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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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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 build-
ings and fed a diet of horsemeat supplemented with vita-
mins and minerals. Histologic findings in the juvenile alliga-
tors 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 pos-
itive for West Nile virus for one of four cases associated
with the 2001 epizootic and three of three cases associat-
ed 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.
W
est 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 alli-
gator 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 mini-
mum temperatures between 0°C and 8°C and maximum
temperatures between 10°C and 18°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 main-
tained at 32.2°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 >
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 min-
erals 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
794 Emerging Infectious Diseases • Vol. 9, No. 7, July 2003
RESEARCH
West Nile Virus in
Farmed Alligators
Debra L. Miller,* Michael J. Mauel,* Charles Baldwin,* Gary Burtle,* Dallas Ingram,*
Murray E. Hines II,* and Kendal S. Frazier*
*University of Georgia, Tifton, Georgia, USA
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, stom-
ach, intestines, and reproductive tract. Fresh tissue speci-
mens were submitted for virus isolation, reverse transcrip-
tion-polymerase chain reaction (RT-PCR), and bacterial
culture. Tissues were also collected in 10% buffered for-
malin, processed, and embedded in paraffin. Five-microm-
eter-thick sections were stained with hematoxylin and
eosin and viewed by light microscopy. Tissues opportunis-
tically collected from an adult clinically normal, free-rang-
ing alligator served as a control.
Multiple aliquots (totaling 1 g) of the ground raw horse-
meat (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 speci-
men. The homogenate was centrifuged for 10 min at 2,000
RPM and 4°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 Vero cells. Inoculated
cells were incubated in a 5% CO
2
atmosphere at 37°C.
Cells were examined each day for viral cytopathic effect
(CPE). If no CPE was observed, aliquots of the first pas-
sage were transferred to a second preformed monolayer of
Vero 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.
Fluorescent-Antibody Testing
Mouse anti–WNV-specific polyclonal antibody
(Centers for Disease Control and Prevention [CDC],
Division of Vector-Borne Infectious Diseases, Fort
Collins, CO) was applied to the chamber and the slide
incubated in 5% CO
2
at 37°C for 30 min. The slide was
rinsed two times for 5 min in a sodium carbonate/bicarbon-
ate buffer (pH 9.3). The slide was then air-dried, followed
by an anti-mouse fluorescein-conjugated antibody, and
incubated as before for 30 min. The slide was washed
twice in carbonate buffer, followed by 5 min in 0.5%
Evans blue counter stain. Slides were dipped in distilled
water, and a glycerin/water mounting media and coverslip
was added. Slides were examined with a fluorescent
microscope. All isolates were tested for WNV. All isolates
were also tested for Eastern equine encephalomyelitis
virus (EEEV) by using a similar protocol. We tested for
EEEV because of its known prevalence within the geo-
graphic area. The EEEV-specific monoclonal antibody
(CDC, Atlanta, GA) was prepared against the New Jersey
1960 strain of EEEV.
RNA Extraction
RNA was extracted from various specimens (fresh tis-
sue, virus isolation homogenate or cell culture lysate, and
formalin-fixed paraffin-embedded tissue). For extraction
from fresh specimens, approximately 1 g of tissue was
placed in a whirlpack bag and homogenized by using a
Stomacher Lab Blender 80 (Tekmar Co., Cincinnati, OH)
with three times the tissue volume of phosphate-buffered
saline (PBS). Three milliliters of the tissue homogenate
was processed with a Rneasy Midi kit (QIAGEN, Inc.,
Valencia, CA) per manufacturers directions. If a virus iso-
lation homogenate or cell culture lysate in Earle’s MEM
was used, approximately 4 mL of the homogenate or lysate
was washed with 5 mL of PBS, the supernatant removed,
and the pellet processed with the Rneasy Midi kit. For
paraffin sections, several 5-µm sections from paraffin
blocks were cut and deparaffinized with xylene. The
xylene was removed, and samples were washed two times
with 100% ethanol for 10 min, once with 95% and once
with 70% ethanol. Samples were incubated overnight at
56°C in 80 µL of proteinase K with 5 mL of Buffer RLT
from the Rneasy Midi kit and then processed per manufac-
turers directions.
RT-PCR
RT-PCR for WNV was performed on the tissues
according to the procedure described by Kuno (6) and
using the RT-nested primer sets described by Johnson et al.
(7). In brief, a RT-PCR mixture was prepared by using the
outside primer set (P1401 – ACCAACTACTGTGGAGTC
and P1845 – TTCCATCTTCACTCTACACT) to amplify a
445-bp product. Forty microliters of the RT-PCR mixture
and 10 µL of sample were dispensed into a 0.2-mL thin
wall PCR tube, and 10 µL of Rnase-free water was added
for a final volume of 50 µL. With the use of a model PTC-
200 thermal cycler (MJ Research, Inc., Waltham,
Massachusetts), cycling conditions for the RT-PCR were
as follows: 53°C for 30 min, followed by 40 cycles of
94°C for 1 min, 53°C for 1 min, 72°C for 1 min, and then
held at 4°C. Ten microliters of RT-PCR first-round product
was used for the nested PCR (nPCR). The nPCR mixture
was prepared by using 40 µL of PCR mixture (now with
Emerging Infectious Diseases • Vol. 9, No. 7, July 2003 795
RESEARCH
the inside primer set [P1485 – GCCTTCATACACAC-
TAAAG and P1732 – CCAATGCTATCACAGACT]) to
amplify a 248-bp product. The cycling conditions for the
nPCR were as described above, but the first ramp was
omitted (53°C for 30 min). A 10-µL aliquot of each reac-
tion with 1 µL of loading buffer added was loaded onto a
1.5 % agarose gel in Tris-borate-EDTA (TBE) buffer and
run at 70 V for approximately 1.5 h.
