Abstract

A 12-year-old male harbor seal (Phoca vitulina), born July 6, 1990 at the National Aquarium in Baltimore, was transferred to the New Jersey State Aquarium in Camden in the spring of 1992. Since then he had been housed in an outdoor exhibit with four other harbor seals and three gray seals (Halichoerus grypus). His medical history was basically unremarkable prior to this problem. The seals receive monthly ivermectin for heartworm prevention. His weight was 184 pounds at the beginning of September 2002. As part of the West Nile Virus Surveillance Program his blood had been included with that of several other seals and all of the penguins and was sent to Cornell Diagnostic Lab in April 2002. All of the results were negative for West Nile Virus (WNV) at that time using serum neutralization.

In September 2002 the harbor seal stopped eating. The next day he had diarrhea (soft, unformed stool), some of which had streaks of blood in it. His physical exam was unremarkable. The fecal was negative for parasites. A direct smear was stained and a large number of gram-positive, spore-containing rods were seen. Blood was drawn and sent to Antech along with a stool culture and also rectal culture for both anaerobic and aerobic cultures. Blood was also drawn for cell counts and chemistries, which were sent to Antech but also performed at the local veterinary hospital for immediate results. Nothing remarkable was detected. The trainers reported the seal occasionally seemed to have abdominal discomfort. Though his socialization and trained behaviors appeared normal, he was refusing food, so it was not possible to give oral medications. While he was restrained in a crate to obtain blood, he was given penicillin G IM and lactated ringers under the blubber. He was offered fresh water in a separate pool and from a running hose. During the next seven days he continued to refuse food, vomited once, and passed some stool every day. By the end of this weeklong period his stool returned to normal consistency. His trained behaviors and socialization with the other seals and trainers continued to remain normal for the most part. He would follow verbal and visual commands but would not come over to station at deck. Besides offering the various fish in his regular diet, including his favorite fish, he was eventually offered live fish but still showed no interest in eating.

Blood work was repeated two days after the initial blood draw and the parenteral fluids and antibiotic injections were continued every other day. The serum electrophoresis (EPH) and blood chemistries were within normal limits (WNL); however, the red cell indices had dropped to low normal and the WBC had dropped from 7,100 to 4,100 indicating a possible viral infection. Antech was asked to performed some viral titers (corona virus, canine distemper, borrelia, parvo virus, leptospirosis and feline infectious peritonitis) on the serum they already had at the lab; however, they refused to perform the canine and feline tests saying it would not be accurate and thus a waste of their time and materials (even though the trainers had pointed out that stray dogs and cats had recently been found in the exhibit).

Seven days after the seal had stopped eating, he passed normal looking stool, but started to exhibit tremors and labored breathing. He had tremors mostly of his head and neck, but also of his front flippers. The first day these were seen, the tremors would come and go, but worsened when he tried to move, heard a noise or seemed afraid. He would then stiffen and also appeared to have a "tetanus" grin. With these new neurological signs happening, more blood was drawn and tested for rabies, tetanus and blood lead. There was nothing in the literature about tetanus in pinnipeds, and calls to colleagues for help indicated that no one had heard of tetanus in pinnipeds, nor did they have other ideas of a likely infectious etiology for these signs, especially since he was part of a closed collection.

The new blood sample taken had 17 percent bands and a WBC, which had dropped to 2,500, and was climbing back up into the normal range. While awaiting blood results from Antech, it was decided to treat the animal for possible tetanus. After locating tetanus antitoxin (Ft. Dodge) from several sources and testing the animal for a possible anaphylactic reaction, he was treated with 60,000 IU given intramuscularly in two different sites. An increased dose of penicillin G was given, after drawing additional blood for a blood culture.

EPH graphs were sent to Carolyn Cray at the University of Miami Comparative Pathology Diagnostic Lab. She indicated that they were showing no apparent immune response to whatever was causing the clinical signs. She felt it would indeed be worthwhile running viral titers to perhaps help point to a direction for the diagnosis, so blood was sent to her to check for calcivirus, corona virus, morbillivirus, feline infectious peritonitis and parvovirus.

Laurie Gage (Six Flags Marine World, Vallejo, CA) was consulted and she said that she had seen similar signs from hyponatremia. Although the sodium levels were in the low normal range, the lactated ringers were switched to normal saline for any parenteral fluids given. She also said she had seen similar signs due to thiamine deficiency and recommended a very large dose of vitamin B-1. She said in her experience the animals with thiamine deficiency respond clinically within 12 hours after the injection.

At this point Marty Haulena (Marine Mammal Center in Sausalito, CA) mentioned that he had heard that Ann Duncan at the Detroit Zoo had a harbor seal with similar problems, which was diagnosed with WNV. Initially Ann was not available, but Cynthia Stadler explained that their seal had stopped eating and shortly thereafter had similar tremors. Nothing was found on blood work so they started treating with antibiotics, an antifungal and aspirin. Later they also treated him with thiamine. He started to improve on the fourth day, and they continued to treat him with anti-inflammatory medication and thiamine. Later they received test results showing he tested positive for WNV. With this information it became important to call Amy Glaser at Cornell Diagnostic Lab to ask her to test blood from our seal for WNV and rabies.

On day eight of the anorexia and the second day of neurologic signs, there were still no answers from the labs. There was no change from the thiamine injection. The differentials were expanded to include other possible etiologies of tremors and tetany such as other toxins (strychnine, thallium, Bufo toad poisoning (Bufo marinus), mushrooms) and viral agents (such as equine encephalitis). In the meantime an anti-inflammatory medication Banamine (flunixin meglumine) IM was started while the vitamin B1 injections and parenteral fluids were continued. At this point the seal finally seemed hungry, but he could neither apprehend nor swallow any fish. Oddly the trainers said he could still snap at flies and was catching some, which was a normal behavior for him. The trainers felt he was drinking some water, but were not sure he could actually swallow it. Assisted feeding with some larger trout was attempted, but that set off gagging and tremors. By the afternoon he appeared to have fewer tremors and twitches.

