Nasal bots carry relevant titers of CWD prions in naturally infected white-tailed deer.

Chronic wasting disease (CWD) is a prion disease affecting farmed and free-ranging cervids. CWD is rapidly expanding across North America and its mechanisms of transmission are not completely understood. Considering that cervids are commonly afflicted by nasal bot flies, we tested the potential of these parasites to transmit CWD. Parasites collected from naturally infected white-tailed deer were evaluated for their prion content using the protein misfolding cyclic amplification (PMCA) technology and bioassays. Here, we describe PMCA seeding activity in nasal bot larvae collected from naturally infected, nonclinical deer. These parasites efficiently infect CWD-susceptible mice in ways suggestive of high infectivity titers. To further mimic environmental transmission, bot larvae homogenates were mixed with soils, and plants were grown on them. We show that both soils and plants exposed to CWD-infected bot homogenates displayed seeding activity by PMCA. This is the first report describing prion infectivity in a naturally occurring deer parasite. Our data also demonstrate that CWD prions contained in nasal bots interact with environmental components and may be relevant for disease transmission.

Characterization of the complete mitochondrial genomes of the zoonotic parasites Bolbosoma nipponicum and Corynosoma villosum (Acanthocephala: Polymorphida) and the molecular phylogeny of the order Polymorphida.

Acanthocephalans of the order Polymorphida mainly parasitic in birds and mammals, are of veterinary, medical and economic importance. However, the evolutionary relationships of its 3 families (Centrorhynchidae, Polymorphidae and Plagiorhynchidae) remain under debate. Additionally, some species of Polymorphida (i.e. Bolbosoma spp. and Corynosoma spp.) are recognized as zoonotic parasites, associated with human acanthocephaliasis, but the mitochondrial genomes for representatives of Bolbosoma and Corynosoma have not been reported so far. In the present study, the complete mitochondrial genomes B. nipponicum and C. villosum (Acanthocephala: Polymorphidae) are reported for the first time, which are 14 296 and 14 241 bp in length, respectively, and both contain 36 genes [including 12 PCGs, 22 tRNA genes and 2 rRNA genes] and 2 non-coding regions (NCR1 and NCR2). The gene arrangement of some tRNAs in the mitogenomes of B. nipponicum and C. villosum differs from that found in all other acanthocephalans, except Polymorphus minutus. Phylogenetic results based on concatenated amino acid (AA) sequences of the 12 protein-coding genes (PCGs) strongly supported that the family Polymorphidae is a sister to the Centrorhynchidae rather than the Plagiorhynchidae, and also confirmed the sister relationship of the genera Bolbosoma and Corynosoma in the Polymorphidae based on the mitogenomic data for the first time. Our present findings further clarified the phylogenetic relationships of the 3 families Plagiorhynchidae, Centrorhynchidae and Polymorphidae, enriched the mitogenome data of the phylum Acanthocephala (especially the order Polymorphida), and provided the resource of genetic data for diagnosing these 2 pathogenic parasites of human acanthocephaliasis.

Review of metazoan parasites of the northern fur seal (Callorhinus ursinus) and the analysis of the gastrointestinal helminth community of the population on St. Paul Island, Alaska.

The northern fur seal (NFS), Callorhinus ursinus (Mammalia: Otariidae), is a marine mammal species included into the IUCN Red List as the vulnerable species which population is dramatically declining. A significant amount of parasitological data collected previously and our recent data allowed us to clarify the list of NFS metazoan parasites and to perform a comprehensive analysis of the gastrointestinal helminth community. Gastrointestinal tracts from 756 NFSs (3- to 4-year-old males) were collected during the annual Aleut subsistence harvests in July-August of 2011-2014 from five separate rookeries on St. Paul Island, Alaska. Totally, 27,625 specimens of helminths and approximately 1000 nasal mites were collected and identified. Detailed analysis of the previously published and newly obtained data revealed 32 species of metazoan parasites, including trematodes (6 species), cestodes (4), nematodes (9), acanthocephalans (9) and arthropods (4). The gastrointestinal helminth community of newly studied NFSs comprised 19 species including trematodes (4), cestodes (3), nematodes (5) and acanthocephalans (7). Temporal changes in the helminth community structure were small but statistically significant. Gastrointestinal helminth infracommunities comprised from 1 to 10 species (average of 4). Small but significant correlation was found between the abundances of acanthocephalans (Corynosoma similis and C. strumosum), nematodes (Contracaecum osculatum, Pseudoterranova spp.) and cestode Diphyllobothrium tetrapterum.