This protocol was repeated on all samples with primer
sets for EEEV and St. Louis encephalitis virus (SLEV).
For the 262-bp EEEV genomic fragment, an outer set of
forward (P4 (EEE-4) - CTAGTTGAGCACAAACACCG-
CA) and reverse (P7 (cEEE-7) - CACTTGCAAGGT-
GTCGTCTGCCCTC) primers, followed by a nested set of
forward (P5 (EEE-5) - AAGTGATGCAAATCCAACTC-
GAC) and reverse (P6 (cEEE-6) - GGAGCCACACG-
GATGTGACACAA) primers, was used (8). The RT-PCR
mixture was similar to that described by Kuno (6). The
thermal cycling parameters varied from those of WNV as
follows: 94°C for 90 s followed by 30 cycles of 94°C for
20 s, 65°C for 35 s, 72°C for 17 s, and then a final elonga-
tion step of 72°C for 4 min. A single RT-PCR procedure
was used for SLEV. The 393-bp genomic fragment was
generated by using forward (SLE727 – GTAGCCGACG-
GTCAATCTCTGTGC) and reverse (SLE119c - ACTCG-
GTAGCCTCCATCTTCATCA) primers and using param-
eters as for WNV (9).
Bacterial Culture
Swabs of individual tissues were streaked onto 5%
bovine blood agar (BBA), Wilkins-Chalgren anaerobe
agar, mycoplasma agar, Lowenstein-Jensen agar slant, and
Hektoen Enteric agar (HE) agar (intestines only). Blood
was inoculated into thioglycolate broth and streaked onto
BBA. Inoculated media were incubated at 30°C with
duplicate blood agar plates incubated in the presence or
absence of 5% CO
2
, with the exception of the anaerobic
cultures, which were incubated at 37°C. The thioglycolate
broth was subcultured onto BBA after 24 h. Plates were
examined each day for growth and subcultured onto BBA
as needed. Bacterial colonies selected from pure cultures
were Gram stained. Cultures were injected into Sensititre
(Trek Diagnostic Systems, Westlake, OH) gram-negative
AP80 or gram-positive AP90 autoidentification plates and
the antibiotic sensitivity plate CMVIECOF and allowed to
incubate for 18 h at 37°C before automated reading of the
reactions per the manufacturers directions. Any isolates
that failed to be identified by the Sensititre system were
identified by using the RapID NF Plus System (Remel,
Norcross, GA) or the API20E system (API Analytab
Products, Plainview, NY).
Results
Clinical Findings
The affected alligators appeared to “star gaze” in the
water just before death, suggesting neurologic lesions (10).
Alligators sometimes became stranded in the dry part of
the pen with loss of leg control and neck spasms. No long-
term signs of stress were noted, and most animals were
eating well until a few days before death. The hatchlings
(approximately 30-cm long at the time of the epizootic)
and juveniles (1–2 m long) seemed to be more severely
affected.
A specific pattern of transmission was not noted in
2001. However, in 2002, the alligator deaths initially
occurred in one building and spread throughout the build-
ing in the opposite direction from that taken to feed and
clean the animals. At least one interruption of chlorine
addition to flush water occurred before the 2002 epizootic.
Deaths were not incurred in the breeding colony, and no
deaths were reported in birds that inhabited the rookery.
Gross Findings
2001
Both Florida and Georgia stock animals were affected,
but, in general, the Florida stock was affected first.
Initially, three juvenile alligators were sent for necropsy
during the 2001 epizootic. In general, the alligators were in
good to excellent body condition. One alligator had
approximately 25 mL of serosanguinous fluid in the peri-
cardial sac and 50 mL yellow serous fluid in the peritoneal
cavity. Two of the three had yellow-tan, caseous necrosis
of the palatine tonsils and multiple caseous yellow-tan
plaques, 2- to 10-mm in diameter, on the mucosal surfaces
of the esophagus, corpus, and pars pylorica. Only scant
ingesta were noted throughout the gastrointestinal (GI)
tract, and the intestinal mucosa was hemorrhagic in rare
instances. The liver and spleen of one alligator had multi-
ple 1- to 3-mm tan foci scattered throughout the parenchy-
ma. One alligator was in poor to moderate body condition
and had scattered bronchiectasis, no ingesta throughout the
GI tract, and mild multifocal serous atrophy of fat. No
other gross lesions were noted.
Approximately 2 months after the 2001 epizootic
began, another juvenile, live alligator was submitted to our
laboratory. The gross lesions were similar to those
described above but with numerous 1- to 3-mm tan foci in
the parenchyma of the liver, spleen, and kidneys.
2002
Three alligators were examined from the fall 2002 epi-
zootic, two juveniles and a hatchling. The two juveniles
had lesions similar to those described in the previous year.
