Troglostrongylus brevior and Troglostrongylus subcrenatus (Strongylida: Crenosomatidae) as agents of broncho-pulmonary infestation in domestic cats
© Brianti et al.; licensee BioMed Central Ltd. 2012
Received: 25 June 2012
Accepted: 12 August 2012
Published: 23 August 2012
Aelurostrongylus abstrusus is currently regarded as the main metastrongyloid infesting domestic cats, whereas the reports of Troglostrongylus spp. in domestic and wild felids largely remain anecdotic. This paper reports on pulmonary infestation caused by Troglostrongylus brevior and Troglostrongylus subcrenatus in two kittens and describes, for the first time, associated clinical presentations and pathological features. Morphometrical, molecular and phylogenetic analyses have also been conducted to differentiate here the examined Troglostrongylus species from A. abstrusus, towards a clearer delineation of metastrongyloids affecting cats.
Two kittens were referred for respiratory distress and hospitalized with a diagnosis of severe aelurostrongylosis, based on the presence of metastrongyloid larvae in the faeces. Despite prompt treatment, kittens died within 48 hours. Both kittens were submitted to necropsy to determine the cause of death.
At necropsy, nematode specimens were found in the trachea, bronchi and bronchioles and were associated with respiratory signs (i.e., dyspnoea, polypnea, severe coughing and nasal discharge). Morphology and measurements of adult parasites found allowed the unequivocal identification of T. brevior and T. subcrenatus, even if first stage larvae were rather similar to those of A. abstrusus. Briefly, T. brevior and T. subcrenatus larvae were shorter in length and lacking the typical knob-like terminal end of A. abstrusus. Molecular and phylogenetic analyses corroborated morphological identification and provided data on mitochondrial and ribosomal DNA genes of T. brevior.
Data presented here indicate that T. brevior and T. subcrenatus may cause major respiratory distress in domestic cats. Consequently, these two species should be included, along with A. abstrusus, in the differential diagnosis of cat bronchopulmonary affections and treatment protocols need to be evaluated. Through research on the biology, epidemiology and control of Troglostrongylus spp. infestations in domestic cats are advisable to implement current knowledge on these neglected metastrongyloids.
KeywordsAelurostrongylus abstrusus Cat Diagnosis Italy Metastrongyloidea Molecular biology Troglostrongylus brevior Troglostrongylus subcrenatus
Nematode infestations of the cardio-pulmonary system of dogs and cats have recently gained thescientific interest of researchers and practitioners, due to the clinical severity of the conditions they cause and to their spread throughout many Europeancountries [1–3]. Among them, Aelurostrongylus abstrusus (Strongylida: Angiostrongylidae) is commonly regarded as the only metastrongyloid of domestic cats [4, 5]. Broncho-pulmonary infestations by A. abstrusus are widespread throughout most European countries with prevalence rates ranging from 1% to 24.4%, according to sampled populations and detection procedures (e.g., copromicroscopy or molecular detection) [2, 6–12]. Actually, other metastrongyloids have been reported as causative agents of respiratory infestation in domestic cats. For instance, Oslerus rostratus (Strongylida: Filaroididae) has been recorded to infest the pulmonary system of cats with prevalence rates up to 24% in Spain [13, 14]. Meanwhile, Troglostrongylus subcrenatus (Strongylida: Crenosomatidae) was retrieved once at the necropsy of a domestic cat from Blantyre (Nyasaland, Malawi)  and, larvae of a yet unclassified Troglostrongylus sp. have recently been identified in faeces of domestic cats from Ibiza (Spain), which exhibited respiratory disease .
The genus Troglostrongylus encompasses four nematode species (i.e., T. brevior T. subcrenatus T. troglostrongylus and T. wilsoni), which are commonly found in the trachea and bronchi of wild felids [17–21]. Albeit scantly studied, Troglostrongylus spp. display an indirect life cycle similar to that of A. abstrusus , with terrestrial molluscs serving as intermediate hosts and small mammals (e.g., mice) as paratenic ones [4, 22, 23]. Larvae of T. brevior may develop in several species of molluscs (i.e., Helicella barbesiana Helicella vestalis Limax flavus Monacha syriaca Retinella nitellina and Theba pisana ), becoming infective from 8 to 40 days depending on the environmental temperature (i.e., between 22°-27°C and between 4°-8°C, respectively) . Nonetheless, the scant scientific information available on the distribution and pathogenicity of Troglostrongylus sp. in domestic cats  might indicate that this infestation occurs sporadically or that it is misdiagnosed with the commonest A. abstrusus.
