/Pleistocene transition.     

Vertical Transmission of Babesia microti in BALB/c Mice: Preliminary Report

Babesia spp. (Apicomplexa, Piroplasmida) are obligate parasites of many species of mammals, causing a malaria-like infection- babesiosis. Three routes of Babesia infection have been recognized to date. The main route is by a tick bite, the second is via blood transfusion. The third, vertical route of infection is poorly recognized and understood. Our study focused on vertical transmission of B. microti in a well-established mouse model. We assessed the success of this route of infection in BALB/c mice with acute and chronic infections of B. microti. In experimental groups, females were mated on the 1^st day of Babesia infection (Group G0); on the 28^th day post infection (dpi) in the post- acute phase of the parasite infection (G28); and on the 90^th and 150^th dpi (G90 and G150 group, respectively), in the chronic phase of the parasite infection. Pups were obtained from 58% of females mated in the post-acute phase (G28) and from 33% of females in groups G90 and G150. Mice mated in the pre-acute phase of infection (G0) did not deliver pups. Congenital B. microti infections were detected by PCR amplification of Babesia 18S rDNA in almost all pups (96%) from the experimental groups G28, G90 and G150. Parasitaemia in the F1 generation was low and varied between 0.01–0.001%. Vertical transmission of B. microti was demonstrated for the first time in BALB/c mice.     

Distribution of small rodents and shrews in a lowland rain forest zone of Nigeria, with observations on their reproductive biology

This paper describes the habitat preferences of small rodents and shrews from April 1984 to March 1985 in four types of vegetation in a lowland rain forest zone of Nigeria. The vegetation types were high forest, scrub, farmlands and teak plantations in Ogba Forest Reserve, Nigeria. A collection of 359 small mammals, consisting of six species of small rodents and four species of shrews, was made in the study area. The small rodents and their percentage composition were Mus musculoides (26.2%), Praomys tullbergi (16.2%), Mastomys natalensis (7.0%), Lophuromys sikapusi (4.5%), Lemniscomys striatus (2.5%), and Arvicanthis niloticus (1.1%). The shrews were Crocidura nigeriae (19.5%), C. crossei (12.3%), C.grandiceps (7.8%) and C. ftavescens manni (3.1%). The vegetational distribution of the small rodents and shrews is discussed. In the high forest, seven species of small mammals were caught; these were Crocidura nigeriae, C. crossei, C.grandiceps, C.flavescens manni, Mus musculoides, Praomys tullbergi and Lophuromys sikapusi. Mus musculoides, Crocidura nigeriae and Praomys tullbergi were more abundant in the high forest. All the species of small mammals recorded in the study area, except Arvicanthis niloticus, were found in the scrub. In the farmlands, all ten species of small mammals were trapped. Mus musculoides was the commonest species in the farmlands. Three species of shrews, Crocidura crossei, C. grandiceps and C.flavescens manni, and also two species of small rodents, Mus musculoides and Mastomys natalensis were caught in the teak plantation. The reproductive data for Mus musculoides, Crocidura nigeriae and Praomys tullbergi show that breeding in these species occurs throughout most months of the year. Fecundity appears to be maximal in the wet season in Mus musculoides and Crocidura nigeriae and in the dry season in the case of Praomys tullbergi.     

Investigating disease severity in an animal model of concurrent babesiosis and Lyme disease

The incidence of babesiosis, Lyme disease and other tick-borne diseases has increased steadily in Europe and North America during the last five decades. Babesia microti is transmitted by species of Ixodes, the same ticks that transmit the Lyme disease-causing spirochete, Borrelia burgdorferi. B. microti can also be transmitted through transfusion of blood products and is the most common transfusion-transmitted infection in the U.S.A. Ixodes ticks are commonly infected with both B. microti and B. burgdorferi, and are competent vectors for transmitting them together into hosts. Few studies have examined the effects of coinfections on humans and they had somewhat contradictory results. One study linked coinfection with B. microti to a greater number of symptoms of overall disease in patients, while another report indicated that B. burgdorferi infection either did not affect babesiosis symptoms or decreased its severity. Mouse models of infection that manifest pathological effects similar to those observed in human babesiosis and Lyme disease offer a unique opportunity to thoroughly investigate the effects of coinfection on the host. Lyme disease has been studied using the susceptible C3H mouse infection model, which can also be used to examine B. microti infection to understand pathological mechanisms of human diseases, both during a single infection and during coinfections. We observed that high B. microti parasitaemia leads to low haemoglobin levels in infected mice, reflecting the anaemia observed in human babesiosis. Similar to humans, B. microti coinfection appears to enhance the severity of Lyme disease-like symptoms in mice. Coinfected mice have lower peak B. microti parasitaemia compared to mice infected with B. microti alone, which may reflect attenuation of babesiosis symptoms reported in some human coinfections. These findings suggest that B. burgdorferi coinfection attenuates parasite growth while B. microti presence exacerbates Lyme disease-like symptoms in mice.