necropsy- killer whale kanduke

The Orca Project Corp 5/2/2012 www.theorcaproject.com

Necropsy Report Killer Whale (Orcinus-orca) Kanduke

Age 25 yrs — SeaWorld of Florida Name: Kanduke (male) (aka Kandu IV, Kandu 4, T9, T009) Species: Killer Whale (Orcinus orca) Source: wild capture, 08-16-1975, Pedder Bay, BC, Canada, age: est. 5 yrs Deceased: 09-20-1990, SeaWorld of Florida, age: est. 25 yrs Reported cause of death (per NMFS MMIR data): Viral Leptomenigitis Necropsy info: Histology- Mike Walsh, DVM, SeaWorld (1990): Histologic and bacterial examination of tissue have not pointed to an exact cause of death, though the opinion of the independent histopathologists who conducted the examinations is that a virus is the most likely cause. Additional independent studies including virus isolation and further histologic examination will be undertaken in order to attempt to pinpoint a virus as a cause of death. If additional pertinent information is obtained, it wi11 be added to the necropsy report. A gastric foreign body was found in the first compartment of the animal's stomach. While the presence of the object (a 55 x 20 x 13 cm collapsed fishing buoy) was visually striking, it was not related to the cause of this animal's death. This object did not appear to impede normal food intake or digestion. Since there was no chance of access to this object while at SeaWorld, it had to have been present prior to the time of the animal's arrival at SeaWorld of Florida. Initial results from first pathologist on 10-12-90 pointed toward a possible viral infection as evidenced by: 1) Meningoencephalitis 2) Lymphoid hyperplasia 3) Enteritis A second set of tissues were submitted to a second histopathologist. Initial phone results indicated similar findings and additional slides containing meninges were sent by the 1st pathologist for review. The second histopathologist's major findings included: 1) Leptomeningitis 2) Chronic cerebral and spinal chord perivasculitis


3) Lymphoid inflammation of intestinal tract Attempts at virus isolation included: Kent G. Osborn, DVM, Zoological Pathology Consult (1990, 1991) Gregory D. Bossart, VMD, Veterinary Reference Laboratories, Inc. (1990, 1991) Sam H. Ridgway DVM, NOSC (1991) Charles D. Buck, Animal Virus Collection Manager, American Type Culture Collection (1991) 1993 Update: Although not reflected or updated in NMFS MMIR, a 1993 study was published—“Isolation of St. Louis encephalitis virus from a killer whale” which indicates the death of Kanduke was caused by the St. Louis encephalitis virus with a mosquito vector. That report is attached at the end of this document. The 1993 published paper was co-authored by: Charles Buck & Grace P. Paulino- Virology Department, American Type Culture Collection Daniel J. Medina & G.D. Hsiung- Department of Laboratory Medicine, Yale University School of Medicine & Virology Laboratory, VA Medical Center Terry W. Campbell & Michael T. Walsh- SeaWorld of Florida, Orlando, FL More on the mosquito vector and virus: Whale and Dolphin Conservation Society: Documents Prove Mosquito-borne Virus Responsible for Captive Orca Death http://www.wdcs-na.org/story_details.php?select=275 Voice of the Orcas: Mosquitoes have killed 2 SeaWorld Orcas: Has Anyone Noticed? https://sites.google.com/site/voiceoftheorcas/home/the-current-story/mosquitoeshavekilled2seaworldorcashastheanyonenoticed Whale and Dolphin Conservation Society: WDCS And Partners Reveal Unseen Threat To Orcas In Captivity http://www.wdcs-na.org/story_details.php?select=305 Voice of the Orcas: John Jett & Jeffrey Ventre reveal "Death by Mosquito" at Marine Mammal Conference with WDCS https://sites.google.com/site/voiceoftheorcas/home/the-current-story/johnjettjeffreyventrerevealdeathbymosquitoatmarinemammalconferencewithwdcs


Poster presented by Dr. John Jett at 2012 Marine Mammal Health Conference- Sarasota, FL: Evidence of Lethal Mosquito Transmitted Viral Disease in Captive Orcinus orca http://www.wdcs-na.org/submissions_bin/fmmhc.pdf Notes: Prior to reforms of the Marine Mammal Protection Act (MMPA) in 1994, holders of marine mammals for public display were required to submit necropsy reports (animal autopsy reports) for deceased animals, making the documents available to the public and scientific community. Presently, marine mammal parks in the U.S. are only required to provide a “cause of death” to the National Oceanic and Atmospheric Administration (NOAA) National Marine Fisheries Service (NMFS) which maintains Marine Mammal Inventory Reports (MMIR). Details of marine mammal deaths are now a closely guarded secret at U.S. entertainment facilities. The Orca Project acquired the following documents from the National Marine Fisheries Service (U.S.A.) via the Freedom of Information Act for deaths that occurred prior to implementation of the 1994 MMPA changes. For more information visit www.theorcaproject.com Necropsy, Autopsy, Veterinarian, NOAA, NMFS, National Oceanic and Atmospheric Administration, National Marine Fisheries Service, MMIR, Marine Mammal Inventory Report, MMPA, Marine Mammal Protection Act, Killer Whale, Orca, Shamu, Death, Die, SeaWorld, Orlando, Florida, Kanduke, Kandu IV, Kandu 4, T9, T009, St. Louis encephalitis, virus, mosquito

D r . Nancy F o s t e r , D i r e c t o r O f f i c e o f P r o t e c t e d S p e c i e s and H a b i t a t

C o n s e r v a t i o n N a t i o n a l M a r i n e F i s h e r i e s S e r v i c e 1335 Eas t -Wes t Highway, Room 8268 S i l v e r S p r i n g , M a r y l a n d 20910

Dear D r . F o s t e r :

P l e a s e f i n d e n c l o s e d t h e p a t h o l o g y r e p o r t f r o m o u r 25+ y e a r o l d k i l l e r w h a l e 00-8701, who d i e d on September 20, 1990. As y o u a r e aware, t h i s a n i m a l had been m a i n t a i n e d a t Sea W o r l d ' o f F l o r i d a f o r t h r e e y e a r s s i n c e h i s a r r i v a l f r o m M a r i n e l a n d o f Canada. The a n i m a l had a n o r m a l c o n s i s t e n t f o o d i n t a k e d u r i n g . h i s s t a y a t Sea Wor ld , w i t h no a p p a r e n t g a s t r o i n t e s t i n a l p r o b l e m s . Age was e s t i m a t e d a t 25+ y e a r s b y c o u n t i n g g r o w t h - l a y e r - g r o u p s i n an a c i d - e t c h e d , 1 o n g i t u d i n a l l y b i s e c t e d t o o t h .