Multifocal Necrotizing Myopathy in Northern Elephant Seal ( Mirounga angustirostris) Pups, San Miguel Island, California.

A field study addressing causes of mortality in freshly dead northern elephant seals ( Mirounga angustirostris, Gill, 1866) was conducted on San Miguel Island, California, in February 2015. Necropsies were performed on 18 pups ranging in age from stillbirths to approximately 7 to 8 weeks. The primary gross diagnoses in these pups included trauma, myopathy, starvation/emaciation, infections, congenital anomalies, and perinatal mortality. However, 6 (33%) had a previously unrecognized myopathy characterized by multiple white streaks that were most obvious within the inner layer of the abdominal wall and the small innermost ventral intercostal muscles. Following histological examination, 2 more pups from San Miguel Island and 6 pups from The Marine Mammal Center (Sausalito, California) were found to have similar lesions. Histologically, the lesions within the skeletal muscles were characterized by a multifocal polyphasic, mild to severe, acute to subacute necrotizing myopathy with mineralization. Acute necrosis and degeneration characterized by pyknotic nuclei, eosinophilic cytoplasm and cytoplasmic vacuolization were found in smooth muscle myocytes within the urinary bladder and digestive system. Degeneration of myocytes was present in the tunica media of a few small- to medium-sized vessels and was characterized by a vacuolar degeneration and occasionally necrosis. This condition has been termed multifocal necrotizing myopathy. A cause of this myopathy was not identified.

Morphological and molecular evidence for synonymy of Corynosoma obtuscens Lincicome, 1943 with Corynosoma australe Johnston, 1937 (Acanthocephala: Polymorphidae).

Corynosoma obtuscens Lincicome, 1943 (Acanthocephala: Polymorphidae) is synonymised with Corynosoma australe Johnston, 1937 based on combined morphological and molecular evidence. Morphological comparison of C. obtuscens (24 males and 27 females) collected from a California sea lion Zalophus californianus (Lesson) in California, USA, with the type-specimens of C. obtuscens and C. australe, and with published data on C. australe collected from different hosts and regions showed no significant differences. The levels of genetic divergence in the cox1 sequences obtained from C. obtuscens from a California sea lion in the present study and C. australe from otariid seals from Argentina and penguins from Brazil ranged between 1.4-1.6% and was considered to represent intraspecific variability. Additionally, cox1 sequences were generated for Andracantha phalacrocoracis (Yamaguti, 1939), Corynosoma semerme (Forssell, 1904), C. strumosum (Rudolphi, 1802), C. validum Van Cleave, 1953 and C. villosum Van Cleave, 1953. Our results revealed inconsistency in the identification of material used as a source of the previously published sequence data for C. obtuscens and C. magdaleni Montreuil, 1958.

Tonsil biopsy to detect chronic wasting disease in white-tailed deer (Odocoileus virginianus) by immunohistochemistry.

Chronic wasting disease (CWD) continues to spread in wild and farmed cervid populations. Early antemortem CWD testing of farmed cervids is of considerable interest to producers and regulatory agencies as a tool to combat this spread. The tissues accessible for antemortem sampling are limited and include biopsy of the tonsil and recto-anal mucosa-associated lymphoid tissue (RAMALT). The sensitivity to detect CWD by immunohistochemistry (IHC)-the regulatory gold standard-using biopsy samples of RAMALT from naturally infected white-tailed deer (WTD) has been determined by several studies. However, similar information is lacking for tonsil biopsy. In this study, two-bite tonsil biopsies from 79 naturally infected farmed WTD were used to determine the diagnostic sensitivity of tonsil IHC compared to the official CWD status based on results from the medial retropharyngeal lymph nodes and obex. IHC detection of CWD by tonsil biopsy was compared to the result and follicle metrics from the contralateral whole tonsil. The sensitivity of two-bite tonsil biopsy for detecting CWD by IHC was 72% overall. When the stage of infection was considered, the sensitivity was 92% for deer in late preclinical infection but only 55% for early preclinical infection. For deer with early preclinical infection, the sensitivity for deer homozygous for the prion protein gene (PRNP) coding for glycine at codon 96 (GG) was 66% but only 30% when heterozygous for the serine substitution (GS). The results indicate that the sensitivity of two-bite tonsil biopsy in WTD, and consequently its potential utility as an antemortem diagnostic, is limited during early infection, especially in WTD heterozygous for the serine substitution at PRNP codon 96.

