203

Neotrop. Helminthol., 8(2), 2014

2014 Asociación Peruana de Helmintología e Invertebrados Afines (APHIA)

ISSN: 2218-6425 impreso / ISSN: 1995-1043 on line

ORIGINAL ARTICLE / ARTÍCULO ORIGINAL

SOME ASPECTS OF THE LIFE HISTORY AND MORPHOLOGY OF STRONGYLOIDES

OPHIDIAE PEREIRA, 1929 (RHABDITIDA: STRONGYLOIDIDAE) IN LIOPHIS MILIARIS

(SQUAMATA: DIPSADIDAE)

ALGUNOS ASPECTOS DE LA HISTORIA DE VIDA Y MORFOLOGÍA DE

STRONGYLOIDES OPHIDIAE PEREIRA, 1929 (RHABDITIDA: STRONGYLOIDIDAE) EN

LIOPHIS MILIARIS (SQUAMATA: DIPSADIDAE)

Vitor Luís Tenório Mati & Alan Lane de Melo

Abstract

Keywords: Experimental and natural strongyloidiasis - ivermectin - reptile - snake - Strongyloides ophidiae life cycle.

Suggested citation Mati, V.L.T. & Melo, A.L. 2014. Some aspects of the life history and morphology of Strongyloides ophidiae

Pereira, 1929 (Rhabditida: Strongyloididae) in Liophis miliaris (Squamata: Dipsadidae). Neotropical Helminthology, vol. 8, n°2,

jul-dec, pp. 203-216.

Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. C.P. 486,

30123-970. E-mail: vitormati@yahoo.com.br

Snake strongyloidiasis was studied in specimens of Liophis miliaris that were experimentally and

naturally infected with Strongyloides ophidiae. Fecal analysis indicated that S. ophidiae

parasitism could last more than three months in the host. Parasite development occurred in snakes

infected via the subcutaneous route, and the prepatent period of the infection was seven days.

These snakes exhibited significant clinical signs and none of the stool analyses were negative.

However, in naturally infected snakes, intermittent results were found in serial fecal tests. A direct

cycle of development was predominant in stool cultures from snakes with both types of infection,

and attempts to eliminate the parasite with ivermectin failed. Enteritis was a common gross

finding in dead snakes. As previous descriptions of S. ophidiae have presented certain

shortcomings, a morphological analysis of the parasite was performed, and clear differences

between this South American species and S. serpentis from North America were observed. There

has been taxonomic uncertainty in the literature as to whether these species of Strongyloides are

indeed distinct. The observations made in L. miliaris provide experimental evidence that the

biology of the parasite in heterothermic hosts is similar to that observed in mammals, and this

species may be considered a potential dipsadid model for the study of snake strongyloidiasis.

Mati & Melo

Life history and morphology of Strongyloides ophidiae

204

Resumen

Palabras clave: Estrongiloidosis natural y experimental – ivermectina – reptil – serpiente - ciclo de vida del Strongyloides

ophidiae.

Se estudió la estrongiloidosis de serpientes en especímenes de Liophis miliaris naturalmente y

experimentalmente infectados con Strongyloides ophidiae. Análisis fecales indicaron que el

parasitismo con el S. ophidiae podría durar más de tres meses en su huésped. El desarrollo del

parásito se produjo en serpientes infectadas por la vía subcutánea, y el período pre-patente de la

infección fue de siete días. Estas serpientes tenían signos clínicos significativos y ninguno de los

análisis de heces fue negativo. Sin embargo, en las serpientes infectadas naturalmente se

encontraron resultados intermitentes en las pruebas fecales seriales. El ciclo directo del desarrollo

fue predominante en los cultivos fecales de serpientes con ambos tipos de infección, y los intentos

de eliminar el parásito con ivermectina fracasaron. En serpientes muertas la enteritis fue un

hallazgo macroscópico frecuente. Como las descripciones anteriores de S. ophidiae tienen

presentado algunas deficiencias, se realizó el análisis morfológico del parásito y se observaron

diferencias claras entre esta especie de América del Sur y S. serpentis de América del Norte. Había

incertidumbre taxonómica en la literatura si estos serían de hecho especies distintas de

Strongyloides. Las observaciones realizadas en L. miliaris han proporcionado evidencias

experimentales de que la biología del parásito en los huéspedes heterotermos es similar al

observado en los mamíferos, y esta especie de dipsadido puede ser considerada como un posible

modelo para el estudio de la estrongiloidosis de serpientes.

Despite advances in taxonomic knowledge

regarding certain species of Strongyloides, the

biology of these parasites and the host diseases

induced by nematodes in reptiles have been

largely ignored. Previous reports of these

species essentially consist of case records (Holt,

1978; Holt et al., 1979; Wiesman & Greve,

1982; Veazey et al., 1994). Singh (1954) detailed

some aspects of the life history of S. mirzai from

the Oriental rat snake, Ptyas mucosus (Linnaeus,

1758), but his analysis of the biology of the

worms was mainly based on nematodes obtained

from coprocultures (i.e., free-living forms).

Indeed, Strongyloides spp. possess unusual life

cycles that should be investigated further,

particularly in snakes. Only parthenogenetic

female nematodes are found as parasitic adults,

although the development of free-living

generations in which both sexes are present can

also occur (Schad, 1989; Viney & Lok, 2007).