796 Emerging Infectious Diseases • Vol. 9, No. 7, July 2003
RESEARCH
The liver and kidneys of the hatchling were pale and mot-
tled tan/brown. Ingesta were scant throughout the GI tract.
The free-ranging alligator was in excellent body condition.
No significant gross changes were noted in its tissues.
Light Microscopic Findings
2001
Tissues of the alligators from the 2001 epizootic were
examined and were similar in two of the three alligators. In
the brain, rare glial nodules that contained occasional het-
erophils were present (Figure 1). The spleen was congest-
ed with moderate diffuse reticuloendothelial hyperplasia
and moderate numbers of heterophils. The tonsil had
severe multifocal coalescing areas of caseous necrosis and
heterophilic inflammation with reactive lymphoid follicu-
lar hyperplasia. In the esophagus, a focally extensive,
mixed ulcerative, and proliferative lesion was present; it
had a marked mixed but predominantly mononuclear
inflammation, colonies of bacteria, and extensive fibrin
deposition. In the liver, multifocal lymphoplasmacytic
aggregates and heterophilic granulomas were present, con-
sisting of caseous necrotic foci with degenerate heterophils
surrounded by an outer layer of macrophages, lympho-
cytes, and heterophils. The lungs were congested with mild
diffuse or patchy lymphoplasmacytic and heterophilic
interstitial infiltrates. The kidney had multifocal het-
erophilic 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, dif-
fuse 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 tis-
sues were as described for the first two alligators.
Tissues from the alligator seen 2 months after the epi-
zootic had similar findings to those of the first two alliga-
tors 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.
2002
Multiple tissues from the two juvenile alligators from
the 2002 epizootic were examined. The tissue changes
were similar to those described for the 2001 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 perivas-
cular infiltration of mild numbers of heterophils (Figure 1).
One alligator had a small focus of macrophages and het-
erophils noted within the endocardium.
Multiple tissues were examined from the hatchling alli-
gator, 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 mul-
tiple, random foci of patchy vacuolar degeneration of the
myocytes and random aggregates of lymphocytes, plasma
cells and heterophils. Mild numbers of mixed inflammato-
ry cells were seen within the intestinal lamina propria. The
remaining tissues were unremarkable. Major pathologic
changes were not observed by light microscopy in the tis-
sues from the free-ranging alligator.
Virus Isolation/RT-PCR
Virus isolation was negative for all animals from the
2001 epizootic. WNV was isolated from tissues from all
animals in the 2002 epizootic. Additionally, all animals
from the 2002 epizootic and one animal from the 2001 epi-
zootic were positive for WNV by RT-PCR from fresh or
Emerging Infectious Diseases • Vol. 9, No. 7, July 2003 797
RESEARCH
Figure 1. Perivascular changes observed within the brain of alliga-
tors infected with West Nile virus (400x). A. Perivascular infiltrates
were composed of primarily lymphocytes, plasma cells, and
macrophages in the hatchling alligator. B. Perivascular infiltrates
were composed of primarily heterophils (arrows) in juvenile alliga-
tors.
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. All tissues tested
negative by RT-PCR for EEEV and SLEV. Retrospective
attempts to culture WNV at both 37°C and room tempera-
ture on FHM, CCO, EPC, and WWS cells were negative.
Aliquots from the horsemeat that was being fed during
the 2002 epizootic tested positive for WNV by RT-PCR
(Figure 2). Aliquots of the horsemeat from two postepi-
zootic shipments were negative for WNV by RT-PCR.
Bacterial Culture
Aeromonas sobria and Edwardsiella tarda were consis-
tently cultured from the intestines. These organisms and
occasionally others (Escherichia coli, Pseudomonas fluo-
rescens, α- and β-hemolytic Streptococcus) were isolated
from various tissues (liver, lung, and kidney) from the alli-
gators dying during the 2001 epizootics and the juveniles
from the 2002 epizootics. Alcaligenes spp. were isolated
from a tonsil swab in one of the animals in 2001.
Salmonella Group D was isolated from the intestines of the
hatchling alligator submitted in 2002.
Discussion
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 bac-
terial cause. Given that both the RT-PCR and virus isola-
tion were positive for WNV, that virus is suspected to be
the underlying cause of both epizootics. Contaminated
horsemeat is the presumed source of the outbreak. We
speculate that the WNV infection led to the alligators’
immune systems’ becoming immunocompromised, which
resulted in the animals being more susceptible to various
environmental stressors and subsequent invasion by
opportunistic pathogens. Failure to isolate virus from the
alligators in 2001 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 cul-
ture 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 envi-
ronmental temperature, this finding was likely coinciden-
tal, especially given that the animals were housed in envi-
ronmentally 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 sup-
ply was less likely to contain virus. Furthermore, all ani-
mals have equal potential for viral exposure through con-
sumption 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 tem-
perature dependent until the animals are several months of
age (11). 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 sec-
ondary invaders.
Extrinsic stressors may have increased certain animals’
susceptibility to the virus or opportunistic pathogens. For
example, the pens where the epizootics originated tended
to be the first to be washed out at 6 a.m., the coolest time
of the day. 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. This cold stressor would serve as a shock to the
animals’ systems. During the 2002 outbreak, an additional
stress was internal construction, undertaken 2 weeks before
the epizootic within the initially affected building. The
environmental (temperature and darkness) control of the
building was maintained during this time, but silence was
not maintained. Additionally, sanitation-related stress may
have occurred during periods of intermittent flushing, such
as over weekends and during pen renovation activities.
