Moraxella oculi sp. nov., isolated from a cow with infectious bovine keratoconjunctivitis.

A novel species of the genus Moraxella was isolated from an ocular swab from a cow with infectious bovine keratoconjunctivitis. 16S rRNA gene sequencing suggested this species was Moraxella bovis (99.59 % nucleotide identity). Average nucleotide identity was calculated using a draft whole genome sequence of this strain compared with type strains of closely related Moraxella species and results established that it represents a new species. The genome size was 2 006 474 nucleotides and the G+C content was 42.51 mol%. The species could not be identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry using a commercial database, confirming the novelty of the strain. We propose the name Moraxella oculi sp. nov. for this new species. The type strain is Tifton1T and has been deposited into the American Type Culture Collection (TSD-373T) and the National Collection of Type Cultures (NCTC), UK Health Security Agency (NCTC 14942T).

Phylogenomic assessment of 23 equid alphaherpesvirus 1 isolates obtained from USA-based equids.

BACKGROUND: Equid alphaherpesvirus 1 (EHV-1) is a global viral pathogen of domestic equids which causes reproductive, respiratory and neurological disease. Few isolates acquired from naturally infected USA-based hosts have been fully sequenced and analyzed to date. An ORF 30 (DNA polymerase) variant (A2254G) has previously been associated with neurological disease in host animals. The purpose of this study was to perform phylogenomic analysis of EHV-1 isolates acquired from USA-based hosts and compare these isolates to previously sequenced global isolates. METHODS: EHV-1 was isolated from 23 naturally infected USA-based equids (6 different states, 15 disease outbreaks) with reproductive (22/23) or neurological disease (1/23). Following virus isolation, EHV-1 DNA was extracted for sequencing using Illumina MiSeq. Following reference-based assembly, whole viral genomes were annotated and assessed. Previously sequenced EHV-1 isolates (n = 114) obtained from global host equids were included in phylogenomic analyses. RESULTS: The overall average genomic distance was 0.0828% (SE 0.004%) for the 23 newly sequenced USA isolates and 0.0705% (SE 0.003%) when all 137 isolates were included. Clade structure was predominantly based on geographic origin. Numerous nucleotide substitutions (mean [range], 179 [114-297] synonymous and 81 [38-120] non-synonymous substitutions per isolate) were identified throughout the genome of the newly sequenced USA isolates. The previously described ORF 30 A2254G substitution (associated with neurological disease) was found in only one isolate obtained from a host with non-neurological clinical signs (reproductive disease), six additional, unique, non-synonymous ORF 30 substitutions were detected in 22/23 USA isolates. Evidence of recombination was present in most (22/23) of the newly sequenced USA isolates. CONCLUSIONS: Overall, the genomes of the 23 newly sequenced EHV-1 isolates obtained from USA-based hosts were broadly similar to global isolates. The previously described ORF 30 A2254G neurological substitution was infrequently detected in the newly sequenced USA isolates, most of which were obtained from host animals with reproductive disease. Recombination was likely to be partially responsible for genomic diversity in the newly sequenced USA isolates.

Adherent-invasive Escherichia coli associated with granulomatous colitis and extraintestinal dissemination in a Sphynx cat.

This case report describes a case of granulomatous colitis (GC) associated with adherent-invasive Escherichia coli (AIEC) with extension to cecum and ileum and dissemination to multiple lymph nodes, the spleen, and brain in a 10-year-old, male Sphynx cat. The cat had an episode of diarrhea 4 months prior to consultation due to sudden blindness. Signs rapidly progressed to ataxia, seizures, and death. Gross and histologic findings were consistent with granulomatous inflammation in all affected organs. In situ hybridization confirmed the presence of intracellular E. coli within enterocytes and infiltrating macrophages, and whole genome sequencing identified virulence traits commonly linked to AIEC strain. This is the first characterization of GC in a cat associated to AIEC resembling the metastatic form of Crohn's disease in humans and GC of dogs. Extraintestinal involvement might provide evidence of the ability of AIEC to promote granulomatous inflammation beyond the gut.

Malakoplakia in the Urinary Bladder of 4 Puppies.

