Co-infecting Reptarenaviruses Can Be Vertically Transmitted in Boa Constrictor.

Boid inclusion body disease (BIBD) is an often fatal disease affecting mainly constrictor snakes. BIBD has been associated with infection, and more recently with coinfection, by various reptarenavirus species (family Arenaviridae). Thus far BIBD has only been reported in captive snakes, and neither the incubation period nor the route of transmission are known. Herein we provide strong evidence that co-infecting reptarenavirus species can be vertically transmitted in Boa constrictor. In total we examined five B. constrictor clutches with offspring ranging in age from embryos over perinatal abortions to juveniles. The mother and/or father of each clutch were initially diagnosed with BIBD and/or reptarenavirus infection by detection of the pathognomonic inclusion bodies (IB) and/or reptarenaviral RNA. By applying next-generation sequencing and de novo sequence assembly we determined the "reptarenavirome" of each clutch, yielding several nearly complete L and S segments of multiple reptarenaviruses. We further confirmed vertical transmission of the co-infecting reptarenaviruses by species-specific RT-PCR from samples of parental animals and offspring. Curiously, not all offspring obtained the full parental "reptarenavirome". We extended our findings by an in vitro approach; cell cultures derived from embryonal samples rapidly developed IB and promoted replication of some or all parental viruses. In the tissues of embryos and perinatal abortions, viral antigen was sometimes detected, but IB were consistently seen only in the juvenile snakes from the age of 2 mo onwards. In addition to demonstrating vertical transmission of multiple species, our results also indicate that reptarenavirus infection induces BIBD over time in the offspring.

Nidovirus-Associated Proliferative Pneumonia in the Green Tree Python (Morelia viridis).

In 2014 we observed a noticeable increase in the number of sudden deaths among green tree pythons (Morelia viridis). Pathological examination revealed the accumulation of mucoid material within the airways and lungs in association with enlargement of the entire lung. We performed a full necropsy and histological examination on 12 affected green tree pythons from 7 different breeders to characterize the pathogenesis of this mucinous pneumonia. By histology we could show a marked hyperplasia of the airway epithelium and of faveolar type II pneumocytes. Since routine microbiological tests failed to identify a causative agent, we studied lung tissue samples from a few diseased snakes by next-generation sequencing (NGS). From the NGS data we could assemble a piece of RNA genome whose sequence was <85% identical to that of nidoviruses previously identified in ball pythons and Indian pythons. We then employed reverse transcription-PCR to demonstrate the presence of the novel nidovirus in all diseased snakes. To attempt virus isolation, we established primary cultures of Morelia viridis liver and brain cells, which we inoculated with homogenates of lung tissue from infected individuals. Ultrastructural examination of concentrated cell culture supernatants showed the presence of nidovirus particles, and subsequent NGS analysis yielded the full genome of the novel virus Morelia viridis nidovirus (MVNV). We then generated an antibody against MVNV nucleoprotein, which we used alongside RNA in situ hybridization to demonstrate viral antigen and RNA in the affected lungs. This suggests that in natural infection MVNV damages the respiratory tract epithelium, which then results in epithelial hyperplasia, most likely as an exaggerated regenerative attempt in association with increased epithelial turnover.IMPORTANCE Novel nidoviruses associated with severe respiratory disease were fairly recently identified in ball pythons and Indian pythons. Herein we report on the isolation and identification of a further nidovirus from green tree pythons (Morelia viridis) with fatal pneumonia. We thoroughly characterized the pathological changes in the infected individuals and show that nidovirus infection is associated with marked epithelial proliferation in the respiratory tract. We speculate that this and the associated excess mucus production can lead to the animals' death by inhibiting normal gas exchange in the lungs. The virus was predominantly detected in the respiratory tract, which renders transmission via the respiratory route likely. Nidoviruses cause sudden outbreaks with high rates of mortality in breeding collections, and most affected snakes die without prior clinical signs. These findings, together with those of other groups, indicate that nidoviruses are a likely cause of severe pneumonia in pythons.

