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Occurrence and Molecular Diagnosis of Cryptosporidiumserpentis in Captive Snakes in São Paulo, BrazilAuthor(s): Anaiá da Paixão Sevá, M.Sc., Michele K. Sercundes, M.Sc., JulianaMartins, M.Sc., Sheila Oliveira de Souza, B.Sc. (Biol), João Batista da Cruz,Ph.D., Cybele Sabino Lisboa, B.Sc. (Biol), Sandra Helena Ramiro Correa, Ph.D.,and Rodrigo Martins Soares, Ph.D.Source: Journal of Zoo and Wildlife Medicine, 42(2):326-329. 2011.Published By: American Association of Zoo VeterinariansDOI: http://dx.doi.org/10.1638/2010-0157.1URL: http://www.bioone.org/doi/full/10.1638/2010-0157.1

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Journal of Zoo and Wildlife Medicine 42(2): 326–329, 2011

Copyright 2011 by American Association of Zoo Veterinarians

OCCURRENCE AND MOLECULAR DIAGNOSIS OF CRYPTOSPO-

RIDIUM SERPENTIS IN CAPTIVE SNAKES IN SAO PAULO, BRAZIL

Anaia da Paixao Seva, M.Sc., Michele K. Sercundes, M.Sc., Juliana Martins, M.Sc., Sheila Oliveira

de Souza, B.Sc. (Biol), Joao Batista da Cruz, Ph.D., Cybele Sabino Lisboa, B.Sc. (Biol), Sandra

Helena Ramiro Correa, Ph.D., and Rodrigo Martins Soares, Ph.D.

Abstract: The present study aimed to determine whether Cryptosporidium oocysts were present in stools from

captive snakes at Fundacao Parque Zoologico (Zoological Park Foundation) in Sno Paulo, Brazil. Two collections

were performed; the first in July 2008 and the second in February 2009. Fecal samples were collected from 74

enclosures that housed 101 individuals of 23 snake species. The stool specimens collected from 16 out of the 74

enclosures (21.6%) contained Cryptosporidium spp. oocysts; all of them were confirmed as Cryptosporidium

serpentis, using molecular techniques. Only in three (18.7%) out of the 16 enclosures with positive samples were

there animals with clinical signs compatible with infection by C. serpentis, such as regurgitation and significant

progressive weight loss. From the results, it was concluded that diagnostic examinations need to be performed

periodically, even on clinically healthy animals, as a preventive measure.

Key words: Cryptosporidium serpentis, snakes, molecular diagnosis, zoological.

BRIEF COMMUNICATION

Cryptosporidiosis is a disease caused by proto-

zoan parasites of the genus Cryptosporidium that

affects different groups of vertebrates. In the

reptile group, cryptosporidiosis is caused by two

species: Cryptosporidium serpentis, which usually

affects snakes, and Cryptosporidium varanii, which

occurs more frequently in lizards.11,12

Infections in snakes are associated with consid-

erable morbidity and mortality,3,6 and individuals

exposed to stress, e.g., captivity, are particularly

susceptible to these infections. Cryptosporidium

serpentis is a parasite that inhabits the gastric

epithelium, causing clinical signs such as anorex-

ia, lethargy, postprandial regurgitation, increased

abdominal volume, and weight loss.2,4 In this

reptile group, cryptosporidiosis generally affects

adult individuals and the clinical course of the

infection is lengthy.6 At the subclinical stage,

cryptosporidiosis is insidious and difficult to

diagnose and may pose a significant risk to

captive individuals due to its transmission poten-

tial.6 This parasite can be detected using conven-

tional coproparasitologic methods, but the

causative agent can only be identified by means

of molecular methods.1

The present study aimed to determine whether

Cryptosporidium oocysts were present in stools

from captive snakes at Fundacao Parque Zoolo-

gico (Zoological Park Foundation), in Sao Paulo,

Brazil, by means of the centrifugation–floatation

test in sucrose and to identify the causative agent

by means of molecular methods.

Occurrences of deaths in snake hatcheries at

this institution between January and July 2007

motivated the present investigation. At that time,

examinations of gastric biopsy and necropsy

specimens confirmed the presence of tissue struc-

tures morphologically compatible with merogenic

forms of Cryptosporidium spp. (Soares, unpubl.

data).

For the present diagnostic survey, fecal samples

were collected from 74 enclosures at two different

times. A total of 101 individuals were distributed

in 74 enclosures, among which 12 were collec tive

enclosures containing two to five snakes of the

same species.

In July 2008, stool specimens were collected

from 47 enclosures, as follows: one enclosure with

a tiger rat snake (Spilotes pullatus), one with a

tropical rattlesnake (Caudisona durissa), two with

western ratsnakes (Pantherophis obsoletus), eight

with corn snakes (Pantherophis guttatus), three with

milk snakes (Lampropeltis triangulum), three with

California kingsnakes (Lampropeltis getula califor-

niae), two with Mexican kingsnakes (Lampropeltis

mexicana), eight with jararacas (Bothropoides jara-

raca), three with red-tailed boas (Boa constrictor),

one with an indigo snake (Drymarchon corais),

From the Departamento de Medicina Veterinaria

Preventiva e Saude Animal, Faculdade de Medicina

Veterinaria e Zootecnia, Universidade de Sao Paulo, Av.

