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Parasitol Res (2007) 100:855–860

DOI 10.1007/s00436-006-0346-1

ORIGINAL PAPER

Thelazia rhodesi (Spirurida, Thelaziidae), bovine eyeworm:


morphological study by scanning electron microscopy
Soraya Naem

Received: 3 September 2006 / Accepted: 13 September 2006 / Published online: 10 November 2006
# Springer-Verlag 2006

Abstract Thelazia rhodesi (Spirurida, Thelaziidae) is a development takes place in the thorax and abdomen of the
parasite of ruminants, which causes ocular infections. insect, and infective stages are present in 18–25 days.
Scanning electron microscopy was used to study the surface Development to adult takes place without migration, and
ultrastructure of adult worms of this nematode. At the the prepatent period is between 3 and 6 weeks. The first
anterior end of both sexes, the buccal opening was stage of Thelazia is very short-lived in the lachrymal
orbicular. Around the mouth, four pairs of cephalic papillae secretion, only surviving a few hours, and transmission
and two lateral amphids were seen. A pair of lateral cervical depends upon the continuous presence of the vectors. For
papillae was present. In the female, the vulva was located at this reason, thelaziasis has a seasonal occurrence according
the anterior part of the body, and the tail end was stumpy to the seasonality of the intermediate hosts (Dunn 1978).
with two phasmids near its extremity. In the male, the tail The adult and larval stages live in eyes causing conjunc-
was blunt, without caudal alae, and curved ventrally. There tivitis, keratitis, lacrimation, ocular discharge, and ulcers
were 14 paired preanal papillae, one single papilla directly (Soulsby 1986; Otranto and Traversa 2005). Bovine
anterior to cloaca, one paired postanal papilla, and two thelaziasis was first reported in Iran by Ebadi in 1951.
phasmids at the posterior end. The spicules were unequal Other investigations indicate the presence of bovine
and dissimilar. The cuticle was transversally striated and thelaziasis in Japan, Ghana, Afghanistan, USA, Canada,
appeared serrated. and Italy (Okoshi and Kitano 1966; Vohradsky 1970; Barus
et al. 1976; Genden and Stoffolano 1980; O’Hara and
Kennedy 1991; Giangaspero et al. 2000).
Introduction Apart from the morphological descriptions (Kikuchi
1976; Guttekova 1987; Choi et al. 1989; Beelitz et al.
Thelazia rhodesi Desmarest, 1822 is a parasite of domestic 1997; Otranto et al. 2003a; Naem 2005) and molecular
and wild ruminants and less commonly of horses (Soulsby findings (Otranto et al. 2001; Tarsitano et al. 2002; Otranto
1986; Anderson 2000). This nematode occurs on the et al. 2003b) that have already demonstrated the differences
surface of the cornea, under the nictitating membrane, in among Thelazia species, no detailed morphological de-
the conjunctival sac and lachrymal duct. The worms are scription of T. rhodesi has been presented. This article
viviparous, and the first-stage larvae are passed by females reports most surface features of adult T. rhodesi by
into the lachrymal secretions where they are ingested by scanning electron microscopy (SEM).
nonbiting Diptera. It has been confirmed that Musca
autumnalis and Musca larvipara are suitable intermediate
hosts (Anderson 2000; Giangaspero et al. 2004). Larval Materials and methods

S. Naem (*) From April to December 2002, 400 cattle were examined at
Department of Pathobiology, College of Veterinary Medicine,
an abattoir located in Tehran City, Iran. The conjunctival
Urmia University,
P.O. Box1177, Urmia, Iran sac and lachrymal duct of both eyes were flushed with
e-mail: sorayanaem@yahoo.com sterile saline solution, and 20 adult worms were removed
856 Parasitol Res (2007) 100:855–860

