Molecular detection of Bartonella species infecting rodents in Slovenia

Rodents, collected in three zoogeographical regions across Slovenia, were tested for the presence of bartonellae using direct PCR-based amplification of 16S/23S rRNA gene intergenic spacer region (ITS) fragments from splenic DNA extracts. Bartonella DNA was detected in four species of rodents, Apodemus flavicollis, Apodemus sylvaticus, Apodemus agrarius and Clethrionomys glareolus, in all three zoogeographic regions at an overall prevalence of 40.4%. The prevalence of infection varied significantly between rodent species and zoogeographical regions. Comparison of ITS sequences obtained from bartonellae revealed six sequence variants. Four of these matched the ITS sequences of the previously recognized species, Bartonella taylorii, Bartonella grahamii, Bartonella doshiae and Bartonella birtlesii, but one was new. The identity of the bartonellae from which the novel ITS sequences was obtained were further assessed by sequence analysis of cell division protein-encoding gene (ftsZ) fragments. This analysis demonstrated that the strain is most likely a representative of possible new species within the genus.     

The use of arboreal runways by the woodland rodents, Apodemus sylvaticus (L.), A. flavicollis (Melchior) and Clethrionomys glareolus (Schreber)

The use of arboreal runways by the rodents, Apodemus sylvaticus, A. flavicollis and Clethrionomys glareolus , was investigated in the deciduous woodland of Woodchester Park, Gloucestershire, England. Longworth live traps were tied to horizontal and inclining branches of trees and shrubs up to 3 m. Arboreality was extensive in all three species, although less in C. glareolus than in either Apodemus species. There was no indication that A. flavicollis consistently used arboreal runways more than A. sylvaticus but the data suggest that the peak of arboreal activity of these congeners occurred in different seasons. In A. sylvaticus , males were more arboreal than females and adults were more arboreal than juveniles. In, 4. flavicollis , juveniles were trapped significantly more often on arboreal runways than adults. The reasons for, and the consequences of, arboreality in small rodents are discussed.     

Wild small mammals are the reservoir hosts of the Bartonella genus bacteria in the south of Moscow region

A total of 103 blood samples collected from wild small mammals captured in the Prioksko-Terrasny Reserve on the south of Moscow region were studied to determine the bartonellae prevalence. The examined species were the yellow-necked mice Apodemus flavicollis (35 samples), the European wood mouse Apodemus uralensis (10 samples), the bank vole Clethrionomys glareolus (51 samples), the house mouse Mus musculus (3 samples), the common vole Microtus arvalis (2 samples), and the shrew Sorex araneus (2 samples). Initially, we obtained 76 bacterial Bartonella-like isolates after plating onto the surface of the solid nutrient media. 66 of them were PCR-positive at least for three of four targets, gltA, ftsZ, ribC and 16S RNA. Thus, the percentage of the infection in the studied community was 64%. Subsequent RFLP assay showed that obtained isolates belonged to the Bartonella grahamii and/or B. taylorii species. In 7 cases we found both bartonellae species in one animal. These data were confirmed by direct sequencing of four ftsZ, four ribC and two gltA amplicons. According to our data, there is no any marked host specificity for these bartonellae species. Now we have laid the bartonellae strain collection consisting of 31 isolates. To our knowledge, this is the first investigation of the bartonellae prevalence in wild small mammals performed in Russia. The comparison of our data with those obtained by European researchers and issues of coinfection by different bartonellae species and host specificity are discussed.     

Experimental adiaspiromycosis of the common vole (Microtus arvalis) and other small wild mammals after intraperitoneal inoculation

After intraperitoneal inoculation ofE. crescens to nine species of wild small mammals, six species of rodents (Apodemus flavicollis, A. sylvaticus, Clethrionomys glareolus, Microtus agrestis, M. arvalis, Mus musculus) developed generalized adiaspiromycosis. The course of experimental infection corresponded to the infections of laboratory animals provoked in the same manner of inoculation. The authors studied the affliction of individual organs, the dynamics of growth of adiaspores in the organs of the abdominal cavity and in the lungs and followed up morphological changes in the adiaspores. In fresh cover glass preparations, the presence of manifestations was demonstrated justifying considerations on the capacity of multiplication of the adiaspiromycosis agent in the host organism.     