Strongyloides ophidiae was the first species of

the genus recorded in reptiles, and the

description was brief and based solely on

parasitic females obtained from the small

intestine of Mastigodryas bifossatus (Raddi,

The tiny nematode species of the genus

Strongyloides Grassi, 1879, most of which live

in the small intestine of the host, are parasites

with great evolutionary success that are found in

all classes of vertebrates with the exception of

fish. The classical list of Strongyloides spp.

reported by Speare (1989) included 52 valid

species in the genus, six of which are parasites of

Reptilia: Strongyloides ophidiae Pereira, 1929;

S. mirzai Singh, 1954; S. gulae Little, 1966; S.

serpentis Little, 1966; S. cruzi Rodrigues, 1968;

and S. darevskyi Sharpilo, 1976. Since that time,

new species of Strongyloides have been

described (Navarro et al., 1989; Viney et al.,

1991; Skerratt, 1995; Sato et al., 2007),

including two new species from reptiles: S.

ophiusensis Roca & Hornero, 1992 and S.

natricis Navarro & Lluch, 1993 (Roca &

Hornero, 1992; Navarro & Lluch, 1993). Thus,

the current number of valid Strongyloides

species is approaching 60, though only eight of

these species (approximately 14%) have been

described in reptile hosts.

INTRODUCTION

205

Neotrop. Helminthol., 8(2), 2014

third-stage filariform larvae (L i) of S. ophidiae

obtained from cultures of the feces of naturally

infected L. miliaris (see details in the following

section). The experimentally infected snakes

were also examined daily, and one specimen was

euthanized to facilitate the recovery of

parthenogenetic females from the small intestine

at 21 days post-infection (DPI), which were then

subjected to morphological analysis. All

procedures were conducted according to the

institutional ethics committee guidelines for

animal research and the resolution n . 1,000 of

the Conselho Federal de Medicina Veterinária

from Brazil (2012).

Parasitological analysis and coproculture

At all time points at which the snakes evacuated

during this study, feces were collected for

qualitative analysis and culture of the nematodes

to obtain free-living forms and L i larvae for

experimental infection. The spontaneous

sedimentation method (Lutz, 1919) was

performed to diagnose and monitor the course of

infection. Five to ten slides were examined per

sample. Additionally, fresh feces were mixed

with moistened vermiculite to prepare

coprocultures, which were incubated at 27°C for

a period of 48 to 72 h.

Obtaining and quantifying L i larvae

Infective and free-living forms were recovered

from coprocultures using the Baermann method,

as modified by Moraes (1948), and the number

of L i S. ophidiae in the suspension obtained was

then determined. Three 25 µl aliquots were

collected with the aid of an automated pipette

and placed on slides as drops. The mean number

of L i was calculated by evaluating these

aliquots, and the suspensions were diluted or

concentrated to reach a final concentration of

approximately 1,000 L i in 0.5 mL of distilled

water.

Recovery of parasites

Only specimens of experimentally infected L.

milliaris, including the euthanized specimen and

the other snakes that died throughout the

experiment, were necropsied to recover

parasites. Their abdominal cavities were

opened, and the viscera were removed and

1820) from the State of São Paulo, Brazil

(Pereira, 1929). In North America, Little (1966)

described the species S. gulae and S. serpentis

from snakes and argued that the latter species

could not be easily morphologically

differentiated from S. ophidiae because the

original description of the Brazilian species

included some deficiencies and indicated that

further studies would be useful to discharge this

taxonomic doubt. The differentiation of these

species remains a topic of discussion, as

appropriate criteria to have not been developed

for this purpose.

With regard to the biology of S. ophidiae, the

available information is not sufficient, although

in a recent report on S. ophidiae in Brazil,

morphological and molecular data on the

parasite were presented (Santos et al., 2010).

Thus, to improve our understanding of the

biology of S. ophidiae, in the present study, some

aspects of the life history of this parasite were

studied based on data from natural and

experimental infections of the common water

snake Liophis miliaris (Linnaeus, 1758).

Additional morphological aspects of S. ophidiae

are described, and the parasitological and

clinical characteristics of strongyloidiasis of

snakes, including details related to the use of

ivermectin in the treatment of infected reptiles,

are discussed.

Snakes and parasites

Specimens of the dipsadid snake L. miliaris (n =

5) weighing 51 ± 23 g naturally infected with S.

ophidiae from Muriaé, State of Minas Gerais,

Southeast Brazil, were studied. Prior to the death

of the reptiles, the parasitological and clinical

characteristics of natural strongyloidiasis in L.

miliaris were evaluated based on daily

coproparasitological testing and inspection of

animals performed in the laboratory. Other

specimens of L. miliaris (n = 6) reared under

laboratory conditions weighing 64 ± 35 g that

were free of parasites according to

parasitological stool evaluation were infected

via the subcutaneous route with 1,000 infective

MATERIAL AND METHODS

206

Mati & Melo

Life history and morphology of Strongyloides ophidiae

The Neotropical snake L. miliaris is a permissive

host for S. ophidiae, and parasitism by this

nematode lasts for a period of at least three

months in these animals, as observed through

fecal analysis of naturally and experimentally

infected specimens throughout the study. The

reptiles that were experimentally infected

provided information regarding the

development of the parasite in the small

intestine, and the prepatent period of the

infection was seven days. Moreover, the L.

miliaris specimens showed clinical signs

(emaciation, listlessness and an increased stool

frequency) that were more significant, and the

results of their coproscopies were constant,

without any negative parasitological tests being

obtained during the time period before treatment

with ivermectin. Conversely, intermittent results

were obtained in parasitological tests of the five

naturally infected snakes, with brief

interruptions between positive tests being

recorded during the course of the experiment.

However, in both groups, only eggs from

parasites were observed in fresh feces, and

rhabditiform larvae were never found.