Malakoplakia in humans most often affects the urinary bladder and is characterized by inflammation with von Hansemann-type macrophages, with or without Michaelis-Gutmann bodies, and is frequently associated with Escherichia coli infection. We describe the microscopic features of malakoplakia in the urinary bladder of 4 puppies. In all cases, the lamina propria of the urinary bladder was markedly expanded by sheets of large, round to polygonal macrophages with intracytoplasmic, periodic acid-Schiff-positive granules and granular inclusions, and rare Prussian blue-positive inclusions. Macrophages were positive for CD18 and Iba1. In 2 cases, Michaelis-Gutmann bodies were detected with hematoxylin and eosin stain and were best demonstrated with von Kossa stain. E. coli infection was confirmed in 2 cases with bacterial culture or polymerase chain reaction (PCR) and sequencing of the bacterial 16S ribosomal RNA gene. Transmission electron microscopy of one case demonstrated macrophages with abundant lysosomes, phagolysosomes, and rod-shaped bacteria. Microscopic features were similar to human cases of malakoplakia. In dogs, the light microscopic characteristics of malakoplakia closely resemble granular cell tumors and histiocytic ulcerative colitis.

Emerging Pathogens and a Current-Use Pesticide: Potential Impacts on Eastern Hellbenders.

Populations of the eastern hellbender Cryptobranchus alleganiensis alleganiensis have been declining for decades, and emerging pathogens and pesticides are hypothesized to be contributing factors. However, few empirical studies have attempted to test the potential effects of these factors on hellbenders. We simultaneously exposed subadult hellbenders to environmentally relevant concentrations of either Batrachochytrium dendrobatidis (Bd) or a frog virus 3-like ranavirus (RV), a combination of the pathogens, or each pathogen following exposure to a glyphosate herbicide (Roundup). Additionally, we measured the ability of the skin mucosome to inactivate Bd and RV in growth assays. We found that mucosome significantly inactivated RV by an average of 40% but had no negative effects on Bd growth. All treatments that included RV exposure experienced reduced survival compared to controls, and the combination of RV and herbicide resulted in 100% mortality. Histopathology verified RV as the cause of mortality in all RV-exposed treatments. No animals were infected with Bd or died in the Bd-only treatment. Our results suggest that RV exposure may be a significant threat to the survival of subadult hellbenders and that Roundup exposure may potentially exacerbate this threat.

Natural Canine Distemper Virus Infection in Linnaeus's 2-Toed Sloths (Choloepus didactylus).

An outbreak of canine distemper virus in a private zoo in eastern Tennessee in July 2016 led to fatal clinical disease in 5 adult, wild-caught Linnaeus's 2-toed sloths (Choloepus didactylus). Clinical signs included hyporexia, lethargy, mucopurulent nasal discharge, and oral and facial ulcers. At necropsy, affected animals had crusts and ulcers on the lips, nose, tongue, and oral cavity. Microscopically, all sloths had widespread, random, hepatic necrosis; lymphoid depletion; and bronchointerstitial pneumonia. The central nervous system did not contain gross or histopathologic lesions in any of the 5 sloths, although immunoreactivity for viral antigen was present within vessel walls. Epithelial cells and histiocytes within numerous organs contained intranuclear and intracytoplasmic inclusions and occasional syncytial cells. Canine distemper virus was confirmed with immunohistochemistry and virus isolation. Viral sequencing identified the novel American-4 strain prevalent in eastern Tennessee wildlife. This is the first pathologic characterization of canine distemper virus infection in sloths (family Choloepodidae, order Pilosa) and emphasizes the significant morbidity and mortality in this species.

GENETIC CHARACTERISTICS OF CANINE DISTEMPER VIRUSES CIRCULATING IN WILDLIFE IN THE UNITED STATES.