A f t e r a no rma l b r e e d i n g p e r i o d f r o m September 15-17, 1990, t h e a n i m a l showed a d e c r e a s e i n a p p e t i t e f o l l o w e d b y a r a p i d d e t e r i o r a t i o n o f i t s c l i n i c a l appearance ( s e e h i s t o r y on p a t h o l o g y r e p o r t ) and d e a t h on September 20, 1990.

H i s t o l o g i c and b a c t e r i a l e x a m i n a t i o n o f t i s s u e have n o t p o i n t e d t o an e x a c t cause o f dea th , t h o u g h t h e o p i n i o n of t h e i n d e p e n d e n t h i s t o p a t h o l o g i s t s who c o n d u c t e d t h e e x a m i n d t i o n s i s t h a t a v i r u s i s t h e mos t l i k e l y cause . A d d i t i o n a l i n d e p e n d e n t s t u d i e s i n c l u d i n g v i r u s i s o l a t i o n 'and f u r t h e r h i s t o l o g i c e x a ~ i l i n a t i o n w i l l b e u n d e r t a k e n i n o r d e r t o a t t e m p t t o p i n p o i n t a v i r u s as a c a u s e o f d e a t h . I f a d d i t i o n a l p e r t i n e n t i n f o r m a t i o n i s o b t a i n e d , i t w i l l be added t o t h e n e c r o p s y r e p o r t .

A g a s t r i c f o r e i g n body was f o u n d i n t h e f i r s t compar tment o f t h e a n i m a l ' s s tomach. W h i l e t h e p r e s e n c e o f t h e o b j e c t ( a 55 x 20 x 13 cm c o l l a p s e d f i s h i n g b o u y ) was v i s u a l l y s t r i k i n g , i t was n o t r e l a t e d t o t h e c a u s e o f t h i s a n i m a l ' s dea th . T h i s o b j e c t d i d n o t appear t o impede no rma l f o o d i n t a k e o r d i g e s t i o n . S i n c e t h e r e was n o chance o f access t o t h i s o b j e c t w h i l e a t Sea Wor ld , i t had t o have been p r e s e n t p r i o r t o t h e t i m e of t h e a n i m a l ' s a r r i v a l a t Sea Wor l d o f F l o r i da.

Sea World. Inc. 7007 Sea World Drive Orlando, FL 32821-8097 (407) 351 -3600 FAX (407) 345-5397

Busch Entertainment

0 * F 0' .-t .r*I":;tR c::;.. <...\'2"',,! '

S i n c e r e l y , a d & .

M i c h a e l T. Walsh, D . V . M .

OMB NO. 0648-0085, EXP 4/30fl1 SN: , ' ASN: LEX: SP: ANREP: FNUM:

FOR NMFS USE ONLY MARINE MAMMAL COLLECTION/INVENTORY REPORT

TYPE OF REPORT: ~nventory/mortality

NAME OF ANIMAL HOLDER: Sea world r Inc DATE OF REPORT: 11/19/90

SPECIES SCIENTlFlC NAME: Orcinus orca COMMON NAME: Killer whale

ANIMAL N A W DENIlFICATlON

- SWF-00-8701

2

n and a d d i d i o n a l r e v i e w o f b r a i n an6

I I I I I I I I I I I I

s E X M

TAKE TYPE

LM

-------

LOCATION OF TAKE PLACE NAME AND

LATITUDE-LONGITUDE

From Marinelanc. of Canada, Niagara Fallsr

Canada

EST BIRTH YEAR

AUTHOR DOCUMENT

#575

DATE TAKEN OR ACQUIRED

1-09-87

COLLECTOR OR SOURCE

N/A

d y l l ~ b l b pe a + \I' u c - v . . -

CURR STAT

D-C

DEATH OR DISPOSITION DATE EXPLANATION

9-20-90

Chronic non- su#fUrpt] v e I e p t o - - ne n a 1 t i s, c e r e b r a ~ e r i v a s c u l i t i s , i n t e s t i n a l lympho-

NECRP' FILED NMFS Yes

C . ~ l a s m a c y t i c , . i n f l a m

SEA WORLD

GROSS NECROPSY REPORT

. .

FACILITY: Sea World of Florida PROSECTOR: Walsh, Campbell, Buesse, Odell

GENUS/SPECIES: Orcinus orca

ID NUMBER: 00-8701 AGE: a+ SEX: Male

DATE OF DEATH: 9-20-90 DATE OF NECROPSY: 9-20-90

EXTERNAL MORPHOMETRICS: (metric only)

WEIGHT: 5,568 kq

TOTAL LENGTH: 675 cm GIRTH AT AXILLA:

GIRTH AT ANUS: See data sheet FLUKE WIDTH:

GIRTH AT UMBILICUS: DORSAL FIN HEIGHT:

HISTORY: This mature male killer whale was obtained from Canada for Sea World's breeding program in 1987. Upon arrival, the animal was noted have numerous worn teeth which did not seem to inhibit eating or normal activity. His overall behavior during this period was similar to breeding males of other species with periods of active involvement with the females during heat cycles followed by separation by the females during non-receptive periods. His health was stable with an occasional slight rise in his serum creatinine. Because of his potential age, dietary adjustments were made by the veterinary staff to avoid age related kidney disease as seen in most older mammals. Following a heavy breeding episode from 9-15-90 - 9-17-90, there was a slight decrease in his appetite and he avoided his caretakers. Attempts at obtaining a pool side fluke blood sample were unsuccessful, so the animal was placed in the medical pool. At this time it was noted that his attitude had rapidly deteriorated. Antibiotics were administered after the blood sample and the water returned to normal levels without incident. Before his planned second treatment it was noted that he was incoherent and not bothering to right himself in the water column. Assistance was provided by use of a stretcher to avoid water inhalation but the animal was nonresponsive and died shortly thereafter.

' I D NO. 00-8701

GENERAL EXTEMAL APPEARANCE: (oral cavity, external nares, skin, eyes) The animal's overall weight appeared grossly normal. The majority of the teeth in all four archades were chronically worn but showed no evidence of infection.

, . SUBDERMAL CONDITION: (blubber, mus'cles; lymph nodes) Blubber layers were adequate. There were no gross .abnormalities in the muscle groups. External lymph nodes were nonremarkable. Refer to morphornetric data sheet.

CRANIAL EXAM: (ears, melon, pterygoid sinus) Ears - no gross abnormalities Melon- II II

Pterygoid sinuses were clean

CENTRAL NERVOUS SYSTEM: (brain, pituitary, spinal cord) Brain - Sections were taken for histology and frozen. The brain was iixed in formalin for possible CAT scan and MRI. No gross lesions were noted.

THORACIC CAVITY: (pleura) No gross lesionswere found and the pleura was clean. There was no gross accumulation of fluid in pleural cavity.

UPPER RESPIRATORY SYSTEM: (nasal sacs, nares, larynx) An ulcerative lesion was found in the caudal exterior portion of the exterior blow hole. This appeared to be limited to a 1.5 x 3cm area where the blow flap contacts to the caudal wall. No other portion of the nasal passage was affected.

