Angiostrongylus chabaudi Biocca, 1957: a new parasite for domestic cats?

Background Natural infection with a species of Angiostrongylus has been reported only once in wildcats from central Italy by Biocca in 1957. The causative species of this infection was identified as Angiostrongylus chabaudi. Following this report, this parasite had never been found in either wild or domestic cats. Findings The lungs and the pulmonary arteries of an adult female cat (Felis silvestris catus), road-killed in Sardinia, Italy, were macroscopically examined and dissected under a light microscope for the presence of parasites. A slender nematode was detected and its morphometrical features were consistent with those of A. chabaudi. Morphological data were supplemented by sequencing of the partial cytochrome oxidase c subunit 1 (cox1) gene, as well as the internal transcribed spacer 2 (ITS2) of the rDNA. Nucleotide sequences displayed 99% homology with the ITS2 sequence [GenBank KM216825.1] of a specimen of Angiostrongylus sp. recovered recently from the pulmonary artery of a wildcat in Germany and 91% with cox1 sequence [GenBank GU138118.1] of Angiostrongylus vasorum. Conclusion The results of the present study indicate, for the first time, that A. chabaudi may also infect domestic cats, and thus should be considered in the diagnosis of metastrongyloid species infecting their cardio-pulmonary system.


Findings
Nematodes affecting the cardio-pulmonary system of pets have recently attracted the interest of researchers due to their increasing distribution in several European countries [1]. This is the case of Aelurostrongylus abstrusus and Eucoleus aerophilus (syn. Capillaria aerophila) in cats and of Angiostrongylus vasorum in dogs [2]. Recent reports of infections caused by other members of the superfamily Metastrongyloidea in domestic cats (e.g., Oslerus rostratus, Troglostrongylus brevior and Troglostrongylus subcrenatus) have further stimulated the interest of the scientific community on these little known species [3][4][5][6][7]. In addition, cats may act as permissive hosts of A. vasorum, although the first-stage larvae of this nematode are not shed in the faeces of experimentally infected animals [8,9]. A natural infection with a species of Angiostrongylus has been reported only once in wildcats from central Italy by Biocca in 1957, who described and named the causative species as Angiostrongylus chabaudi [10]. Following this report, the species has never been found in either wild or domestic cats. Fifty-seven years after its description, we report the presence of A. chabaudi in a domestic cat from Sardinia together with the first molecular characterization of this little known angiostrongylid infecting cats.
The material originated from an adult female cat, road-killed in the municipality of Villacidro, province of Cagliari, Sardinia, Italy (39°27'33"N, 8°44'02E"). Upon inspection of the pulmonary arteries, a slender nematode was retrieved. This parasite was washed in saline solution and subsequently mounted on a slide with glycerol, for microscopical observation. Light microscopy images and measurements were taken using a digital image processing system (Olympus BX41; Soft Imaging solution GMBH LG20, Munster, Germany). The parasite was identified to the species level using morphological keys [10,11]. A faecal sample was also collected from the rectum of the cat and processed by Baermann method for the detection of first-stage larvae of broncho-pulmonary nematodes [7]. The genetic identity of the cat (i.e., Felis silvestris silvestris, Felis silvestris lybica, Felis silvestris catus or hybrid) was assessed molecularly on a muscle sample as described elsewhere [12].
Following parasite identification, DNA was extracted from the mid-body of the nematode using a commercial kit (High Pure PCR Template Preparation kit, Roche Diagnostics, Mannheim, Germany). Partial mithocondrial cytochrome c oxidase subunit 1 (cox1) gene and the internal transcribed spacer 2 (ITS2) of the rRNA gene were amplified, as previously described [13,14]. PCR amplicons were purified using an Exo SAP-IT kit (Amersham Biosciences) and sequenced through an external service (MWG Eurofins), using the same primers as for the PCR. Nucleotide sequences were compared with those available in GenBank® using the basic local alignment search tool (BLAST) analysis (National Centre for Biotechnology Information, ncbi.nlm.nih.gov). In order to investigate the relationships among metastrongyloids affecting wild and domestic carnivores, sequences of cox1 and ITS2 were analysed with those available in GenBank™. The evolutionary history was inferred for ITS2, using the maximum parsimony method using the Subtree-Pruning-Regrafting (SPR) algorithm with the software MEGA 6 [15]. The bootstrap consensus trees inferred from over 8,000 replicates were taken to represent the evolutionary history of the taxa analysed. The ITS2 sequence of Nematodirus battus was used as an outgroup.
