New Zealand Sea Lion Epidemic January-February 1998

Preliminary report June 1998, by: Wildlife Health Centre, Institute of Veterinary, Animal and Biomedical Sciences, Massey University & Department of Conservation (DoC):

Padraig Duignan (pathologist), Jane Hunter (bacteriologist), Maurice Alley (pathologist), Keith Thompson (pathologist), Joanne Meers (virologist), Stanley Fenwick (bacteriologist), Nadine Gibbs (biologist, IVABS), Nicholas Gales (pinniped ecologist, DoC), Simon Childerhouse (pinniped ecologist, DoC), Ian Garthwaite (toxicologist) and Per Madie (Public health).

Epidemiology Clinical Signs Samples Collected Gross Pathology Histopathology

Bacteriology Virology Biotoxins Parasitology Discussion

1. Epidemiology

Background

The primary breeding rookeries for New Zealand sea lions, Phocarctos hookeri, are located in the subantarctic Auckland Islands of Enderby, Dundas, and Figure of Eight. A small breeding rookery is also on Campbell Island (Childerhouse and Gales, 1998). The total sea lion population is estimated at between 12,000 and 14,000 animals. A team of DoC biologists were present on the Auckland Islands between January 13th and February 27th as part of an ongoing study of sea lion biology. The base camp for the research group was at Sandy Bay on Enderby Island. The following is an account of a mortality event that took place in the breeding colonies over that period. Clinical signs and gross pathological changes in the animals are described. Use has been made of field records and a video tape recorded during the event. This report also covers the results of a preliminary investigation of samples of sea lions collected by the expedition during and after the epidemic.

Life History

Adult males establish territories from late November and pregnant females begin to arrive on rookeries during the first week of December. Most pups are born from early December to mid-January. Mating occurs 7 to 10 days after the pups are born and most adult males return to sea by late February. The females forage at sea and return to suckle their pups every 24 to 48 hours and spend a similar amount of time ashore. There is no discernible diurnal pattern to their arrival or departure so that approximately 50% of the females are ashore at any given time. At Sandy Bay, between 200 and 400 adult females are on the beach at any time. By four weeks of age, the pups begin to explore and swim attended by their mothers. Lactation continues for between 8 and 12 months (see Gales, 1995).

Mortality Event

When the research team arrived at base camp pup mortality at Sandy Bay, Enderby, was found to be low this year compared to previous years (2% vs 11%). No diseased animals were observed at that locality and pup weights were in the expected range. The daily mortality rate began to rise around January 20th and peaked on Jan 30th. By February 10th the rate had decreased to the same level as January 20th. On 27 January, an overnight desertion of the beach by adult females was observed although some of the females did return to the colony with time. A number of females and males were observed either sick or dead. Some had swelling of the ventral neck region.

On 21 January, the team visited Dundas Island (9km from Enderby), where the largest sea lion breeding colony establishes each year. An unusual level of pup mortality (3 times normal level) was noted and most of the deaths appeared to have occurred in the previous ten days. Based on surveys, 1748 +/- 90 live pups and 625 dead pups (36% mortality) were recorded. During live captures for telemetry studies, some adult females were also found to have swellings in the neck region.

The duration of the mortality event at Dundas Island appeared to have been between approximately 12 January to 7 February, and at Sandy Bay between 20 January and 8 February. Certainly by 22 February, when the research team left the Auckland Islands, the event was virtually over and mortality had returned to normal levels (Fig.2).

The final tally of dead pups on 20 February was 1606 (53%). This is a minimum estimate because on the final count on Dundas, 700 pups were unaccounted for. They presumably had either dispersed away from the island or had died and were washed out to sea.

Because adult sea lions disperse widely and an unknown proportion are at sea at any given time, it is not possible to acquire meaningful survey data. A total of 74 females and 11 males were found dead on the islands. Adults are presumed to have died at sea but the number involved is not known.

