Detection of Pneumocystis and Morphological Description of Fungal Distribution and Severity of Infection in Thirty-Six Mammal Species.

Pneumocystis spp. are thought to adapt to the lungs of potentially all mammals. However, the full host range, fungal burden and severity of infection are unknown for many species. In this study, lung tissue samples originating from 845 animals of 31 different families of eight mammal orders were screened by in situ hybridization (ISH) using a universal 18S rRNA probe for Pneumocystis, followed by hematoxylin and eosin (H&E) staining for determining histopathological lesions. A total of 216 (26%) samples were positive for Pneumocystis spp., encompassing 36 of 98 investigated mammal species, with 17 of them being described for the first time for the presence of Pneumocystis spp. The prevalence of Pneumocystis spp. as assessed by ISH varied greatly among different mammal species while the organism load was overall low, suggesting a status of colonization or subclinical infection. Severe Pneumocystis pneumonia seemed to be very rare. For most of the Pneumocystis-positive samples, comparative microscopic examination of H&E- and ISH-stained serial sections revealed an association of the fungus with minor lesions, consistent with an interstitial pneumonia. Colonization or subclinical infection of Pneumocystis in the lung might be important in many mammal species because the animals may serve as a reservoir.

Comparative analysis of genome-scale, base-resolution DNA methylation profiles across 580 animal species.

Methylation of cytosines is a prototypic epigenetic modification of the DNA. It has been implicated in various regulatory mechanisms across the animal kingdom and particularly in vertebrates. We mapped DNA methylation in 580 animal species (535 vertebrates, 45 invertebrates), resulting in 2443 genome-scale DNA methylation profiles of multiple organs. Bioinformatic analysis of this large dataset quantified the association of DNA methylation with the underlying genomic DNA sequence throughout vertebrate evolution. We observed a broadly conserved link with two major transitions-once in the first vertebrates and again with the emergence of reptiles. Cross-species comparisons focusing on individual organs supported a deeply conserved association of DNA methylation with tissue type, and cross-mapping analysis of DNA methylation at gene promoters revealed evolutionary changes for orthologous genes. In summary, this study establishes a large resource of vertebrate and invertebrate DNA methylomes, it showcases the power of reference-free epigenome analysis in species for which no reference genomes are available, and it contributes an epigenetic perspective to the study of vertebrate evolution.

The Amphibian Heart.

Currently, there are more than 8200 amphibian species described, including the orders Anura (frogs and toads), Caudata (salamanders and newts) and Gymnophiona (caecilians). Amphibians have 3 heart chambers: 2 atria and 1 ventricle. Their heart anatomy, histology, and physiology are reviewed. The basic morphology of the heart is similar in all amphibians with some differences due to their lifestyle. Blood flow, blood mixing, and blood oxygenation show variation due to interindividual and interspecific differences. Finally, different diagnostic methods to investigate the amphibian heart are described and reported amphibian heart diseases are summarized, including genetic, congenital, infectious, and neoplastic heart diseases.

Serological Evidence That SARS-CoV-2 Has Not Emerged in Deer in Germany or Austria during the COVID-19 Pandemic.

Spillover of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) to North American white-tailed deer (Odocoileus virginianus) has been documented. However, it is unclear if this is a phenomenon specific to North American deer or is a broader problem. We evaluated pre and pandemic exposure of German and Austrian deer species using a SARS-CoV-2 pseudoneutralization assay. In stark contrast to North American white-tailed deer, we found no evidence of SARS-CoV-2 exposure.

Porcine Circoviruses and Herpesviruses Are Prevalent in an Austrian Game Population.

