Relaxation of selective constraints shapes variation of toll-like receptors in a colonial waterbird, the black-headed gull.

Nonspecific innate immune response is activated by toll-like receptors (TLRs), which recognize conserved molecular motifs characteristic for a broad spectrum of pathogens. In this study, we examined nucleotide substitution patterns and allelic diversity at five TLR genes in a wild nonpasserine bird, the black-headed gull Chroicocephalus ridibundus. We hypothesized that balancing selection can maintain high allelic diversity of TLR genes in the black-headed gull because of its ecological characteristics, coloniality, and migratoriness, which are associated with increased exposure and transmission of pathogens. Although we found moderately high levels of sequence polymorphism (8-49 haplotypes retrieved per locus within a sample of 60 individuals), most of these haplotypes were recorded at low frequencies within our study population. At the same time, we found no convincing evidence for the role of balancing selection in the maintenance of this variation (Tajima's D < 0.5), and sites with a significant excess of nonsynonymous mutations (dN/dS > 1) were recorded only at two loci (TLR5 and TLR7). This pattern is consistent with relaxation of selective constraints, where most mutations are slightly deleterious and usually removed by purifying selection. No differences in the diversity and nucleotide substitution rates were found between endosomal loci responsible for viral RNA sensing and loci responsible for the recognition of extracellular pathogens. Our study provides the first information on evolutionary mechanisms shaping polymorphism of TLRs in a species from Lari suborder (gulls and allies) and suggests that TLR genes may be poorly responsive to ecological and life-history characteristics of hosts.

Ecotype Variation in Trace Element Content of Hard Tissues in the European Roe Deer (Capreolus capreolus).

Animals living in anthropogenic habitats bear a multitude of costs, which are directly or indirectly associated with human activities. Among others, an elevated exposure to environmental pollution can have negative consequences for wildlife populations. We examined the differences in the concentrations of trace elements between the field and forest ecotype of the European roe deer (Capreolus capreolus). Naturally, roe deer inhabited various types of woodlands (forest ecotype), but within the last century, they adapted to life in a human-transformed agricultural areas (field ecotype), which could be associated with an increased exposure to pollution. In this study, we measured concentrations of seven trace metals (barium, copper, iron, lead, manganese, strontium, zinc) and fluoride in skull bones and permanent teeth of more than 230 roe deer from 8 study plots in East-Central Europe. We found that field roe deer had higher concentrations of four trace metals (copper, iron, lead, strontium) and fluoride compared with forest roe deer. These differences were consistent with variations in the general level of environmental contamination within the study plots, as assessed with trace element content in wild plants. Our study indicates that bone and teeth of the European roe deer can be used as a valid indicator of environmental pollution. Also, we expect that elevated exposure of field roe deer to environmental pollution can have negative consequences for wild populations of this species, as well as for the consumers of venison.

Brown Hare's (Lepus europaeus) Histone H1 Variant H1.2 as an Indicator of Anthropogenic Stress.

From the liver tissues of brown hare individuals that lived in two various habitats, i.e., the agricultural region with the predominant farms and the industrial area near a metallurgical plant, histones H1 were analyzed to compare their within and between population variability. Furthermore, because agricultural production emits mainly organic pollutants and metallurgical industry is a primarily source of inorganic contaminations, we wanted to check how the brown hare individuals are sensitive for both agents. Among brown hare H1 histones, the histone H1.2 was determined as heterogeneous due to its varied mobility in two-dimensional SDS-polyacrylamide gel. The obtained electrophoretic patterns contained differently moving single spots of histone H1.2 and also its double spots have a similar rate of electrophoretic mobility. Based on this, two homozygous phenotypes (slowly migrating 2a and faster moving 2b) and a heterozygous phenotype (2a2b) was distinguished. The relatively low variable (CV < 0.25) and comparably abundant (p > 0.05) histone H1.2 homozygous phenotypes form a heterozygous phenotype in a similar proportion, at a ratio approximating 0.5. Although the brown hare population originating from agricultural area displayed a slight excess of heterozygous individuals 2a2b (F = - 0.04), it was conformed to the Hardy-Weinberg assumption (χ2 = 0.035, p = 0.853). Compared with this population, a sevenfold reduced frequency of the phenotype 2b and above tenfold increase of a heterozygosity (F = - 0.53) was observed in the brown hare population inhabiting the vicinity of metallurgical plant. Therefore, this population did not fit to the Hardy-Weinberg law (χ2 = 5.65, p = 0.017). Despite the negligible genetic differentiation (FST = 0.026) between brown hare populations inhabiting areas with different anthropogenic pressure, a statistically significant difference in the distribution of their phenotypes (χ2 = 6.01, p = 0.049) and alleles (χ2 = 6.50, p = 0.013) was noted. The collected data confirm that the brown hare species is sensitive for environmental quality and may serve as a good indicator of habitat conditions related to both organic pollution emitted by agricultural activities (PIC = 0.48) and inorganic contamination originating from metallurgical processes (PIC = 0.49). These difference in the environmental quality might be assessed by estimation of genetic variability among the brown hare populations, based on the phenotypes distribution of histone H1 variant H1.2, the protein that was not so far employed as a molecular marker of anthropogenic stress.