from the eyes of eight naturally infected cattle. Seventeen Results


adult T. rhodesi (13 females and 4 males) were selected and
washed in 2% sodium cacodylate buffer (pH 7.2). The The cephalic region was similar in both sexes. The mouth
nematodes were cleared with lactophenol solution (glycer- was orbicular and had no lips, with its anterior edge turned
in, lactic acid, phenol, and distilled water), and identifica- back and cut into two festoons, starting with a small
tion of the species was confirmed on the basis of light capsule 10–14.5 μm wide at the buccal opening. Around
microscopic examination with reference to keys (Ershov the mouth, four pairs of submedian small, conoidal cephalic
1956; Yamaguti 1961; Skrjabin et al. 1967; Skrjabin 1969; papillae and two lateral amphidal apertures were seen
Chabaud 1975; Lichtenfels 1975; Soulsby 1986). The (Fig. 1). Also, two lateral cervical papillae, one on each
worms were next placed in 4% glutaraldehyde. The speci- side, 350–384 μm from the anterior end, were noticed
mens were then transferred from Iran to the EM Facility at (Figs. 2, 3, and 5). At the anterior end of both sexes, an
the Department of Pathology and Molecular Medicine, excretory pore was seen (Fig. 4).
McMaster University, Canada, for SEM study. The nema- The females were 12.5–20.5 mm long and 300–500 μm
todes were put into fresh 4% glutaraldehyde for 2 h, wide. At the anterior end of the body, the vulva was located
postfixed in 1% osmium tetraoxide in cacodylate buffer in the esophageal region, 505.2–536.3 μm from the
(pH 7.2) at 4°C for 1 h, and dehydrated through a series of cephalic region (Figs. 5 and 6). The pattern of the cuticle
increasing ethanol concentrations (50, 75, 96, and 100%). around the vulva was different (Fig. 6). Also, there was a
They were dried with liquid CO2 in a critical point drier small papilla close to the vulva on the lateral side of the
apparatus (Critical Point Drier, Model # 2800, Ladd female’s body (Figs. 7 and 8). At the posterior end, the anal
Research Industries, Burlington, VT, USA). Each worm pore was seen (Fig. 9), and the tail end was stumpy with
was then placed on a numbered aluminum stab, which was two phasmids near its extremity (Figs. 9 and 10).
coated with gold in an ion-coating sputter (Sputter Coater, The cuticular surface showed coarse transverse stria-
Model # E5100, Polaron Instruments, USA) and then tions in both sexes, giving them a serrated appearance
viewed with a JEOL JSM-840 Scanning Electron Micro- (Figs. 11 and 12). The bands of striation at the anterior,
scope operated at 15 kV. Photographs were taken with a middle, and posterior portions were 15, 20, and 30 μm
digital acquisition system for analog SEMs (GW Electron- apart, respectively. The cuticle showed some tumor-like
ics, Norcross, GA, USA). masses at the posterior end of one female worm (Figs. 13
and 14).
The males were 7.5–14.5 mm long and 420–475 μm
wide. The tail was blunt, curved ventrally, and without

Fig. 1 Scanning electron mi-


crograph of the anterior end of
the male Thelazia rhodesi,
showing mouth, with its anterior
edge turned back and cut into
two festoons (arrows), cephalic
papillae (stars), and amphidal
apertures (A). Bar 10 μm
Fig. 2 Scanning electron
micrograph of the anterior end
of the male Thelazia rhodesi,
showing cervical papilla (CP).
Bar 100 μm
Fig. 3 Scanning electron mi-
crograph of anterior end of the
male Thelazia rhodesi, showing
a higher magnification of cervi-
cal papilla (CP) and cuticular
transverse striations (TS).
Bar 10 μm
Fig. 4 Scanning electron
micrograph of the anterior end
of the male Thelazia rhoesi,
showing excretory pore (EP)
and cuticular transverse
striations (TS). Bar 10 μm
Parasitol Res (2007) 100:855–860 857

Fig. 5 Scanning electron mi-


crograph of the anterior end of
the female Thelazia rhodesi,
showing cervical papillae (CP)
and vulva (V). Bar 100 μm
Fig. 6 Scanning electron micro-
graph of the female Thelazia
rhodesi, showing a higher mag-
nification of the vulva (V), cuticle
around the vulva (star), and
cuticular transverse striations
(TS). Bar 10 μm
Fig. 7 Scanning electron micro-
graph of the female Thelazia
rhodesi, showing cervical papilla
(arrowhead), vulva (V), and a
small papilla close to vulva (ar-
row). Bar 100 μm
Fig. 8 Scanning electron micro-
graph of the female Thelazia
rhodesi, showing a higher mag-
nification of a small papilla close
to the vulva (arrowhead) and
cuticular transverse striations
(TS). Bar 10 μm
Fig. 9 Scanning electron micro-
graph of the posterior end of the
female Thelazia rhodesi, show-
ing anal pore (AP) and phasmid
(Ph). Bar 10 μm
Fig. 10 Scanning electron mi-
crograph of the posterior end of
the female Thelazia rhodesi,
showing a higher magnification
of phasmids (Ph). Bar 10 μm

caudal alae, and the spicules were unequal and dissimilar In this study, T. rhodesi shows an internal circle of two
(Figs. 15 and 16). There were 14 paired preanal papillae, labial festoons, an external circle of four pairs of cephalic
one single papilla directly anterior to the cloaca, one paired papillae, and two lateral amphids. Gibbons (1986) also
postanal papilla (Figs. 16, 17, and 18), and two nipple- showed the internal circle of labial papillae in Thelazia
shaped phasmids at the posterior end (Figs. 17 and 18). gulosa. In Thelazia skrjabini, the head has two circles of
papillae: the inner circle with six papillae and the outer
circle with four papillae. Amphids are at both lateral sides
Discussion (Kikuchi 1976). T. californiensis shows an internal circle of
six very slightly reduced papillae, and an external circle of
This paper reports most surface features of adult T. rhodesi eight well developed, symmetrically spaced papillae, each
(Spirurida, Thelaziidae) using SEM. papilla of the external circle being situated at the same level
The cephalic characteristics of spiruroids present a on the head (Chitwood and Wehr 1934). Choi et al. (1989)
varied and complex picture. Different species belonging to observed two large head papillae on the mouth opening of
this genus are characterized according to the morphological the male T. callipaeda, which were absent in the females
description of Yamaguti (1961). Among the Thelazia and were distinct from the cephalic papillae in their
species, Thelazia callipaeda and Thelazia californiensis morphology and orientation. In another investigation on
were known to cause human thelaziasis (Miyazaki 1991). T. callipaeda, several pairs of the nipple-shaped cephalic
The arrangement and the number of the cephalic papillae in papillae were reported (Arizono et al. 1976). SEM
T. californiensis appear to be the most generalized of any observations of the female T. callipaeda showed only one
known spiruroid of nematode (Chitwood and Wehr 1934). pocket-shaped amphid at the anterior end (Choi et al.
858 Parasitol Res (2007) 100:855–860

