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

Systemic Helicobacter infection and associated mortalities in endangered Grand Cayman blue iguanas (Cyclura lewisi) and introduced green iguanas (Iguana iguana).

The Blue Iguana Recovery Programme maintains a captive breeding and head-starting program for endangered Grand Cayman blue iguanas (Cyclura lewisi) on Grand Cayman, Cayman Islands. In May 2015, program staff encountered two lethargic wild Grand Cayman blue iguanas within the Queen Elizabeth II Botanic Park (QEIIBP). Spiral-shaped bacteria were identified on peripheral blood smears from both animals, which molecular diagnostics identified as a novel Helicobacter species (provisionary name Helicobacter sp. GCBI1). Between March 2015 and February 2017, 11 Grand Cayman blue iguanas were identified with the infection. Two of these were found dead and nine were treated; five of the nine treated animals survived the initial infection. Phylogenetic analysis of the 16S rRNA gene suggests Helicobacter sp. GCBI1 is most closely related to Helicobacter spp. in chelonians. We developed a Taqman qPCR assay specific for Helicobacter sp. GCBI1 to screen tissue and/or blood samples from clinical cases, fecal and cloacal samples from clinically healthy Grand Cayman blue iguanas, including previously infected and recovered iguanas, and iguanas housed adjacent to clinical cases. Fecal and/or cloacal swab samples were all negative, suggesting that Grand Cayman blue iguanas do not asymptomatically carry this organism nor shed this pathogen per cloaca post infection. Retrospective analysis of a 2014 mortality event affecting green iguanas (Iguana iguana) from a separate Grand Cayman location identified Helicobacter sp. GCBI1 in two of three cases. The source of infection and mode of transmission are yet to be confirmed. Analysis of rainfall data reveal that all infections occurred during a multi-year dry period, and most occurred shortly after the first rains at the end of seasonal drought. Additionally, further screening has identified Helicobacter sp. GCBI1 from choanal swabs of clinically normal green iguanas in the QEIIBP, suggesting they could be asymptomatic carriers and a potential source of the pathogen

Post-mortem findings in southern right whales Eubalaena australis at Península Valdés, Argentina, 2003-2012

Between 2003 and 2012, 605 southern right whales (SRW; Eubalaena australis) were found dead along the shores of Península Valdés (PV), Argentina. These deaths included alarmingly high annual losses between 2007 and 2012, a peak number of deaths (116) in 2012, and a significant number of deaths across years in calves-of-the-year (544 of 605 [89.9%]; average = 60.4 yr-1). Post-mortem examination and pathogen testing were performed on 212 whales; 208 (98.1%) were calves-of-the-year and 48.0% of these were newborns or neonates. A known or probable cause of death was established in only a small number (6.6%) of cases. These included ship strike in a juvenile and blunt trauma or lacerations (n = 5), pneumonia (n = 4), myocarditis (n = 2), meningitis (n = 1), or myocarditis and meningitis (n = 1) in calves. Ante-mortem gull parasitism was the most common gross finding. It was associated with systemic disease in a single 1-2 mo old calf. Immunohistochemical labeling for canine distemper virus, Toxoplasma gondii and Brucella spp., and PCR for cetacean morbillivirus (CeMV), influenza A, and apicomplexan protozoa were negative on formalin-fixed, paraffin-embedded lung and brain samples from a subset of whales; PCR for Brucella spp. was positive in a newborn/neonate with pneumonia. Skin samples from whales with gull parasitism were PCR negative for CeMV, poxvirus, and papillomavirus. This is the first long-term study to investigate and summarize notable post-mortem findings in the PV SRW population. Consistent, significant findings within or between years to explain the majority of deaths and those in high-mortality years remain to be identifiedInst. de PatobiologíaFil: McAloose, DeniseWildlife Conservation Society Zoological Health Program; Estados Unidos. Southern Right Whale Health Monitoring Program; ArgentinaFil: Rago, Virginia. Southern Right Whale Health Monitoring Program; Argentina. Wildlife Conservation Society Wildlife Health & Health Policy Program; Estados UnidosFil: Di Martino, Matías. Southern Right Whale Health Monitoring Program; Argentina. Wildlife Conservation Society Wildlife Health & Health Policy Program; Estados UnidosFil: Chirife, AndreaSouthern Right Whale Health Monitoring Program; ArgentinaFil: Olson, Sarah. Wildlife Conservation Society Wildlife Health & Health Policy Program; Estados UnidosFil: Beltramino, Lucas. Southern Right Whale Health Monitoring Program; ArgentinaFil: Pozzi, Luciana Melina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Nacional Patagónico; Argentina. Southern Right Whale Health Monitoring Program; Argentina. Fundación Patagonia Natural; ArgentinaFil: Musmeci, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Nacional Patagónico; Argentina. Southern Right Whale Health Monitoring Program; Argentina. Fundación Patagonia Natural; ArgentinaFil: la Sala, Luciano Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Bahia Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biologia, Bioquimica y Farmacia. Catedra de Parasitologia Clinica; ArgentinaFil: Mohamed, Nadia. Southern Right Whale Health Monitoring Program; ArgentinaFil: Sala, Juan Emilio. Southern Right Whale Health Monitoring Program; ArgentinaFil: Bandieri, Lucas. Southern Right Whale Health Monitoring Program; ArgentinaFil: Andrejuk, Julian. Southern Right Whale Health Monitoring Program; ArgentinaFil: Tomaszewicz, Ania. Wildlife Conservation Society Zoological Health Program; Estados UnidosFil: Tomaszewicz, Ania. Wildlife Conservation Society Zoological Health Program; Estados UnidosFil: Seimon, Tracie. Wildlife Conservation Society Zoological Health Program; Estados UnidosFil: Sironi, Mariano. Southern Right Whale Health Monitoring Program; Argentina. Instituto de Conservación de Ballenas; ArgentinaFil: Samartino, Luis Ernesto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: Rowntree, Victoria. Southern Right Whale Health Monitoring Program; Argentina. University of Utah. Department of Biology; Estados UnidosFil: Uhart, Marcela M. Southern Right Whale Health Monitoring Program; Argentina. Wildlife Conservation Society Wildlife Health & Health Policy Program; Estados Unidos. University of California. School of Veterinary Medicine. One Health Institute; Argentin