Highly Pathogenic Avian Influenza A(H5N1) Virus Clade 2.3.4.4b Infections in Wild Terrestrial Mammals, United States, 2022.

We describe the pathology of natural infection with highly pathogenic avian influenza A(H5N1) virus of Eurasian lineage Goose/Guangdong clade 2.3.4.4b in 67 wild terrestrial mammals throughout the United States during April 1‒July 21, 2022. Affected mammals include 50 red foxes (Vulpes vulpes), 6 striped skunks (Mephitis mephitis), 4 raccoons (Procyon lotor), 2 bobcats (Lynx rufus), 2 Virginia opossums (Didelphis virginiana), 1 coyote (Canis latrans), 1 fisher (Pekania pennanti), and 1 gray fox (Urocyon cinereoargenteus). Infected mammals showed primarily neurologic signs. Necrotizing meningoencephalitis, interstitial pneumonia, and myocardial necrosis were the most common lesions; however, species variations in lesion distribution were observed. Genotype analysis of sequences from 48 animals indicates that these cases represent spillover infections from wild birds.

Association of Environmental Factors with Seasonal Intensity of Erysipelothrix rhusiopathiae Seropositivity among Arctic Caribou.

Several caribou (Rangifer tarandus) populations have been declining concurrently with increases in infectious diseases in the Arctic. Erysipelothrix rhusiopathiae, a zoonotic bacterium, was first described in 2015 as a notable cause of illness and death among several Arctic wildlife species. We investigated epidemiologic and environmental factors associated with the seroprevalence of E. rhusiopathiae in the Arctic and found that seropositivity was highest during warmer months, peaking in September, and was highest among adult males. Summer seroprevalence increases tracked with the oestrid index from the previous year, icing and snowing events, and precipitation from the same year but decreased with growing degree days in the same year. Seroprevalence of E. rhusiopathiae varied more during the later years of the study. Our findings provide key insights into the influence of environmental factors on disease prevalence that can be instrumental for anticipating and mitigating diseases associated with climate change among Arctic wildlife and human populations.

Mycoplasma ovipneumoniae in Wildlife Species beyond Subfamily Caprinae.

Elucidating the emergence of Mycoplasma ovipneumoniae-associated respiratory disease in ruminants requires identification of the pathogen host range. This bacterium was thought to be host restricted to subfamily Caprinae, but we describe its identification in healthy moose, caribou, and mule deer and diseased mule and white-tailed deer, all species in subfamily Capreolinae.

First isolation of Brucella pinnipedialis and detection of Brucella antibodies from bearded seals Erignathus barbatus.

Brucella species infecting marine mammals was first reported in 1994 and in the years since has been documented in various species of pinnipeds and cetaceans. While these reports have included species that inhabit Arctic waters, the few available studies on bearded seals Erignathus barbatus have failed to detect Brucella infection to date. We report the first isolation of Brucella pinnipedialis from a bearded seal. The isolate was recovered from the mesenteric lymph node of a bearded seal that stranded in Scotland and typed as ST24, a sequence type associated typically with pinnipeds. Furthermore, serological studies of free-ranging bearded seals in their native waters detected antibodies to Brucella in seals from the Chukchi Sea (1990-2011; 19%) and Svalbard (1995-2007; 8%), whereas no antibodies were detected in bearded seals from the Bering Sea or Bering Strait or from captive bearded seals.

Sarcocystis arctica (Apicomplexa: Sarcocystidae): ultrastructural description and its new host record, the Alaskan wolf (Canis lupus).

Sarcocystis sarcocysts are common in muscles of herbivores but are rare in muscles of carnivores. Here, we report sarcocysts in the muscles of a gray wolf (Canis lupus) from Alaska, USA, for the first time. Sarcocysts extracted from the tongue of the wolf were up to 900 µm long and slender and appeared to have a relatively thin wall by light microscope. By transmission electron microscopy, the sarcocyst wall most closely resembled "type 9c," and had a wavy parasitophorous vacuolar membrane folded as pleomorphic villar protrusions (vp), with anastomoses of tips. The vp and the ground substance (gs) layer were smooth without tubules or granules. The gs was up to 2.0 µm thick. The total width of the wall including vp and the gs was 3.5 µm. The vp were up to 1.5 µm long. Mature sarcocysts contained numerous bradyzoites and few metrocytes. The bradyzoites were 9.5 µm long and 1.5 µm wide, and contained all organelles found in Sarcocystis bradyzoites with at least two rhoptries. Molecular characterization showed the highest identity for 18S rRNA, 28S rRNA, ITS-1, and cox1 sequences of Sarcocystis arctica of the Arctic fox (Vulpes lagopus) from Norway. The ultrastructure of S. arctica from the fox is unknown. Here, we provide ultrastructure of S. arctica from the Alaskan wolf for the first time. The definitive host of S. arctica remains unknown.

