Health Effects of Patagial Wing Tags in Red Kites (Milvus milvus) in the UK.

Patagial wing tags are commonly used for identification of Red Kites (Milvus milvus) for postrelease monitoring, as they are easy to apply, affordable, permanent, and are apparently safe. The Red Kite was successfully reintroduced in the UK in the second half of the 20th century and postrelease health surveillance has been achieved through radio and satellite tracking, monitoring nest sites, and pathologic investigation of Red Kites found dead. This study reports on pathologic findings associated with the use of patagial wing tags in three of 142 (2.1%) wing-tagged Red Kites examined postmortem since the beginning of the reintroduction project in 1989. In these three Red Kites the presence of the patagial wing tags was associated with inflammatory lesions. Further surveys of the potential short- and longer-term negative effects of patagial wing tags on Red Kites and other birds are advocated; the future use of patagial wing tags in raptors should be carefully monitored.

Prevalence and Molecular Analysis of Encephalomyocarditis Virus-2 in the Hazel Dormouse.

The hazel dormouse (Muscardinus avellanarius) population in the UK continues to decline due to habitat loss, despite reintroductions of captive-bred individuals being conducted nationally for over 30 years. Disease surveillance of captive-bred and wild dormice is performed to identify novel and existing disease threats which could impact populations. In this study, we firstly investigated cause of death in seven hazel dormice found dead in England, through next-generation sequencing identifying a virus closely related to a wood mouse encephalomyocarditis virus-2 (EMCV-2). Subsequently, lung tissue samples from 35 out of 44 hazel dormice tested positive for EMCV-2 RNA using a reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and Sanger sequencing methods developed in this study. Formalin-fixed tissues available for nine hazel dormice which tested positive for EMCV-2 RNA were examined microscopically. Three cases showed moderate interstitial pneumonia with minimal to mild lymphoplasmacytic myocarditis, but no evidence of encephalitis. However, the presence of possible alternative causes of death in these cases means that the lesions cannot be definitively attributed to EMCV-2. Here, we report the first detection of EMCV-2 in hazel dormice and conclude that EMCV-2 is likely to be endemic in the hazel dormouse population in England and may be associated with clinical disease.

The risk from SARS-CoV-2 to bat species in england and mitigation options for conservation field workers.

The newly evolved coronavirus, SARS-CoV-2, which has precipitated a global COVID-19 pandemic among the human population, has been shown to be associated with disease in captive wild animals. Bats (Chiroptera) have been shown to be susceptible to experimental infection and therefore may be at risk from disease when in contact with infected people. Numerous conservation fieldwork activities are undertaken across the United Kingdom bringing potentially infected people into close proximity with bats. In this study, we analysed the risks of disease from SARS-CoV-2 to free-living bat species in England through fieldworkers undertaking conservation activities and ecological survey work, using a qualitative, transparent method devised for assessing threats of disease to free-living wild animals. The probability of exposure of bats to SARS-CoV-2 through fieldwork activities was estimated to range from negligible to high, depending on the proximity between bats and people during the activity. The likelihood of infection after exposure was estimated to be high and the probability of dissemination of the virus through bat populations medium. The likelihood of clinical disease occurring in infected bats was low, and therefore, the ecological, economic and environmental consequences were predicted to be low. The overall risk estimation was low, and therefore, mitigation measures are advisable. There is uncertainty in the pathogenicity of SARS-CoV-2 in bats and therefore in the risk estimation. Disease risk management measures are suggested, including the use of personal protective equipment, good hand hygiene and following the existing government advice. The disease risk analysis should be updated as information on the epidemiology of SARS-CoV-2 and related viruses in bats improves. The re-analysis may be informed by health surveillance of free-living bats.

Two Novel Adenoviruses in Free-Living British lizards.

