Assessing bat droppings and predatory bird pellets for vector-borne bacteria: molecular evidence of bat-associated <Emphasis Type="Italic">Neorickettsia</Emphasis> sp. in Europe

In Europe, several species of bats, owls and kestrels exemplify highly urbanised, flying vertebrates, which may get close to humans or domestic animals. Bat droppings and bird pellets may have epidemiological, as well as diagnostic significance from the point of view of pathogens. In this work 221 bat faecal and 118 bird pellet samples were screened for a broad range of vector-borne bacteria using PCR-based methods. Rickettsia DNA was detected in 13 bat faecal DNA extracts, including the sequence of a rickettsial insect endosymbiont, a novel Rickettsia genotype and Rickettsia helvetica. Faecal samples of the pond bat (Myotis dasycneme) were positive for a Neorickettsia sp. and for haemoplasmas of the haemofelis group. In addition, two bird pellets (collected from a Long-eared Owl, Asio otus, and from a Common Kestrel, Falco tinnunculus) contained the DNA of a Rickettsia sp. and Anaplasma phagocytophilum, respectively. In both of these bird pellets the bones of Microtus arvalis were identified. All samples were negative for Borrelia burgdorferi s.l., Francisella tularensis, Coxiella burnetii and Chlamydiales. In conclusion, bats were shown to pass rickettsia and haemoplasma DNA in their faeces. Molecular evidence is provided for the presence of Neorickettsia sp. in bat faeces in Europe. In the evaluated regions bat faeces and owl/kestrel pellets do not appear to pose epidemiological risk from the point of view of F. tularensis, C. burnetii and Chlamydiales. Testing of bird pellets may provide an alternative approach to trapping for assessing the local occurrence of vector-borne bacteria in small mammals.     

Comparative Analysis of the Amino Acid Spectrum of Blood Plasma in Chiroptera (<Emphasis Type="Italic">Vespertilio murinus</Emphasis> L., 1758 and <Emphasis Type="Italic">Myotis dasycneme</Emphasis> B., 1825) in the Fauna of the Ural Mountains

The article presents the results of a new comparative analysis of free amino acids in the blood plasma of representatives of insectivorous Chiroptera (Mammalia: Vespertilionidae) in the fauna of the Ural Mountains: the pond bat (Myotis dasycneme Boie, 1825) and the parti-colored bat (Vespertilio murinus Linnaeus, 1758). This is the first study to show the species variability of free amino acids in resident and migratory species of bats from different ecosystems of the Ural Region.     

Species structure and dynamics of bat communities (Chiroptera: Vespertilionidae) hibernating in artificial caves of Samara Luka

Long-term data on the composition and absolute and relative abundance of eight bat species hibernating in artificial caves of Samara Luka are considered. About 13 000 hibernating individuals are annually accounted there. The relative abundance of the species decreased in the following order: Myotis brandtii (35%), Plecotus auritus (20%), M. daubentonii (13%), M. mystacinus (13%), Eptesicus nilssonii (8%), M. dasycneme (8%), and M. nattereri (2%). Singular E. serotinus were recorded not every year. The number of hibernating animals in pits was proportional to their size and the number of shelters. Bats used previously chosen shelters and were never found in adjacent caves. The maximum species diversity (H’) and evenness (E) were observed in bat communities in medium-sized caves. One of the main factors of increasing abundance of hibernating bats in abandoned pits is their gradual colonization by the animals.     

The isozyme spectra of lactate dehydrogenase in the tissues of the raccoon dog <Emphasis Type="Italic">Nyctereutes procyonoides</Emphasis> in the autumn

An electrophoretic assay of lactate dehydrogenase (EC 1.1.1.27) isozymes in the tissue homogenates of cardiac and skeletal muscles, kidney, lungs, spleen, and liver of the raccoon dog Nyctereutes procyonoides from two different geographic zones, viz., northwestern Russia and Poland, as well as the Arctic blue fox Alopex lagopus L. and the red fox Vulpes vulpes L. was performed during the preparatory period to the winter season. Raccoon dogs, which hibernate under natural conditions, differ from other canids (the red fox and Arctic blue fox) to which they are close taxonomically by their body weight and by the higher proportion of aerobic H subunits of lactate dehydrogenase in all organs except for the heart. A higher content of “fast” anode fractions, lactate dehydrogenase-1 and lactate dehydrogenase-2, in the heart, kidney, lungs, liver, and spleen was detected in the raccoon dogs from the northern region compared to those from the southern geographic zone. The shift in the reaction catalyzed by lactate dehydrogenase towards the production of pyruvate indicates that this metabolite is necessary for the synthesis of fatty acids during lipogenesis in the autumn.     