LOWER RESPIRATORY SYSTEM: (trachea, bronchi, lungs, lymph nodes) Tracheal epithelium was brown in color. . . .

CARDIOVASCULAR SYSTEM: (heart, aorta, major vessels) No gross abnormalities were noted, valves were clean, and no myocardial discoloration was noted.

PAGE .3 ID NO. 00-8701

ABDOMINAL CAVITY: ( lymph nodes). Abdominal lymph nodes appeared slightly enlarged- No abnormal fluid was present.

DIGESTIVE s~s~~~:(esophagus,stomach, intestine, cecum, rectum, lymph nodes) There ' was a circular- band of mucosal ulceration in the caudal, p o r t i o n of the esophagus. Cytology was n e g a t i v e for Candida. The stomach contained a 55 x 20 x lScm deflated, brittle, fishing buoy which was discolored dark brown. Raised lettering was present on the buoy. The buoy apparently o r i g i n a t e d from a Norway company. A dozen small stones and a small piece of wood 2 x 8cm were' found in the stomach. NQ ulcerations were present i n the stomach chambers. Numerous l-2m white circular areas were diffusely scattered through the duodenum resembling peyers patches,

LIVER: (biliary system) The liver tissue was normal in color though the serosa Was more opaque and khickened as is cccn in older animals. 'l'he texture of the liver parenchyma appeared fibrotic.

i

PANCREAS ! -. .- No g r o s s lesions.

SPLEEN: No g r o s s lesions, s erosa was thickened.

REPRODUCTIVE 8YBTEM: (testicles, ovaries) A normal mature male r e p r o d u c t i v e tract was found. A pen i l e scar 10 cm long w a s p r c c c n t on the left lateral surface, with no obvious problems noted.

URINARY SYSTEMr (kidneys, ureter, bladder, urethra) No gross lesions noted in any segment. Kidneys were normal in color and t h e u r i n a r y bladder c o n t a i n e d 20cc of urine. Urine color was straw colored.

ADRENAL GLANDS ? No gross lesions.

SKELETAL SYSTEM: No gross lesions.

PARASITE SUMMARY , None observed.

S P E C I A L TESTS issues were taken for virus isolation, metal and pesticide analysis.

ID NO. 00-8701

GROBB BUMMARY

1) Ventral pneumonitis - acute 2) Esophag,itis - ulcerative - chronic 3) Gastric foreign body - chronic

. ,

TENTATIVE DIAGNOSIS:

Open until bacteriology and histology. The rapid clinical decline of this individual would suggest an infectious or toxic influence.

CONCLUSIONS: (after histology & clinical pathology review)

See attached sheet.

- 7 d k DATE : - SIGNED: m&///dA I d-""t

Conclusions 11/13/90

Bacterial: There was no evidence of septicemia from tissue bacterial culture. Normal bacteria were found in the upper respiratory and colon area. ,

Histology: Initial results from first pathologist on 10-12-90, pointed toward a possible viral infection as evidenced by

1) Meningoencephalitis 2) Lymphoid hyperplasia 3) Enteritis

A second set of tissues were submitted to a second histopathologist. Initial phone results indicated similar findings and additional slides containing meninges were. sent by the 1st pathologist for review.

The second histopathologist8s major findings included 1) Leptomeningitis 2) Chronic cerebral and spinal chord perivasculitis 3) Lymphoid inflammation of intestinal tract

Additional tissues were requested on 11-13-90 which will be sent for processing.

CAT and MRI exam of brain - No lesions were noted.

Comments: The acute clinical decline of this animal suggested the possibility of an infectious or toxic etiology. The relative absence of any lethal lesions noted at necropsy also suggested that the diagnosis would depend on the histopathology evaluation. Findings by independent histopathologists resulted in similar findings with emphasis placed on the intestinal tract, brain and lymphoid tissue.

Unfortunately if the lesions noted are viral in origin, discovery of the exact virus may be difficult. Frozen tissues will be submitted for viral isolation. Additional examination of the brain tissue will be initiated by a third party in an attempt to locate other potential lesion sites.

The ventral pneumonitis and esophagitis while evident grossly were not serious enough to be related to the cause,of death. 'The foreign body recovered from the first compartment of the stomach, while visually surprising, did not cause the death of the animal. This is further evidenced by the fact that it was present for at least three years without any clinical signs of gastrointestinal abnormality.

Zoological ~ a t h o l o g y consult K e ~ i t G. Osborn, DVM

Histopathology Report , ,

Z P C # r 90SWMX :1OA Submitted b y ; Sea World, Florida D a t e Received: 10-04-90 '

D a t e Reported: 10-12-90

ID Species: Prcinus srca Common : killer whale Breed : House : 4h8701

S e x : male Age: adult

1. brain, cerebrum, cerebel lum, spinal cord, inflammation, perivascular infiltrates, lymphocytic infiltrate plus minimal neutrophil infiltrates, c a u s e undetermined, possible virus, see comment.

2. meninges, inflammation, lymphoid Infiltrate plus neutrophilic infiltrate

3. peripheral plus mesenikric 1j.mph nodes, interfbllicular lymphoid hyperplasia, c a u s e undetermined, possible virus, see cornmen t

4. spleen, lymphoid hyperplasia

5. trachea, submutasa, inflammation, mild plasma cell infiltrate plus rare neutrophils

6. intestine, possible duodenum, mucosa, T i b r o s i s plus mild, chronic intlammation

7. intestine, possible duodenum, serosa, mild acute inflammation

8. ec;ophagus, ulcer plus chronic intlammatian, lymphacytic infiltrate plus minirnal/mild neutrophil infiltrate, c a u s e undetermined, possible gastric reflux, see comment

9. skin, blowhole?, ulcer plus moderately severe chronic a c t i v e inflammation, lyrnphopla~macytic infiltrate plus neutrophils, etiology undetermined

10. heart, inflammation, lymphocytic plus neutrophilic infiltrate, see comment

~ i ~ r o s c w ~ i c fhrnrnery (slide number in parentheses)

T.haa following tissues were examined rnicro~copically and found t o b e essentially normal! brachial plexus (11,14), adrenal ( 1 3 ) , glandular stomach (141, and skeletal muscle (9).

,' Lesi'ons and/or notable microscopic findings a r e present , n the

following tiesue61 brain (cerebrum (11. cerebeldum with meninges (21) and spinal cord (2) : Perlvascular cuffs consisting primarily af lymphocytes with minimal o r n o accompanying neutrophils are present in the cerebrum section, in the cerebellar white matter and in the spinal cord gray matter. There is marked involvement of the cere.bellar meninges as well. Within these lymphoid aggregetes, the cells are medium-sized lymphocytes and there a r e rare mitotic Yiqures.