During the annual hunt in a privately owned Austrian game population in fall 2019 and 2020, 64 red deer (Cervus elaphus), 5 fallow deer (Dama dama), 6 mouflon (Ovis gmelini musimon), and 95 wild boars (Sus scrofa) were shot and sampled for PCR testing. Pools of spleen, lung, and tonsillar swabs were screened for specific nucleic acids of porcine circoviruses. Wild ruminants were additionally tested for herpesviruses and pestiviruses, and wild boars were screened for pseudorabies virus (PrV) and porcine lymphotropic herpesviruses (PLHV-1-3). PCV2 was detectable in 5% (3 of 64) of red deer and 75% (71 of 95) of wild boar samples. In addition, 24 wild boar samples (25%) but none of the ruminants tested positive for PCV3 specific nucleic acids. Herpesviruses were detected in 15 (20%) ruminant samples. Sequence analyses showed the closest relationships to fallow deer herpesvirus and elk gammaherpesvirus. In wild boars, PLHV-1 was detectable in 10 (11%), PLHV-2 in 44 (46%), and PLHV-3 in 66 (69%) of animals, including 36 double and 3 triple infections. No pestiviruses were detectable in any ruminant samples, and all wild boar samples were negative in PrV-PCR. Our data demonstrate a high prevalence of PCV2 and PLHVs in an Austrian game population, confirm the presence of PCV3 in Austrian wild boars, and indicate a low risk of spillover of notifiable animal diseases into the domestic animal population.

Outbreak of Cronobacter turicensis in European brown hares (Lepus europaeus).

This is the first report of acute deaths in five European brown hares (Lepus europaeus) attributed to mucoid and necrotizing typhlocolitis caused by genetically different Cronobacter (C.) turicensis strains in northeastern Austria. As this opportunistic pathogen is mainly known for causing disease in immunocompromised humans and neonates, this previously unrecognized potential for a spill over from a wildlife reservoir to humans warrants further attention.

Management of Suspected Cases of Feline Immunodeficiency Virus Infection in Eurasian Lynx (Lynx lynx) During an International Translocation Program.

The Eurasian lynx (Lynx lynx) population in Switzerland serves as a source for reintroductions in neighboring countries. In 2016-2017, three lynx from the same geographical area were found seropositive for feline immunodeficiency virus (FIV) in the framework of an international translocation program. This novel finding raised questions about the virus origin and pathogenicity to lynx, the emerging character of the infection, and the interpretation of serological results in other lynx caught for translocation. Archived serum samples from 84 lynx captured in 2001-2016 were retrospectively tested for FIV antibodies by Western blot. All archived samples were FIV-negative. The three seropositive lynx were monitored in quarantine enclosures prior to euthanasia and necropsy. They showed disease signs, pathological findings, and occurrence of co-infections reminding of those described in FIV-infected domestic cats. All attempts to isolate and characterize the virus failed but serological data and spatiotemporal proximity of the cases suggested emergence of a lentivirus with antigenic and pathogenic similarities to FIV in the Swiss lynx population. A decision scheme was developed to minimize potential health risks posed by FIV infection, both in the recipient and source lynx populations, considering conservation goals, animal welfare, and the limited action range resulting from local human conflicts. Development and implementation of a cautious decision scheme was particularly challenging because FIV pathogenic potential in lynx was unclear, negative FIV serological results obtained within the first weeks after infection are unpredictable, and neither euthanasia nor repatriation of multiple lynx was acceptable options. The proposed scheme distinguished between three scenarios: release at the capture site, translocation, or euthanasia. Until April 2021, none of the 40 lynx newly captured in Switzerland tested FIV-seropositive. Altogether, seropositivity to FIV was documented in none of 124 lynx tested at their first capture, but three of them seroconverted in 2016-2017. Diagnosis of FIV infection in the three seropositive lynx remains uncertain, but clinical observations and pathological findings confirmed that euthanasia was appropriate. Our experiences underline the necessity to include FIV in pathogen screenings of free-ranging European wild felids, the importance of lynx health monitoring, and the usefulness of health protocols in wildlife translocation.

Discovery of a phylogenetically distinct poxvirus in diseased Crocodilurus amazonicus (family Teiidae).

A novel poxvirus was discovered in Crocodilurus amazonicus (Teiidae) presenting with a debilitating skin disease. The generated first genome sequence of a reptilian poxvirus revealed the closest phylogenetic relationship to avipoxviruses, highlighting potential virus exchanges between avian and reptilian species.

Widespread occurrence of the non-pathogenic hare calicivirus (HaCV Lagovirus GII.2) in captive-reared and free-living wild hares in Europe.