Pups: Few clinical signs were noted on Enderby Is. Pups were judged either to be normal or close to death. No pups were recognized as being at an early or intermediate stage of illness. Approximately 10 pups were seen on this island dragging their hind limbs. Pups in extremis lay with their necks outstretched and stiff, their mouths partly open and had laboured breathing. At the start of the event, dying pups were in good body condition, but as the event progressed, a greater proportion of the pups appeared to be starving, due possibly to female abandonment. On Dundas the clinical signs were similar, although more pups appeared to have bacterial infections as indicated by purulent discharges from eyes and superficial abscesses and ulcers.

Adults: The clinical signs in adults on land were quite variable and included the following:

2. Some adults had discrete, raised focal lesions in the skin and superficial blubber that were firm and red on cut surface. In some animals that were observed repeatedly, the skin lesions appeared first on the ventrum and spread within a matter of hours to the thorax and dorsal surface Fig.4

3. Some females were stiff and lethargic and, in some cases, dragged their hind limbs. These animals were also less alert than normal and allowed close approach by observers.

Clinical signs and/or gross lesions were seen in a relatively small number of adults (18%) during the epidemic and in few animals after the peak of mortality (Table 1). Many of the adults found dead did not have any gross lesions.

Table 1. Signs of clinical illness or gross pathology in adult New Zealand sea lions.

The Auckland Islands are remote, and transport between them and the New Zealand mainland is limited by weather conditions. There are no permanent facilities on the islands, and all scientific equipment must be carried to and from the islands on each visit. Sampling of sea lions was therefore by necessity constrained by these factors. There were only two opportunities to collect fresh samples and have them returned relatively quickly to the mainland. On the first occasion (Jan 28th) a helicopter flew out with sampling material and had to return to the mainland by the following afternoon. This interval allowed samples to be collected from three adult females and four pups. Three entire pups were also packaged for shipping to Massey University. Milk and swabs were collected from one female that had a draining neck swelling. The second opportunity to send samples off the island was by ship on February 22 by which time the mortality event was essentially over. On this occasion, fixed tissue samples from two adults and three pups were collected. In addition serum and, in some cases, milk were collected from 26 apparently healthy females. Fresh faecal samples were also collected for bacteriology.

All post mortem examinations were conducted in the field in less than ideal conditions apart from three pups that were shipped frozen to Massey University. In total, 10 pups, one sub-adult female, and 6 adult females were examined from the Auckland islands. Four of these females were close to death and were euthanased on humane grounds. One adult female was also sampled on Campbell Island.

Pups: Of the 10 pups, 5 were in poor body condition with marked to severe depletion of the blubber layer and marked dehydration. The remaining pups were in fair or moderate body condition throughout the entire epidemic. One pup had ulceration of the anal muco- cutaneous junction and one had a stifle abscess/suppurative arthritis. The remaining pups had no significant gross lesions.

Adults: The sub-adult female was in excellent body condition. There was focal haemorrhage in the blubber and sub-cutaneous tissues and multifocal hepatic abscessation. Body condition was adequate for 6 adults and very poor for one. Of the 4 adults euthanased on Enderby Island, one had cellulitis of the right side of the neck and multifocal skin lesions on the ventral surface. The cellulitis was characterised by the proliferation of edematous and haemorrhagic tissue in the region of the parotid salivary gland and retropharyngeal lymph node. The skin lesions were 1 cm diameter, raised, red and firm that exuded blood-stained fluid on cut surface. There were no other lesions. A second female that was extremely weak and had been observed convulsing, was found to have multifocal skin lesions as described but no other significant gross lesions. A third female was also weak and had numerous skin lesions but no internal abnormalities. The fourth female was in very poor body condition and appeared reluctant to move but there were no significant gross findings in this case. One female that was found dead at the outset of the mortality had numerous skin lesions as described and swollen congested lungs. A second dead female had a subcutaneous abscess on one pelvic limb but no other significant lesions. No lesions were recorded for the female found dead on Campbell Is.

