Higher antibody titres against Pseudogymnoascus destructans are associated with less white-nose syndrome skin lesions in Palearctic bats.

Introduction: Serological tests can be used to test whether an animal has been exposed to an infectious agent, and whether its immune system has recognized and produced antibodies against it. Paired samples taken several weeks apart then document an ongoing infection and/or seroconversion. Methods: In the absence of a commercial kit, we developed an indirect enzyme-linked immunosorbent assay (ELISA) to detect the fungus-specific antibodies for Pseudogymnoascus destructans, the agent of white-nose syndrome in bats. Results and Discussion: Samples collected from European Myotis myotis (n=35) and Asian Myotis dasycneme (n=11) in their hibernacula at the end of the hibernation period displayed 100% seroprevalence of antibodies against P. destructans, demonstrating a high rate of exposure. Our results showed that the higher the titre of antibodies against P. destructans, the lower the infection intensity, suggesting that a degree of protection is provided by this arm of adaptive immunity in Palearctic bats. Moreover, P. destructans infection appears to be a seasonally self-limiting disease of Palearctic bats showing seroconversion as the WNS skin lesions heal in the early post-hibernation period.

Case report: Filarial infection of a parti-coloured bat: Litomosa sp. adult worms in abdominal cavity and microfilariae in bat semen.

Background: Filarial infections have been understudied in bats. Likewise, little is known about pathogens associated with the reproductive system in chiropterans. While semen quality is critical for reproductive success, semen-borne pathogens may contribute to reproductive failure. Methods: For the first time we performed electroejaculation and used computer-assisted semen analysis to provide baseline data on semen quality in a parti-coloured bat (Vespertilio murinus). Results: The semen quality values measured in the V. murinus male appeared high (semen concentration = 305.4 × 106/mL; progressive and motile sperm = 46.58 and 60.27%, respectively). As an incidental finding, however, microfilariae were observed in the bat semen examined. At necropsy, eight adult filarial worms, later genetically identified as Litomosa sp., were found in the peritoneal cavity, close to the stomach, of the same particoloured bat male dying as a result of dysmicrobia and haemorrhagic gastroenteritis in a wildlife rescue centre. Histopathology revealed microfilariae in the testicular connective tissue and the epidydimal connective and fat tissues. A PCR assay targeting cytochrome c oxidase subunit 1 confirmed that adult worms from the peritoneal cavity and testicular microfilariae were of the same filarial species. Mildly engorged argasid mite larvae attached to the bat skin proved negative for filarial DNA and the adult filarial worms proved negative for endosymbiont Wolbachia. Conclusion: While the standard filarial life cycle pattern involves a vertebrate definitive host and an invertebrate vector, represented by a blood-sucking ectoparasite, our finding suggests that microfilariae of this nematode species may also be semen-borne, with transmission intensity promoted by the polygynous mating system of vespertilionid bats in which an infected male mates with many females during the autumn swarming. Presence of microfilariae may be expected to decrease semen quality and transmission via this route may challenge the success of reproductive events in females after mating. Further investigation will be necessary to better understand the bat-parasite interaction and the life cycle of this filarial worm.

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.

Transcriptomic responses of bat cells to European bat lyssavirus 1 infection under conditions simulating euthermia and hibernation.

BACKGROUND: Coevolution between pathogens and their hosts decreases host morbidity and mortality. Bats host and can tolerate viruses which can be lethal to other vertebrate orders, including humans. Bat adaptations to infection include localized immune response, early pathogen sensing, high interferon expression without pathogen stimulation, and regulated inflammatory response. The immune reaction is costly, and bats suppress high-cost metabolism during torpor. In the temperate zone, bats hibernate in winter, utilizing a specific behavioural adaptation to survive detrimental environmental conditions and lack of energy resources. Hibernation torpor involves major physiological changes that pose an additional challenge to bat-pathogen coexistence. Here, we compared bat cellular reaction to viral challenge under conditions simulating hibernation, evaluating the changes between torpor and euthermia. RESULTS: We infected the olfactory nerve-derived cell culture of Myotis myotis with an endemic bat pathogen, European bat lyssavirus 1 (EBLV-1). After infection, the bat cells were cultivated at two different temperatures, 37 °C and 5 °C, to examine the cell response during conditions simulating euthermia and torpor, respectively. The mRNA isolated from the cells was sequenced and analysed for differential gene expression attributable to the temperature and/or infection treatment. In conditions simulating euthermia, infected bat cells produce an excess signalling by multitude of pathways involved in apoptosis and immune regulation influencing proliferation of regulatory cell types which can, in synergy with other produced cytokines, contribute to viral tolerance. We found no up- or down-regulated genes expressed in infected cells cultivated at conditions simulating torpor compared to non-infected cells cultivated under the same conditions. When studying the reaction of uninfected cells to the temperature treatment, bat cells show an increased production of heat shock proteins (HSPs) with chaperone activity, improving the bat's ability to repair molecular structures damaged due to the stress related to the temperature change. CONCLUSIONS: The lack of bat cell reaction to infection in conditions simulating hibernation may contribute to the virus tolerance or persistence in bats. Together with the cell damage repair mechanisms induced in response to hibernation, the immune regulation may promote bats' ability to act as reservoirs of zoonotic viruses such as lyssaviruses.

