Parasitol Res (2014) 113:2103–2111
DOI 10.1007/s00436-014-3860-6
ORIGINAL PAPER
Protostrongylus pulmonalis (Frölich, 1802) and P. oryctolagi
Baboš, 1955 (Nematoda: Protostrongylidae), parasites of the lungs
of European hare (Lepus europaeus L.) in France: morphological
and molecular approaches
Célia Lesage & Damien Jouet & Cécile Patrelle &
Jean-Sébastien Guitton & Anouk Decors & Hubert Ferté
Received: 27 January 2014 / Accepted: 12 March 2014 / Published online: 1 April 2014
# Springer-Verlag Berlin Heidelberg 2014
Abstract Pulmonary protostrongyliasis of hare is a parasitic
disease caused by nematodes belonging to the genus
Protostrongylus (Nematoda, Protostrongylidae). During sur-
vey of wildlife disease in the South-East of France, pathologic
examination of lungs from European hares found dead or
hunter-killed between 2009 and 2012 was performed. Adult
male worms were morphologically characterized and the iden-
tification confirmed by molecular biology (D2 domain of the
28S and ITS2 of rDNA). Two different species were identi-
fied: the first one, Protostrongylus pulmonalis, is identical
with the haplotype previously deposited in GenBank. Based
on morphological criteria of copulatory bursa of adult male
worms (especially length of spicules and gubernaculum struc-
ture), we identified a second species found in France as
Protostrongylus oryctolagi. This is the first report of
P. oryctolagi in France from European hare and rabbit.
P. oryctolagi was isolated from 248 hares and 3 rabbits in
the South of France. P. pulmonalis was isolated from four
hares found dead in the Northern France and from one hare in
the South, which was co-parasitized by P. oryctolagi and
P. pulmonalis. It’s the first coinfection observed with these
two species from a lung of hare in France.
C. Lesage : D. Jouet (*) : C. Patrelle : H. Ferté
EA 4688 « VECPAR », UFR de Pharmacie, Université de Reims
Champagne-Ardenne, 51 rue Cognacq-Jay, 51096 Reims, France
e-mail: damien.jouet@univ-reims.fr
C. Lesage : A. Decors
Direction des études et de la recherche, Office National de la Chasse
et de la Faune Sauvage, 5 rue de Saint Thibaud, 78610 Auffargis,
France
J.<S. Guitton
Office National de la Chasse et de la Faune Sauvage, 39 boulevard
Albert Einstein - CS 42355, 44323 Nantes, France
Keywords Protostrongyliasis . Protostrongylus oryctolagi .
Protostrongylus pulmonalis . Lagomorphs . Molecular
approach . France
Introduction
Pulmonary protostrongyliasis is a parasitic disease that can
affect domestic and wild ruminants and Lagomorphs
(Panayotova-Pencheva 2005; Laakkonen et al. 2006;
Anderson 2000). Animal is contaminated after ingestion of
infective larvae (third-stage larva or L3) which are released in
the field by an intermediate host previously identified as snails
(Joyeux and Gaud 1946; Grewal et al. 2003). Typically, the
clinical aspect of infection is characterized by bilateral bron-
chopneumonia and subpleural lesions (Battisti et al. 2000).
Extensive lesions could modify pulmonary function and de-
teriorate general condition and health status. Therefore, the
ability to escape predators could be reduced and animal could
be more susceptible to secondary bacterial infections (Kralka
and Samuel 1984; Keith et al. 1986; Pajersky et al. 1992;
Murray et al. 1998). In Europe, pulmonary helminthiasis is
frequently encountered and occasionally implicated in the
cyclic decline of hares’ populations (Bull 1964; Chroust
et al. 2012).
This parasitic disease is caused by nematodes belonging to
the genus Protostrongylus (Nematoda, Protostrongylidae).
This genus includes 29 species belonging to three subgenera.
According to Boev (1975), seven species are observed in
Lagomorphs: Protostrongylus pulmonalis (Frölich, 1802)
Goble and Dougherty 1943; Protostrongylus terminalis
(Passerini 1884) Schulz, Orlow and Kutass, 1933;
Protostrongylus kamenskyi Schulz, 1930; Protostrongylus
cuniculorum (Joyeux and Gaud 1946) Schutz and
2104
Kadenatsii, 1949; Protostrongylus tauricus Schutz and
Kadenatsii, 1949, Protostrongylus boughtoni Goble and
Dougherty 1943, and Protostrongylus oryctolagi Baboš 1955.
In 1884, Passerini identified P. terminalis in Italy but gave
inappropriate criteria for species diagnosis. This species has
subsequently been reported by Shimalov in Belarus
(Shimalov 2001). Whether this species has been repeatedly
questioned, including confusion on morphological char-
acters between P. terminalis and other species such as
P. pulmonalis and Protostrongylus rufescens (Goble and
Dougherty 1943; Schutz and Kadenatsii 1949), it now
appears that the species described by Passerini in 1884
is distinct from other species (Baboš 1961; 1962; Boev
1975).
