West nile virus and alligators

Seven alligators were submitted to the Tifton Veterinary Diagnostic and Investigational Laboratory for necropsy during two epizootics in the fall of and The alligators were raised in temperature-controlled buildings and fed a diet of horsemeat supplemented with vitamins and minerals. Histologic findings in the juvenile alligators were multiorgan necrosis, heterophilic granulomas, and heterophilic perivasculitis and were most indicative of septicemia or bacteremia.

Histologic findings in a hatchling alligator were random foci of necrosis in multiple organs and mononuclear perivascular encephalitis, indicative of a viral cause. West Nile virus was isolated from submissions in Reverse transcription-polymerase chain reaction RT-PCR results on all submitted case samples were positive for West Nile virus for one of four cases associated with the epizootic and three of three cases associated with the epizootic.

RT-PCR analysis was positive for West Nile virus in the horsemeat collected during the outbreak but negative in the horsemeat collected after the outbreak. Emerg Infect Dis. Document Type:. Journal Article ;. Collection s :. Emerging Infectious Diseases. In the liver, multifocal lymphoplasmacytic aggregates and heterophilic granulomas were present, consisting of caseous necrotic foci with degenerate heterophils surrounded by an outer layer of macrophages, lymphocytes, and heterophils.

The lungs were congested with mild diffuse or patchy lymphoplasmacytic and heterophilic interstitial infiltrates. The kidney had multifocal heterophilic granulomas. The pars pylorica region of the stomach had multifocal mucosal abscesses and moderate diffuse lymphoplasmacytic and heterophilic infiltrates of the lamina propria. The small intestine had moderate, diffuse mucosal and submucosal infiltrates of lymphocytes, heterophils, and plasma cells and multifocal areas of acute necrosis associated with bacteria.

The remaining tissues appeared within normal limits. Special stains for fungi and acid-fast bacteria were negative. A population of primarily gram-negative and fewer gram-positive bacteria was observed in the heterophilic granulomas. The third alligator had primarily pulmonary changes.

The airways contained moderate numbers of heterophils, occasional mucous plugs with degenerate inflammatory cells, and scattered bacterial colonies. The remaining tissues were as described for the first two alligators. Tissues from the alligator seen 2 months after the epizootic had similar findings to those of the first two alligators with the addition of rare, small caseating granulomas within the lungs.

The granulomas contained numerous large macrophages and multinucleated cells. Acid-fast stains demonstrated low numbers of slender, beaded, acid-fast positive bacilli consistent with mycobacteria. Multiple tissues from the two juvenile alligators from the epizootic were examined. The tissue changes were similar to those described for the epizootic except that the inflammatory component was primarily heterophils.

The meninges within the brain and all spinal cord sections except those from the sacral spinal cord had stasis of heterophils within the blood vessels and perivascular infiltration of mild numbers of heterophils Figure 1.

One alligator had a small focus of macrophages and heterophils noted within the endocardium. Multiple tissues were examined from the hatchling alligator, and lesions differed from the previous submissions on the basis of cellular composition of the inflammatory cell infiltrates.

Lymphoplasmacytic perivascular cuffs were present throughout the brain and meninges Figure 1. Rarely, heterophils were admixed within the cuffs.

Similar changes were not seen within the spinal cord. Random foci of necrosis were seen within the liver, pancreas, and tonsil. Mild to moderate perivascular infiltrates of lymphocytes, plasma cells, and heterophils were seen within the kidney and heart, and similar but fewer numbers of infiltrates were seen within the pulmonary interstitium.

The heart had multiple, random foci of patchy vacuolar degeneration of the myocytes and random aggregates of lymphocytes, plasma cells and heterophils. Mild numbers of mixed inflammatory cells were seen within the intestinal lamina propria. The remaining tissues were unremarkable. Major pathologic changes were not observed by light microscopy in the tissues from the free-ranging alligator. Figure 2.

West Nile virus WNV reverse transcription-polymerase chain reaction results from epizootic die-offs in farm-raised alligators. The expected amplicon is bp. Lane 1, a bp molecular weight ladder. Lane 2, the Virus isolation was negative for all animals from the epizootic. WNV was isolated from tissues from all animals in the epizootic. Additionally, all animals from the epizootic and one animal from the epizootic were positive for WNV by RT-PCR from fresh or formalin-fixed, paraffin-embedded tissues Figure 2.

In general, liver was the most likely tissue to yield positive results. Positive results were not obtained from any of the tissues from the free-ranging alligator. Aeromonas sobria and Edwardsiella tarda were consistently cultured from the intestines. Alcaligenes spp. Salmonella Group D was isolated from the intestines of the hatchling alligator submitted in The histologic findings from the hatchling alligator were most suggestive of a viral etiology, whereas those of the older alligators were most suggestive of a primary bacterial cause.

Contaminated horsemeat is the presumed source of the outbreak. Failure to isolate virus from the alligators in may have been due to the inability of the virus to propagate in the four cell lines used FHM, CCO, EPC, and WWS cells , as determined by retrospective culture attempts, rather than absence of virus.

Two important points to examine further are time of year and age of affected animals. Both epizootics occurred in the late fall to early winter. Although the epizootics appeared to be correlated with the first abrupt drop in environmental temperature, this finding was likely coincidental, especially given that the animals were housed in environmentally controlled barns. The most likely factor in the time of year is correlation with the occurrence of WNV infection in horses.

Historically, horses become infected with WNV during the mosquito season summer through early fall. Undiagnosed WNV-infected animals sold for food would most likely end up in the food supply during the late summer and early fall months. As was found in this study, deaths traced to consumption of contaminated food would taper off in late fall or early winter as the food supply was less likely to contain virus.