The parasite specimen was a female with a slender and elongated body (16.9 mm long and 195.8 μm wide). The anterior extremity was attenuated, whilst the posterior was rounded and curved forward ( Figure 1). The cuticle was longitudinally striated and slightly dilated in the anterior end ( Figure 1B). The buccal aperture, small and circular, was situated in the terminal position, surrounded by six perityls (Figure 2A). Two pairs of sensorial papillae (two ventral and two dorsal), each represented by two symmetrical conical protuberances, were noted posteriorly to the perityls ( Figure 2A). The oesophagus was 276.6 μm long and clavated. The excretory pore opened just behind the oesophageal-intestinal junction, at 361.5 μm from the apical end. The vulva was sub-terminal and the anus was posterior to the vulvar aperture at 160.6 μm and at 43.4 μm, respectively, from the caudal end ( Figures 1C and 2B). Eggs (mean length 49.6 ± 4.2 μm; mean width 33.4 ± 0.9 μm) were seen inside the terminal tract of the uterus and in the ovijector ( Figure 1C). The morphometrical features of the above parasite were consistent with those of A. chabaudi (Table 1) reported in the original description [10]. At the Baermann examination, only first-stage larvae of Troglostrongylus brevior were found and molecularly confirmed (data not shown). According to the molecular identification of the genotype, the cat was identified as pure domestic cat (F. silvestris catus).
The BLASTn of cox1 sequences herein obtained displayed a nucleotide homology of 91% with A. vasorum [GenBank GU138118.1], 89% with Angiostrongylus cantonensis [GenBank GU138111.1] 88% with A. abstrusus    1]. Analysis of the cox1 sequences clustered A. chabaudi in the clade including A. vasorum but this was not optimal due to the small number of sequences available (data not shown). Conversely, ITS2 phylogeny clearly indicated that A. chabaudi clustered within the clade of all other Angiostrongylus species available in public databases (Figure 3).
Fifty-seven years after its original description [10], A. chabaudi is reported here for the first time in a domestic cat from Sardinia. In addition, the present study represents the first report, ever, of an angiostrongylid of the genus Angiostrongylus naturally infecting domestic cat. The recovery of this nematode is of importance as it suggests that cats may also be infected by metastrongyloids localizing in the cardio-vascular system. Morphological features and size of the parasite found in the present survey are similar to those reported in the original description of A. chabaudi [10] (Table 1). Although eggs were observed in the uterus of the specimen herein studied, the absence of specific first-stage larvae at the Baermann test indicates that the parasite was an immature or unfertilized female, thereby explaining its smaller size compared to that previously recorded [10]. The morphometry of the parasite here described differs from that of A. vasorum in dogs and wildlife hosts (Table 1) [16], in that four sensorial papillae are present in the anterior region posteriorly to perityls, and the distance between the vulvar aperture and the caudal extremity (i.e., 160.6 μm) is significantly shorter than in other species of Angiostrongylus but similar to that of A. chabaudi (170-210 μm) ( Table 1). These morphological differences were also clearly supported by the nucleotide homology with sequences from other species (i.e., up to 91% for cox1 Figure 3 Phylogenetic tree based on ITS2 sequences for different species of metastrongyloid nematodes retrieved from GenBank. The evolutionary history was inferred using the Maximum Parsimony method. The tree was obtained using the Subtree-Pruning-Regrafting (SPR) algorithm. sequences with that of A. vasorum). The phylogenetic analyses, based on ITS2, supported their morphological identification, in that sequences derived from this nematode were clustered within those belonging to the genus Angiostrongylus.
In the original description, A. chabaudi was considered typical for wildcats, having been found in 85% of a sample from central Italy, but not in stray cats and dogs or in other wildlife species (i.e., Meles meles and Vulpes vulpes) from the same area [10].
Here A. chabaudi is reported for the first time in a domestic cat. No data are available on the presence of this nematode in wildcats from Sardinia, which belong to another subspecies (F. silvestris lybica) than that found in continental Italy [17]. Therefore, at least for Sardinia, it is not yet possible to infer that wildcats may be regarded as wild reservoirs of A. chabaudi. On the other hand, according to Biocca's study [10], wildcats may be highly infected by A. chabaudi. In addition, the ITS2 sequence of A. chabaudi herein characterized displayed a very high nucleotide homology (99%) with that of Angiostrongylus sp. collected from a wildcat in Germany. Also, both nematodes above were collected from the pulmonary artery, which is the same site of collection of A. chabaudi as described by Biocca [10].
The data presented here suggest that a careful inspection of the vascular system of lungs should be undertaken during necropsies of domestic and wild cats. Also, firststage larvae of A. chabaudi have not been described so far [10] and thus their morphological delineation from those of other metastrongyloids affecting felids warrants investigation. Additional studies should elucidate the life history of A. chabaudi, determine its distribution and impact of this little known parasite on the health of wild and domestic cats.