Fig. 11 Scanning electron


micrograph of the female
Thelazia rhodesi, showing
cuticular transverse striations
(TS). Bar 10 μm
Fig. 12 Scanning electron mi-
crograph of the posterior end of
the female Thelazia rhodesi,
showing cuticular transverse
striations (TS). Bar 10 μm
Fig. 13 Scanning electron
micrograph of the female
Thelazia rhodesi, showing
cuticular tumor-like masses
(arrows). Bar 100 μm
Fig. 14 Scanning electron mi-
crograph of the female Thelazia
rhodesi, showing a higher mag-
nification of cuticular tumor-like
masses (stars) and cuticular
transverse striations (TS). Bar
10 μm

1989), while Otranto et al. (2003a) observed two amphids These papillae were paired, simple, and similar in shape.
in lateral position and four submedian papillae on both The cervical papillae are often very small and inconspicu-
sexes of T. callipaeda. Nematode amphids exist in a variety ous, which has led to the erroneous conclusion that they are
of forms and sizes, some probably being purely chemo- absent in some species. The larger, conspicuous cervical
sensory while some being photoreceptive, with an associ- papillae vary in form from thin, needle-like appendages to
ated gland (McLaren 1974). The functional type of the complex structures with denticulate posterior borders. The
amphids of T. rhodesi is yet unknown. In this study, two position, size, and shape of the cervical papillae are used as
cervical papillae at the anterior end of the worm were seen. taxonomic characters. It is likely, in view of their structure

Fig. 15 Scanning electron micro-


graph of the posterior end of the
male Thelazia rhodesi, showing
spicules (S), precloacal papilla
(PrCP), postcloacal papilla (PoCP),
and phasmid (Ph). Bar 10 μm
Fig. 16 Scanning electron micro-
graph of the posterior end of the
male Thelazia rhodesi, showing
spicule (S), one single papilla di-
rectly anterior to cloaca (black
arrow), precloacal papilla (PrCP),
and postcloacal papilla (PoCP).
Bar 10 μm
Fig. 17 Scanning electron
micrograph of the posterior end
of the male Thelazia rhodesi,
showing precloacal papilla
(PrCP), cloaca (C), postcloacal
papilla (PoCP), and phasmid (Ph).
Bar 10 μm
Fig. 18 Scanning electron micro-
graph of the posterior end of the
male Thelazia rhodesi, showing
postcloacal papillae (PoCP) and
phasmids (Ph). Bar 10 μm
Parasitol Res (2007) 100:855–860 859

and position, that they function as mechanoreceptors and helping the worm to maintain itself in a suitable
(Gibbons 1986). These organs determine whether the environment (McLaren 1976).
nematode can pass through a small space (McLaren
1976). Acknowledgements The author deeply thanks Mr. Samani and Mr.
Gerami, Faculty of Veterinary Medicine, Tehran University, Iran, who
Thelazia species have morphological differences such were of enormous assistance in the collection of material examined in
as the location of the vaginal opening, the number of this study. The author wishes to express her appreciation to Professor
cuticular transverse striations, and the number of caudal Larry A. Arsenault, head of the electron microscope facility,
papillae. The results of the present study indicate that the Department of Pathology and Molecular Medicine, McMaster Uni-
versity, Canada, for his assistance in providing all facilities during the
vulva was located 505.2–536.3 μm from the anterior end study. Thanks are due to Mr. Ernie Spitzer, chief technician, and all
of T. rhodesi. The vulva of T. skrjabini was located 0.4– the technicians in the electron microscope facility for their help. The
0.7 mm from the anterior end and protruded (Kikuchi author is also grateful to Dr. Shigehiko Uni, Osaka City University,
1976). Another investigation showed that the vulva of Japan, for translating the Japanese article and helpful comments.
Finally, the author is especially obliged to Dr. Christine M. Budke,
Thelazia lacrymalis was situated 593 μm from the anterior Texas A&M University, USA, for her continuous support and critical
end (Beelitz et al. 1997). In a study which was carried out review of the manuscript.
by Otranto et al. (2003a) on T. callipaeda, the vaginal
opening was located at 62.0–162.2 μm anterior to the
esophago–intestinal junction.
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