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Parasitology ResearchFounded as Zeitschrift fürParasitenkunde ISSN 0932-0113 Parasitol ResDOI 10.1007/s00436-014-4053-z

Feline lungworm Oslerus rostratus(Strongylida: Filaridae) in Italy: first casereport and histopathological findings

Emanuele Brianti, Gabriella Gaglio,Ettore Napoli, Luigi Falsone, AlessioGiannelli, Giada Annoscia, AntonioVarcasia, et al.

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ORIGINAL PAPER

Feline lungworm Oslerus rostratus (Strongylida: Filaridae)in Italy: first case report and histopathological findings

Emanuele Brianti & Gabriella Gaglio & Ettore Napoli & Luigi Falsone & Alessio Giannelli &Giada Annoscia & Antonio Varcasia & Salvatore Giannetto & Giuseppe Mazzullo &

Domenico Otranto

Received: 22 June 2014 /Accepted: 23 July 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract Oslerus rostratus syn. Anafilaroides rostratus(Strongylida: Filaroididae) is a metastrongyloid transmittedby snails, which localizes in peri-bronchial tissues and in thelung parenchyma of wild as well as domestic cats. In Europe,this nematode has been reported only on two occasions, beingdiagnosed in cats from Majorca Island and in northern Spain.Here, we describe a case of O. rostratus infection in anecropsied 4-year-old cat in Sicily (southern Italy). At theinspection of lungs, slender and greyish nematodes (fourfemales and two males) were found embedded in the peri-bronchial tissues and in the bronchial walls. Parasites weremorphological andmolecularly identified asO. rostratus, withtheir 18S sequences being identical among them and showinga high homology (99 %) with those available in public data-bases. At the histology, nematodes were encapsulated in apseudo-cystic formation surrounded by an interstitial inflam-matory process and fibrous tissue. Lung lesions were mainlyrepresented by peri-luminal fibrosis, hyperplasia and hyper-trophy of the bronchial mucosa and glands, respectively. Thisfirst record of O. rostratus infection from Italy indicates thatthis parasite should be included in the differential diagnosis offeline of lungworm infection.

Keywords Oslerus rostratus . Cat . Italy . Gross anatomy .

Histopathology .Molecular biology

Introduction

Feline lungworms infect diverse districts of the respiratorysystem of domestic and wild felids (Brianti et al. 2014).Nematode species within this group include members of thesuperfamilyMetastrongyloidea, which are characterized by anindirect life cycle and require intermediate (i.e. snails or slugs)and paratenic (i.e. amphibians, birds, reptiles and rodents)hos t s fo r the i r t r ansmiss ion (Anderson 2000) .Aelurostrongylus abstrusus (Strongylida: Angiostrongylidae)is regarded as the main species infecting domestic cats, whileother metastrongyloids, such as Troglostrongylus brevior(Strongylida: Crenosomatidae) and Oslerus rostratus (syn.Anafilaroides rostratus) (Strongylida: Filaridae) have beenconsidered for long time of minimal importance (Anderson2000; Bowman et al. 2002; Traversa and Di Cesare 2013).Recently, cases of infection by T. brevior, alone or associatedwith A. abstrusus in domestic cats have been reported in Italy(Annoscia et al. 2014; Brianti et al. 2012; Di Cesare et al.2014; Giannelli et al. 2014; Tamponi et al. 2014) and Spain(Jefferies et al. 2010), indicating that these infections havebeen most likely misdiagnosed or overlooked in the past(Otranto et al. 2013).

As regard to O. rostratus, after its first description from acat in Jerusalem, Palestine (Gerichter 1949), it was shown tobe highly frequent in cats from Sri Lanka (Seneviratna 1955)and Hawaii (Ash 1970), as well as in bobcats (Lynx rufus)from the USA (Klewer 1958; Watson et al. 1981). In Europe,O. rostratus has been reported in feral cats from MajorcaIsland (Spain) (Millán and Casanova 2009) and, in co-infection with A. abstrusus, in a cat from northern Spain(Juste et al. 1992).