Subcellular localization of isozymes of NAD-dependent malate dehydrogenase in sugar beet <Emphasis Type="Italic">Beta vulgaris</Emphasis> L.

Subcellular localization of isozymes of NAD-dependent malate dehydrogenase (MDH) in sugar beet was studied. Isozymes ss and ll controlled by loci Mdh2 and Mdh3, respectively, were shown to locate in mitochondria, whereas isozyme pp controlled by locus Mdh1, in microbodies. All examined samples lack hybrid MDH isozymes, which could testify to the interaction between products of nonallelic Mdh genes. This can be explained by the localization of nonallelic isozymes in various compartments of the cell and organelles.     

Coronavirus and paramyxovirus in bats from Northwest Italy

Background

Bat-borne virus surveillance is necessary for determining inter-species transmission risks and is important due to the wide-range of bat species which may harbour potential pathogens. This study aimed to monitor coronaviruses (CoVs) and paramyxoviruses (PMVs) in bats roosting in northwest Italian regions. Our investigation was focused on CoVs and PMVs due to their proven ability to switch host and their zoonotic potential. Here we provide the phylogenetic characterization of the highly conserved polymerase gene fragments.

Results

Family-wide PCR screenings were used to test 302 bats belonging to 19 different bat species. Thirty-eight animals from 12 locations were confirmed as PCR positive, with an overall detection rate of 12.6% [95% CI: 9.3–16.8]. CoV RNA was found in 36 bats belonging to eight species, while PMV RNA in three Pipistrellus spp. Phylogenetic characterization have been obtained for 15 alpha- CoVs, 5 beta-CoVs and three PMVs; moreover one P. pipistrellus resulted co-infected with both CoV and PMV. A divergent alpha-CoV clade from Myotis nattereri SpA is also described. The compact cluster of beta-CoVs from R. ferrumequinum roosts expands the current viral sequence database, specifically for this species in Europe. To our knowledge this is the first report of CoVs in Plecotus auritus and M. oxygnathus, and of PMVs in P. kuhlii.

Conclusions

This study identified alpha and beta-CoVs in new bat species and in previously unsurveyed Italian regions. To our knowledge this represents the first and unique report of PMVs in Italy. The 23 new bat genetic sequences presented will expand the current molecular bat-borne virus databases. Considering the amount of novel bat-borne PMVs associated with the emergence of zoonotic infections in animals and humans in the last years, the definition of viral diversity within European bat species is needed. Performing surveillance studies within a specific geographic area can provide awareness of viral burden where bats roost in close proximity to spillover hosts, and form the basis for the appropriate control measures against potential threats for public health and optimal management of bats and their habitats.

     

Managing anthropogenic driven range expansion behaviourally: Mediterranean bats in desert ecosystems

Bat abundance and activity in deserts is affected by the distribution of water sources. Kuhl’s pipistrelle (Pipistrellus kuhlii) is one of several bat species that penetrated the Negev desert from Mediterranean habitats following anthropogenic development, and potentially competes with desert dwelling bats of the same guild. Pipistrellus kuhlii is an obligate drinker requiring a clear ‘swoop zone’ to drink. Thus, to reduce the competitive load that P. kuhlii has on desert dwelling species, we tested the effectiveness of obstructions above water surfaces, which we hypothesised would reduce the drinking ability of P. kuhlii, thus providing desert species a competitive edge. We obstructed the water surface of a swimming pool and successfully prevented P. kuhlii from drinking. Next, we manipulated natural water sources used by bats for foraging. This was done on two spatial and temporal scales: on 11 adjacent ephemeral water pools in Nahal Zin where each treatment was applied for several nights and on eight isolated natural pools in the Negev where treatments were alternated every 20 min. Manipulation of adjacent pools had no effect on activity levels of bats, but in isolated pools, the obstruction reduced the proportion of P. kuhlii activity by up to 15%. Although this tool cannot be used for management without further examination of its long-term effects on other bats and wildlife, this study does demonstrate its feasibility. Conservation schemes aiming to mitigate effects of undesirable species can be designed by identifying ecological differences between competing species, and manipulating the environment accordingly.     