Age-Related Patterns in Trace Element Content Vary Between Bone and Teeth of the European Roe Deer (Capreolus capreolus).

Game animals, such as the roe deer (Capreolus capreolus), have long been used as bioindicators of environmental contamination. Most ecotoxicological research on ungulates has focused on trace element content in soft tissues and antlers. Also, only fragmentary information exists about whether and how trace element concentrations vary with the age of wild-living animals and whether these age-related patterns are similar for different types of tissues. The purpose of this study was to measure concentrations of seven trace metals (barium, copper, iron, lead, manganese, strontium, zinc) and fluoride in bone and teeth of roe deer and to determine whether significant variation is evident with individual age. For this purpose, we collected permanent molars and fragments of mandible bone from more than 130 female roe deer in Central Poland. We found that concentrations of four trace elements (barium, manganese, zinc, and fluoride) in teeth of deer showed positive linear relationships with individual age. No such trends were recorded for trace element content in bone. We suggest that these striking differences in age-related patterns of trace element bioaccumulation between bone and permanent teeth of roe deer might be explained by higher turnover rate and constant remodelling of bone tissue. The results suggest that analysis of permanent teeth may be useful for assessing throughout-life intoxication by environmental pollution in the roe deer and possibly in other mammal species. Our study reinforces the need to carefully account for age-related variation in ecotoxicological research on wild-living animals.

Spatial and temporal variation of lead, cadmium, and zinc in feathers of great tit and blue tit nestlings in Central Poland.

In this study, we examined heavy-metal concentrations in feathers of nestling great tits Parus major and blue tits Cyanistes caeruleus at two different sites (urban parkland vs. deciduous forest) located in the Lódz agglomeration in relation to interyear variation. We found that tit species did not differ significantly in lead and cadmium concentrations. Zinc concentration was significantly higher in blue tits. We also found that lead and cadmium levels in blue tit nestlings and the level of lead in great tit nestlings were higher in the parkland site than in the woodland site. We explain habitat variation in heavy-metal concentrations in feathers of nestlings by different levels of contamination at study sites. For both tit species, significant variation in heavy-metal amounts accumulated by nestlings was found between years with the lowest value in a year with the lowest value of rainfall. We suggest that the interyear variation may be accounted for by differences in rainfall, thus influencing quantities of trace elements bioavailable in the environment.

Total antioxidant capacity of feces of mammalian herbivores and carnivores.

The total antioxidant capacities (TAC) of feces of mammalian herbivores and carnivores were compared. TAC were estimated using three different methods: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical (ABTS*) reduction, 1,1-diphenyl-2-picrylhydrazyl radical (DPPH*) reduction, and ferric reducing antioxidant power (FRAP). TAC of 18 herbivorous species were generally higher with respect to 16 carnivorous species [(14.21 +/- 6.72) vs. (9.45 +/- 7.32) mmol Trolox equivalents/kg feces; P < 0.05] in the FRAP assay. The ABTS* reduction assay indicated that the TAC originating from "fast" reacting antioxidants were higher in the herbivores than in carnivores [(17.92 +/- 7.18) vs. (12.22 +/- 8.5) mmol Trolox equivalents/kg feces; P < 0.05], while a reverse trend was observed for TAC originating from "slowly" reacting antioxidants [(20.68 +/- 4.85) vs. (24.68 +/- 6.87) mmol Trolox equivalents/kg feces].

Boginia virus, a newfound hantavirus harbored by the Eurasian water shrew (Neomys fodiens) in Poland.