Concerning the status of other species, P. pulmonalis was
reported in hare (Lepus sp.) and rabbit (Oryctolagus
cuniculus) in Germany, Finland, Sweden, England, Austria,
Italy, Iberian Peninsula, and Czech Republic (Chroust et al.
2012; Laakkonen et al. 2006; Battisti et al. 2000; Casanova
et al. 1999; Costantini et al. 1990; Nickel and Gottwald 1979;
Joyeux and Gaud 1946; Soveri and Valtonen 1983; Soveri
et al. 1992). This species was described under several names,
including Protostrongylus commutatus (Diesing, 1851), now
considered a synonym of P. pulmonalis. In Russia,
P. kamenskyi was frequently found and usually associated
with P. pulmonalis in a process of co-infestation in the same
lung of hare (Lepus timidus). Shulz (1930) is the first one to
report event of co-infestation by two species of
Protostrongylus: P. pulmonalis and P. kamenskyi. In 1946,
P. cuniculorum was isolated from hare (Lepus sp.) and rabbit
(Oryctolagus cuniculus) in Italy and France. P. cuniculorum
has morphological similarity with P. rufescens (Leuckart,
1865), species found in domestic ruminants. Hence,
P. cuniculorum was regarded as a variety of P. rufescens
specific to Lagomorph. Indeed, Joyeux and Gaud (1946)
showed that sheep cannot be contaminated by P. rufescens
var. cuniculorum isolated from Lagomorph. In 1949, Schutz
and Kadenatsii considered the variety P. cuniculorum as a
valid species. P. tauricus has only been isolated from Lepus
europaeus in Caucasa, Austria, Yugoslavia, Hungary, and
Iberian Peninsula (Casanova et al. 1999). Finally, the descrip-
tion of P. oryctolagi was performed once from Oryctolagus
cuniculus in Hungary (Baboš 1955). The last species
P. boughtoni, was only found in North America, from subspe-
cies of the American hare, Lepus americanus (Keith et al.
1985, 1986; Sovell and Holmes 1996).
In France, the first report of verminous pneumonia in hare
and rabbit was done by Railliet (1890), who identified species
of lungworms as P. commutatus (synonym of P. pulmonalis).
However, the description of worms given by this author
corresponded to P. cuniculorum according to Joyeux and
Gaud (1946), who isolated this parasite from hares and wild
rabbits in South-East of France.
Parasitol Res (2014) 113:2103–2111
Various studies on the molecular analysis were performed
in order to investigate the phylogenetic relationships within
Protostrongylidae. These studies focused on the D2 and inter-
nal transcribed spacer (ITS) of ribosomal DNA (Bryan et al.
2010; Carreno et al. 2012; Carreno and Nadler 2003; Ezenwa
et al. 2010; Gajadhar et al. 2000; Gerhold et al. 2010; Huby-
Chilton et al. 2006; Jenkins et al. 2005; Kutz et al. 2013;
Mitchell et al. 2011; Murakami et al. 2011; Tanabe et al.
2010) and mitochondrial cytochrome oxidase 1 (cox1)
(Asmundsson et al. 2008; Hoberg et al. 2005; Jabbar et al.
2013; Mortenson et al. 2006).
In this study, we compared the genetic sequences from both
D2 and ITS2, known as specific markers for Nematodes
(Campbell et al. 1995; Chilton 2004; Chilton et al. 1995,
1997, 2006; Gasser 1997; Gouÿ de Bellocq et al. 2001;
Hoste et al. 1993), in order to confirm morphological identi-
fication of worms.
This work aims to bring update contribution to the study of
the parasites responsible for pulmonary protostrongyliasis in
Lagomorphs under natural conditions in France, and to iden-
tify the species involved, by both morphological and molec-
ular approaches.
Materials and methods
Sample collection
Samples were collected in the context of the network activity
SAGIR, responsible for monitoring of wildlife diseases at the
national level. This network is based on a national collabora-
tion of field agents composed primarily of hunters and tech-
nical staff of departmental veterinary laboratories. The wild
lagomorphs found dead between 2009 and 2012 were trans-
mitted to the local veterinary laboratories for postmortem
examination. After examination, positive lungs stored at
−20 °C before sending to our laboratory.
A second collection was set up during hunting seasons of
hares hunter-killed in several departments in South-East of
France in 2010 and 2012: Ardèche, Gard, Hérault, Tarn, and
Vaucluse.
Parasite examination
The larvae infestations were detected by the modified
Baermann technique (Beane and Hobbs 1983; Forrester and
Lankester 1997) and lungworm isolated by dissection of the
defrosted pulmonary tracts. After tearing, tissues were vigor-
ously shaked in a water-filled jar, and worms and larvae
accumulated on the bottom were observed under a stereomi-
croscope according to the technique developed by Skirnisson
and Kolářová (2008). The larvae and/or adult worms collected
were preserved in 90% ethanol.
Parasitol Res (2014) 113:2103–2111 2105