Canine distemper virus (CDV) is a highly contagious disease of wild and domestic mammals. Maintenance of CDV among wildlife plays an important role in the disease epidemiology. Wild animals, including raccoons (Procyon lotor) and gray foxes (Urocyon cinereoargenteus), serve as reservoirs of CDV and hamper the control of the disease. Recently, we discovered that at least three different CDV lineages (America-3 [Edomex], America-4, and America-5] that are genetically different from the available vaccine strains are circulating in domestic dogs in the United States. Because wildlife serve as a reservoir for the virus, it is important to determine if wildlife play a role in the maintenance and spread of these lineages. To determine the genetic characteristics of circulating strains of CDV in wildlife in various geographic regions in the United States, we studied the nucleotide sequences of the hemagglutinin (H) gene of 25 CDV strains detected in nondomestic species. The species included were free-ranging wildlife: three fishers (Martes pennanti), six foxes, one skunk (Mephitis mephitis), 10 raccoons, two wolves (Canis lupus), and one mink (Neovison vison). Strains from two species in managed care, one sloth (Choloepus didactylus) and one red panda (Ailurus fulgens), were also evaluated. Phylogenetic analysis of the H genes indicated that in addition to America-3, America-4, and America-5 lineages, there are at least two other lineages circulating in US wildlife. One of these includes CDV nucleotide sequences that grouped with that of a single CDV isolate previously detected in a raccoon from Rhode Island in 2012. The other lineage is independent and genetically distinct from other CDV strains included in the analysis. Additional genetically variable strains were detected, mainly in raccoons, suggesting that this species may be the host responsible for the genetic variability of newly detected strains in the domestic dog population.

SAFETY OF AND HUMORAL IMMUNE RESPONSE TO THE MERIAL RECOMBITEK CANINE DISTEMPER VIRUS VACCINE IN MANED WOLVES (CHRYSOCYON BRACHYURUS).

This study evaluated the safety of and humoral response to the Merial Recombitek® recombinant canine distemper virus (rCDV) vaccine in maned wolves (n = 9, age 2-9 yr). All maned wolves had prior history of annual vaccination with the Merial Purevax® ferret rCDV vaccine. Serum neutralization (SN) to CDV was measured prior to initial vaccination with the rCDV Recombitek vaccine followed by a booster vaccination at 4-6 wk. Final SN titers were obtained at 13 wk post initial vaccination. The maned wolves developed no observable adverse side effects through the study. Pre-Recombitek vaccination SN titers ranged from negative to 1: 8. Postvaccination CDV titers ranged from negative to 1: 8, and were therefore below the range of that considered protective in domestic dogs.

TIGER (PANTHERA TIGRIS) AND DOMESTIC CAT (FELIS CATUS) IMMUNE RESPONSES TO CANARYPOX-VECTORED CANINE DISTEMPER VACCINATION.

Two methods for delivering a canarypox-vectored canine distemper vaccine to tigers (Panthera tigris) and domestic cats (Felis catus) were investigated. Eight tigers were divided randomly into two vaccination groups: subcutaneous injection or topical tonsillar application. Each tiger received 2 ml of canine distemper virus (CDV) vaccine (Merial Ferret Distemper Vaccine). Blood was collected from tigers on days 0, 21, 35 or 37, and 112 post-initial vaccination (PIV). Domestic cats were divided randomly into four treatment groups: saline injection (negative controls), low- and high-dose oral, and subcutaneous vaccinates. Blood was collected from domestic cats on days 0, 7, 21, and 28 and 165 or 208 PIV. Sera were tested for CDV antibodies by virus neutralization. All individuals were seronegative at the beginning of the study. One tiger vaccinated subcutaneously developed a titer of 32 by day 35, which reduced to 16 by day 112. Another tiger vaccinated by tonsillar application developed a titer of 8 on day 112. All other tigers remained seronegative. Cats that received saline injection or oral vaccination remained seronegative at each sampling time. Domestic cats vaccinated subcutaneously developed titers ranging from 4 to >128 by day 28, and those re-bled at day 166 had titers of 16 or 64. The disparity in response between domestic cats and tigers may be due to species differences or it may represent a dose-dependent effect. Subcutaneous vaccination with canarypox-vectored Purevax Ferret Distemper® is safe and elicits persistent antibody titers in domestic cats vaccinated parenterally.

RED PANDAS' (AILURUS FULGENS) SEROLOGICAL RESPONSE TO CANARYPOX-VECTORED CANINE DISTEMPER VACCINES.

Red pandas (Ailurus fulgens) are susceptible to canine distemper, with a number of reported vaccine-induced canine distemper cases. Canarypox-vectored recombinant canine distemper vaccines (PureVax Ferret Distemper [PFD] and Recombitek CDV [rCDV]) provide protection without inoculating a live distemper virus, but there are currently no published data regarding these vaccines' safety and efficacy in red pandas. One hundred twenty-two serum samples were collected from 50 captive red pandas and analyzed for antibodies to canine distemper. All naïve red pandas (n = 20) had negative titers. Naïve pandas receiving two PFD vaccinations had either negative or intermediate titers (n = 4). In contrast, naïve pandas receiving a series of two or three rCDV vaccinations (n = 14) had greater antibody responses. Red pandas vaccinated with PFD >12 mo since their last vaccination and a rCDV booster vaccination showed the highest titers observed. We recommend red pandas be administered a series of at least three recombinant vaccine (PDF or rDCV) vaccinations, followed by annual booster vaccinations.

