Christine K. Johnson

Prey choice and habitat use drive sea otter pathogen exposure in a resource-limited coastal system.

The processes promoting disease in wild animal populations are highly complex, yet identifying these processes is critically important for conservation when disease is limiting a population. By combining field studies with epidemiologic tools, we evaluated the relationship between key factors impeding southern sea otter (Enhydra lutris nereis) population growth: disease and resource limitation. This threatened population has struggled to recover despite protection, so we followed radio-tagged sea otters and evaluated infection with 2 disease-causing protozoal pathogens, Toxoplasma gondii and Sarcocystis neurona, to reveal risks that increased the likelihood of pathogen exposure. We identified patterns of pathogen infection that are linked to individual animal behavior, prey choice, and habitat use. We detected a high-risk spatial cluster of S. neurona infections in otters with home ranges in southern Monterey Bay and a coastal segment near San Simeon and Cambria where otters had high levels of infection with T. gondii. We found that otters feeding on abalone, which is the preferred prey in a resource-abundant marine ecosystem, had a very low risk of infection with either pathogen, whereas otters consuming small marine snails were more likely to be infected with T. gondii. Individual dietary specialization in sea otters is an adaptive mechanism for coping with limited food resources along central coastal California. High levels of infection with protozoal pathogens may be an adverse consequence of dietary specialization in this threatened species, with both depleted resources and disease working synergistically to limit recovery.

ASSOCIATION OF AN UNUSUAL MARINE MAMMAL MORTALITY EVENT WITH PSEUDO-NITZSCHIA SPP. BLOOMS ALONG THE SOUTHERN CALIFORNIA COASTLINE

During 2002, 2,239 marine mammals stranded in southern California. This unusual marine mammal stranding event was clustered from April to June and consisted primarily of California sea lions (Zalophus californianus) and long-beaked common dolphins (Delphinus capensis) with severe neurologic signs. Intoxication with domoic acid (DA), a marine neurotoxin produced during seasonal blooms of Pseudo-nitzschia spp., was suspected. Definitively linking harmful algal blooms to large-scale marine mammal mortalities presents a substantial challenge, as does determining the geographic extent, species composition, and potential population impacts of marine mammal die-offs. For this reason, time series cross-correlation analysis was performed to test the temporal correlations of Pseudo-nitzschia blooms with strandings occurring along the southern California coastline. Temporal correlations were identified between strandings and blooms for California sea lions, long-beaked common dolphins, and short-beaked common dolphins (Delphinus delphis). Similar correlations were identified for bottlenose dolphins (Tursiops truncatus) and gray whales (Eschrichtius robustus), but small sample sizes for these species made associations more speculative. The timing of the blooms and strandings of marine mammals suggested that both inshore and offshore foraging species were affected and that marine biotoxin programs should include offshore monitoring sites. In addition, California sea lion-strandings appear to be a very sensitive indicator of DA in the marine environment, and their monitoring should be included in public health surveillance plans.

Impact of the California Lead Ammunition Ban on Reducing Lead Exposure in Golden Eagles and Turkey Vultures

Predatory and scavenging birds may be exposed to high levels of lead when they ingest shot or bullet fragments embedded in the tissues of animals injured or killed with lead ammunition. Lead poisoning was a contributing factor in the decline of the endangered California condor population in the 1980s, and remains one of the primary factors threatening species recovery. In response to this threat, a ban on the use of lead ammunition for most hunting activities in the range of the condor in California was implemented in 2008. Monitoring of lead exposure in predatory and scavenging birds is essential for assessing the effectiveness of the lead ammunition ban in reducing lead exposure in these species. In this study, we assessed the effectiveness of the regulation in decreasing blood lead concentration in two avian sentinels, golden eagles and turkey vultures, within the condor range in California. We compared blood lead concentration in golden eagles and turkey vultures prior to the lead ammunition ban and one year following implementation of the ban. Lead exposure in both golden eagles and turkey vultures declined significantly post-ban. Our findings provide evidence that hunter compliance with lead ammunition regulations was sufficient to reduce lead exposure in predatory and scavenging birds at our study sites.