Dx: brain, cerebrum, cerebellum, spinal c o r d , inflammation, perivascular infiltrates, lymphocytic infiltrate plus minimal nautrophil infiltrates, cause undetermined, possfble virus, see comment.

D x t meninges, inflammation, lymphoid infiltrate plus neutrophilic infiltrate

prostate (31: T h e prostate itself has no recognized lesions, but there is a focal aggregate of lymphacytes in the periprastatic adipase/connectlve tissue.

testicle (3): There i s mild/moderate epermatogenesis, otherwise no lesions a r e recognized (NLR).

peripheral lymph node ( 4 ) I T w o lymph node sections present here have widely expanded cortex, due t o a reactive interfollicular lymphoid population. Follicles a r e relatively inconspicuous, being compressed by the large interfal licular population to an area just beneath t h e cortex. The follicles are not themselves obviously reactive. The interfollicular lymphoid population consists primarily of moderate t o m a d ~ r a t e l y l a r g ~ lymphocytes, admixed with smaller numbers of small lymphocytes and a-t lymphablasts. M i totlc figures are common in these areas, o n e o r more per oil immersion field. Medullary cords have'moderate lymphoplasmacytic populations.

D X I peripheral lymph node, interfoll lcular lymphoid hyperplaeia, cause undetermined, possible virus, see corrmen t Arr,+ivm< p: 'hn Try_? oroi-2:

\ A A A A A ~ ~ * ~ ~ ~ ~ ~ P ~ ~ c ) ~ ~ ~ P I * . . ~ ~ ~

luns (5.8L: There a r e occasional loose aggregates of amell lymphocytas within the parenchymal interstitial tissue, sometimes near bronchioles. CIlso present are scattered pigment-laden macropheges. T h e eubmucosa of s o m e bronchi contains mild plasma cell populations, Moderate congestion i s present in the section in s l i d e 8.

duodenum ( 6 ) I This section o f intestine, which came from a container labeled duodenum, has a marked lamina proprial population of plasma cells, as wall a s a lymphoid nodule in the deep mucosal lamina propria.

m B e e Th.is node ha% the s a m e appearance as the peripheral lymph node^.

trachea (7): In this section, the superficial tracheal mucosa i s missing, possibly due to artifactual loss. T h e aubmucosa contains a mild loose plasmacytic inf iltrete and scattered neutrophi 1s.

Dx I trachea, submucoea, inflammation, mild plasma cell infiltrate plus rare neutraphile

intestine. n o t otherwise specified (NOS) ( 7 ) 8 This section of tissue has e smooth muscle wall UP several layers and a mucasa that contains crypts lined by columnar e p i the1 ium. There a r e some submucosa 1 glanda w i t h cuboidal epithelium, suggestive of Brunner's glands, evidence that thih; may be a aectian af duodenum. T h e rnucosa is moderately heavily infiltrated by fibrous tissue which distorts the expected orientation and spacing of the crypts. In addition, there is a d i f fuse , mild mixed inflammatory mucosal infiltrate consisting of plasma cells, lymphacytes ond neutrophils. The csro6a

_ o f t h i s section has mild inflammation, with edema and a neutrophilic inflammatory infiltrate.

Dx: intestine, possible duodenum, mucosa, f i b r o s i ~ plus mild, chronic inflammation, see comment

Dx : intestine, possible duodenum, serosa, mild acute inflammation

skin, possibly blow hole (7)r This skin section is ulcerated on one side and contains chronic active inflammation, c o n s i ~ t i n g of 1ymphoplasmacyticinfiltratea admixed with neutrophils in the superficial dermis, plus large aggregates at neutrophils at the surface. No etiologic agents a r e recognized.,

Dx I skin, blowhole?, ulcer p l u s moderately severe c h r o n i c active inflammation, lymphoplasmacytic infiltrate plus neutrophils, etiology undetermined

kin (1918 This s k i n section has mild/minimal lymphoplasmacytic infiltrates in t h e superficial dermis.

spleen (12): There is moderate l ymphoid hyperplasia. without reactive follicles, suggesting expansion o f periarteriolar lymphcid sheaths. T h e cell populations are morphologically similar to those described in the lymph nodes, and have a$ similar amount of mitotic figures.

Dx: spleen, lymphoid hyperplasia

k idney (12) : The kidney cortex has widespread moderate tubular dilatation, suggestive of a "shock kidney". Glcimerular morphology varies, with often owollen, a t times hypercellular glomerular tufte, and variable hypertrophy of Bowman's capsule parietal epithelium. The Bowman's capsule basement membranes are variably thickened (minimal t o moderate).

1 iwer (13) r Mild centrllobular degeneration i s p r e s ~ n t in thi's section, otherwise NLR.

Avoah node NOS (13) r Morphology is similar t o that described for peripheral lymph nodes.

- t NO epithelium i s present in this section of esophagus. The luminal surface i s lined by a dense cellular infiltrate which overlies densely callagenaus, almaet hyalinized submucosa. The cellular infiltrate consists o'f a pleomorphic reactive lymphoid population, in which there are scattered mitotic Sigures;. Rare small aggregates of neutrophils are also present, generally near t h e luminal surface.

Dx r esophagus, ulcer plus ctironic inflammation, lymphocytic infiltrate plus minimel/mild neutrophil Infiltrate, cause undetermined, possible gastric reflun, see comment

heart (9): In this heart section there i s mild/moderate nuclear size variatian and mild/minimal intracellular myocardial' fiber pigment, probably lipafuscin. O f note a r e scattered aggregates of . inf lammatory cel Is that generally are placed only. around the margin of t h e section, giving some suggestion that they a r e artifactual "floaters" from other tissue sections. These aggregates consist of lymphocytes and nmaller numbers of

1 ,rr,+ivmi p: 1, err,. Oroi~P,: \ A A A A A ! . * ~ ~ ~ ~ ~ P ~ ~ C ) ~ ~ ~ ~ : . ~ ~ ~

neu t roph i 15, admixed w i t h e ry th rocy tes .