Roost characteristics as indicators for heterothermic behavior of forest-dwelling bats

Many forest-dwelling bats spend their diurnal inactivity period in tree cavities. During this time bats can save energy through heterothermy. A heterothermic response (torpor) is characterized by a lowered body temperature, reduced metabolic rate, and reduction of other physiological processes, and can be influenced by the microclimatic conditions of roost cavities. The thermal and physical characteristics of roosts used by the sympatric, ecologically, and morphologically similar bat species Myotis bechsteinii, M. nattereri, and Plecotus auritus were compared. These three species differ in their heterothermic behavior, with the lowest skin temperatures observed for P. auritus. Therefore, we hypothesized that roosts occupied by the three species should differ in roost characteristics and microclimatic conditions, whereby P. auritus should select colder and thermally less stable roosts. The results showed that horizontal depth of the cavity, diameter of the roost tree, and microclimatic conditions within roosts differed among species. Roosts of P. auritus had the lowest horizontal depth, lowest thermal stability, and lowest mean minimum roost temperatures. Height of the roost, diameter of the roost tree, and vertical depth were also shown to influence microclimatic conditions. With increasing diameter of the tree and increasing horizontal depth, mean minimum roost temperature increased and thermal stability improved. Furthermore, with ascending height above ground insulation and mean roost temperatures increased. Our results imply that species such as P. auritus, which use pronounced torpor as a primary energy saving strategy, prefer colder cavities that support their heterothermic strategy.     

Seasonal activity pattern and habitat use by the Kuhl’s pipistrelle (<Emphasis Type="Italic">Pipistrellus kuhlii</Emphasis>) in an arid environment

We studied the seasonal activity pattern of the Kuhl’s pipistrelle Pipistrellus kuhlii in eight sites of the Bou Hedma National Park, central Tunisia, from June 2010 to June 2011, using both mistnetting and acoustic bat detection. Pipistrelles were captured all year-round only at water bodies. Captures peaked in late spring and early autumn. Pregnant females were observed from March to June, lactating females only in June, and flying juveniles from June to August. Echolocation calls and buzzes were recorded in all sites throughout the year. However, activity varied significantly among months and sites. Activity peaked in June and September after a noteworthy decline in August; the minimum was recorded in December and January. Bats mainly foraged at water bodies and around streetlamps. This result supports the previously reported major role of water bodies for bats in arid environments. Bats also foraged in the open acacia forest in summer, during the flowering period of Acacia Acacia tortilis raddiana. The steppe was the least used habitat. Social calls were mainly recorded in autumn around streetlamps, suggesting a swarming behaviour. Despite being a common bat species in the area, P. kuhlii should deserve conservation efforts that would benefit other bat species.     

Ecomorph Evolution in <Emphasis Type="Italic">Myotis</Emphasis> (Vespertilionidae,Chiroptera)

The genus Myotis is unique among mammals in its high taxonomic diversity and global distribution. Their phylogenetic relationships reflect biogeographic affinities rather than phenotypes. Myotis diverged from other bats in the early Miocene, with a subsequent split between Old and New World lineages about 19 million years ago. Similar ecomorphs (‘Leuconoe’ [near-water hunters], ‘Myotis’ [gleaners], ‘Selysius’ [aerial hawkers]) emerged independently in different lineages of Myotis. We retrieved the probable ancestral ecomorph for each lineage. Phenetic diversity was estimated from the analysis of body and skull traits. It seems that evolution of Myotis fluctuated between ‘Leuconoe’, ‘Selysius’, and larger ‘Myotis’.     

<Emphasis Type="Italic">Malassezia</Emphasis> spp. in Acoustic Meatus of Bats (<Emphasis Type="Italic">Molossus molossus</Emphasis>) of the Amazon Region,Brazil

The yeasts of the Malassezia genus are opportunistic microorganisms and can cause human and animal infections. They are commonly isolated from the skin and auricular canal of mammalians, mainly dogs and cats. The present study was aimed to isolate Malassezia spp. from the acoustic meatus of bats (Molossus molossus) in the Montenegro region, “Rondônia”, Brazil. From a total of 30 bats studied Malassezia spp. were isolated in 24 (80%) animals, the breakdown by species being as follows (one Malassezia sp. per bat, N = 24): 15 (62.5%) M. pachydermatis, 5 (20.8%) M. furfur, 3 (12.5%) M. globosa and 1 (4.2%) M. sympodialis. This study establishes a new host and anatomic place for Malassezia spp., as it presents the first report ever of the isolation of this genus of yeasts in the acoustic meatus of bats.     

