Douglas E. Skilling

Infectious disease and the decline of Steller sea lions (Eumetopias jubatus) in Alaska, USA: insights from serologic data.

Serologic data were examined to determine whether infectious disease may have played a role in the decline of Steller sea lions (Eumetopias jubatus) in the Gulf of Alaska and Aleutian Islands, USA. Available published data, unpublished data, and recent collections (1997–2000) were compared and reviewed. Data were stratified by geography to compare the declining western Alaskan population in the Aleutian Islands through eastern Prince William Sound to the increasing population in southeastern Alaska. Prevalences of antibodies from the 1970s to the early 1990s were noted for Leptospira interrogans, Chlamydophila psittaci, Brucella spp., phocid herpesvirus-1, and calciviruses. Serum samples collected from 1997–2000 were tested for antibodies to these agents as well as to marine mammal morbilliviruses, canine parvovirus, and canine adenovirus-1 and −2. Conclusions could not be drawn about changes in antibody prevalence to these agents during the decline of Steller sea lions, however, because data were incomplete or not comparable as a result of inconsistencies in testing techniques. Despite these shortcomings, results provided no convincing evidence of significant exposure of Steller sea lions to morbilliviruses, Brucella spp., canine parvovirus, or L. interrogans. Steller sea lions have been exposed to phocid herpesviruses, caliciviruses, canine adenovirus, and C. psittaci or to cross-reactive organisms in regions of both increasing and decreasing sea lion abundance. Based on similar antibody prevalence estimates from the increasing and decreasing populations, these agents are unlikely to have been the primary cause of the population decline. They may have contributed to the decline or impeded population recovery, however, because of undetected mortality and morbidity or reductions of fecundity and body condition in animals under other stresses. Systematic monitoring for disease agents and their effects is needed to determine whether infectious disease currently plays a role in the decline and lack of recovery of Steller sea lions.

Inhibition of Vesivirus Infections in Mammalian Tissue Culture with Antisense Morpholino Oligomers

Caliciviruses infect and cause disease in animals and humans. They are nonenveloped, positive-stranded RNA viruses with a genome of approximately 7.5 kb that encodes viral proteins in three open reading frames (ORF). Antisense oligomers targeting one of the three ORF of caliciviruses of the genus Vesivirus significantly inhibit viral replication in tissue culture. Porcine kidney and African green monkey kidney cells were infected with Vesivirus isolates SMSV-13 and PCV Pan-1. Phosphorodiamidate morpholino oligomers (PMO) with sequence complementary to the AUG translation start site regions of ORF1, ORF2, and ORF3 were evaluated for their effect on viral titer. Scrape-loading delivered PMO to 50%-70% of the cells of the two cell lines, as measured by fluorescence microscopy and flow cytometry. A PMO targeting ORF3 caused a significant increase in viral titer. A PMO targeting ORF2, a scrambled PMO control sequence, and an unrelated PMO antisense sequence did not alter viral titer. Various PMO sequences antisense to an upstream region of ORF1 were effective in reducing viral titer up to 80% in a dose-dependent and sequence-specific manner. The extent of viral titer reduction was proportional to the delivery of PMO to cells. These observations demonstrate that antisense PMO can disrupt caliciviral gene function in a nucleic acid sequence-specific manner and are potentially effective antiviral agents.

MORTALITY OF HARBOR SEAL PUPS AT DIFFERENT SITES IN THE INLAND WATERS OF WASHINGTON

We examined the mortality rates and causes of death of harbor seal (Phoca vitulina) pups in three regions of the inland waters of Washington (USA) in 1984. One hundred eight pups were collected during 239 searches of the shoreline areas near harbor seal haulout sites or through public reports. Minimum neonatal (up to 1 mo after birth) mortality rates at these regions ranged from 12% to 26% of the pups born. Neonatal mortality was highest in the Strait of Juan de Fuca; 33 of the estimated 105 (31%) pups born at the primary site died. Causes of death varied by location. In southern Puget Sound predation by coyotes (Canis latrans) was the primary cause of death, accounting for eight of 43 (19%) of the dead pups examined; starvation was the next most common cause of death. Mortality at study sites in the Strait of Juan de Fuca was related to premature parturition; 19 of 49 (39%) of the pups found dead were born prematurely. Nine species of bacteria were identified in samples taken from 42 pups; Proteus sp. and Escherichia coli were the most common.

Characterization of a new calicivirus isolated from feces of a dog

SummaryCanine calicivirus (CaCV), isolated from feces of a dog with diarrhea, was readily propagated in cultures of canine cells and in a dolphin cell line. Serologic evidence indicated many dogs in at least one geographic area had been infected with CaCV, but its role as an etiologic agent of disease was not established. In cell culture most CaCV virions were strongly cell-associated making purification difficult. CaCV was established as a member of the Caliciviridae by morphology and physicochemical properties of virions (density, sedimentation rate, single major polypeptide, RNA genome size), although some of the properties differed slightly from those of previously described caliciviruses; evidence was also obtained for caliciviral RNA species in infected cells. Based on tests with antisera to numerous caliciviruses and presumed caliciviruses, CaCV appeared to be not closely related to any previously described virus except the stunting syndrome agent of chickens.

Naturally-occurring leptospirosis in northern fur seals (Callorhinus ursinus).