On the ninth day of anorexia and the third day of neurologic signs he seemed weak and exhausted and to have twitching most of the time including involuntary eye blinking. He was producing foam around the mouth and eyes from the constant twitching. Most of his locomotion on land was abnormal looking, and his hind flippers seemed to be affected although not as severely as his front flippers and head and neck. We had blocked off the pool, fearing he may drown and filled a shallow pool for him. Since he was normally fed in the water, we decided to try to feed him in the regular pool. We thought if we could make this as routine as possible for him, he might eat. Additionally, we hoped gravity might help when he was positioned in the water to be fed. First we prepared for emergency evacuation in case he could not swim. Despite his tremors he swam rather normally and had no problem entering or exiting the pool, however, he still could not eat in the water, although he was acting very hungry. After numerous attempts the curator was eventually successful in force-feeding two herring, although he later regurgitated one of them.

By day 10 we had ruled out lead toxicity, his red cell indices were dropping below normal and his WBC had nearly recovered. There was no improvement in his now constant tremors and twitching. We had no diagnosis or response to treatment and were considering euthanasia. Showing no other changes, he died later that day.

Necropsy the following day by Perry Habecker (University of Pennsylvania, School of Veterinary Medicine, Department of Pathobiology, New Bolton Center, PA) was mostly unremarkable except for two things: "The lungs are purple-brown and have interlobular emphysema...The capsules of the kidneys are difficult to remove." Tissues were sent out for histopathology and also for testing for rabies and WNV at the state lab. A week or two later the rabies and WNV PCR were both reported negative. The pathologist reported that he had seen several horses with similar brain lesions that initially tested negative and later positive for WNV with a double-nested PCR, so he sent tissues to the USDA lab in Ames, Iowa.

The histopathology was returned shortly after the original pathology report:

1.  Lung: interlobular emphysema, moderately severe, diffuse

2.  Brain: meningoencephalitis, lymphocytes, minimal to mild, multifocal

3.  Spinal cord: poliomyelitis, nonsuppurative, moderately sever, diffuse

Almost a month after the seal had died, we received a positive test for WNV. "Spinal cord--strongly positive for WNV antigen" and "Brain tissue POS for WNV via RT-PCR at USDA NVSL in Ames, IA." Another week after that report we received the report from Cornell that the blood was POS 1:40 on WNV SN and also positive on WNV PCR. Since then, we have retested all of the remaining seals and penguins and found no others have seroconverted.

Subsequent conversations with Ann Duncan about her harbor seal revealed similar signs although the duration was different. That seal didn't eat for a day and started showing tremors on day one, which were worse the second day. Besides the tremors, twitches, and hyperesthesia, he appeared less able to hear and see and seemed confused. He improved on the third day and appeared clinically normal on the fourth day with normal appetite and activities. The tremors resumed three days later; however, they remained mild. Between day 12 and day 15 of the illness the seal had sporadic episodes of sneezing with bloody discharge from the right nostril. This was considered to be a separate problem and worked up and treated. On day 23 the harbor seal developed dyspnea and the primary problem was assessed to be pneumonia or nasal occlusion. Additional antibiotic therapies were added; however, his illness progressed rapidly, and he died on day 25. The serum collected on day two of his illness was submitted to Cornell for WNV serological testing. This sample was positive on serum neutralization at a titer of >1:640 and positive on PCR. The sample was negative for St. Louis encephalitis at a 1:20 dilution.

Histopathological evaluation of tissues determined the cause of death to be subacute bronchopneumonia caused by bacteria. Overall interpretation of histopathological findings is a pneumonia secondary to stress and immunocompromise caused by the West Nile Virus. Brain, spleen, kidney, heart, lymph node and esophagus were sent for immunohistochemical staining and were found positive for WNV using a double-nested PCR.

A second seal at the Detroit Zoological Institute had very mild tremors of the head and neck on the third day of seal #1's illness but appeared completely normal the next day. Serum was collected on day two and was positive for WNV on serum neutralization at a titer of 1:8192 and positive on PCR.

Serological screening of three other seals housed in the same enclosure demonstrated that all three seroconverted during the summer of 2002. Serum neutralization titers ranged from 1:8192 to 1:16,384.

Further work

Fabio Del Piero (University of Pennsylvania, School of Veterinary Medicine, Department of Pathobiology, New Bolton Center, PA) has a special interest in viruses is preparing a paper to more specifically describe the histological lesions found in this case. He will compare the lesions seen with those seen in horses, and has included some photos of the histology.

Acknowledgements

The authors wish to thank the husbandry staff members of the New Jersey State Aquarium and the veterinary and husbandry staff of the Detroit Zoological Institute for providing observations and answers regarding husbandry conditions and for assisting in medical examinations and treatments. Paul Callé (Wildlife Conservation Society, Bronx, NY), Laurie Gage (Six Flags Marine World, Vallejo, CA), Frances M.D. Gulland and Martin Haulena (both from The Marine Mammal Center in Sausalito, CA) offered helpful suggestions for a rule out list. We also want to thank Amy Glaser at the Cornell Diagnostic Lab for the WNV testing of these and subsequent samples from the collections as part of the WNV Surveillance Program. Carolyn Cray from the University of Miami Comparative Pathology Diagnostic Lab was very helpful in interpreting the EPH graphs.