Higher white-nose syndrome fungal isolate yields from UV-guided wing biopsies compared with skin swabs and optimal culture media.

BACKGROUND: North American bat populations have suffered severe declines over the last decade due to the Pseudogymnoascus destructans fungus infection. The skin disease associated with this causative agent, known as white-nose syndrome (WNS), is specific to bats hibernating in temperate regions. As cultured fungal isolates are required for epidemiological and phylogeographical studies, the purpose of the present work was to compare the efficacy and reliability of different culture approaches based on either skin swabs or wing membrane tissue biopsies for obtaining viable fungal isolates of P. destructans. RESULTS: In total, we collected and analysed 69 fungal and 65 bacterial skin swabs and 51 wing membrane tissue biopsies from three bat species in the Czech Republic, Poland and the Republic of Armenia. From these, we obtained 12 viable P. destructans culture isolates. CONCLUSIONS: Our results indicated that the efficacy of cultures based on wing membrane biopsies were significantly higher. Cultivable samples tended to be based on collections from bats with lower body surface temperature and higher counts of UV-visualised lesions. While cultures based on both skin swabs and wing membrane tissue biopsies can be utilised for monitoring and surveillance of P. destructans in bat populations, wing membrane biopsies guided by UV light for skin lesions proved higher efficacy. Interactions between bacteria on the host's skin also appear to play an important role.

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.

Detection of Leptospira species in bat cadavers, Czech and Slovak Republics.

Kidney samples from 300 bat cadavers from the Czech and Slovak Republics were tested for Leptospira DNA using PCR and sequencing of three genes (lipL32, flab, and 16S ribosomal RNA). Overall detection rate was 4.7% and two bat species (Myotis myotis and Nyctalus noctula) were PCR-positive for at least one gene. Detected Leptospira sequences were similar to L. interrogans and L. borgpetersenii, and included a potentially novel species related to L. weilii.

Species-Specific Molecular Barriers to SARS-CoV-2 Replication in Bat Cells.

Bats are natural reservoirs of numerous coronaviruses, including the potential ancestor of SARS-CoV-2. Knowledge concerning the interaction between coronaviruses and bat cells is sparse. We investigated the ability of primary cells from Rhinolophus and Myotis species, as well as of established and novel cell lines from Myotis myotis, Eptesicus serotinus, Tadarida brasiliensis, and Nyctalus noctula, to support SARS-CoV-2 replication. None of these cells were permissive to infection, not even the ones expressing detectable levels of angiotensin-converting enzyme 2 (ACE2), which serves as the viral receptor in many mammalian species. The resistance to infection was overcome by expression of human ACE2 (hACE2) in three cell lines, suggesting that the restriction to viral replication was due to a low expression of bat ACE2 (bACE2) or the absence of bACE2 binding in these cells. Infectious virions were produced but not released from hACE2-transduced M. myotis brain cells. E. serotinus brain cells and M. myotis nasal epithelial cells expressing hACE2 efficiently controlled viral replication, which correlated with a potent interferon response. Our data highlight the existence of species-specific and cell-specific molecular barriers to viral replication in bat cells. These novel chiropteran cellular models are valuable tools to investigate the evolutionary relationships between bats and coronaviruses. IMPORTANCE Bats are host ancestors of several viruses that cause serious disease in humans, as illustrated by the ongoing SARS-CoV-2 pandemic. Progress in investigating bat-virus interactions has been hampered by a limited number of available bat cellular models. We have generated primary cells and cell lines from several bat species that are relevant for coronavirus research. The various permissivities of the cells to SARS-CoV-2 infection offered the opportunity to uncover some species-specific molecular restrictions to viral replication. All bat cells exhibited a potent entry-dependent restriction. Once this block was overcome by overexpression of human ACE2, which serves at the viral receptor, two bat cell lines controlled well viral replication, which correlated with the inability of the virus to counteract antiviral responses. Other cells potently inhibited viral release. Our novel bat cellular models contribute to a better understanding of the molecular interplays between bat cells and viruses.

Blood Parasites and Health Status of Hibernating and Non-Hibernating Noctule Bats (Nyctalus noctula).

