Joseph K. Gaydos

Densovirus associated with sea-star wasting disease and mass mortality

Significance Sea stars inhabiting the Northeast Pacific Coast have recently experienced an extensive outbreak of wasting disease, leading to their degradation and disappearance from many coastal areas. In this paper, we present evidence that the cause of the disease is transmissible from disease-affected animals to apparently healthy individuals, that the disease-causing agent is a virus-sized microorganism, and that the best candidate viral taxon, the sea star-associated densovirus (SSaDV), is in greater abundance in diseased than in healthy sea stars. Populations of at least 20 asteroid species on the Northeast Pacific Coast have recently experienced an extensive outbreak of sea-star (asteroid) wasting disease (SSWD). The disease leads to behavioral changes, lesions, loss of turgor, limb autotomy, and death characterized by rapid degradation (“melting”). Here, we present evidence from experimental challenge studies and field observations that link the mass mortalities to a densovirus (Parvoviridae). Virus-sized material (i.e., <0.2 μm) from symptomatic tissues that was inoculated into asymptomatic asteroids consistently resulted in SSWD signs whereas animals receiving heat-killed (i.e., control) virus-sized inoculum remained asymptomatic. Viral metagenomic investigations revealed the sea star-associated densovirus (SSaDV) as the most likely candidate virus associated with tissues from symptomatic asteroids. Quantification of SSaDV during transmission trials indicated that progression of SSWD paralleled increased SSaDV load. In field surveys, SSaDV loads were more abundant in symptomatic than in asymptomatic asteroids. SSaDV could be detected in plankton, sediments and in nonasteroid echinoderms, providing a possible mechanism for viral spread. SSaDV was detected in museum specimens of asteroids from 1942, suggesting that it has been present on the North American Pacific Coast for at least 72 y. SSaDV is therefore the most promising candidate disease agent responsible for asteroid mass mortality.

Paratuberculosis in key deer (Odocoileus virginianus clavium).

Paratuberculosis was diagnosed in an endangered Key deer (Odocoileus virginianus clavium) in November 1996. Between 10 April 1997 and 28 September 2000, the Key deer population was monitored for infection with Mycobacterium avium subsp. paratuberculosis by necropsy of available carcasses (n = 170), fecal cultures, and serology. One additional clinically affected Key deer was discovered in July 1998, and M. avium subsp. paratuberculosis was cultured from the feces of one live, asymptomatic deer. The results of this study provided sufficient evidence to consider the Key deer herd infected with M. avium subsp. paratuberculosis at very low prevalence.

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.

INNATE RESISTANCE TO EPIZOOTIC HEMORRHAGIC DISEASE IN WHITE-TAILED DEER

Differences in innate disease resistance at the sub-species level have major implications for wildlife management. Two subspecies of white-tailed deer, Odocoileus virginianus borealis and O. virginianus texanus were infected with epizootic hemorrhagic disease (EHD) viruses. These viruses are highly virulent pathogens of white-tailed deer and are endemic within the range of O. virginianus texanus but not within the range of O. virginianus borealis. Two experimental infections were performed. Five O. virginianus texanus fawns and five O. virginianus borealis fawns were infected with 107.1 median tissue culture infective doses (TCID50) of EHD virus, serotype 1 and five of each subspecies were infected with 107.1 TCID50 of EHD virus, serotype 2. Infections with both EHD virus serotypes caused severe clinical disease and mortality in O. virginianus borealis fawns, whereas disease was mild or nondetectable in O. virginianus texanus fawns. Virus titers and humoral immune response were similar in both subspecies suggesting that differences in innate disease resistance explain the differences seen in clinical disease severity. In white-tailed deer, innate disease resistance may vary at the subspecies level. Should this phenomenon occur in other species, these findings have major implications for managing wildlife populations, both endangered and non-endangered, using tools such as translocation and captive propagation.

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.

AEROBIC BACTERIAL ISOLATIONS FROM HARBOR SEALS (PHOCA VITULINA) STRANDED IN WASHINGTON: 1992–2003

Abstract Bacterial cultures collected over 12 yr from stranded harbor seal (Phoca vitulina) pups and weanlings located in the North Puget Sound and San Juan Islands region of Washington were analyzed retrospectively to determine the most common pathogenic isolates and to describe their antimicrobial resistance patterns. Culture attempts (n = 58) from wounds, umbilici, ears, conjunctiva, nares, oral lesions, and feces yielded 134 pathogenic isolates that represented 17 genera. The majority of isolates were Gram-negative (n = 87; 65%) and of the tested isolates were most susceptible to amikacin (n = 76; 99%) and gentamicin (n = 76; 97%) and least susceptible to ampicillin (n = 76; 26%). Of the Gram-positive isolates tested (n = 29), all were susceptible to amoxicillin/clavulanic acid. The most frequent isolates were Escherichia coli (17%), β-hemolytic Streptococcus spp. (15%), Enterococcus spp. (11%), and Pseudomonas aeruginosa (11%), with all four exhibiting resistance to more than 50% of the antimicrobials tested. The variety of organisms isolated, the variation in either Gram-negative or Gram-positive predominance, and the multiple drug resistance patterns observed suggest that when treating stranded harbor seals, culture and sensitivity testing are warranted and that antibiotic therapy should be based on results.

