WEST NILE VIRUS INFECTION IN FARMED AMERICAN ALLIGATORS (ALLIGATOR MISSISSIPPIENSIS) IN FLORIDA

96

Journal of Wildlife Diseases, 41(1), 2005, pp. 96–106� Wildlife Disease Association 2005

WEST NILE VIRUS INFECTION IN FARMED AMERICAN ALLIGATORS(ALLIGATOR MISSISSIPPIENSIS) IN FLORIDA

Elliott R. Jacobson,1,5 Pamela E. Ginn,1 J. Mitchell Troutman,1 Lisa Farina,1 Lillian Stark,2

Kaci Klenk,3,4 Kristen L. Burkhalter,3 and Nicholas Komar3

1 College of Veterinary Medicine, University of Florida, Gainesville, Florida 32610, USA2 Florida Department of Health Bureau of Laboratories, Tampa, Florida 33612, USA3 Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado80522-2089, USA4 Current address: USDA National Wildlife Research Center, Fort Collins, Colorado 80521,USA5 Corresponding author (email: [email protected])

ABSTRACT: In September and October 2002, an epizootic of neurologic disease occurred at analligator farm in Florida (USA). Three affected American alligators (Alligator mississippiensis)were euthanatized and necropsied, and results confirmed infection with West Nile virus (WNV).The most significant microscopic lesions were a moderate heterophilic to lymphoplasmacyticmeningoencephalomyelitis, necrotizing hepatitis and splenitis, pancreatic necrosis, myocardial de-generation with necrosis, mild interstitial pneumonia, heterophilic necrotizing stomatitis, and glos-sitis. Immunohistochemistry identified WNV antigen, with the most intense staining in liver,pancreas, spleen, and brain. Virus isolation and RNA detection by reverse transcription–poly-merase chain reaction confirmed WNV infection in plasma and tissue samples. Of the tissues,liver had the highest viral loads (maximum 108.9 plaque-forming units [PFU]/0.5cm3), whereasbrain and spinal cord had the lowest viral loads (maximum 106.6 PFU/0.5cm3 each). Virus titersin plasma ranged from 103.6 to 106.5 PFU/ml, exceeding the threshold needed to infect Culexquinquefasciatus mosquitoes (105 PFU/ml). Thus, alligators may serve as a vertebrate amplifyinghost for WNV.

Key words: Alligator mississippiensis, American alligator, infection, molecular virology, pa-thology, West Nile Virus.

INTRODUCTION

West Nile virus (WNV) is a mosquito-borne flavivirus (Family Flaviviridae) thatis transmitted by various species of adultCulex mosquitoes to a variety of mammalsand birds (Hayes, 1989). It emerged inNorth America in 1999 when it infectedand caused the deaths of people, horses,and birds in New York (USA) (Komar,2000). By the year 2001, WNV had spreadinto the southeastern US, including Flor-ida (USA) (Blackmore et al., 2003). Thecontinued expansion of WNV in NorthAmerica presents an emerging threat tohuman and animal health in the WesternHemisphere.

Reptiles are known to be infected withseveral mosquito-borne viruses. Westernequine encephalomyelitis virus (FamilyTogaviridae) was isolated from blood andfound to overwinter in garter snakes(Thamnophis spp.; Thomas and Eklund,1962; Gebhardt et al., 1973) and the Texas

tortoise (Gopherus berlandieri; Bowen,1977). Japanese encephalitis virus (FamilyFlaviviridae) was isolated from Chinese ratsnakes (Elaphe rufodorsata) in Korea (Leeet al., 1972). In a survey in Venezuela, tegulizards (Tupinambis nigropunctatus) werefound to have antibody against easternequine encephalomyelitis (EEE; FamilyTogaviridae) and Venezuelan encephalitisviruses (Family Togaviridae; Walder et al.,1984). In crocodilians, seropositive wildAmerican alligators (Alligator mississip-piensis) for EEE virus (Karstad, 1961) andfarmed Nile crocodiles (Crocodylus nilo-ticus) in Israel for WNV (Steinman et al.,2003) have been reported. Recently, mor-tality associated with WNV infection wasreported in farmed American alligators inGeorgia, USA (Miller et al., 2003). Thereare no additional reports of WNV in rep-tiles (Komar, 2003). Here we report anoutbreak of WNV infection in farmedAmerican alligators in Florida. Viremia

JACOBSON ET AL.—WEST NILE VIRUS INFECTION IN AMERICAN ALLIGATORS 97

data are presented that implicate theAmerican alligator as a potential amplify-ing host for WNV in southeastern NorthAmerica.

