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320
Journal of Zoo and Wildlife Medicine 32(3): 320–323, 2001
Copyright 2001 by American Association of Zoo Veterinarians
INFECTIOUS DISEASE SEROLOGIC SURVEY IN FREE-RANGING
VENEZUELAN ANACONDAS (EUNECTES MURINUS)
Paul P. Calle, V.M.D., Dipl. A.C.Z.M., Jesu´s Rivas, Ph.D., Marı´a Mun˜oz, Lic.,
John Thorbjarnarson, Ph.D., William Holmstrom, B.A., and William B. Karesh, D.V.M.
Abstract: Reptiles can harbor pathogenic microorganisms asymptomatically and serve as potential reservoirs of
infection for humans, domestic animals, and other reptiles. Infectious diseases are also problematic for free-ranging
reptile populations and are an important consideration in reptile reintroduction and translocation projects. There have
been limited serologic studies of free-ranging reptiles for evidence of exposure to potential pathogens. In the present
study, serum or plasma samples from five male and five female free-ranging Venezuelan anacondas (Eunectes murinus)
were screened for antibodies to eastern, western, and Venezuelan equine encephalitis viruses, vesicular stomatitis virus,
ophidian paramyxovirus, 19 Leptospira interrogans serovars, and Cryptosporidium serpentes. Antibodies to these agents
were not detected, or antibody titers were low and possibly nonspecific. These results for the limited number of
anacondas surveyed suggest that they do not serve as significant reservoirs for these infectious agents at this location.
Key words: Anaconda, Eunectes murinus, cryptosporidia, health assessment, serology, virology.
INTRODUCTION
Reptiles may harbor viral, bacterial, and proto-
zoal pathogens asymptomatically and serve as res-
ervoirs of infection for humans, domestic animals,
and other reptiles. Potential pathogens include such
arboviruses of economic importance in agricultural
species as vesicular stomatitis (VS) virus and Vene-
zuelan, eastern, and western equine encephalitis
(VEE, EEE, and WEE, respectively) viruses.
1,19,30,31
Leptospira interrogans is a bacterium that can be
harbored by reptiles and can affect agricultural spe-
cies, humans, and reptiles.
12,16,21,32,34
Other patho-
gens, such as ophidian paramyxovirus
6,19
and Cryp-
tosporidium serpentes,
5,13,33
are specific to snakes
and have caused considerable morbidity and mor-
tality in captive specimens.
Infectious diseases are also factors in the health
of free-ranging populations
7–9,28
and must be con-
sidered when attempting reptilian reintroduction or
translocation.
18
Reports of serologic studies docu-
menting exposure of free-ranging reptile popula-
tions to potential pathogens are limited.
3,12,15,20,28,34
As part of a long-term study of the biology and
conservation of anacondas (Eunectes murinus)in
From the Department of Clinical Care, Wildlife Health
Sciences (Calle), International Programs (Thorbjarnarson),
the Department of Herpetology (Holmstrom), and the De-
partment of Field Veterinary Studies (Karesh), Wildlife
Conservation Society, Bronx, New York 10460-1099,
USA; and Profauna, Ministerio del Ambiente y de Los
Recursos Naturales, Renovables, Caracas, Venezuela (Ri-
vas, Mun˜oz). Present addresses (Mun˜oz): Universidad Si-
mo´n Bolivar, Departamento Estudios Ambientales, Sarte-
nejas, Caracas 1080-A, Venezuela; (Rivas): National Geo-
graphic Television, 1145 17th Street NW, Washington,
D.C. 20036-4688, USA.
the Venezuelan llanos,
2,24–26
health assessments
were performed in conjunction with implantation of
radiotelemetry equipment in free-ranging anacon-
das in March 1992.
2,22,25
Results of hematologic,
biochemical, vitamin, mineral, toxicologic, and par-
asite studies of these snakes have been reported
previously.
2
Serum and heparinized plasma samples
obtained during the project were utilized for viral,
bacterial, and protozoal serologic assays to inves-
tigate the prevalence of exposure to potential path-
ogens in the study population.
MATERIALS AND METHODS
Five male and five female anacondas were col-
lected during the 1992 dry season at the site of an
ongoing study of anaconda ecology and conserva-
tion (Hato El Cedral in the seasonally flooded Ven-
ezuelan llanos).