In this study, we describe two novel adenoviruses isolated from (i) a common lizard (Zootoca vivipara) found dead and examined post-mortem and (ii) pooled samples from free-living sand lizards (Lacerta agilis agilis). Sequencing indicated the two were closely related atadenovirus strains which were distinct from previously recorded adenoviruses in lizards. Adenoviruses are not always associated with disease in squamates, but morbidity and mortality have been reported. These are the first known cases of adenovirus infection in free-living native British lizards, and further monitoring will be necessary to elucidate the implications of these possible pathogens for vulnerable populations of native reptiles.

Eimeria spp. in captive-reared corncrakes (Crex crex): results of a GeneScan assay consistent with high prevalence of infection and extra-intestinal life stages.

Eimeria crecis and Eimeria nenei have been detected in association with enteric disease ("coccidiosis") in the corncrake (Crex crex: Family Rallidae, Order Gruiformes). Both parasite species are common in apparently healthy free-living corncrakes, but captive-bred juvenile birds reared for reintroduction appeared particularly susceptible to clinical coccidiosis. We investigated the occurrence and relative pathogenicity of these Eimeria species in this juvenile corncrake population and developed a diagnostic species-specific polymerase chain reaction (PCR) for their identification. PCR amplification and sequencing of 18S rDNA were performed on genomic DNA extracted from samples of corncrake intestine, liver and spleen. Sequences generated were used to design a GeneScan diagnostic PCR assay targeting a species-specific TTA indel located within the 18S rDNA - the results suggested this assay was more sensitive than the 18S rDNA/amplicon sequencing approach. Eimeria sp. DNA (consistent with Eimeria sp. infection) was detected at a high prevalence and E. crecis was the predominant species. Each Eimeria species was detected in cases with and without histological evidence of coccidiosis: parasite detection was not statistically associated with disease. In addition to intestinal tissue, liver and spleen samples were positive for Eimeria sp. DNA. Its detection in tissues other than intestine is unusual and a novel finding in corncrakes, although extra-intestinal infection occurs with closely related Eimeria species in cranes (Family Gruidae, Order Gruiformes). Eimeria sp. infection of corncrakes appears typically to be chronic, and to exhibit extra-intestinal spread: as in cranes, these characteristics may be adaptations to the host's migratory nature.

Protecting Free-Living Dormice: Molecular Identification of Cestode Parasites in Captive Dormice (Muscardinus avellanarius) Destined for Reintroduction.

The success of any population translocation programme relies heavily on the measures implemented to control and monitor the spread of disease. Without these measures, programmes run the risk of releasing immunologically naïve species or, more dangerously, introducing novel infectious agents to native populations. As a precaution, a reintroduction programme for the common or hazel dormouse, Muscardinus avellanarius, in England screens dormice before release following captive breeding. Using PCR sequencing of a range of genes, we tested whether the same species of tapeworm(s) were present in captive and free-living dormice. Whilst only Rodentolepis straminea were identified in free-living dormice, cestode ova found in a captive individual produced a molecular match closely related to Hymenolepis microstoma and a previously unrecorded Rodentolepis species. To prevent putting at risk the free-living population, we recommended the continued treatment of dormice showing tapeworm infection before release. Our work demonstrates how molecular techniques can be used to inform reintroduction programmes, reduce risk from disease and increase chances of reintroduction success.

Using Qualitative Disease Risk Analysis for Herpetofauna Conservation Translocations Transgressing Ecological and Geographical Barriers.

Through the exploration of disease risk analysis methods employed for four different UK herpetofauna translocations, we illustrate how disease hazards can be identified, and how the risk of disease can be analysed. Where ecological or geographical barriers between source and destination sites exist, parasite populations are likely to differ in identity or strain between the two sites, elevating the risk from disease and increasing the number and category of hazards requiring analysis. Simplification of the translocation pathway through the avoidance of these barriers reduces the risk from disease. The disease risk analysis tool is intended to aid conservation practitioners in decision making relating to disease hazards prior to implementation of a translocation.

Bringing Back a Healthy Buzz? Invertebrate Parasites and Reintroductions: A Case Study in Bumblebees.