The Lagovirus genus comprises both pathogenic viruses as European brown hare syndrome virus (EBHSV- GII.1) and rabbit hemorrhagic disease viruses (RHDV-GI.1 and RHDV2-GI.2), that principally infect European brown hares (Lepus europeaus) and European rabbits (Oryctolagus cuniculus), respectively, causing severe necrotic hepatitis, spleen enlargement and disseminated haemorrhage. This genus includes also non-pathogenic agents, such as rabbit calicivirus (RCV-E1 - GI.3) and the non-pathogenic hare Lagovirus, provisionally named hare calicivirus (HaCV - GII.2). The latter had been identified for the first time in 2012 in the gut contents and faeces of healthy young hares raised in a breeding farm. In this study, we further investigated the presence of HaCV by testing the intestinal tract of 621 wild hares collected between 2010 and 2018 in Northern and Central Italy, and in 2011 in Austria, Germany and Spain. These wild hares were found dead for causes other than EBHS or were healthy hares shot during the hunting season. Forty-three out of 322 hare samples from Italy and 14 out of 299 samples from Austria and Germany were positive for HaCV-GII.2 by RT-PCR using universal primers for lagoviruses and primers specific for HaCV. Sequence analysis of the full capsid protein gene conducted on 12 strains representative of different years and locations indicated that these viruses belong to the same, single cluster as the prototype strain initially identified at the hares' farm (HaCV_Bs12_1). The relatively high level of genetic variation (88% nt identity) within this cluster suggests HaCVs may have been circulating widely in Europe for some time.

First-time application of a PCR-based clonality assay in a large cohort of non-domestic felines.

Feline lymphoma, one of the most important malignant tumors in domestic cats, is also increasingly diagnosed in non-domestic felines, most notably, African lions (Panthera leo). The gold standard for the diagnosis of lymphoma is histopathological evaluation. As an additional tool, the PCR for antigen receptor gene rearrangement (PARR) has been established. To support the diagnosis on a molecular level, the PCR-based clonality assay is designed to distinguish between reactive and neoplastic lymphocyte populations. In general, PARR primers are used to target complete immunoglobulin heavy chain V-D-J (IGH-VDJ) and T-cell receptor gamma V-J (TRG-VJ) chain gene rearrangements. In this study, we validated the primer sets used in routine diagnostics of domestic cats for the application in non-domestic felines. Clonality testing was used in 41 non-domestic feline species and the results were interpreted in the light of their clinical history and their pathology. In total, clonality could be detected in 8 non-domestic felines (19.4%), including 3 lymphoma cases confirmed by histopathology. These results confirmed the successful application of domestic feline-specific PARR primers in non-domestic feline species. Diagnostic sensitivity and specificity of the clonality assay were 100% and 88%, respectively. Finally, the overall diagnostic accuracy was 89%.

The Ratio of Linoleic and Linolenic Acid in the Pre-hibernation Diet Influences NFκB Signaling in Garden Dormice During Torpor.

The fatty acid composition of a pre-hibernation diet can influence the depth and duration of metabolic suppression achieved by hibernators. More specifically, a diet high in n-6 polyunsaturated fatty acids (PUFAs) relative to n-3 PUFAs is essential to maximize torpor expression. However, few studies have investigated how diets with different n-6/n-3 PUFA ratios change stress-inducible cell signaling. Garden dormice (Eliomys quercinus) were fed one of three diets designed with different ratios of n-6 PUFA linoleic acid (LA) and n-3 PUFA linolenic acid (ALA). Then, NFκB signaling was assessed in the white adipose, brown adipose, and liver tissues of euthermic and hibernating dormice via multiplex and RT-qPCR analyses of relative protein and transcript levels, respectively. Dormice fed a high LA diet regulated NFκB signaling in a protective manner in all tissues. NFκB signaling was generally decreased in the high LA group, with significant decreases in the protein levels of NFκB mediators IKKα/ß, IκBα, and downstream pro-apoptotic protein FADD. Liver and white adipose from torpid dormice fed a high LA diet increased sod2 expression relative to the other diets or relative to euthermic controls, indicating protection against ROS generated from potentially increased ß-oxidation of n-6 PUFAs. The low LA diet increased biomarkers for apoptosis relative to other diets and relative to euthermia, suggesting low LA diets may be detrimental to hibernator health. Overall, this study suggests that changes in the ratio of n-6/ n-3 PUFAs in the diet influences apoptotic and antioxidant responses in white adipose, brown adipose, and liver of hibernating garden dormice.

