Spatial Venomics─Cobra Venom System Reveals Spatial Differentiation of Snake Toxins by Mass Spectrometry Imaging.

Among venomous animals, toxic secretions have evolved as biochemical weapons associated with various highly specialized delivery systems on many occasions. Despite extensive research, there is still limited knowledge of the functional biology of most animal toxins, including their venom production and storage, as well as the morphological structures within sophisticated venom producing tissues that might underpin venom modulation. Here, we report on the spatial exploration of a snake venom gland system by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), in combination with standard proteotranscriptomic approaches, to enable in situ toxin mapping in spatial intensity maps across a venom gland sourced from the Egyptian cobra (Naja haje). MALDI-MSI toxin visualization on the elapid venom gland reveals a high spatial heterogeneity of different toxin classes at the proteoform level, which may be the result of physiological constraints on venom production and/or storage that reflects the potential for venom modulation under diverse stimuli.

Suid alphaherpesvirus 1 of wild boar origin as a recent source of Aujeszky's disease in carnivores in Germany.

BACKGROUND: The high susceptibility of carnivores to Suid Alphaherpesvirus 1 [SuAHV1, synonymous pseudorabies virus (PrV)], renders them inadvertent sentinels for the possible occurrence of Aujeszky's disease (AD) in domestic and wild swine populations. The aim of this study was to epidemiologically analyse the occurrence of PrV infections in domestic and wild animals in Germany during the last three decades and to genetically characterise the causative PrV isolates. METHODS: PrV in dogs was detected using standard virological techniques including conventional and real time PCR, virus isolation or by immunohistochemistry. Available PrV isolates were characterized by partial sequencing of the open gC reading frame and the genetic traits were compared with those of archived PrV isolates from carnivores and domestic pigs from Germany before the elimination of AD in the domestic pig population. RESULTS: During 1995 and 2022, a total of 38 cases of AD in carnivores, e.g. dogs and red foxes, were laboratory confirmed. Sequencing and subsequent phylogenetic analysis of PrV isolates established a strong connection between AD cases in carnivores and the occurrence of PrV infections in European wild boars in the end phase of and after elimination of AD from the domestic pig population. While PrV infections occur at low numbers but regularly in hunting dogs, interestingly, PrV was not observed in grey wolves in Germany. In none of 682 dead-found grey wolves and wolf-dog hybrids tested from Germany during 2006-2022 could PrV infection be detected by molecular means. CONCLUSIONS: Although PrV has been eliminated from domestic pigs, spillover infections in domestic and wild carnivores should always be expected given the endemic presence of PrV in wild pig populations. Since detection of PrV DNA and virus in carnivores is sporadic even in areas with high seroprevalence of PrV in wild pigs, it may not reflect the full diversity of PrV.

Streptococcus catagoni sp. nov., isolated from the respiratory tract of diseased Chacoan peccaries (Catagonus wagneri).

Novel catalase-negative, Gram-stain-positive, beta-haemolytic, coccus-shaped organisms were isolated from Chacoan peccaries that died from respiratory disease. The initial API 20 Strep profiles suggested Streptococcus agalactiae with acceptable identification scores, but the 16S rRNA gene similarity (1548 bp) to available sequences of streptococci was below 98 %. Next taxa of the genus Streptococcus, displaying highest similarities to the strains from this study, were S. bovimastitidis NZ1587T (97.5 %), S. iniae ATCC 29178T (97.5 %), S. hongkongensis HKU30T (97.4 %), S. parauberis DSM 6631T (97.1 %), S. penaeicida CAIM 1838T (97.1 %), S. pseudoporcinus DSM 18513T (97.0 %), S. didelphis DSM 15616T (96.6 %), S. ictaluri 707-05T (96.6 %), S. uberis JCM 5709T (96.5 %) and S. porcinus NCTC 10999T (96.4 %). All other Streptococcus species had sequence similarities of below 96.4 %. A sodA gene as well as whole genome-based core genome phylogeny of three representative strains and 145 available Streptococcus genomes confirmed the unique taxonomic position. Interstrain average nucleotide identity (ANI) and amino acid identity (AAI) values were high (ANI >96 %; AAI 100%), but for other streptococci clearly below the proposed species boundary of 95-96 % (ANI <75 %; AAI <83 %). Results were confirmed by genome-to-genome distance calculations. Pairwise digital DNA-DNA hybridization estimates were high (>90 %) between the novel strains, but well below the species boundary of 70 % for closely related Streptococcus type strains (23.5-19.7 %). Phenotypic properties as obtained from extended biochemical profiles and MALDI-TOF mass spectrometry supported the outstanding rank. Based on the presented molecular and physiological data of the six strains, we propose a novel taxon for which we suggest the name Streptococcus catagoni sp. nov. with the type strain 99-1/2017T (=DSM 110457T=CCUG 74072T) and five reference strains.