Age-dependent flea (Siphonaptera) parasitism in rodents: a host's life history matters

We studied age-dependent patterns of flea infestation in 7 species of rodents from Slovakia (Apodemus agrarius, A. flavicollis, A. sylvaticus, A. uralensis, Clethrionomys glareolus, Microtus arvalis, and M. subterraneus). We estimated the age of the host from its body mass and expected the host age-dependent pattern of flea abundance, the level of aggregation, and prevalence to be in agreement with theoretical predictions. We expected that the mean abundance and the level of aggregation of fleas would be lowest in hosts of smallest and largest size classes and highest in hosts of medium size classes, whereas pattern of variation of prevalence with host age would be either convex or asymptotic. In general, mean abundance and species richness of fleas increased with an increase in host age, although the pressure of flea parasitism in terms of number of fleas per unit host body surface decreased with host age. We found 2 clear patterns of the change in flea aggregation and prevalence with host age. The first pattern demonstrated a peak of flea aggregation and a trough of flea prevalence in animals of middle age classes (Apodemus species and C. glareolus). The second pattern was an increase of both flea aggregation and flea prevalence with host age (both Microtus species). Consequently, we did not find unequivocal evidence for the main role of either parasite-induced host mortality or acquired resistance in host age-dependent pattern of flea parasitism. Our results suggest that this pattern can be generated by various processes and is strongly affected by natural history parameters of a host species such as dispersal pattern, spatial distribution, and structure of shelters.     

Acquisition of nonspecific Bartonella strains by the northern grasshopper mouse (Onychomys leucogaster)

Rodent-associated Bartonella species are generally host-specific parasites in North America. Here evidence that Bartonella species can 'jump' between host species is presented. Northern grasshopper mice and other rodents were trapped in the western USA. A study of Bartonella infection in grasshopper mice demonstrated a high prevalence that varied from 25% to 90% by location. Bartonella infection was detected in other rodent species with a high prevalence as well. Sequence analyses of gltA identified 29 Bartonella variants in rodents, 10 of which were obtained from grasshopper mice. Among these 10, only six variants were specific to grasshopper mice, whereas four were identical to variants specific to deer mice or 13-lined ground squirrels. Fourteen of 90 sequenced isolates obtained from grasshopper mice were strains found more commonly in other rodent species and were apparently acquired from these animals. The ecological behavior of grasshopper mice may explain the occurrence of Bartonella strains in occasional hosts. The observed rate at which Bartonella jumps from a donor host species to the grasshopper mouse was directly proportional to a metric of donor host density and to the prevalence of Bartonella in the donor host, and inversely proportional to the same parameters for the grasshopper mouse.     

Within‐host interactions shape virulence‐related traits of trematode genotypes

Within‐host interactions between co‐infecting parasites can significantly influence the evolution of key parasite traits, such as virulence (pathogenicity of infection). The type of interaction is expected to predict the direction of selection, with antagonistic interactions favouring more virulent genotypes and synergistic interactions less virulent genotypes. Recently, it has been suggested that virulence can further be affected by the genetic identity of co‐infecting partners (G × G interactions), complicating predictions on disease dynamics. Here, we used a natural host–parasite system including a fish host and a trematode parasite to study the effects of G × G interactions on infection virulence. We exposed rainbow trout (Oncorhynchus mykiss) either to single genotypes or to mixtures of two genotypes of the eye fluke Diplostomum pseudospathaceum and estimated parasite infectivity (linearly related to pathogenicity of infection, measured as coverage of eye cataracts) and relative cataract coverage (controlled for infectivity). We found that both traits were associated with complex G × G interactions, including both increases and decreases from single infection to co‐infection, depending on the genotype combination. In particular, combinations where both genotypes had low average infectivity and relative cataract coverage in single infections benefited from co‐infection, while the pattern was opposite for genotypes with higher performance. Together, our results show that infection outcomes vary considerably between single and co‐infections and with the genetic identity of the co‐infecting parasites. This can result in variation in parasite fitness and consequently impact evolutionary dynamics of host–parasite interactions.     