Development and validation of a portable, point-of-care canine distemper virus qPCR test.

Canine distemper virus (CDV) is a multi-host pathogen that can cause significant mortality in domestic, wild terrestrial and marine mammals. It is a major conservation threat in some endangered species. Infection can result in severe respiratory disease and fatal encephalitis. Diagnosis and disease monitoring in wildlife, and differentiation of CDV from rabies (a life-threatening zoonotic disease that can produce similar neurologic signs), would benefit from the availability of a portable, point-of-care (POC) diagnostic test. We therefore developed a quantitative RT-PCR assay for CDV using shelf-stable, lyophilized reagents and target-specific primers and probes for use with the handheld Biomeme two3™ qPCR thermocycler. Biomeme's extraction methodology, lyophilized reagents, and thermocycler were compared to our standard laboratory-based methods to assess sensitivity, efficiency and overall test performance. Results using a positive control plasmid for CDV showed comparable sensitivity (detection of 50 copies) and PCR efficiency between the two platforms, and CDV detection was similar between platforms when tested using a modified live CDV vaccine. Significantly higher Ct values (average Ct = 5.1 cycles) were observed using the Biomeme platform on known CDV positive animal samples. CDV detection using the Biomeme platform was similar in 25 of 26 samples from suspect CDV cases when compared to standard virology laboratory testing. One false positive was observed that was negative upon retest. The Biomeme methodology can be adapted for detection of specific targets, and this portable technology saves time by eliminating the need for local or international sample transport for laboratory-based diagnostics. However, results of our testing suggest that decreased diagnostic sensitivity (higher Ct values) relative to laboratory-based methods was observed using animal samples, so careful validation and optimization are essential. Portable qPCR platforms can empower biologists and wildlife health professionals in remote and low-resource settings, which will greatly improve our understanding of CDV disease ecology and associated conservation threats in wildlife.

Purifying selection shaping the evolution of the Toll-like receptor 2 TIR domain in brown hares (Lepus europaeus) from Europe and the Middle East.

Toll-like receptors (TLRs) are transmembrane proteins of the innate immune system, composed of the ectodomain involved in pathogen recognition and the intracellular Toll/interleukin-1 receptor (TIR) domain important for downstream signal transduction. Here, we analyze the genetic variability of TIR nucleotide and amino-acid sequences of the TLR2 gene in 243 brown hares from Europe and the Middle East and tested for the presence of selection signals and spatial structuring. TLR2 TIR domain sequences were PCR amplified and sequenced, while genotyping was performed by phasing. Genetic diversity indices were calculated in DnaSP and Arlequin, while presence of selection signals was tested using MEGA and the Datamonkey web server. The presence of spatial patterns in TIR sequence distribution was tested by spatial Principal Component Analysis (sPCA) in adegenet. A total of 13 haplotypes were revealed with haplotype diversity of 0.424, and nucleotide diversity (π) of 0.00138. Two spatial clusters were revealed: "Anatolia/Middle East" and "Europe". In Anatolia the two most prevalent amino-acid variants, A and B (the latter being the most ancestral) were maintained at similar frequencies; but in Europe a shift in genotype frequencies was observed as well as a higher number of nonsynonymous substitutions giving rise to novel amino-acid protein variants originating from the evolutionarily younger protein variant. Molecular diversity (haplotype and nucleotide diversity) indices were significantly higher in the "Anatolia/Middle East" cluster. A signal of purifying selection was detected acting on the TIR sequences.

Regulation of Peroxisome Proliferator-Activated Receptor Pathway During Torpor in the Garden Dormouse, Eliomys quercinus.