Entomological identification of the post-mortem colonization of wolf cadavers in different decomposition stages.

Human-driven biodiversity loss is progressively becoming a problem with dramatic consequences for the conservation of vital ecosystems. The increasing number of illegal killings of the grey wolf (Canis lupus, Linnaeus, 1758), a threatened species, displays the need for investigation and prosecution of such offences. Forensic entomology makes use of the knowledge about necrophagous insects to estimate a minimum time-since-death interval of the deceased person or animal, which can give important information on a possible perpetrator. The cadaver fauna along five decomposition stages of wolves in Germany was investigated in the period 2014-2021. The insects from 70 wolf cadavers, originating from all over Germany, were provided by the Leibniz Institute for Zoo and Wildlife Research Berlin. The accumulated degree day (ADD) model was applied for the post-mortem interval estimation on wolf cadavers for the first time. A total of 20 coleopteran species and 14 different dipteran species were discovered and identified. Almost 99 % of all insect specimens were from the order of Diptera, and beetles (Coleoptera) accounted for only 1 % of the cadaver fauna. The blowflies (Calliphoridae) are of particular importance for forensic issues, accounting for about 66 % of all families. Carrion beetles (Silphidae) were found as the second most abundant family (about 21 %). In addition, combining all cases, a steadily increasing insect species richness S was detected from early decay to advanced decay (fresh S = 8; bloated S = 12; active decay S = 21; advanced decay S = 34). In the following remains stage, the species number decreased again (S = 24). However, no significant difference in the number of species was found between the stages of decay when the cases were considered individually. The temporal pattern of insect appearance was found to be congruent with those of previous studies. Furthermore, a time of death was determined for each case and compared to the pathologist's estimates. This study provides insights into the arthropod fauna of wolf remains for the first time, applies the ADD-Model for post-mortem interval estimation, and discusses the suitability of forensic entomology for wildlife death investigations.

Co-infections: Simultaneous detections of West Nile virus and Usutu virus in birds from Germany.

The emergence of West Nile virus (WNV) and Usutu virus (USUV) in Europe resulted in significant outbreaks leading to avifauna mortality and human infections. Both viruses have overlapping geographical, host and vector ranges, and are often co-circulating in Europe. In Germany, a nationwide bird surveillance network was established to monitor these zoonotic arthropod-borne viruses in migratory and resident birds. In this framework, co-infections with WNV and USUV were detected in six dead birds collected in 2018 and 2019. Genomic sequencing and phylogenetic analyses classified the detected WNV strains as lineage 2 and the USUV strains as lineages Africa 2 (n = 2), Africa 3 (n = 3) and Europe 2 (n = 1). Preliminary attempts to co-propagate both viruses in vitro failed. However, we successfully cultivated WNV from two animals. Further evidence for WNV-USUV co-infection was obtained by sampling live birds in four zoological gardens with confirmed WNV cases. Three snowy owls had high neutralizing antibody titres against both WNV and USUV, of which two were also positive for USUV-RNA. In conclusion, further reports of co-infections in animals as well as in humans are expected in the future, particularly in areas where both viruses are present in the vector population.

Replication of Rift Valley Fever Virus in Amphibian and Reptile-Derived Cell Lines.

