A reduced SNP panel optimised for non-invasive genetic assessment of a genetically impoverished conservation icon, the European bison.

The European bison was saved from the brink of extinction due to considerable conservation efforts since the early twentieth century. The current global population of > 9500 individuals is the result of successful ex situ breeding based on a stock of only 12 founders, resulting in an extremely low level of genetic variability. Due to the low allelic diversity, traditional molecular tools, such as microsatellites, fail to provide sufficient resolution for accurate genetic assessments in European bison, let alone from non-invasive samples. Here, we present a SNP panel for accurate high-resolution genotyping of European bison, which is suitable for a wide variety of sample types. The panel accommodates 96 markers allowing for individual and parental assignment, sex determination, breeding line discrimination, and cross-species detection. Two applications were shown to be utilisable in further Bos species with potential conservation significance. The new SNP panel will allow to tackle crucial tasks in European bison conservation, including the genetic monitoring of reintroduced populations, and a molecular assessment of pedigree data documented in the world's first studbook of a threatened species.

Effect of Translocation on Host Diet and Parasite Egg Burden: A Study of the European Bison (Bison bonasus).

For the purpose of nature management and species conservation, European bison (Bison bonasus) are being increasingly reintroduced into nature reserves across Europe. The aim of this study was to investigate European bison's adaptability to new areas through the study of their parasite-EPG (eggs per gram feces) and dietary diversity during twelve months after translocation. We compared the parasite-EPG from introduced European bison in Lille Vildmose, Denmark, with the parasite-EPG from populations from Bornholm, Denmark, and Bialowieza Forest, Poland. From March 2021 to February 2022, fecal samples were collected from three populations. Samples from Lille Vildmose were examined through flotation, sedimentation, the Baermann technique, and nanopore sequencing. Fecal samples from Bornholm and Bialowieza were examined through flotation and sedimentation. Nanopore sequencing of DNA from 63 European bison's fecal samples collected during March-September in Lille Vildmose identified 8 species of nematodes within the digestive tract of the European bison, with Haemonchus contortus being the most frequently observed. In Lille Vildmose, a significantly higher excretion of nematode-EPG was observed during the summer period than in the spring, autumn, and winter. In addition, monthly differences in the excretion of nematode eggs were found, with this being significantly higher in June than in the months during autumn and winter (October-February). Significant differences in the nematode-EPG were only found between the excretion of nematode eggs in Bialowieza Forest when compared to that of Lille Vildmose, with significantly higher excretion in Lille Vildmose (October-November). The results indicate that the development rates for nematodes may be affected by changes in temperature, with increasing temperatures speeding up their development time. Independent of this study design, wildlife vets together with the gamekeepers managing the herd found it necessary to treat the herd with antiparasitics for practical and animal welfare reasons in relation to translocation. Furthermore, 79 plant taxa were identified in the diet of the European bison. The broadest diet was observed in March suggesting that the European bison quickly adapted to their new habitat. The results suggest a seasonal shift in their diet, with this being most apparent from March to April.

In Search of Species-Specific SNPs in a Non-Model Animal (European Bison (Bison bonasus))-Comparison of De Novo and Reference-Based Integrated Pipeline of STACKS Using Genotyping-by-Sequencing (GBS) Data.

The European bison is a non-model organism; thus, most of its genetic and genomic analyses have been performed using cattle-specific resources, such as BovineSNP50 BeadChip or Illumina Bovine 800 K HD Bead Chip. The problem with non-specific tools is the potential loss of evolutionary diversified information (ascertainment bias) and species-specific markers. Here, we have used a genotyping-by-sequencing (GBS) approach for genotyping 256 samples from the European bison population in Bialowieza Forest (Poland) and performed an analysis using two integrated pipelines of the STACKS software: one is de novo (without reference genome) and the other is a reference pipeline (with reference genome). Moreover, we used a reference pipeline with two different genomes, i.e., Bos taurus and European bison. Genotyping by sequencing (GBS) is a useful tool for SNP genotyping in non-model organisms due to its cost effectiveness. Our results support GBS with a reference pipeline without PCR duplicates as a powerful approach for studying the population structure and genotyping data of non-model organisms. We found more polymorphic markers in the reference pipeline in comparison to the de novo pipeline. The decreased number of SNPs from the de novo pipeline could be due to the extremely low level of heterozygosity in European bison. It has been confirmed that all the de novo/Bos taurus and Bos taurus reference pipeline obtained SNPs were unique and not included in 800 K BovineHD BeadChip.