This paper reports on pulmonary infestation by T. brevior and T. subcrenatus in two kittens and describes, for the first time, associated clinical presentations and pathological features. Additionally, morphometric, molecular and phylogenetic analyses have also been conducted to differentiate the Troglostrongylus species here examined from A. abstrusus towards a clearer delineation of metastrongyloids affecting cats.
A 3-month-old female privately owned kitten (case 1), and a 3-month-old female stray kitten (case 2) were referred to the Faculty of Veterinary Medicine of Messina (Sicily region, Italy) on April 2010 and June 2011. Both animals exhibited respiratory signs such as dyspnoea, polypnea, severe coughing and nasal discharge (see Additional file 1 and Additional file 2). In addition, case 1 had developed inappetence and lethargy in the 48 h prior to referral while case 2 was dehydrated and in poor general condition at the moment of the clinical examination. Thoracic radiographs, performed only for case 2, showed diffuse increase of radiograph density and marked bronchial pattern in diaphragmatic lobes. Leucocytosis (i.e., 26.3 × 103/μl in case 1, and 20.9 × 103/μl in case 2) was diagnosed while biochemistry was within normal limits in both cases. Toxocara cati eggs were detected at the copromicroscopy using the flotation method in case 1 and of nematode larvae, resembling those of A. abstrusus, were retrieved by the Baermann technique in both cases. Kittens were both hospitalized with a diagnosis of severe aelurostrongylosis and antiparasitic drugs (imidacloprid 10 mg/kg/moxidectin 1 mg/kg spot-on in case 1, and fenbendazole 50 mg/kg PO once every 24 h in case 2) were administered. Both kittens also underwent systemic therapy with intravenous fluids, antibiotics (amoxicillin 20 mg/kg IV twice every 24 h and enrofloxacin 5 mg/kg IV once every 24 h) and placed in an oxygen chamber but they did not show any improvement with exitus within 24-48 h after hospitalization. Animals were not enrolled in any experimental trial. Kittens were hospitalized and keep according to Animal Welfare and Good Clinical Practice (VICH GL9-GCP, 2 "Good Clinical Practice", CVMP, June 2000) guidelines. In both cases necropsy was requested and authorized by the owners.
Pathological findings and parasite identification
Carcasses of both kittens were subjected to necropsy. Parasitic specimens collected from lungs were washed in saline solution and stored in 70% ethanol (case 1) and in formalin (case 2). Worms were individually mounted on slides by the glycerol-ethanol method and microscopically observed. Microscopic images and measures were taken by using a digital image processing system (AxioVision rel. 4.8, Carl Zeiss, Germany). Parasites were sexed and identified at the species level by morphometrical and morphological keys [15, 17, 23]. Slide-mounted specimens of both sexes were stored in the parasite collection of the Faculty of Veterinary Medicine of Messina. In addition, gross anatomical findings, anatomical localization of parasites, and their morphometric features were also compared with those from A. abstrusus from a road-killed stray cat (E.B. unpublished observations).
Molecular procedures and analyses
The molecular identification was performed by extracting genomic DNA from two parasite specimens of case 1 using a commercial kit (DNeasy Blood & Tissue Kit, Qiagen, GmbH, Hilden, Germany) in accordance with the manufacturer’s instructions. Unfortunately, all attempts to extract genomic DNA from nematodes retrieved from case 2 failed, probably because the specimens were kept in formalin.
GenBank TM accession numbers (AN) of the Metastrongyloidea used in the phylogenetic analyses for 18S rRNA and the internal transcribed spacer 2 (ITS2) genes
Neovison vison (=Mustela vison)
Felis silvestris catus
Felis silvestris catus
Odocoileus hemionus hemionus
Sequences were determined from both strands (using the same primers individually as for the PCR) and the electropherograms verified by eye. In order to ensure open reading frames, the nucleotide sequence of pcox 1 was conceptually translated into amino acid sequence using the invertebrate mitochondrial code MEGA5 software . Sequences for all genes were compared with those available in the GenBank™ database by Basic Local Alignment Search Tool (BLAST) . In order to investigate the relationships among metastrongyloids affecting wild and domestic carnivores, sequences of 18S and ITS2 were analysed with those available in GenBankTM (Table 1) and the evolutionary history was inferred using the neighbour-joining method . The bootstrap consensus trees inferred from over 7,000 replicates were taken to represent the evolutionary history of the taxa analysed . Branches corresponding to partitions reproduced in less than 50% bootstrap were collapsed and the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test was shown next to the branches . Phylogenetic analyses were conducted in MEGA5 software . Sequences of Nippostrongylus brasiliensis (Strongylida: Heligmonellidae) and Nematodirus battus (Strongylida: Molineidae) were used as outgroups. The nucleotide sequences analyzed in this paper are available in the GenBankTM database under accession numbers: JX290562 (18S); JX290563 (cox 1); JX290564 (ITS2).