Rift Valley fever phlebovirus (RVFV) is a zoonotic arthropod-borne virus, which has led to devastating epidemics in African countries and on the Arabian Peninsula. Results of in-vivo, in-vitro and field studies suggested that amphibians and reptiles may play a role as reservoir hosts of RVFV, promoting its maintenance during inter-epidemic periods. To elucidate this hypothesis, we examined two newly established reptile-derived cell lines (Egyptian cobra and Chinese pond turtle) and five previously generated reptile- and amphibian-derived cell lines for their replicative capacity for three low- and high-pathogenic RVFV strains. At different time points after infection, viral loads (TCID50), genome loads and the presence of intracellular viral antigen (immunofluorescence) were assessed. Additionally, the influence of temperatures on the replication was examined. Except for one cell line (read-eared slider), all seven cell lines were infected by all three RVFV strains. Two different terrapin-derived cell lines (Common box turtle, Chinese pond turtle) were highly susceptible. A temperature-dependent replication of RVFV was detected for both amphibian and reptile cells. In conclusion, the results of this study indicate the general permissiveness of amphibian and reptile cell lines to RVFV and propose a potential involvement of terrapins in the virus ecology.

Re-Emergence and Spread of Haemorrhagic Septicaemia in Germany: The Wolf as a Vector?

Since 2010, outbreaks of haemorrhagic septicaemia (HS) caused by Pasteurella (P.) multocida capsular type B (PmB) emerged in Germany. In 2017, we noticed a close spatiotemporal relationship between HS outbreak sites and wolf (Canis lupus) territories. Thus, the main objectives of our study were to investigate the molecular epidemiology of German PmB-HS-isolates and to assess the role of wolves as putative vectors of this pathogen. We collected 83 PmB isolates from HS outbreaks that occurred between 2010 and 2019 and sampled 150 wolves, which were found dead in the years 2017 to 2019, revealing another three PmB isolates. A maximum-likelihood-based phylogeny of the core genomes of 65 PmB-HS-isolates and the three PmB-wolf-isolates showed high relatedness. Furthermore, all belonged to capsular:LPS:MLST genotype B:L2:ST122RIRDC and showed highly similar virulence gene profiles, but clustered separately from 35 global ST122RIRDC strains. Our data revealed that German HS outbreaks were caused by a distinct genomic lineage of PmB-ST122 strains, hinting towards an independent, ongoing epidemiologic event. We demonstrated for the first time, that carnivores, i.e., wolves, might harbour PmB as a part of their oropharyngeal microbiota. Furthermore, the results of our study imply that wolves can carry the pathogen over long distances, indicating a major role of that animal species in the ongoing epidemiological event of HS in Germany.

In action-an early warning system for the detection of unexpected or novel pathogens.

Proactive approaches in preventing future epidemics include pathogen discovery prior to their emergence in human and/or animal populations. Playing an important role in pathogen discovery, high-throughput sequencing (HTS) enables the characterization of microbial and viral genetic diversity within a given sample. In particular, metagenomic HTS allows the unbiased taxonomic profiling of sequences; hence, it can identify novel and highly divergent pathogens such as viruses. Newly discovered viral sequences must be further investigated using genomic characterization, molecular and serological screening, and/or in vitro and in vivo characterization. Several outbreak and surveillance studies apply unbiased generic HTS to characterize the whole genome sequences of suspected pathogens. In contrast, this study aimed to screen for novel and unexpected pathogens in previously generated HTS datasets and use this information as a starting point for the establishment of an early warning system (EWS). As a proof of concept, the EWS was applied to HTS datasets and archived samples from the 2018-9 West Nile virus (WNV) epidemic in Germany. A metagenomics read classifier detected sequences related to genome sequences of various members of Riboviria. We focused the further EWS investigation on viruses belonging to the families Peribunyaviridae and Reoviridae, under suspicion of causing co-infections in WNV-infected birds. Phylogenetic analyses revealed that the reovirus genome sequences clustered with sequences assigned to the species Umatilla virus (UMAV), whereas a new peribunyavirid, tentatively named 'Hedwig virus' (HEDV), belonged to a putative novel genus of the family Peribunyaviridae. In follow-up studies, newly developed molecular diagnostic assays detected fourteen UMAV-positive wild birds from different German cities and eight HEDV-positive captive birds from two zoological gardens. UMAV was successfully cultivated in mosquito C6/36 cells inoculated with a blackbird liver. In conclusion, this study demonstrates the power of the applied EWS for the discovery and characterization of unexpected viruses in repurposed sequence datasets, followed by virus screening and cultivation using archived sample material. The EWS enhances the strategies for pathogen recognition before causing sporadic cases and massive outbreaks and proves to be a reliable tool for modern outbreak preparedness.