Suspected True Hermaphroditism in a Free-ranging Gray Wolf (Canis lupus) in Interior Alaska, USA.

Hermaphroditism or intersexuality occurs endogenously in most mammal species. We document the behavior and physical and anatomic appearance of a suspected true hermaphroditic free-ranging wild gray wolf (Canis lupus) in Denali National Park and Preserve, Alaska, USA.

Evidence of alphaherpesvirus infections in Alaskan caribou and reindeer.

BACKGROUND: The reindeer (Rangifer tarandus tarandus) industry in Alaska began with animals imported from Siberia (Russia) in the 1890's. Cervid herpes virus 2 (CvHV2) is endemic in reindeer in Scandinavia. We sought to determine if the same virus, or similar herpesviruses, were circulating in Alaskan reindeer and caribou (Rangifer tarandus granti). Serum samples from 292 reindeer were collected during annual reindeer handlings (1988-2005) near Nome, Alaska. In 2005, swab samples were collected from 40 calves from this herd, near Nome, Alaska. In 2007, ocular and nasal swab samples were collected from 30 apparently healthy reindeer calves near Wales, Alaska. Samples of plasma and white blood cells were collected from three Alaskan caribou herds, Mulchatna (n = 24), Teshekpuk (n = 34) and the Western Arctic (n = 87) in 2009. RESULTS: Of 292 reindeer samples tested by ELISA for antibodies against alphaherpesvirus (bovine herpesvirus 1 as antigen), seroprevalence was 47% (136/292) and adult reindeer had higher seroprevalence than yearlings. The overall seroprevalence for caribou was 60% (87/145), with no significant differences among caribou herds. A virus neutralization test of 20 samples from both reindeer and caribou showed that ELISA positive samples always neutralized CvHV2 to a greater extent than BoHV1 or elk herpesvirus (ElkHV), indicating that CvHv2 is the most likely virus circulating. PCR of nasal and ocular swabs sampled from 30 reindeer calves in Wales, Alaska (2007) yielded four CvHV2 positive samples. PCR amplicons of the expected size (294 bp) were obtained from 2 of the 36 buffy coats samples from caribou, and the amplicon sequences were consistent with CvHV2. CONCLUSIONS: This study shows that Alaskan reindeer and Caribou are infected with an alphaherpesvirus. Based on sequence similarity, CvHV-2 is the most likely virus. Further studies should be conducted to determine the impact of this infection on the health of these animals.

Multilocus Sequence Typing of Mycoplasma ovipneumoniae Detected in Dall's Sheep (Ovis dalli dalli) and Caribou (Rangifer tarandus grantii) in Alaska, USA.

In 2018, Mycoplasma ovipneumoniae was detected in free-ranging caribou (Rangifer tarandus grantii) and Dall's sheep (Ovis dalli dalli) in Alaska, US. Evaluation of additional nasal swabs and archived tissues for M. ovipneumoniae suggested that this bacterium was widespread geographically and temporally in populations of both species. Multilocus sequence typing of four loci identified a single, novel, apparently stable strain type of M. ovipneumoniae in 11 Dall's sheep and 15 caribou in multiple populations across Alaska sampled over a period of 15 yr (2004-19). This strain type differs from those detected to date from wild or domestic sheep (Ovis aries) or goats (Capra aegagrus hircus) tested in Alaska or the lower 48 states. Although the population health implications of this strain are unknown, it has not been associated with population-wide mortality events. The presence of this strain does not decrease the potential risk from the introduction of a pathogenic M. ovipneumoniae strain associated with severe disease in other wildlife populations; therefore, continued monitoring for signs of disease and additional strains is important.

LABORATORY CONCORDANCE STUDY FOR THE MOLECULAR DETECTION OF MYCOPLASMA OVIPNEUMONIAE.