MATERIALS AND METHODS

Animals

Between early September and mid-October2002, a central Florida alligator farm (28�32�N,80�56�W) had a large die-off of alligators thatwere up to 1.3 m in length and up to 3 yr old.Alligators were kept at a constant ambient tem-perature of 32 C in water-filled igloo-shaped orrectangular concrete pens, which on averageheld approximately 200 animals of approxi-mately the same size and age. The igloo-shapedpens had 848–897 l of water and the rectan-gular pens had 799–3,464 l of water. Pens werekept dark and completely enclosed with the ex-ception of narrow gaps around the entry doorfor feeding or maintenance. Pens were provid-ed with chlorine-treated well water that wasdrained and refilled three times a week. Alli-gators were maintained on a diet of groundbeef and alligator chow (Burris Alligator Food,Burris Mill and Feed, Franklinton, Louisiana,USA).

Pens with affected and unaffected alligatorswere randomly dispersed. Mortality was high-est in the youngest alligators, with up to 56 dy-ing per pen, whereas older alligators died at upto 32 per pen. Approximately 300 from a pop-ulation of 9,000 (3.33% of the total population)died over a 2-mo period. Clinical signs includedanorexia, lethargy, intention tremors, swimmingon their sides, spinning in the water, and op-isthotonus. Death occurred 24–48 hr after on-set of clinical signs. All alligators on the farmwere hatched from wild collected eggs thatwere purchased from the Florida Departmentof Fish and Wildlife Conservation Commission.

Three of the youngest alligators (�30.5 cmlong and less than 1 mo old) with signs of neu-rologic disease were submitted alive to the Uni-versity of Florida College of Veterinary Medi-cine (Gainesville, Florida) for physical exami-nation and pathologic investigations. Blood wasobtained from the supravertebral vein and plas-ma was removed and frozen at �70 C.

Pathology

The three alligators were euthanatized withan intracoelomic cavity injection of pentabar-bitol (80 mg/kg of body weight). Tissues col-lected at necropsy and examined histologicallywere heart, lung, liver, spleen, kidney, brain,spinal cord, adrenal gland, pharyngeal mucosa,small intestine, pancreas, and tongue. Tissues

were fixed in 10% neutral phosphate-bufferedformalin, routinely processed, sectioned at 5�m, and stained with hematoxylin and eosin.Tissues from major organs were also frozen andstored at �80 C for virus isolation and viralRNA detection by reverse transcription–poly-merase chain reaction (RT-PCR).

Immunohistochemistry (IHC) was used toidentify tissues infected with WNV and to de-termine cellular tropism of the virus. Formalin-fixed paraffin-embedded 5-�m sections ofbrain, spinal cord, liver, lung, spleen, kidney,small intestine, pancreas, and pharyngeal mu-cosa were mounted on positively chargedslides. A mouse monoclonal antibody to WNV(BioReliance, Rockville, Maryland, USA) dilut-ed 1:200 in Dako antibody diluent (Dako Cor-poration, Carpinteria, California, USA) servedas the primary antibody and a standard im-munoperoxidase protocol was implemented ac-cording to the manufacturer’s directions (DakoEnvision System, Dako Corporation) using3,3’-diaminobenzidine as the chromogen andhematoxylin as a counterstain. Before incuba-tion with the primary antibody, tissue sectionswere treated with 40 �l of proteinase K (DakoCorporation) diluted in 2 ml of Tris HCl bufferat room temperature for 5 min, followed byperoxidase blocking for 5 min at room temper-ature in 0.03% H2O2. A section of equine brainfrom a WNV-infected horse served as a positivecontrol. Substitution of the primary antibodywith normal mouse serum served as a negativecontrol.