Molecular survey of tick-borne pathogens reveals a high prevalence and low genetic variability of Hepatozoon canis in free-ranging grey wolves (Canis lupus) in Germany.

Wild carnivores are important hosts for ixodid ticks and presumed reservoirs for several tick-borne pathogens (TBPs) of medical and veterinary importance. However, little is known about the role that the European grey wolf (Canis lupus) plays in the enzootic cycle of TBPs. The recent recolonization of Central European lowland, including some regions in Germany, by the grey wolf, opened up an excellent opportunity for studying the impact of the wolf population on pathogen diversity and transmission dynamics. Therefore, in the present study, we investigated spleen samples collected from 276 grey wolves in 11 federal states in Germany for common TBPs by molecular methods. In total, 127 grey wolves (46.0 %) were found to be positive for Hepatozoon canis. Only two genetic variants of this protozoan parasite (herein designated as G1 and G2) were found to circulate among the grey wolves, which can be potentially shared between populations of domestic and other wild carnivores in the country. Two grey wolves were positive for Anaplasma phagocytophilum (0.7 %), and both were co-infected with H. canis G1 genotype. The presence of other pathogens could not be confirmed by PCR and sequencing. This study represents the first one reporting H. canis in a grey wolf population worldwide, and it provides highly relevant information, which may contribute to a better understanding of the epidemiology of TBPs and the pathogen transmission dynamics among the reintroduced population of grey wolves and other carnivores.

West Nile Virus Epidemic in Germany Triggered by Epizootic Emergence, 2019.

One year after the first autochthonous transmission of West Nile virus (WNV) to birds and horses in Germany, an epizootic emergence of WNV was again observed in 2019. The number of infected birds and horses was considerably higher compared to 2018 (12 birds, two horses), resulting in the observation of the first WNV epidemy in Germany: 76 cases in birds, 36 in horses and five confirmed mosquito-borne, autochthonous human cases. We demonstrated that Germany experienced several WNV introduction events and that strains of a distinct group (Eastern German WNV clade), which was introduced to Germany as a single introduction event, dominated mosquito, birds, horse and human-related virus variants in 2018 and 2019. Virus strains in this clade are characterized by a specific-Lys2114Arg mutation, which might lead to an increase in viral fitness. Extraordinary high temperatures in 2018/2019 allowed a low extrinsic incubation period (EIP), which drove the epizootic emergence and, in the end, most likely triggered the 2019 epidemic. Spatiotemporal EIP values correlated with the geographical WNV incidence. This study highlights the risk of a further spread in Germany in the next years with additional human WNV infections. Thus, surveillance of birds is essential to provide an early epidemic warning and thus, initiate targeted control measures.

Lethal pneumonia in a captive juvenile chimpanzee (Pan troglodytes) due to human-transmitted human respiratory syncytial virus (HRSV) and infection with Streptococcus pneumoniae.

BACKGROUND: During an outbreak of respiratory disease in captive chimpanzees (Pan troglodytes), gorillas (Gorilla gorilla), Bornean orangutans (Pongo pygmaeus), and red-capped mangabeys (Cercocebus torquatus) also staff members showed non-specific upper respiratory signs. One infant female chimpanzee with severe respiratory symptoms died despite immediate medical treatment and was submitted for necropsy. METHODS: Routine post mortem, histological and bacteriological examinations were conducted. Additionally lung tissue samples form the chimpanzee and swab samples from the staff members and the other primates were examined by PCR. RESULTS: A severe catarrhal to purulent bronchopneumonia and an interstitial pneumonia were found and human respiratory syncytial virus (HRSV) as well as Streptococcus pneumoniae was detected in lung samples by PCR. Swab samples from one animal keeper revealed the same HRSV sequence as of the chimpanzee. CONCLUSIONS: Therefore, it is suggested that the outbreak of respiratory disease within a zoological institution was due to transmission of HRSV between both human and primates.

Comparing erosion and organ accumulation rates of lead and alternative lead-free ammunition fed to captive domestic ducks.