Dx : hea r t , in f lammat ion , lyrnphocytic p l u s n e u t r o p h i l i c i n f i l t r a t e , see comment

Cornmen t The disease process recognized i n these s e c t i o n s t h a t i s most

l i k e l y r e l a t e d t o t h e an ima l ' s death i 6 a widesprmad neneuppurat ive men ingoencepha l i t i s . The predominant ly lymphoid n a t u r e o f t h e i n f lammatory i n f i l t r a t e s i n t h e ce.ntra1 nervous system p a i n t e away from a b a c t e r i a l cause, g i v i n g evidence, i n s t e a d f o r a c e l l mediated r e a c t i o n . Fu r the r suppor t f o r a ce l l -med ia ted immune response, which c o u l d occur i n a p o t e n t i a l v i r u s i n f e c t i o n , i s t h e n a t u r e o f t he lymphoid hyperp las ia t h a t i 6 described in t h e lymph nadea and spleen. This r s a r t i a n appears ta be o c c u r r i n g i n areas t h a t a r e the normal homing s i t e s af T lymphocytes. 1 have seen s i m i l a r r e a c t i o n 5 i n o ther an imals w i t h systemic v i r a l i n f e c t i o n . 4s D r . Walsh and I discussed over the t e l e p h ~ n o , d e f i n i t i v e e t i o l o g i c a l d iagnos is is d i f f i c u l t . I agree w i t h Dr. Walmh's s ~ ~ g g ~ ~ t i n n t h a t e;nrnlngy t ~ q t i n g fnr ~ n r ~ p h r s l n r n y n r a r d i t i c l vf t -11~ AS

an i m p o r t a n t r u l e o u t . Beyond t h a t , c u l t u r e f o r a mystery v i r u s can be ex t remely e l u s i v e . I wcruld recommend ma in ta in ing t h e f rozen ~arnp lee o f t i s s u e ( b r a i n and lymph node i n p a r t i c u l a r ) a t -70 degrees F u n t i l such t ime as c u l t u r m a t tempts migh t be c a r r i e d out,

From t h e v e r b a l h i s t o r y 1 r e c e i v s d from Dr. Walsh, t h e esophagus s e c t i o n w i t h u l c e r a t i o n probably c o r r e 6 p ~ n d s t o t h e

'distal esophageal u l c e r recognized a t necropey. The g r o s s l y I - e e e g ~ i r e d rthiCe Qsei i~ +he duadrntlm p~.nhnhlr,a wmra bko rnrolc a t lamina p r o p r i a l f i b r o s i c descr ibed i n the s e c t i o n present hero. These may be the s i t e s o f p rev ious i n f l ammat ion and p o s s i b l e u l c e r s t h a t have s i n c e healed. T h e h e a r t s e c t i o n in f lammat ion , as mentioned above, Ls o n l y p resent a long the edges, making i t d i f f i c u l t t o say w i t h essurance t h a t these in f lammatory aggregates w e r e a c t u a l l y assoc ia ted with the h e a r t . I w i l l g e t recuts o f t h i s s l i d e , and have a s k e d D r . bdalsh t o send an a d d i t i o n a l h e a r t s e c t i o n .

VETERINARY REFERENCE LABORATORIES

Dr. M I C ~ S P ~ TI k ' a l s h S e a Wcrld 7087 S e s W o r l d Crlve Clrlando, FL

Dear Lt. Y s l s k , :

I recently reviewEd t h e hist~pathtlogy o f a case f r o n t h e U n ~ v e r E ~ t y o f Miami D c p a r t ~ ; ~ n ' t o f C c t ~ p a r a t i v e Path01 o g y (CF-9B- 4540-10 g l a s s $ l i d e s ) ana a case identified f r o n t h e San b l e g o Z o o (RP-43E3-1 g l a s s 5 3 a d a ) . T h e s e s l i d e s represented t ~ s s u e s from a v e p ~ r t e d pale killer w h a J ~ ,

Case RP-4323 c n n t a j n s t w o 9 e c t i a r 1 s o f c e n t r a l n e r v o u s system t i s s u e ~ h i l e CF-08-4540 c o n S a a n s t i c s u e & ft-om t h e central end peripheral nervous s y s t e a s , gartr@in+estinal trac* aneluding 1 ~ v e r * , praaary lyrrphoic! t i s s u e s , - s k e l e t a l muscle , a d r e n a l gland bnd v s r i o u s epitheljal tissues some raesemb1ing possible s u p e r f i c i a l t i s s u e s o f t h e nasal sac r e g i o n ,

R s one o f many consultants on t h i s case 1 w i l l review o n l y t h e significant macroscopic l e s i o n s .

T h e lepraaeninges of' t h e cerebellum o f f?P-4323 are characterized by moderate m u l t l f oral in91 l t r s t e s o f primarily 1 y m p h a c y t e s , p l a s r a cells, and sparse numbers 17 h i s t i o c y t r s and neutrophiis. f h o r o 1s s l s o miJd nultlfocal her~rrhege. Meningeal tissue i s not present w i t h the o t h c ~ CNS t i s s u e submit ted .

7he cerebrum and s p i n a l rord g r a y matter have m5ld t o ~ s d @ r a t e perlva5culer c u f f i n g ~f priaarlly l y n p h a c y t e r and p i a s p a cells. Gcrasi tna3 spinal c o r d neurons c o n t a i n 1 ipofuscin and there i c mild multifocal hemoPrtiage.

The single sect103 o f s m a l l i h t e r t i n e eKanineb 1s characterized by b o t h e ~ t e n s l Y R superflcicl and d e e p l a n ~ n a praprjal- populations o f l y ~ p h o i d cells includina l y n p h ~ b l a s t s , well- di f f e r e n t i a t c d 1 y @ p h b ~ ' j t e b srtd p l a ~ n a eel 1 5 .

D r e Michael T . Wafsh t4c~vernber 13, 1 9 4 8 p a g e 2

mvltlple ~ e c t ~ o n 5 tf lymph node and ~ p l e e n are r~:amaned. Some ' l y m p h n c d e ~ ar-e reP,&rkahly abncrmal charac,teri z e d b y l ymphoid d e p j e t ion o r both g e t - s ; ~ r , a l fell z c 2 e s ant: parafolllcular r b n e s , Medullary 5 1 n u s r s in some n o d e s contazn numerous h i s t i o r y t e s and e ~ s ~ n o p h ~ l s . One n o d e has moderate wultifocal hemosiCerosis w i t h porafollicuJar hyperplasaa. The s p l e e n hss rcgderate reactive c f r a n 3 ~ s and fccal t l 5 ~ c 5 i d e ~ t s : 5 ,

T h e 1 I v e r has m i I d centrilobular vacuolar d ~ 5 e n e r a t ion o f h e p a t o c y t ~ s and m ~ l d extramedul!ery hehatopoiests.

Tf-rc f > r s t and s e r h n C b t @ a S c h coinpar.tnfr~ts a r e characterize6 by m r l d t o a o d ~ r a t e m u l i ; i f o c a l superfacial submuc@sal f j m p h ~ p f a ~ a & c y t > ~ i n f ~ l t r a t e ~ . There 1s a l s o f ~ c a j R U C O S ~ ~ Prorron, vacuolar degeneration and w i l d lyprphocytic e ~ o c y t o s a ~ in one section of first stcraach clr distal esophagus . The third stomach comparte fr l t has similar m i 1 6 chronic ~ n f l a m m a t ~ ~ n ,

Sectlonb o f nonintegumsn~ary epithelia3 ti s s u e s h a v e moderate d i f f u s e vacucmlar epithellsl degeneration Nath n o d r r a t e associated submuccrsal rnf iltrates DT pramari 1 y lymphocytes, histiocytes and p l e s n a cells. Methenamane Y i 1 cler s f d i n s demonstrate numerous small s e p t a t e , occaoionally branching, fungel hyphae jn only the supcrP1ciaJ epithelial layers and n n t generally asscciated ~ i t h rnf l a n n a t o r y cell ~ n f iltrate5.