Immunohistochemistry also was used to de-termine the presence of chlamydial antigen. Amonoclonal antibody (Virostat 1631, Virostat,Portland, Maine) that recognizes chlamydialLPS antigen including serotypes of Chlamydiatrachomatis, Chlamydophila psittaci, and Chla-mydophila pneumoniae, was used as the pri-mary antibody. The working dilution was 1:10in blocking buffer and the IHC procedure wasthe same as above. Liver from a puff adder (Bi-tis arietans) with chlamydiosis served as thepositive control (Jacobson et al., 2002).

Microbiology

Blood, portions of lung, and swabs of the oralcavity were obtained from the alligators andcultured for Mycoplasma alligatoris using pre-viously described methods (Brown et al., 2001).

Portions of frozen heart, liver, lung, kidney,spinal cord, and brain from the three alligatorswere processed for WNV isolation by plaqueassay in Vero cells as previously described (Pa-nella et al., 2001). Plasma samples also weresubmitted and were treated as clarified tissuehomogenates. Viral titers in all specimens were

98 JOURNAL OF WILDLIFE DISEASES, VOL. 41, NO. 1, JANUARY 2005

FIGURE 1. Oral cavity of a hatchling alligator withnecrotizing pharyngitis. Fibrinous material and exu-date can be seen covering the base of the tongue andpalate.

quantified by plaque assay of serial 10-fold di-lutions.

Infected cell culture supernatants and tissuehomogenates were examined for the presenceof WNV RNA using a TaqMan RT-PCR assay(Lanciotti et al., 2000). RNA was extractedfrom a 140-�l aliquot of each sample using aQIAamp Viral RNA mini Kit (Qiagen Inc, Va-lencia, California) according to manufacturer’sinstructions. The RT-PCR was performed usingthe ABI Prism 7000 Sequence Detection Sys-tem (Applied Biosystems, Foster City, Califor-nia) or the Bio-Rad Icycler IQ� Real-TimeDetection System (Bio-Rad, Hercules, Califor-nia) according to manufacturers’ specifications.Selected viral harvests from plaque assays wereidentified as WNV using a WNV-specific anti-gen detection assay (Medical Analysis Systems,Inc., Camarillo, California; Ryan et al., 2003).

RESULTS

Clinical findings

The three alligators weighed 110.4 gm,191.8 gm, and 238.3 gm respectively. Nomuscle atrophy was evident and no lesionswere seen affecting the integumentary sys-tem. The eyes were clear and a blink re-sponse was present. Each alligator had amildly distended abdomen and all had fi-brinonecrotic material in the oral cavityjust cranial to the glottal flap (Fig. 1). Twoalligators were listless and depressed. Onealligator exhibited intention tremors, indi-

cating central nervous system (CNS) dis-ease.

Pathologic findings

The coelomic cavities of each alligatorcontained moderately sized fat bodies andapproximately 3–5 ml of clear yellow fluid.The liver in each case was slightly enlargedwith rounded edges and mottled yellow tored. The spleens and areas of the myocar-dium were mottled tan to red. All otherorgans were grossly within normal limits.

Light microscopic abnormalities weresimilar for the three alligators. The liverwas characterized by randomly scatteredindividual or clusters of necrotic hepato-cytes accompanied by mild infiltrates ofheterophils. Kupffer cells often containedcell debris (Fig. 2A). Portal regions hadmild bile ductular hyperplasia and fibrosisand a mild to moderate infiltrate of het-erophils, lymphocytes, and plasma cells.Occasional hepatocytes contained lipidvacuoles. The myocardium had mild infil-trates of plasma cells, lymphocytes, heter-ophils, and macrophages. Scattered singleto small groups of degenerating and ne-crotic myocardial fibers characterized byhypereosinophilia, fragmentation, and oc-casional nuclear pyknosis were present. Inthe kidneys, rare tubular epithelial cellswere necrotic. The renal interstitium hadminimal infiltrates of lymphocytes andplasma cells. The small intestine had amild to moderate infiltrate of lymphocytes,plasma cells, and heterophils and mild en-terocyte necrosis and hyperplasia. Thepancreas had widespread necrosis of aci-nar cells (Fig. 2B). The adrenal glandswere moderately edematous and had amild heterophilic infiltrate and mild indi-vidual cell necrosis. The pharyngeal mu-cosa was mildly hyperplastic with multi-focal epithelial cell necrosis and mild in-traepithelial heterophilic infiltrates. Insome areas, there were extensive areas ofulceration covered with a dense layer ofnecrotic cellular debris. The submucosawas edematous with mild to moderateperivascular infiltrates of lymphocytes and


