Mitochondrial Diversity and Phylogenetic Relationship of Eight Native Bulgarian Sheep Breeds.

The geographical, geomorphological, and climatic characteristics of Bulgaria are particularly favorable for animal breeding and, above all, for pastoral farming and sheep breeding. These conditions created prerequisites for the creation of about 30 unique local breeds of sheep. In this study we investigated the genetic diversity of eight of the most popular Bulgarian native breeds, based on the sequence analysis of a part of the mitochondrial D-loop region. An almost entire mitochondrial DNA (mtDNA) D-loop region (1180 bp) was amplified and sequenced. The obtained results showed the presence of a large number of haplotypes-225, belonging to two main haplogroups. The majority of samples showed a high prevalence of the European haplogroup B (95.2%) while the remaining individuals were assigned to haplogroup A (4.8%). None of the other reported mitochondrial haplogroups were observed. The number of polymorphic sites, nucleotide and haplotype diversity was high (240, 0.01237, and 0.9968, respectively), which is evidence for multiple maternal origins in all populations. The Tajima D-test value in all the study populations was -1.905 (p < 0.05), indicating that the abundance of rare alleles was most likely due to population expansion after a recent bottleneck. The Median joining network showed that almost all haplotypes belonging to haplogroup B formed a star-like network, which revealed a weak genetic differentiation and a large gene flow between the Bulgarian native breeds.

Phylogeographic Aspects of Bat Lyssaviruses in Europe: A Review.

During the last few decades, bat lyssaviruses have become the topic of intensive molecular and epidemiological investigations. Since ancient times, rhabdoviruses have caused fatal encephalitis in humans which has led to research into effective strategies for their eradication. Modelling of potential future cross-species virus transmissions forms a substantial component of the recent infection biology of rabies. In this article, we summarise the available data on the phylogeography of both bats and lyssaviruses in Europe and the adjacent reg ions, especially in the contact zone between the Palearctic and Ethiopian realms. Within these zones, three bat families are present with high potential for cross-species transmission and the spread of lyssaviruses in Phylogroup II to Europe (part of the western Palearctic). The lack of effective therapies for rabies viruses in Phylogroup II and the most divergent lyssaviruses generates impetus for additional phylogenetic and virological research within this geographical region.

Bat-derived cells use glucose as a cryoprotectant.

Evolution of heterothermy in environments with variable temperatures has allowed bats to survive food scarcity during seasonal climatic extremes by using torpor as a hibernation strategy. The controlled reduction of body temperature and metabolism through complex behavioural and physiological adaptations at organismal, organ, cellular and molecular levels includes the ability of tissues and cells to adapt to temperature alterations. Based on the prediction that cells of different tissues cultured in vitro would differ in their ability to withstand freezing and thawing of the medium, we determined the survival rate of bat-derived cells following exposure to -20 °C for 24 h in media with no cryoprotective agents or medium supplemented by glucose in concentration range 0-3333 mM. Cell survival rates were determined in relation to availability of glucose in the medium, organ origin, cell concentration and bat species. In general, increased glucose helped cells survive at sub-zero temperatures, though concentrations up to 80-fold higher than those found in chiropterans were needed. However, cells in glucose-free phosphate buffered saline also survived, suggesting that other mechanisms may be contributing to cell survival at low temperatures. Highest in vitro viability was observed in nervus olfactorius-derived cell cultures, with high survival rates and rapid re-growth under optimal conditions after exposure to -20 °C. Kidney cells from different bat species showed comparable overall survival rate patterns, though smaller chiropteran species appeared to utilise lower glucose levels as a cryoprotectant than larger species. Our in vitro data provide evidence that cells of heterothermic bats can survive sub-zero temperatures and that higher glucose levels in important tissues significantly improve hibernation survival at extremely low temperatures.

Molecular Detection and Phylogenetic Analysis of Deformed Wing Virus and Sacbrood Virus Isolated from Pollen.