Evaluation of Targeted Next-Generation Sequencing for Detection of Bovine Pathogens in Clinical Samples.

The laboratory diagnosis of infectious diseases, especially those caused by mixed infections, is challenging. Routinely, it requires submission of multiple samples to separate laboratories. Advances in next-generation sequencing (NGS) have provided the opportunity for development of a comprehensive method to identify infectious agents. This study describes the use of target-specific primers for PCR-mediated amplification with the NGS technology in which pathogen genomic regions of interest are enriched and selectively sequenced from clinical samples. In the study, 198 primers were designed to target 43 common bovine and small-ruminant bacterial, fungal, viral, and parasitic pathogens, and a bioinformatics tool was specifically constructed for the detection of targeted pathogens. The primers were confirmed to detect the intended pathogens by testing reference strains and isolates. The method was then validated using 60 clinical samples (including tissues, feces, and milk) that were also tested with other routine diagnostic techniques. The detection limits of the targeted NGS method were evaluated using 10 representative pathogens that were also tested by quantitative PCR (qPCR), and the NGS method was able to detect the organisms from samples with qPCR threshold cycle (CT ) values in the 30s. The method was successful for the detection of multiple pathogens in the clinical samples, including some additional pathogens missed by the routine techniques because the specific tests needed for the particular organisms were not performed. The results demonstrate the feasibility of the approach and indicate that it is possible to incorporate NGS as a diagnostic tool in a cost-effective manner into a veterinary diagnostic laboratory.

Phylogenetic analysis of the wild-type strains of canine distemper virus circulating in the United States.

BACKGROUND: Canine distemper (CD) is a highly contagious, systemic, viral disease of dogs seen worldwide. Despite intensive vaccination in developed countries, recent reports suggest both the re-emergence and increased activity of Canine distemper virus (CDV) worldwide, including the United States. CDV is an RNA virus of the genus Morbillivirus within the family Paramyxoviridae. Viral genomic RNA encodes six structural proteins. Of the six structural proteins, the hemagglutinin (H) gene has the greatest genetic variation and is therefore a suitable target for molecular epidemiological studies. The majority of neutralizing epitopes are found on the H protein, making this gene also important for evaluation of changes over time that may result in antigenic differences among strains. The aim of this study was to determine the phylogenetic relationship of CDV strains circulating in the US. METHODS: Fifty-nine positive canine distemper virus samples collected from dogs from different regions and states from 2014 to 2017 were sequenced with a targeted next-generation sequencing (NGS) method. The sequences of the H, F, and P genes and the matrix-fusion (M-F) intergenic region of the amplified CDVs were analyzed individually. RESULTS: Sequence analysis of the H gene revealed that there are at least 3 different lineages of CDV currently circulating in the US. These lineages include America-3 (Edomex), America-4, and a clade that was previously reported in association with an outbreak in Wyoming, which was linked to a domestic dog-breeding facility in Kansas in 2010. These lineages differ from the historically identified lineages in the US, including America-1, which contains the majority of the vaccine strains. Genetic differences may result in significant changes to the neutralizing epitopes that consequently may lead to vaccine failure. Phylogenetic analyses of the nucleotide sequences obtained in this study of the F and P genes and the M-F intergenic region with sequences from the GenBank database produced similar findings to the H gene analysis. CONCLUSIONS: The CDV lineages currently circulating in the US differ from the historically identified lineages America-1. Continuous surveillance is required for monitoring circulating CDV strains in the US, to prevent potential vaccine breakthrough events.

SEROSURVEY, HEMATOLOGY, AND CAUSES OF MORTALITY OF FREE-RANGING AMERICAN MARTENS ( MARTES AMERICANA) IN MICHIGAN.