FIGURE 2. Organs from an alligator with West Nile virus infection. (A) Liver. Note the area of hepato-cellular necrosis and infiltrates of leukocytes. H&E stain. Bar�200 �m. (B) Pancreas. Note the extensivenecrosis of pancreatic acinar cells. H&E stain. Bar�100 �m.

plasma cells. The lungs were characterizedby a moderate heterophilic to lymphoplas-macytic interstitial pneumonia and mildrespiratory epithelial cell hyperplasia. Thespleen had a moderate infiltrate of heter-

ophils and plasma cells and was depletedof lymphocytes and contained largeamounts of cellular debris frequentlyphagocytosed by numerous macrophages.A moderate to severe heterophilic to lym-


phoplasmacytic meningoencephalomyelitisaffected the brain and spinal cord (Fig.3A). Vessels were surrounded by densecuffs of mononuclear leukocytes and het-erophils, which extended to a milder de-gree into the neuropil (Fig. 3B). Both greyand white matter were affected. The cer-ebellum was affected to a more severe de-gree than other areas of the brain. In someareas, the meninges and Virchow-Robinspaces were heavily infiltrated primarily byheterophils.

Immunohistochemistry

Immunostaining indicated the presenceof WNV antigen in multiple tissues. In theliver, degenerating and necrotic hepato-cytes, Kupffer cells, and some leukocyteswithin sinusoids contained antigen. Smallnumbers of macrophages within the in-flamed pharyngeal mucosa contained an-tigen. Large numbers of splenic macro-phages contained antigen. The pancreashad widespread labeling of acinar cells.Occasional enterocytes and leukocyteswithin the lamina propria of the small in-testine contained antigen as well as smallnumber of renal tubular epithelial cells. Inthe brain and spinal cord, small numbersof neurons and mononuclear leukocytes inperivascular regions and within the neu-ropil were labeled (Fig. 4). Immunohisto-chemical staining for chlamydial antigenwas negative.

Microbiology

Blood, lung, and throat swabs from eachalligator submitted for M. alligatoris iso-lation yielded no growth.

All tissue homogenates tested positivefor WNV RNA. Viral plaques were de-tected after 3 days of culture, and identityof WNV was confirmed by antigen detec-tion. Of the tissues, liver had the highestviral loads (maximum 108.9 plaque formingunits [PFU]/0.5cm3), whereas brain andspinal cord had the lowest viral loads (max-imum 106.6 PFU/0.5cm3 each (Table 1).Viral loads in plasma ranging from 103.6 to

106.5 PFU/ml confirmed viremia in each ofthe alligators.

DISCUSSION

There is a paucity of information oncauses of mortality in both farmed andwild alligators. Some information is avail-able for crocodiles being farmed in Aus-tralia, New Guinea, Africa, and Thailand(Foggin, 1987; Ladds and Sims, 1990;Buenviaje et al., 1994; Youngprapakorn etal., 1995; Huchzermeyer, 2003). For themost part, alligator farmers in Florida andelsewhere in the US do not routinely sub-mit dead and dying alligators for thoroughpostmortem evaluations. Such is the casefor the alligators of the present case re-port. At the time of the outbreak at thefarm in Florida, the owner received infor-mation that other farms were affected.However, no alligators were submitted forpostmortem evaluations from these farms.