7he h c a r - t has rriId niultifccal arrt~rstitisl f i b v r . 5 1 ~ and t h e t c s t i c l t i s are characterized by seninifertus tubular atrophy. Dnly occasional tubular l u n e n s c b n t a i n mature spermatozpa.

7 h o section o f lung examined has mild m u l t ifccal i r l t E r s t i t i a J inf i l traters t f p r i m a ~ r l y l y e p h o i d cells, There is also f c r e l p u l ~ o n a r y h e n c r ~ - h a g € ,

The a d r e n a l g1;hd h ~ s c o d e r a t e diffuse lipid depletion prinarily o f t h e zona g l o n ~ r u l c s a Gnu fhsicklts.

) Page 2 o f 3

F r o m t h e t ~ s r . u e a , e ) : a n i n ~ d the cause o f t h i 6 whale's death 1 3 , a t hf'z t , speculat 1 : ' ~ . Rddlticnal CNP t i s s u e w i t h a s s o c ~ a t e d a r r l ~ r ~ g o s 5t,ou!d t e e x e s i n ~ d t o t r y t o 6 r c c r t s i n t h e e x t ~ n t and p o 5 3 ; t . 1 ~ ~ t l o l t)gv ~f t h e ci-~ronic fir*,-sgppurative l a p t o m e n i n f i ~ t s ant5 rt'~r*on~c r e v e t :'<I] ant2 s r ~ n a 1 E C Y ~ p e r i v & ~ c u 1 i t 1 5 . Whi 1 e e ? i c , ! o g ~ t egerli;s a v e r i o t prhsent I ~ I t h e s e tisclcres, t h r h i r t t l o g i c ~ a t t p r r i c ~ . ~ y r ~ c ~ t / l . ~ e c f a bjral c- r - s o m e p r o t t ~ : o z n l n f e c t ~ ~ r ' ~ ~ .

t l c t c t l c - ~ ~ . f ~ f ~ . ~ ' l $ f r ' l c C , I S S U P s h o u l d a160 br e u a m l n ~ d tc, try t c , c ? c t i ~ - o l n c Yt'!e c h ~ l n g ~ f y r e s e t ~ t r * ~ f l r c t mat-C.~c'i gut- 35 s c c ~ , ? t e c ' f c c n l 1 y f i p h c ~ l d h y ~ e r p l a s ~ a or a c51ffuse 1 ) a ~ p r , o p j a s r : z , t sr ~faf l a n a ~ t c r ) . f r c c e s s .

t h s C t i 3 G , ~ I E / O Y e f i t E P I C l ~ s j o t ; ~ W F ~ S ~ x t o n s i v ~ , funct i c lna l P I D P I S F c s f S ~ I E a s 5 c i z i i , t ~ 6 organs aey h a v e tee13 c o r ~ t r ~ b u t o r b ~ t o r s i r l thrs a ~ i r n a l ~ s death.

Page 3 o f 3

March 15, 1991

Dr. Nancy Foster Director National Marine Fisheries Service ,

Protected Species and Habitat Conservation 1335 East West Highway Room 8268 Silver Spring, MD 20910

Re: Necropsy report/SWF-00-8701

Dear Dr. Foster:

Our letter dated November 19, 1991, which transmitted the gross necropsy and pathology reports for the Killer whale identified as SWF-00-8701, stated that additional histologic and viral studies would be conducted in an attempt to pinpoint a specific virus as the cause of death of this animal.

These studies (attached), have been completed. Visual examination of the formalin fixed brain and re-examination of other brain sites histologically were conducted by three independent investigators. A fourth independent specialist received samples of all tissues for in-depth viral screening. A causative virus w'as not detected by the histopathologist or isolated by the viral specialist.

Therefore, the diagnosis made in the necropsy report conclusion dated 11/13/90 remains unchanged.

Mike Walsh, D.V.M.

Sea World, Inc. 7007 Sea World Drive Orlando, FL 32821-8097 (407) 351 -3600 FAX (407) 345-5397

k s c h Entertainment Corporation ON( Or It![ IN I ICUSLI I HI>\, I s COVIILNIL>.

Za~lqJiroI Pathology Co~rru l t Kent 6. O s b o m , DVM

H i 8 tupathalagy Report

ZPC # r 90SWMX.lOR t7DDENRUR Submitted by1 S w a World, F l a r t d a Date Reported; 3-12-91

ID Species; QrcJnus orca Sex: male Common t killer wheile Age: a d u l t RI-eed : House r 4fi87O1

c)-i a Q-

b r a i n , cerebrum, cearebel lum, i n f lammatian, perivaseular Infiltrates, lymphocytic inf i l t r e t c p l u s minimal n a u t r o p h i l 1nflltratBsp cause undetermined, possible virus. s e n c o m m e n t .

R d c J i t i o n ~ l brain sections submitted ta m r by Dr. S a m Ridgway. conta in l e s i o n s s i m i l a r t o thwre a l r e a d y described in the i n i t i a l report o n October 12, 1990. &da$tional hear t sect ions submitted ta m e by Dr. Mike Welsh have no recognized les ions. Thw i n i t i a l l y recognized p e r i p t ~ e r a ) Inflammatory i n . f i 1 t r e t e probably w Q r a "floaters':, as suspec'teb shd suqgemtad when those rections were i r ~ i t i b l l~ d n c ; c r r i b ~ d . W i t h no slgnif i c a n t d i f f s r e n c e m in the b r a i n Sect ions, the original r.uptrrt s t a n d s , in which thig bnimal's pr.irnary. problem i s a n o n s u p p u r a t i v e neningoencephali tls, v e r y likely of v i r a l etiology.

March 8 , 1901

Dr. Hlcheal T. Walsh Sea World 7007 Sea World Drive Orlsnbo, F l o r i d a

Pear Dr. Walsh:

I recent ly revjeved the hlstoputhology of a t ¶ d i t i ~ n r l Centtad nervoue system tissue fxom a k i l l e r whale (BW-00-6701). HY original p a t h o l o g i c i r n p r e ~ ~ i o n remain6 essentially unchanged,

The cerebellar leptomeninges +re cbatacterieed my m i l d t o m o d e r a t e rnul t i foca l I n f i l t r a t e 8 of primarily lymphooytea a n d o c c a s ~ o n a l platma cells and hiutlocytes, There $8 8160 m i l d multifocal meningeal congestion and hemorrhgge,

The brainatem, cerebrum, end cexebel lum are characterized by mild to moderate perivascvlar infiltrate6 of both Jerge and emall ~ y m p n o c y t e ~ and occasional piasma cells and histiocytea, S c a t t e r e d neuron6 contain lipoSu$ci~+ I n addition, there are infrequent pyknot i c neuron8+ This l 3 often associat .ad with anoxia.