Tick-borne zoonotic bacteria in wild and domestic small mammals in northern Spain

The prevalence and diversity of tick-borne zoonotic bacteria (Borrelia spp., Anaplasma phagocytophilum, Coxiella burnetii, and spotted fever group rickettsiae) infecting 253 small mammals captured in the Basque Country (Spain) were assessed using PCR and reverse line blot hybridization. Trapping sites were selected around sheep farms (study 1, 2000 to 2002) and recreational parks (study 2, 2003 to 2005). The majority of the studied mammals (162) were wood mice (Apodemus sylvaticus), but six other different species were also analyzed: yellow-necked mice (Apodemus flavicollis), shrews (Crocidura russula and Sorex coronatus), bank voles (Clethrionomys glareolus), domestic mice (Mus domesticus), and moles (Talpa europaea). The results showed an infection rate ranging from 10.7% to 68.8%, depending on the small mammal species. One C. russula shrew and one A. sylvaticus mouse gave positive reactions for A. phagocytophilum, and C. burnetii was detected in two domestic mice and one A. sylvaticus mouse in a farm. The DNA of Borrelia spp. was detected in 67 animals (26.5%), most of them presenting positive hybridization with the probe for Borrelia sp. strain R57, the new Borrelia species previously detected in small mammals in our region. Furthermore, a second PCR and reverse line blot hybridization specific for B. burgdorferi sensu lato revealed the presence of Borrelia afzelii in 6.3% of C. glareolus voles and 14.3% of S. coronatus shrews. All small mammals were negative for spotted fever group rickettsiae. These results highlight the relevance of small mammals as reservoirs of some zoonotic bacteria.     

Host ecology determines the relative fitness of virus genotypes in mixed-genotype nucleopolyhedrovirus infections

Mixed‐genotype infections are common in many natural host–parasite interactions. Classical kin‐selection models predict that single‐genotype infections can exploit host resources prudently to maximize fitness, but that selection favours rapid exploitation when co‐infecting genotypes share limited host resources. However, theory has outpaced evidence: we require empirical studies of pathogen genotypes that naturally co‐infect hosts. Do genotypes actually compete within hosts? Can host ecology affect the outcome of co‐infection? We posed both questions by comparing traits of infections in which two baculovirus genotypes were fed to hosts alongside inocula of the same or a different genotype. The host, Panolis flammea, is a herbivore of Pinus sylvestris and Pi. contorta. The pathogen, PfNPV (a nucleopolyhedrovirus), occurs naturally as mixtures of genotypes that differ, when isolated, in pathogenicity, speed of kill and yield. Single‐genotype infection traits failed to predict the ‘winning’ genotypes in co‐infections. Co‐infections infected and caused lethal disease in more hosts, and produced high yields, relative to single‐genotype infections. The need to share with nonkin did not cause fitness costs to either genotype. In fact, in hosts feeding on Pi. sylvestris, one genotype gained increased yields in mixed‐genotype infections. These results are discussed in relation to theory surrounding adaptive responses to competition with nonkin for limited resources.     

Fungal infections of the phytoplankton: seasonality, minimal host density, and specificity in a mesotrophic lake

Phytoplankton infections by fungal parasites in the upper, mixed layer of a mesotrophic northern temperate lake were analysed according to the following parameters: host and parasite species, host population density and prevalence of infection, resting spore formation by the parasite, and the lowest host density at which parasites appeared. The phytoplankton taxa recorded included the Cyanobacteria, Dinomastigota, Chrysophyceae, Bacillariophyceae, Chlorophyceae, Cryptophyceae and Haptophyceae, but infection was never found in the last two classes. The parasites belonged almost exclusively to the monocentric Chytridiomycetes. Fungal epidemics occurred at all times of the year. Parasites appeared at population densities as low as about 1 cell ml⁻¹ in some host species, with infection prevalence sometimes exceeding 80%. The proportion of the total phytoplankton biovolume infected by fungi was usually much <1%, but occasionally reached 10%. Parasitism proved to be highly species-specific, with one parasite species usually infecting only one host species. In the case of Zygorhizidium planktonicum , which infected both Asterionella formosa and Synedra acus , there is evidence that two species-specific formae speciales , each infecting only one of these two host species, are present in the lake.     