Digestive tract nematode infections in non-native invasive American mink with the first molecular identification of Molineus patens.

Parasites may negatively affect hosts condition, especially when infection intensity is high. Species introduced to a new habitat are often less exposed to a parasite pressure but may accumulate parasites in time. American mink (Neovison vison) introduced to Europe, Asia, and South America is an example of such invasive species. We analysed nematode prevalence and digestive tract infection intensity in 796 feral American mink from Poland. The analyses were performed separately for stomach, duodenum, small intestine and large intestine. Parasite species identification was performed using molecular methods based on highly conserved nuclear 18S rRNA gene and supplemented with morphological analysis. In total, we collected 26,852 nematodes and 98.6% of them were isolated from mink stomachs. We found positive association between infection intensity in stomach and other parts of digestive tract. Nematode prevalence was estimated at 63.8% and average infection intensity per one American mink at 52.9 (range from 1 to 1118). If the stomach results were theoretically and intentionally omitted the prevalence was 5 times lower (12.7%) and infection intensity 14 times lower (3.7; range 1-50). We identified two nematode species in digestive tracts of American mink: Aonchotheca putorii and Molineus patens. The 18S rRNA gene sequence of Molineus patens has been reported for the first time. The results showed that Aonchotheca putorii is a dominating nematode in the invasive American mink and that it inhabits stomach intensively and preferably.

The first case of autochthonous subcutaneous dirofilariasis (Dirofilaria repens) in a dog from Bialowieza (NE Poland) and possible threat posed to inhabitants of Bialowieza Primeval Forest area.

We present the first case of dirofilariasis in a dog from Bialowieza village located in the primeval European forest-Bialowieza Primeval Forest (NE Poland). Molecular analysis of adult nematode specimens isolated from subcutaneous tissue confirmed the infection with Dirofilaria repens. An adult male dog has not travelled out of the Bialowieza village for at least five years; therefore, we assume this is the autochthonous case of the disease. We discuss possible inter- and intra-species transmission routes of dirofilariasis on this territory, which is inhabited by diverse community of wild carnivores, domestic animals, and humans. We also discuss the likely sources of the disease in this, highly biodiverse unique European forest complex. We underline the lack of attention to this problem and its importance for veterinary, wildlife, and human health safety.

Multispecies reservoir of Spirometra erinaceieuropaei (Cestoda: Diphyllobothridae) in carnivore communities in north-eastern Poland.

BACKGROUND: Spirometra erinaceieuropaei is a diphylobothriid tapeworm with a complex life-cycle including definitive, intermediate and paratenic (transport) hosts. Multiple routes of parasite transmission often make it impossible to determine what type of host a specific infected animal is considered to be. Spargana larvae cause sparganosis, a severe food- and water-borne disease mainly found in Asia. In Poland, Spirometra sp. was reported in large carnivores in Bialowieza Primeval Forest for the first time in the 1940s and was recently confirmed as S. erinaceieuropaei in several mammals and snakes using molecular methods. METHODS: In total, 583 carcasses of 9 carnivore species were necropsied between 2013 and 2019 in north-eastern (NE) Poland. The larvae of S. erinaceieuropaei (spargana) were isolated from subcutaneous tissue, counted, and preserved for genetic analyses. We calculated the prevalence and intensity of infection. To assess spatial variation in S. erinaceieuropaei infection probability in NE Poland, we applied a generalized additive model (GAM) with binomial error distribution. To confirm the species affiliation of isolated larvae, we amplified a partial fragment of the 18S rRNA gene (240 bp in length). RESULTS: Spirometra larvae were found in the subcutaneous tissue of 172 animals of 7 species and confirmed genetically as S. erinaceieuropaei. The overall prevalence in all studied hosts was 29.5% with a mean infection intensity of 14.1 ± 33.8 larvae per individual. Native European badgers and invasive raccoon dogs were characterized by the highest prevalence. An analysis of parasite spread showed a spatially diversified probability of infection with the highest values occurring in the biodiversity hot spot, Bialowieza Primeval Forest. CONCLUSIONS: Our study revealed that various mammal species (both native and non-native) can serve as S. erinaceieuropaei reservoirs. The frequency and level of infection may differ between selected hosts and likely depend on host diversity and habitat structure in a given area. Further studies are needed to assess the distribution of the parasite throughout Europe and the environmental and biological factors influencing infection severity in wild mammals.

