J. Parasitol., 96(3), 2010, pp.

000–000
F American Society of Parasitologists 2010

MORPHOLOGICAL REDESCRIPTION OF DIROFILARIA IMMITIS

Adriano P. Furtado, Francisco T. V. Melo, Elane G. Giese, and Jeannie N. dos Santos*
Laboratório de Biologia Celular e Helmintologia ‘‘Profa. Dra. Reinalda Marisa Lanfredi,’’ Instituto de Ciências Biológicas, Universidade Federal do
Pará, Guamá, Belém, Pará, Brasil, 66075-110. e-mail: jeannie@ufpa.br

; ABSTRACT: Morphological descriptions of Dirofilaria immitis are scarce. For this reason, we carried out morphologic studies using
< both light and scanning electron microscopy for this filaroid species. Morphometric and morphological data were compatible with
previous descriptions of D. immitis, but several anatomical structures are described by scanning electron microscopy for the first time,
such as details of the cuticular striations, positioning of amphids, visualization of anal and vulvar opening, descriptions of deirids,
lateral line, the pair of phasmids in the posterior end in females, and visualization of a small pair of latero-terminals papillae in the
posterior end in males.

Dirofilaria immitis is the most common filaroid found REDESCRIPTION

parasitizing dogs, cats, and other carnivores throughout the
world. It is transmitted by various species of mosquitoes (Insecta:
Diptera: Culicidae) (McCall et al., 2008). This parasite is the
causative agent for ‘‘heartworm disease’’ and occurs primarily in
the right ventricle and adjacent pulmonary artery.
Using light microscopy, several authors (López-Neyra, 1947;
Yamaguti, 1962; Yorke and Maplestone, 1962) have described D.
immitis. However, scanning electron microscopy (SEM) has
become an important tool that can add new taxonomic characters
to previous species descriptions made by light microscopy
(Chagas-Moutinho et al., 2007; Torres et al., 2007), as well as
contributing to descriptions of new species (Santos et al., 2008;
Giese et al., 2009). Few reports have described D. immitis using
this technique, although Wong and Brummer (1978) conducted a
comparative study of filarial longitudinal ridges, and included D.
immitis, while Rodrigues-Silva et al. (1999) described the rugosae
area and the posterior end of male D. immitis. The present report
is a redescription of D. immitis by SEM.
MATERIALS AND METHODS
Filaroids were collected from the right ventricle and pulmonary artery
of domestic pet dogs in the cities of Salvaterra (00u459120S, 48u319000W)
and São Sebastião da Boa Vista (01u439050S, 49u319450W), Marajo Island,
Para State, Brazil. Routinely, domestic dogs with visceral leishmaniasis
are killed by public health agencies (SESPA and SESMA) in these cities.
These agencies authorized 30 necropsies of dogs (20 in Salvaterra and 10
in São Sebastião da Boa Vista) so that we could undertake the present
investigation.
The collected worms were washed immediately in phosphate buffered
saline, pH 7.4, and fixed in hot AFA (2% acetic acid, 3% formaldehyde,
and 95% ethanol 70 degrees GL) at 60 C.
For light microscopy, 2-cm sections of the anterior and posterior ends
of 18 females and 7 male D. immitis were observed using a light
microscope, equipped with a camera lucida, after dehydration in ethanol
series (70–100%), then cleared in Aman’s lactophenol for 72 hr.
Measurements are given in millimeters, followed by ranges in paren-
theses.
A male and female filaroid from each municipality were deposited
in the Helminthological Collection of the Instituto Oswaldo Cruz,
Rio de Janeiro, Brazil (CHIOC no. 35658A/B and CHIOC no.
35659A/B).
For SEM, 2-cm sections of the anterior and posterior ends of 10
males and females were processed according the methods of Mafra and
Lanfredi (1998) and observed using a JEOL JSM 6390 LV scanning
microscope.
Received 28 May 2009; revised 17 August 2009, 19 October 2009, 23