With regard to the observations related to

coprocultures, the eggs laid by parasitic forms

gave rise to embryos, which enabled the

initiation of direct or indirect developmental

cycles after hatching. Forms corresponding to

both types of developmental cycles were found

in the samples. In the predominant direct cycle,

rhabditiform larvae of S. ophidiae grew and then

transformed into L i larvae after two molts,

while in the indirect cycle, three distinct stages

(rhabditiform larvae, juveniles and adult female

f r ee -l iv in g f or ms ) w er e o b se rv ed ,

corresponding to a total four molts, but no free-

living male adults were found. Additionally, the

successful subcutaneous infection of L. miliaris

using L i S. ophidiae that had developed in

coprocultures indicates their viability and ability

to migrate through host tissues.

Over the course of the three-month follow-up

period, none of the five naturally infected snakes

died, whereas deaths were observed among the

examined for helminths. The mucosa of the

intestines was scraped, and the obtained material

was transferred to 0.85% saline in a Petri dish.

Representative adult nematodes were collected,

fixed in 10% formalin and diaphanized in

lactophenol.

Morphology

Additional morphological data were obtained

for the eggs, filariform larvae and free-living and

parasitic females of S. ophidiae. Examination of

the nematode specimens was performed on glass

slides without permanent preparation to permit

handling of the worms and allow better

assessment of specific morphological

characteristics. Adult parasites were drawn with

aid of a camera lucida attached to a light

microscope. Measurements of morphological

characteristics were performed with a

curvimeter (Tokyo Sakurai, Japan) or were

carried out directly during microscopic

evaluation utilizing a micrometer grid in the

ocular eyepiece. To evaluate the most

representative specimens, the morphological

structures of parasitic females were assessed in

specimens of S. ophidiae recovered from

untreated snakes, particularly from one snake

that was euthanized at 21 DPI. Specific

identification was performed according to the

original description and others data from

different authors (Pereira, 1929; Singh, 1954;

Wiesman & Greve, 1982; Little, 1966; Navarro

& Lluch, 1993; Vicente et al., 1993; Santos et al.,

2010; Mati et al., 2013), and nematode

specimens were deposited in the collection of

the Department of Parasitology, UFMG (DPIC

2526 and 2527).

Ivermectin treatment

Two treatment regimens with ivermectin were

tested in three snakes subjected to experimental

infection. At 45 DPI, two reptiles received a

-1

single dose of the drug (0.2 mg·kg ,

subcutaneously), and another specimen received

the same dose twice after an interval of seven

days. The other two experimentally infected

snakes that had not been euthanized were

monitored and used as controls for this

therapeutic trial.

Details of the life history of S. ophidiae

RESULTS

207

Neotrop. Helminthol., 8(2), 2014

the snakes following drug administration

(<10% of slides examined showed parasite eggs

after treatment).

Morphological data

Strongyloides ophidiae Pereira, 1929 (Table 1,

Figs. 1–4).

Host: Liophis miliaris (new host).

o o

Locality: Muriaé (21 7'29''S, 42 23'24''W), State

of Minas Gerais, Brazil (new locality).

Other hosts and localities: Mastigodryas

bifossatus (type host) and Oxyrhopus guibei

from the State of São Paulo, Brazil (Pereira,

1929; Santos et al., 2010).

Site of infection: Small intestine.

Parasitic females (Figs. 1, 3): The body is

experimentally infected reptiles (one untreated

animal at 71 DPI (1/2) and all animals treated

with ivermectin at 76, 79 and 88 DPI (3/3)). In

the post-mortem examinations, enteritis with

abundant mucus, particularly in the duodenum,

was the most significant finding of the

macroscopic analysis. Furthermore, during

necropsies, parthenogenetic parasitic females

were observed within the mucosa of the small

intestine and presented normal development,

even in snakes that received anthelminthic

treatment, which died between 31 and 43 days

after the initiation of drug administration. Thus,

the ivermectin treatment applied under the

conditions of the present study cannot be

considered effective, although positive results

were less common in the stool examinations of

Figures 1–4. Morphological features of Strongyloides ophidiae observed under light microscopy. (1) General view of the

parasitic female. Note the ovaries that spiral around the intestine and intrauterine eggs. Bar = 100 µm. (2) General view of a free-

living female. Bar = 50 µm. (3) Detail of the tail of a parasitic female in lateral view. Bar = 25 µm. (4) Detail of the tail, in lateral

view, of a free-living female. Bar = 50 µm.

208

Mati & Melo

Life history and morphology of Strongyloides ophidiae

row of eggs, commonly 6 or 7. Eggs close to the

vulva are often already undergoing cleavage,

containing larvae in some cases. The lips of the

vulva are prominent and are situated

immediately anterior to the middle of the body.

The tail is elongate–conoid and is comparatively

long and sharply pointed.

Infective filariform larvae: Thin form with a

filariform esophagus whose length extends for

approximately 47% of the body. The tail is

notched.

Eggs: Ellipsoidal with a very thin wall. In fecal

material, the eggs are embryonated and

sometimes already contain completely

developed larva (Fig. 5). The length and width of

the eggs are 48–59 and 27–38 μm, respectively.

In the uteri of parasitic females, the eggs are

smaller with a length and width of 35–45 and

19–24 μm, respectively.

Table 1 provides a comparison of the

cylindrical and thin. At the anterior end, there is a

circumoral elevation, and the mouth opens into a

long and cylindrical esophagus extending for

one-third of the total length (0.31 – 0.34). Lips

are absent. The reproductive system is

amphidelphic with equal uteri and ovaries

reflexed upon themselves, with one loop

occurring near the esophagus and the other loop

close to the anal region. The ovarian tubules

spiral around the intestine; the anterior tube

exhibits two-and-a-half spirals, and the posterior

is partially spiraled. The uteri are short and

contain between 6 and 12 eggs, which are

generally segmented when located in the vulva

region. The vulva is slightly protuberant and is

located close to the posterior third of the

parasite's body. The tail is abruptly tapered but

has a fine point.