Furthermore, all animals have equal potential for viral exposure through consumption because individual packages of horsemeat are combined before mixing with the vitamin supplements and being divided between all barns.

In general, reptiles achieve immunocompetence at an early age often in a matter of days , but this immunocompetence may be temperature dependent until the animals are several months of age This fact may partially explain why the hatchling alligators tended to die from the viral infection, whereas the juveniles tended to die from infections caused by secondary invaders.

For example, the pens where the epizootics originated tended to be the first to be washed out at 6 a. During the first abrupt drop in environmental temperature, the first wash water was possibly cooler because of colder water in the line between the boiler and the pens. Forty alligators with LPSA and 41 controls were examined.

In the treatment group, Of the skin sections within the treatment group that had no LPSA lesions, 7. N2 - West Nile virus WNV is known to affect captive populations of alligators and, in some instances, cause significant mortalities.

AB - West Nile virus WNV is known to affect captive populations of alligators and, in some instances, cause significant mortalities. Overview Fingerprint. Water samples were added to cm 3 tissue culture flasks Corning, Corning, NY 1 mL per flask containing Vero cell monolayers. Flask media were replaced on day 6 postinfection.

Flasks were checked daily for cytopathic effect CPE through day 10 postinfection. Blood samples 0. These mixtures were then tested by plaque assay. Controls employed BA-1 only cell viability control , serum-free virus mixture with BA-1 only to enumerate PFU in the challenge dose of virus , and West Nile hyperimmune mouse ascitic fluid diluted mixture with virus to verify challenge virus identity.

Daily viremia titers. No injected alligators died of the infection. Tankmate viremia onset ranged from 12 to 24 days after infection. Because we stopped routine daily bleeding after day 15 postinfection, the exact viremia onset days of two alligators in this group are unknown. Also, the average duration of viremia for these alligators cannot be calculated. Two alligators in this group died of WNV infection after 12 or 13 days of viremia.

Viremia developed in the other tankmate on day 16 postinfection. Because of the absence of daily bleeding, the duration of viremia is not precisely known.

Of 29 viremic alligators, 24 had detectable viral loads in their cloacae Table 1. All five remaining infected alligators became viremic on the last 1 to 2 days of swabbing or after daily swabbing ceased, so no positive swabs can be reported from them. Viral shedding was detected within 3 days of detectable viremia and, in some instances, was detected on the same day as viremia onset.

Of 29 infected alligators, 2 died, and WNV was detected in their tissues Table 2. No virus was isolated from the seven alligators that recovered from infection. No infectious virus or viral RNA was detected in water samples. Sample volumes were each 0. In some southern states, alligator farms contribute to the economy as agricultural producers and tourist attractions.

A typical operation raises 3, alligators each year. The market value of raw products e. Beginning in , alligator farms in at least four different states suffered substantial economic losses due to WNV outbreaks in young alligators. Public health risks involved in these large outbreaks and the eventual culling of thousands of young alligators are also substantial. We have shown that sick juvenile alligators carry high viral loads in tissues, which poses a threat to handlers, processors, and consumers, although this risk has not been quantified beyond one reported case in Idaho of human West Nile fever in a handler of imported Florida juvenile alligators.

Furthermore, all infected alligators in our study shed WNV from the cloaca, which poses another possible threat to other alligators and to handlers. Although tankmates in our study became infected at a high rate, we cannot conclude with certainty that cloacal shedding is the cause of this direct transmission.

Direct transmission likely plays an important role in the epizootiology of WNV infection in farmed alligators but has not been documented in wild alligators 19 ; E. However, we now know that high levels of viremia develop in young alligators, so WNV infection could likely lead to mosquitoborne transmission as well. In general, viremia reached titers considered to be infectious to Culex quinquefasciatus mosquitoes with the NY99 strain of WNV 5.

Godsey, pers. Numerous species of mosquitoes feed on reptiles as well as birds and mammals and thus could be vectors from alligators to people In , Tait et al. Although neutralizing antibody circulation is only one part of immune function, previous studies have suggested that multiple aspects of the ectothermic immune system may be affected by body temperature, which is directly affected by environmental temperature 27 — Transmission of WNV by means other than mosquitoes has been shown in humans 30 — 32 , mice 33 , and birds 25 , 34 , although some modes of transmission are poorly understood.

All 12 injected and 7 of 12 orally inoculated alligators became viremic. Other transmission routes could include bloodborne transmission, although wounds were observed on only two alligators during the experiment, or direct transmission by contaminated water droplets sprayed onto the conjunctiva or other mucous membranes.

Although we apparently sampled water that was too dilute to detect WNV particles, at discrete moments, pockets of highly concentrated virus particles in the water could exist and lead to transmission.

Infectious saliva could also contribute to direct transmission, but this factor was not examined in this study. These data confirm the observations on the farms that WNV infection kills some alligators.

Because of infectious virus in their tissues, these dead alligators represent a potential health threat to handlers, alligator meat consumers, and other alligators. Infectious virus was not isolated from tissues of seven alligators that recovered from infection, which suggests that surviving alligators do not pose a health threat after viremia and cloacal shedding cease within 4 weeks postinfection.

In summary, juvenile alligators may be competent hosts for WNV. This study showed that juvenile alligators have adequate viremia levels high-titer and long-lasting for viral transmission by mosquitoes. Coupled with multiple routes of infection, alligators may play a role in WNV ecology, especially in areas where the density of young alligators is high.

Her main interests are zoonotic diseases with current focus on WNV ecology. We thank the St.