October 2009, 21 January 2010; accepted 22 January 2010.
* To whom correspondence should be addressed.
DOI: 10.1645/GE-2178.1
Dirofilaria immitis
(Figs. 1–19; Table I)
General diagnosis: Elongated worm, filiform, with cephalic extremity
slightly thin and rounded (Figs. 1, 4). Terminal oral opening, circular,
without lips, surrounded by 4 pairs of small cephalic papillae and 2 lateral
amphids (Fig. 5). Amphids presenting basal opening directed to posterior
region of body (Fig. 6). Esophagus divided in muscular and glandular
regions, but without clear demarcation between these regions (Fig. 1).
Small lateral deirids situated between nerve ring and esophageal–intestine
junction (Fig. 1), in center of cuticular depression, next to lateral line,
slightly dislocated for ventral region. Deirids with a filamentous structure,
base wider than apex, and longitudinal concavity. Apex trifurcated
(Figs. 7, 8). Body surface with delicate transversal striation (Fig. 9).
Cuticular striations random in cephalic extremity (around cephalic
papillae, amphids, and oral opening) (Fig. 5) and in posterior extremity
of females (near phasmids) (Fig. 14). Absent cuticular transversal
striations surrounding deirids (Figs. 7, 8), vulvar opening (Fig. 12), and
anus (Fig. 13). Inconspicuous lateral lines constituted by discrete
discontinuous ridges in anterior and posterior portion of filarids
(Fig. 10), and by 2 or 3 parallel continuous lines throughout body
(Fig. 11).
Males: Body: 137.6 (119.4–162.3) length, 0.41 (0.3–0.5) width.
Esophagus 1.21 (1.08–1.46) length, 0.11 (0.09–0.13) width. Nerve ring to
anterior end 0.40 (0.35–0.47), in first third of esophagus. Deirids to
anterior end 0.57 (0.29–0.77) (Figs. 7, 8). Ventral rugosae area in spiraled
posterior end of males with cuticular parallel ridges of varied length.
Ridges follow longitudinal axis of body in larger spirals (Fig. 15),
gradually dislocating from transversal position in smaller spirals
(Fig. 16). In region of larger spirals, number of ridges varies from 7 to
11. Each ridge composed of small units formed of prolongated cuneiform
cuticular elevations, with base alleviated contour, rectilinear apex of
varied length, shorter when perpendicular to common fine cuticular
transversal striations throughout body (Fig. 17). Numerous rounded
cuticular elevations in a line at end of each ridge and in spaces between
these ridges (Fig. 17). Similar structures also observed laterally from
rugosae area to dorsal region of worm, but more random. Paired and
unpaired pre-cloacal papillae, varying from 1 to 6 in number (Figs. 2, 3,
18). Small pair of ad-cloacal papillae located lateral to cloacal opening
(Figs. 3, 18). Five pairs of post-cloacal papillae: 1 pair ventral, 1 large pair
ventro-lateral, 2 small pairs ventro-lateral, 1 pair terminal (Figs. 2, 3, 18
[and insert]). Pair of ventral phasmids between last 2 pair post-cloacal
papillae (Figs. 2, 3, 18). Large spicule 0.34 (0.31–0.39), lying in channel
formed by small spicule 0.17 (0.15–0.19) (Fig. 19). Gubernaculum absent.
Tail: 0.10 (0.09–0.11).
Female: Body: 218.3 (177.0–272.0) length, 0.49 (0.40–0.61) width.
Esophagus length 1.29 (1.05–1.57), 0.12 (0.09–0.17) width (Fig. 1). Nerve
ring to anterior end 0.41 (0.33–0.52), in first third of esophagus (Fig. 1).
Deirids to anterior end 0.70 (0.56–0.97). Vulva next to anterior end, with
an elliptical and transverse vulvar opening, delimited by fine, but not
prominent, lips, without papillae or fringes (Fig. 12) located at 2.68 (2.23–
3.26) to anterior end, just behind esophageal–intestine junction (Fig. 1).
Rounded terminal end. Anus sub-terminal with incurved opening
(Fig. 13), toward posterior extremity, situated 0.17 (0.15–0.27) to
posterior end. Pair of latero-ventral phasmids with circular openings
(Fig. 14). Viviparous, didelphic, and opisthodelphic ovaries.