Genomic Footprints of Recovery in the European Bison.

After extinction in the wild in the beginning of the 20th century, the European bison has been successfully recovered in 2 distinct genetic lines from only 12 and 7 captive founders. We here aimed at characterizing the levels of realized inbreeding in these 2 restored lines to provide empirical insights into the genomic footprints left by population recovery from a small number of founders. To that end, we genotyped 183 European bison born over the last 40 years with the Illumina BovineHD beadchip that contained 22 602 informative autosomal single-nucleotide polymorphisms after data filtering. We then identified homozygous-by-descent (HBD) segments and classified them into different age-related classes relying on a model-based approach. As expected, we observed that the strong and recent founder effect experienced by the 2 lines resulted in very high levels of recent inbreeding and in the presence of long HBD tracks (up to 120 Mb). These long HBD tracks were associated with ancestors living approximately from 4 to 32 generations in the past, suggesting that inbreeding accumulated over multiple generations after the bottleneck. The contribution to inbreeding of the most recent groups of ancestors was however found to be decreasing in both lines. In addition, comparison of Lowland individuals born at different time periods showed that the levels of inbreeding tended to stabilize, HBD segments being shorter in animals born more recently which indicates efficient control of inbreeding. Monitoring HBD segment lengths over generations may thus be viewed as a valuable genomic diagnostic tool for populations in conservation or recovery programs.

The first case of genetically confirmed sparganosis (Spirometra erinaceieuropaei) in European reptiles.

Sparganosis is a zoonosis caused by the spargana (larvae) of Spirometra sp. (Diphyllobothriidae). Reptiles are particularly important vectors for the transmission of this parasite in Asia; however, their role in sparganosis spread in European wildlife is unrecognized. We investigated the infection of reptiles with Spirometra sp. in NE Poland, where several mammalian hosts have been identified recently and in the past. Of the 59 dead reptiles, plerocercoids were found in two grass snakes (Natrix natrix) from the Bialowieza Primeval Forest (BPF). The Spirometra erinaceieuropaei species was genetically confirmed using the evolutionary conserved nuclear 18S rRNA gene, and then compared to GenBank deposits. The sequences were identical to previously investigated Spirometra sp. found in Eurasian badger and wild boar from BPF. Our finding is the first genetically confirmed record of Spirometra sp. in reptiles in Europe. Since reptiles are often a component of mammalian diet, they can be a source of Spirometra tapeworm infection in European wildlife; however, further studies are needed to investigate the prevalence of infection in reptiles and other non-mammalian hosts.

Update of the helminth fauna in Eurasian lynx (Lynx lynx) in Poland.

The Eurasian lynx (Lynx lynx) is a strictly protected species of large carnivore in Poland. It inhabits forest complexes in north-eastern Poland and the Carpathian region in southern Poland. The status of the lynx within Poland requires special attention because its range decreased between 1980 and 2001 and has not yet recovered. One of the factors negatively affecting lynx populations is diseases, particularly parasites. The helminth fauna of the Eurasian lynx is not fully known in Poland. Previous coprological studies revealed that Polish lynx have been infected with seven species of nematodes, three species of cestodes, and one species of trematode. In this study, we present new data based on examination of opportunistically collected lynx carcasses. The aim of the study was to complement data on the helminth fauna of Eurasian lynx inhabiting Poland based on morphological and molecular analysis of parasites. Four species of cestodes-Taenia lynciscapreoli, Mesocestoides lineatus, Spirometra sp., and Taenia krabbei-were found for the first time in Eurasian lynx from Poland and three previously reported species of nematodes-Ancylostoma tubaeforme, Toxascaris leonina, and Toxocara cati-were confirmed. Larvae of Trichinella britovi were also detected in Eurasian lynx in Poland for the first time.