Many ill alligators at the affected farmin Florida exhibited signs of CNS disease.Little is known about the causes of CNSdisease in alligators and other crocodilians.Adult alligators dying in Lake Griffin (306deaths), a lake in central Florida, from De-cember 1998 through November 2000were found to have clinical signs of CNSdisease that included swimming in circles,inability to submerge, lethargy, weakness,unresponsiveness, slow reflexes, draggingthe dorsal surfaces of the hind feet, headtilt, and anisocoria (Schoeb et al., 2002).Histopathology identified necrosis of thetorus semicircularis of the midbrain. Thelesions seen in the brains of the three al-ligators of this report revealed inflamma-tory infiltrates that were distinct from thelesions seen in the alligators in Lake Grif-fin.

Because mycoplasmosis is a recognizeddisease in farmed alligators in Florida, andcan infect the CNS causing weakness andlimb paresis (Clippinger et al., 2000), at-tempts were made to culture mycoplasmafrom tissues and blood of the submittedalligators. Mycoplasma was not isolated.Although hepatitis can be seen in alligator


FIGURE 3. Cerebrum of an alligator with meningoencephalitis due to West Nile virus infection. (A) Notethe meningeal infiltrate of heterophils (H). H&E stain. Bar�200 �m. (B) Note the dense perivascular cuffof mononuclear leukocytes and heterophils. H&E stain. Bar�50 �m.


FIGURE 4. Brain of an alligator with West Nilevirus encephalitis. Note labeling of neurons and leu-kocytes within the neuropil. Immunoperoxidase stain.Bar�50 �m.

TABLE 1. West Nile viral loads determined by Vero plaque assay for plasma and selected tissues of threealligators.

AlligatorPlasma

(log10 PFU/mL)

Tissue (log10 PFUa/0.5 cm3)

Heart Liver Lung Kidney Spinal cord Brain

ABC

6.06.53.6

7.97.68.4

8.98.98.8

8.97.17.9

8.38.38.3

6.65.35.2

NTb

5.26.6

a PFU � plaque forming units.b NT � not tested.

mycoplasmosis, the hepatitis seen in thealligators in the current case report wasnecrotizing and primarily heterophiliccompared to that in mycoplasmosis, whichconsists of a variable portal to periportallymphocytic hepatitis. The pneumonia inalligator mycoplasmosis is predominantlyproliferative compared to interstitial inWNV-infected alligators in this report andthe alligators infected with WNV had myo-cardial necrosis compared to fibrinouspericarditis in alligators infected with my-coplasma. Joint disease seen in alligatorswith mycoplasmosis (Clippinger et al.,2000; Brown et al., 2001; Pye et al., 2001)was not seen in the alligators of this report.

Because Chlamydia has been identifiedand believed to be responsible for hepa-titis in farmed Nile crocodiles in South Af-rica (Huchzermeyer et al., 1994), an IHCstaining procedure using a monoclonal an-

tibody against Chlamydia LPS antigen wasused to assay for the presence of this path-ogen. This monoclonal antibody has beenused to detect chlamydial antigen in otherreptiles (Jacobson et al., 2002). No label-ing for chlamydial antigen was present intissues of alligators of this report.

At the time the sick alligators in thisstudy were evaluated by us, there were nopublished accounts of WNV disease in al-ligators or other reptiles. Attempts to showthat reptiles and amphibians may contrib-ute to WNV transmission as amplifyinghosts in North America failed using threeother species of reptiles and the Americanbullfrog (Rana catesbiana) (Klenk and Ko-mar, 2003). Still, because of WNV activityin birds, horses, and humans in Florida(Blackmore et. al., 2003), an investigationinto the possible role of WNV in the out-break of neurological disease in alligatorswas warranted. At approximately the sametime a large die-off of farmed alligatorswas associated with WNV infection inGeorgia (Miller et al., 2003).

Histologic lesions and positive labelingfor WNV IHC in the Florida alligatorswere similar to that reported in birds withWNV in that similar organs were affectedby necrosis and lymphoplasmacytic to het-erophilic inflammation (Steele et al.,2000). The lesions reported in birds, how-ever, were more severe in some organs(CNS, heart, kidney, intestines) than thoseseen in the alligators of this report. WestNile virus-infected alligators in Georgia(Miller et al., 2003) had lesions in the CNSand in some cases, parenchymal organs,


that were similar to the alligators in thisreport; however, we did not observe le-sions attributed to systemic bacterial infec-tions as was documented in the alligatorsfrom Georgia.