INVESTIGATION OF THE TRACHEAL MUCOCILIARY CLEARANCE IN SNAKES WITH AND WITHOUT BOID INCLUSION BODY DISEASE AND LUNG PATHOLOGY.

Pneumonia is a common complication of boid inclusion body disease (BIBD) in snakes. The tracheal mucociliary apparatus of eight boas ( Boa constrictor) and two pythons ( Python regius, Morelia viridis) was examined to assess whether absent or reduced mucociliary clearance could be a predisposing factor. Nine of the examined snakes were positive for BIBD by detection of inclusion bodies and three had lung pathologies other than the formation of inclusion bodies. A considerable individual variation of ciliary beat frequency (CBF, 3.0 ± 0.75 Hz to 7.8 ± 1.27 Hz), transport speed (23.1 ± 12.56 µm/sec to 189.2 ± 41.17 µm/sec), and transport direction (-12.5° ± 11.43° to 36.1° ± 7.53°) was found. CBFs of the BIBD-affected snakes with or without lung pathologies were markedly lower than ranges published for birds or mammals, but the net transport speeds and directions lay well within. The present investigation does therefore not reveal any signs of an inadequate mucociliary clearance in BIBD-affected snakes.

Analysis of a European general wildlife health surveillance program: Chances, challenges and recommendations.

In a One Health perspective general wildlife health surveillance (GWHS) gains importance worldwide, as pathogen transmission among wildlife, domestic animals and humans raises health, conservation and economic concerns. However, GWHS programs operate in the face of legal, geographical, financial, or administrative challenges. The present study uses a multi-tiered approach to understand the current characteristics, strengths and gaps of a European GWHS that operates in a fragmented legislative and multi-stakeholder environment. The aim is to support the implementation or improvement of other GWHS systems by managers, surveillance experts, and administrations. To assess the current state of wildlife health investigations and trends within the GWHS, we retrospectively analyzed 20 years of wildlife diagnostic data to explore alterations in annual case numbers, diagnosed diseases, and submitter types, conducted an online survey and phone interviews with official field partners (hunting administrators, game wardens and hunters) to assess their case submission criteria as well as their needs for post-mortem investigations, and performed in-house time estimations of post-mortem investigations to conduct a time-per-task analysis. Firstly, we found that infectious disease dynamics, the level of public awareness for specific diseases, research activities and increasing population sizes of in depth-monitored protected species, together with biogeographical and political boundaries all impacted case numbers and can present unexpected challenges to a GWHS. Secondly, we found that even a seemingly comprehensive GWHS can feature pronounced information gaps, with underrepresentation of common or easily recognizable diseases, blind spots in non-hunted species and only a fraction of discovered carcasses being submitted. Thirdly, we found that substantial amounts of wildlife health data may be available at local hunting administrations or disease specialist centers, but outside the reach of the GWHS and its processes. In conclusion, we recommend that fragmented and federalist GWHS programs like the one addressed require a central, consistent and accessible collection of wildlife health data. Also, considering the growing role of citizen observers in environmental research, we recommend using online reporting systems to harness decentrally available information and fill wildlife health information gaps.

Acute mortality in California tiger salamander (Ambystoma californiense) and Santa Cruz long-toed salamander (Ambystoma macrodactylum croceum) caused by Ribeiroia ondatrae (Class: Trematoda).