Whether brood stock source had an affect on the sus-
ceptibility of the animals is not clear. Although Florida
stock animals were those initially affected, this finding
was likely coincidental because of their location in the
pens. The pens that were more exposed to external stres-
798 Emerging Infectious Diseases • Vol. 9, No. 7, July 2003
RESEARCH
Figure 2. West Nile virus (WNV) reverse transcription-polymerase
chain reaction results from epizootic die-offs in farm-raised alliga-
tors. The expected amplicon is 248 bp. Lane 1, a 100-bp molecu-
lar weight ladder. Lane 2, the positive WNV control. Lane 3, fresh
tissue samples from a juvenile alligator in the 2002 epizootic. Lane
4, virus isolation cell homogenate from a juvenile alligator in the
2002 epizootic. Lane 5, horsemeat that was being fed to alligators
during the 2002 epizootic. Lane 6, initial postepizootic horsemeat
shipment. Lanes 7, 8, and 9, formalin-fixed, paraffin-embedded tis-
sues of juvenile alligators in 2001 and 2002. Lane 10, fresh tissue
from a wild alligator. Lane 11, negative WNV control.
sors contained Florida animals. Additionally, most animals
in the production unit are from Florida brood stock.
Several management recommendations were suggested
to the producer. The primary recommendation was to stop
feeding horsemeat and switch to another food source such
as beef or fish. We also recommended that the water tem-
perature be reduced to 29.4°C in an attempt to reduce the
stress of rapid growth and perhaps produce an environment
less conducive for viremia. To date, neither of these rec-
ommendations has been implemented, but subsequent
horsemeat shipments have tested negative. Future investi-
gation will include the testing of the eggs from the brood
stock, clinically healthy animals, rookery birds, and free-
ranging alligators to explore the epidemiology of this virus
in ectotherms.
Acknowledgments
We thank the staff of The University of Georgia Tifton
Veterinary Diagnostic and Investigational Laboratory for help in
processing the samples.
Dr. Miller is an assistant professor in the Department of
Pathology at the University of Georgia (UGA) College of
Veterinary Medicine. She works as a veterinary pathologist at the
UGA Tifton Veterinary Diagnostic and Investigational
Laboratory. Her research interests are in wildlife disease and
reproduction.
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Address for correspondence: Debra L. Miller, University of Georgia,
Veterinary Diagnostic and Investigational Laboratory, Tifton, GA 31793,
USA; fax: 229-386-7128; email: dmiller@tifton.uga.edu
Emerging Infectious Diseases • Vol. 9, No. 7, July 2003 799
RESEARCH
The opinions expressed by authors contributing to this journal do
not necessarily reflect the opinions of the Centers for Disease
Control and Prevention or the institutions with which the authors
are affiliated.
... WNV is maintained by an enzootic cycle between susceptible bird species and competent mosquitoes, particularly Culex species mosquitoes [9], as vectors. A wide variety of mammals and reptiles [10][11][12], including humans and horses, can occasionally become infected with WNV. Generally, these species do not develop sufficient viremia to sustain transmission and are considered incidental or dead-end-hosts [13,14]. ...
Article
Full-text available
West Nile virus (WNV) infections were first detected in Germany in 2018, but information about WNV seroprevalence in horses is limited. The study's overall goal was to gather information that would help veterinarians, horse owners, and veterinary-, and public health-authorities understand the spread of WNV in Germany and direct protective measures. For this purpose, WNV seroprevalence was determined in counties with and without previously registered WNV infections in horses, and risk factors for seropositivity were estimated. The cohort consisted of privately owned horses from nine counties in Eastern Germany. A total of 940 serum samples was tested by competitive panflavivirus ELISA (cELISA), and reactive samples were further tested by WNV IgM capture ELISA and confirmed by virus neutralization test (VNT). Information about potential risk factors was recorded by questionnaire and analyzed by logistic regression. A total of 106 serum samples showed antibodies against flaviviruses by cELISA, of which six tested positive for WNV IgM. The VNT verified a WNV infection for 54 samples (50.9%), while 35 sera neutralized tick-borne encephalitis virus (33.0%), and eight sera neutralized Usutu virus (7.5%). Hence, seroprevalence for WNV infection was 5.8% on average and was significantly higher in counties with previously registered infections (p = 0.005). The risk factor analysis showed breed type (pony), housing in counties with previously registered infections, housing type (24 h turnout), and presence of outdoor shelter as the main significant risk factors for seropositivity. In conclusion, we estimated the extent of WNV infection in the resident horse population in Eastern Germany and showed that seroprevalence was higher in counties with previously registered equine WNV infections.
... Several encephalitis viruses as well as West Nile Virus have been detected in free-ranging lizards, snakes, turtles, and crocodylians (Farfán-Ale et al., 2006;Mendoza-Roldan et al., 2021). Although population-level effects of mosquito-borne viruses have not been reported for reptilian hosts, West Nile Virus is suspected as the cause of the deaths of hundreds of captive alligators in Florida (Miller et al., 2003;Jacobson et al. 2005), and has been reported in wild and captive crocodiles (Steinman et al., 2003;Machain-Williams et al., 2013;Dahlin et al., 2016) West Nile and other mosquito-borne viruses are potential threats to wild populations of reptiles in reptilian biodiversity hotspots, such as the Galápagos Islands (Kilpatrick et al., 2006;Bataille et al., 2009). ...