As part of a respiratory pathogen survey of Alaska wildlife, we conducted a concordance study to assess Mycoplasma ovipneumoniae detection among three different PCR assays using a total of 346 nasal swabs sampled from four species (Dall's sheep, Ovis dalli dalli; mountain goats, Oreamnos americanus; caribou, Rangifer tarandus granti; and moose, Alces alces gigas), and two taxonomic subfamilies (Bovidae subfamily Caprinae and Cervidae subfamily Capreolinae). A federal research laboratory performed two PCR assays (LM40 and intergenic spacer region [IGS]), and a state diagnostic laboratory performed the third (universal Mycoplasma [UM]). Overall concordance was good, ranging from 93% to 99%, which was probably a result of low detection rate of M. ovipneumoniae. Due to differences in positive agreement, the quality of concordance between LM40 and both IGS and UM was considered fair. However, the quality of concordance between IGS and UM was excellent. All three PCR methods detected M. ovipneumoniae in a non-Caprinae species (caribou), and the LM40-PCR assay also detected M. ovipneumoniae in additional Caprinae species. The LM40-PCR assay detected M. ovipneumoniae in a larger number of samples than did the other two assays (IGS, UM). Because of potential differences in detection rates, it is critical to consider test parameters when evaluating a host population for the presence of M. ovipneumoniae.

Adenovirus Hemorrhagic Disease in Moose (Alces americanus gigas) in Alaska, USA.

In 1993, an epizootic of adenovirus hemorrhagic disease (AHD) caused the death of at least 1,000 mule deer (Odocoileus hemionus) in California, US. Since then, numerous cervid species throughout the US have had deaths confirmed to be caused by AHD. In 2015, the death of two captive moose (Alces americanus gigas) calves marked the first recognized AHD-caused deaths in Alaska, a state in which moose are important economically as well as for food security and cultural identity. Both cases were characterized by systemic vasculitis with endothelial cell intranuclear inclusion bodies, pulmonary edema, petechial hemorrhages, and enterotyphlocolitis.

Draft Genome Sequence of a Novel Mycoplasma Species Identified from the Respiratory Tract of an Alaska Moose (Alces alces gigas).

The mycoplasmas represent a large and diverse group of bacteria, many of which are pathogens of humans and animals. Here, we describe a draft genome sequence of a novel Mycoplasma species. This novel Mycoplasma species has potential to cause false-positive PCR results for Mycoplasma ovipneumoniae, a respiratory-associated pathogen of ruminants.

Patterns and processes of pathogen exposure in gray wolves across North America.

The presence of many pathogens varies in a predictable manner with latitude, with infections decreasing from the equator towards the poles. We investigated the geographic trends of pathogens infecting a widely distributed carnivore: the gray wolf (Canis lupus). Specifically, we investigated which variables best explain and predict geographic trends in seroprevalence across North American wolf populations and the implications of the underlying mechanisms. We compiled a large serological dataset of nearly 2000 wolves from 17 study areas, spanning 80° longitude and 50° latitude. Generalized linear mixed models were constructed to predict the probability of seropositivity of four important pathogens: canine adenovirus, herpesvirus, parvovirus, and distemper virus-and two parasites: Neospora caninum and Toxoplasma gondii. Canine adenovirus and herpesvirus were the most widely distributed pathogens, whereas N. caninum was relatively uncommon. Canine parvovirus and distemper had high annual variation, with western populations experiencing more frequent outbreaks than eastern populations. Seroprevalence of all infections increased as wolves aged, and denser wolf populations had a greater risk of exposure. Probability of exposure was positively correlated with human density, suggesting that dogs and synanthropic animals may be important pathogen reservoirs. Pathogen exposure did not appear to follow a latitudinal gradient, with the exception of N. caninum. Instead, clustered study areas were more similar: wolves from the Great Lakes region had lower odds of exposure to the viruses, but higher odds of exposure to N. caninum and T. gondii; the opposite was true for wolves from the central Rocky Mountains. Overall, mechanistic predictors were more informative of seroprevalence trends than latitude and longitude. Individual host characteristics as well as inherent features of ecosystems determined pathogen exposure risk on a large scale. This work emphasizes the importance of biogeographic wildlife surveillance, and we expound upon avenues of future research of cross-species transmission, spillover, and spatial variation in pathogen infection.

Escherichia albertii in wild and domestic birds.

Escherichia albertii has been associated with diarrhea in humans but not with disease or infection in animals. However, in December 2004, E. albertii was found, by biochemical and genetic methods, to be the probable cause of death for redpoll finches (Carduelis flammea) in Alaska. Subsequent investigation found this organism in dead and subclinically infected birds of other species from North America and Australia. Isolates from dead finches in Scotland, previously identified as Escherichia coli O86:K61, also were shown to be E. albertii. Similar to the isolates from humans, E. albertii isolates from birds possessed intimin (eae) and cytolethal distending toxin (cdtB) genes but lacked Shiga toxin (stx) genes. Genetic analysis of eae and cdtB sequences, multilocus sequence typing, and pulsed-field gel electrophoresis patterns showed that the E. albertii strains from birds are heterogeneous but similar to isolates that cause disease in humans.