Free-living females (Figs. 2, 4): Small and

presenting a rhabditoid esophagus. The

reproductive system is didelphic with opposed

and reflected ovaries. The uteri contain a single

Figure 5. Egg of Strongyloides ophidiae containing completely developed larva in fresh feces. Bar = 50 µm.

209

Neotrop. Helminthol., 8(2), 2014

Strongyloides

ophidiae

Strongyloides

mirzai

Strongyloides

serpentis

Strongyloides

gulae

Strongyloides

natricis

Host Mastigodryas

bifossatus

Oxyrhopus

guibei

Liophis

miliaris

Ptyas

mucosus

Chondropython

viridis

Natrix cyclopion

cyclopion

Natrix cyclopion

cyclopion

Natrix

maura

Author(s)

Pereira (1929)

Santos et al. (2010)

Present study

Singh (1954)

Wiesman & Greve

(1982)

Little (1966)

Little (1966) Navarro &

Lluch (1993)

Parasitic female (n)

Not set

(n = 10)

Not set

(n = 27)

(n = 15)

(n = 10)

Total length

2,700–3,600

4,700 (3,525–5,372)

3,260 (2,750–3,670)

2,670–3,690

3,890–4,230

3,170 (2,400–3,700)

2,170 (1,800–2,400) 4,128

Length of the esophagus

1,050–1,130

1,633 (1,426–1,902)

1,060 (850–1,180)

880–1,070

880–1,050

1,280 (890–1,500)

850 (710–1,000) 1,165

Length of the esophagus/

total length*

0.31–0.39

0.36 (0.35–0.40)

0.33 (0.31–0.34)

0.29–0.33

0.23–0.25

0.40 (0.37–0.41)

0.39 (0.39–0.42) 0.28

Distance between the

mouth and vulva

1,900–2,400

2,292 (1,759–2,620)

2,170 (1,650–2,450)

1,750–2,530

2,240–2,680

2,180 (1,700–2,500)

1,510 (1,200–1,700) 2,743

Distance between the mouth and

vulva/total length*

0.67–0.70

0.49 (0.48–0.50)

0.67 (0.60–0.68)

0.66–0.69

0.56–0.63

0.69 (0.54–0.71)

0.70 (0.67–0.71) 0.66

Length of the tail

70–100

105 (64–124)

99 (70–117)

63–90

86–90

75 (50–100)

77 (60–95) 90

Length of the tail/total length

0.03

0.02

0.03

0.02

0.02 (0.02–0.03)

0.04 (0.03–0.04) 0.02

Width

51 (41–58)

42 (35–55)

35–50

40 (30–50)

34 (30–40) 49

Shape of the anterior and posterior

ovaries†

Incomplete

description, but

with spiral(s).

AO spiraled twice.

PO with partial

spiral.

AO with two-and-a-

half spirals. PO with

partial spiral.

AO usually

spiraled three

times. PO with

one spiral.

AO usually spiraled

twice, occasionally

straight. PO usually

straight, sometimes

with partial spiral

AO usually spiraled

once, occasionally

straight. PO usually

straight, occasionally

with one spiral

AO usually

spiraled twice.

PO usually

with one spiral

Number of eggs in the uteri

6–7

2–5

7.4 (6–13)

Up to 11

Up to 10

Up to 6

8–10

Free-living female ‡ (n)

(n = 10)

Not set

(n = 15) §

Total length

826 (712–1,089)

860 (670–950)

760–890

960 (710–1,100)

Length of the esophagus

149 (117–261)

130 (120–150)

113–126

137 (130–145)

Length of the esophagus/total length

0.18 (0.16–0.24)

0.15 (0.14–0.20)

0.14–0.15

0.14 (0.13–0.18)

Distance between

the mouth and vulva

413 (345–561)

490 (435–605)

396–467

495 (440–540)

Distance between the mouth and

vulva/total length

- 0.50 (0.48–0.52) 0.57 (0.51–0.64) 0.52 - 0.52 (0.49–0.62) -

Length of the tail - 100 (94–109) 101 (87–114) 65–75 - 95 (85–100) -

Length of the tail/total length - 0.12 (0.10–0.13) 0.12 (0.12–0.13) 0.08–0.09 - 0.10 (0.09–0.12) -

Table 1. Morphological and morphometric data for parasitic and free-living females, filariform larvae and eggs of the five species of Strongyloides found in snakes

worldwide. Measurements are presented in micrometers (µm), and the provided values are means, with ranges shown in parentheses.

Table 1. Continuation.

210

Mati & Melo

Life history and morphology of Strongyloides ophidiae

Width

37 (30–53)

42 (37–45)

37–47

48 (46–50)

Number of eggs in the uteri

6.2 (5–7)

Filariform larvae (n)

(n = 30)

(n = 25)

(n = 32) §

Total length

486 (422–603)

505 (430–550)

- -

480 (430–520)

Length of the esophagus

171 (112–254)

245 (235–260)

- -

230 (220–240)

Length of the esophagus/total length

0.35 (0.27–0.42)

0.49 (0.47–0.55)

0.48 (0.46–0.51)

Length of the tail

63 (31–103)

52 (47–61)

48 (45–55)

Length of the tail/total length

0.13 (0.07–0.17)

0.10 (0.10–0.11)

0.10 (0.09–0.11)

Width - 15 (12–19) 13 (12–16) - - 12.5 (12–14) -

Eggs

Characteristics of eggs when passed in

fresh stools

- In cleavage. In cleavage and/or

fully embryonated.

Embryonated.

Larvae also

found in feces.

In cleavage. - Possibly larvae

and not eggs in

feces.