Viral loads in alligator tissues were sim-ilar in magnitude to viral loads detected infatal cases of WNV infection in experi-mentally infected corvids (Komar et al.,2003). One notable difference was thehigher virus titer in liver of affected alli-gators compared to the liver of crows,which have lower viral loads comparedwith other organs (Steele et al., 2000; Ko-mar et al., 2003).

The finding of viremia greater than 105.0

PFU/ml plasma suggests that WNV-infect-ed alligators may be infectious to mosqui-toes. This level of viremia was shown to bethe threshold above which Culex pipiensand C. quinquefasciatus mosquitoes be-come infected (Turell et al., 2000; Sardeliset al., 2001). Culex quinquefasciatus is oneof the principle vectors of WNV in Florida(Blackmore et. al., 2003), suggesting thatalligators, like some bird species, mayserve as a vertebrate amplifying host. Thisdiffers from dead-end hosts such as hu-mans and horses in which the virus is un-likely to be transmitted to mosquitoes toperpetuate the transmission cycle. It issuspected that certain arboviruses such aswestern equine encephalomyelitis viruscan overwinter in certain poikilothermssuch as tortoises and snakes (Gebhardt etal., 1973; Bowen, 1977). This may also oc-cur with WNV in alligators and thus needsto be investigated.

Alligators are derived from the samephylogenetic lineage common to birds(Hedges, 1994). Alligators and certainbirds may share biologic attributes that al-low WNV to replicate to infectious levelsin these hosts. Not all birds, however, de-velop infectious viremias (Komar, 2003;Komar et al., 2003), and of the 23 speciesof crocodilians, we do not know the rangeof their susceptibility to WNV. Since dif-ferential pathogenicity of M. alligatoris hasbeen demonstrated in crocodilians, with

Siamese crocodiles (Crocodylus siamensis)resistant to pathogenic effects (Pye et al.,2001), similar differential pathogenicity incrocodilians may be seen with WNV. Forinstance, although serum neutralization re-sults indicated that 70% of Nile crocodilessampled at two farms in Israel were sero-positive for exposure to WNV, no mortalitywas seen (Steinman et al, 2003).

Florida is one of several southeasternstates in the US where alligators arefarmed for meat and hides. In Florida,there are approximately 49 alligator farm-ing operations. Of these, 19 have morethan 1,000 alligators at any one time. Atthese farms, alligators are generally rearedunder highly controlled environmentalconditions to achieve maximal growthrates in the shortest period of time. Sincegrowth rates are directly influenced by en-vironmental temperature, they are oftenraised in pens maintained at an ambienttemperature of 32 C. The alligators in thecurrent report were from pens kept at thistemperature for up to 3 yr while they ma-tured. Because alligators are typicallyhoused in large numbers, they are alsokept in the dark to reduce aggression anddamage to their hides. These conditionsare in contrast to fluctuating environmen-tal conditions that wild alligators encoun-ter throughout their life. The body tem-perature of wild alligators typically fluctu-ates both daily and seasonally. Since alli-gators are from a temperate climate,winter months are typically cool with min-imum growth rates. Although the affectedfarm was adjacent to a tourist attractionwhere adult alligators were kept outdoorsunder fluctuating environmental condi-tions, no deaths or signs of CNS diseasewere seen in these older and larger alli-gators.

Body temperature affects the immunesystem of all reptiles (Ambrosius 1976;Cooper et al., 1988) and environmentaltemperatures directly affect body temper-ature of alligators. Environmental temper-ature is known to affect both the humoralresponse to antigens and graft rejection in


reptiles (Cooper et al., 1988). Althoughthere is little scientific information on theeffect of varying body temperatures onsusceptibility to infection with differentpathogens, there is some evidence to sup-port the notion of an ideal body temper-ature for infection of reptiles with certainpathogens. For instance, a paramyxovirusknown to infect snakes optimally replicatesin cell cultures at 27–33 C, with less effi-cient replication at 37 C (Clark et al.,1979). Studies are needed to investigatethe interplay between environmental tem-perature and age on the ability of WNV toinfect alligators, establish a viremia, andcause lesions in the CNS.