Transmissibility of caprine scrapie in ovine transgenic mice.

BACKGROUND: The United States control program for classical ovine scrapie is based in part on the finding that infection is typically spread through exposure to shed placentas from infected ewes. Transmission from goats to sheep is less well described. A suitable rodent model for examining the effect of caprine scrapie isolates in the ovine host will be useful in the ovine scrapie eradication effort. In this study, we describe the incubation time, brain lesion profile, glycoform pattern and PrPSc distribution patterns in a well characterized transgenic mouse line (Tg338) expressing the ovine VRQ prion allele, following inoculation with brain from scrapie infected goats. RESULTS: First passage incubation times of caprine tissue in Tg338 ovinized mice varied widely but second passage intervals were shorter and consistent. Vacuolation profiles, glycoform patterns and paraffin-embedded tissue blots from terminally ill second passage mice derived from sheep or goat inocula were similar. Proteinase K digestion products of murine tissue were slightly smaller than the original ruminant inocula, a finding consistent with passage of several ovine strains in previous reports. CONCLUSIONS: These findings demonstrate that Tg338 mice propagate prions of caprine origin and provide a suitable baseline for examination of samples identified in the expanded US caprine scrapie surveillance program.

Update on the prevalence of the hookworm, Uncinaria lucasi, in northern fur seals (Callorhinus ursinus) on St. Paul Island, Alaska, 2011.

Prevalence of hookworms (Uncinaria lucasi Stiles, 1901) was determined in the northern fur seal (Callorhinus ursinus Linnaeus, 1758) on St. Paul Island (SPI), Alaska in July and August, 2011. Three of 61 (4.9%) dead pups harbored 1 to 13 adult hookworms each in their intestines. Parasitic larvae (L(3)) of hookworms were recovered from the blubber of 4 of 133 (3%) of subadult males (SAMs) examined. One parasitic L(3) was detected from each infected SAM. Adult U. lucasi (n = 3) were found in the intestine of 1 of 105 SAMs examined (0.95%). This is the first documented finding of adult U. lucasi in SAMs of the northern fur seals. Continued low prevalence of hookworms the last several years parallels the tremendous decline in the number of fur seals on SPI over a similar time period.

Morphology, molecular characterization and phylogeny of Bolbosoma nipponicum Yamaguti, 1939 (Acanthocephala: Polymorphidae), a potential zoonotic parasite of human acanthocephaliasis.

Human acanthocephaliasis is a rare parasitic zoonosis mainly caused by acanthocephalans belonging to the genera Acanthocephalus, Bolbosoma, Corynosoma, Macracanthorhynchus, and Moniliformis. In the present paper, the juveniles of Bolbosoma nipponicum Yamaguti, 1939 collected from the northern fur seal Callorhinus ursinus (Linnaeus) (Mammalia: Carnivora) in Alaska, USA were precisely identified based on morphological characters and genetic data. Their detailed morphology was studied using light and, for the first time, scanning electron microscopy. The molecular characterization of the nuclear genes [small ribosomal subunit (18S) and large ribosomal subunit (28S)] and the mitochondrial cytochrome c oxidase subunit 1 (cox1) sequence data of B. nipponicum are provided for the first time. Moreover, in order to clarify the phylogenetic relationships of the genus Bolbosoma and the other genera in the family Polymorphidae, phylogenetic analyses were performed integrating different nuclear (18S + ITS+28S) and mitochondrial (cox1) sequence data using maximum likelihood (ML) and Bayesian inference (BI). The phylogenetic results showed that Bolbosoma has a sister relationship with Corynosoma, and also revealed that Southwellina is sister to Ibirhynchus + Hexaglandula. Our molecular phylogeny also indicated a possible host-switch pattern during the evolution of the polymorphid acanthocephalans. The ancestors of polymorphid acanthocephalans seem to have originally parasitized fish-eating waterfowl in continental habitats, then extended to fish-eating marine birds in brackish water and marine habitats, and finally, opportunistically infected the marine mammals.