All adult male worms isolated from each hare and rabbit
were morphologically analyzed to identify parasite species.
Posterior ends of worms (copulatory bursa with spicules) were
cleared and preserved in Amman lactophenol between slide
and cover slide. For some of them, the anterior part of worm
was also mounted. The species identification was established
on caudal bursa according to the features proposed by Boev
(1975), especially morphology and measurements of
gubernaculum (whole and legs) and spicules.
Molecular analyses
The middle part of the body of some worms mounted and
larvae were individualized and preserved into a sterile vial
with 95 % ethanol for molecular biology (Table 1). At least
one worm from each animal found dead and collected by the
SAGIR network was analyzed by molecular biology. DNA
extraction was performed by a QIAamp DNA mini kit
(Qiagen, Hilden, Germany) according to the manufacturer’s
instructions. During the first step (tissue lysis), the middle part
of worms was crushed one by one using a piston pellet (Treff,
Degersheim, Switzerland). DNA was eluted in 100 μl of the
elution buffer provided. Sequencing of the D2 domain of the
28S and the second internal transcribed spacer (ITS2) of the
ribosomal DNA were used for the identification of worms.
Polymerase chain reaction (PCR) was performed in a 50 μl
volume using 5 μl of DNA and 50 pmol of each of the
primers. The PCR mix contained (final concentrations)
10 mM Tris HCl (pH 8.3), 1.5 mM MgCl2, 50 mM KCl,
0.01 % Triton X-100, 200 μM dNTP each base, and 1.25 units
of Taq polymerase (Eppendorf, Germany). D2 domain was
amplified using primers C2’ (5′-GAAAAGAACTTTGRAR
AGAGA-3′) and D2 (5′-TCCGTGTTTCAAGACGGG-3′)
(Gouÿ de Bellocq et al. 2001). ITS2 was amplified with
NC1 (5′-ACGTCTGGTTCAGGGTTGTT-3′) and NC2 (5′-
ATGCTTAAGTTCAGCGGGT-3′) previously described by
Gasser et al. (1993). The initial denaturation at 94 °C for
3 min was followed by 40 cycles of denaturation at 94 °C
for 30 s, annealing at 40 °C (D2) or 50 °C (ITS2) for 1 min,
and extension at 68 °C for 1 min with a final elongation time
of 10 min at 68 °C. PCR products were directly sequenced in
both directions with the primers used for DNA amplification
(Genoscreen, France). Our sequences were deposited in
GenBank under accession numbers KJ450993 to KJ451018.
Sequence alignment was performed using the ClustalW
routine included in the MEGA version 5 software (Tamura
et al. 2011) and checked by eye. The D2 domain of the rDNA
was used for tree construction using sequences obtained dur-
ing this study and sequences of Protostrongylinae available in
GenBank: P. pulmonalis (EU595590), P. boughtoni
(EU595595), Protostrongylus rushi (EU595598),
Protostrongylus stilesi (EU595599), P. rufescens
(EU595600), Protostrongylus rupicaprae (EU595601), and
Orthostrongylus macrotis (EU595592). Sequences of
Muelleriinae, Muellerius capillaris (AY292798), and
Cystocaulus ocreatus (EU595593) were set as outgroup.
Phylogenetic trees were constructed using the neighbor-
joining (NJ), the maximum likelihood (ML), and minimum
evolution (ME) methods using the MEGA 5 software. For all
NJ, ML, and ME analyses, the most appropriate nucleotide
substitution model was determined, gaps were treated as miss-
ing data and internal node support was assessed by
bootstrapping over 500 replicates. ITS2 was used to estimate
pairwise distance between Protostrongylus species.

