Since 1990, one and two year old monk seals (Monachus shauinslandi), at French Frigate Shoals in the Northwestern Hawaiian Islands, have been exhibiting signs of emaciation and failure to thrive. Although a specific cause for this occurrence has escaped identification, limited food resources may be involved because relocation to areas with more abundant food and less intraspecies interaction has facilitated recovery of affected animals. To help assess the possible risk of spreading infectious disease from French Frigate Shoals to other monk seal breeding locations, a virological survey of monk seals on French Frigate Shoals was carried out in the Spring of 1992. Nasal swabs, rectal swabs and buffy coats from nineteen, emaciated and healthy, monk seals were examined for the presence of viruses. Viral isolation attempts in Vero monkey kidney and porcine kidney cell lines were unsuccessful. Hybridization experiments using a group-specific calicivirus cDNA probe against RNA extracted from the swabs and buffy coat samples indicated that all animals tested possessed nucleic acid sequences which were recognized by the probe. Electron microscopy on selected hybridization positive samples confirmed the presence of viral particles consistent with calicivirus morphology. Although caliciviruses have been reported to infect and are associated with disease in a wide variety of marine mammals, the causal relationship between this non-cultivatable calicivirus infecting the monk seals on French Frigate Shoals and the seals' failure to thrive has not been determined.

Introduction

The Hawaiian Monk Seal (Monachus schauinslandi) is an endangered phocid pinniped with a population distribution restricted mainly to the Northwest Hawaiian Islands. Studies to enhance monk seal population recovery have focused on, haul-out patterns, population structure, reproductive patterns, and a variety of survival factors (5,6,7,12). Adult male aggression toward adult female and immature seals has been identified as a primary survival factor (7). A limited number of reports on monk seal.

Since 1990, immature monk seals at French Frigate Shoals in the Northwest Hawaiian Islands have exhibited signs of failure to thrive. Although causal relationships are unclear, adult male aggression and its effect on food intake may play a role. Relocation efforts and female pup enhancement programs in similar situations have resulted in an increase in food intake of affected animals and a decrease in negative intraspecies interactions (6).

Virtually nothing is known about the virology of the monk seal. Only serological evidence exists to indicate that virus infection occurs within the population. Smith and co-workers have reported monk seal serum neutralizing antibodies against two caliciviruses, San Miguel sea lion virus type 1 (SMSV-1) and vesicular exanthema of swine virus type I (VESV-I) (10). To further assess the possible risk of spreading infectious disease among monk seal populations, and add to the knowledge base of monk seal pathobiology, a virological survey was carried out at French Frigate Shoals in the Spring of 1992.

Because evidence exists for the possibility of calicivirus infection within the monk seal population, a recently developed calicivirus-specific cDNA hybridization probe was utilized (8). While some caliciviruses can be cultivated in tissue culture, a large number of calicivirus-like agents have resisted in vitro propagation. Until recently, the only method to detect caliciviruses that resist growth in tissue culture was by expensive and time-consuming electron microscopic examination of field samples. The cDNA probe provides a rapid means of detecting calicivirus RNA and does not depend on lengthy and often unsuccessful attempts to identify viral presence, by laboratory propagation or electron microscopy.

Methods

Nineteen immature monk seals at French Frigate Shoals were captured and sampled. Based on field observations, these individuals represented clinically normal (six), undersized (nine), and emaciated (four) conditions. Nasal and rectal swabs were collected from all 19 monk seals and blood was collected from 17 of the 19 animals. The swabs were immediately placed in tissue culture media and frozen in liquid nitrogen. Blood samples, clotted and un-clotted (containing EDTA), were placed on ice.

All swab samples were passaged in both Vero cells and Porcine Kidney 15 cells. The inoculated cell lines were observed for evidence of cytopathic effect. Three blind passages were performed for each sample (9).

RNA was extracted from all swab samples. In addition, RNA from buffy coats, prepared from the 17 blood samples collected, was extracted. The prepared samples were probed using a calicivirus-specific cDNA hybridization probe in a dot hybridization assay (8).

Five samples were examined by direct electron microscopy in order to confirm the results obtained by the hybridization assay. The electron microscopy samples were negatively stained with phosphotungstic acid (9).

Results

All nasal and rectal swab samples were negative for viral cytopathology after three blind passages in Vero and Porcine Kidney cells. The hybridization assay, however, indicated that all but six swabs had RNA sequences which were recognized by the cDNA probe (Figure 1). Furthermore, one buffy coat sample was hybridization-positive. Every monk seal tested was found to have at least a rectal swab or nasal swab that was calicivirus RNA positive.

Five swab samples that were positive in the hybridization assay were examined by direct electron microscopy. Every sample contained viral particles consistent with calicivirus morphology. No other particles consistent with virus morphology was observed.

Figure 1. Results of a Calicivirus cDNA Hybridization Assay from Normal, Undersized, and Emaciated Monk Seals at French Frigate Shoals, Northwest Hawaiian Islands.

When body condition of the sampled animals was taken into account, no difference was seen with respect to calicivirus infection between normal and affected seals (Figure 1). The only positive buffy coat sample, however, was obtained from a clinically normal animal.

Discussion

Caliciviruses have been isolated from, or detected in, a phylogenetically diverse variety of marine and terrestrial animals, including insects, mollusks, fish, birds, mammals and man (3,10). This is the first report of a caliciviral, or any viral, infection in the Hawaiian monk seal.

A large group of caliciviruses, the candidate caliciviruses, have resisted propagation in tissue culture and can only be detected, until recently, by direct visualization using electron microscopy. The majority of known candidate caliciviruses have been implicated in enteric and hepatic diseases of food animals, and man. Furthermore, human caliciviruses are a common cause of "food poisoning" outbreaks in infants, adults, and the elderly (3).

The cDNA hybridization probe used in this study has detected all caliciviruses tested, both cultivatable and non-cultivatable. The probe has not cross-reacted with any other virus family assayed (8). Based on the positive results of the hybridization assay and confirmation on selected samples by electron microscopy, the monk seal population at French Frigate Shoals is infected with and shedding a calicivirus which resists propagation.

The relationship between this infection and the failure to thrive syndrome is unclear. Animals with normal, undersized, and emaciated body conditions appear to be equally infected. The only buffy coat sample to be positive, however, was from a normal animal. Calicivirus presence in the buffy coat indicates a viremia, which would be consistent with either a persistent infection or an early stage of virus infection accompanied by secondary spread to target organs. Unfortunately, the role caliciviruses play in monk seal disease requires more controlled epidemiological studies. It may benefit the monk seal population to continue to monitor the distribution and movement of this new calicivirus throughout the Northwest Hawaiian Islands.

References

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