Prof. Dr. Orlando Marques de Paiva, 87, CEP 05508–

270, Sao Paulo, SP, Brasil (Seva, Sercundes, Martins,

Souza, Correa, Soares); Fundacao Parque Zoologico de

Sao Paulo, Avenida Miguel Stefano, 4241, CEP 04301-

905, Sao Paulo, SP, Brasil (Lisboa, da Cruz). Corre-

spondence should be directed to Dr. Soares

(rosoares@usp.br).

326

three with ball pythons (Python regius), one with an

Indian python (Python molurus), one with a

rainbow boa (Epicrates alvarezi), two with rainbow

boas (Epicrates crassus), three with Amazon tree

boas (Corallus hortulanus), and five with green

anacondas (Eunectes murinus) (Table 1).

In February 2009, stool specimens were col-

lected from 27 enclosures, as follows: one with

Lichtenstein’s green racer (Philodryas olfersii), four

with jararacas (B. jararaca), one with a jararacussu

(Bothrops jararacussu), two with red-tailed boas

(Boa constrictor), one with a black false boa

(Pseudoboa nigra), six with ball pythons (Py.

regius), one with a blood python (Python curtus),

two with Brazilian rainbow boas (Epicrates cen-

chria), eight with green anacondas (Eu. murinus),

and one with a Brazilian smooth snake (Hydro-

dynastes gigas) (Table 1).

The stool samples were preserved by dilution

with 2.0% potassium dichromate, refrigerated and

sent to the Laboratory of Parasitic Diseases,

Department of Preventive Veterinary Medicine,

School of Veterinary Medicine and Zootechnics,

University of Sao Paulo, Sao Paulo, Brazil. After

microscopic detection of Cryptosporidium spp.

oocysts that were cleared by centrifugation–floa-

tation in sucrose solution, the oocysts were

isolated for nested polymerase chain reaction

(PCR) to be performed using specific primers

directed toward the 18S ribosomal unit.10 The

oocyst DNA was extracted and purified using a

previously described protocol.10 The products

from the nested PCR were sequenced in order to

Table 1. Results from the survey for Cryptosporidium serpentis in stool of captive snakes at Fundacao ParqueZoologico of Sao Paulo, Brazil.

HostNo. of positiveenclosuresa

No. ofcollectiveenclosuresb

Clinicalsignsc

Surveyed in Jul 2008

Tiger rat snake (Spilotes pullatus) 0/1

Tropical rattlesnake (Caudisona durissa) 1/1 Yes

Western ratsnake (Pantherophis obsoletus) 0/2

Corn snake (Pantherophis guttatus) 5/8

Milk snake (Lampropeltis triangulum) 1/3

California kingsnake (Lampropeltis getula californiae) 0/3

Mexican kingsnake (Lampropeltis mexicana) 1/2 Yes

Jararaca (Bothropoides jararaca) 1/8

Red-tailed boa (Boa constrictor) 0/3

Indigo snake (Drymarchon corais) 0/1

Ball python (Python regius) 0/3

Indian python (Python molurus) 0/1 1

Rainbow boa (Epicrates alvarezi) 0/1

Brazilian rainbow boa (Epicrates cenchria) 1/2 2 Yes

Amazon tree boa (Corallus hortulanus) 0/3 1

Green anaconda (Eunectes murinus) 2/5d 3

Surveyed in Feb 2009

Lichtenstein’s green racer (Philodryas olfersii) 0/1

Jararaca (Bothropoides jararaca) 0/4

Jararacussu (Bothrops jararacussu) 1/1

Red-tailed boa (Boa constrictor) 1/2

Black false boa (Pseudoboa nigra) 0/1

Ball python (Python regius) 1/6

Blood python (Python curtus) 0/1 1

Rainbow boa (Epicrates crassus) 0/2

Green anaconda (Eunectes murinus) 1/8e 4

Brazilian smooth snake (Hydrodynastes gigas) 0/1

a No. of enclosures positive for the presence of C. serpentis/total no. of enclosures surveyed.b No. of collective enclosures among the total of enclosures surveyed.c Species in which clinical signs of cryptosporidiosis were observed (Yes).d The two positive enclosures were collective.e The positive enclosure had only one animal.

DA PAIXAO ET AL.—CRYPTOSPORIDIUM SERPENTIS IN CAPTIVE SNAKES IN BRAZIL 327

determine the parasite genotypes and species,

using the Big Dye Terminator kit (Applied

Biosystems, Foster City, California 94404,

USA). The sequences were compared with ho-

mologous sequences available in GenBank for

molecular identification of the parasites.

As shown in the coproparasitologic examina-

tions, stool samples from 16 out of the 74

enclosures (21.6%) were found to contain Cryp-

tosporidium spp. (Table 1). All the microscopy-

positive samples were analyzed and sequenced

using PCR. The results from the molecular

analyses confirmed all the samples as C. serpentis.