To better understand the clinical pathology, diseases, and causes of mortality of reintroduced American martens ( Martes americana) in Michigan, a study was conducted from 2011 to 2015 in the Upper and Lower Peninsulas of Michigan. Samples obtained from live trapping ( n = 58) or harvested carcasses ( n = 34) were serologically tested for select pathogens. Antibodies against Toxoplasma gondii and canine distemper virus were detected in 58 and 3.4% of samples, respectively. All samples were seronegative for Leptospira spp. and negative for Dirofilaria immitis antigen. Urine samples tested for Leptospira spp. via immunofluorescent antibody assay ( n = 7), polymerase chain reaction ( n = 6) , or both ( n = 3) were all negative. Parvovirus DNA was detected in 9.1% of small intestine samples ( n = 22) collected from carcasses and in 3.7% of fecal samples ( n = 27) collected during live trapping. Complete blood counts ( n = 64) and serum biochemistries ( n = 63) were obtained from 49 live-trapped martens. Biochemical parameters found to be significantly different ( P < 0.05) between genders were calcium, creatinine, glucose, and phosphorus. There was no significant difference between genders for any hematologic parameter. Significant differences ( P < 0.05) between summer and winter seasons were found in total estimated white blood cell count, neutrophils, lymphocytes, monocytes, alkaline phosphatase, bicarbonate, calcium, creatinine, globulin, glucose, phosphorus, potassium, sodium, and total protein. There was no significant difference in blood cell count or serum biochemistry values between radio-collared ( n = 17) and noncollared ( n = 47) martens. Animals seropositive for T. gondii were found to have significantly higher ( P < 0.05) eosinophil and globulin levels than seronegative animals. The primary natural cause for mortality of radio-collared American martens was predation. Histologic examinations revealed a high percentage (60%) of martens with verminous or granulomatous pneumonia.

SEROLOGIC RESPONSE TO CANINE DISTEMPER VACCINATION IN CAPTIVE LINNAEUS'S TWO-TOED SLOTHS ( CHOLOEPUS DIDACTYLUS) AFTER A FATAL CANINE DISTEMPER VIRUS OUTBREAK.

Canine distemper virus (CDV) affects many wild and captive, nondomestic species worldwide but has not been previously reported in Xenarthra. Paucity of information on vaccination safety and efficacy presents challenges for disease prevention in captive collections. CDV infections and subsequent mortalities in five captive Linnaeus's two-toed sloths ( Choloepus didactylus) in eastern Tennessee are reported. Clinical signs included oculonasal discharge, oral ulcerations, and diarrhea, and the diagnosis was confirmed by necropsy, histopathology, immunohistochemistry, virus isolation, and polymerase chain reaction. Viral sequencing identified the strain to be consistent with a new CDV lineage currently affecting domestic dogs and wildlife in Tennessee. Seven sloths were examined and vaccinated using a recombinant CDV vaccine on days 0 and 21. Subsequent blood samples showed increased titers in 3/4 sloths. Based on the outbreak and serologic findings postvaccination without adverse effects, the authors recommend recombinant CDV vaccination in sloths exposed to known carriers of CDV.

Ranavirus could facilitate local extinction of rare amphibian species.

There is growing evidence that pathogens play a role in population declines and species extinctions. For small populations, disease-induced extinction may be especially probable. We estimated the susceptibility of two amphibian species of conservation concern (the dusky gopher frog [Lithobates sevosus] and boreal toad [Anaxyrus boreas boreas]) to an emerging pathogen (ranavirus) using laboratory challenge experiments, and combined these data with published demographic parameter estimates to simulate the potential effects of ranavirus exposure on extinction risk. We included effects of life stage during pathogen exposure, pathogen exposure interval, hydroperiod of breeding habitat, population carrying capacity, and immigration in simulations. We found that both species were highly susceptible to ranavirus when exposed to the pathogen in water at environmentally relevant concentrations. Dusky gopher frogs experienced 100 % mortality in four of six life stages tested. Boreal toads experienced 100 % mortality when exposed as tadpoles or metamorphs, which were the only life stages tested. Simulations showed population declines, greater extinction probability, and faster times to extinction with ranavirus exposure. These effects were more evident with more frequent pathogen exposure intervals and lower carrying capacity. Immigration at natural rates did little to mitigate effects of ranavirus exposure unless immigration occurred every 2 years. Our results demonstrate that disease-induced extinction by emerging pathogens, such as ranavirus, is possible, and that threat may be especially high for species with small population sizes. For the species in this study, conservation organizations should incorporate ranavirus surveillance into monitoring programs and devise intervention strategies in the event that disease outbreaks occur.