Length x width (eggs from stools) - 76 (40–86) x 44 (37–

48)

56 (48–59) x 33 (27–

38)

53–62 x 27–31 - - - -

Length x width (intrauterine eggs in

parasitic females)

38 x 15–23 52 (48–54) x 32 (30–

34)

40 (35–45) x (19–24) - 55–64 x 24–

51 (44–55) x 24 (23–

26)

60 (54–70) x 24 (23–

26)

50 x 29

Table 1. Continuation.

* Ratios were calculated when not directly provided in the source study.

† AO = anterior ovary and PO = posterior ovary.

‡ Free-living males have only been observed for S. mirzai and S. serpentis/S. gulae thus far. Therefore, no analysis of this stage was performed. See details in Singh (1954) and Little (1966).

§ According to Little (1966), the filariform larvae and free-living stages of S. serpentis and S. lutrae are indistinguishable.

211

Neotrop. Helminthol., 8(2), 2014

constitute a substantial problem for

herpetoculture, favoring the transmission of the

nematode. Direct cycles appear to be related to a

higher occurrence of gastrointestinal nematodes

in captive reptiles, for which maintenance

conditions are sometimes inadequate and may

result in depression of the immune system of

these animals, thus permitting the invasion of

opportunistic pathogens and the propagation of

diseases such as strongyloidiasis, which is

considered a common problem in pets and in

commercial reptile breeding (Klingenberg,

1993).

Furthermore, the semiological data obtained in

L. miliaris infected with S. ophidiae were similar

to findings reported in the literature regarding

the infection of other snakes with Strongyloides

spp. (Holt, 1978; Holt et al., 1979; Wiesman &

Greve, 1982), although ureteritis and nephritis,

which have previously been observed during

strongyloidiasis in snakes (Veazey et al., 1994),

were not evaluated in the present work. Infection

with S. ophidiae was most likely correlated with

the death of the snakes, including those that were

experimentally infected and were treated with

ivermectin. Dehydration and electrolyte

imbalances are possible mechanisms leading to

the death of reptiles harboring these nematodes,

but the parasites also appear to favor the

occurrence of fatal secondary bacterial

infections (Holt et al., 1979). The authors of this

last study speculated as to whether bacterial

pneumonia observed during strongyloidiasis in

snakes would be correlated with pulmonary

migration after larval cutaneous penetration,

similar to what is observed in the parasitism of

other vertebrates. The experimental

subcutaneous infection of six specimens of L.

miliaris with S. ophidiae and the subsequent

development of the parasite supports the idea

that larvae of the nematode are able to migrate in

host tissues.

Concerning parasitological diagnosis, it is

noteworthy that parasite eggs were observed in

the feces during the infection of L. miliaris with

S. ophidiae, while larvae have previously been

observed during the coproscopy of snakes

infected with Strongyloides spp. (Klingenberger

morphological characteristics of the parasitic

and free-living females, infective filariform

larvae and eggs of S. ophidiae that were

analyzed in the present study with the

descriptions available in the literature for other

Strongyloides species from snakes. Data on

species from lizards (S. cruzi, S. darevskyi and S.

ophiusensis) are not shown but they can easily be

differentiated from reptilian Strongyloides

based on criteria such as morphometric aspects,

the greater number of eggs in their uteri and their

more sharply pointed tails compared with

species from lizards (Mati et al., 2013).

Parasitic richness in snakes, particularly

Neotropical snakes, is high and has generally

been underestimated (Dobson et al., 2008; Pinto

et al., 2012). Despite some advances in the

taxonomy of these parasites, their biology

remains poorly understood, including for

Strongyloides spp. Indeed, L. miliaris is known

to harbor a wide variety of helminths (Travassos

et al., 1969; Santos & Tayt-Son Rolas, 1973;

Vicente et al., 1993; Pinto et al., 2012).

However, this is the first report describing this

dipsadid as a host for S. ophidiae, which is the

only species of the genus found in snakes in the

neotropics thus far (Pereira, 1929; Santos et al.,

2010).

There is also likely a deficit in our knowledge of

the taxonomy of Strongyloides spp. from snakes.

However, there is even less available

in formation regard ing h ost-parasi te

relationships and the clinical aspects of infection

with these species of nematodes. Despite the

potential limitations related to the number of

animals studied, in the present study,

experimental infection of snakes with

Strongyloides was performed, and the prepatent

period was established. The life cycle of S.

ophidiae is short, as embryonated eggs of the

parasite were found in the feces of

experimentally infected animals beginning on

day seven of infection and thereafter. This

characteristic, while similar to the life cycles of

other Strongyloides species from reptiles, can

DISCUSSION

212

Ivermectin is a broad-spectrum drug that is

widely used in mammals to treat many types of

intestinal nematodes, including Strongyloides

(Benz et al., 1989; Campbell et al., 1989). In

addition, the administration of ivermectin to

snakes has been considered safe at doses of 0.2

to 0.4 mg/kg (Lawrence, 1984; Luppi et al.,

2007; Aiello & Moses, 2013). However, toxic

effects have been observed in chameleons (Széll

et al., 2001), and due to the occurrence of

adverse events ranging from mild ataxia to

paralysis and death, ivermectin is prescribed for

turtles only at low dosages (Teare & Bush, 1983;

Aiello & Moses, 2013). The data obtained in the

present study also indicated that the use of this

drug for the treatment of strongyloidiasis in

reptiles must be considered with caution, as

three snakes treated with the tested regimen

were not found to be cured and subsequently

died (although ivermectin cannot be directly

incriminated as the cause of death of these

animals). Therefore, the benefits of the use of

ivermectin during strongyloidiasis in reptiles,

including snakes, may not outweigh the risks.

After 48-72 h of culture, a free-living generation

(immature and female adults) of S. ophidiae was

observed, although a predominance of

homogonic (or direct) development was noted.