We do not know how WNV infected thealligators at the farm. Miller et al (2003)provided evidence that horsemeat fed toalligators in Georgia in 2002 was contam-inated with WNV genetic material, sug-gesting that contaminated feed may havebeen the source for late fall epizootics oc-curring in 2001 and 2002. They suggestedthat WNV-infected horses sold for foodconsumption may have inadvertently beenfed to alligators, and linked the late fallseasonality of WNV outbreaks in farmedalligators with the peak of WNV infectionin horses. In contrast, no horsemeat (onlybeef and alligator chow) was fed to the al-ligators that are the subject of this report.At the Florida farm, frozen beef was de-frosted in the open and may have beencontaminated by feces of wild birds. Nu-merous bird species have been shown toshed WNV per cloaca, presumably leadingto fecal contamination (Langevin et al.,2001; Komar et al., 2003).

The epizootics in Florida and Georgiaoccurred during September through Oc-tober, the peak period for WNV infectionin horses in Florida. Mosquitoes havebeen seen feeding upon alligators at otheralligator farms in Florida, but the speciesof mosquitoes feeding upon alligators andtheir ability to transmit WNV await furtherstudies. Because of the high viral titers intissues of these alligators, WNV may beshed through the feces into the water, re-

sulting in horizontal transmission. Al-though we did not attempt to determineviral shedding in the feces of the alligatorswe necropsied, this has now been ob-served in experimentally infected alligatorsthat shed virus per cloaca and infectedpen-mates in the absence of mosquitoes(Klenk et al., 2004). All three WNV-in-fected alligators had oral lesions. Althoughthe exact cause of these lesions is un-known, virus may have been able to infectthe alligators through damaged mucosaltissues.

Mosquitoes may have infected alligatorswith WNV, followed by subsequent spreadbetween alligators. Morbidity and mortal-ity in the affected farm appeared to be re-stricted to certain pens, suggesting that di-rect transmission among alligators mayhave been occurring. Mosquito trappingstudies are ongoing at the affected farm toidentify the species and determine whichfeed upon alligators.

Further work is needed to better un-derstand the impact of WNV on farmedand wild alligators. Experimental transmis-sion studies of juvenile alligators takenfrom Florida farms have confirmed that al-ligators are susceptible to WNV infection,develop high levels of WNV viremia ca-pable of infecting mosquitoes, and occa-sionally develop clinical signs as a result ofthe infection (Klenk et al., 2004).

The effect of WNV in wild alligators isunknown. It is rare to find dead alligatorsin the wild unless there is massive mortal-ity. Die-offs that are seen are generally inpopulations being monitored for biologicstudies or are being harvested. If younganimals were at a greater risk to illness anddeath from WNV than adults, die-offs inthese young animals would most likely bemissed. In the absence of recognized die-offs, serologic surveys are a very practicalmethod of monitoring populations for out-breaks of disease. Recently, an indirect en-zyme-linked immunosorbent assay was de-veloped and validated for determining an-tibodies to WNV in alligators (Jacobson etal., 2005), a tool that will be useful in eval-


uating the impact of WNV on alligatorpopulations. A serologic survey fromnorthern to southern Florida indicated lowseroprevalence to WNV in wild popula-tions of alligators (Jacobson et al., 2005).This probably indicates that epizootics ofdisease or mortality events due to WNV inwild alligators have not occurred at thesesampling sites. This survey will serve as abaseline for future seroepidemiologicstudies.

ACKNOWLEDGMENTS

We are grateful to G. Eldred, J. Velez, D.Kazanis, R. Krueger, M. Casteneda, and R.Schofield for laboratory assistance. We alsothank F. S. Kennedy, Veterinary Pathology Ser-vices, for initially describing an encephalitis ina farmed alligator that led to case submissionsof the alligators in this report to the Universityof Florida. Published as University of FloridaCollege of Veterinary Medicine, Journal Series636.

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Received for publication 13 April 2004.

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WEST NILE VIRUS INFECTION IN FARMED AMERICAN ALLIGATORS (ALLIGATOR MISSISSIPPIENSIS) IN FLORIDA