Lead poisoning of birds of prey from ingestion of ammunition lead has been well documented. Alternative, lead-free ammunition is widely available, but the toxicokinetics of other metals in birds are poorly understood. We tested the erosion of lead, copper, zinc, iron and brass by feeding domestic Pekin ducks (Anas platyrhynchos forma domestica) defined numbers of small metal pellets. The accumulation of these metals was analysed in the breast muscle, brain, pancreas, liver and kidney. Four weeks after application, the ducks were euthanized and necropsied, internal organs tested for metal accumulation and gizzard pellets collected and weighed to record loss by erosion. Degree of erosion was highest in zinc pellets (81% mass loss), followed by iron (46%) and lead (45%) and was only marginal in copper and brass. Only lead showed highly elevated levels of accumulation in organs compared to controls.

Tox-positive Corynebacterium ulcerans in hedgehogs, Germany.

Toxigenic Corynebacterium ulcerans may cause both respiratory and cutaneous diphtheria in humans. As a zoonotic emerging pathogen it has been isolated from a wide variety of animals living in captivity, such as livestock, pet, zoo and research animals and additionally in a large number of different wild animals. Here we report the isolation of tox-positive C. ulcerans in four hedgehogs with cutaneous diphtheria and pneumonia, respectively.

Novel Polyomaviruses in Mammals from Multiple Orders and Reassessment of Polyomavirus Evolution and Taxonomy.

As the phylogenetic organization of mammalian polyomaviruses is complex and currently incompletely resolved, we aimed at a deeper insight into their evolution by identifying polyomaviruses in host orders and families that have either rarely or not been studied. Sixteen unknown and two known polyomaviruses were identified in animals that belong to 5 orders, 16 genera, and 16 species. From 11 novel polyomaviruses, full genomes could be determined. Splice sites were predicted for large and small T antigen (LTAg, STAg) coding sequences (CDS) and examined experimentally in transfected cell culture. In addition, splice sites of seven published polyomaviruses were analyzed. Based on these data, LTAg and STAg annotations were corrected for 10/86 and 74/86 published polyomaviruses, respectively. For 25 polyomaviruses, a spliced middle T CDS was observed or predicted. Splice sites that likely indicate expression of additional, alternative T antigens, were experimentally detected for six polyomaviruses. In contrast to all other mammalian polyomaviruses, three closely related cetartiodactyl polyomaviruses display two introns within their LTAg CDS. In addition, the VP2 of Glis glis (edible dormouse) polyomavirus 1 was observed to be encoded by a spliced transcript, a unique experimental finding within the Polyomaviridae family. Co-phylogenetic analyses based on LTAg CDS revealed a measurable signal of codivergence when considering all mammalian polyomaviruses, most likely driven by relatively recent codivergence events. Lineage duplication was the only other process whose influence on polyomavirus evolution was unambiguous. Finally, our analyses suggest that an update of the taxonomy of the family is required, including the creation of novel genera of mammalian and non-mammalian polyomaviruses.

Complete Draft Genome Sequence of an Extended-Spectrum ß-Lactamase-Producing Citrobacter freundii Strain Recovered from the Intestine of a House Sparrow (Passer domesticus) in Germany, 2017.

Here, we announce the genome of an extended-spectrum ß-lactamase-producing Citrobacter freundii strain isolated from the cecum of a house sparrow that was found dead in Berlin-Lichtenberg, Germany, in 2017. This isolate exhibits increased MICs for several antimicrobials and a comprehensive set of acquired resistance determinants potentially involved in horizontal gene transfer.

Population expansion and individual age affect endoparasite richness and diversity in a recolonising large carnivore population.

The recent recolonisation of the Central European lowland (CEL) by the grey wolf (Canis lupus) provides an excellent opportunity to study the effect of founder events on endoparasite diversity. Which role do prey and predator populations play in the re-establishment of endoparasite life cycles? Which intrinsic and extrinsic factors control individual endoparasite diversity in an expanding host population? In 53 individually known CEL wolves sampled in Germany, we revealed a community of four cestode, eight nematode, one trematode and 12 potential Sarcocystis species through molecular genetic techniques. Infections with zoonotic Echinococcus multilocularis, Trichinella britovi and T. spiralis occurred as single cases. Per capita endoparasite species richness and diversity significantly increased with population size and changed with age, whereas sex, microsatellite heterozygosity, and geographic origin had no effect. Tapeworm abundance (Taenia spp.) was significantly higher in immigrants than natives. Metacestode prevalence was slightly higher in ungulates from wolf territories than from control areas elsewhere. Even though alternative canid definitive hosts might also play a role within the investigated parasite life cycles, our findings indicate that (1) immigrated wolves increase parasite diversity in German packs, and (2) prevalence of wolf-associated parasites had declined during wolf absence and has now risen during recolonisation.