However, free-living male nematodes were not

recovered, indicating that these forms are rare or

short-lived. This observation should be further

investigated in the future, given that in other

studies on this species of parasite, there has been

no mention of free-living male worms during the

indirect cycle of development (Pereira, 1929;

Santos et al., 2010). In the life cycle of S. mirzai,

free-living males and females are first seen on

the second and third days, but the males die out

by the fourth day, while the females continue to

live for a week. It was observed that prior to

these early deaths, during the act of copulation,

three to five male worms coil around a single

free-living S. mirzai female, and fertilization is

carried out by the male that is closest to the vulva

region (Singh, 1954).

In addition to the characteristics of the free-

living cycle of S. ophidiae, considering the

migration of the parasite, its location (small

1993; Holt, 1978; Holt et al., 1979). However,

the detection of larvae or eggs in feces can

depend on the Strongyloides species (Little,

1966). Eggs have also been observed during

fecal analysis of parasitism by S. mirzai (Singh,

1954; Wiesman & Greve, 1982), S. gulae and S.

serpentis (Little, 1966).

The higher frequency of negative parasitological

results, together with the less substantial clinical

manifestations and the absence of deaths in

naturally infected snakes, may indicate a lower

parasite burden in these animals as well as the

presence of a presumptive chronic infection that

is better balanced in terms of the host–parasite

relationship. This is an area that requires further

investigation, especially considering the

possibility of false-negative tests in veterinary

practice. The alternation between positive and

negative results in these snakes is similar to what

is observed in mammalian infections with

Strongyloides spp. (Nielsen & Mojon, 1987;

Schad et al., 1997; Melo et al., 2012). These

findings have been explained in experimental

strongyloidiasis in marmosets by the occurrence

of a dynamic host–parasite relationship in which

the constant search for equilibrium may result in

greater or lower worm fecundity in response to

changes in the environment (Melo et al., 2012).

Even in human strongyloidiasis, which has been

much better studied, the fecal diagnostic

techniques that are currently available are

thought to possess low sensibility, and it is

ideally recommended that several samples be

collected on different days (Siddiqui & Berk,

2001), which is very difficult to perform under

some conditions due to intrinsic animal

characteristics. The negative tests results

obtained in the present study may be related to a

reduction in the reproductive capacity of

parthenogenetic females, as sterile worms are

commonly found during chronic experimental

infections of dogs with a canine isolate of S.

stercoralis (Schad et al., 1997).

Given that there are no specific data on the

efficacy of ivermectin use to treat reptile

strongyloidiasis, the effects of treatment with

this drug during the experimental infection of L.

miliaris with S. ophidiae were evaluated.

Mati & Melo

Life history and morphology of Strongyloides ophidiae

213

Neotrop. Helminthol., 8(2), 2014

latter species exhibits an anterior ovary that

spirals twice around intestine and a posterior

ovary that is usually straight, occasionally also

partially spiraling around the intestine) as well

as the number of eggs in the uteri (between 6 and

12 eggs in S. ophidiae and up to 10 in S.

serpentis), the mean ratio of the length of

esophagus to the total length (lower in S.

ophidiae) and the length of the tail (lower in S.

serpentis), as shown in table 1. Considering

these morphological differences and the

geographical distance between the records of S.

ophidiae and S. serpentis, we believe that our

findings contribute to resolving any hesitation

about the validity of both species from the

American continent. Santos et al. (2010)

identified S. ophidiae from Oxyrhopus guibei

and presented the first molecular analysis of

Strongyloides from snakes, in addition to

morphological and morphometric data.

However, the presentation of specific

morphological criteria to diagnose these two

species was not the goal of their study. In the

parasitic females studied by these researchers,

the anterior ovary spiraled twice around the

intestine, while the posterior ovary formed a

partial spiral, and the uteri contained 2 to 5 eggs.

The total length of these worms was greater than

was observed by Pereira (1929) and in the

present study, but these variations may be due to

the different hosts involved, as the effects of the

host immune response on the morphology and

total length of parasitic females of S. ratti and S.

venezuelensis, which are species from rodents

that have been widely studied, have been

established (Kimura et al., 1999; Baek et al.,

2003; Gazzinelli & Melo, 2008).

Regarding species from the Old World, S.

mirzai, which is also morphologically close to S.

ophidiae and S. serpentis, differs from the

species analyzed in the present study in terms of

the shape of its ovaries (showing an anterior

ovary with three spirals and a posterior ovary

with one complete spiral), its proportionally

shorter esophagus (lower mean value of the ratio

of the length of the esophagus to the total

length), the greater number of intrauterine eggs

found in parasitic females and the shorter tail

length observed in free-living females of the

intestine) in the host and the probable variations

in the fecundity of parasitic females over the

course of infection, there is evidence that the

biology of Strongyloides in heterothermic hosts

is similar to that observed in mammals. Thus,

previous speculations regarding the parasitism

of reptiles with S tron gyloide s were

experimentally confirmed.

The parasitic and free-living forms of S.

serpentis were adequately studied when they

were originally described. However, given the

fact that only the parasitic form of S. ophidiae

was described and certain shortcomings of this

description, Little (1966) stated that additional

studies would be needed to ensure that these two

species of Strongyloides are indeed distinct

species. To resolve this doubt and to

complement the existing information (Pereira,

1929; Santos et al., 2010), eggs, filariform

larvae and free-living females of S. ophidiae

obtained from fecal cultures were also studied.

Despite deficiencies in the description of

parasitic females of S. ophidiae, the

identification of our specimens as this species

was made possible by examining the originally

described morphological characteristics

(Pereira, 1929; see table 1), which coincided

with our findings and were also attributable to

the close phylogenetic relationship between the

known host species (dipsadid snakes) and the

proximity of areas in which the parasite occurs

(restricted to southeastern Brazil thus far). In

descriptions of S. ophidiae, the ovaries have

been reported to be spiraled, but their form,

which is a key feature in the taxonomy of the

genus Strongyloides, was not described in detail,

given that it was not an important taxonomic

criterion at that time. Nevertheless, although the

original description of the species is incomplete,

it is not inconsistent with the characteristics of

the parasite herein studied.