Reintroductions can play a key role in the conservation of endangered species. Parasites may impact reintroductions, both positively and negatively, but few case studies of how to manage parasites during reintroductions exist. Bumblebees are in decline at regional and global scales, and reintroductions can be used to re-establish extinct local populations. Here we report on how the risks associated with parasites are being managed in an ongoing reintroduction of the short-haired bumblebee, Bombus subterraneus, to the UK. Disease risk analysis was conducted and disease risk management plans constructed to design a capture-quarantine-release system that minimised the impacts on both the bumblebees and on their natural parasites. Given that bumblebee parasites are (i) generalists, (ii) geographically ubiquitous, and (iii) show evidence of local adaptation, the disease risk management plan was designed to limit the co-introduction of parasites from the source population in Sweden to the destination site in the UK. Results suggest that this process at best eliminated, or at least severely curtailed the co-introduction of parasites, and ongoing updates of the plan enabled minimization of impacts on natural host-parasite dynamics in the Swedish source population. This study suggests that methods designed for reintroductions of vertebrate species can be successfully applied to invertebrates. Future reintroductions of invertebrates where the parasite fauna is less well known should take advantage of next-generation barcoding and multiple survey years prior to the start of reintroductions, to develop comprehensive disease risk management plans.

A Comparison of Disease Risk Analysis Tools for Conservation Translocations.

Conservation translocations are increasingly used to manage threatened species and restore ecosystems. Translocations increase the risk of disease outbreaks in the translocated and recipient populations. Qualitative disease risk analyses have been used as a means of assessing the magnitude of any effect of disease and the probability of the disease occurring associated with a translocation. Currently multiple alternative qualitative disease risk analysis packages are available to practitioners. Here we compare the ease of use, expertise required, transparency, and results from, three different qualitative disease risk analyses using a translocation of the endangered New Zealand passerine, the hihi (Notiomystis cincta), as a model. We show that the three methods use fundamentally different approaches to define hazards. Different methods are used to produce estimations of the risk from disease, and the estimations are different for the same hazards. Transparency of the process varies between methods from no referencing, or explanations of evidence to justify decisions, through to full documentation of resources, decisions and assumptions made. Evidence to support decisions on estimation of risk from disease is important, to enable knowledge acquired in the future, for example, from translocation outcome, to be used to improve the risk estimation for future translocations. Information documenting each disease risk analysis differs along with variation in emphasis of the questions asked within each package. The expertise required to commence a disease risk analysis varies and an action flow chart tailored for the non-wildlife health specialist are included in one method but completion of the disease risk analysis requires wildlife health specialists with epidemiological and pathological knowledge in all three methods. We show that disease risk analysis package choice may play a greater role in the overall risk estimation of the effect of disease on animal populations involved in a translocation than might previously have been realised.

Analyzing disease risks associated with translocations.

Translocations of species are expected to be used increasingly to counter the undesirable effects of anthropogenic changes to ecosystems, including loss of species. Methods to assess the risk of disease associated with translocations have been compiled in a comprehensive manual of disease-risk analysis for movement of domestic animals. We used this manual to devise a qualitative method for assessing the probability of the occurrence of disease in wild animals associated with translocations. We adapted the method such that we considered a parasite (any agent of infectious or noninfectious disease) a hazard if it or the host had crossed an ecological or geographical barrier and was novel to the host. We included in our analyses hazards present throughout the translocation pathway derived from the interactions between host immunity and the parasite, the effect of parasites on populations, the effect of noninfectious disease agents, and the effect of stressors on host-parasite interactions. We used the reintroduction of Eurasian Cranes (Grus grus) to England to demonstrate our method. Of the 24 hazards identified, 1 was classified as high risk (coccidia) and 5 were medium risk (highly pathogenic avian influenza virus, Mycobacterium avium, Aspergillus fumigatus, tracheal worms [Syngamus sp. and Cyathostoma sp.], and Tetrameres spp.). Seventeen other hazards were considered low or very low risk. In the absence of better information on the number, identity, distribution, and pathogenicity of parasites of wild animals, there is uncertainty in the risk of disease to translocated animals and recipient populations. Surveys of parasites in source and destination populations and detailed health monitoring after release will improve the information available for future analyses of disease risk. We believe our method can be adapted to assess the risks of disease in other translocated populations.