Pups: Pneumonia was the most significant histological lesion in three pups. In two pups this was classified as mild acute interstitial pneumonia of unknown aetiology while the third pup had mild suppurative bronchopneumonia associated with nematode parasites. One of the pups with interstitial pneumonia also had moderate lymphoid depletion in peripheral lymph nodes and it had acute suppurative cellulitis affecting the stifle region. One pup had suppurative encephalitis with neutrophilic perivascular cuffing affecting primarily the thalamus and mid brain while a second pup and had mild neuronal degeneration with formation of axonal spheroids. One pup had acute suppurative lymphadenitis. The remaining four pups had no significant lesions.

Adults: The sub-adult female had hepatic abscessation and random multifocal hepatic necrosis, mild suppurative bronchopneumonia and mild lymphoid depletion. Parasitic bronchopneumonia was also present in two adult females. The skin lesions were characterized as focal acute suppurative dermatitis and arteritis with oedema and haemorrhage. The lesions were located deep in the dermis and often extended into the superficial blubber. The inflammatory response was centred on blood vessels and there was fibrinoid necrosis of arterioles associated with large numbers of small pleomorphic gram negative bacteria. Similar bacteria were distributed in the surrounding tissues and associated with fibrin and a marked neutrophilic infiltration. There was frequently severe fibrinopurulent lymphadenitis of superficial lymph nodes and tonsils often associated with bacteria. Severe suppurative lymphadenitis with extension to surrounding tissues accounted for the neck swelling in one female. In two animals there was severe acute haemorrhagic and suppurative pneumonia that, in one case at least, involved most of the lung field. The inflammation was associated with acute fibrinoid necrosis of pulmonary arterioles with intra-lesional bacteria as before. In one of the euthanased females, in addition to dermal and lymphoid changes, there was hepatic atrophy, acute adrenal cortical haemorrhage and mild focal thalamic haemorrhage.

Bacteriological examination has been carried out at Massey University on 92 sea lion samples including faeces, organs, and swabs from lesions. Several organisms were isolated in mixed culture and those identified include a variety of Salmonella serotypes, Gemella haemolysans, haemolytic and nonhaemolytic coliforms, Bacillus spp., Oligella urealytica, Pseudomonas sp., Gardnerella vaginalis. In addition, some very small, slow-growing organisms were isolated in mixed culture from 3 adults (Enderby) and one pup (Dundas). One isolate has been provisionally identified as a Campylobacter-like species by the Enteric Reference Laboratory, ESR, Porirua, and work is continuing on other isolates.

Salmonella spp. were isolated as follows:
Dundas Island:
Superficial swabs: 1 of 3 live pups positive - S. cerro.
Tissues: 5 of 6 dead pups positive: S. newport, S. derby , S. enteriditis PT 4, PT 8 and untypable.

Enderby Island:
Tissues: Adults: 4 of 6 positive - S. enteritidis PT 8, PT 4, S. cerro.
Pups: 3 of 3 positive - S. enteritidis PT 8, S. derby and S. typhimurium
phage type untypable.
Serotyping was carried out by the Enteric Reference Laboratory, ESR, Porirua.

Faecal samples collected on 16-17 February from Enderby Island (20), Dundas
Island (3) and Campbell Island (18) were cultured for salmonellae. One of the 20 samples from Enderby Island was positive (S. derby). No Salmonella sp. were cultured from 3 seabirds, a dusky dolphin and fur seal by-caught near the Auckland islands around the time of the sea lion epidemic.

Tissue samples from the sea lions have been tested for the presence of viruses by Auckland University (AU), Massey University (MU) and the Central Animal Health Laboratory (CAHL), Wallaceville, in New Zealand and by the Institute of Virology, and Erasmus University Hospital, Rotterdam, The Netherlands (EU).

Tests on tissues have included electron microscopy, inoculation and passaging on cell cultures (VERO, SekC, SeFB and seal PBMC), inoculation of ferrets, IgG and IgM ELISA's and PCR.