Coexistence of multiple coronaviruses in several bat colonies in an abandoned mineshaft

Since the 2002–2003 severe acute respiratory syndrome(SARS) outbreak prompted a search for the natural reservoir of the SARS coronavirus, numerous alpha- and betacoronaviruses have been discovered in bats around the world. Bats are likely the natural reservoir of alpha- and betacoronaviruses, and due to the rich diversity and global distribution of bats, the number of bat coronaviruses will likely increase. We conducted a surveillance of coronaviruses in bats in an abandoned mineshaft in Mojiang County, Yunnan Province, China, from 2012–2013. Six bat species were frequently detected in the cave: Rhinolophus sinicus, Rhinolophus affinis, Hipposideros pomona, Miniopterus schreibersii, Miniopterus fuliginosus, and Miniopterus fuscus. By sequencing PCR products of the coronavirus RNA-dependent RNA polymerase gene(Rd Rp), we found a high frequency of infection by a diverse group of coronaviruses in different bat species in the mineshaft. Sequenced partial Rd Rp fragments had 80%–99% nucleic acid sequence identity with well-characterized Alphacoronavirus species, including Bt CoV HKU2, Bt CoV HKU8, and Bt CoV1,and unassigned species Bt CoV HKU7 and Bt CoV HKU10. Additionally, the surveillance identified two unclassified betacoronaviruses, one new strain of SARS-like coronavirus, and one potentially new betacoronavirus species. Furthermore, coronavirus co-infection was detected in all six bat species, a phenomenon that fosters recombination and promotes the emergence of novel virus strains. Our findings highlight the importance of bats as natural reservoirs of coronaviruses and the potentially zoonotic source of viral pathogens.     

Coronavirus Infection and Diversity in Bats in the Australasian Region

Following the SARS outbreak, extensive surveillance was undertaken globally to detect and identify coronavirus diversity in bats. This study sought to identify the diversity and prevalence of coronaviruses in bats in the Australasian region. We identified four different genotypes of coronavirus, three of which (an alphacoronavirus and two betacoronaviruses) are potentially new species, having less than 90% nucleotide sequence identity with the most closely related described viruses. We did not detect any SARS-like betacoronaviruses, despite targeting rhinolophid bats, the putative natural host taxa. Our findings support the virus-host co-evolution hypothesis, with the detection of Miniopterus bat coronavirus HKU8 (previously reported in Miniopterus species in China, Hong Kong and Bulgaria) in Australian Miniopterus species. Similarly, we detected a novel betacoronavirus genotype from Pteropus alecto which is most closely related to Bat coronavirus HKU9 identified in other pteropodid bats in China, Kenya and the Philippines. We also detected possible cross-species transmission of bat coronaviruses, and the apparent enteric tropism of these viruses. Thus, our findings are consistent with a scenario wherein the current diversity and host specificity of coronaviruses reflects co-evolution with the occasional host shift.     

West nile virus antibodies in bats from New Jersey and New York

Eighty-three serum samples were obtained from big brown (Eptesicus fuscus), little brown (Myotis lucifugus), and northern long-eared (Myotis septentriotalis) bats (Chiroptera: Vespertilionidae), from New Jersey and New York (USA) between July and October 2002. Samples were analyzed for neutralizing antibodies to West Nile virus (WNV) and St. Louis encephalitis (SLE) virus. One little brown bat and one northern long-eared bat tested positive for WNV neutralizing antibodies. No bats had antibodies to SLE virus. This was the first large-scale investigation of WNV infection in bats in New Jersey. Additional work is needed to determine the effects of WNV on bat populations.     

Isoenzyme spectra of lactate dehydrogenase in tissues of mammals of the Mustelidae family

A comparative analysis of distribution of lactate dehydrogenase (LDH) isoenzymes in the heart, kidneys, lungs, spleen, liver and skeletal muscle was performed in 4 species of carnivorous mammals (Mustelidae family): American mink (Mustela vison Schr.), polecat (Mustela putorius L.), sable (Martes zibellina L.) and pine marten (Martes martes L.). It was found that in the mink, unlike pure terrestrial mustelids (polecat, sable, pine marten), the anaerobic LDH-5 fraction was predominant in the LDH isoenzyme spectrum in the liver, skeletal muscle, lungs and spleen. In all species studied, aerobic B(H) subunits of LDH were found to prevail in the heart and kidney, whereas anaerobic A(M) subunits prevailed in the liver and spleen. The tissue- and species-specific features of LDH isoenzyme spectra revealed in different mustelids reflect the biochemical adaptation of the animals to environmental conditions.     