Dead or alive: animal sampling during Ebola hemorrhagic fever outbreaks in humans

There are currently no widely accepted animal surveillance guidelines for human Ebola hemorrhagic fever (EHF) outbreak investigations to identify potential sources of Ebolavirus (EBOV) spillover into humans and other animals. Animal field surveillance during and following an outbreak has several purposes, from helping identify the specific animal source of a human case to guiding control activities by describing the spatial and temporal distribution of wild circulating EBOV, informing public health efforts, and contributing to broader EHF research questions. Since 1976, researchers have sampled over 10,000 individual vertebrates from areas associated with human EHF outbreaks and tested for EBOV or antibodies. Using field surveillance data associated with EHF outbreaks, this review provides guidance on animal sampling for resource-limited outbreak situations, target species, and in some cases which diagnostics should be prioritized to rapidly assess the presence of EBOV in animal reservoirs. In brief, EBOV detection was 32.7% (18/55) for carcasses (animals found dead) and 0.2% (13/5309) for live captured animals. Our review indicates that for the purposes of identifying potential sources of transmission from animals to humans and isolating suspected virus in an animal in outbreak situations, (1) surveillance of free-ranging non-human primate mortality and morbidity should be a priority, (2) any wildlife morbidity or mortality events should be investigated and may hold the most promise for locating virus or viral genome sequences, (3) surveillance of some bat species is worthwhile to isolate and detect evidence of exposure, and (4) morbidity, mortality, and serology studies of domestic animals should prioritize dogs and pigs and include testing for virus and previous exposure.

Further Evidence for Bats as the Evolutionary Source of Middle East Respiratory Syndrome Coronavirus

ABSTRACT The evolutionary origins of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) are unknown. Current evidence suggests that insectivorous bats are likely to be the original source, as several 2c CoVs have been described from various species in the family Vespertilionidae. Here, we describe a MERS-like CoV identified from a Pipistrellus cf. hesperidus bat sampled in Uganda (strain PREDICT/PDF-2180), further supporting the hypothesis that bats are the evolutionary source of MERS-CoV. Phylogenetic analysis showed that PREDICT/PDF-2180 is closely related to MERS-CoV across much of its genome, consistent with a common ancestry; however, the spike protein was highly divergent (46% amino acid identity), suggesting that the two viruses may have different receptor binding properties. Indeed, several amino acid substitutions were identified in key binding residues that were predicted to block PREDICT/PDF-2180 from attaching to the MERS-CoV DPP4 receptor. To experimentally test this hypothesis, an infectious MERS-CoV clone expressing the PREDICT/PDF-2180 spike protein was generated. Recombinant viruses derived from the clone were replication competent but unable to spread and establish new infections in Vero cells or primary human airway epithelial cells. Our findings suggest that PREDICT/PDF-2180 is unlikely to pose a zoonotic threat. Recombination in the S1 subunit of the spike gene was identified as the primary mechanism driving variation in the spike phenotype and was likely one of the critical steps in the evolution and emergence of MERS-CoV in humans. IMPORTANCE Global surveillance efforts for undiscovered viruses are an important component of pandemic prevention initiatives. These surveys can be useful for finding novel viruses and for gaining insights into the ecological and evolutionary factors driving viral diversity; however, finding a viral sequence is not sufficient to determine whether it can infect people (i.e., poses a zoonotic threat). Here, we investigated the specific zoonotic risk of a MERS-like coronavirus (PREDICT/PDF-2180) identified in a bat from Uganda and showed that, despite being closely related to MERS-CoV, it is unlikely to pose a threat to humans. We suggest that this approach constitutes an appropriate strategy for beginning to determine the zoonotic potential of wildlife viruses. By showing that PREDICT/PDF-2180 does not infect cells that express the functional receptor for MERS-CoV, we further show that recombination was likely to be the critical step that allowed MERS to emerge in humans. Global surveillance efforts for undiscovered viruses are an important component of pandemic prevention initiatives. These surveys can be useful for finding novel viruses and for gaining insights into the ecological and evolutionary factors driving viral diversity; however, finding a viral sequence is not sufficient to determine whether it can infect people (i.e., poses a zoonotic threat). Here, we investigated the specific zoonotic risk of a MERS-like coronavirus (PREDICT/PDF-2180) identified in a bat from Uganda and showed that, despite being closely related to MERS-CoV, it is unlikely to pose a threat to humans. We suggest that this approach constitutes an appropriate strategy for beginning to determine the zoonotic potential of wildlife viruses. By showing that PREDICT/PDF-2180 does not infect cells that express the functional receptor for MERS-CoV, we further show that recombination was likely to be the critical step that allowed MERS to emerge in humans.