Chapter
Most of the more than 11,000 extant species of nonavian reptiles are squamates (lizards and snakes); there are about 360 extant species of turtles, 26 crocodylians, and one rhynchocephalian. Although the diversity of reptiles is greatest in the tropics, many species occur in temperate regions and a few have geographic ranges that extend north of the Arctic Circle. Antarctica is the only continent with no extant reptiles. Oviparity is the ancestral mode of reproduction, but viviparity has evolved repeatedly among squamates. Both genetic sex determination (XX/XY and ZW/ZZ) and environmental sex determination are represented, and genetic, environmental, and non-genetic maternal factors interact in some species. Environmental sex-determination is universal in crocodylians, widespread among turtles, and present in some clades of squamates. Parental care is universal among crocodylians and is present in some species of squamates and turtles. Ectothermy, an ancestral character, is central to the biology of reptiles, and is responsible for their low metabolic rates and their high efficiency of secondary production. Lizards typically eat daily and consume many small prey items, whereas snakes eat less frequently and consume larger prey items relative to their body size. Low metabolic rates make small body sizes energetically feasible for ectotherms, and more than half of the extant species of lizards are smaller than nearly all mammals and birds. Among squamates, the mode of predation – from sit-and-wait to widely foraging – has a strong phylogenetic component and correlates with many elements of ecology, morphology, physiology, and behavior. Many species of snakes and a few lizards are venomous, and some snakes are poisonous because they sequester toxins from their prey. Although most species of reptiles have little economic value, they are important components of energy and nutrient flow in terrestrial ecosystems. Habitat loss, pollution, invasive species, disease, and global climate change affect many species. The life histories of most large species of turtles, lizards, snakes, and crocodylians depend on prolonged adult survival and reproduction, and these species are vulnerable to commercial exploitation.
... Monitoring the prevalence and extent of these outbreaks is easily done by PCR using water, soil, or tissue samples [17][18][19]. For example, farmed alligators are known to be an environmental reservoir for West Nile virus [38]; however, the extent to which alligators immunologically respond to the virus with specific IgY production remains unknown. Also, while there are examples of other crocodilians being sero-positive for the human pathogen Leptospira, and suggestions that humans may acquire Leptospira from crocodilians [39][40][41]. ...
Article
Full-text available
Little is known about the disease ecology of American alligators (Alligator mississippiensis), and especially how they respond immunologically to emerging infectious diseases and zoonotic pathogens. In this study, we examined serum samples collected from wild alligators in Florida (2010–2011) and South Carolina (2011–2012, 2014–2017) for antibody responses to multiple bacteria. Immunoglobulin Y (IgY) was purified from serum to generate a mouse monoclonal antibody (mAb AMY-9) specific to the IgY heavy chain. An indirect ELISA was then developed for quantifying antibody responses against whole cell Escherichia coli, Vibrio parahaemolyticus, Vibrio vulnificus, Mycobacterium fortuitum, Erysipelothrix rhusiopthiae, and Streptococcus agalactiae. In Florida samples the primary differences in antibody levels were between January–March and late spring through summer and early fall (May–October), most likely reflecting seasonal influences in immune responses. Of note, differences over the months in antibody responses were confined to M. fortuitum, E. rhusiopthiae, V. vulnificus, and E. coli. Robust antibody responses in SC samples were observed in 2011, 2014, and 2015 against each bacterium except E. coli. All antibody responses were low in 2016 and 2017. Some of the highest antibody responses were against V. parahaemolyticus, M. fortuitum, and E. rhusiopthiae. One SC alligator estimated to be 70+ years old exhibited the highest measured antibody response against V. parahaemolyticus and M. fortuitum. By combining data from both sites, we show a clear correlation between body-mass-indices (BMI) and antibody titers in all six of the bacteria examined. Our study provides a critical antibody reagent and a proof-of-concept approach for studying the disease ecology of alligators in both the wild and in captivity.
... In 1995, 60 out of 74 farmed American alligators in a Florida farm died from a novel mycoplasma species (Clippinger et al. 2000). Farmed American alligators have had mortality incidents associated with West Nile virus in 2002 in Florida, USA (Jacobson et al. 2005), and in 2003 in Georgia, USA (Miller et al. 2003). Other crocodilian species have shown to be asymptomatic with West Nile virus infection, such as captive Morelet's crocodiles in Mexico (Farfán-Ale et al. 2006;Machain-Williams et al. 2013), or to show mild symptoms such as the saltwater crocodile (Isberg et al. 2019). ...
Book
This book aims to be a comprehensive review of the literature on the conservation genetics of the New World crocodilians, from the biological and demographical aspects of the living species to the application of molecular techniques for conservation purposes. It covers the current status of the molecular genetics applied to phylogenetics, phylogeography, diversity, kinship and mating system, and hybridization, as well its implications for decision making with regards to the conservation of these species at academic and governmental levels. This book can be used as a guide for graduate and undergraduate students to understand how conservation genetics techniques are carried out and how they can help preserve not only crocodilians but also other living species.