Two almost identical haplotypes of 725 base pairs

in length were found and submitted to GenBank

under the accession numbers HQ537111 and

HQ537112. These sequences are, respectively,

99.8% and 100% similar to Cryptosporidium ser-

pentis AF151376.

The 12 enclosures that were found to contain

samples positive for Cryptosporidium spp. from the

first collection were composed as follows: one

with a tropical rattlesnake (Ca. durissa), five with

corn snakes (P. guttatus), one with a milk snake (L.

triangulum), one with a Mexican kingsnake (L.

mexicana), one with a jararaca (B. jararaca), one

with a Brazilian rainbow boa (E. cenchria), and

two with green anacondas (Eu. murinus). The four

enclosures that were found to contain samples

positive for Cryptosporidium spp. from the second

collection were distributed as follows: one with a

jararacussu (Bothrops jararacussu), one with a red-

tailed boa (Boa constrictor), one with a ball python

(Py. regius), and one with a green anaconda (Eu.

murinus) (Table 1).

Out of the 16 enclosures found to contain

samples that were positive for Cryptosporidium

spp. in both samplings, only three (18.7%) had

animals with clinical signs compatible with infec-

tion by C. serpentis: one tropical rattlesnake (Ca.

durissa), one Mexican kingsnake (L. mexicana),

and one rainbow boa (E. crassus). The clinical

signs observed were regurgitation and significant

progressive weight loss.

The presence of C. serpentis in 25.5% of the

enclosures from the first collection and in 14.8%of the enclosures from the second collection

suggests that this parasite had widespread distri-

bution throughout the enclosures. The high

prevalence of this parasite is due to its direct life

cycle, the persistence of the oocysts, and efficient

transmission between hosts, particularly in cap-

tivity.9 Transmission of Cryptosporidium spp. is

through the fecal–oral route, and usually occurs

by means of direct ingestion of infective oocysts

in stools or ingestion of food or water contami-

nated by infective forms of the agent.5 The stress

experienced by captive snakes increases their

susceptibility to infections.7 It is important to

emphasize that the animals investigated in the

present study drank water supplied by the public

water company, and sometimes the same tools

were used to clean the water troughs in all

enclosures, as well as to remove the feces.

Since resistant forms of this parasite are only

intermittently eliminated, individuals with nega-

tive tests may be false negatives: that is, they are

infected by the parasite but do not necessarily

eliminate the oocysts. These animals could be

asymptomatic hosts. It is important to emphasize

that only one stool sample was collected from

each enclosure. Because of the intermittent elim-

ination of Cryptosporidium oocysts in feces, re-

peated coproparasitologic tests are required in

order to identify the infection. Only with sequen-

tial negative results and a compatible general

clinical stage is it possible to affirm that an animal

is free from infection by this parasite.6 Moreover,

in the subclinical stage of this infection, the

number of oocysts eliminated may be low, which

thus may produce a misleading diagnosis.6

The subclinical stage was most prevalent in the

present study, given that 81.2% (13 of 16) of the

enclosures where the parasite was found con-

tained animals that did not show clinical symp-

toms suggestive of cryptosporidiosis. This

infection in snakes is frequently insidious.6 In

turn, the infection may be subclinical for several

months in other reptiles.9

The absence of clinical signs in most of the

animals in the present study and the wide

dissemination of the parasite explain why unno-

ticed infection affected 21.3% of the animals. This

finding confirms the highly transmissible nature

of this infection.

On the other hand, the detection of clinical

signs in three infected snakes indicates the

importance of this parasitic infection, which

debilitates the host animal and may cause its

death. It is important to point out that the

Cryptosporidium species found was C. serpentis,

and that it is well known that reptiles infected by

this parasite may develop a chronic infection and

die.11

The findings demonstrate that subclinical in-

fection is highly prevalent among these animals.

Thus, periodic tests are essential. Early diagnosis

of subclinical C. serpentis infection makes it

possible to use emergency prevention and control

actions, such as isolation of snakes in captive

328 JOURNAL OF ZOO AND WILDLIFE MEDICINE

populations, and careful handling of the animals

to reduce the incidence of pathogens, thereby

promoting hatchery enhancement and preventing

economic losses.

Cryptosporidium serpentis also affects other rep-

tile species. Therefore, its presence in snakes

demonstrates that preventive measures to avoid

dissemination of the parasite to other reptiles are

essential. Infections by Cryptosporidium have been

reported in up to 57 different reptile species.8

Because 21.6% of the enclosures investigated

were found to be positive for the presence of this

agent, it is concluded that captive animals must be

handled very carefully. Furthermore, diagnostic

tests should be periodically carried out, even

among clinically healthy animals, as a preventive

measure.

Acknowledgments: Dr. Rodrigo M. Soares was

suported by a productivity fellowship from

CNPq.

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Received for publication 8 September 2010

DA PAIXAO ET AL.—CRYPTOSPORIDIUM SERPENTIS IN CAPTIVE SNAKES IN BRAZIL 329