Differential levels of n-6 and n-3 essential polyunsaturated fatty acids (PUFAs) are incorporated into the hibernator's diet in the fall season preceding prolonged, multi-days bouts of torpor, known as hibernation. Peroxisome proliferator-activated receptor (PPAR) transcriptional activators bind lipids and regulate genes involved in fatty acid transport, beta-oxidation, ketogenesis, and insulin sensitivity; essential processes for survival during torpor. Thus, the DNA-binding activity of PPARα, PPARδ, PPARγ, as well as the levels of PPARγ coactivator 1α (PGC-1α) and L-fatty acid binding protein (L-FABP) were investigated in the hibernating garden dormouse (Eliomys quercinus). We found that dormice were hibernating in a similar way regardless of the n-6/n-3 PUFA diets fed to the animals during the fattening phase prior to hibernation. Further, metabolic rates and body mass loss during hibernation did not differ between dietary groups, despite marked differences in fatty acid profiles observed in white adipose tissue prior and at mid-hibernation. Overall, maintenance of PPAR DNA-binding activity was observed during torpor, and across three n-6/n-3 ratios, suggesting alternate mechanisms for the prioritization of lipid catabolism during torpor. Additionally, while no change was seen in L-FABP, significantly altered levels of PGC-1α were observed within the white adipose tissue and likely contributes to enhanced lipid metabolism when the diet favors n-6 PUFAs, i.e., high n-6/n-3 ratio, in both the torpid and euthermic state. Altogether, the maintenance of lipid metabolism during torpor makes it likely that consistent activity or levels of the investigated proteins are in aid of this metabolic profile.

Positive selection and precipitation effects on the mitochondrial NADH dehydrogenase subunit 6 gene in brown hares (Lepus europaeus) under a phylogeographic perspective.

Previous studies in hares and jackrabbits have indicated that positive selection has shaped the genetic diversity of mitochondrial genes involved in oxidative phosphorylation, which may affect cellular energy production and cause regional adaptation to different environmental (climatic) pressures. In the present study, we sequenced the NADH dehydrogenase subunit 6 (MT-ND6) gene of 267 brown hares (L. europaeus) from Europe and Asia Minor and tested for positive selection and adaptations acting on amino acid sequences (protein variants). Molecular diversity indices and spatial clustering were assessed by DnaSP, Network, and Geneland, while the presence of selection signals was tested by codeml in PAML, and by using the Datamonkey Adaptive Evolution web server. The SPSS software was used to run multinomial regression models to test for possible effects of climate parameters on the currently obtained protein variants. Fifty-eight haplotypes were revealed with a haplotype diversity of 0.817, coding for 17 different protein variants. The MT-ND6 phylogeographic pattern as determined by the nucleotide sequences followed the earlier found model based on the neutrally evolving D-loop sequences, and reflected the earlier found phylogeographic Late Pleistocene scenario. Based on several selection tests, only one codon position consistently proved to be under positive selection. It did occur exclusively in the evolutionarily younger hares from Europe and it gave rise to several protein variants from the southeastern and south-central Balkans. The occurrence of several of those variants was significantly favored under certain precipitation conditions, as proved by our multinomial regression models. Possibly, the great altitudinal variation in the Balkans may have lead to bigger changes in precipitation across that region and this may have imposed an evolutionarily novel selective pressure on the protein variants and could have led to regional adaptation.

First report of hare treponematosis seroprevalence of European brown hares (Lepus europaeus) in the Czech Republic: seroprevalence negatively correlates with altitude of sampling areas.