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FIGURES 1–3. Dirofilaria immitis by light microscopy. (1) Anterior end of female, showing the nerve ring, esophagus, deirids, first portion of intestine,
and the vulvar opening. Bar 5 200 mm. (2) Lateral view of male posterior end, showing the large and small spicules, pre-cloacal papillae, large ventro-
lateral post-cloacal papillae, and the phasmids. Bar 5 100 mm. (3) Ventral view of male posterior end, with the pre-cloacal papillae, a small pair of ad-
cloacal papillae, the cloacal opening, 4 pairs of post-cloacal papillae (1 pair ventral, 1 large pair ventro-lateral, and 2 small pairs ventro-lateral), a pair of
ventral phasmids, and large and small spicules. Bar 5 100 mm.

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FIGURES 4–8. Dirofilaria immitis by scanning electron microscopy. (4) Anterior end, showing the vulva (arrow). Bar 5 500 mm. (5) Detail of the
anterior end, with oral opening (arrow), 4 pairs of cephalic papillae (arrow heads), and amphids (asterisks). Bar 5 20 mm. (6) Detail of amphid (arrow),
with the anfidial opening (arrow head). Bar 5 5 mm. (7) Detail of a totally everted deirid (arrow), located in the center of cuticular depression. Bar 5
2 mm. (8) Detail of a partially everted deirid (arrow). Bar 5 5 mm.

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FIGURES 9–16. Dirofilaria immitis by scanning electron microscopy. (9) Detail of fine transversal cuticular striation (arrow heads). Bar 5 5 mm. (10)
Lateral line in anterior portion of the body. Bar 5 5 mm. (11) Inconspicuous lateral line throughout length of body (arrow heads). Bar 5 50 mm. (12)
Vulva opening (arrow), without cuticular striations surrounding the structure (arrow head). Bar 5 10 mm. (13) Anal aperture (arrow), without cuticular
striations surrounding the structure (arrow head). Bar 5 10 mm. (14) Posterior extremity of female, showing the phasmids (arrow head). Bar 5 5 mm. (15)
Rugosae area of males, with cuticular ridges (arrow heads) longitudinally disposed in the large spirals. Bar 5 20 mm. (16) Detail of the rugosae area of
males, showing the cuticular ridges (arrow heads) transversally disposed in the smaller spirals. Bar 5 20 mm.

Taxonomic summary Remarks

Host: Canis familiaris (Linnaeus, 1758) (Carnivora: Canidae).
Site of infection: Pulmonary artery and right ventricle.
Locality: Salvaterra (00u459120S, 48u319000W) and São Sebastião da
Boa Vista (01u439050S, 49u319450W), Marajo Island, Para State, Brazil.
Deposition of specimens: Four voucher specimens in Helminthological
Collection of the Instituto Oswaldo Cruz, Rio de Janeiro, Brazil (CHIOC
no. 35658A/B and CHIOC no. 35659A/B).
Several new anatomical variations have been described for D. immitis.
For example, Murata et al. (2003) and Sano et al. (2005) examined D.
immitis from leopards and penguins in a Japanese zoo. They reported the
presence of delicate longitudinal cuticular ridges along the entire length of
the body using ordinary light microscopy. Nonetheless, both reports
showed that these filarids also presented molecular characteristics similar
to the D. immitis from canids. These data disagree with those of Wong and

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FIGURES 17–19. Dirofilaria immitis by scanning electron microscopy. (17) Rugosae area, with segmented cuticular ridges, consisting of elevated
cuneiform cuticle (arrows), elevated round cuticle (arrow heads). Bar 5 10 mm. (18) Ventral view of male posterior end, revealing paired pre-cloacal
papillae (larger arrows), a pair of small ad-cloacal papillae (smaller arrows), a pair of post-cloacal ventral papillae (p), large post-cloacal ventro-lateral
papillae (P), and ventral phasmids (arrow heads). Bar 5 30 mm. Detail (insert) of 1 terminal papilla in male. Bar 5 5 mm. (19) Details of large (arrow) and
small spicules (arrow head). Bar 5 30 mm.

TABLE I. Comparison of Dirofilaria immitis morphometric parameters.