The importance of Laelaps agilis C.L. Koch 1836 (Mesostigmata: Parasitiformae) as a vector of Hepatozoon sylvatici Coles 1914 (Sporozoa: Haemogregarinidae) (author's transl)]

The importance of the mite Laelaps agilis (Parasitiformae) for the transmission of the Hepatozoon species living in the blood of Apodemus sylvaticus and Apodemus flavicollis (Hepatozoon sylvatici) is examined. With this mite the experimental transfer of the parasite to bred hosts of the same species was successful five times; for once, the transmission to a non-specific host (Clethrionomys glareolus) was also possible. Besides, an eventual stimulating effect emanating from the gonads of female mites to the development of parasites can be considered. Differences in the morphology of schizonts in the bone marrow and in the liver of infected Apodemus flavicollis are especially pointed out.     

Cross-species regulation of malaria parasitaemia in the human host

Longitudinal genetic analysis of the composition of malaria parasites infecting humans has demonstrated that individuals living in endemic areas are chronically infected with multiple genotypes and species of Plasmodium. The accumulation of infections is a consequence of superinfection from the bites of many infected anopheline mosquitoes. The clinical outcome of infection is determined by the host's ability to regulate the density of malaria parasites in the blood. Interestingly, most infections do not cause symptoms of malarial disease after a degree of immunity is acquired. Here, we review data from the first genetic study of the longitudinal dynamics of multiple Plasmodium species and genotypes in humans. The data show that the total parasite density of Plasmodium species oscillates around a threshold and that peaks of infection with each species do not coincide. We propose that malaria parasitaemia is controlled in a density-dependent manner in these semi-immune children. This implies that a cross-species mechanism of parasite regulation exists. A model of how multiple immune responses could act in concert to explain these within host dynamics is discussed in relation to known regulatory mechanisms.     

An experimental test of the independent action hypothesis in virus–insect pathosystems

The ‘independent action hypothesis’ (IAH) states that each pathogen individual has a non-zero probability of causing host death and that pathogen individuals act independently. IAH has not been rigorously tested. In this paper, we (i) develop a probabilistic framework for testing IAH and (ii) demonstrate that, in two out of the six virus–insect pathosystems tested, IAH is supported by the data. We first show that IAH inextricably links host survivorship to the number of infecting pathogen individuals, and develop a model to predict the frequency of single- and dual-genotype infections when a host is challenged with a mixture of two genotypes. Model predictions were tested using genetically marked, near-identical baculovirus genotypes, and insect larvae from three host species differing in susceptibility. Observations in early-instar larvae of two susceptible host species support IAH, but observations in late-instar larvae of susceptible host species and larvae of a less susceptible host species were not in agreement with IAH. Hence the model is experimentally supported only in pathosystems in which the host is highly susceptible. We provide, to our knowledge, the first qualitative experimental evidence that, in such pathosystems, the action of a single virion is sufficient to cause disease.     

Additional observations to the biology of Hepatozoon erhardovae Krampitz 1964 in bank voles from the south-western Styria and from areas around the Neusiedlersee (Burgenland) (author's transl)]

The incidence of Hepatozoon erhardovae in the blood of bank voles (Clethrionomys glareolus) captured in the investigated areas is examined. The biology of schizonts, their distribution in the lobes of the lung and the further development of natural infections in vertebrate hosts kept strictly free of ectoparasites are observed. The accumulation of gametocytes in larvae of the tick Ixodes ricinus and of the mite Neotrombicula zachvatkini after sucking on strongly infected bank voles is pointed out. Occasionally a parasitaemia is produced in the non-specific host (Apodemus flavicollis), but only with a specific transmitter (Megabothris turbidus).     

Recombination, Diversity and Allele Sharing of Infectivity Proteins Between Bartonella Species from Rodents