E. Brianti (*) :G. Gaglio : E. Napoli : L. Falsone : S. Giannetto :G. MazzulloDipartimento di Scienze Veterinarie, Università degli Studi diMessina, Polo Universitario Annunziata, 98168 Messina, Italye-mail: ebrianti@unime.it

A. Giannelli :G. Annoscia :D. OtrantoDipartimento di Medicina Veterinaria, Università degli Studi di Bari,Valenzano, Bari, Italy

A. VarcasiaLaboratorio di Parassitologia, Ospedale Didattico Veterinario,Dipartimento di Medicina Veterinaria, Università degli Studi diSassari, Sassari, Italy

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Although adult metastrongyloids greatly differ amongthem for their anatomical localization in the lungs, theiraetiological diagnosis through identification of first-stage lar-vae (L1s) in the faeces of the hosts is challenged by theirmorphological similarities (Brianti et al. 2014). Here, wereport the first case of infection by O. rostratus in a domesticcat from southern Italy, along with histopathological andmolecular findings.

Materials and methods

On June 2013, a 4-year-old male stray cat died for roadcausalities in the urban area of Messina (38.185481° N,15.556279° E, northern Sicily), was necropsied at theDepartment of Veterinary Sciences of the University ofMessina. During the necropsy, multiple injuries (i.e. headtrauma, fractures and abdominal haemorrhages) were ob-served, and its death was attributed to cardiovascular andnervous failure. Adult parasites, namely Dipylidium caninum,Taenia taeniaeformis and Toxocara cati, were found in theintestine (data not shown). On gross examination, lungs werewidely consolidated and oedematous (Fig. 1a), and severallong and slender nematodes were found either embedded inthe parenchyma or in the peri-bronchial tissues (Fig. 1b and c).These worms were beneath the lung tissues, being very diffi-cult to isolate. Anyhow, a total of six portions of almostcomplete parasites were collected from the apical (n=1) andcaudal lobes (n=5) and were used for the morphologicalidentification. In addition, fragments were fixed in 70 % al-cohol, until being molecular analysed using a protocol de-scribed elsewhere (Brianti et al. 2012). Samples of lung pa-renchyma (1.5 cm×1.5 cm) along with parasites were fixed in10 % buffered formalin and routinely paraffin embedded for

histological examination. Finally, the whole respiratory pack-age (i.e. trachea and lungs) was flushed with saline solutionand the sediment analysed for the presence of other lungwormspecies (Falsone et al. 2014), while faeces collected from therectum of the animal were analysed through flotation tech-nique (MAFF 1986) for parasitic eggs and larvae.

Results

The six portions of the parasites collected from the lungsbelonged to four gravid females and two mature males. Thelongest portion of female was 25 mm in length (range from 19to 25 mm), while males were from 10 to 15 mm long.Nematodes were characterized by a rounded anterior edgewith a retracted oral opening (Fig. 2a) a club-shaped oesoph-agus (from 384 to 390 μm in length and from 95 to 101 μm inwidth), consisting of a narrow anterior and a bulbous posteriorpart (Fig. 2a). The intestine was dark brown and tubular,typically running straight through the body worm or, in thefemale, coiled around the uteri. The vulva opening was in theposterior end near the anus (Fig. 2b) and fully developedlarvae surrounded by a thin shell were observed in the distalportion of the uteri and in the vagina. Newly hatched larvae(330–348 μm long and 20–22 μm wide) were featured by arounded head, a central oral opening, surrounded by a ring andcontinuing into a cylindrical buccal capsule (Fig. 2c and d).Their tail (32–35 μm in length) was undulated, bearing aventral incision deeper than the dorsal. However, the shapeof tail was not constant and slight variations were observed inits morphology.