The specimens of S. ophidiae that were

evaluated in this study were different from S.

serpentis in terms of the shape of the ovaries (the

former species exhibits an anterior ovary with

two-and-a-half and a posterior ovary that

partially spirals around the intestines, while the

214

Mati & Melo

Life history and morphology of Strongyloides ophidiae

Ivermectin and abamectin. Springer-

Verlag, New York, U.S.A., pp. 215–229.

Campbell, WC, Leaning, WHD & Seward, RL.

1989. Use of ivermectin in horses. In:

Campbell, WC (ed.). Ivermectin and

abamectin. Springer-Verlag, New York,

U.S.A., pp. 234–244.

Dobson, A, Lafferty, KD, Kuris, AM, Hechinger,

RF & Jetz, W. 2008. Homage to Linnaeus:

how many parasites? How many hosts?

Proceedings of the National Academy of

Sciences, vol. 105, pp. 11482–11489.

Gazzinelli, SEP & Melo, AL. 2008. Interação

entre Strongyloides venezuelensis e

Schistosoma mansoni em camundongos

da linhagem AKR/J. Revista de Ciências

Médicas e Biológicas, vol. 7, pp.

149–155.

Holt, PE. 1978. A case of Strongyloides

infestation in a speckled king snake.

Veterinary Record, vol. 102, pp. 404–405.

Holt, PE, Cooper JE, & Needham, JR. 1979.

Strongyloides infection in snakes: three

case reports. Veterinary Record, vol. 104,

pp. 213–214.

Kimura, E, Shinotoku, Y, Kadosaka, T,

Fujiwara, M, Kondo, S & Itoh, M. 1999. A

second peak of egg excretion in

Strongyloides ratti-infected rats: its

origin a nd bio l o g ical me a n ing.

Parasitology, vol. 119, pp. 221–226.

Klingenberg, RJ. 1993. Understanding reptile

pa r a s it es : A b a s i c ma n u a l fo r

herpetoculturists and veterinarians.

Advanced Vivarium Systems, Lakeside,

U.S.A.

Lawrence, K. 1984. Ivermectin as an

ectoparasiticide in snakes. Veterinary

Record, vol. 115, pp. 441–442.

Little, MD. 1966. Seven new species of

Strongyloides ( N e m a t o d a ) f ro m

Louisiana. Journal of Parasitology, vol.

52, pp. 85–97.

Luppi, MM, Costa, MET, Malta, MCC, Tinoco,

HP & Motta, ROC. 2007. Tratamento de

Rhabdias labiata com levamisol e

ivermectina em jiboias (Boa constrictor

amarali). Veterinária Notícias, vol. 13, pp.

61–65.

Lutz, A. 1919. O Schistosomum mansoni e a

South American species.

Additional morphological characteristics of S.

ophidiae from the present study, confirm that

this species from South America is distinct from

S. serpentis from North America. Strongyloides

ophidiae has been the only species of the genus

recorded in snakes in Brazil thus far, and its

biology was completely unknown previously.

Thus, the data on the biology of S. ophidiae in L.

miliaris obtained in this work contribute to a

better understanding of reptile strongyloidiasis,

including experimentally demonstrating that the

biology of Strongyloides in heterothermic hosts

is similar to that observed in mammals.

Considering the susceptibility of this

Neotropical snake to the parasite and the ease of

its handling and maintenance, L. miliaris may be

considered a potential model for the study of

snake strongyloidiasis.

We thank Tiago de Oliveira Lima for kindly

providing and assisting with snakes and

Wanderlany Amâncio Martins for technical

support.

AKNOWLEDGMENTS

Aiello, SE & Moses, MA (eds.). 2013. The

Merck Veterinary Manual Online (Last

full review/revision 2011), accessed in

August 23, 2013, <http://www.merckvet-

manual.com/mvm/index.jsp?cfile=htm/b

c/171410.htm>

Baek, BK, Islam, MK, Kim, BS, Lim, CW, Hur,

J, Oluoch, AO, Kim, CH & Kakoma, I.

2003. Characterization of the protective

response against a homologous challenge

infection with Strongyloides venezue-

lensis in rats. Veterinary Parasitology, vol.

113, pp. 217–227.

Benz, GW, Roncalli, RA & Gross SJ. 1989. Use

of ivermectin in cattle, sheep, goats, and

swine. In: Campbell, WC (ed.).

BIBLIOGRAPHICS REFERENCES

215

Neotrop. Helminthol., 8(2), 2014

ophiusensis s p . n . ( N e m a t o d a :

Strongyloididae), parasite of an insular

lizard, Podarcis pityusensis (Sauria:

Lacertidae). Folia Parasitologica, vol. 39,

pp. 369–373.

Santos, JC & Tayt-Son Rolas, FJ. 1973. Sobre

alguns cestóides de Bothrops e de Liophis

miliaris. Atas da Sociedade de Biologia do

Rio de Janeiro, vol. 17, pp. 35–40.

Santos, KR, Carlos, BC, Paduan, KS, Kadri,

SM, Barrella, TH, Amarante, MR,

Ribolla, PE & Silva, RJ. 2010.

M o r p h o l o g i c a l a n d m o l e c u l a r

characterization of Strongyloides

ophidiae (Nematoda, Strongyloididae).

Journal of Helminthology, vol. 84, pp.

136–142.