Leidy (1856) apud Lent

and Teixeira-de-Freitas
Data for this work (1937) López-Neyra (1947)
Taxonomic parameters Female Male Female Male Female Male

Body length 218.3 (177–272) 137.6 (119–162) 150–300 120–180 210–310 120–200
Body width 0.49 (0.40–0.61) 0.41 (0.34–0.5) 1.0–1.3 0.6–0.9 1.0–1.3 0.6–0.7
Esophagus length 1.29 (1.05–1.57) 1.21 (1.08–1.46) . . 1.2–1.5 .
Esophagus width 0.12 (0.09–0.17) 0.11 (0.09–0.13) . . . .
Nerve ring 0.41 (0.33–0.52) 0.40 (0.35–0.47) . . 0.4 .
Deirids 0.70 (0.56–0.97) 0.57 (0.29–0.77) . . . .
Vulvae 2.68 (2.23–3.26) . 2.3–3.4 . 2.1–3.4 .
Tail 0.17 (0.15–0.27) 0.10 (0.09–0.11) . . 0.18–0.21 0.09–0.10
Large spicule . 0.34 (0.31–0.39) . 0.3 . 0.3
Small spicule . 0.17 (0.15–0.19) . 0.17–0.22 . 0.17–0.26
Male cloacal papillae .
distribution Unpaired and paired pre-cloacal Unpaired pre-cloacal (4–
(1–6); 1 pair ad-cloacal; 5 pairs 5); 3–5 pairs post-
post-cloacal (1 pair ventral, cloacal (first and fourth
1 large pair ventro-lateral, laterally dislocated
2 small pairs ventro-lateral, papillae) + frequently a
1 pair latero-terminal) unpaired papillae +
unique terminal papillae

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 0 THE JOURNAL OF PARASITOLOGY, VOL. 96, NO. 3, JUNE 2010
Brummer (1978), who described D. immitis without longitudinal cuticular
ridges using SEM. Because of these discrepancies, we carried out a detailed
morphologic study in D. immitis using light microscopy and SEM.
Several characteristics have been previously described for the cephalic
end of D. immitis (Wong and Brummer, 1978). These include the circular
and terminal oral opening without lips, the number and distribution of
cephalic papillae and amphids, and transverse cuticular striations.
In the present work, pre-cloacal papillae were found in varying numbers
(1–6), and may be both unpaired and paired. A pair of ad-cloacal papillae,
i.e., the first and second pair of post-cloacal papillae, were observed in all
specimens by light microscopy and SEM. The third and fourth pair of
post-cloacal papillae were best observed by light microscopy. In contrast,
terminal post-cloacal papillae were best seen by SEM. The pre-cloacal
papillae distribution is similar to the descriptions of Wong and Brummer
(1978) and Rodrigues-Silva et al. (1999), although López-Neyra (1947)
described a different distribution of post-cloacal papillae for D. immitis.
Based on this variability, Rodrigues-Silva et al. (1999) concluded that the
cloacal papillae distribution cannot be considered as a reliable taxonomic
character for D. immitis.
The general characteristic of spiraled posterior end in males, including
the rugosae area (arrangement of longitudinal and rounded cuticular
striations), are similar to Wong and Brummer (1978) descriptions. Our
observations, however, differ from those of Sano et al. (2005) in
positioning of male phasmids. Using light microscopy, the latter authors
observed phasmids situated near the tail tip. However, with light
microscopy and SEM, we found these structures to be positioned
ventrally. These differences could be artifactual or erroneous interpreta-
tions of penguin filarid morphology observed by light microscopy.
Data presented in the present study show that D. immitis found in the
thoracic organs (pulmonary arteries and right ventricle) of domestic dogs
have morphological characteristics compatible with the descriptions and
measures reported previously by others authors (López-Neyra, 1947;
Yamaguti, 1962; Yorke and Maplestone, 1962; Anderson and Bain, 1976).
However, we have also added several new features for D. immitis using
SEM, thereby providing a much more robust characterization of the
species.
ACKNOWLEDGMENTS
The authors thank the Para State Secretary of Public Health (Secretaria
Estadual de Saúde Pública—SESPA, 7a Regional de Proteção Social) and
the Municipal Secretaries of Health (Secretaria Municipal de Saúde) in
Salvaterra and São Sebastião da Boa Vista for permission to undertake
this study; Dra. Reinalda Marisa Lanfredi (in memoriam); Dr. William
Leslie Overal for language revision; Dra. Adriana Lanfredi Rangel and
Dr. Marcos André Vannier Santos (CPqGM—FIOCRUZ) provided
technical assistance in the scanning electron microscopy; and Dr. Gerald
W. Esch and anonymous referees for valuable comments and suggestions.
Brazilian financial support was from PROCAD-CAPES, FAPESPA, and
CNPq.
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