The male edge lacked of bursa and pairs of post-analpapillae were evident (Fig. 2e), as well sub-equal spicules(109–111 μm in length and 119–121 μm the shorter and the

Fig. 1 aMacroscopic features oflungs presenting with areas ofconsolidation and irregularity ofthe surface; parasites were foundwithin the b lung parenchyma orc sub-pleural

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longer, respectively) and the gubernaculum (33 μm×14 μm)(Fig. 2e).

Morphology and measurements of the parasites aboveand of their larvae were all consistent with thoseof O. rostratus, as reported by Gerichter (1949) andSeneviratna (1959a). The morphological identificationwas also confirmed by the molecular amplificationand sequencing of a partial 18S ribosomal RNA (rRNA)gene sequence (1,708 bp). The nucleotide sequence(KM035792), examined by BLAST tool, displayed 99 %

homology with the sequence (GU946678) of O. rostratusfrom a bobcat (L. rufus) deposited in GenBank (Jefferieset al. 2010).

At the histological study, lung lesions were mainly repre-sented by interstitial peri-luminal fibrosis, hyperplasia of thebronchial mucosa and glandular hypertrophy. The presence ofthe parasites was recorded in different microscopic fields.They were mainly located in the interstitium between thefascia and the bronchial cartilage (Fig. 3a and b). The parasitesoccurred in pseudo-cystic formations (Fig. 3c) surrounded by

Fig. 2 Light microscopy of Oslerus rostratus. a Cephalic region of afemale, lateral view; note the retracted position of the oral opening(arrowhead). b Caudal region of a female, lateral view; note the terminalposition of the vulva near the anus. c, d Newly hatched first-stage larvae.

e Caudal region of a male, ventral view; note the absence of the caudalbursa and short and sub-equal spicules. Scale bars=100 μm. A anus, Eeggs, G gubernaculum, I intestine, O oesophagus, P post-anal papillae, Sspicules, V vulva aperture

Fig. 3 Histological sections(haematoxylin and eosin stain) oflung tissue parasitized by Oslerusrostratus. a, b Parasites(arrowhead) were located in theperi-bronchial tissue between thefascia and the bronchial cartilage.c, d Parasites were included in apseudo-cystic formationsurrounded by interstitialinflammation and fibrousreaction. Scale bars=100 μm. BLbronchial lumen, BM bronchialmucosa, C cartilage, F fibrousreaction, IC inflammatory cells,ML muscolaris layer, PC pseudo-cystic reaction

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an interstitial inflammatory process and characterized bypolymorphonucleated cells, as well as a fibrous tissue reaction(Fig. 3d). No other parasites species were found in the lungs orin the sediments of flush. Although fully developed larvaewere detected in the uteri and vagina of female worms, nolarvae were found in the cat faeces.

Discussion

This is the first report of O. rostratus infection in Italy.According to published records, O. rostratus occurred with alimited prevalence in Palestine, where it was found only in oneout of the seventy-three cats examined by Gerichter (1949).On the other hand, its prevalence is much larger in othergeographical areas of Asia (Sri Lanka) and Europe (MajorcaIsland), where it was reported in over 60 and 24 % of exam-ined cats, respectively (Millán and Casanova 2009;Seneviratna 1955). The life cycle of O. rostratus does notdiffer from that of A. abstrusus and T. brevior, in that it maysuccessfully develop into a wide range of mollusc species (i.e.Laevicaulis alte, Mariella dussumieri, Achatina fulica andHelix aspersa) in about 20–56 days (Seneviratna 1959b).Accordingly, O. rostratus may share both intermediate hostsand ecological niches with other metastrongyloid species.Additionally, mice and chickens serving as paratenic hosts(Seneviratna 1959b) further contribute to its dispersion andtransmission to cats.

L1s of O. rostratus are highly susceptible to extreme tem-peratures, since they die at 55 °C in 1 minute, and they cannotwithstand freezing overnight (Seneviratna 1959a). This indi-cates that the distribution of this species is likely to be limitedto semi-tropical or temperate zones. However, the reports ofO. rostratus infections in wild felids (e.g. bobcat and lynx)inhabiting continental climatic zones (i.e. northern USA)(Klewer 1958; Watson et al. 1981) did not support the hypo-thetical distribution above.