Immunogenicity, Safety, and Anti-Viral Efficacy of a Subunit SARS-CoV-2 Vaccine Candidate in Captive Black-Footed Ferrets (Mustela nigripes) and Their Susceptibility to Viral Challenge.

A preliminary vaccination trial against the emergent pathogen, SARS-CoV-2, was completed in captive black-footed ferrets (Mustela nigripes; BFF) to assess safety, immunogenicity, and anti-viral efficacy. Vaccination and boosting of 15 BFF with purified SARS-CoV-2 S1 subunit protein produced a nearly 150-fold increase in mean antibody titers compared to pre-vaccination titers. Serum antibody responses were highest in young animals, but in all vaccinees, antibody response declined rapidly. Anti-viral activity from vaccinated and unvaccinated BFF was determined in vitro, as well as in vivo with a passive serum transfer study in mice. Transgenic mice that received BFF serum transfers and were subsequently challenged with SARS-CoV-2 had lung viral loads that negatively correlated (p < 0.05) with the BFF serum titer received. Lastly, an experimental challenge study in a small group of BFF was completed to test susceptibility to SARS-CoV-2. Despite viral replication and shedding in the upper respiratory tract for up to 7 days post-challenge, no clinical disease was observed in either vaccinated or naive animals. The lack of morbidity or mortality observed indicates SARS-CoV-2 is unlikely to affect wild BFF populations, but infected captive animals pose a potential risk, albeit low, for humans and other animals.

Review of research on hookworms (Uncinaria lucasi Stiles, 1901) in northern fur seals (Callorhinus ursinus Linnaeus, 1758).

The objective of this article is to review knowledge on the hookworm Uncinaria lucasi Stiles, 1901 in northern fur seals, Callorhinus ursinus Linnaeus, 1758. Emphasis is placed on research on this host-parasite system in the Pribilof Islands, AK, USA where the bulk of the studies has been performed.

OPPORTUNISTIC SURVEILLANCE OF CAPTIVE AND FREE-RANGING BIGHORN SHEEP (OVIS CANADENSIS) IN COLORADO, USA, FOR TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES.

Bighorn sheep (Ovis canadensis) are predicted to have a degree of susceptibility to the transmissible spongiform encephalopathies (TSE) chronic wasting disease and scrapie. We opportunistically screened 127 captive bighorn sheep and 152 free-ranging bighorn sheep in Colorado, US for the presence of TSE over a period of 35 yr. None of the animals demonstrated clinical signs, gross pathology, histopathology, or immunohistochemical staining patterns suggestive of TSE.

Regional and Distant Metastasis of Oral Squamous Cell Carcinoma in a Male Castrated Llama: A Case Report.

A 19-year-old male castrated llama presented with a 3-year history of tooth root abscesses and an osseous mass on the left mandible. Surgical excision of the affected teeth and mass was performed, and histopathologic review indicated that the mass was an oral squamous cell carcinoma. The patient was admitted to the hospital again 4 months later in severe respiratory distress with submandibular edema, and bicavitary effusion. Necropsy results revealed regional and distant metastatic squamous cell carcinoma.

Detection of two dissimilar chronic wasting disease isolates in two captive Rocky Mountain elk (Cervus canadensis) herds.

