Herpesvirus skin disease in free-living common frogs Rana temporaria in Great Britain.

Infectious disease is a significant driver of global amphibian declines, yet despite this, relatively little is known about the range of pathogens that affect free-living amphibians. Recent detection of the tentatively named Ranid herpesvirus 3 (RHV3), associated with skin disease in free-living common frogs Rana temporaria in Switzerland, helps to address this paucity in knowledge, but the geographic distribution and epidemiology of the pathogen remains unclear. Syndromic surveillance for ranid herpesvirus skin disease was undertaken throughout Great Britain (GB), January 2014 to December 2016. Reports of common frogs with macroscopic skin lesions with a characteristic grey appearance were solicited from members of the public. Post-mortem examination was conducted on one affected frog found dead in 2015 at a site in England. In addition, archived samples from an incident involving common frogs in England in 1997 with similar macroscopic lesions were further investigated. Transmission electron microscopy identified herpes-like virions in skin lesions from both the 1997 and 2015 incidents. RHV3, or RHV3-like virus, was detected in skin lesions from the 2015 case by PCR and sequencing. Our findings indicate that herpesvirus skin disease is endemic in common frogs in GB, with widespread distribution at apparently low prevalence. Further research into the role of host immunity, virus latency and the significance of infection to host survival is required to better understand the epidemiology and impact of cutaneous herpesvirus infections in amphibian populations.

First cases of squirrelpox in red squirrels (Sciurus vulgaris) in Scotland.

Squirrelpox, caused by a poxvirus, is a major threat to the remaining UK red squirrel population. The spread of antibody-positive grey squirrels has been monitored in the UK for the past decade. In 2005 grey squirrels that had been exposed to the virus appeared in the south of Scotland for the first time, followed approximately two years later by the appearance of squirrelpox disease in the local red squirrels. Four squirrels were examined. They all had gross external lesions and histological lesions typical of squirrelpox disease, but no significant internal lesions. The diagnosis was confirmed by PCR, electron microscopy and serology.

Systematic assessment of the impact of adenovirus infection on a captive reintroduction project for red squirrels (Sciurus vulgaris).

PCR was used to amplify adenoviral DNA, and transmission electron microscopy (TEM) to detect adenovirus particles in tissue and intestinal content samples from red squirrels (Sciurus vulgaris) associated with a reintroduction study on Anglesey (North Wales), from other populations on the island and from stock held at the Welsh Mountain Zoo, 38 km to the east. Samples were collected during the routine surveillance postmortem examinations of all 60 red squirrels with carcases retrieved in a suitable condition between 2004 and 2010, including 29 captive and 31 free-living animals. Following significant clusters of mortality in captive red squirrels, adenovirus was identified retrospectively in faecal material from 12 of 13 (92 per cent) examined carcases from squirrels captive on Anglesey, and 14 of 16 (88 per cent) from the Welsh Mountain Zoo. Virus was identified in 13 of 31 (42 per cent) free-living wild animals, with evidence of both subclinical and clinically significant enteric adenoviral infections in wild squirrels. Without ancillary PCR and TEM testing, the extent of adenovirus infection in such populations would have been underestimated. Screening protocols that include examinations for adenovirus should, therefore, be part of the routine biosecurity measures protecting reintroduction or captive breeding programmes for red squirrels.

Epidemiological and postmortem findings in 262 red squirrels (Sciurus vulgaris) in Scotland, 2005 to 2009.

Postmortem and virological examinations for squirrelpox virus (SQPV) were carried out on 262 red squirrels (Sciurus vulgaris) found dead or moribund in Scotland between September 2005 and July 2009, to determine the likely causes of death and highlight factors that might be threats to the red squirrel population. Most of the squirrels were submitted from Dumfries and Galloway, and 71 per cent of them were adults. Road traffic accidents, squirrelpox, trauma or starvation were responsible for death in a large proportion (73 per cent) of the squirrels. Thin or emaciated body condition was associated with deaths resulting from pneumonia SQPV infection and starvation, and with the presence of external parasites. There were differences between age groups with regard to the cause of death; a large proportion of juveniles died of starvation, whereas a large proportion of subadults and adults died in road traffic accidents. SQPV infection was associated with the presence of external parasites, but was not associated with the sex of the animals.

Mortality in red squirrels (Sciurus vulgaris) associated with exudative dermatitis.

Red squirrels (Sciurus vulgaris) found dead or dying on the Isle of Wight and the island of Jersey were suffering from exudative, ulcerative dermatitis and superficial staphylococcal pyoderma. The principal gross lesions were on the lips, eyelids and feet and showed similarities to those of squirrelpox. The histopathological lesions were also similar and, although there was no ballooning degeneration of epidermal cells, intracytoplasmic inclusions resembling those seen in poxvirus infections were present. Examination of lesions by electron microscopy failed to identify any virions, and PCR analysis for squirrelpox virus proved negative. The skin lesions also resembled those of mange, but although numerous mites were present in the fur these were mostly Dermacarus sciurinus with small numbers of Metalistrophorus pagenstecheri. The occurrence of these species on red squirrels in Britain is confirmed, but neither is pathogenic and they were not considered to have been involved in the pathogenesis of the dermatitis, the primary cause of which was not established.

Retrospective detection by negative contrast electron microscopy of faecal viral particles in free-living wild red squirrels (Sciurus vulgaris) with suspected enteropathy in Great Britain.

Transmission electron microscopy identified adenovirus particles in 10 of 70 (14.3 per cent) samples of large intestinal content collected at postmortem examination from free-living wild red squirrels (Sciurus vulgaris) across Great Britain between 2000 and 2009. Examination was limited to cases in which an enteropathy was suspected on the basis of predetermined macroscopic criteria such as semi-solid or diarrhoeic faeces, suspected enteritis or the presence of intussusception. In most cases, meaningful histological examination of enteric tissue was not possible due to pronounced autolysis. Two (2.9 per cent) of the samples were negative for adenovirus but were found to contain rotavirus particles, a novel finding in this species.

Improved spectrophotometric assay for beta-lactam residues in kidney tissue.

This paper describes a detection system for beta-lactams using a commercially prepared carboxypeptidase enzyme (CPase) and a substrate system in which lactic acid is cleaved from a synthetic peptide, N alpha-N epsilon-diacetyl-L-lysyl-d-alanyl-d-lactic acid. The lactate is itself oxidized by lactate dehydrogenase to form NADH. Oxidized NAD+ is regenerated by diaphorase with the simultaneous reduction of the colourless 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride hydrate (INT) indicator substrate to produce a red-mauve colour that is proportional to CPase activity. The presence of beta-lactams decreases the intensity of colour produced. The lower limit of detection for benzyl penicillin (Pen G) by this system is 20 ng g-1 compared with 50 ng g-1 by the same assay but using a R-d-ala-d-ala substrate from a commercial kit.