Anatomical localization and morphometrical features of adult stages of Aelurostrongylus abstrusus , Troglostrongylus brevior and Troglostrongylus subcrenatus
Tail (anus-caudal end)
Aelurostrongylus abstrusus (Gerichter, 1949)
Bronchi and bronchioles
Male (n = 3)
Female (n = 6)
Trachea and bronchi
Male (n = 7)
Female (n = 15)
Mean measurements (microns) of first stage larvae (L1) of Aelurostrongylus abstrusus , Troglostrongylus brevior and Troglostrongylus subcrenatus
Species (number of specimens)
First stage larvae (L1)
Aelurostrongylus abstrusus (n = 40)
399.1 ( ± 11.3)
18.5 ( ± 1.2)
Troglostrongylus brevior (n = 40)
339.3 ( ± 14.1)
18.6 ( ± 1)
Troglostrongylus subcrenatus (n = 10)
280.7 ( ± 17.9)
15.5 ( ± 1.7)
Parasites collected from case 2 were stout and filiform nematodes of medium size with a cuticle conspicuously inflated at the anterior end. The oesophagus was club-shaped. The excretory pore opening coincided with the ventral cuticular groove at about the middle of the oesophagus length (Figure 3B). A total of 15 females and seven males were identified. Females (20–24 mm in length and 486–542 μm in width) presented a conical and bluntly pointed tail (Figure 3D; Table 2) with the vulva opening posterior to the middle of body and the anus at 178–189 μm from the caudal end (Figure 3D). The uteri were filled with larvated eggs. Males (9–10 mm in length and 285–305 μm in width) presented an undivided bursa composed of a single dorsal stout ray, bearing papillae at the tip and externo-dorsal rays originating independently. Postero- and medio-lateral rays were fused over half or more of the length, antero-lateral rays were single and ventral rays fused for almost their extension (Figure 3F). The spicules (613–686 μm in length) were equal in size and shape, slender and finely striated except for their distal edges. Each spicule was split into two spikes (Figure 3H). First stage larvae (280.7 μm in length and 15.5 μm in width) had a rhabditoid oesophagus and pointed tail bearing typical dorsal and ventral cuticular spines (Figure 4B; Table 3). Morphology and measurements of adult and larval nematodes collected from case 2 were all consistent with those of T. subcrenatus[15, 31].
Morphological and morphometrical features of adult parasites collected from cases 1 and 2 are reported in Table 2 and compared to those of A. abstrusus. In spite of the differences in anatomical localization and body size of adult parasites, first stage larvae of A. abstrusus, T. brevior and T. subcrenatus are rather similar, except for their slightly differing body lengths and the knob-like terminal end of A. abstrusus (see Figure 4 and Table 3).
PCR amplification of each target region characterized for nematodes from case 1 resulted in amplicons ranging from ~400 bp (pcox 1) to ~1700 bp (18S) and ~630 bp (ITS2). The BLAST analysis of pcox 1 gene sequences showed the closest homology (91%) with that of Necator americanus (GenBank accession number: AF303151) whereas ITS2 revealed a high homology (99%, fragment of 89 bp) when compared with Angiostrongylus cantonensis and Angiostrongylus vasorum (Strongylida: Angiostrongylidae) (GenBank accession numbers: HQ540551 and GU045376, respectively). The 18S sequence here produced was identical (homology of 100%) to Troglostrongylus sp. (GenBank accession number: GU946677).