Spillover and pandemic properties of zoonotic viruses with high host plasticity

Most human infectious diseases, especially recently emerging pathogens, originate from animals, and ongoing disease transmission from animals to people presents a significant global health burden. Recognition of the epidemiologic circumstances involved in zoonotic spillover, amplification, and spread of diseases is essential for prioritizing surveillance and predicting future disease emergence risk. We examine the animal hosts and transmission mechanisms involved in spillover of zoonotic viruses to date, and discover that viruses with high host plasticity (i.e. taxonomically and ecologically diverse host range) were more likely to amplify viral spillover by secondary human-to-human transmission and have broader geographic spread. Viruses transmitted to humans during practices that facilitate mixing of diverse animal species had significantly higher host plasticity. Our findings suggest that animal-to-human spillover of new viruses that are capable of infecting diverse host species signal emerging disease events with higher pandemic potential in that these viruses are more likely to amplify by human-to-human transmission with spread on a global scale.

Novel and Canine Genotypes of Giardia duodenalis in Harbor Seals (Phoca vitulina richardsi)

Abstract Feces of harbor seals (Phoca vitulina richardsi) and hybrid Glaucous-winged/Western gulls (Larus glaucescens/ occidentalis) from Washington States inland marine waters were examined for Giardia spp. and Cryptosporidium spp. to determine whether genotypes carried by these wildlife species were the same as those that commonly infect humans and domestic animals. Using immunomagnetic separation followed by direct fluorescent antibody detection, Giardia spp. cysts were detected in 42% (41/97) of seal fecal samples. Giardia spp.–positive samples came from 90% (9/10) of the sites, and the prevalence of positive seal fecal samples differed significantly among study sites. Fecal samples collected from seal haulout sites with >400 animals were 4.7 times more likely to have Giardia spp. cysts than were samples collected at smaller haulout sites. In gulls, a single Giardia sp. cyst was detected in 4% (3/78) of fecal samples. Cryptosporidium spp. oocysts were not detected in any of the seals or gulls tested. Sequence analysis of a 398 base pair (bp) segment of Giardia duodenalis DNA at the glutamate dehydrogenase (GDH) locus suggested that 11 isolates originating from seals throughout the region were a novel genotype and 3 isolates obtained from a single site in south Puget Sound were the G. duodenalis canine genotype D. Real-time TaqMan polymerase chain reaction (PCR) amplification and subsequent sequencing of a 52 bp small subunit ribosomal DNA region from novel harbor seal genotype isolates showed sequence homology to canine genotypes C and D. Sequence analysis of the 52 bp small subunit ribosomal DNA products from the 3 canine genotype isolates from seals produced mixed sequences that could not be evaluated.

Lead Exposure in Free-Flying Turkey Vultures Is Associated with Big Game Hunting in California

Predatory and scavenging birds are at risk of lead exposure when they feed on animals injured or killed by lead ammunition. While lead ammunition has been banned from waterfowl hunting in North America for almost two decades, lead ammunition is still widely used for hunting big game and small game animals. In this study, we evaluated the association between big game hunting and blood lead concentration in an avian scavenger species that feeds regularly on large mammals in California. We compared blood lead concentration in turkey vultures within and outside of the deer hunting season, and in areas with varying wild pig hunting intensity. Lead exposure in turkey vultures was significantly higher during the deer hunting season compared to the off-season, and blood lead concentration was positively correlated with increasing wild pig hunting intensity. Our results link lead exposure in turkey vultures to deer and wild pig hunting activity at these study sites, and we provide evidence that spent lead ammunition in carrion poses a significant risk of lead exposure to scavengers.