Epizootiology and pathologic findings associated with a newly described adenovirus in the red squirrel, Sciurus vulgaris.

An infectious disease caused by Squirrelpox virus has contributed to the decline of red squirrels, Sciurus vulgaris, in the British Isles. Because of the heightened disease surveillance activity in red squirrels, adenovirus infection with associated mortality has been detected. Adenoviral disease is described in other rodent species usually associated with stressors. Here we 1) describe the pathologic findings in red squirrels found dead with adenoviral infection and gastrointestinal disease, and 2) investigate the epizootiology of the disease through pathologic investigation, scanning surveillance, and virologic studies. Ten red squirrels involved in conservation studies were diagnosed with adenoviral infection by electron microscopy or PCR. All squirrels exhibited diarrhea and small intestinal inflammation or hemorrhage was evident in seven cases. Lesions indicative of splenic lymphocytolysis were observed in one squirrel and leukocytic hepatitis in another. No adenovirus was detected in grey squirrels, Sciurus carolinensis, inhabiting the same forest area, but previous serologic studies showed that grey squirrels cannot be discounted as a reservoir of the virus. Scanning surveillance showed that 12% of 493 red squirrels had diarrheal disease and two of 13 free-living red squirrels with diarrheal disease had adenovirus infection. Adenoviral disease in declining free-living wild red squirrel populations in the British Isles occurs at a detectable frequency and its impact on the conservation of this species deserves further attention.

Poxviral disease in red squirrels Sciurus vulgaris in the UK: spatial and temporal trends of an emerging threat.

The squirrel poxvirus (SQPV) is the probable mediator of apparent competition between the introduced invading gray squirrel (Sciurus carolinensis) and the red squirrel (Sciurus vulgaris) in the UK, and modeling studies have shown that this viral disease has had a significant impact on the decline of the red squirrel in the UK. However, given our limited understanding of the epidemiology of the disease, and more generally the effects of invasive species on parasite ecology, there is a need to investigate the transmission dynamics and the relative pathogenicity of the virus between species. We aimed to increase our knowledge of these processes through an empirical study in which we: (i) used pathological signs and transmission electron microscopy (TEM) to diagnose SQPV disease in red squirrels found dead during scanning surveillance between 1993 and 2005; (ii) detected antibody to SQPV using an enzyme-linked immunosorbent assay (ELISA) in the same animals; and (iii) mapped cases of the disease, and the gray squirrel distribution, using a geographical information system. We analyzed the distribution of cases of SQPV disease according to woodland type, a measure of squirrel density. SQPV disease occurred only in areas of England also inhabited by seropositive gray squirrels, and as the geographical range of gray squirrels expanded, SQPV disease occurred in these new gray squirrel habitats, supporting a role for the gray squirrel as a reservoir host of the virus. There was a delay between the establishment of invading gray squirrels and cases of the disease in red squirrels which implies gray squirrels must reach a threshold number or density before the virus is transmitted to red squirrels. The spatial and temporal trend in SQPV disease outbreaks suggested that SQPV disease will have a significant effect on Scottish populations of red squirrels within 25 years. The even spread of cases of disease across months suggested a direct rather than vector-borne transmission route is more likely. Eight juvenile and sub-adult free-living red squirrels apparently survived exposure to SQPV by mounting an immune response, the first evidence of immunity to SQPV in free-living red squirrels, which possibly suggests a changing host-parasite relationship and that the use of a vaccine may be an effective management tool to protect remnant red squirrel populations.

Genomic characterization of a novel poxvirus contributing to the decline of the red squirrel (Sciurus vulgaris) in the UK.