Based on electron microscopy (MU, AU, EU) there were no viral particles observed in skin lesions or in cell cultures. Cell cultures have also been negative for cytopathic viruses (MU, CAHL) and haemagglutinating viruses (WRC) after the fifth passage. Based on PCR, there were no indications of infection by either morbilliviruses or herpesviruses (Phocid herpsevirus I and II). Initial weakly positive samples could not be confirmed by hybridization with specific probes for morbilliviruses (EU). Passage of material in ferrets did not show any indication of viral infection, either by clinical signs or by serology (EU).

Serology and virus isolation results from the Foreign Animal Diseases Diagnostic Laboratory, U.S. Department of Agriculture, Plum Island, New York, are pending.

Samples were examined at the Toxinology and Food Safety Laboratory,
AgResearch, Ruakura, New Zealand by Dr Ian Garthwaite.

Blood, stomach contents, liver and kidney from a number of affected animals,
and a single milk sample were analysed by ELISA assay for the following
toxins and toxin groups :-

• Brevetoxin, including the neurotoxic shellfish poisoning toxins.
• Saxitoxin, and other members of the paralytic shellfish poisoning group of toxins.
• Domoic Acid, the causative agent of amnesic shellfish poisoning.
• Okadaic acid, a diarrhetic shellfish toxin.

Extracts were prepared and analysed by the neuroblastoma cell culture assay which detects cytotoxicity, and the presence of sodium channel active toxins (NSP, PSP and other toxins). No cytotoxicity or sodium channel activity were detected.

Mouse bio-assays have been run and no lipid soluble toxins were detected in the combined NSP/DSP screening procedure. No mouse deaths were observed after 24 hours.

Post-epidemic samples from sea lions (stomach contents, liver, milk) as well as stomach contents and liver from sea birds found dead in the vicinity of the Auckland Islands are under investigation at Ruakura.

One of the pups examined at Massey University had a heavy burden of hookworms (1428) in the intestine. This pup was in poor body condition, markedly dehydrated, and it had suppurative encephalitis associated with Salmonella enteritidis PT8.

Faecal samples from Enderby Is. (n = 18), Dundas Is (n = 3), and Campbell Is. (n = 15) were examined for parasites. Protostrongylid larvae were found in one sample from each of Enderby and Dundas.

The conclusions that may be drawn from this investigation are limited by the fact that sampling was opportunistic and only a relatively small number of animals were sampled. In particular, few animals were examined at the onset and peak of the event. The results to date do not confirm any aetiology for the mortality event. Thus, in the absence of an unequivocal aetiology any conclusions can only be speculative. There are three possible scenarios:

1. An infectious agent acting alone.
2. A marine algal biotoxin.
3. A combination of these and possibly including El Nino-related disturbance of the ecosystem.

It is unlikely that the epidemic was caused by any known infectious agent acting alone for the following reasons:

1. There are no consistent gross or histological lesions and no lesions indicative of agents known to cause epidemics among marine mammals such as morbilliviruses, influenza viruses etc.
2. There are no viral isolates or indications of viral infection.
3. A variety of potentially pathogenic bacteria were isolated but there is little consistency in isolates between animals.

A variety of Salmonella serotypes were isolated from several animals; however, based on the available tissue samples it was not always possible to associate isolates with histopathology lesions. The role that salmonellosis played in the epidemic was probably that of an opportunistic invader in which it was responsible for the death of some animals. Salmonella enteritidis was isolated from the brain of the Dundas pup with encephalitis and has previously been reported as the cause of meningomyeloencephalitis in a northern fur seal (Callorhinus ursinus) pup found dead on St. George Is. Alaska (Stroud and Roelke, 1980). Salmonellae were also isolated from several adult sea lions and may have been the cause of lymphadenitis and vasculitis. Salmonella newport was isolated previously from a captive New Zealand fur seal (Arctocephalus forsteri) that had enteritis (Cordes and O'Hara, 1979). Intestinal samples were available from only three pups examined here but they did not have enteritis.