This paper is the first of all reports on clinical signs and gross lesions associated with T. brevior and T. subcrenatus infestation in two domestic kittens. The delineation between these two species of metastrongyloids and the most known A. abstrusus is pivotal for the aetiological diagnosis of helminthic broncho-pulmonary disease of cats. These two troglostrongylids have been neglected for a long time or underestimated and deserve further investigations to assess their actual geographical distribution and clinical significance, especially in young cats. Indeed, Troglostrongylus spp. have been reported as causative agents of respiratory conditions in many species of wild felids (e.g., Lynx rufus, Panthera pardus Panthera tigris) [17–21], but their occurrence in domestic cats was considered negligible . Recently, a Troglostrongylus sp. detected in domestic cats exhibiting respiratory signs renewed the scientific interest on this genus . The 18S sequences herein produced for T. brevior and those derived from the parasitic specimens recently identified  provided an a posteriori aetiological identification for the causative agents of the cases above from Spain. This evidence might suggest that Troglostrongylus spp. are more diffuse than currently estimated and that the limited number of records of T. brevior and T. subcrenatus infesting domestic cats could be due to the close morphological features shared among their L1s (i.e., the parasitic stage mostly used for the diagnosis) with those of A. abstrusus. On the other hand, it cannot be ruled out that troglostrongylosis in domestic cats is limited to areas where cats are more frequently exposed to intermediate and/or paratenic hosts. Furthermore, molluscs of genera (i.e., Helicella spp. Limax spp. and Theba spp.) are common in the locality where both cats reported in this paper came from .
Accordingly, almost all data available on lungworms affecting cats are based on copromicroscopical findings [6–12], which might lead to a misdiagnosis unless a thorough morphological and morphometric analysis is undertaken. Even if not easily achievable, an accurate evaluation of morphometrical features (i.e., total length and tail morphology) of L1s may lead to a discrimination among T. brevior T. subcrenatus and A. abstrusus, being larvae of Troglostrongylus spp. shorter in length and devoid of the typical knob-like terminal end (see Figure 4 and Table 3).
Molecular and phylogenetic analyses presented here provide, for the first time, data on mitochondrial and ribosomal gene sequences of T. brevior, giving a dataset for its identification. Based on the analysis of the 18S gene, the aetiological cause of infestation in cats from Ibiza was T. brevior. The phylogenetic analysis of 18S further confirms the molecular analysis, clustering T. brevior within other Troglostrongylus spp. infecting domestic carnivores. Nonetheless, further studies should be undertaken to differentiate T. brevior and T. subcrenatus molecularly in order to better understand their pathogenicity, biology and bionomics (including the role of different species of molluscs as intermediate hosts) and infer their potential spread throughout Europe. A molecular characterization of these two species might contribute to assessing a specific and sensitive molecular tool for the detection of both species in faeces and pharyngeal swabs, as recently validated for A. abstrusus.
The greater body size of T. brevior and T. subcrenatus in comparison with A. abstrusus and their anatomical localisation in the upper respiratory airways (i.e., bronchioles and large bronchi) also reflects differences in the presentations and severity of clinical signs they cause in cats. Indeed, both clinical cases here described exhibited severe respiratory distress and had a lethal outcome due to respiratory failure. Accordingly, most of the lung damage observed at the gross examination was associated with the presence and action of nematode specimens. This clinical picture differs from that caused by A. abstrusus, which generally induces minor signs and is reported as an often self-limiting parasite [1, 5, 34]. Nonetheless, the clinical presentation caused by Troglostrongylus spp. and A. abstrusus may overlap in simultaneously infested animals, thus making any definitive aetiological diagnosis based on respiratory signs difficult.
Troglostrongylus brevior and T. subcrenatus share similar biology with A. abstrusus, all involving mollusc intermediate hosts in their life cycle [4, 17, 22] and thus, most likely, occupying the same ecological niches and occurring simultaneously in a given host population, as confirmed by co-infestation in the same cat population from Ibiza . Due to their capacity of developing to the infective stage under different climatic conditions in many species of intermediate hosts, troglostrongylids may have a potential broader distribution than currently believed. Indeed, larvae of T. brevior show the highest resistance in the environment, developing to the infective stage at low temperatures (i.e., 4-8°C in 40 days) but not A. abstrusus, even when kept up to 7 months at the same conditions . Under optimal conditions (i.e., 22-27°C) T. brevior displays the shortest development time (i.e., 8 days) known for any metastrongyloid species , suggesting that this species may potentially express a higher parasitic pressure for feline populations than A. abstrusus.
Data presented here indicate that T. brevior and T. subcrenatus may occur in domestic cats causing overt respiratory disease. Moreover, these two species should be included, along with A. abstrusus, in the differential diagnosis of bronchopulmonary affections in cats. Although the delineation of L1s among metastrongyloids is achievable using morphometrical criteria, molecular biology tools should be considered and implemented toward enhancing current knowledge on the biology, epidemiology and control of Troglostrongylus spp. infestations in domestic cats.
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