Pervasive introgression facilitated domestication and adaptation in the Bos species complex.

Species of the Bos genus, including taurine cattle, zebu, gayal, gaur, banteng, yak, wisent and bison, have been domesticated at least four times and have been an important source of meat, milk and power for many human cultures. We sequence the genomes of gayal, gaur, banteng, wisent and bison, and provide population genomic sequencing of an additional 98 individuals. We use these data to determine the phylogeny and evolutionary history of these species and show that the threatened gayal is an independent species or subspecies. We show that there has been pronounced introgression among different members of this genus, and that it in many cases has involved genes of considerable adaptive importance. For example, genes under domestication selection in cattle (for example, MITF) were introgressed from domestic cattle to yak. Also, genes in the response-to-hypoxia pathway (for example, EGLN1, EGLN2 and HIF3a) have been introgressed from yak to Tibetan cattle, probably facilitating their adaptation to high altitude. We also validate that there is an association between the introgressed EGLN1 allele and haemoglobin and red blood cell concentration. Our results illustrate the importance of introgression as a source of adaptive variation and during domestication, and suggest that the Bos genus evolves as a complex of genetically interconnected species with shared evolutionary trajectories.

Complex Admixture Preceded and Followed the Extinction of Wisent in the Wild.

Retracing complex population processes that precede extreme bottlenecks may be impossible using data from living individuals. The wisent (Bison bonasus), Europe's largest terrestrial mammal, exemplifies such a population history, having gone extinct in the wild but subsequently restored by captive breeding efforts. Using low coverage genomic data from modern and historical individuals, we investigate population processes occurring before and after this extinction. Analysis of aligned genomes supports the division of wisent into two previously recognized subspecies, but almost half of the genomic alignment contradicts this population history as a result of incomplete lineage sorting and admixture. Admixture between subspecies populations occurred prior to extinction and subsequently during the captive breeding program. Admixture with the Bos cattle lineage is also widespread but results from ancient events rather than recent hybridization with domestics. Our study demonstrates the huge potential of historical genomes for both studying evolutionary histories and for guiding conservation strategies.

Novel Graphical Analyses of Runs of Homozygosity among Species and Livestock Breeds.

Runs of homozygosity (ROH), uninterrupted stretches of homozygous genotypes resulting from parents transmitting identical haplotypes to their offspring, have emerged as informative genome-wide estimates of autozygosity (inbreeding). We used genomic profiles based on 698 K single nucleotide polymorphisms (SNPs) from nine breeds of domestic cattle (Bos taurus) and the European bison (Bison bonasus) to investigate how ROH distributions can be compared within and among species. We focused on two length classes: 0.5-15 Mb to investigate ancient events and >15 Mb to address recent events (approximately three generations). For each length class, we chose a few chromosomes with a high number of ROH, calculated the percentage of times a SNP appeared in a ROH, and plotted the results. We selected areas with distinct patterns including regions where (1) all groups revealed an increase or decrease of ROH, (2) bison differed from cattle, (3) one cattle breed or groups of breeds differed (e.g., dairy versus meat cattle). Examination of these regions in the cattle genome showed genes potentially important for natural and human-induced selection, concerning, for example, meat and milk quality, metabolism, growth, and immune function. The comparative methodology presented here permits visual identification of regions of interest for selection, breeding programs, and conservation.

Early cave art and ancient DNA record the origin of European bison.

The two living species of bison (European and American) are among the few terrestrial megafauna to have survived the late Pleistocene extinctions. Despite the extensive bovid fossil record in Eurasia, the evolutionary history of the European bison (or wisent, Bison bonasus) before the Holocene (<11.7 thousand years ago (kya)) remains a mystery. We use complete ancient mitochondrial genomes and genome-wide nuclear DNA surveys to reveal that the wisent is the product of hybridization between the extinct steppe bison (Bison priscus) and ancestors of modern cattle (aurochs, Bos primigenius) before 120 kya, and contains up to 10% aurochs genomic ancestry. Although undetected within the fossil record, ancestors of the wisent have alternated ecological dominance with steppe bison in association with major environmental shifts since at least 55 kya. Early cave artists recorded distinct morphological forms consistent with these replacement events, around the Last Glacial Maximum (LGM, ∼21-18 kya).