BACKGROUND: Guided by decades-old reports of hantaviral antigens in the Eurasian common shrew (Sorex araneus) and the Eurasian water shrew (Neomys fodiens) in European Russia, we employed RT-PCR to analyze lung tissues of soricine shrews, captured in Boginia, Huta Dlutowska and Kurowice in central Poland during September 2010, 2011 and 2012. FINDINGS: In addition to Seewis virus (SWSV), which had been previously found in Eurasian common shrews elsewhere in Europe, a genetically distinct hantavirus, designated Boginia virus (BOGV), was detected in Eurasian water shrews captured in each of the three villages. Phylogenetic analysis, using maximum likelihood and Bayesian methods, showed that BOGV formed a separate lineage distantly related to SWSV. CONCLUSIONS: Although the pathogenic potential of BOGV and other recently identified shrew-borne hantaviruses is still unknown, clinicians should be vigilant for unusual febrile diseases and clinical syndromes occurring among individuals reporting exposures to shrews.

Avian feathers as bioindicators of the exposure to heavy metal contamination of food.

The aim of this study was to determine the possibility of using feathers of blue tit nestlings to assess the level of endogenous accumulation of lead. For this purpose we conducted an experiment with lead application to randomly chosen nestlings from eight randomly drawn broods. Five days after the exposure, feathers of lead-treated nestlings had significantly higher lead concentrations than control nestlings. This result suggests that feathers can be used as reliable non-destructive bioindicators to assess the level of heavy metals originating from contaminated food, which is of great significance for comparative studies on ecological consequences of pollution.

Phylogeny of Shrew- and Mole-Borne Hantaviruses in Poland and Ukraine.

Earlier, we demonstrated the co-circulation of genetically distinct non-rodent-borne hantaviruses, including Boginia virus (BOGV) in the Eurasian water shrew (Neomys fodiens), Seewis virus (SWSV) in the Eurasian common shrew (Sorex araneus) and Nova virus (NVAV) in the European mole (Talpa europaea), in central Poland. To further investigate the phylogeny of hantaviruses harbored by soricid and talpid reservoir hosts, we analyzed RNAlater®-preserved lung tissues from 320 shrews and 26 moles, both captured during 1990-2017 across Poland, and 10 European moles from Ukraine for hantavirus RNA through RT-PCR and DNA sequencing. SWSV and Altai virus (ALTV) were detected in Sorex araneus and Sorex minutus in Boginia and the Bialowieza Forest, respectively, and NVAV was detected in Talpa europaea in Huta Dlutowska, Poland, and in Lviv, Ukraine. Phylogenetic analyses using maximum-likelihood and Bayesian methods showed geography-specific lineages of SWSV in Poland and elsewhere in Eurasia and of NVAV in Poland and Ukraine. The ATLV strain in Sorex minutus from the Bialowieza Forest on the Polish-Belarusian border was distantly related to the ATLV strain previously reported in Sorex minutus from Chmiel in southeastern Poland. Overall, the gene phylogenies found support long-standing host-specific adaptation.

A Novel Hantavirus of the European Mole, Bruges Virus, Is Involved in Frequent Nova Virus Coinfections.

Hantaviruses are zoonotic viruses with a complex evolutionary history of virus-host coevolution and cross-species transmission. Although hantaviruses have a broad reservoir host range, virus-host relationships were previously thought to be strict, with a single virus species infecting a single host species. Here, we describe Bruges virus, a novel hantavirus harbored by the European mole (Talpa europaea), which is the well-known host of Nova virus. Phylogenetic analyses of all three genomic segments showed tree topology inconsistencies, suggesting that Bruges virus has emerged from cross-species transmission and ancient reassortment events. A high number of coinfections with Bruges and Nova viruses was detected, but no evidence was found for reassortment between these two hantaviruses. These findings highlight the complexity of hantavirus evolution and the importance of further investigation of hantavirus-reservoir relationships.

Isolation and partial characterization of a highly divergent lineage of hantavirus from the European mole (Talpa europaea).

Genetically distinct hantaviruses have been identified in five species of fossorial moles (order Eulipotyphla, family Talpidae) from Eurasia and North America. Here, we report the isolation and partial characterization of a highly divergent hantavirus, named Nova virus (NVAV), from lung tissue of a European mole (Talpa europaea), captured in central Poland in August 2013. Typical hantavirus-like particles, measuring 80-120 nm in diameter, were found in NVAV-infected Vero E6 cells by transmission electron microscopy. Whole-genome sequences of the isolate, designated NVAV strain Te34, were identical to that amplified from the original lung tissue, and phylogenetic analysis of the full-length L, M and S segments, using maximum-likelihood and Bayesian methods, showed that NVAV was most closely related to hantaviruses harbored by insectivorous bats, consistent with an ancient evolutionary origin. Infant Swiss Webster mice, inoculated with NVAV by the intraperitoneal route, developed weight loss and hyperactivity, beginning at 16 days, followed by hind-limb paralysis and death. High NVAV RNA copies were detected in lung, liver, kidney, spleen and brain by quantitative real-time RT-PCR. Neuropathological examination showed astrocytic and microglial activation and neuronal loss. The first mole-borne hantavirus isolate will facilitate long-overdue studies on its infectivity and pathogenic potential in humans.