Chronic wasting disease (CWD) continues to spread in both wild and captive cervid herds in North America and has now been identified in wild reindeer and moose in Norway, Finland and Sweden. There is limited knowledge about the variety and characteristics of isolates or strains of CWD that exist in the landscape and their implications on wild and captive cervid herds. In this study, we evaluated brain samples from two captive elk herds that had differing prevalence, history and timelines of CWD incidence. Site 1 had a 16-year history of CWD with a consistently low prevalence between 5% and 10%. Twelve of fourteen naïve animals placed on the site remained CWD negative after 5 years of residence. Site 2 herd had a nearly 40-year known history of CWD with long-term environmental accrual of prion leading to nearly 100% of naïve animals developing clinical CWD within two to 12 years. Obex samples of several elk from each site were compared for CWD prion strain deposition, genotype in prion protein gene codon 132, and conformational stability of CWD prions. CWD prions in the obex from site 2 had a lower conformational stability than those from site 1, which was independent of prnp genotype at codon 132. These findings suggest the existence of different CWD isolates between the two sites and suggest potential differential disease attack rates for different CWD strains.

Experimental oral transmission of chronic wasting disease to red deer (Cervus elaphus elaphus): early detection and late stage distribution of protease-resistant prion protein.

Chronic wasting disease (CWD), an important emerging prion disease of cervids, is readily transmitted by intracerebral or oral inoculation from deer-to-deer and elk-to-elk, suggesting the latter is a natural route of exposure. Studies of host range susceptibility to oral infection, particularly of those species found in habitats where CWD currently exists are imperative. This report describes the experimental transmission of CWD to red deer following oral inoculation with infectious CWD material of elk origin. At 18 to 20 months post-inoculation, mild to moderate neurological signs and weight loss were observed and animals were euthanized and tested using 3 conventional immunological assays. The data indicate that red deer are susceptible to oral challenge and that tissues currently used for CWD diagnosis show strong abnormal prion (PrP(CWD)) accumulation. Widespread peripheral PrP(CWD) deposition involves lymphoreticular tissues, endocrine tissues, and cardiac muscle and suggests a potential source of prion infectivity, a means of horizontal transmission and carrier state.

Causes of mortality in northern elephant seal pups on San Miguel Island, California.

In February 2015, we conducted a field study of causes of mortality of northern elephant seal (Mirounga angustirostris) pups on San Miguel Island, California. Autopsies were performed on 18 freshly dead pups. Ages of pups ranged from stillborn to 6-8 wk. Gross and histologic lesions included trauma (9 of 18 pups), multifocal necrotizing myopathy (8 of 18), starvation with emaciation (7 of 18), congenital anomalies (3 of 18), bacterial infections (3 of 18), and perinatal mortality (stillbirths and neonates; 2 of 18). Trauma and emaciation or starvation were the most significant contributors to death. Bacterial infections included hemolytic Escherichia coli isolated from the lungs of 2 pups with pneumonia. Additionally, non-hemolytic Streptococcus sp. and hemolytic E. coli were isolated from the liver of an emaciated pup that had mild multifocal suppurative hepatitis. Other lesions, including a previously described necrotizing myopathy, congenital anomalies, and bacterial infections, were detected concurrently in cases with starvation and/or emaciation or trauma.

Complex Virome in a Mesenteric Lymph Node from a Californian Sea Lion (Zalophus Californianus) with Polyserositis and Steatitis.

An emaciated subadult free-ranging California sea lion (Csl or Zalophus californianus) died following stranding with lesions similar to 11 other stranded animals characterized by chronic disseminated granulomatous inflammation with necrotizing steatitis and vasculitis, involving visceral adipose tissues in the thoracic and peritoneal cavities. Histologically, affected tissues had extensive accumulations of macrophages with perivascular lymphocytes, plasma cells, and fewer neutrophils. Using viral metagenomics on a mesenteric lymph node six mammalian viruses were identified consisting of novel parvovirus, polyomavirus, rotavirus, anellovirus, and previously described Csl adenovirus 1 and Csl bocavirus 4. The causal or contributory role of these viruses to the gross and histologic lesions of this sea lion remains to be determined.

Primary pneumonic plague contracted from a mountain lion carcass.

