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A Second Captive Orca, Taku, Dies from Virus Transmitted By Mosquito Vector

A Second Captive Orca, Taku, Dies from Virus Transmitted By Mosquito Vector

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Published by jmventre
Shockingly, another SeaWorld orca, Taku, has died from a mosquito transmitted virus. As confirmed in this peer reviewed scientific journal article, in 2007, this killer whale died rapidly at a young age from the West Nile Virus. This is significant as it proves that the death of Kanduke, in 1990, also by a mosquito transmitted St. Louis Encephalitis, was not an anomaly.
Shockingly, another SeaWorld orca, Taku, has died from a mosquito transmitted virus. As confirmed in this peer reviewed scientific journal article, in 2007, this killer whale died rapidly at a young age from the West Nile Virus. This is significant as it proves that the death of Kanduke, in 1990, also by a mosquito transmitted St. Louis Encephalitis, was not an anomaly.

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Published by: jmventre on Oct 15, 2011
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West Nile VirusInfection in Killer Whale, Texas,USA, 2007
Judy St. Leger, Guang Wu, Mark Anderson,Les Dalton, Erika Nilson, and David Wang
In 2007, nonsuppurative encephalitis was identi
edin a killer whale at a Texas, USA, marine park. PanviralDNA microarray of brain tissue suggested West Nile virus(WNV); WNV was con
rmed by reverse transcription PCRand sequencing. Immunohistochemistry demonstratedWNV antigen within neurons. WNV should be considered incases of encephalitis in cetaceans.
W
est Nile virus (WNV) is a single-stranded RNAvirus of the genus
Flavivirus
that is transmitted by mosquitoes. In humans and animals, WNV has beenassociated with a spectrum of clinical conditions fromasymptomatic infections to sudden death. These have beenidenti
ed in a variety of animal species. Among marinemammals, WNV infection has been reported in a harbor seal (
Phoca vitulina
) (
1
). We describe WNV infectionin a killer whale (
Orcinus orca
) and seroprevalence inconspeci
c cohort and noncohort groups.
The Study
In 2007, a 14-year-old male killer whale at a marine park in San Antonio, Texas, USA, died suddenly withoutnotable premonitory signs. On gross examination,mild multifocal meningeal hyperemia and petechial parenchymal hemorrhage were noted in the right cerebrumand cerebellum. The left hemisphere of the brain appearednormal. Focally extensive tan discoloration and
 brosiswere present in the right accessory lung lobe with associatedhemorrhage and congestion. Both lung lobes were mildlyand diffusely heavy and wet. All thoracic and abdominallymph nodes were moderately enlarged and edematous.The second gastric chamber displayed numerous chronicand active ulcerations of 1.5–2 cm. Fresh and buffered 10%formalin-
xed specimens were collected. Fresh tissues werestored at –80°C. Tissues
xed in 10% buffered formalinwere processed routinely and stained with hematoxylin andeosin for histologic examination.Histologic review demonstrated moderate multifocalsubacute vasculitis and nonsuppurative encephalitis.In
ammatory lesions of the central nervous system werefocused in gray matter of the medulla oblongata, pons,mesencephalan, and cerebellum. Lesions were bilateral but more severe on the right side. Meninges demonstratedmoderate focally extensive and multifocal areas of acutemeningeal congestion and hemorrhage. Mild multifocallymphocytic in
ltrates expanded the leptomeninges.Blood vessels demonstrated mild to moderate acutenecrosis and lymphocytic and contained plasmacytic andneutrophilic in
ltrates within vascular walls. Encephalitiswas characterized by perivascular lymphocytes and fewer  plasma cells expanding the Virchow-Robbins spaces.Small, scattered, perivascular ring hemorrhages werenoted. A few multifocal loosely arranged glial noduleswere within cerebral white matter.Predominant lesions in the lungs were areas of chronic and active abscessation amid a focally extensivearea of mixed in
ammation and
 brosis. There wasmoderate diffuse acute pulmonary edema and congestion.Gastric ulcerations were present in the
rst gastricchamber and were chronic and active. They werecharacterized by central ulcerations with necrosis anda mixed in
ammatory in
ltrate surrounded by variable
 brosis and a rim of epithelial hyperplasia. Changes inspleen, lymph node, and kidney included acute edema,congestion, and vascular dilation.Conventional diagnostic assays were performed for aerobic, anaerobic, and fungal microbes in liver, lung,kidney, cerebrospinal
uid, and brain. All yielded minimalgrowth of 
 Escherichia coli
.The
nal diagnosis was fulminant peracute bacteremiaand septicemia secondary to a primary viral infectionassociated with nonsuppurative encephalitis. Publishedetiologic considerations for cetacean nonsuppurativeencephalitis include morbillivirus and protozoal infections(
2
). A DNA microarray with highly conserved sequencesfrom >1,000 viruses was selected to screen for known andnovel viruses (
3
). Total RNA was extracted from brain tissueand hybridized to a microarray as described (
4
). Analysisof the resulting hybridization pattern demonstrated a stronghybridization signal to many oligonucleotide probes onthe microarray from the family
Flaviviridae
, in particular to WNV. Consensus reverse transcription PCR primers(
5
) targeting WNV were used to con
rm the microarrayresults. Sequencing of the 261-bp amplicon (GenBank accession no. HQ610502) yielded a sequence with 99% ntidentity and 100% aa acid identity to WNV strain OK03(GenBank accession no. EU155484.1), a strain originallyidenti
ed in Oklahoma, USA.
Emerging Infectious Diseases www.cdc.gov/eid Vol. 17, No. 8, August 2011 1531Author af 
liations: SeaWorld, San Diego, California, USA (J. St.Leger, E. Nilson); Washington University School of Medicine, St.Louis, Missouri, USA (G. Wu, D. Wang); University of California atDavis, Davis, California, USA (M. Anderson); and SeaWorld, SanAntonio, Texas, USA (L. Dalton)DOI: 10.3201/eid1708.101979
 