The diagnosis In t h i s came remains unchanged, The e t i o a o g y of the chronic non-suppurative leptCUncnlzIgiti6 and ce,xbbral, cerebs31arr and brain stem mononuclear pcrivasculitis i s unknovn. Infsct ioue agents were not ddentifled In the tlssue sections examined. The hietologdc ? a t t e r n 01 those lesions i e suggestive of ei v i r a l Infmctfon, however distinctive v i r a 1 inclusion bodies were no t ot~acrved,

I f y o u have any additivnal q u e s t i o n s p l e a a s do n o t hesitate to c o n t a c t me.

o s s a r t , V . M . D .

. . Dr. Mike Walsh .. . : ': . . : veterinarian . . .

Sea world of Florida . . ' ; 7007 Sea World Drive Orlando, Fl 3 2 8 0 9 . ! . . .

Fax: 407 363 2316

Dear Dr. Walsh: 1 i I ThanK you very much for giving m e the opportunity to

examine the brain of your Male Orca (673 cm i n lehgth and 5568 kg died of 2 0 September, l99Omz 1 have a e ~ t i o n e d the b+in and have, as yet, found no certain sbnormalitles. The aura over the convexity hemispheres appeared relatively opaque and thickened compared ts other normal killer whales brains that I have exdined. This may have simply been the result o f the whales advanced age or to i n f e c t i o n present at death or at earLier times, There was no gross evidence of inflammation or swelling on the surface of the brain tissue- X trust that the histological epecimene eubmittsd to pathologists will clarify the issue of infectLen.

Sincerely,

I Sam H. Ridgway D m , PhD NOSC i Code 5107 San Diego, CA 92152 Fax: 619 553 1355 Tell 619 553 1374 .

American 7Jpe Culture Collection

March 15, 1991

Terry W. campbell Sea World 7007 Sea World Drive Orlando, FL 32821

Dear D r . Campbell::

We have completed our initial screening of the frozen necropsy samples you provided and have isolated no virus. This screening conaisLs uf Lwo passages in GP 1 (dolphin) c e l l u , PrMK (morrkey) cells, HiLu (mink) cells and CE (9-10 day old chick embryos). We chose not to filter these samples prior to first passage and several (as shown below) became contaminated (4s evidenced by growth of presumed bacteria on blood agar plates) . Supernatants from the first passage were filtered through a 0.45 urn filter prior to second passage.

Sea World h o p s y samples for !ins Isolatioa

4A870i heart 4A8701 spleen (A8751 l ~ v e r 4b6791 kldney (A87CI d ~ o d e n u t I A 8 1 C l ~ r a i n 4ARlOi ~esenteric iynpb 4AP;O: I y l p n node (A8701 pancreas 4A870i !ung

9 : ? t i e s a r p l e nas oniy been passased once on C3. 6 A i t . Grcvth cn b i m d aaar. - . :I .;oils ~ b n o r m a i [SF-i or. i s t passaga)

Only two of the samples gave any indication t h a t a viral agent might be present. The first passage brain for 4A8701 caused questionable cytopathic effect on the SP-1 cells but we have found neural tissues often produce toxic effect on cell culture and the second passage was normal. The second passage kidney produced questionable hemagglutination on passage 2 in CE but a third passage was normal.

We w i l l , a s part of my rcsearch, cunLinue to test the above mentioned samples and will let you know if any v i r u s are l a t e r i c o l a t e d . Thank yo for the opportunity to work with you on these samples .

S i n c e r e l y ,

Charles D. Buck Animal Virus Collection Manager

Clinical and Diagnostic Virology, 1 (1993) 109-112 © 1993 Elsevier Science Publishers B.V. All rights reserved 0928-0197/93/$06.00

DIAVIR 00011

Clinical and Diagnostic Virology

Isolation of St. Louis encephalitis virus from a killer whale

Charles Buck a, Grace P. Pau l ino a, Daniel J. Med ina b.c, G.D. Hsiung b,c, Terry W. Campbel l d and Michael T. Walsh d

a Virology Department, American Type Culture Collection, Rockville, MD 20852, USA, b Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT, USA, c Virology Laboratory,

VA Medical Center, West Haven, CT, USA and d Sea World of Florida, Orlando, FL, USA

(Received 20 November 1992; revision received and accepted 26 January 1993)

Abstract We report the isolation of St. Louis Encephalitis (SLE) virus from a mature male

killer whale (Orcinus orca). This represents the first isolation of SLE virus from a marine mammal. The animal presented with reduced appetite, rapidly became lethargic and subsequently died. Virus-induced CPE was observed in a dolphin cell line, SP-1K (ATCC CCL 78), inoculated with brain, kidney, and lung tissues obtained at necropsy. Electron microscopy of infected SP-1 K cells revealed the presence of virions having morphology and size resembling members of the Flaviviridae. Final identification as SLE virus was made by neutralization and immunofluorescence staining tests.

Key words: Flavivirus; Orcinus orca; Encephalitis; Marine mammal

Introduction

Case Report

A mature male killer whale (Orcinus orca), estimated to be 25 or more years old, presented with reduced appetite. Within 24 h, the animal became lethargic. The animal died in September, 1990, within 48 h after the onset of clinical signs, while under veterinary care. The necropsy was unremarkable, but the histopathology examination reported a non-suppurative meningoencephalitis.

Laboratory Studies

Virus isolation. Frozen samples of brain, lung, kidney and several other organs were sent to the ATCC Virology Laboratory as part of an ongoing screen for viruses of cetaceans. All tissues were thawed, mixed with sterile sand and tissue culture medium, and aseptically ground with a mortar and pestle. The suspensions were clarified by low-speed centrifugation (500 x g for 10 min) and the supernatants were used to

Correspondence to." C. Buck, Virology Department, ATCC, 12301 Parklawn Drive, Rockville, MD 20852, USA.

Archived at The Orca Projectwww.theorcaproject.com

110

inoculate primary African Green Monkey kidney cells, SP-1K (a dolphin cell line, ATCC CCL 78) cells and bovine turbinate (ATCC CRL 1390) cells. Upon initial inoculation, bacterial contaminations were noted in all cultures. All first passage cultures were frozen, thawed and blind passaged on fresh cells in the presence of 100 #g/ml gentamycin sulfate. Possible CPE was noted in SP-1K cells inoculated with the brain, kidney and lung samples. These samples were passed through a 0.2-#m filter and a third passage was made on SP-1K cells. A definitive CPE was then observed from the brain, lung and kidney samples; cells became rounded and refractile and detached from the flask. No further attempts were made to isolate the agent from the bovine turbinate and primary monkey kidney cells.