BACKGROUND: Primary pneumonic plague is a rare but often fatal form of Yersinia pestis infection that results from direct inhalation of bacteria and is potentially transmissible from person to person. We describe a case of primary pneumonic plague in a wildlife biologist who was found deceased in his residence 1 week after conducting a necropsy on a mountain lion. METHODS: To determine cause of death, a postmortem examination was conducted, and friends and colleagues were interviewed. Physical evidence was reviewed, including specimens from the mountain lion and the biologist's medical chart, camera, and computer. Human and animal tissues were submitted for testing. Persons in close contact (within 2 meters) to the biologist after he had developed symptoms were identified and offered chemoprophylaxis. RESULTS: The biologist conducted the necropsy in his garage without the use of personal protective equipment. Three days later, he developed fever and hemoptysis and died approximately 6 days after exposure. Gross examination showed consolidation and hemorrhagic fluid in the lungs; no buboes were noted. Plague was diagnosed presumptively by polymerase chain reaction and confirmed by culture. Tissues from the mountain lion tested positive for Y. pestis, and isolates from the biologist and mountain lion were indistinguishable by pulsed-field gel electrophoresis. Among 49 contacts who received chemoprophylaxis, none developed symptoms consistent with plague. CONCLUSIONS: The biologist likely acquired pneumonic plague through inhalation of aerosols generated during postmortem examination of an infected mountain lion. Enhanced awareness of zoonotic diseases and appropriate use of personal protective equipment are needed for biologists and others who handle wildlife.

Antemortem detection of PrPCWD in preclinical, ranch-raised Rocky Mountain elk (Cervus elaphus nelsoni) by biopsy of the rectal mucosa.

Antemortem biopsy of the rectal mucosa was evaluated as a method for the preclinical diagnosis of chronic wasting disease (CWD) in a herd of ranch-raised Rocky Mountain elk (Cervus elaphus nelsoni) quarantined because of exposure to CWD. Biopsy samples were obtained from 41 elk during the winter of 2005-2006 and from 26 elk from that herd still alive and available for testing during the winter of 2006-2007. Samples were examined for PrP(CWD), the protein marker for CWD infection, by immunohistochemistry. PrP(CWD) was detected in follicles of the rectoanal mucosa-associated lymphoid tissue in biopsy samples from 1 elk with clinical signs of chronic wasting disease and 5 clinically normal elk. The diagnosis was confirmed in all 6 animals by postmortem analysis of brain and peripheral lymph nodes. PrP(CWD) was also observed in the submucosal plexus and myenteric plexus of the enteric nervous system, and in close association with nonmyelinated mucosal and submucosal nerve fibers. In antemortem rectal biopsy samples from positive animals, immunostaining was consistently observed in approximately 60% of the mucosa-associated lymphoid tissue follicles if 10 or more total follicles per biopsy were present for evaluation. Most antemortem biopsy samples obtained from elk younger than 6.5 years contained at least 10 follicles per rectal mucosal biopsy. These findings support the analysis of antemortem biopsy of the rectal mucosa samples as part of an integrated strategy to manage chronic wasting disease in Rocky Mountain elk.

Impact of age and sex of Rocky Mountain elk (Cervus elaphus nelsoni) on follicle counts from rectal mucosal biopsies for preclinical detection of chronic wasting disease.

To determine if the number of rectal lymphoid follicles decreases with respect to age and sex relative to diagnosis of chronic wasting disease (CWD), rectal biopsies (n = 1,361) were taken from captive Rocky Mountain elk (Cervus elaphus nelsoni) at 4 ranches in the western United States between 2005 and 2008. Rectal tissues were stained with a monoclonal antibody (F99/97.6.1), which selectively stains the abnormal isoform of the prion protein associated with CWD of elk. The number of lymphoid follicles obtained from typical biopsy tissues decreased with the age of the animal. The acceptable number of lymphoid follicles for detection of CWD was not considered to be a problem in elk up to 8.5 years of age, but in elk over 8.5 years of age, the follicle count was considered to be low. Sex of the animal had no effect on the number of lymphoid follicles observed in each age group. Rectal biopsies were an accurate test to diagnose preclinical stages of CWD in elk but may be best suited to elk that are less then 8.5 years of age.