Assessment of clinical pathology and pathogen exposure in sea otters (Enhydra lutris) bordering the threatened population in Alaska.

Northern sea otter (Enhydra lutris kenyoni) abundance has decreased dramatically over portions of southwest Alaska, USA, since the mid-1980s, and this stock is currently listed as threatened under the Endangered Species Act. In contrast, adjacent populations in south central Alaska, USA, and Russia have been stable to increasing during the same period. Sea otters bordering the area classified in the recent decline were live-captured during 2004–2006 at Bering Island, Russia, and the Kodiak Archipelago, Alaska, USA, to evaluate differences in general health and current exposure status to marine and terrestrial pathogens. Although body condition was lower in animals captured at Bering Island, Russia, than it was at Kodiak, USA, clinical pathology values did not reveal differences in general health between the two regions. Low prevalences of antibodies (>5%) were found in Kodiak, USA, and on Bering Island, Russia, to Toxoplasma gondii, Sarcocystis neurona, and Leptospira interrogans. Exposure to phocine herpesvirus-1 was found in both Kodiak, USA (15.2%), and Bering Island, Russia (2.3%). Antibodies to Brucella spp. were found in 28% of the otters tested on Bering Island, Russia, compared with only 2.7% of the samples from Kodiak, USA. Prevalence of exposure to Phocine distemper virus (PDV) was 41% in Kodiak, USA, but 0% on Bering Island, Russia. Archived sera from southwest and south-central Alaska dating back to 1989 were negative for PDV, indicating exposure occurred in sea otters in Kodiak, USA, in recent years. Because PDV can be highly pathogenic in naïve and susceptible marine mammal populations, tissues should be examined to explore the contribution of this virus to otter deaths. Our results reveal an increase in exposure to pathogens in sea otters in Kodiak, Alaska, USA, since the 1990s.

Comparative Analysis of Three Brevetoxin-Associated Bottlenose Dolphin (Tursiops truncatus) Mortality Events in the Florida Panhandle Region (USA)

In the Florida Panhandle region, bottlenose dolphins (Tursiops truncatus) have been highly susceptible to large-scale unusual mortality events (UMEs) that may have been the result of exposure to blooms of the dinoflagellate Karenia brevis and its neurotoxin, brevetoxin (PbTx). Between 1999 and 2006, three bottlenose dolphin UMEs occurred in the Florida Panhandle region. The primary objective of this study was to determine if these mortality events were due to brevetoxicosis. Analysis of over 850 samples from 105 bottlenose dolphins and associated prey items were analyzed for algal toxins and have provided details on tissue distribution, pathways of trophic transfer, and spatial-temporal trends for each mortality event. In 1999/2000, 152 dolphins died following extensive K. brevis blooms and brevetoxin was detected in 52% of animals tested at concentrations up to 500 ng/g. In 2004, 105 bottlenose dolphins died in the absence of an identifiable K. brevis bloom; however, 100% of the tested animals were positive for brevetoxin at concentrations up to 29,126 ng/mL. Dolphin stomach contents frequently consisted of brevetoxin-contaminated menhaden. In addition, another potentially toxigenic algal species, Pseudo-nitzschia, was present and low levels of the neurotoxin domoic acid (DA) were detected in nearly all tested animals (89%). In 2005/2006, 90 bottlenose dolphins died that were initially coincident with high densities of K. brevis. Most (93%) of the tested animals were positive for brevetoxin at concentrations up to 2,724 ng/mL. No DA was detected in these animals despite the presence of an intense DA-producing Pseudo-nitzschia bloom. In contrast to the absence or very low levels of brevetoxins measured in live dolphins, and those stranding in the absence of a K. brevis bloom, these data, taken together with the absence of any other obvious pathology, provide strong evidence that brevetoxin was the causative agent involved in these bottlenose dolphin mortality events.