Past climate changes, population dynamics and the origin of Bison in Europe.

BACKGROUND: Climatic and environmental fluctuations as well as anthropogenic pressure have led to the extinction of much of Europe's megafauna. The European bison or wisent (Bison bonasus), one of the last wild European large mammals, narrowly escaped extinction at the onset of the 20th century owing to hunting and habitat fragmentation. Little is known, however, about its origin, evolutionary history and population dynamics during the Pleistocene. RESULTS: Through ancient DNA analysis we show that the emblematic European bison has experienced several waves of population expansion, contraction, and extinction during the last 50,000 years in Europe, culminating in a major reduction of genetic diversity during the Holocene. Fifty-seven complete and partial ancient mitogenomes from throughout Europe, the Caucasus, and Siberia reveal that three populations of wisent (Bison bonasus) and steppe bison (B. priscus) alternately occupied Western Europe, correlating with climate-induced environmental changes. The Late Pleistocene European steppe bison originated from northern Eurasia, whereas the modern wisent population emerged from a refuge in the southern Caucasus after the last glacial maximum. A population overlap during a transition period is reflected in ca. 36,000-year-old paintings in the French Chauvet cave. Bayesian analyses of these complete ancient mitogenomes yielded new dates of the various branching events during the evolution of Bison and its radiation with Bos, which lead us to propose that the genetic affiliation between the wisent and cattle mitogenomes result from incomplete lineage sorting rather than post-speciation gene flow. CONCLUSION: The paleogenetic analysis of bison remains from the last 50,000 years reveals the influence of climate changes on the dynamics of the various bison populations in Europe, only one of which survived into the Holocene, where it experienced severe reductions in its genetic diversity. The time depth and geographical scope of this study enables us to propose temperate Western Europe as a suitable biotope for the wisent compatible with its reintroduction.

Sparganosis in wild boar (Sus scrofa) - Implications for veterinarians, hunters, and consumers.

From February to March 2016 we found plerocercoids of Spirometra sp. in four wild boar hunted in Bialowieza Primeval Forest, north-eastern Poland. Plerocercoids were located subcutaneously and in muscle tissue. A sequence of a nuclear 18S rRNA gene was used for genetic specification of the samples. The analyzed gene fragment showed 100% identity with the Spirometra erinacei sequence. Thus, the emerge of human sparganosis due to consumption of undercooked or smoked wild boar meat is likely in the areas where wild boar is an approved food source, especially in the absence of routine guidelines for vets. It has become a priority to inform the public about possibilities and consequences of this zoonosis.

Genome-wide association study for posthitis in the free-living population of European bison (Bison bonasus).

BACKGROUND: About 5-6% of the European bison (Bison bonasus) males are affected by posthitis (necrotic inflammation of the prepuce) and die in the wild forest. Despite many years of study, pathogenesis of this disease has not yet been determined. The main aim of the study was to find SNP markers significantly associated with the incidence of posthitis and mine the genome for candidate genes potentially involved in the development of the disease. RESULTS: It was shown that relatively small number of SNPs effects reached genome-wide significance after false discovery rate (FDR) correction. Among 25 significant markers, the highest effects were found for two SNPs (rs110456748 and rs136792896) located at the distance of 23846 bp and 37742 bp, respectively, from OR10A3 gene (olfactory receptor genes), known to be involved in atopic dermatitis in humans. It was also observed that five other significant SNP markers were located in the proximity of candidate genes involved in severe diseases of skin tissue and cancer/tumour development of epithelial or testicular germ cells, which suggest their potential participation in the posthitis. The 25 investigated SNPs showed marked differences in allelic and genotypic frequencies between the healthy and affected bison groups. CONCLUSIONS: The 2 Mb region of the BTA15 chromosome is involved in genetic background of posthitis and should be closer examined to find causal mutations helpful in better understanding of the disease ethology and to control its incidence in the future.

The first report of sparganosis (Spirometra sp.) in Eurasian badger (Meles meles).