EVALUATION OF TWO CANINE DISTEMPER VIRUS VACCINES IN CAPTIVE TIGERS (PANTHERA TIGRIS).

Canine distemper virus (CDV) has caused clinical disease and death in nondomestic felids in both captive settings and in the wild. Outbreaks resulting in high mortality rates in tigers (Panthera tigris) have prompted some zoos to vaccinate tigers for CDV. In this study, six tigers received a recombinant canarypox-vectored CDV vaccine (1 ml s.c.) and were revaccinated with 3 ml s.c. (mean) 39 days later. Blood collection for CDV antibody detection via serum neutralization was performed on (mean) days 0, 26, and 66 post-initial vaccination. No tigers had detectable antibodies at days 0 or 26, and only two tigers had low (16 and 32) antibody titers at day 66. Eight additional tigers received a live, attenuated CDV vaccine (1 ml s.c.) on day 0 and were revaccinated with 1 ml s.c. (mean) 171 days later. Blood collection for CDV antibody detection via serum neutralization was performed on (mean) days 0, 26, 171, and 196. Seven of eight tigers receiving the live, attenuated vaccine had no detectable titers prior to vaccination, but all animals had titers of >128 (range 128-1,024) at day 26. At 171 days, all tigers still had detectable titers (geometric mean 69.8, range 16-256), and at 196 days (2 wk post-revaccination) all but two showed an increase to >128 (range 32-512). To determine safety, an additional 41 tigers were vaccinated with 2 ml of a recombinant vaccine containing only CDV components, and an additional 38 tigers received 1 ml of the live, attenuated vaccine, administered either subcutaneously or intramuscularly; no serious adverse effects were noted. Although both vaccines appear safe, the live, attenuated vaccine produced a stronger and more consistent serologic response in tigers.

CANINE DISTEMPER VIRUS ANTIBODY TITERS IN DOMESTIC CATS AFTER DELIVERY OF A LIVE ATTENUATED VIRUS VACCINE.

Three methods for delivering a live attenuated canine distemper virus (CDV) vaccine to domestic cats ( Felis catus ) were investigated, as models for developing vaccination protocols for tigers (Panthera tigris). Twenty domestic cats were randomly divided into four treatment groups: saline injection (negative controls); and oral, intranasal, and subcutaneous vaccinates. Cats were injected with saline or a CDV vaccine (Nobivac DP, Merck) at wk 0 and 4. Blood and nasal swabs were collected at wk 0 (prior to the initial vaccination) and weekly thereafter for 9 wk. Urine samples were collected on wk 1 to 9 after initial vaccination. Forty-nine weeks following the initial vaccination series, three cats from the subcutaneous group and three cats from the intranasal group were revaccinated. Blood was collected immediately prior, and 7 and 21 days subsequent to revaccination. Nasal swabs and urine samples were collected from each cat prior to wk 49 revaccination and daily for 7 days thereafter. Nasal swabs and urine were analyzed by quantitative PCR for vaccine virus presence. Sera were tested for CDV antibodies by virus neutralization. All cats were sero-negative for CDV antibodies at the beginning of the study, and saline-injected cats remained sero-negative throughout the study. A dramatic anamnestic response was seen following wk 4 subcutaneous vaccinations, with titers peaking at wk 6 (geometric mean = 2,435.5). Following wk 49 revaccination, subcutaneous vaccinates again mounted impressive titers (wk 52 geometric mean = 2,048). Revaccination of the intranasal group cats at wk 49 produced a small increase in titers (wk 52 geometric mean = 203). CDV viral RNA was detected in six nasal swabs but no urine samples, demonstrating low viral shedding postvaccination. The strong antibody response to subcutaneous vaccination and the lack of adverse effects suggest this vaccine is safe and potentially protective against CDV infection in domestic cats.

Sequencing of emerging canine distemper virus strain reveals new distinct genetic lineage in the United States associated with disease in wildlife and domestic canine populations.