The genome of a virulent squirrelpox virus (SQPV) isolate was characterized in order to determine its relationship with other poxviruses. Restriction enzyme analysis suggested a genome length of approximately 158 kb, whilst sequence analysis of the two ends of the genome indicated a G + C composition of approximately 66 %. Two contiguous stretches of 23 and 37 kb at the left-hand and right-hand ends of the genome, respectively, were sequenced allowing the identification of at least 59 genes contained therein. The partial sequence of a further 15 genes was determined by spot sequencing of restriction fragments located across the genome. Phylogenetic analysis of 15 genes conserved in all the recognized genera of the subfamily Chordopoxvirinae confirmed that the SQPV does not group within the family Parapoxvirinae, but instead partitions on its own in a separate clade of the poxviruses. Analysis of serum from British woodland rodents failed to find any evidence of SQPV infection in wood mice or bank voles, but for the first time serum samples from grey squirrels in the USA were found to contain antibody against SQPV.

Bone assessment of free-living red squirrels (Sciurus vulgaris) from the United Kingdom.

Metabolic bone disease has been reported in free-living red squirrels (Sciurus vulgaris) in the United Kingdom but the prevalence of this disease is unknown. In this study the bone quality of free-living red squirrels in the UK was assessed by radiology and bone densitometry. The study comprised 20 red squirrels found dead and submitted to the Zoological Society of London (UK) between 1997 and 1998, 10 were from the Isle of Wight (IoW), where gray squirrels (Sciurus carolinensis) are absent, and 10 were from Cumbria (Cu), where gray squirrels are present. Gray squirrels are considered potential competitors for red squirrels. Radiologic evaluation of humerus, femur, tibia, radius, and ilium revealed a slightly lower bone density and thinner cortices in red squirrels from the IoW when compared with those from Cu. Dual-energy X-ray absorptiometry was used to measure bone mineral content and density of the isolated right humerus and femur of 19 of the 20 red squirrels. The bone densitometry study reinforced the radiographic findings. The IoW specimens had lower bone mineral density values, although statistical significance (P<0.05) between animals from the IoW and Cu was only reached for the proximal epiphysis of the femur and between males from the IoW and males from Cu for the proximal epiphysis of the humerus. A highly positive correlation (r>0.94) was found when the bone mineral content and density between the femur and the humerus among groups and within each group were compared, showing a uniform level of mineralization between upper and lower limbs. These findings suggested generalized bone loss for the IoW red squirrels that may be compatible with some degree of osteopenia. Within the wide range of causes that lead to osteopenia, malnutrition (especially protein deficiency), calcium and copper deficiencies, and genetic factors remain as possible etiologies.

Oral disease in free-living red squirrels (Sciurus vulgaris) in the United Kingdom.

Ninety-one red squirrels (Sciurus vulgaris) found dead in the UK between January 1994 and August 1998 were necropsied at the Institute of Zoology (London, UK); their oral cavities were examined visually, and in eight cases, radiographically. Four red squirrels, which had evidence of oral disease when necropsied as part of a mortality survey, also were examined. A low prevalence (prevalence = 0.033, SE = 0.02, n = 91) of oral disease was found in free-living red squirrels. In only two cases was oral disease the probable cause of death. Attrition of the check teeth (three cases) and overgrowth of the incisors (four cases) were the most common lesions found. Partial anodontia was recorded in one squirrel.

A novel poxvirus lethal to red squirrels (Sciurus vulgaris).

A parapoxvirus has been implicated in the decline of the red squirrel in the United Kingdom. Virus was isolated from an outbreak of lethal disease in red squirrels in the north-east of England. Experimental infection of captive-bred red squirrels confirmed that this virus was the cause of the severe skin lesions observed. Electron microscopic examination of the virus showed that it had a morphology typical of parapoxviruses whilst preliminary sequence data suggested a genomic G+C composition of approximately 66 %, again similar to that found in other parapoxviruses. However Southern hybridization analysis failed to detect three known parapoxvirus genes, two of which have been found so far only in the genus parapoxvirus. Comparative sequence analysis of two other genes, conserved across the eight recognized chordopoxvirus genera, suggests that the squirrel virus represents a previously unrecognized genus of the chordopoxvirus.