Among many pathogens and pests, honey bee viruses are known as one of the most common cause of diseases in honey bee colonies. In this study, we demonstrate that pollen grains and bee bread are potential sources of viral DNA. We extracted DNA from 3 types of pollen samples: directly provided by beekeepers (n = 12), purchased from trade markets (n = 5), and obtained from honeycombs (bee bread, n = 10). The extracted DNA was used for molecular detection (RT-PCR analysis) of six of the most widely distributed honey bee viruses: deformed wing virus, sacbrood virus, acute bee paralysis virus, black queen cell virus, Kashmir bee virus, Israeli acute paralysis virus, and chronic bee paralysis virus. We successfully managed to establish only the deformed wing virus (DWV) and the sacbrood virus (SBV), with different distribution frequencies depending on the territory of the country. The phylogenetic analyses of Bulgarian isolates were performed with the most similar sequences available in molecular databases from other countries. Phylogenies of Bulgarian viral strains demonstrated genetically heterogeneous populations of DWV and relatively homogenous populations of SBV. In conclusion, the results obtained from the current study have shown that pollen is a valuable source for molecular detection of honey bee pathogens. This allows epidemiological monitoring of honey bee diseases at a regional and a national level.

Bats at an Altitude above 2000 m on Pirin Mountain, Bulgaria.

The study describes a pilot survey on bats in the highest areas of Pirin Mountain. The methods included examining subfossil bone remains, mist-netting, and recording echolocation calls. The study was conducted in August 2002 and 2013 and from 2019 to 2020. While in general, bat diversity tends to decrease with increasing altitude due to harsher environmental conditions, the present study, despite a short period, reveals high diversity. Twenty species, more than half of the Bulgarian bat fauna, were detected. The recording and analysis of vocal signatures proved to be the best way to inventory bat diversity. At least 13 species were detected by this method. Vespertilio murinus and Tadarida teniotis together make up more than 60% of all reliably determined echolocation sequences. Significant activity was found for Myotis myotis/blythii, Plecotus auritus, Eptesicus serotinus, and E. nilssonii. The registration of the latter species is of considerable faunistic interest. It was previously only known from a single specimen at one location in the country. The sex and age structure of the bat assemblage suggests that it is likely a swarming assemblage. The area is the highest swarming location in Europe. The results provide valuable information on bat ecology and behaviour, which can be used to inform management and protection efforts.

Performance of bat-derived macrophages at different temperatures.

Heterothermy, as a temperature-dependent physiological continuum, may affect host-pathogen interactions through modulation of immune responses. Here, we evaluated proliferation and functional performance of a macrophage cell line established from the greater mouse-eared (Myotis myotis) bat at 8, 17.5, and 37°C to simulate body temperatures during hibernation, daily torpor and euthermia. Macrophages were also frozen to -20°C and then examined for their ability to proliferate in the immediate post-thaw period. We show that bat macrophages can proliferate at lower temperatures, though their growth rate is significantly slower than at 37°C. The cells differed in their shape, size and ability to attach to the plate surface at both lower temperatures, being spheroidal and free in suspension at 8°C and epithelial-like, spindle-shaped and/or spheroidal at 17.5°C. While phagocytosis at temperatures of 8 and 17.5°C amounted to 85.8 and 83.1% of the activity observed at 37°C, respectively, full phagocytic activity was restored within minutes of translocation into a higher temperature. Bat-derived macrophages were also able to withstand temperatures of -20°C in a cryoprotectant-free cultivation medium and, in the immediate post-thaw period, became viable and were able to proliferate. Our in vitro data enhance understanding of macrophage biology.

A new species of Spinturnix (Acari: Spinturnicidae) from the Turkestani long-eared bat Otonycteris leucophaea (Chiroptera: Vespertilionidae) in Kazakhstan.

The female, male and protonymph of Spinturnix otonycterisi Dundarova & Orlova, sp. nov. (Acari: Spinturnicidae) are described from specimens collected on the enigmatic and rarely captured vespertilionid bat Otonycteris leucophaea (Severcov, 1873) in South Kazakhstan. A key for the identification of the species of Spinturnix von Heyden, 1826 found in Kazakhstan is provided.