Contrarily to A. abstrusus and T. brevior which are fre-quently found in young kittens (Brianti et al. 2014), also as aneffect of the potential vertical transmission of the latter species(Brianti et al. 2013), O. rostratus infection has never beendiagnosed in cats younger than 6 months of age (Seneviratna1958), likely as an effect of the long prepatent period (about78 days) (Seneviratna 1958). Accordingly, the case here re-ported was a 4-year-old cat. Unfortunately, because of thestray condition and of the sudden death of the animal, it wasnot possible to collect data on the presence of clinical signsassociated with the infection. However, since no plugs ofmucous were detected around the nose or in the lumen oftrachea and bronchi at the necropsy, the animal had mostlikely a normal breathing, as previously observed in bothnatural and experimental infected cats (Seneviratna 1958).Since O. rostraus lives in the peri-bronchial tissues and in

the bronchial walls, the lesions it causes are different fromthose of other worms that live in the bronchial lumen (i.e.T. brevior) or in the lung parenchyma (i.e. A. abstrusus).Indeed, the principal lesions observed in the case here reportedwere mainly represented by interstitial peri-luminal fibrosis,hyperplasia of the bronchial mucosa and glandular hypertro-phy. This pathological condition is different from thatcaused by T. brevior which is featured by a catarrhalbronchitis with massive exudate that, in some cases,may obliterate the lumen of bronchi (Giannelli et al.2014). The catarrhal bronchitis is less evident even duringA. abstrusus infection, which is mainly characterized byalveolar wall thickening with a variable number of inflam-matory cells in the alveolar septa and lumina (Schnyderet al. 2014). Nonetheless, in mixed T. brevior andA. abstrusus infections, the pathologic picture may beeven more complicated by a catarrhal bronchitis and in-terstitial pneumonia (Traversa et al. 2014).

Interestingly, in the case here reported, parasites weredelimited by pseudo-cystic formations surrounded by inflam-matory cells and fibrous tissue reaction. A similar finding wasobserved in both naturally and experimentally infected cats inthe so-called “regressive stage” of O. rostratus infection, inwhich worms are encapsulated by a fibrous tissue capsulesurrounded by a great accumulation of leucocytes(Seneviratna 1958). This strong fibrous reaction may explainthe reason why any larvae were detected in the faeces of theinfected animal, even if female worms were filled with L1s inthe uterus. A similar finding was also reported in another caseof A. abstrusus and O. rostratus co-infection, in which onlyL1s of A. abstrusus where detected in the intestinal content ofthe infected cat, while those ofO. rostratuswhere only seen inthe uteri of a damaged female found embedded in the lungparenchyma (Juste et al. 1992). These findings suggest that inthe “regressive stage”, O. rostratus infection may be featuredby the absence of larvae in the faeces of affected animals,which makes more challenging its diagnosis. In addition,while L1s are 300–340 μm long soon after their release fromfemale nematodes (Gerichter 1949), those in faeces are 335–412 μm due to a distinct grow that occurs during their passagefrom trachea to intestine (Seneviratna 1959a). Therefore, mea-sures of O. rostratus L1s overlap with those of othermetastrongyloids, thus making the delineation among specieseven more challenging, unless accurate morphometrical keysor molecular biology tools are employed for a proper identi-fication. Worthy of note, this case was recorded in a stray catliving in an urbanized area where no other wild felids arepresent (e.g. wild cats), which may act as reservoirs of meta-strongyloid species (Falsone et al. 2014). This suggests thatO. rostratusmay circulate in the urban environment. Hence, itwould be advisable that the presence of other metastrongyloidspecies different from A. abstrusus should be always consid-ered in the differential diagnosis of cat lungworm affections

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and properly addressed through microscopical, molecular bi-ology and, when applicable, necroscopical methods.

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