In early September 2019, a morbidity and mortality event affecting California tiger salamanders (Ambystoma californiense) and Santa Cruz long-toed salamanders (Ambystoma macrodactylum croceum) in late stages of metamorphosis was reported at a National Wildlife Refuge in Santa Cruz County, California, U.S.A. During the postmortem disease investigation, severe integumentary metacercarial (Class: Trematoda) infection, associated with widespread skin lesions, was observed. Planorbid snails collected from the ponds of the refuge within seven days of the mortality event were infected with Ribeiroia ondatrae, a digenetic trematode that can cause malformation and death in some amphibians. We suggest sustained seasonal high-water levels due to active habitat management along with several years of increased rainfall led to increased bird visitation, increased over-wintering of snails, and prolonged salamander metamorphosis, resulting in a confluence of conditions and cascading of host-parasite dynamics to create a hyper-parasitized state.

Pathology of Lagovirus europaeus GI.2/RHDV2/b (Rabbit Hemorrhagic Disease Virus 2) in Native North American Lagomorphs.

Rabbit hemorrhagic disease, a notifiable foreign animal disease in the US, was reported for the first time in wild native North American lagomorphs in April 2020 in the southwestern US. Affected species included the desert cottontail (Sylvilagus audubonii), mountain cottontail (Sylvilagus nuttallii), black-tailed jackrabbit (Lepus californicus), and antelope jackrabbit (Lepus alleni). Desert cottontails (n=7) and black-tailed jackrabbits (n=7) collected in April and May 2020 were necropsied at the US Geological Survey National Wildlife Health Center and tested positive for Lagovirus europaeus GI.2, also known as rabbit hemorrhagic disease virus 2 (GI.2/RHDV2/b), by real-time PCR at the US Department of Agriculture's Foreign Animal Disease Diagnostic Laboratory. Gross and microscopic lesions were similar to those reported in European rabbits (Oryctolagus cuniculus) and other hare (Lepus) species with GI.2/RHDV2/b infection; they included epistaxis (12/13; 92%); massive hepatocellular dissociation (14/14; 100%) and necrosis or apoptosis (11/11; 100%); pulmonary congestion (12/12; 100%), edema (12/13; 92%), and hemorrhage (11/12; 92%); and acute renal tubular injury (3/8; 38%). As in previous reports, massive hepatocellular dissociation and necrosis or apoptosis was the most diagnostically distinct finding. As North American Sylvilagus and Lepus species appear to be susceptible to fatal GI.2/RHDV2/b infection, additional work is needed to understand the host range, pathogenicity, and potential population effects of GI.2/RHDV2/b in North America.

Analysis of Archival Specimens Confirms White-Nose Syndrome in Little Brown Bats (Myotis lucifugus) from New York, USA, in Spring 2007.

White-nose syndrome (WNS), an emerging fungal disease of North American bats, was first diagnosed in January 2008, although mortality and photodocumentation suggest the disease might have been present earlier. Using archived samples, we describe a definitive case of WNS in little brown bats (Myotis lucifugus) from New York, US, in spring 2007.

Experimental challenge of a North American bat species, big brown bat (Eptesicus fuscus), with SARS-CoV-2.

The recently emerged novel coronavirus, SARS-CoV-2, is phylogenetically related to bat coronaviruses (CoVs), specifically SARS-related CoVs from the Eurasian bat family Rhinolophidae. As this human pandemic virus has spread across the world, the potential impacts of SARS-CoV-2 on native North American bat populations are unknown, as is the ability of North American bats to serve as reservoirs or intermediate hosts able to transmit the virus to humans or to other animal species. To help determine the impacts of the pandemic virus on North American bat populations, we experimentally challenged big brown bats (Eptesicus fuscus) with SARS-CoV-2 under BSL-3 conditions. We inoculated the bats both oropharyngeally and nasally, and over the ensuing three weeks, we measured infectivity, pathology, virus concentrations in tissues, oral and rectal virus excretion, virus transmission, and clinical signs of disease. We found no evidence of SARS-CoV-2 infection in any examined bat, including no viral excretion, no transmission, no detectable virus in tissues, and no signs of disease or pathology. Based on our findings, it appears that big brown bats are resistant to infection with the SARS-CoV-2. The potential susceptibility of other North American bat species to SARS-CoV-2 remains to be investigated.