Establishing a baseline for tick surveillance in Alaska: Tick collection records from 1909-2019.

The expanding geographic ranges of tick species that are known pathogen vectors can have implications for human, domestic animal, and wildlife health. Although Alaska is home to several hard tick species, it has historically been outside of the range of the most common medically important ticks in the contiguous United States and western Canada. To assess the status of tick species establishment in the state and to provide a baseline for tracking future change in the distribution of ticks, we reviewed and compiled historical tick records and summarized recent tick occurrence records collected through the development of the Alaska Submit-A-Tick Program and through tick drag sampling at sentinel sites in southcentral Alaska. Between 1909-2019, there were 1190 tick records representing 4588 individual ticks across 15 species in Alaska. The majority of ticks were species historically found in Alaska: Haemaphysalis leporispalustris, Ixodes angustus, Ixodes auritulus, Ixodes howelli, Ixodes signatus, and Ixodes uriae. Over half of all tick records in the state were collected in the last 10 yr. During this time, the number of tick records and the number of tick species recorded in Alaska each year has increased substantially. Between 2010-2019, there were 611 tick records representing 1921 individual ticks. The most common hosts for reported ticks were domestic animals (n = 343, 56 %) followed by small wild mammals (n = 147, 24 %), humans (n = 49, 8%), and wild birds (n = 31, 5%). Less than 5% of records (n = 25) were of unattached ticks found in the environment. Since 2007, non-native tick species have been documented in the state every year, including Amblyomma americanum, Dermacentor andersoni, Dermacentor occidentalis, Dermacentor variabilis, Ixodes pacificus, Ixodes ricinus, Ixodes scapularis, Ixodes texanus, and Rhipicephalus sanguineus sensu lato (s.l.). Almost half of the records (n = 68, 48 %) of non-native tick species from 2010 to 2019 represented ticks found on a host (usually a dog or a human) that had traveled outside of Alaska in the two weeks prior to collection. However, A. americanum, D. variabilis, I. pacificus, I. texanus, and R. sanguineus s.l. have been found on humans and domestic animals in Alaska without reported recent travel. In particular, there is evidence to suggest that there is local establishment of R. sanguineus s.l. in Alaska. A tick species historically found in the state, I. angustus was frequently found on human and dogs, suggesting a potential role as a bridge vector of pathogens. Given the inconsistency of tick monitoring in Alaska over the past century, it is difficult to draw many conclusions from temporal trends in the data. Continued monitoring through the Alaska Submit-A-Tick Program will allow a more accurate assessment of the changing risk of ticks and tick-borne diseases in the state and provide information for setting clinical and public health guidelines for tick-borne disease prevention.

The biogeography of the caribou lungworm, Varestrongylus eleguneniensis (Nematoda: Protostrongylidae) across northern North America.

Varestrongylus eleguneniensis (Nematoda; Protostrongylidae) is a recently described species of lungworm that infects caribou (Rangifer tarandus), muskoxen (Ovibos moschatus) and moose (Alces americanus) across northern North America. Herein we explore the geographic distribution of V. eleguneniensis through geographically extensive sampling and discuss the biogeography of this multi-host parasite. We analyzed fecal samples of three caribou subspecies (n = 1485), two muskox subspecies (n = 159), and two moose subspecies (n = 264) from across northern North America. Protostrongylid dorsal-spined larvae (DSL) were found in 23.8%, 73.6%, and 4.2% of these ungulates, respectively. A portion of recovered DSL were identified by genetic analyses of the ITS-2 region of the nuclear rDNA or the cytochrome oxidase c subunit I (COI) region of the mtDNA. We found V. eleguneniensis widely distributed among caribou and muskox populations across most of their geographic prange in North America but it was rare in moose. Parelaphostrongylus andersoni was present in caribou and moose and we provide new geographic records for this species. This study provides a substantial expansion of the knowledge defining the current distribution and biogeography of protostrongylid nematodes in northern ungulates. Insights about the host and geographic range of V. eleguneniensis can serve as a geographically extensive baseline for monitoring current distribution and in anticipating future biogeographic scenarios under a regime of accelerating climate and anthropogenic perturbation.

Organochlorine contaminant concentrations in blubber of young Steller sea lion (Eumetopias jubatus) are influenced by region, age, sex, and lipid stores.

