West Nile virus in farmed alligators - Research

Emerging Infectious Diseases, July, 2003 by Debra L. Miller, Michael J. Mauel, Charles Baldwin, Gary Burtle, Dallas Ingram, Murray E. Hines, II., Kendal S. Frazier

Seven alligators were submitted to the Tifton Veterinary Diagnostic and Investigational Laboratory for necropsy during two epizootics in the fall of 2001 and 2002. 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 2002. 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 2001 epizootic and three of three cases associated with the 2002 epizootic. RT--PCR analysis was positive for West Nile virus in the horsemeat collected during the 2002 outbreak but negative in the horsemeat collected after the outbreak.

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West Nile virus (WNV) has been reported in a variety of species but primarily endotherms. Arboviruses have been reported to affect ectotherms, and in some cases ectotherms are thought to serve as a reservoir (1-4). The mode of transmission of the arbovirus to ectotherms has often been presumed to be through ingestion or a bite from the insect carrier (5).

During the fall of 2001 and 2002, two epizootics occurred among captive alligators on a south Georgia alligator farm that houses over 10,000 animals. Approximately 250 alligators died between November and December 2001, and >1,000 alligators died in 2002. These epizootics tended to occur approximately 2 weeks after the first abrupt drop in ambient temperature, which occurred both years in mid-October and was characterized by minimum temperatures between 0[degree]C and 8[degrees]C and maximum temperatures between 10[degrees]C and 18[degrees]C for a period of 1 to 3 days.

Methods

Animals and Housing

Animals were housed in six barns that were divided into 10 pens; each pen contained approximately 100-200 alligators. The nursery animals are obtained either as eggs from Florida or as hatchlings from onsite breeders.

All pens are cleaned in the morning starting at 6 a.m. An automatic flushing system is used to drain the pens, flush them, and fill them with clean water. Well water is chlorinated with an automated system that injects chloride gas into the water. The water is then piped into a central collecting area and heated. The water temperature is maintained at 32.2[degrees]C year-round, and the buildings are kept dark to reduce environmental stress on the animals. The reduced stress and warm environment allow continued growth (i.e., growth of [greater than or equal to] 1 m per year rather than 0.30 m per year).

Alligators are fed in the mid- to late afternoon. The diet consists of 95% ground raw horsemeat (obtained frozen from a source in Pennsylvania) to which vitamins and minerals are added in a pelleted alligator diet carrier. The ingredients are thoroughly mixed in a large commercial mixer. The source of the horsemeat has remained constant since 1985. The source of the vitamins and minerals has varied, based upon availability.

The breeding population is maintained in a separate fenced enclosure on the premises. This enclosure is a native swampland and therefore subjected to ambient weather conditions. A rookery was recently established in the breeding area by native birds. Attempts to depopulate the rookery (using U.S. Department of Agriculture-approved methods) have been unsuccessful. The alligators eat fledglings and older birds that fall from the nests and branches or otherwise get within reach. Alligators do not nest under the rookery. No mosquito control is practiced on the farm.

Tissue Collection

Animals were seen moribund or dead upon arrival at the laboratory. Blood was collected from the occipital sinus or caudal vein of live animals. Gross observations were made, and the animals were humanely euthanized. Tissues were collected from the eye, thyroid gland, lymph node, lung, heart, brain, spinal cord, kidney, liver, spleen, pancreas, adrenal gland, gallbladder, tonsil, trachea, stomach, intestines, and reproductive tract. Fresh tissue specimens were submitted for virus isolation, reverse transcription-polymerase chain reaction (RT-PCR), and bacterial culture. Tissues were also collected in 10% buffered formalin, processed, and embedded in paraffin. Five-micrometer-thick sections were stained with hematoxylin and eosin and viewed by light microscopy. Tissues opportunistically collected from an adult clinically normal, free-ranging alligator served as a control.

Multiple aliquots (totaling 1 g) of the ground raw horsemeat (without additives) that was being fed during the 2002 epizootic (October and November) were collected and processed for RT-PCR. Subsequent aliquots from postepizootic horsemeat shipments (in December and January,) were similarly processed.

Virus Isolation

A 10% homogenate in Earle's minimal essential media (MEM) containing gentamicin was made of each specimen. The homogenate was centrifuged for 10 min at 2,000 RPM and 4[degrees]C. The supernatent was filtered and spread onto a preformed monolayer of Vero cells. In 2001, fathead minnow (FHM), white sturgeon skin (WSS), epithelioma papillosum caprini (EPC), and channel catfish ovary (CCO) cells were used instead of the Veto cells. Inoculated cells were incubated in a 5% C[O.sub.2] atmosphere at 37[degrees]C. Cells were examined each day for viral cytopathic effect (CPE). If no CPE was observed, aliquots of the first passage were transferred to a second preformed monolayer of Veto cells (FHM, WSS, EPC, and CCO cells in 2001) on day 7. If no CPE was observed after a second 7 days of passage, the culture was considered negative. Monolayers demonstrating viral CPE were passaged to chambered slides. The slides were fixed in cold methanol, and a West Nile fluorescent-antibody test was conducted to confirm the isolate.