AVIAN VACUOLAR MYELINOPATHY OUTBREAKS AT A SOUTHEASTERN RESERVOIR

Avian vacuolar myelinopathy (AVM) is a neurologic disease of unknown etiology that affects bald eagles (Haliaeetus leucocephalus), American coots (Fulica americana), and several species of waterfowl. An unidentified neurotoxin is suspected as the cause of AVM, which has been documented at several reservoirs in the southeastern United States. We conducted diagnostic and epidemiologic studies annually during October–March from 1998–2004 at Clarks Hill/Strom Thurmond Lake on the Georgia/South Carolina border to better understand the disease. Avian vacuolar myelinopathy was confirmed or suspected as the cause of morbidity and mortality of 28 bald eagles, 16 Canada geese (Branta canadensis), six American coots, two great-horned owls (Bubo virginianus), and one killdeer (Charadrius vociferus). Active surveillance during the outbreaks yielded annual average prevalence of vacuolar lesions in 17–94% of coots, but not in 10 beavers (Castor canadensis), four raccoons (Procyon lotor), and one gray fox (Urocyon cinereoargenteus) collected for the study. Brain lesions were not apparent in 30 Canada geese collected and examined in June 2002. The outbreaks at this location from 1998–2004 represent the most significant AVM-related bald eagle mortality since the Arkansas epornitics of 1994–95 and 1996–97, as well as the first confirmation of the disease in members of Strigiformes and Charadriiformes.

Top 10 Principles for Designing Healthy Coastal Ecosystems Like the Salish Sea

Like other coastal zones around the world, the inland sea ecosystem of Washington (USA) and British Columbia (Canada), an area known as the Salish Sea, is changing under pressure from a growing human population, conversion of native forest and shoreline habitat to urban development, toxic contamination of sediments and species, and overharvest of resources. While billions of dollars have been spent trying to restore other coastal ecosystems around the world, there still is no successful model for restoring estuarine or marine ecosystems like the Salish Sea. Despite the lack of a guiding model, major ecological principles do exist that should be applied as people work to design the Salish Sea and other large marine ecosystems for the future. We suggest that the following 10 ecological principles serve as a foundation for educating the public and for designing a healthy Salish Sea and other coastal ecosystems for future generations: (1) Think ecosystem: political boundaries are arbitrary; (2) Account for ecosystem connectivity; (3) Understand the food web; (4) Avoid fragmentation; (5) Respect ecosystem integrity; (6) Support nature’s resilience; (7) Value nature: it’s money in your pocket; (8) Watch wildlife health; (9) Plan for extremes; and (10) Share the knowledge.

Does human proximity affect antibody prevalence in marine-foraging river otters (Lontra canadensis)?

The investigation of diseases of free-ranging river otters (Lontra canadensis) is a primary conservation priority for this species; however, very little is known about diseases of river otters that forage in marine environments. To identify and better understand pathogens that could be important to marine-foraging river otters, other wildlife species, domestic animals, and humans and to determine if proximity to human population could be a factor in disease exposure, serum samples from 55 free-ranging marine-foraging river otters were tested for antibodies to selected pathogens. Thirty-five animals were captured in Prince William Sound, Alaska (USA), an area of low human density, and 20 were captured in the San Juan Islands, Washington State (USA), an area characterized by higher human density. Of 40 river otters tested by indirect immunofluorescent antibody test, 17.5% were seropositive (titer ≥320) for Toxoplasma gondii. All positive animals came from Washington. Of 35 river otters tested for antibodies to Leptospira interrogans using the microscopic agglutination test, 10 of 20 (50%) from Washington were seropositive (titer ≥200). None of the 15 tested animals from Alaska were positive. Antibodies to Neospora caninum (n=40), Sarcocystis neurona (n=40), Brucella abortus (n=55), avian influenza (n=40), canine distemper virus (n=55), phocine distemper virus (n=55), dolphin morbillivirus (n=55), porpoise morbillivirus (n=55), and Aleutian disease parvovirus (n=46) were not detected. Identifying exposure to T. gondii and L. interrogans in otters from Washington State but not in otters from Alaska suggests that living proximal to higher human density and its associated agricultural activities, domestic animals, and rodent populations could enhance river otter exposure to these pathogens.

CROSS-PROTECTION BETWEEN EPIZOOTIC HEMORRHAGIC DISEASE VIRUS SEROTYPES 1 AND 2 IN WHITE-TAILED DEER

Viruses in the epizootic hemorrhagic disease (EHD) serogroup are the most frequent cause of hemorrhagic disease in the southeastern United States, but nothing is known about cross-protection between the two EHD serotypes (EHDV-1 and EHDV-2) present in this region. We experimentally tested whether deer surviving EHDV-2 infection would be protected against subsequent infection with EHDV-1, and used field data to examine the possibility of reciprocal cross-protection. Eleven white-tailed deer fawns (Odocoileus virginianus) were experimentally infected with EHDV-2 and later challenged with EHDV-1. Two EHDV-2-naïve fawns also were infected with EHDV-1. Deer were monitored via physical examination, complete blood counts, clotting profiles, viral isolation, and serology, and each animal was assigned a quantitative clinical disease severity score based on presence of certain physical and clinical parameters. Infection of naïve controls with EHDV-1 caused severe clinical disease and death of both fawns, whereas deer previously infected with EHDV-2 exhibited no or minimal signs of disease. Thus, infection with EHDV-2 conferred protection against disease caused by subsequent EHDV-1 infection. Although prior EHDV-2 exposure protected deer from severe clinical disease, it did not prevent infection nor viremia indicating they could still act as virus amplifying hosts. These experimental infections suggest that EHDV-1 and 2 may exist in a state of mutual permissiveness.