2,24–26
The snakes were manually re-
strained, physical examinations were performed,
and blood samples were obtained from the ventral
coccygeal vein.
2
Serum and heparinized plasma
samples were separated by centrifugation, trans-
ported to the USA, and frozen at
2
70
8
C until an-
alyzed.
2
Samples were then assessed for exposure
to various infectious agents.
Antibody titers to VEE, EEE, and WEE viruses
were determined for all the anacondas by hemag-
glutination inhibition (HI) testing.
14,29
The HI titer
was the reciprocal of the highest dilution of sample
that inhibited 8 hemagglutinating units of virus. A
positive result was defined as a titer of
$
20. Sera
from seven anacondas (four female and three male)
were screened for titers to VS virus serovars Indi-
ana and New Jersey by serum neutralization (SN)
testing.
11
The SN titer was the reciprocal of the
highest dilution of sample that inhibited cytopathic
effect. A positive result was defined as a titer of
321
CALLE ET AL.—VENEZUELAN FREE-RANGING ANACONDA SEROLOGY
Table 1. Positive antibody titers to Leptospira interrogans serovars and Venezuelan equine encephalitis (VEE) virus
in free-ranging Venezuelan anacondas (Eunectes murinus). All anacondas tested had negative antibody titers to 14
other L. interrogans serovars, eastern equine encephalitis virus, western equine encephalitis virus, vesicular stomatitis
virus, ophidian paramyxovirus, and Cryptosporidium serpentes (data not shown).
Anaconda
no.
L. interrogans serovars
a
icterohaemorrhagiae/
copenhageni autumnalis bratislava
icterohaemorrhagiae/
icterohaemorrhagiae kennewicki VEE virus
b
49
73
75
76
78
79
100
100
100
100
200
100
200
200
100
100
100
100 100
20
a
Antibody titers determined by microscopic agglutination testing.
b
Antibody titers determined by hemagglutination inhibition testing.
$
8. Testing for VEE, EEE, WEE, and VS virus
antibodies was performed at the Texas Veterinary
Medical Diagnostic Laboratory (College Station,
Texas 77841, USA).
Samples from all 10 anacondas were tested at the
University of Florida College of Veterinary Medi-
cine (Gainesville, Florida 32610, USA) for ophid-
ian paramyxovirus antibody titers with an HI test
developed and validated for snakes.
23
HI titers were
the reciprocal of the highest dilution of sample that
inhibited agglutination. Negative results were de-
fined as titers of
#
20, titers of 40–80 were consid-
ered suspicious, and titers of
.
80 were considered
positive.
Antibody titers to L. interrogans serovars cani-
cola, hardjo, grippotyphosa, icterohaemorrhagiae/
copenhageni, pomona, australis, autumnalis, bal-
lum, bataviae, bratislava, icterohaemorrhagiae/icte-
rohaemorrhagiae, javanica, pyrogenes, sejroe, sa-
xkoebing, szwajizak, tarassovi, kennewicki, and
wolffi were determined for all anacondas by micro-
scopic agglutination testing (MAT) performed at
the Diagnostic Laboratory, New York State College
of Veterinary Medicine (Ithaca, New York 14852,
USA).
4,10,27
Samples were considered positive if
$
50% of the live Leptospira antigen/cells aggluti-
nated at the screening dilution of 1:100. Titers of
samples reacting at this dilution were determined
by assay of serial dilutions of sample.
Samples from five female and three male ana-
condas were analyzed at the Animal Diagnostic
Laboratory (Baltimore, Maryland 21228, USA) for
C. serpentes antibody titers by an indirect enzyme-
linked immunosorbent assay (ELISA) developed
and validated for snakes.
13
Positive results were de-
fined as an absorbance value greater than 3 SDs
above the mean absorbance value of the negative
control samples.
RESULTS
Positive results are listed in Table 1. All 10 an-
acondas had negative HI titers of 10 for EEE and
WEE viruses. Nine had negative VEE virus titers
of 10, and one had a titer of 20 (Table 1). All seven
anacondas tested had negative titers for VS virus
serovars Indiana and New Jersey. All anacondas
had negative HI titers of
#
10 for ophidian para-
myxovirus. Five anacondas were seropositive with
titers of 100 or 200 for one to four L. interrogans
serovars (Table 1). All 10 anacondas had negative
ELISA titers for C. serpentes.