BACKGROUND: The aim of this study was to quantify the seroprevalence of hare treponematosis in European brown hare (Lepus europaeus) populations in the Czech Republic and to test for an association between treponematosis prevalence and the altitude of the areas in which hares were sampled. We tested 289 serum samples of brown hares collected between 2015 and 2017. The sampling areas included 12 districts (73 villages) distributed throughout the Czech Republic. Serum samples were tested for the presence of antibodies against the causative agent of hare treponematosis (Treponema paraluisleporidarum ecovar Lepus, TPeL) using two serological tests for human syphilis that cross-react with TPeL: the Treponema pallidum hemagglutination assay (TPHA) and the fluorescent treponemal antibody absorption (FTA-ABS) test. To account for the imperfect diagnostic sensitivity and specificity of each test, apparent prevalence estimates of TPeL were converted to true prevalence estimates using the Rogan Gladen estimator. The correlation between TPeL true seroprevalence and altitude of sampling areas was analyzed using Pearson's correlation coefficient at three levels of spatial resolution: (1) four groups, each composed of two merged districts, with ≥20 samples collected, differing in their altitude median (206, 348, 495, and 522 m above sea level); (2) separately tested eight districts, where ≥20 samples were collected per district; and (3) 27 groups composed of villages of the same altitude level distributed across the whole dataset. RESULTS: One hundred and seven of the 289 samples were seropositive to both tests, the FTA-ABS test was positive for an additional 47 samples. Seropositive samples were found in all 12 districts. True seroprevalence of TPeL in the sampled hares was 52% (95% confidence interval 46 to 58%). A statistically significant negative correlation between TPeL seroprevalence and altitude was identified at the district level (Pearson's r = - 0.722, p = 0.043). CONCLUSIONS: Between 2015 and 2017 hare treponematosis was present at a relatively high prevalence in brown hares in all 12 districts in the Czech Republic where sampling was carried out. The seroprevalence of TPeL in brown hares was negatively correlated with the altitude of the areas in which hares were sampled.

Gastrointestinal parasite infestation in the alpine mountain hare (Lepus timidus varronis): Are abiotic environmental factors such as elevation, temperature and precipitation affecting prevalence of parasite species?

Information concerning factors regulating Alpine mountain hare (Lepus timidus varronis) populations such as host-parasite interactions is missing as only a few parasitological surveys exist of this subspecies. Parasites are not only dependent on their host but also on suitable environmental conditions for infestation. Abiotic environmental factors have an important regulating role on parasites in mammals. It is estimated that the elevation range of parasites is likely to shift in response to alternate host movement and changes in climate. Here we assess the parasitic infestation in the Alpine mountain hare by analysing the parasites in faeces and comparing the parasite infestation at different elevation ranges and at varied weather conditions for two years in the Austrian Alps. Almost half of the faecal samples were free of parasites (46.2%, n = 52). Most frequent was the infection by Coccidia (46.2%), whereas stomach intestine strongylids, Trichuris spp, and Cestoda were only found in 9.6% of all faeces. Hence, only Coccidia may be prevalent enough to regulate Alpine mountain hare populations in the Austrian Alps. Elevation had a significant positive effect on the infection of animals by Trichuris spp, whereas temperature had a significant negative effect on the infection by any parasite traceable in faeces and, when looking at the parasite groups individually, on Coccidia.

Characterization of mecC gene-carrying coagulase-negative Staphylococcus spp. isolated from various animals.

The presence of the methicillin resistance gene mecC in coagulase-negative Staphylococcus spp. (CoNS) is scarce. The aim of this study was to characterize mecC-positive CoNS isolated from various wild and domestic animals. The presence of the mecC gene was screened in 4299 samples from wild animals and domestic animals. Fifteen coagulase-negative staphylococci, that displayed a cefoxitin-resistant phenotype, were tested mecC-positive by PCR. Antimicrobial susceptibility testing was performed for all isolates. The 15 isolates were genotyped by sequencing of the entire class E mec gene complex (blaZ-mecC-mecR1-mecI), the ccrA and ccrB recombinase genes and other determinants within the type XI SCCmec element. DNA microarray analysis was performed and five selected isolates were additionally whole genome sequenced and analyzed. S. stepanovicii (n = 3), S. caprae (n = 1), S. warneri (n = 1), S. xylosus (n = 1) and S. sciuri (n = 9) were detected. All but the S. sciuri isolates were found to be susceptible to all non-beta lactams. The entire class E mec gene complex was detected in all isolates but ccrA and ccrB genes were not identified in S. stepanovicii and S. xylosus. The genes erm(B) and fexA (n = 4, each) were the most predominant non-beta lactam resistance genes detected in the S. sciuri isolates. Even though the presence of the mecC gene among CoNS is a rare observation, this study further expands our knowledge by showing that the mecC gene, including its allotypes, are present in more staphylococcal species from different animal species than has been previously described.