The alpha-Proteobacterium Bartonella is a common parasite of voles and mice, giving rise to short-lived (4 weeks to 2 months) infections. Here, we report high sequence diversity in genes of the VirB/VirD type IV secretion system (T4SS), amongst Bartonella from natural rodent populations in NE Poland. The VirB5 protein is predicted to consist of three conserved alpha helices separated by loops of variable length which include numerous indels. The C-terminal domain includes repeat stretches of KEK residues, reflecting underlying homopolymeric stretches of adenine residues. A total of 16 variants of VirB5, associated with host identity, but not bacterial taxon, were identified from 22 Bartonella isolates. One was clearly a recombinant from two others, another included an insertion of two KEK repeats. The virB5 gene appears to evolve via both mutation and recombination, as well as slippage mediated insertion/deletion events. The recombinational units are thought to be relatively short, as there was no evidence of linkage disequilibrium between virB5 and the bepA locus only 5.5 kb distant. The diversity of virB5 is assumed to be related to immunological role of this protein in Bartonella infections; diversity of virB5 may assist persistence of Bartonella in the rodent population, despite the relatively short (3-4 weeks) duration of individual infections. It is clear from the distribution of virB5 and bepA alleles that recombination within and between clades is widespread, and frequently crosses the boundaries of conventionally recognised Bartonella species.     

Ixodes ricinus, transmitted diseases and reservoirs

The tick Ixodes ricinus has been recorded in most Italian regions especially in thermo-mesophilous woods and shrubby habitats where the relative humidity allow the tick to complete its 3 year developmental cycle, as predicted for the European climatic ranges. This tick acts both as vector and reservoir for a series of wildlife zoonotic pathogens, especially the agents of Lyme diseases, Tick borne encephalitis and Human Granulocytic Ehrlichiosis, which are emerging in most of Europe. To assess the spatial distribution of these pathogens and the infection risk for humans and animals within the territory of the Province of Trento, we carried out a long term study using a combination of eco-epidemiological surveys and mathematical modelling. An extensive tick collection with a GIS based habitat suitability analysis allowed us to identify the areas where tick occurs at various density. To identify the areas with higher infection risk, we estimated the values of R0 for Borrelia burgdorferi s.l., TBE virus and Anaplasma phagocytophila under different ecological conditions. We assessed the infection prevalence in the vector and in the wildlife reservoir species that play a central role in the persistence of these infections, ie the small mammals A. flavicollis and C. glareolus. We also considered the double effect of roe deer (Capreolus capreolus) which act as reservoir for A. phagocytophila but is an incompetent host for B. burgdorferi and TBE virus, thus reducing the infection prevalence in ticks of these last two pathogens. Infection prevalence with B. burgdorferi and A. phagocytophila in the vector was assessed by PCR screening 1212 I. ricinus nymphs collected by dragging in six main study areas during 2002. The mean infection prevalence recorded was 1.32% for B. burgdorferi s.l. and 9.84% for A. phagocytophila. Infection prevalence in nymphs with TBE virus, as assessed in a previous study was 0.03%. Infection prevalence in rodents was assessed by screening (with ELISA and PCR) tissues and blood samples collected from 367 rodent individuals trapped extensively during 2002 within 6 main study areas. A. flavicollis (N=238) was found to be infected with all three pathogens investigated, with infection prevalence ranging from 3.3% for TBE virus to 11.7% for A. phagocytophila, and 16.6% with B. burgdorferi s.l. C. glareolus (N=108) showed an infection prevalence of 6.5% with A. phagocytophila and 12.7% with B. burgdorferi s.l., while no individuals were infected with TBE virus. We also screened 98 spleen samples collected from roe deer with PCR, resulting in a mean prevalence of infection with A. phagocytophila of 19.8%. Using a deterministic model we explored the condition for diseases persistence under different rodent and roe deer densities. R0 values resulted largely above 1 for B. burgdorferi s.l. in the vast majority of the areas classified as suitable for I. ricinus occurrence in Trentino, while the condition for TBE persistence appeared to be more restricted by a combination of climatic condition and host densities.     

A mathematical epidemiological model of gram-negative Bartonella bacteria: does differential ectoparasite load fully explain the differences in infection prevalence of Rattus rattus and Rattus norvegicus?

We postulate that the large difference in infection prevalence, 24% versus 5%, in R. norvegicus and R. rattus, respectively, between these two co-occurring host species may be due to differences in ectoparasite and potential vector infestation rates. A compartmental model, representative of an infectious system containing these two Rattus species and two ectoparasite vectors, was constructed and the coefficients of the forces of infection determined mathematically. The maximum difference obtained by the model in the prevalence of Bartonella in the two Rattus species amounts to 4.6%, compared to the observed mean difference of 19%. Results suggest the observed higher Bartonella infection prevalence in Rattus norvegicus compared to Rattus rattus, cannot be explained solely by higher ectoparasite load. The model also highlights the need for more detailed biological research on Bartonella infections in Rattus and the importance of the flea vector in the spread of this disease.     