Table 1 Samples from the study used for molecular and morphological analyses

Parasite species Host Stage Locality Taxa

Protostrongylus sp. 1 Lepus europaeus Adult, male Jura (F) LIE1

Adult, male Aisne (F) SEU3, SEU4
Adult, female Hérault (F) KEC1, KEC2a
Adult, male Hérault (F) KEC3, KEC6a
Larvae Savoie (F) SEU47, SEU49
Protostrongylus sp. 2 Lepus europaeus Adult, female Charente (F) SEU5, SEU7
Adult, male Charente (F) SEU6
Adult, male Gard (F) KEC8, KEC9, SEU60
Adult, female Gard (F) KEC10, KEC11
Adult, female Hérault (F) KEC4, KEC7a
Adult, male Ardèche (F) SEU61
Adult, male Tarn (F) SEU18
Adult, male Vaucluse (F) SEU10
Adult, male Bouches du Rhône (F) SEU9
Oryctolagus cuniculus Adult, male Bouches du Rhône (F) SEU8
a
Isolated from the same pulmonary tract of hare
2106 Parasitol Res (2014) 113:2103–2111

Results to the two subgenera of Protostrongylus: Protostrongylus sp.1

Morphological analyses
Two parasite species were isolated in France, separated ac-
cording to their morphological characters and named
Protostrongylus sp.1 and Protostrongylus sp.2. They belong
to the Pulmostrongylus subgenus (Fig. 1a, b) and
Protostrongylus sp.2 to the Protostrongylus subgenus
(Fig. 1c, d), which differs by the edge of legs of crura of the
gubernaculum, smooth for the first one (Fig. 1b) and with
tubercles for the second (Fig.1f, g). The measurements of
worms, isolated during our study, are presented (Table 2)

Fig. 1 Copulatory bursa and

accessory pieces of males of P.
a b
(Pul.) pulmonalis (a, b) and P. (P.)
oryctolagi (c–h). a caudal bursa.
b smooth gubernaculum. c–e
caudal bursa. f, g gubernaculum
with tubercles. h head of
gubernaculum

c d

e f

g h
Parasitol Res (2014) 113:2103–2111 2107

[145–150]
Table 2 Measurements of males of Protostrongylus isolated from Lagomorphs, according to Boev (1975) and completed with samples from this study (x=average; n=number of specimens analyzed)

[90–112]
and compared to other species of Protostrongylus from

[32–84]
[45–51]

[68–84]
[43–65]

[72–84]

[80–86]
n=12
x=48

x=78
(μm)
Lagomorphs according to the key of Boev (1975).

Legs

n=3
Specimens belonging to the subgenus Pulmostrongylus
(Protostrongylus sp.1) and found in France corresponds with
appendages in head
P. (Pul.) pulmonalis (Frölich, 1802) for morphological criteria
(Table 2). They differ from P. kamenskyi by the length of their
Number of

[3–10] spicules (<200 μm) and from P. boughtoni by their legs of

gubernaculum comprised between 32 to 68 μm in length.

n=12
[4–7]

[6–8]
n=3
x=6 Specimens belonging to subgenus Protostrongylus

2
2
3

3
3
(Protostrongylus sp.2) were identified as P. (P.) oryctolagi
Number of edges

Baboš 1955 according to the princeps description of Baboš

(1955) and integrated in the key proposed by Boev (1975).
of crura

Our specimens differ from P. cuniculorum and P. terminalis

n=12
[3–6]