Thesis
L'objectif était d'évaluer le risque de circulation enzootique du virus West Nile (WN) chez le cheval en Camargue, région dans laquelle ce virus a déjà causé plusieurs épizooties. L'épidémiologie de la maladie de WN est très complexe, du fait de l'implication potentielle d'un grand nombre d'espèces de vecteurs et d'hôtes. De par sa transmission principalement vectorielle, la circulation du virus WN est fortement influencée par des facteurs environnementaux. L'espèce équine a été choisie comme témoin de la circulation du virus WN, car le cheval est particulièrement sensible à l'infection par ce virus. La méthode appliquée est basée sur l'utilisation du lien direct existant entre l'environnement et la circulation enzootique du virus WN, par l'étude de la séropositivité (IgG) chez le cheval. Dans les deux premières études présentées, certains facteurs de risque environnementaux ont été identifiés, comme des classes d'occupation du sol (zones agricoles hétérogènes, végétation inondée) ou des indices de paysage (Indice d'Imbrication et de Juxtaposition), ayant conduit à l'élaboration d'une carte de risque pour cette circulation dans le bassin méditerranéen français. Des facteurs de risque individuels, comme la race, l'âge et l'activité du cheval, ont également été identifiés. Dans la troisième étude présentée, des hypothèses de transmission du virus en Camargue ont été testées. Dans la région d'étude, le virus serait introduit par les oiseaux migrateurs et amplifié par Culex modestus et plusieurs espèces d'oiseaux compétentes. L'effet de dilution n'aurait pas d'impact sur l'amplification du virus en Camargue. Le virus serait transmis au cheval par C. modestus et C. pipiens.
Article
West Nile virus (WNV) is a neurotropic flavivirus that can cause acute febrile illness leading to neuroinvasive disease. Depression is a well-described outcome following infection, but the underlying pathogenic mechanisms are unknown. Proinflammatory cytokines play important roles in WNV infection, but their role in depression post-WNV remains unstudied. This research aimed to retrospectively evaluate associations between proinflammatory cytokines and new onset depression in a WNV cohort. Participants with asymptomatic WNV infection were significantly less likely to report new onset depression when compared to those with symptomatic disease. Participants with encephalitis and obesity were significantly more likely to report new onset depression post-infection. Based on univariate analysis of 15 antiviral or proinflammatory cytokines, depression was associated with elevated MCP-1 and decreased TNFα, whereas G-CSF was significantly elevated in those with a history of neuroinvasive WNV. However, no cytokines were statistically significant after adjusting for multiple comparisons using the Bonferroni method. While symptomatic WNV infection, encephalitis, and obesity were associated with new onset depression following infection, the role of proinflammatory cytokines requires additional studies. Further research involving paired acute-convalescent samples, larger sample sizes, and additional data points would provide additional insight into the impact of the inflammatory response on WNV-mediated depression.
Article
West Nile virus (WNV) overwintering is poorly understood and likely multifactorial. Interest in alligators as a potential amplifying host arose when it was shown that they develop viremias theoretically sufficient to infect mosquitoes. We examined potential ways in which alligators may contribute to the natural ecology of WNV. We experimentally demonstrated that alligators are capable of WNV amplification with subsequent mosquito infection and transmission capability, that WNV-infected mosquitoes readily infect alligators and that water can serve as a source of infection for alligators but does not easily serve as in intermediate means for transmission between birds and alligators. These findings indicate potential mechanisms for maintenance of WNV outside of the primary bird-mosquito transmission cycle.
Chapter
Although reptiles have often been overlooked in research, information on viruses of reptiles has been growing steadily in recent decades as has our understanding of the importance of these animals in the ecosystem. As ectotherms, their immune systems are dependent on temperature, among other factors, and interactions between infection and disease are complex and dependent on host, pathogen, and environmental factors. This chapter provides an overview of the viruses described in reptiles so far, as well as insight into some of the diseases caused by viruses in this group of animals. It also discusses the reptile immune system and the host reaction to infection. Influences of the environment on development of disease are in many cases not well understood, and this chapter includes a discussion of some important progress in this field. Studies of the effects of viruses on wild, pet, and farmed reptiles are limited, but indicate that viral disease can strongly affect individual populations in the wild, and that human action and the animal trade likely play a role in disease epidemiology.
Chapter
Since its introduction to North America in 1999, West Nile virus (WNV) has established itself as an endemic pathogen with regular seasonal outbreaks. The single-stranded RNA flavivirus is primarily transmitted by mosquitoes in the genus Culex and maintained in an enzootic transmission cycle by a diverse assemblage of avian hosts. Humans, equines, and other mammals serve as incidental or dead-end hosts. WNV is a significant threat to public health, with estimates indicating that more than seven million individuals have been infected. Although the majority of these individuals are asymptomatic, approximately 20% develop a febrile illness or neuroinvasive disease, the latter associated with high rates of mortality in the elderly and immunocompromised. Disease-associated pathology of the central nervous system is prevalent not only during the acute phase of WNV infection but also as significant long-term sequelae. Although vaccine and therapeutic research progressed over the last 20 years, no agents are licensed for use in humans, and treatment depends on supportive care. Mitigation efforts are instead directed towards the elimination and control of mosquito vectors. Future research will need to leverage technological and epidemiological advances to overcome a host of challenges in order to alleviate the immense economic and human costs of this endemic zoonotic disease.