The bacterial contaminants, although not identified, do not at this time appear related to the animal's death. Many of the necropsy samples received by ATCC for viral screening from cetaceans have bacterial or fungal contaminants. This is due in part to the cetacean's large size and insulating layer of blubber which make it impossible to quickly cool the animal after death. As a result, bacteria from the intestine and other non-sterile areas quickly multiply and spread throughout the peritoneal cavity.

Electron microscopy. When infected SP-1K cells showed distinct CPE, they were removed from the flask and fixed with 2% glutaraldehyde for two hours. Fixed cell suspensions were sent to the VA Virology Laboratory in West Haven, Connecticut by overnight mail. The cells were postfixed in osmium tetroxide and embedded in Spurr's resin as described previously (Hsiung, 1982). Thin sections were cut, stained with uranyl acetate and lead citrate, and examined with a transmission electron microscope (Fig. 1). For each of the three (brain, lung and kidney) SP-1K samples, numerous enveloped virus particles 40-50 nm in size were seen in the cytoplasm. Each particle had an electron-dense core 25 nm in diameter; virus particles budding from the plasma membrane were also observed (Fig. 1, inset). With the noted size and morphology, the isolate was tentatively identified as a member of the Flaviviridae family.

Final identification. A virus preparation propagated from the brain sample, having a titer of 104.5 tissue culture infectious doses (TCIDso) was inactivated by exposure to 20% chloroform for two hours. A neutralization test on the same virus preparation was performed in SP-1K cells. Antisera to Japanese encephalitis virus (ATCC VR- 1259AF) suppressed the appearance of CPE for 72 h, but CPE subsequently devel- oped. In contrast, antisera to St. Louis encephalitis virus (ATCC VR-1265AF) com- pletely neutralized the infectious virus through the entire 7 days incubation. In addition, acetone-fixed infected and non-infected SP-1K cells were sent to Dr. Karabatsos at the CDC Arbovirus Diseases Branch of the Division for Vector-Borne Infectious Disease, CDC in Fort Collins, CO, for confirmation. Using monoclonal SLE type-specific antibody (Roehrig et al., 1983), the three isolates were identified as St. Louis encephalitis virus by the immunofluorescent staining test (Nick Karabatsos, personal communication). It should be noted that, although two strains of SLE are available through the ATCC, neither has been propagated at the ATCC during the past four years that the marine mammal screening program has been active. Consequently, it is very unlikely that the three SLE isolates, from a single animal, were the result of cross-contamination from existing stocks.


111

Fig. I. Electron micrograph of SLE virus-infected SP-1K cell showing numerous cytoplasmic virus particles. Inset: virus budding into cytoplasmic vacuole.

Discussion

We have been unable to find any reference to SLE virus infecting marine mammals in the current literature (McLean and Bowen, 1980; Buck and Schroeder, 1990; Kennedy-Stospkopf, 1990; Monath, 1990). Since 1933, when the SLE virus was first isolated from a human brain, there have been many recognized epidemics of St. Louis encephalitis in the United States. The clinical cases associated with these outbreaks, and the intervening endemic periods, have been most frequent in California, Texas, Florida, Ohio, Illinois, and Indiana (Monath, 1990).

SLE is a member of the Japanese Encephalitis (JE) antigenic subgroup of the Flaviviridae (Wengler, 1991). Wild birds appear to be the primary (maintaining) vertebrate host, and the virus is transmitted by mosquitoes from infected viremic birds to susceptible birds. When conditions are suitable (large numbers of viremic birds and large numbers of mosquitoes), transmission to incidental hosts such as humans and horses occurs. Although several members of the JE subgroup are


112

pathogenic for humans, only the prototype JE strain is economically important as a disease of domestic animals (Fenner, 1987). SLE virus does not cause clinical disease in horses and other domestic mammals (Monath, 1990). Even in humans where SLE can cause severe encephalitis, many cases of SLE go undiagnosed or are subclinical (Monath, 1990).

In the present case, the killer whale apparently contracted SLE virus during a recognized SLE outbreak in Florida (CDC, 1990a,b). It is likely that, as with humans, killer whales are incidental hosts in the SLE cycle. Other cetaceans in the vicinity of the infected animal showed no illness consistent with SLE and were seronegative for SLE antibody (data not presented). The absence of reports associating illness or death of any marine mammal with SLE or other member of the Japanese encephalitis group further suggests that infections of marine mammals by infected mosquitoes are quite rare or, as in the case of many terrestrial mammals, most SLE infections are subclinical.

This report of an unexpected isolation of St. Louis Encephalitis virus from a cetacean demonstrates the importance of viral screening in animal species where the knowledge of viral pathogens is limited. The EM examination provided a rapid presumptive identification of an unknown agent which was confirmed by antibody neutralization and immunofluorescent staining tests. Studies are currently under way to identify two additional agents recently isolated in cell culture from other marine mammal specimens.

Acknowledgements

This work was funded in part by Sea World, Inc. The authors thank Sigrid Klein for her assistance in the Electron Microscopy.

Sea World of Florida Technical Contribution 9213-F.

References

Buck, C. and Schroeder, P. (1990) Public Health Significance of Marine Mammal Disease. In: L. Dierauf (Ed.), CRC Handbook of Marine Mammals Medicine, CRC Press, Boca Raton, pp. 163-173.

CDC (1990a) Arboviral Surveillance-United States, 1990. Morbidity and Mortality Weekly Report, 39, 593-598.

CDC (1990b) St. Louis Encephalitis-Florida and Texas, 1990. Morbidity and Mortality Weekly Report, 39, 757-759.

Fenner, F., Bachmann, P., Gibbs, E., Murphy, F., Studdert, M. and White, D. (1987) Veterinary Virology, Academic Press, New York, pp. 470-471.

Hsiung, G.D. (1982) Diagnostic Virology, Third edition, Yale University Press. New Haven, pp. 71-75. Kennedy-Stopkopf, S. (1990) Viral Diseases of Marine Mammals. In: L. Dierauf (Ed.), CRC Handbook

of Marine Mammal Medicine, CRC Press, Boca Raton, pp. 97-114. McLean, R. and Bowen, G. (1980) Vertebrate Hosts. In: T. Monath (Ed.), St. Louis Encephalitis, American

Public Health Association, Washington DC, pp. 381-450. Monath, T. (1990) Flaviviruses In: B. Field and D. Knipe (Eds.) Virology, Raven Press, New York,

pp. 763-814. Roehrig, J., Mathews, J. and Trent, D. (1983) Identification of epitopes or the E glycoprotein of Saint

Louis encephalitis virus using monoclonal antibodies. Virology, 128, 118-126. Wengler, G. (1991) Flaviviridae. In: R. Francki, C. Fauquet, D. Knudson and F. Brown (Eds.),

Classification and Nomenclature of Viruses: Fifth Report, Springer, New York, pp. 223-227.