[4–6]
x=5
by the head of gubernaculum where no ears are present, three

5
Gubernaculum

appendages can be observed (Fig. 1h), and legs of

Total length

gubernaculum more than 75 μm long. In addition, they differ

[216–288]
[300–400]

[800–940]

[153–200]
[170–260]

[230–300]
[75–180]

from P. tauricus by the length of spicules, less than 400 μm

n=110
x=850

x=210
(μm)

n=3

long (Table 2). We therefore consider our specimens belong-

ing to P. (P.) oryctolagi Baboš 1955, even if this species had
[538–560]

[170–190]

[250–380]

[280–310]
[360–468]
[260–305]
[175–290]
[275–318]

never been recognized in Lagomorphs since the Baboš’

[90–198]
Spicules

n=120
x=180

x=330

description.
(μm)

n=3

Sequencing and molecular analysis

Esophagus

[464–481]
[210–660]

[276–330]
[255–350]

[288–342]
[350–480]

[230–280]
x=420

x=260
n=15
(μm)

Concerning sequences of Protostrongylinae deposited in

n=4

390

GenBank, seven species of Protostrongylus are available for

D2 domain of rDNA. Among these sequences, four corre-
[154–176]
[40–189]

[90–130]
[62–114]

spond to parasites of wild ruminants (P. rupicaprae,

x=120
Width

n=13
(μm)

232

310
216

P. rufescens, P. stilesi, and P. rushi) and two correspond to

lungworms of Lagomorphs (P. pulmonalis and P. boughtoni).
[21–26.5]

We also added the sequence of Orthostrongylus macrotis to

[6–23.5]
[43–47]
[16–45]

[23–28]

[35–45]
[35–54]
Length

n=11

our analysis. If the classification of Boev (1975) grouped this

x=24
(mm)

genus with Neostrongylus in the Neostrongylinae, morpho-

logical and molecular analyzes confirmed the membership of
Oryctolagus cuniculus

Oryctolagus cuniculus

this species to the Protostrongylus genus, as described by

Lepus americanus

Lepus europaeus
Lepus europaeus

Lepus europaeus

Dikmans in 1931 (Carreno and Hoberg 1999; Asmundsson

Lepus timidus

et al. unpublished).
Lepus sp.

Lepus sp.

The molecular analysis of the D2 domain confirmed that

Hosts

worms isolated from lungs of hares and rabbit in France

belonged to the Protostrongylus genus (Fig. 2). Moreover,
our analyses clearly showed that DNA sequences are separat-
P (Pul.) pulmonalis
P (Pul.) pulmonalis
P (Pul.) kamenskyi

P (P.) cuniculorum
P (Pul.) boughtoni

ed into two distinct clades, corresponding to the species sep-

P (P.) oryctolagi
P (P.) oryctolagi

P (P.) tauricus

arated on morphological criteria: Protostrongylus sp.1, corre-

(this study)

(this study)

sponding to haplotypes of adults from Jura (LIE1), from

Species

Hérault (KEC2, KEC3, KEC6), from Aisne (SEU3, SEU4)

and larvae from Savoie (SEU47, SEU49); and
Protostrongylus sp.2. corresponding to adults from Charente
Edges of crura: smooth

(SEU5, SEU6, SEU7), from Gard (KEC8, KEC9, KEC10,

Edges of crura: with

KEC11, SEU60), from Hérault (KEC4, KEC7), from Ardèche

Pulmostrongylus

Protostrongylus
Shape of crura

(SEU61), from Tarn (SEU18), from Vaucluse (SEU10), and

tubercles
Subgenus

from Bouches du Rhône (SEU8, SEU9) (Fig. 2, Table 1).