Temporal and Spatial Patterns of <Emphasis Type="Italic">Bartonella</Emphasis> Infection in Black-tailed Prairie Dogs (<Emphasis Type="Italic">Cynomys ludovicianus</Emphasis>)

We describe the temporal dynamics and spatial distribution of Bartonella in black-tailed prairie dogs (Cynomys ludovicianus) based on a longitudinal study conducted in 20 black-tailed prairie dog (BTPD) colonies in Boulder County, CO from 2003 to 2005. Bartonella infection was widely distributed in all colonies with an overall prevalence of 23.1%, but varied by colony from 4.8% to 42.5% and by year from 9.1 to 39.0%, with a marked increase in Bartonella activity in 2005. Levels of bacteremia varied from 40 to 12,000 colony forming units (CFU) per milliliter of BTPD blood, but were highly skewed with a median of 240 CFU. Bartonella infection rates were unimodal with respect to BTPD body mass, first increasing among growing juveniles, then declining among adults. Infection rates exhibited a sigmoidal response to body mass, such that 700g may prove to be a useful threshold value to evaluate the likelihood of Bartonella infection in BTPDs. Bartonella prevalence increased throughout the testing season for each year, as newly emerged juveniles developed bacteremia. Data from recaptured animals suggest that Bartonella infections did not persist in individual BTPDs, which may explain the relatively low prevalence of Bartonella in BTPDs compared to other rodent species. No association was found between Bartonella prevalence and host population density. Prevalence did not differ between males and females. The spatio-temporal pattern of Bartonella infection among colonies suggests epizootic spread from northern to central and southern portions of the study area. The potential significance of the BTPD-associated Bartonella for public health needs to be further investigated.     

Patterns of coexistence: ectoparasites on small mammals in northern Fennoscandia

The total aestival ectoparasitic burden of six small mammal species ( Sorex araneus, Clethrionomys glareolus, C. rutilus, C. rufocanus, Microtus agrestis , and M. oeconomus ) was investigated in terms of frequency distribution, frequency of occurrence, species diversity and joint occurrences. The mammals were collected in northern Fennoscandia during peak density years. The frequency distribution of the ectoparasites was best described as negative binomial on C. glareolus, M. agrestis , and M. oeconomus but not so on S. araneus, C. rutilus and C. rufocanus. The distribution did not fit the Poisson distribution in any species. The percentage of S. araneus that had ectoparasites was 49%, and of the microtidae species, 73-96% had ectoparasites. The median number of ectoparasites on the vole species was between 2 and 9 specimens of 1 or 2 species. There was a significant, positive correlation between the number of ectoparasitic species and the total number of individuals on all host species. Pairs of ectoparasitic species occurring together more or less often than expected by chance were found on all host species. However, the pairs rarely repeated themselves on the same host species under different environmental conditions, or on other host species under similar circumstances. Differences in total infestation between reproductive categories and sexes were observed in M. agrestis but not in S. araneus and C. glareolus.     

Hantaviruses in small wild living mammals in Czechoslovakia. Results of a 1983-1989 study

During a 7- year period were hunted small wild living mammals and examined by ELISA and RIA techniques for the presence of hantavirus antigen and/or antibodies by MFA. In total 3,050 animals of 16 species caught in 9 out of 10 regions of Czechoslovakia, were examined. The proportion of positive animals was 4.4%. To the positive ones with the serotype 2 (Western type) belonged the following: M. arvalis, C. glareolus, P. subterraneus, A. sylvaticus, A. flavicollis. To the Eastern serotype: A. agrarius in eastern Slovakia, A. flavicollis in North of Bohemia and A. sylvaticus in South of Moravia. The repeatedly examined localities were found to be either repeatedly positive or repeatedly negative. The antigen titres in the lungs of M. arvalis were constant irrespective of sex and season of capture. They were, however, much higher in young animals, whereas the proportion of positivities was higher in adults ones. The titres of antigen in the lungs of C. glareolus never exceeded those of M. arvalis.