Whole-Genome Sequence of a Novel Hantavirus Isolated from the European Mole (Talpa europaea).

The complete genome sequence of Nova virus, a novel hantavirus isolated from a European mole (Talpa europaea) captured in central Poland, was determined. The availability of this sequence will facilitate the search for other mole-borne hantaviruses and will accelerate the acquisition of new knowledge about their phylogeography and evolutionary origin.

Co-circulation of soricid- and talpid-borne hantaviruses in Poland.

Previously, we reported the discovery of a genetically distinct hantavirus, designated Boginia virus (BOGV), in the Eurasian water shrew (Neomys fodiens), as well as the detection of Seewis virus (SWSV) in the Eurasian common shrew (Sorex araneus), in central Poland. In this expanded study of 133 shrews and 69 moles captured during 2010-2013 in central and southeastern Poland, we demonstrate the co-circulation of BOGV in the Eurasian water shrew and SWSV in the Eurasian common shrew, Eurasian pygmy shrew (Sorex minutus) and Mediterranean water shrew (Neomys anomalus). In addition, we found high prevalence of Nova virus (NVAV) infection in the European mole (Talpa europaea), with evidence of NVAV RNA in heart, lung, liver, kidney, spleen and intestine. The nucleotide and amino acid sequence variation of the L segment among the SWSV strains was 0-18.8% and 0-5.4%, respectively. And for the 38 NVAV strains from European moles captured in Huta Dlutowska, the L-segment genetic similarity ranged from 94.1%-100% at the nucleotide level and 96.3%-100% at the amino acid level. Phylogenetic analyses showed geographic-specific lineages of SWSV and NVAV in Poland, not unlike that of rodent-borne hantaviruses, suggesting long-standing host-specific adaptation. The co-circulation and distribution of BOGV, SWSV and NVAV in Poland parallels findings of multiple hantavirus species co-existing in their respective rodent reservoir species elsewhere in Europe. Also, the detection of SWSV in three syntopic shrew species resembles spill over events observed among some rodent-borne hantaviruses.

Hematology and plasma biochemistry values of great cormorant (Phalacrocorax carbo sinensis) nestlings.

Hematology and plasma biochemistry values were determined in 92 free-living Great Cormorant (Phalacrocorax carbo sinensis) chicks at Jeziorsko reservoir, central Poland. Percentage distribution of leukocytes, packed cell volume, plasma concentrations of hemoglobin and basic biochemical parameters were evaluated. These values may be treated as reference ranges for free-living Great Cormorant nestlings.

Characterization of Tula virus from common voles (microtus arvalis) in Poland: evidence for geographic-specific phylogenetic clustering.

Tula virus (TULV), a recently identified arvicolid rodent-borne hantavirus, is harbored by the European common vole (Microtus arvalis) in Central Russia and the Czech and Slovak Republics. We report the isolation and characterization of this hantavirus from M. arvalis captured in Poland, a country where human disease caused by hantaviruses has not been recognized. Of 34 arvicolid rodents (24 Clethrionomys glareolus, 9 M. arvalis, 1 Pitymys sp.) captured in Lodz and Tuszyn, Poland, during June to September 1995, sera from 3 M. arvalis and 3 C. glareolus contained IgG antibodies to Puumala virus (PUUV), as determined by an indirect immunofluorescent antibody assay. Alignment and comparison of the 1852-nucleotide S segment and a 1676-nucleotide region of the G2 glycoprotein-encoding M segment, amplified from lung tissues of two hantavirus-seropositive M. arvalis, revealed 83.9-85.2% and 82.3-83.5% sequence similarity, respectively, with TULV strains from Central Russia and the Czech and Slovak Republics. A > 98% sequence conservation was found at the amino acid level. Phylogenetic analysis indicated that the newly found TULV strains from Poland were closely related to, but distinct from, TULV from elsewhere in Europe.