Zebra Alphaherpesviruses (EHV-1 and EHV-9): Genetic Diversity, Latency and Co-Infections.

Alphaherpesviruses are highly prevalent in equine populations and co-infections with more than one of these viruses' strains frequently diagnosed. Lytic replication and latency with subsequent reactivation, along with new episodes of disease, can be influenced by genetic diversity generated by spontaneous mutation and recombination. Latency enhances virus survival by providing an epidemiological strategy for long-term maintenance of divergent strains in animal populations. The alphaherpesviruses equine herpesvirus 1 (EHV-1) and 9 (EHV-9) have recently been shown to cross species barriers, including a recombinant EHV-1 observed in fatal infections of a polar bear and Asian rhinoceros. Little is known about the latency and genetic diversity of EHV-1 and EHV-9, especially among zoo and wild equids. Here, we report evidence of limited genetic diversity in EHV-9 in zebras, whereas there is substantial genetic variability in EHV-1. We demonstrate that zebras can be lytically and latently infected with both viruses concurrently. Such a co-occurrence of infection in zebras suggests that even relatively slow-evolving viruses such as equine herpesviruses have the potential to diversify rapidly by recombination. This has potential consequences for the diagnosis of these viruses and their management in wild and captive equid populations.

A potentially fatal mix of herpes in zoos.

Pathogens often have a limited host range, but some can opportunistically jump to new species. Anthropogenic activities that mix reservoir species with novel, hence susceptible, species can provide opportunities for pathogens to spread beyond their normal host range. Furthermore, rapid evolution can produce new pathogens by mechanisms such as genetic recombination. Zoos unintentionally provide pathogens with a high diversity of species from different continents and habitats assembled within a confined space. Institutions alert to the problem of pathogen spread to unexpected hosts can monitor the emergence of pathogens and take preventative measures. However, asymptomatic infections can result in the causative pathogens remaining undetected in their reservoir host. Furthermore, pathogen spread to unexpected hosts may remain undiagnosed if the outcome of infection is limited, as in the case of compromised fertility, or if more severe outcomes are restricted to less charismatic species that prompt only limited investigation. We illustrate this problem here with a recombinant zebra herpesvirus infecting charismatic species including zoo polar bears over at least four years. The virus may cause fatal encephalitis and infects at least five mammalian orders, apparently without requiring direct contact with infected animals.

Epidemiology, control and management of an EBHS outbreak in captive hares.

Here we describe an outbreak of European brown hare syndrome (EBHS) in a captive hare population. The EBHS outbreak occurred in March 2009, at the beginning of the breeding season. Overall mortality was 53% out of an original population of 61 animals. Animals between five and eleven months showed a significantly higher mortality rate than other age classes. Pregnant females either aborted their foetuses and survived or died pregnant. All foetuses (n=10) of the pregnant hares were PCR positive for EBHSV. Only one offspring born during the outbreak survived. Shortly after the outbreak, the surviving hares developed a specific anti-EBHSV titre between 1:80 and 1:2560, which dropped to 1:10-1:160 nine months later. Hares between one and three years of age developed a significantly higher titre than hares younger than one year or older than four years. Offspring born after the outbreak showed a lower titre of 1:10, indicating passive antibody transfer via placenta and milk. After two months, the titre was not detectable any longer. In December 2009, the captive population was vaccinated against EBHS virus with inactivated virus prepared from the organs of infected hares. The titres after the first vaccination ranged from 1:10 to 1:640, and after the second vaccination from 1:10 to 1:320. To estimate the effect of EBHS on reproduction, we compared the breeding seasons 2008 and 2009. Several possible sources of infection of the colony are discussed, but the definite cause could not be determined.