DISCUSSION
The anacondas studied concentrate in small bod-
ies of water during the dry season but disperse
broadly (over
.
3,000 m
2
) across the flooded sa-
vanna during the rainy season.
24,25
The anacondas’
ranges are within an active ranch housing cattle,
horses, and capybara (Hydrochoerus hydrochaer-
is).
24
Reptiles may serve as reservoir hosts for such
zoonotic arboviruses of importance in domestic ag-
ricultural species as VEE, WEE, EEE, and VS vi-
ruses.
1,19,20,30,31
None of the anacondas tested had el-
evated titers to any of these viruses. The one ana-
conda with a VEE titer of 20 may represent a true
VEE titer or may result from cross-reaction in the
HI test with antibodies to a related virus. The se-
rologic test was not validated for this species, so
the titer could have resulted from a nonspecific re-
sponse in the test assay.
Ophidian paramyxovirus can cause severe mor-
bidity and mortality in infected snakes and has
caused multiple devastating outbreaks in captive
snake collections but has not yet been identified in
a free-ranging snake population.
6,19,23
There was no
322
JOURNAL OF ZOO AND WILDLIFE MEDICINE
serologic evidence of exposure to this virus in these
anacondas.
Leptospira interrogans spirochetes can infect
reptiles, humans, and agricultural and other domes-
tic animals and are often associated with aquatic or
moist environments.
12,16,21,32,34
Reptiles are usually
not clinically ill when infected by Leptospira spi-
rochetes. A number of seropositive snakes of sev-
eral species, including captive snakes in Brazil,
have been reported (with titers of up to 6,400).
17
The low-level titers detected in the anacondas in
the present study may represent early infection,
waning titers from previous infection, antibody re-
sponses to Leptospira antigens present in ingested
prey items, or antibodies to Leptospira serovars that
were not included in the serologic panel. Alterna-
tively, rather than being diagnostic for active infec-
tion with a pathogenic Leptospira serovar, they may
be nonspecific responses resulting from cross-re-
action with the nonpathogenic saprophyte Lepto-
spira biflexa, as has been reported in chelonians.
However, this type of cross-reaction has not yet
been documented in snake species.
3,16,21,34
Cryptosporidium serpentes infection of captive
snakes has resulted in chronic morbidity and even-
tual mortality of affected specimens.
5,13
Cryptospo-
ridium spp. have also been documented in many
free-ranging reptile species, although morbidity and
mortality have not been recognized in these free-
ranging individuals.
33
The apparent lack of C. ser-
pentes exposure in this anaconda population sug-
gests that the disease may not be present at this
time but could have health consequences if it were
to be introduced.
Infectious diseases are important factors for the
health of both captive and free-ranging reptile pop-
ulations. Many infectious disease outbreaks have
been documented in captive reptile collec-
tions.
5,6,18,19
Although the impact of infectious dis-
eases in free-ranging populations is not as well doc-
umented, its importance is being increasingly rec-
ognized.
7–9,18,28
The results of this limited survey for
serologic evidence of exposure to some bacteria,
viruses, and protozoa suggest that these anacondas
do not serve as significant reservoirs for selected
arboviruses, ophidian paramyxovirus, C. serpentes,
or pathogenic Leptospira spirochetes at this loca-
tion.
Acknowledgments: This study was funded by
grants from The Convention on International Trade
in Endangered Species; Profauna, the Department
of Wildlife Management, Venezuelan Ministry of
the Environment; and the Wildlife Conservation
Society’s Freed Foundation Species Survival Fund
and Departments of International Programs, Field
Veterinary Studies, and Clinical Care. We are grate-
ful to the Coporacio´n Venezolana de Ganaderia and
are especially indebted to Pedro Azuaje for his hos-
pitality and assistance. We also acknowledge Drs.
A. Angulo, T. Graczyk, E. Jacobson, and P. Mc-
Donough for technical assistance.
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Received for publication 17 May 2001