Climate Change Could Increase the Geographic Extent of Hendra Virus Spillover Risk

Disease risk mapping is important for predicting and mitigating impacts of bat-borne viruses, including Hendra virus (Paramyxoviridae:Henipavirus), that can spillover to domestic animals and thence to humans. We produced two models to estimate areas at potential risk of HeV spillover explained by the climatic suitability for its flying fox reservoir hosts, Pteropus alecto and P. conspicillatus. We included additional climatic variables that might affect spillover risk through other biological processes (such as bat or horse behaviour, plant phenology and bat foraging habitat). Models were fit with a Poisson point process model and a log-Gaussian Cox process. In response to climate change, risk expanded southwards due to an expansion of P. alecto suitable habitat, which increased the number of horses at risk by 175–260% (110,000–165,000). In the northern limits of the current distribution, spillover risk was highly uncertain because of model extrapolation to novel climatic conditions. The extent of areas at risk of spillover from P. conspicillatus was predicted shrink. Due to a likely expansion of P. alecto into these areas, it could replace P. conspicillatus as the main HeV reservoir. We recommend: (1) HeV monitoring in bats, (2) enhancing HeV prevention in horses in areas predicted to be at risk, (3) investigate and develop mitigation strategies for areas that could experience reservoir host replacements.     

Peroxidase isozymes in wheat infected withTilletia controversa Kühn and rye infected withPuccinia recondita Rob. exDesm

Peroxidase isozymes were studied in the case of two diseases:Tilletia controversa in wheat andPuccinia recondita in rye. The method employed was that of disc-electrophoresis on acrylamid gel. In the case of systemic disease (Tilletia controversa) no change was found in the isozyme pattern until a concentration of pathogen took place in the ears that began to form. Proofs are given that the new isozyme in unmatured ears comes from the pathogen. In the case of rye infected withPuccinia recondita a new isozyme was found; the latter does not originate in the pathogen, its origin is being discussed.     

Molecular Phylogeny of Serotines (Mammalia,Chiroptera, <Emphasis Type="Italic">Eptesicus</Emphasis>): Evolutionary and Taxonomical Aspects of the <Emphasis Type="Italic">E. serotinus</Emphasis> Species Group

This article provides a phylogenetic analysis of five nuclear and mitochondrial cytochrome b genes of palaearctic serotines. Nuclear data yield five monophyletic clades: Botta’s serotine and the South African long-tailed house bat E. hottentottus; the common serotine bat (including all studied E. serotinus subspecies and andersoni form of undefined status) and the meridional serotine E. isabellinus; the Gobi serotine, including Bobrinski’s serotine; the northern bat; and New World serotines. We found latest taxonomic decisions regarding mirza and pachyomus questionable and needing further revision. The significant inconsistency between mitochondrial and nuclear phylogenies obtained for genes of different inheritance systems suggests repetitive introgression events in the evolution of the genus.     

Bats (Chiroptera,Vespertillionidae) from the Cis-Urals and South Urals (Republic of Bashkortostan)

New and previously published data on the distribution of 12 bat species in the Cis-Urals and South Urals (Republic of Bashkortostan) are summarized. Data on their subspecies status are given. There is a need to clarify the taxonomic status of the pipistrelle bats Pipistrellus pipistrellus/pygmaeus. In terms of composition, the bat fauna of the Republic of Bashkortostan is a variant of the Central European fauna. It was revealed that the territory in question marks the eastern limits of distribution of Nyctalus leisleri and P. pipistrellus/pygmaeus. An important feature of the territory of Bashkortostan is the presence of places of mass wintering of bats of natural origin: more than 980 karst caves used by bats not only in winter, but also during the period of activity. According to the results of captures in 14 caves, data on the frequency and relative abundance of sedentary bat species in the Republic of Bashkortostan are given.     

Green Fluorescent Protein Expression in <Emphasis Type="Italic">Pseudogymnoascus destructans</Emphasis> to Study Its Abiotic and Biotic Lifestyles

Pseudogymnoascus destructans (Pd) is the etiologic agent of bat White-nose syndrome, a disease that has caused the unprecedented reduction in the hibernating bat populations across eastern North America. The Pd pathogenesis appears to be a complex adaptation of fungus in its abiotic (caves and mines) and biotic (bats) environments. There is a general lack of experimental tools for the study of Pd biology. We described the successful expression of codon-optimized synthetic green fluorescent protein sGFP in Pd. The sGFP(S65T) gene was first fused in frame with the Aspergillus nidulans promoter in the tumor-inducing plasmid pRF-HUE, and the resulting plasmid pHUE-sGFP(S65T) was transformed into Pd by Agrobacterium tumefaciens-mediated transformation system. The integration of sGFP(S65T) in Pd genome was analyzed by PCR, and single integration frequency of approximately 66% was confirmed by Southern hybridization. Fluorescent microscopy and flow cytometric analyses of two randomly selected transformants with single integration revealed high expression of sGFP in both spores and hyphal structures. The biology of mutants as judged by sporulation, growth rate, and urease production was not altered indicating sGFP is not toxic to Pd. Thus, we have generated a valuable tool that will facilitate the elucidation of Pd biology, ecology, and pathogenicity in real time.