BACKGROUND: Recent outbreaks of canine distemper have prompted examination of strains from clinical samples submitted to the University of Tennessee College of Veterinary Medicine (UTCVM) Clinical Virology Lab. We previously described a new strain of CDV that significantly diverged from all genotypes reported to date including America 2, the genotype proposed to be the main lineage currently circulating in the US. The aim of this study was to determine when this new strain appeared and how widespread it is in animal populations, given that it has also been detected in fully vaccinated adult dogs. Additionally, we sequenced complete viral genomes to characterize the strain and determine if variation is confined to known variable regions of the genome or if the changes are also present in more conserved regions. METHODS: Archived clinical samples were genotyped using real-time RT-PCR amplification and sequencing. The genomes of two unrelated viruses from a dog and fox each from a different state were sequenced and aligned with previously published genomes. Phylogenetic analysis was performed using coding, non-coding and genome-length sequences. Virus neutralization assays were used to evaluate potential antigenic differences between this strain and a vaccine strain and mixed ANOVA test was used to compare the titers. RESULTS: Genotyping revealed this strain first appeared in 2011 and was detected in dogs from multiple states in the Southeast region of the United States. It was the main strain detected among the clinical samples that were typed from 2011-2013, including wildlife submissions. Genome sequencing demonstrated that it is highly conserved within a new lineage and preliminary serologic testing showed significant differences in neutralizing antibody titers between this strain and the strain commonly used in vaccines. CONCLUSION: This new strain represents an emerging CDV in domestic dogs in the US, may be associated with a stable reservoir in the wildlife population, and could facilitate vaccine escape.

Rapid and sensitive detection of canine distemper virus by one-tube reverse transcription-insulated isothermal polymerase chain reaction.

BACKGROUND: Canine distemper virus (CDV) has been associated with outbreaks of canine infectious respiratory disease in shelters and boarding kennel environments. POCKITTM Nucleic Acid Analyzer is a field-deployable device capable of generating automatically interpreted insulated isothermal polymerase chain reaction (iiPCR) results from extracted nucleic acid within one hour. In this study, reverse transcription iiPCR (RT-iiPCR) was developed to facilitate point-of-need diagnosis of CDV infection. RESULTS: Analytical sensitivity (limit of detection 95%) of the established CDV RT-iiPCR was about 11 copies of in vitro transcribed RNA per reaction. CDV RT-iiPCR generated positive signals from CDV, but not Bordetella bronchiseptica, canine parvovirus, canine herpesvirus, canine adenovirus 2, canine influenza virus (subtype H3N8), canine parainfluenza virus, and canine respiratory coronavirus. To evaluate accuracy of the established reaction in canine distemper clinical diagnosis, 110 specimens from dogs, raccoons, and foxes suspected with CDV infection were tested simultaneously by CDV RT-iiPCR and real-time RT-PCR. CDV RT-iiPCR demonstrated excellent sensitivity (100%) and specificity (100%), compared to real-time RT-PCR. CONCLUSIONS: The results indicated an excellent correlation between RT-iiPCR and a reference real time RT-PCR method. Working in a lyophilized format, the established method has great potential to be used for point-of-care diagnosis of canine distemper in animals, especially in resource-limited facilities.

High susceptibility of the endangered dusky gopher frog to ranavirus.

Amphibians are one of the most imperiled vertebrate groups, with pathogens playing a role in the decline of some species. Rare species are particularly vulnerable to extinction because populations are often isolated and exist at low abundance. The potential impact of pathogens on rare amphibian species has seldom been investigated. The dusky gopher frog Lithobates sevosus is one of the most endangered amphibian species in North America, with 100-200 individuals remaining in the wild. Our goal was to determine whether adult L. sevosus were susceptible to ranavirus, a pathogen responsible for amphibian die-offs worldwide. We tested the relative susceptibility of adult L. sevosus to ranavirus (103 plaque-forming units) isolated from a morbid bullfrog via 3 routes of exposure: intra-coelomic (IC) injection, oral (OR) inoculation, and water bath (WB) exposure. We observed 100% mortality of adult L. sevosus in the IC and WB treatments after 10 and 19 d, respectively. Ninety-five percent mortality occurred in the OR treatment over the 28 d evaluation period. No mortality was observed in the control treatment after 28 d. Our results indicate that L. sevosus is susceptible to ranavirus, and if adults in the wild are exposed to this pathogen, significant mortality could occur. Additionally, our study demonstrates that some adult amphibian species can be very susceptible to ranavirus, which has been often overlooked in North American studies. We recommend that conservation planners consider testing the susceptibility of rare amphibian species to ranavirus and that the adult age class is included in future challenge experiments.