Contaminant exposure is particularly important for species and populations of conservation concern, such as the Steller sea lion (Eumetopias jubatus). We used blubber samples (n = 120) to determine organochlorine concentrations, including polychlorinated biphenyls (PCBs) and dichloro-diphenyl-trichloroethane (DDTs), and blood samples (n = 107) to estimate total body lipids based on the hydrogen isotope dilution method. We assessed the influence of age, sex, condition, and geographic area on contaminant concentrations in blubber and contaminant body load. The concentration of ΣPCBs was highest in pups (<6 months) from the Aleutian Islands, and the concentrations in males were higher than females in all regions. The ΣPCBs and ΣDDTs concentrations and loads decreased with increasing mass in pups, however, there were no regional or sex differences in contaminant load. Within each of the five age classes, the concentrations of ΣPCBs and ΣDDTs decreased with increasing mass, but overall these OCs increased with age. Further, accounting for the lipid content, a potential proxy for energy balance, in the animal load reduced or removed the regional and sex effects present in age models for contaminants. We propose, that adjusting OCs concentration by the lipid content of the blubber sample alone may not fully account for the variability in OC concentrations associated with differences in condition or energy states between young Steller sea lions.

Correction: Novel insights into serodiagnosis and epidemiology of Erysipelothrix rhusiopathiae, a newly recognized pathogen in muskoxen (Ovibos moschatus).

[This corrects the article DOI: 10.1371/journal.pone.0231724.].

Novel insights into serodiagnosis and epidemiology of Erysipelothrix rhusiopathiae, a newly recognized pathogen in muskoxen (Ovibos moschatus).

BACKGROUND: Muskoxen are a key species of Arctic ecosystems and are important for food security and socio-economic well-being of many Indigenous communities in the Arctic and Subarctic. Between 2009 and 2014, the bacterium Erysipelothrix rhusiopathiae was isolated for the first time in this species in association with multiple mortality events in Canada and Alaska, raising questions regarding the spatiotemporal occurrence of the pathogen and its potential impact on muskox populations. MATERIALS AND METHODS: We adapted a commercial porcine E. rhusiopathiae enzyme-linked immunosorbent assay to test 958 blood samples that were collected from muskoxen from seven regions in Alaska and the Canadian Arctic between 1976 and 2017. The cut-off between negative and positive results was established using mixture-distribution analysis, a data-driven approach. Based on 818 samples for which a serological status could be determined and with complete information, we calculated trends in sample seroprevalences in population time-series and compared them with population trends in the investigated regions. RESULTS: Overall, 219/818 (27.8%, 95% Confidence Interval: 24.7-31.0) samples were classified as positive for exposure to E. rhusiopathiae. There were large variations between years and regions. Seropositive animals were found among the earliest serum samples tested; 1976 in Alaska and 1991 in Canada. In Alaskan muskoxen, sample seroprevalence increased after 2000 and, in two regions, peak seroprevalences occurred simultaneously with population declines. In one of these regions, concurrent unusual mortalities were observed and E. rhusiopathiae was isolated from muskox carcasses. In Canada, there was an increase in sample seroprevalence in two muskox populations following known mortality events that had been attributed to E. rhusiopathiae. CONCLUSION: Our results indicate widespread exposure of muskoxen to E. rhusiopathiae in western Canada and Alaska. Although not new to the Arctic, we documented an increased exposure to the pathogen in several regions concurrent with population declines. Understanding causes for the apparent increased occurrence of this pathogen and its association with large scale mortality events for muskoxen is critical to evaluate the implications for wildlife and wildlife-dependent human populations in the Arctic.

Mycoplasma ovipneumoniae Associated with Polymicrobial Pneumonia in a Free-Ranging Yearling Barren Ground Caribou (Rangifer tarandus granti) from Alaska, USA.

Mycoplasma ovipneumoniae has been reported in association with respiratory disease in the wild only in members of the subfamily Caprinae of the family Bovidae. We identified M. ovipneumoniae in a cervid: a free-ranging barren ground caribou (Rangifer tarandus granti) yearling with polymicrobial bronchopneumonia.

Determination of Thiafentanil in Plasma Using LC-MS.

A new method of analysis has been developed and validated for the determination of thiafentanil in plasma. After protein precipitation, samples were separated on an XBridge BEH C18 column and quantified using mass spectrometry. The mobile phase was a mixture of water with 0.1% formic acid and acetonitrile with 0.1% formic acid (90:10). The standard curve ranged from 0.1 to 25 ng/mL. Intra- and Inter-assay variability for thiafentanil was less than 10%, and the average recovery was greater than 95%. The lower limit of quantification was 0.1 ng/mL. This is the first validated method for thiafentanil analysis in plasma.