All sequences of Protostrongylus sp.1 are 100 % homolo-
gous with the haplotype of P. pulmonalis (EU595590), and
2108 Parasitol Res (2014) 113:2103–2111

Fig. 2 Phylogenetic tree based KEC9

on the D2 domain of rDNA of KEC10
Protostrongylinae constructed KEC8
using the neighbor-joining KEC7
method (Tamura-Nei model). KEC4
Sequences of Muelleriinae 98/78/98 SEU61
(Cystocaulus ocreatus and Protostrongylus oryctolagi
SEU60
Muellerius capillaris) were set as
outgroup. The scale shows the SEU9
number of nucleotide 63/66/60 SEU8
substitutions per site between SEU7
DNA sequences. The node SEU6
support is given in neighbor- KEC11
joining, maximum likelihood, and Protostrongylus rupicaprae (EU595601)
minimum evolution bootstraps
Orthostrongylus macrotis (EU595592)
Protostrongylus rufescens (EU595600)

99/99/99 Protostrongylus stilesi (EU595599)

Protostrongylus rushi (EU595598)
Protostrongylus boughtoni (EU595595)

76/57/78 EU595590
LIE1
84/79/83 KEC2
KEC3 Protostrongylus pulmonalis
KEC6
63/54/67
SEU3
SEU4
SEU47
SEU49
Muellerius capillaris (AY292798)
Cystocaulus ocreatus (EU595593)

0.02

differ from other species of the subgenus Pulmostrongylus ITS2 was also used to confirm the membership of both
available in GenBank (P. rupicaprae and P. boughtoni) haplotypes (Protostrongylus sp.1 and Protostrongylus sp.2) to
(Fig. 3), confirming their membership to this species, and thus two distinct species by estimating pairwise distance between
supporting the identification made previously on morpholog- specimens. Results are congruent with those previously ob-
ical criteria. tained by analyzing of the D2 domain and on morphological
Based on morphological criteria, specimens of our study features.
belonging to the subgenus Protostrongylus (Protostrongylus
sp.2) were identified as P (P.) oryctolagi Baboš 1955 Parasitic prevalence
Sequences of these specimens are 100 % homologous and
differ from other sequences of the subgenus Protostrongylus P. pulmonalis was detected in five hares found dead between
available in GenBank (P. rufescens and P. ruschi) (Fig. 3). No 2009 and 2013: two hares in Aisne, one hare in each following
sequences of P. oryctolagi are actually available in GenBank departments: Savoie, Jura, and Hérault (Fig. 4). P. oryctolagi
and it’s the first molecular description of this species. has been observed mainly in South of France between 2009
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2
3 6 6 7 8 9 9 9 0 0 0 1 1 2 3 4 4 5 5 6 6 8 9 9 0 1
0 0 4 5 5 1 2 7 3 5 8 8 9 3 3 2 7 4 5 7 8 9 1 7 0 9

P. oryctolagi G G A A T A A A A C G T G C T C G A T T T C G A A G
P. rupicaprae A T C T C A
O. macrotis A T G A A T G A T
P. rufescens G A T A C A T G A
P. stilesi A R A T G A T A
P. rushi G T A T A C T G A
P. boughtoni G G G A T A T G A
P. pulmonalis G G A T A A T G A