Co-existence of bats with a wide range of infectious agents relates to their co-evolutionary history and specific physiology. Here, we examined blood samples collected during hibernation and the post-hibernation period to assess the influence of trypanosomes and babesias on the health status of 50 Noctule bats (Nyctalus noctula) using nested PCR. The impact of blood parasites on health was assessed by analysis of haematology and blood chemistry parameters in 21 bats. Prevalence of trypanosomes (Trypanosoma dionisii and T. vespertilionis) and babesia (Babesia vesperuginis) was 44% and 8%, respectively. Analysis of blood parameters indicated impact of babesia on acid-base balance. Blood chemistry parameters showed a significant decrease in total dissolved carbon dioxide and bicarbonate, increased anion gap, and no change in blood pH, suggesting compensated metabolic acidosis. Adverse effects of babesia were only apparent in hibernating bats. Our results suggest differences in the pathogenicity of trypanosomes and babesia in bats. While trypanosomes in general had no significant impact on the health status, we observed alterations in the blood acid-base balance in Babesia-infected bats during hibernation. Despite being infected, Babesia-positive bats survived hibernation without showing any clinical signs.

One or two pups - optimal reproduction strategies of common noctule females.

BACKGROUND: The success of animal reproduction is impacted by a trade-off between energetic costs and mortality associated with immediate vs. future reproductive attempts. The reproductive strategies of European insectivorous bats differ from common mammalian standards due to the use of delayed fertilisation. Phenology of bat reproduction, including length of pregnancy, which may vary in the same species at different latitudes, between years at the same site or between individuals within a colony, is influenced by ecological conditions. To assess factors influencing the course of pregnancy, we evaluated levels of blood progesterone in 20 female common noctule bats Nyctalus noctula. The bats were individually tagged and randomly divided into two groups with different hibernation ending points (i.e. a control group vs. a treatment group with one-week longer hibernation). Following emergence from hibernation, the bats were kept in a wooden box at a stable temperature of 22 °C. RESULTS: The majority of females gave birth to a single neonate (65%), but one female aborted her pups 2 days before the first successful births of other females. Based on development of progesterone concentration, we were able to define a number of different reproduction strategies, i.e. females with single offspring or twins, and females with supposed resorption of one embryo (embryonic mortality after implantation of the developing fertilised egg). Progesterone levels were much higher in females with two embryos during the first part of gestation and after birth. Progesterone levels were at their highest mid-gestation, with no difference between females carrying one or two foetuses. Length of gestation differed significantly between the two groups, with the longer hibernation (treatment) group having a roughly two-day shorter gestation period. CONCLUSIONS: Female N. noctula are able to manipulate their litter size to balance immediate and future reproduction success. The estimated gestation length of approx. 49-days appears to be standard for N. noctula, with females optimising their thermoregulatory behaviour to keep the length of gestation as close to the standard as possible.

Low-level pathogen transmission from wild to farmed salmonids in a flow-through fish farm.

While the potential effects of pathogens spread from farmed fish to wild populations have frequently been studied, evidence for the transmission of parasites from wild to farmed fish is scarce. In the present study, we evaluated natural bacterial and parasitic infections in brown trout (Salmo trutta m. fario) collected from the Cerná Opava river (Czech Republic) as a potential source of infections for rainbow trout (Oncorhynchus mykiss) reared in a flow-through farm system fed by the same river. The prevalence of bacterial and protozoan infections in farmed fish was comparable, or higher, than for riverine fish. Despite this, none of the infected farmed fish showed any signs of severe diseases. Substantial differences in metazoan parasite infections were observed between wild and farmed fish regarding monogeneans, adult trematodes, nematodes, the myxozoan Tetracapsuloides bryosalmonae found in riverine fish only, and larval eye-fluke trematodes sporadically found in farmed fish. The different distribution of metazoan parasites between brown and rainbow trout most probably reflects the availability of infected intermediate hosts in the two habitats. Despite the river being the main water source for the farm, there was no significant threat of parasite infection to the farmed fish from naturally infected riverine fish.

Comparison of diagnostic methods for Tetracapsuloides bryosalmonae detection in salmonid fish.