To further support a WNV diagnosis, we performedimmunohistochemical staining on brain tissue. The immuno- peroxidase stain used was a commercial rabbit polyclonalantibody (BioRelience Corp., Rockville, MD, USA)with peroxidase-tagged goat antirabbit immunoglobulinG (DakoCytomation, Carpinteria, CA, USA) bridgeand 3-amino-9-ethylcarbazole (DakoCytomation) as thechromogen. This staining demonstrated abundant WNVantigen within the cytoplasm of a small number of neuronsand glial cells and in fewer macrophages in the brain tissue(Figure).We evaluated WNV exposure within the same cohort,as well as a geographically distant cohort of whales byusing serologic testing. All testing was performed at thesame laboratory by using a standard plaque-reductionneutralization test. In this assay, a 90% neutralizationcutoff was used (
). A 90% plaque-reduction titer >10 wasconsidered positive. Serum from the affected whale and5 cohort killer whales from the same marine park in SanAntonio as well as 5 whales housed at another facility inOrlando, Florida, USA, were evaluated. In each facility, theanimals have regular contact with each other. The facilitiesare geographically separated so the animals do not haveexposure to those in the other park. All 6 animals fromTexas had 90% plaque-reduction titers >10, ranging from40 to 80. The 5 whales housed together in Orlando had nomeasurable titer.
Conclusions
We demonstrate that WNV can infect and causedisease in killer whales. These
ndings broaden the knownhost tropism of WNV to include cetaceans in additionto previously known pinnipeds. Although we cannotde
nitively attribute the cause of death of this whale toWNV, the observed lesions are consistent with those caused by WNV in other animals. The serologic results demonstratethat subclinical infections can occur and that exposurecan be variable. We did not determine speci
c dates of exposure for these populations. Both Bexar County, Texas,and Orange County, Florida, have had WNV in wildlifesince 2002. We continue annual serology on previouslynegative animals to document seroconversion. Mosquitomanagement practices are similar in both facilities andhave been expanded since this diagnosis. Differences inWNV prevalence or mosquito numbers may have played arole in the different serologic results.Health evaluations of free-ranging and captivecetaceans should include WNV serology to assess exposurerates. This report focuses on killer whales, but the “loa
ng” behavior (stationary positioning at the water’s surface)is commonly seen in many coastal dolphins, therebyincreasing the likelihood of mosquito bites and exposure toWNV. Serologic screening of bottlenose dolphins (
Tursiopstruncatus
) from the Indian River Lagoon demonstratedWNV titers (
). WNV-associated disease in these animalshas not been reported. Active screening for WNV mayenhance diagnostic investigations.As with many species of birds and mammals, WNVinfection carries a risk for zoonotic transmission. Untilthe implications of this infection in marine mammals are better understood, biologists and veterinarians workingwith cetaceans should consider this possibility. Potentialviral shedding can occur through the oropharygeal cavityand feces as well as through blood and organs duringnecropsies.Finally, our study demonstrates the broad applicabilityof using panviral microarray-based diagnostics. Eventhough PCR diagnostics are well developed for WNV, theagent was not initially considered as a potential pathogenin this species. Panviral microarray can be used not only toidentify novel viruses but also to detect unsuspected agents.
This work was funded by National Institutes of Health grantU01 AI070374.Dr St. Leger is the director of pathology and research for SeaWorld Parks and Entertainment, serves on the editorial boardof the journal Veterinary Pathology, and is a board member of the CL Davis Foundation. Her primary research interests arecomparative pathology and infectious disease investigations of marine and aquatic animals.
References
1. Del Piero F, Stremme DW, Habecker PL, Cantile C. West Nile
avi-virus polioencephalomyelitis in a harbor seal (
Phoca vitulina
). VetPathol. 2006;43:58–61. doi:10.1354/vp.43-1-58
DISPATCHES
1532 Emerging Infectious Diseases www.cdc.gov/eid Vol. 17, No. 8, August 2011Figure. Brain specimen from killer whale (
Orcinus orca 
)with West Nile virus infection that died at a marine park, SanAntonio, Texas, USA, 2007. Neurons and glial cells demonstrateabundant intracytoplasmic West Nile viras antigen. Blood vesseldemonstrates mild vasculitis and perivascular lymphocytic in
ltrate.Original magni
cation ×200.
 