Fig. 3 Sequence variations observed in the D2 domain of the ribosomal DNA of Protostrongylinae. Identical bases are represented by a dot (.).
Nucleotide nomenclature: R=A or G; W=A or T. Only variable sites, with sequence positions given above, are shown
Parasitol Res (2014) 113:2103–2111
Fig. 4 Geographical distribution
of two species of Protostrongylus
isolated in Lagomorphs in France.
P (Pul.) pulmonalis was isolated
in little gray areas, P (P.)
oryctolagi in dark gray areas and
both species in the striped area.
No samples were taken in white
areas. In the figure, abbreviations
correspond to French department
names where parasite samples
were collected. A Aisne, Ar
Ardèche, Br Bouches du Rhône,
Ch Charente, Gd Gard, Hr
Hérault, Jr Jura, Sv Savoie, Tr
Tarn, Vc Vaucluse
and 2012 on 248 hares and three rabbits: 45 hunted hares (45/
100) and one found dead in Ardèche, 21 hunted hares in Gard
(21/85), two rabbits found dead and 111 hunted hares (111/
213) in Tarn, 19 hunted hares in Vaucluse (19/36), one hare
and one rabbit found dead in Bouches du Rhône, two hares
found dead in Charente, 48 hunted hares (48/120) in Hérault.
One hare found dead in Hérault was coinfected by the two
species: P. pulmonalis and P. oryctolagi.
Discussion
In different countries, pulmonary helminthiasis is frequently
encountered and occasionally implicated in the cyclic decline
of hares’ populations (Bull 1964; Chroust et al. 2012). In
France, only two studies were conducted on Lagomorphs in
order to isolate and identify species of lungworms responsible
for the disease; the first one in 1890 by Raillet who incriminate
P. commutatus (synonym of P. pulmonalis), and the second
one by Joyeux and Gaud in 1946, who isolated P. cuniculorum
from hare and wild rabbits in South East of the country. Our
investigation has been to identify the causative agents of
pulmonary protostrongyliasis and conducted in the framework
2109
to estimate their potential role in the death of many hares in
Southern France. Before, Protostrongylus tauricus and
P. cuniculorum were considered as the causative agents of
the disease in France and in neighboring countries, especially
in Spain (Casanova et al. 1999; Joyeux and Gaud 1946).
According to morphological criteria defined by Boev (1975)
and used by Panayotova-Pencheva to describe species isolated
from ruminants (Panayotova-Pencheva 2011), these species
are very closed to P. oryctolagi, and diagnosis only based on
male may not be sufficient, particularly when there are few
individuals to take into account the measurements of spicules
and gubernaculum considered as necessary for the specific
identification. In 1999, Casanova isolated only four males of
P. tauricus from lungs of Lepus europaeus. Confusion be-
tween species based on morphological features is also
possible.
In this study, by morphological and molecular approaches,
we thus confirm the presence of two species: P. (Pul.)
pulmonalis and P. (P.) oryctolagi as causative agents of
protostrongyliasis in Lagomorphs in France. In view of the
results obtained and in previous studies, the geographical
location of these species seems to correspond to the following
distribution: species belonging to the Pulmostrongylus
2110
subgenus are preferentially localized in septentrional and
montain areas (e.g. P. (Pul.) pulmonalis in Northern France
and in Scandinavia (Laakkonen et al. 2006)); the species
belonging to the Protostrongylus subgenus in Southern area
(e.g. P. (P.) cuniculorum P. (P.) tauricus and P. (P.) oryctolagi
isolated in Southern France, Italy, and Spain (Battisti et al.
2000; Casanova et al. 1999)). However, we isolated once
P. (Pul) pulmonalis in the South of France. It was a case of
co-parasitism with P. (P.) oryctolagi and this event, described
here for the first time in France, was never repeated thereafter.
The first description of co-parasitism for Protostrongylinae
was reported by Schulz in 1930 and concern two species of
the subgenus Pulmostrongylus : P. pulmonalis and
P. kamenskyi. Several hypotheses could explain the presence
of P. pulmonalis in South of France: the first hypothesis is that
P. pulmonalis was endemic in Southern France and, with the
introduction of the species P. (P). oryctolagi, this species have
gradually disappeared by a phenomenon of competition. This
assumption is not likely because of the presence in the past of
another species of the subgenus Protostrongylus,
P. cuniculorum. The second hypothesis would be an introduc-
tion of hare populations parasited by P. pulmonalis from
North. The low prevalence observed could be explained by a
phenomenon of species selection in intermediate and defini-
tive host. These hypotheses remain to be confirmed by further
works of parasites in Lagomorphs, but also by a research and
parasitological examination of intermediate hosts
(Gateropods) picked up in the fields.
In the future, regarding to the ambiguous morphological
features of protostrongylids nematodes in Lagomorphs, it
seems that a molecular approach is necessary to complete
the morphological identification, including those based on
female worms in order to obtain strong and comparable
contributions.
Acknowledgments Financial support was forthcoming in the form of a
PhD grant from the French Game and Wildlife Agency (Office National
de la Chasse et de la Faune Sauvage, ONCFS). The authors would like to
thank the members of the network SAGIR: the staff of hunters, hunting
Federations, environmental officers of ONCFS, and the local administra-
tive laboratories of veterinary analyses of Ardèche, Gard, Herault, Tarn,
Lozère, Vaucluse, Jura, Aisne, Charente, Bouches du Rhône, and Karin
Lemberger for their technical assistance. We are grateful to Scott Harris
for having kindly reviewed the English.
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