Active surveillance for antibodies confirms circulation of lyssaviruses in Palearctic bats.

BACKGROUND: Palearctic bats host a diversity of lyssaviruses, though not the classical rabies virus (RABV). As surveillance for bat rabies over the Palearctic area covering Central and Eastern Europe and Siberian regions of Russia has been irregular, we lack data on geographic and seasonal patterns of the infection. RESULTS: To address this, we undertook serological testing, using non-lethally sampled blood, on 1027 bats of 25 species in Bulgaria, the Czech Republic, Poland, Russia and Slovenia between 2014 and 2018. The indirect enzyme-linked immunosorbent assay (ELISA) detected rabies virus anti-glycoprotein antibodies in 33 bats, giving an overall seroprevalence of 3.2%. Bat species exceeding the seroconversion threshold included Myotis blythii, Myotis gracilis, Myotis petax, Myotis myotis, Murina hilgendorfi, Rhinolophus ferrumequinum and Vespertilio murinus. While Myotis species (84.8%) and adult females (48.5%) dominated in seropositive bats, juveniles of both sexes showed no difference in seroprevalence. Higher numbers tested positive when sampled during the active season (10.5%), as compared with the hibernation period (0.9%). Bat rabies seroprevalence was significantly higher in natural habitats (4.0%) compared with synanthropic roosts (1.2%). Importantly, in 2018, we recorded 73.1% seroprevalence in a cave containing a M. blythii maternity colony in the Altai Krai of Russia. CONCLUSIONS: Identification of such "hotspots" of non-RABV lyssavirus circulation not only provides important information for public health protection, it can also guide research activities aimed at more in-depth bat rabies studies.

Patterns of mtDNA introgression suggest population replacement in Palaearctic whiskered bat species.

Secondary contacts can play a major role in the evolutionary histories of species. Various taxa diverge in allopatry and later on come into secondary contact during range expansions. When they meet, their interactions and the extent of gene flow depend on the level of their ecological differentiation and the strength of their reproductive isolation. In this study, we present the multilocus phylogeography of two cryptic whiskered bat species, Myotis mystacinus and M. davidii, with a particular focus on their putative sympatric zone. Our findings suggest that M. mystacinus and M. davidii evolved in allopatry and came into secondary contact during range expansions. Individuals in the area of secondary contact, in Anatolia and the Balkans, have discordant population assignments based on the mitochondrial and the nuclear datasets. These observed patterns suggest that the local M. mystacinus populations hybridized with expanding M. davidii populations, which resulted in mitochondrial introgression from the former. In the introgression area, M. mystacinus individuals with concordant nuclear and mitochondrial genotypes were identified in relatively few locations, suggesting that the indigenous populations might have been largely replaced by invading M. davidii. Changing environmental conditions coupled with ecological competition is the likely reason for this replacement. Our study presents one possible example of a historical population replacement that was captured in phylogeographic patterns.

Hibernation temperature-dependent Pseudogymnoascus destructans infection intensity in Palearctic bats.

White-nose syndrome (WNS) is a fungal disease caused by Pseudogymnoascus destructans that is devastating to Nearctic bat populations but tolerated by Palearctic bats. Temperature is a factor known to be important for fungal growth and bat choice of hibernation. Here we investigated the effect of temperature on the pathogenic fungal growth in the wild across the Palearctic. We modelled body surface temperature of bats with respect to fungal infection intensity and disease severity and were able to relate this to the mean annual surface temperature at the site. Bats that hibernated at lower temperatures had less fungal growth and fewer skin lesions on their wings. Contrary to expectation derived from laboratory P. destructans culture experiments, natural infection intensity peaked between 5 and 6°C and decreased at warmer hibernating temperature. We made predictive maps based on bat species distributions, temperature and infection intensity and disease severity data to determine not only where P. destructans will be found but also where the infection will be invasive to bats across the Palearctic. Together these data highlight the mechanistic model of the interplay between environmental and biological factors, which determine progression in a wildlife disease.