WNV in Killer Whale
2. Miller DL, Ewing RY, Bossart GD. Emerging and resurging diseas-es. In: Dierauf LA, Gulland FMD, editors. CRC handbook of marinemammal medicine. 2nd ed. Boca Raton (FL): CRC Press; 2001. p.15–30.3. Wang D, Urisman A, Liu Y-T, Springer M, Ksiazek TG, ErdmanDD, et al. Viral discovery and sequence recovery using DNA micro-arrays. PLoS Biol. 2003;1:E2. doi:10.1371/journal.pbio.00000024. Mihindukulasuriya KA, Wu G, St. Leger J, Nordhausen RW, WangD. Identi
cation of a novel coronavirus from a beluga whale us-ing a pan-viral microarray. J Virol. 2008;82:5084–8. doi:10.1128/JVI.02722-075. Kuno G. Universal RT-PCR diagnostic for arboviruses. J VirolMethods. 1998;72:27–41. doi:10.1016/S0166-0934(98)00003-26. Beaty BJ, Calisher CH, Shope RS. Arboviruses. In: Schmidt NJ,Emmons RW, editors. Diagnostic procedures for viral, rickettsialand chlamydial infections. 6th ed. Washington: American PublicHealth Association; 1989. p. 797–856.7. Schaefer AM, Reif JS, Goldstein JD, Ryan CN, Fair PA, BossartGD. Serological evidence of exposure to selected viral, bacterial,and protozoal pathogens in free-ranging Atlantic bottlenose dolphins(
Tursiops truncatus
) from the Indian River Lagoon, Florida, andCharleston, South Carolina. Aquatic Mammals. 2009;35:163–70.doi:10.1578/AM.35.2.2009.163Address for correspondence: Judy St. Leger, 500 SeaWorld Dr., SanDiego, CA 92109, USA, email: judy.st.leger@seaworld.com
Emerging Infectious Diseases www.cdc.gov/eid Vol. 17, No. 8, August 2011 1533

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