Diagnostic accuracy of pathogen detection depends upon the selection of suitable tests. Problems can arise when the selected diagnostic test gives false-positive or false-negative results, which can affect control measures, with consequences for the population health. The aim of this study was to compare sensitivity of different diagnostic methods IHC, PCR and qPCR detecting Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease in salmonid fish and as a consequence differences in disease prevalence. We analysed tissue from 388 salmonid specimens sampled from a recirculating system and rivers in the Czech Republic. Overall prevalence of T. bryosalmonae was extremely high at 92.0%, based on positive results of at least one of the above-mentioned screening methods. IHC resulted in a much lower detection rate (30.2%) than both PCR methods (qPCR32: 65.4%, PCR: 81.9%). While qPCR32 produced a good match with IHC (60.8%), all other methods differed significantly (p < .001) in the proportion of samples determined positive. Both PCR methods showed similar sensitivity, though specificity (i.e., the proportion of non-diseased fish classified correctly) differed significantly (p < .05). Sample preservation method significantly (p < .05) influenced the results of PCR, with a much lower DNA yield extracted from paraffin-embedded samples. Use of different methods that differ in diagnostic sensitivity and specificity resulted in random and systematic diagnosis errors, illustrating the importance of interpreting the results of each method carefully.

Urinary shedding of leptospires in palearctic bats.

Leptospirosis is a bacterial zoonotic infection of worldwide occurrence. Bats, like other mammalian reservoirs, may be long-term carriers that maintain endemicity of infection and shed viable leptospires in urine. Direct and/or indirect contact with these Leptospira shedders is the main risk factor as regards public health concern. However, knowledge about bat leptospirosis in the Palearctic Region, and in Europe in particular, is poor. We collected urine from 176 specimens of 11 bat species in the Czech Republic, Poland, Republic of Armenia and the Altai Region of Russia between 2014 and 2019. We extracted DNA from the urine samples to detect Leptospira spp. shedders using PCR amplification of the 16S rRNA and LipL32 genes. Four bat species (Barbastella barbastellus n = 1, Myotis bechsteinii n = 1, Myotis myotis n = 24 and Myotis nattereri n = 1) tested positive for Leptospira spp., with detected amplicons showing 100% genetic identity with pathogenic Leptospira interrogans. The site- and species-specific prevalence range was 0%-24.1% and 0%-20%, respectively. All bats sampled in the Republic of Armenia and Russia were negative. Given the circulation of pathogenic leptospires in strictly protected Palearctic bat species and their populations, non-invasive and non-lethal sampling of urine for molecular Leptospira spp. detection is recommended as a suitable surveillance and monitoring strategy. Moreover, our results should raise awareness of this potential disease risk among health professionals, veterinarians, chiropterologists and wildlife rescue workers handling bats, as well as speleologists and persons cleaning premises following bat infestation.

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.

Field study indicating susceptibility differences between salmonid species and their lineages to proliferative kidney disease.

Tetracapsuloides bryosalmonae (Myxozoa: Malacosporea) is the causative agent of proliferative kidney disease (PKD), which affects both wild and farmed salmonid fish. The objective of this study was to outline differences in susceptibility to PKD in different salmonid species, hybrids and breeding lineages. Susceptibility to T. bryosalmonae infection was established based on cumulative mortality, pathological findings and detection of T. bryosalmonae in the kidney using immunohistochemistry and molecular methods. Determination of pure and hybrid individuals of different species in the genus Salvelinus, and dissimilarity of rainbow trout lineages, was performed using traditional polymerase chain reaction (PCR) and microsatellite analyses. Rainbow trout displayed higher disease severity compared with brook trout and Alsatian charr. Moreover, the results indicated differences in infection susceptibility, not only among different salmonid species but also among different lineages of charr and rainbow trout. Our study indicated that some salmonid species and even different lineages of the same species are more suitable for farming under PKD pressure.

Health Surveillance of Wild Brown Trout (Salmo trutta fario) in the Czech Republic Revealed a Coexistence of Proliferative Kidney Disease and Piscine Orthoreovirus-3 Infection.

The population of brown trout (Salmo trutta fario) in continental Europe is on the decline, with infectious diseases confirmed as one of the causative factors. However, no data on the epizootiological situation of wild fish in the Czech Republic are currently available. In this study, brown trout (n = 260) from eight rivers were examined for the presence of viral and parasitical pathogens. Salmonid alphavirus-2, infectious pancreatic necrosis virus, piscine novirhabdovirus (VHSV) and salmonid novirhabdovirus (IHNV) were not detected using PCR. Cell culturing showed no viruses as well, and serological analysis of 110 sera did not detect any specific antibodies against VHSV or IHNV. Fish from two rivers were positive for the presence of piscine orthoreovirus-3 (PRV-3), subtype PRV-3b. However, none of the PRV-3-positive fish showed gross pathologies typically associated with PRV infections. By far the most widespread pathogen was Tetracapsuloides bryosalmonae which was confirmed in each of the examined locations, with a prevalence of up to 65% and 100%, as established by immunohistochemistry and PCR, respectively. Furthermore, up to 43.8% of fish showed signs of proliferative kidney disease caused by T. bryosalmonae, suggesting that this parasite is a main health challenge for brown trout in the Czech Republic.