Spirometra sp. is a diphyllobothriid cestode which reproduces mainly in cat-like carnivores and canids. Several animal species that are not definitive hosts function as paratenic hosts, in which plerocercoids migrate to other tissues causing sparganosis. In this paper we describe the first case of sparganosis (Spirometra sp.) in Eurasian badger (Meles meles). It was found in an adult female Eurasian badger killed on the road in the Bialowieza Primeval Forest (north-eastern Poland) in April 2013. At necropsy, 128 complete and 40 fragments of plerocercoids (spargana) were found and were located subcutaneously, mainly on the hind legs and along the spine. The average length of spargana was 87±38mm. No adult Spirometra sp. tapeworms were found in the animal intestine, indicating that the investigated badger was a paratenic host for Spirometra sp. Analysis of 18S rRNA gene sequences combined with morphological examination confirmed affiliation of the concerned plerocercoids to genus Spirometra.

Depauperate genetic variability detected in the American and European bison using genomic techniques.

A total of 929 polymorphic SNPs in EB (out of 54, 000 SNPs screened using a BovineSNP50 Illumina Genotyping BeadChip), and 1, 524 and 1, 403 polymorphic SNPs in WB and PB, respectively, were analysed. EB, WB and PB have all undergone recent drastic reductions in population size. Accordingly, they exhibited extremely depauperate genomes, deviations from genetic equilibrium and a genome organization consisting of a mosaic of haplotype blocks: regions with low haplotype diversity and high levels of linkage disequilibrium. No evidence for positive or stabilizing selection was found in EB, WB and PB, likely reflecting drift overwhelming selection. We suggest that utilization of genome-wide screening technologies, followed by utilization of less expensive techniques (e.g. VeraCode and Fluidigm EP1), holds large potential for genetic monitoring of populations. Additionally, these techniques will allow radical improvements of breeding practices in captive or managed populations, otherwise hampered by the limited availability of polymorphic markers. This result in improved possibilities for 1) estimating genetic relationships among individuals and 2) designing breeding strategies which attempt to preserve or reduce polymorphism in ecologically relevant genes and/or entire blocks.

Over-seasonal and sex-independent expression of kappa casein gene (CSN3) in mammalian blood lymphocytes.

After observing the nonspecific expression of the alpha S1 casein gene in peripheral blood mononuclear cells of goats, we continued to trace illegitimate gene-expression patterns in mammals. Six mammalian species (Bos taurus, Capra hircus, Ovis aries, Equus caballus, Sus scrofa, and Bison bonasus) were analyzed for the presence of kappa casein in peripheral blood mononuclear cells and Ficoll-isolated lymphocytes. To detect potential relationships between the expression pattern and reproductive status of the individual, the animals within species represented different sex, age, and physiological status (immature and reproducing males; immature, pregnant, lactating, and sterile females). Species-specific primers were designed for the two-step post reverse transcriptional-polymerase chain reaction. The products were sequenced and a Blast search performed. Transcripts of kappa casein were successfully detected in the peripheral blood mononuclear cells and lymphocytes of three of the species (goat, cow, and European bison). The presence of the transcripts was unrelated to sex, physiological status, or age of the animals.

[Multiplex minisequencing applied in detection of human functional CYP21 gene mutations]. / Zastosowanie metody multipleksowego minisekwencjonowania (multiplex minisequencing) do wykrywania mutacji w ludzkim genie CYP21.

We analyzed seven most common mutations within the CYP21B gene, responsible for congenital adrenal hyperplasia (CAH), using the minisequencing method. Functional CYP21B gene sequences were amplified with the pair of specific primers that pevented amplification of pseudogene CYP21P or pseudogene CYP21P/active CYP21 hybrids. Multiplex minisequencing (SNaPShot PCR) assay was performed with fluorescent dideoxynucleotides ([F]ddNTPs) and originally designed primers, claiming seven most common mutation sites responsible for the CAH symptoms. Using the method we detected five novel substitutions of unknown effect on the CAH course in five out of seven analyzed mutation sites. Compared to classic SNPs analyzing methods, especially single SNP detection, multiplex minisequencing is the same highly specific and sensitive but much faster one. The method is recommended for any population screened for known mutations.