Chapter
Advancements in genomic techniques have greatly increased the scope of research into many aspects of crocodilian evolution and biology. As cold-blooded amniotes, crocodilians hold a unique evolutionary position for understanding all vertebrate lineages. Applying up-to-date genomic techniques to crocodilian genomes can have far-reaching implications for understanding whole-genome evolution, adaptation and veterinary medicine. The slow rates of genomic mutations in crocodilians also mean we can use them as a way to gain insights into ancient genomes. Currently, only a few complete crocodilian genomes are publicly available; however this is rapidly changing thanks to the work of several laboratories around the world. This chapter will outline the existing available genomic research into biological systems such as innate immunity and sensory perception, as well as disease susceptibility and possible applications in human medicine. We will also outline industry and conservation implications of having high-quality crocodilian genomes for complex trait analysis and identifying ancient hybridization events.
Article
Full-text available
Specific and sensitive reverse transcription-PCR (RT-PCR) assays were developed for the detection of eastern, western, and Venezuelan equine encephalitis viruses (EEE, WEE, and VEE, respectively). Tests for specificity included all known alphavirus species. The EEE-specific RT-PCR amplified a 464-bp region of the E2 gene exclusively from 10 different EEE strains from South and North America with a sensitivity of about 3,000 RNA molecules. In a subsequent nested PCR, the specificity was confirmed by the amplification of a 262-bp fragment, increasing the sensitivity of this assay to approximately 30 RNA molecules. The RT-PCR for WEE amplified a fragment of 354 bp from as few as 2,000 RNA molecules. Babanki virus, as well as Mucambo and Pixuna viruses (VEE subtypes IIIA and IV), were also amplified. However, the latter viruses showed slightly smaller fragments of about 290 and 310 bp, respectively. A subsequent seminested PCR amplified a 195-bp fragment only from the 10 tested strains of WEE from North and South America, rendering this assay virus specific and increasing its sensitivity to approximately 20 RNA molecules. Because the 12 VEE subtypes showed too much divergence in their 26S RNA nucleotide sequences to detect all of them by the use of nondegenerate primers, this assay was confined to the medically important and closely related VEE subtypes IAB, IC, ID, IE, and II. The RT-PCR-seminested PCR combination specifically amplified 342- and 194-bp fragments of the region covering the 6K gene in VEE. The sensitivity was 20 RNA molecules for subtype IAB virus and 70 RNA molecules for subtype IE virus. In addition to the subtypes mentioned above, three of the enzootic VEE (subtypes IIIB, IIIC, and IV) showed the specific amplicon in the seminested PCR. The practicability of the latter assay was tested with human sera gathered as part of the febrile illness surveillance in the Amazon River Basin of Peru near the city of Iquitos. All of the nine tested VEE-positive sera showed the expected 194-bp amplicon of the VEE-specific RT-PCR-seminested PCR.
Article
Full-text available
The development and application of nucleic acid sequence-based amplification (NASBA) assays for the detection of West Nile (WN) and St. Louis encephalitis (SLE) viruses are reported. Two unique detection formats were developed for the NASBA assays: a postamplification detection step with a virus-specific internal capture probe and electrochemiluminescence (NASBA-ECL assay) and a real-time assay with 6-carboxyfluorescein-labeled virus-specific molecular beacon probes (NASBA-beacon assay). The sensitivities and specificities of these NASBA assays were compared to those of a newly described standard reverse transcription (RT)-PCR and TaqMan assays for SLE virus and to a previously published TaqMan assay for WN virus. The NASBA assays demonstrated exceptional sensitivities and specificities compared to those of virus isolation, the TaqMan assays, and standard RT-PCR, with the NASBA-beacon assay yielding results in less than 1 h. These assays should be of utility in the diagnostic laboratory to complement existing diagnostic testing methodologies and as a tool in conducting flavivirus surveillance in the United States.
Article
Serum from 2,294 wild birds, 128 mammals, 22 reptiles, and 96 amphibians caught from May 1965 to June 1966 in Israel were examined for antibodies against Eastern equine encephalomyelitis. Semliki Forest, Sindbis, Langat, Israel turkey meningoencephalitis, and West Nile (WN) viruses by hemagglutination-inhibition (HI) tests. The incidence of positive serum was 14.4% for wild birds, 5% for mammals, and 9% for reptiles. No antibodies were detected in the amphibian serum; 73% of the positive avian serum had antibodies against group B viruses. The rest of the positive serum was divided among that positive for group A and that positive for both group A and group B viruses. Serologic conversions in sentinel chickens confirmed the activity of WN virus in the Hadera region during the period studied. Use of acetone extraction followed by kaolin treatment of serum minimized the number of falsely positive results obtained in the HI tests. Good correlation was found between results obtained in HI tests and indirect fluorescent-antibody staining of WN antigen among serum samples from 116 Columbidae examined by these two methods.