Surveys for Salmonellae in marine mammals have shown that these bacteria are prevalent in many regions including California (Gilmartin et al., 1979; F. Gulland, pers comm), the Pribilof Islands, Alaska (Jellison and Milner, 1958), and the British Isles (Baker et al. 1995). They are associated with disease but may also be isolated from apparently healthy animals. Thus, the isolation of these bacteria from New Zealand sea lions may be an indication of the normal bacterial flora of this species that under certain circumstance can invade the host and cause death. The reservoir host for Salmonellae in the Auckland Is. is unknown. Based on preliminary data it is possible that adult sea lions may be carriers for some serotypes because faecal samples collected post epidemic on Enderby contained S. derby. Further work on archived samples and from samples collected in the future will be required to determine their origin and role in sea lion mortality.

Based on histopathology, a currently-unidentified small gram negative pleomorphic organism was associated with vasculitis and septicemia in three adults. However, this organism was present in mixed culture and has proved difficult to isolate and identify. The identity of this organism is under investigation at Massey University and ESR, Porirua. It is also unknown whether it is part of the normal flora of sea lions which under certain circumstances becomes invasive and pathogenic. Animals that are immunocompromised or those in which the normal mucosal barriers are impaired may be susceptible to fulminating infection by this, or some other, organism.

The second and third scenarios are that the event was caused by a marine algal biotoxin acting alone or in combination with opportunistic infections and perhaps alterations in prey availability or distribution caused by El Niño. No biotoxin has been identified in the samples collected to date; however, this may reflect the lack of samples rather than the absence of a toxin. For example, no water samples or plankton samples were able to be collected from the Aucklands shelf waters at the time of the epidemic. Neither was it possible to collect samples from sea lions that died at sea. No stomach contents were available for analysis from animals that died during the peak of mortality.

There is a possibility that an algal bloom occurred in the Auckland Islands region during January 1998. Landsat imagery, provided by the NZ National Institute of Water and Atmospheric Research, shows high chlorophyll concentrations in surface water layers around the Auckland Islands in January . However, it is not known whether this phenomenon is a regular or a rare occurrence in that subantarctic region. Coincidentally, there were red tides at other locations around the New Zealand mainland between January and March that caused clinical illness in people and was the likely cause of fish, sea bird, and fur seal deaths in affected areas (Chang, 1998).

A possible scenario for the event is that if, because of prey availability, females had to forage for longer and further from the rookeries this year than in previous years, or that sickness or death-either pathogen or biotoxin-induced- resulted in longer or ineffective foraging sorties, or none, then at least some pups would suffer malnutrition and predisposition to secondary bacterial or parasitic infections that would otherwise be innocuous. Alternatively, sub-lethal exposure to a biotoxin by ingestion or inhalation (Chang, 1998) may have induced immunosuppression (Bossart et al., 1998) or mucosal damage that facilitated the proliferation and invasion of commensal bacteria.

The accumulation of pathogenic bacteria in the colonies could also be facilitated by the "podding" or huddling behaviour of pups on the beach. Thus, high concentrations of pathogenic bacteria could spread rapidly from sick pups to pups that appeared to be in good body condition.

Further work on existing sea lion samples is underway and a winter expedition to further sample the population is proposed. A workshop will be held in Wellington in June to review the results in greater depth.

Acknowledgments

We thank Department of Conservation Auckland Islands expedition members who observed and gathered samples under difficult circumstances. DoC mainland staff supported the logistics of the Auckland Islands expedition, and organised the rapid dispatch of samples to Massey University. We also thank Ian Garthwaite of AgResearch for the biotoxin assay results; Prof. A. D.M.E. Osterhaus and Dr. Marco van de Bildt of Erasmus University and Dr. Julian Motha of the Central Animal Health Laboratory, Wallaceville, for virology results; and Carolyn Nicol, Enteric Reference Laboratory, ESR, Porirua, for bacterial serotyping. Thanks also to Dr.s J taylor, T. Gruijer and M. Pearson from the School of Biological Sciences at Auckland University.

References

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