Sato, H, Torii, H, Une, Y & Ooi, HK. 2007. A

new rhabditoid nematode species in Asian

sciurids, distinct from Strongyloides

robustus in North American sciurids.

Journal of Parasitology, vol. 93, pp. 1476-

1486.

Schad, GA. 1989. Morphology and life history

of Strongyloides stercoralis. In: Grove, DI

(ed.). Strongyloidiasis: a major

roundworm infection of man. Taylor and

Francis, London, U.K., pp. 85–104.

Schad, GA, Thompson, F, Talham, G, Holt, D,

Nolan, TJ, Ashton, FT, Lange, AM &

Bhopale, VM. 1997. Barren female

Strongyloides stercoralis from occult

chronic infections are rejuvenated by

transfer to parasite-naive recipient hosts

and give rise to an autoinfective burst.

The Journal of Parasitology, vol. 83, pp.

785–791.

Siddiqui AA & Berk SL. 2001. Diagnosis of

Strongyloides stercoralis infection.

Clinical Infectious Diseases, vol. 33, pp.

1040–1047.

Singh, SN. 1954. Studies on the morphology and

life-history of Strongyloides mirzai n. sp.

from snakes in India. Journal of

Helminthology, vol. 28, pp. 25–34.

Skerratt, LF. 1995. Strongyloides spearei n. sp.

(Nematoda: Strongyloididae) from the

common wombatVombatus ursinus

(Marsupialia: Vombatidae). Systematic

Parasitology, vol. 32, pp 81-89.

schistosomose, segundo observações

feitas no Brasil. Memórias do Instituto

Oswaldo Cruz, vol. 11, pp. 121–155.

Mati, VLT, Pinto, HA & Melo, AL. 2013.

Strongyloides cruzi (Rhabditida:

Strongyloididae) in Ophiodes striatus

(Squamata: Anguidae) from Brazil: new

host and locality records with taxonomic

comments on Strongyloides of lizards.

Neotropical Helminthology, vol. 7, pp.

327–333.

Melo, AL, Mati, VLT & Martins, WA. 2012.

Callithrix penicillata as a nonhuman

primate model for strongyloidiasis.

Primates, vol. 53, pp. 303–309.

Moraes, RG. 1948. Contribuição para o estudo

do Strongyloides stercoralis e da

estrongiloidíase no Brasil. Revista do

Serviço Especial de Saúde Pública, vol. 1,

pp. 507–624.

Navarro, P & Lluch, J. 1993. Strongyloides

natricis sp. n. (Strongyloididae), un

nouveau nématode parasite de Natrix

maura (L. 1758) (Colubridae) en

Espagne. Annales de Parasitologie

Humaine et Comparée, vol. 68, pp.

136–138.

Navarro, P, Lluch, J & Izquierdo, S. 1989.

Strongyloides mascomai sp . n.

(Strongyloididae): un nouveau nématode

parasite de Rana perezi Seoane, 1885

(Amphibia: Ranidae) de l'est de

l'Espagne. Annales de Parasitologie

Humaine et Comparée, vol. 64, pp.

315–318.

Nielsen, PB & Mojon, M. 1987. Improved

diagnosis of Strongyloides stercoralis by

seven consecutive stool specimens.

Zentra l b lat t für Bakte r i olo g ie,

Mikrobiologie und Hygiene, vol. 263, pp.

616–618.

Pereira, C. 1929. Strongyloides ophidiae n. sp.

Boletim Biológico, vol. 22, pp. 16–18.

Pinto, HA, Mati, VLT & Melo, AL. 2012. New

hosts and localities for trematodes of

snakes (Reptilia: Squamata) from Minas

Gerais State, southeastern Brazil.

Comparative Parasitology, vol. 79, pp.

238–246.

Roca, V & Hornero, MJ. 1992. Strongyloides

216

Mati & Melo

Life history and morphology of Strongyloides ophidiae

Medicine, vol. 25, pp. 119–122.

Vicente, JJ, Rodrigues, HO, Gomes, DC &

Pinto, RM. 1993. Nematóides do Brasil.

Parte III. Nematóides de répteis. Revista

Brasileira de Zoologia, vol. 10, pp.

19–168.

Viney, ME & Lok, JB. 2007. Strongyloides spp.

In: WormBook, The Online Review of C.

elegans Biology, accessed in March 13,

2014,<http://www.wormbook.org/chapte

rs/www_genomesStrongyloides/genome

sStrongyloides.html>

Wiesman, MJ & Greve, JH. 1982. Strongyloides

mirzai infection in a green tree python.

Journal of the American Veterinary

Medical Association, vol. 181, pp.

1329–1330.

Speare, R. 1989. Identification of species of

Strongyloides. In: Grove, DI (ed.).

Strongyloidiasis: a major roundworm

infection of man. Taylor and Francis,

London, U.K., pp. 11–84.

Széll, Z, Sréter, T & Varga, I. 2001. Ivermectin

toxicosis in a chameleon (Chamaeleo

senegalensis) infected with Foleyella

furcata. Journal of Zoo and Wildlife

Medicine, vol. 32, pp.115–117.

Teare, JA & Bush M. 1983. Toxicity and efficacy

of ivermectin in chelonians. Journal of the

A m er i c a n Ve t e r in a r y M e d i c a l

Association, vol. 183, pp. 1195–1197.

Travassos, L, Freitas, JFT & Kohn, A. 1969.

Trematódeos do Brasil. Memórias do

Instituto Oswaldo Cruz, vol. 67, pp.

1–866.

Veazey, RS, Stewart, TB & Snider III, TG.

1994. Ureteritis and nephritis in a

Burmese Python (Python molurus

bivitattus) due to Strongyloides sp.

infection. Journal of Zoo and Wildlife

Received March 14, 2014.

Accepted June 22, 2014.