A 4-year study of Northern fur seal (Callorhinus ursinus) leptospirosis in the Bering Sea has shown that in newborn pups Leptospira pomona is associated with a multiple hemorrhage syndrome. Adults may develop an interstitial nephritis and shed organisms in the urine. The herd prevalence, based on microscopic slide agglutination tests, ranged between 7.0% and 15.4% for adult females and 3-4 year old bachelor bulls, whereas nursing pups averaging 4 months of age had a prevalence of 2%. These results are used to conclude that leptospirosis is not acquired primarily on the breeding rookeries but rather is more frequently acquired subsequent to the pups leaving the rookeries, presumably through the food chain during their first pelagic cycle.

Vesivirus viremia and seroprevalence in humans

Pathogenic caliciviruses of the genus Vesivirus circulate in oceanic ecosystems and spread to and among terrestrial mammals. Isolation of Vesivirus from natural and laboratory infections in humans led to this investigation of Vesivirus seroprevalence and viremia. Sera from four groups were tested for antibodies to Vesivirus as follows: blood donors whose units were cleared for donation, blood donors whose units were not accepted for donation solely because of elevated blood liver alanine aminotransferase (ALT) concentrations, patients with clinical hepatitis of unknown but suspected infectious cause, and patients with clinical hepatitis of unknown cause but associated with blood transfusion or dialysis. Additionally, sera were tested for Vesivirus genome by three methods: dot‐blot and two reverse transcription‐polymerase chain reaction (RT‐PCR) methods. The calculated seroprevalence against Vesivirus virions within these groups (N = 765) was 12%, 21%, 29%, and 47%, respectively (P < 0.001 for group differences). Additionally, 11 (9.8%) of 112 sera tested yielded RT‐PCR amplicons that by nucleotide sequence were distinct from each other and related to known Vesivirus. These data indicate that some blood donors in the population tested have serologic evidence of previous Vesivirus infection and some also have Vesivirus viremia. These results justify further investigation of an association between Vesivirus infection and illness in humans. J. Med. Virol. 78:693–701, 2006.

SEROLOGY AND VIROLOGY OF THE BOWHEAD WHALE (BALAENA MYSTICETUS L.)

Sera from four bowhead whales (Balaena mysticetus L.) were examined for the presence of specific antibodies, and tissue and swab samples from six and four animals respectively were processed for isolation of viruses and for initiation of bowhead whale cell cultures. All sera were negative for antibodies to nine serovars of Leptospira interrogans and to 21 orthomyxovirus subtypes and a paramyxovirus (Newcastle disease virus). All sera were positive, however, for neutralizing antibodies to one or more calicivirus serotypes. Two untyped adenoviruses were isolated from colon samples of two different whales, but neutralizing antibodies to the agents could not be demonstrated in any sera. Three primary bowhead whale cell cultures were derived from kidney (two cultures) and testis (one culture), from three individual whales.

Ultrastructure of newly recognized caliciviruses of the dog and mink

SummaryTwo recently recognized viruses obtained from a dog with glossitis and from mink with hemorrhagic pneumonia were characterized by electron microscopy. The results of the negative-stained preparations indicated that the viruses were structurally compatible with the calicivirus group.

Isolation of Primate Calicivirus Pan paniscus Type 1 from a Douc Langur (Pygathrix nemaeus L.)

Calicivinuses have been associated with an array of disease manifestations that are being identified increasingly in a number of animal species (Smith, 1983, U.S.D.A. Foreign Animal Disease Report No. 11-3, pp. 8-16). Pigs, marine mammals, marine fishes, cats, dogs, cattle, mink, and primates (including human beings) are species from which caliciviruses have been recovered (Smith et a!., 1983, J. Am. Vet. Med. Assoc. 183: 1223-1225; Cubitt and Barrett, 1984, J. Gen. Viro!. 65: 1123-1126; Barlough et a!., 1985, Avian/Exotic Pract., in press). Al! four caliciviruses isolated thus far from species of nonhuman primates (pygmy chimpanzee (Pan paniscus), silver leaf langur (Presbytis cristata), spider monkey (Ateles fusciceps), and lowland gorilla (Gorilla gorilla)-a!! residing at the primate facility of the San Diego Zoo) belong to a single novel serotype, tentatively designated as primate calicivirus Pan paniscus type 1 (PCV-Pan 1) (Smith et al., 1983, Science 221: 79-80; Smith et al., 1985, Am. J. Vet. Res., in press). Although this virus has been recovered from a vesicular lesion (pygmy chimpanzee) and from a case of chronic gingivitis (spider monkey), conclusive etiologica! links to these or to any other specific disease process(es) have not been demonstrated. This communication reports the first isolation

Isolation of reptilian calicivirus Crotalus type 1 from feral pinnipeds.

Ten virus isolates were obtained from three species of marine mammals sampled on San Miguel Island (California, USA) and 1,200 km north on Rogue Reef (Oregon, USA) during tagging operations in 1986–87. Seven of these 10 were derived from 30 sampled Steller sea lion (Eumetopias jubatus pups, while two of 10 were isolated from one of 19 sampled California sea lion (Zalophus californianus californianus pups, and the remaining isolate was derived from 30 sampled northern fur seal (Callorhinus ursinus) pups. All 10 isolates were identified as belonging to a single serotype, reptilian calicivirus Crotalus type 1 (RCV Cro-1), previously isolated from both healthy and diseased snakes and frogs in a California zoologie collection. The marine samples also showed that nine of 30 Steller sea lion pups, one of 19 California sea lion pups and zero of 30 fur seal pups were producing type specific neutralizing antibodies to RCV Cro-1. This represents the first reported instance of the isolation from marine sources of a calicivirus originally isolated from a terrestrial species.