Article
A series of experiments on the role of lizards as overwintering hosts of Japanese encephalitis virus (JEV) was carried out. Two species of lizards, T. tachydromoides and E. latiscutatus, 2 species of mosquitoes, Cx. p. fatigans and Cx. p. pallens, and 2 strains of JEV, JaGAr#01 and JaGAr 19461, were used in this study. Firstly transmission of JEV from infected mosquitoes to uninfected lizards and from infected lizards to normal mice by the bite of mosquitoes was demonstrated successfully. Cx. pipiens group mosquitoes were found to feed readily on lizards as compared to Cx. tritaeniorhynchus, the primary vector of JEV in Japan. Secondly simulated hibernation of JEV in lizards was carried out under indoor and outdoor conditions. In the outdoor hibernation, lizards were injected with JEV on October 14, 1968, entered in hibernation on October 19 and were recovered from hibernation on April 10, 1969. Viremias were demonstrated in the lizards for a few weeks in late April. Thirdly JEV isolation and HI antibody detection were attempted from blood samples of field-caught reptiles, 7 species of snakes and 3 species of lizards and among amphibians, 2 species of frogs. HI antibody against JEV was found at a rate of 14.3% from E. latiscutatus and 4.0% from T. tachydromoides, though JEV was not isolated from all the blood samples of these cold-blooded animals. The roles of lizards as overwintering hosts of JEV were discussed.
Article
Experimental infection of four species of snakes, Rhabdophis tigrinus tigrinus, Elaphe quadrivirgata, Elaphe climacophora and Agkistrodon halys, and five species of lizards, Takydromus tachydromoides, Eumeces latiscutatus, Eumeces barbouri, Eumeces marginatus oshimensis and Gekko japonicus, with Japanese encephalitis virus (JEV) was carried out. Evidence of JEV multiplication in snakes was not obtained at least under the conditions used in the present study. All lizards except G. japonicus were infected with JEV by ip injection of virus suspension. The minimum infectious dose for a lizard was around 10(3) MLD50/0.05 ml, and this dose was considered to be proportional to the virus dose which is injected into a host by a vector mosquito at a single bite. Temperature dependence of JE virus growth in the lizards was demonstrated. JEV multiplied slower at 20 degrees C than at 26 degrees C, though the peak titers of viremia were equivalent in both groups of lizards kept at 20 degrees C and 26 degrees C. E. latiscutatus developed viremia with ip injection of a partially attenuated strain, Nakayama NIH which could not infect adult mice by peripheral inoculation. T. tachydromoides and E. latiscutatus were also infected by oral feeding of JEV infected mosquitoes. E. latiscutatus was infected by oral feeding of only one infected mosquito.
Article
Garter snakes (Thamnophis spp.) have been considered to possibly play an important role in the ecology of western equine encephalitis (WEE) virus. Serological tests (hemagglutination-inhibition, complement-fixation, neutralization test in mice, and plaque neutralization) to detect antibody in these reptiles following laboratory exposure t this virus have, in our experience, been unsatisfactory. A new test, the snake globulin precipitation (SGP) test, has been developed and we consider it to be reliable in detecting antibody in WEE virus-infected garter snakes. Antibody has been detected in these snakes over 4.5 years following inoculation with WEE virus. The SGP test should be a valuable tool in obtaining further information regarding the possible role of these cold-blooded vertebrates in the ecology of this important arbovirus.
Article
A selected number of PCR protocols were evaluated to determine if they could serve as a universal protocol for detecting and identifying all arboviruses. In this study, four parameters that affect the efficacy of RT-PCR (RNA extraction method, choice of reverse transcriptase, choice of DNA polymerase and thermocycling program) were evaluated in combination. The most optimal combination of those parameters employed use of silica gel membrane spin column, RAV-2 reverse transcriptase, Tth DNA polymerase, and a simple modification of a published thermocycling program. By this modified protocol, viral RNA could be amplified satisfactorily with more than 50 pairs of primers designed for diagnosis of arboviruses representing five families. The sensitivity and specificity obtained by this universal protocol were comparable to those obtained by the original protocol for each primer pair tested; and for some primers, improved sensitivity was observed. It was also found that a simple modification of a suggested protocol of a commercial RT-PCR kit could produce nearly identical results and serve as another universal protocol. With the use of a universal diagnostic reverse transcriptase-polymerase chain reaction (RT-PCR) protocol, simultaneous screening of clinical or biological specimens against a large number of RNA viruses belonging to many families can be performed more efficiently for etiologic determination in the situations complicated by the difficulty of differential diagnosis. Furthermore, such a universal protocol facilitates reducing the cost of PCR-based diagnostic operation and standardizing the qualities of PCR-based diagnosis within an institution or among collaborating institutions. A logical strategy is to conduct diagnosis in two stages by using broadly group-reactive primers in the first stage to narrow the range of possible etiologic agents and using virus-specific primers in the second stage for identification. Before such a strategy is employed, however, more group-reactive primers for a large number of arboviruses, for which no such primers currently exist, must be made available. Furthermore, the best pair or pairs of primers need to be selected for each virus for the second stage of the strategy.
Article
A traditional single-stage reverse transcription-polymerase chain reaction (RT-PCR) procedure is effective in determining West Nile (WN) virus in avian tissue and infected cell cultures. However, the procedure lacks the sensitivity to detect WN virus in equine tissue. We describe an RT-nested PCR (RT-nPCR) procedure that identifies the North American strain of WN virus directly in equine and avian tissues.