Validity of genus Perostrongylus Schlegel, 1934 with new data on Perostrongylus falciformis (Schlegel, 1933) in European badgers, Meles meles (Linnaeus, 1758): distribution, life-cycle and pathology

Abstract

Background: A century of debates on the taxonomy of members of the Metastrongyloidea Molin, 1861 led to many reclassifications. Considering the inconstant genus assignation and lack of genetic data, the main aim of this study was to support the validity of the genus Perostrongylus Schlegel, 1934, previously considered a synonym of Aelurostrongylus Cameron, 1927, based on new molecular phylogenetic data and to understand its evolutionary relationships with other metastrongyloid nematodes.

Results: Specimens of lungworm collected from European badgers in Germany, Romania and Bosnia and Herzegovina were morphologically and molecularly (rDNA, cox1) characterized. From a phylogenetic standpoint, Perostrongylus is grouped with high support together with the genera Filaroides van Beneden, 1858 and Parafilaroides Dougherty, 1946 and includes probably two species: Perostrongylus falciformis (Schlegel, 1933), a parasite of Meles meles in Europe and P. pridhami (Anderson, 1962), a parasite of Neovison vison in North America. Perostrongylus and Aelurostrongylus are assigned to different clades. Aelurostrongylus becomes a monotypic genus, with the only species Aelurostrongylus abstrusus (Railliet, 1898). In addition, we provide morphological and morphometric data for the first-stage (L1), second-stage (L2), and third-stage (L3) larvae of P. falciformis and describe their development in experimentally infected Cornu aspersum snails. The pathological and histopathological lesions in lungs of infected European badgers are also described. This is the first record of P. falciformis in Romania.

Conclusions: Molecular phylogenetic and morphological data support the validity of the genus Perostrongylus, most probably with two species, P. falciformis in European badgers and P. pridhami in minks in North America. The two genera clearly belong to two different clades: Perostrongylus is grouped together with the genera Filaroides and Parafilaroides (both in the family Filaroididae Schulz, 1951), whereas Aelurostrongylus belongs to a clade with no sister groups.

Keywords: Aelurostrongylus; European badgers; Metastrongyloidea; Perostrongylus; mustelids.

Fig. 1

Anterior end of Perostrongylus falciformis. a Cylindrical part of the oesophagus. b Bulbous region of the oesophagus. Scale-bar: 100 μm

Fig. 2

Posterior end of female P. falciformis. a Vulva. b Anus. Scale-bar: 50 μm

Fig. 3

Larvated eggs are clearly visible inside the uterus. Scale-bar: 100 μm

Fig. 4

Free L1 larva (center) and eggs with larvae of P. falciformis. Scale-bar: 100 μm

Fig. 5

Light microscopy and schematic representation of the posterior end of the males of Perostrongylus falciformis. a Copulatory bursa, dorsal view (inset: schematic representation: a1, a2, lateral lobes; v, ventral ray; vv, ventro-ventral branch of ventral ray; vl, ventro-lateral branch of ventral ray; l, lateral ray; el, externo-lateral part of lateral ray; ml, medio-lateral part of lateral ray; pl, posterio-lateral branch of lateral ray; ed, externo-dorsal ray; md, median-dorsal ray; s, spicules; g, gubernaculum) b Copulatory bursa, lateral view. Scale-bars: 50 μm

Fig. 6

Spicules and gubernaculum of P. falciformis. a Knob-shaped anterior end of the spicule. b The bent caudal half of the spicule. c Gubernaculum. Scale-bar: 50 μm

Fig. 7

Larval stages of P. falciformis. a First-stage larva and detail of the posterior extremity (a1) and well-visible anus (arrowhead). b Second-stage larva and detail of the posterior extremity (b1) and oesophago-intestinal junction (arrow). c Third-stage larva and details of the anterior (c1) and posterior (c2) extremities and oesophago-intestinal junction (arrow). Scale-bars: 50 μm

Fig. 8

Phylogenetic relationships of P. falciformis (red) with A. abstrusus (blue) and other metastrongyloid nematodes. Phylogenetic analysis based on ITS2 and partial 28S rDNA (domains D2-D3) sequences of metastrongyloid genera. Trees are constructed using Bayesian inference. Posterior probability values are shown next to the nodes; branches with values < 0.5 are not shown. The scale-bar indicates the number of substitutions per site

Fig. 9

Phylogenetic tree (Bayesian inference) using cox1 sequences for P. falciformis (red), A. abstrusus (blue) and species of other metastrongyloid genera. Posterior probability values are shown next to the nodes; branches with values < 0.5 are not shown. The scale-bar indicates the number of substitutions per site

Fig. 10

Gross lesions produced by P. falciformis. a Adults in nodules in the subpleural space (white arrows) and the presence of alveolar emphysema (black arrow). b Adults in the pulmonary parenchyma (white arrows)

Fig. 11

Histological cross- and tangential sections of P. falciformis in the lung parenchyma. a, b. Viable P. falciformis adults (asterisks) coiled in the lung parenchyma and surrounded by a mild leukocyte reaction (many macrophages, occasional multinucleate giant cells, some lymphocytes and eosinophils) (black arrows) and smooth muscle hyperplasia. The alveolar walls are moderately expanded by the above described inflammatory cell population, with occasional alveolar wall rupture (arrowhead) and emphysema. c, d Detail of the marked areas in a and b. The parasites have a thin smooth cuticle (c) and pseudocoelom, coelomyarian-polymyarian musculature (m), intestine (i) with granular pigment and large uterus (u) filled with developing larvae (lv). The leukocyte reaction is also visible (black arrow in c). e, f. Free P. falciformis larvae (with thin walls and granular content) (black arrowhead in f) are present in the bronchiolar (b) and peribronchiolar spaces, associated with prominent inflammatory reaction consisting of many macrophage (some laden with hemosiderin) (asterisk in f), epithelioid cells and multinucleate giant cells (red arrowhead in f) and few eosinophils and fibroblast. Degenerated parasites (black arrow in e) induce a marked inflammatory response consisting of ill-defined granulomas (asterisks in e) and a locally-extensive interstitial lympho-histiocytic and eosinophilic pneumonia (arrowheads in e); H&E staining, ×20 (a, b and f), ×40 (c), ×100 (d) and ×10 (e). Scale-bars: a, b, f, 100 μm; c, 50 μm; d, 25 μm; e, 200 μm