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A rare cardiopulmonary parasite of the European badger, Meles meles: first description of the larvae, ultrastructure, pathological changes and molecular identification of Angiostrongylus daskalovi Janchev & Genov 1988
© The Author(s). 2016
- Received: 14 June 2016
- Accepted: 22 July 2016
- Published: 2 August 2016
Angiostrongylus daskalovi is a rare cardiopulmonary nematode infecting badgers. The parasite was described in 1988 and, since then, found only once in mustelids in Europe. The present study aims to report new cases of patent A. daskalovi infection in badgers from northern Romania and to provide new information on its ultrastructure, molecular diagnosis, and pathology.
Eight road-killed or hunted badgers originating from Maramureș and Alba counties in Romania were collected and necropsied. Adults and larvae of cardio-pulmonary nematodes were collected and examined by light and scanning electron microscopy (SEM). Genomic DNA was extracted from adults and first-stage larvae (L1). PCR amplification of the internal transcribed spacer 2 (ITS2, ∼500 bp) of the rRNA gene was performed. Amplicons were purified, sequenced, and compared to those available in the GenBank database. Histopathological examination of the lungs was performed and lesions described.
The necropsy revealed the presence of nematodes in the pulmonary arteries of three animals. All parasites were mature adults and the coproscopic examination showed the presence of eggs and L1 larvae in all three positive animals. Light microscopy examination confirmed the morphological and morphometric similarity of parasites to Angiostrongylus daskalovi. SEM highlighted the typical angiostrongylid structure of the rays of the copulatory bursa and the anterior extremity, with the presence of six sensory papillae surrounding the mouth opening in which a triangular tooth was visible. The first-stage larva (L1) of A. daskalovi is described here for the first time. Histopathological examination of the lungs showed chronic interstitial verminous pneumonia due to the presence of adult parasites. Molecular analysis showed 100 % nucleotide similarity to an Angiostrongylus sp. isolate originating from a badger from Spain, tentatively identified as A. daskalovi.
Our study unequivocally demonstrates the presence of A. daskalovi in European badgers from Romania, provides the first description of the larvae and reveals new data about the ultrastructure of adult parasites and their pathological impact, contributing to the understanding of the phylogenetic relationships with other congeneric species.
- Angiostrongylus daskalovi
- Meles meles
- Molecular analysis
The family Mustelidae is the richest group within the order Carnivora, comprising five subfamilies: Lutrinae (otters), Melinae (European badgers), Mellivorinae (honey badgers), Taxidiinae (American badgers) and Mustelinae (weasels, tayra, wolverines, martens, polecats) . According to recent multigene phylogenetic analysis, the family is split into four major clades and three monotypic lineages . Among all these mustelids, the European badger (Meles meles) is spread throughout Europe and in some parts of the Middle East. It is an opportunistic omnivorous species; its diet includes a broad range of animals and plants. This varied diet exposes the badger to the risk of contamination by a wide variety of cysts, eggs, larvae or intermediate hosts of certain parasites. Of these, cardiopulmonary nematodes represent a particular group, several species being reported in mustelids. The genus Aelurostrongylus Cameron, 1927 contains two species that are rarely reported in badgers in Europe: A. falciformis Schlegel, 1933 in Italy, Germany, Norway and Great Britain [3–6] and A. pridhami Anderson, 1962 in Spain. Two other metastrongyloid species belonging to the genus Angiostrongylus Kamensky, 1905 have been recorded in badgers. Angiostrongylus daskalovi Janchev & Genov, 1988 was described from the pulmonary arteries of the European badger (M. meles) in the north-central region of Bulgaria  and more recently in Spain . Additionally, Angiostrongylus vasorum (Baillet, 1866) was identified in the cardiopulmonary system of badgers in Switzerland, Italy and Spain [9–12]. Another species, Angiostrongylus gubernaculatus Dougherty, 1946, was described from the right ventricle of the Californian badger, Taxidea taxus neglecta in California and the California Channel Islands, the United States [13, 14].
Apart from these two genera, other lung nematodes have been reported in European badgers including Crenosoma sp., C. vulpis (Dujardin, 1845) and C. melesi Janchev & Genov, 1988, as well as the trichuroid nematode Capillaria aerophila Creplin, 1839 [3, 4, 12, 15, 16]. Cardiopulmonary nematodes have also been reported in other mustelids like the stoat (Mustela erminea) and weasel (Mustela nivalis) infected with A. vasorum [17, 18] and the European pine marten (Martes martes) and the beech marten (Martes foina) infected with A. daskalovi .
The European badger is considered the typical host for A. daskalovi . This nematode has a poorly known geographical distribution, so far being recorded only in Bulgaria and Spain. Moreover, the life-cycle and the host spectrum are incompletely known; the larvae are unknown and the pathological aspects have never been described. Due to these shortcomings, it is important to add new data regarding the infection caused by this nematode species. In this context, the present paper reports the first cases of patent A. daskalovi infection in badgers in Romania, emphasizing the ultrastructure of adult parasites and morphology of the L1 larval stage, molecular characterization, and pathological changes.
Sample origin and collection
Morphometric features of A. daskalovi and comparative data for other Angiostrongylus spp. identified in mustelids
Angiostrongylus sp. (badger)
Janchev & Genov 
Gerrikagoitia et al. 
Costa et al. 
(n = 7)
Body length (mm)
19.4 ± 7.7
Body width (μm)
243 ± 20
Distance from excretory pore to cephalic end (μm)
409 ± 25
Oesophagus length (μm)
333 ± 18
Spicule length (μm)
346 ± 24
(n = 16)
Body length (mm)
25.0 ± 14.0
Body width (μm)
345 ± 18
Distance from excretory pore to cephalic end (μm)
447 ± 102
Oesophagus length (μm)
368 ± 17
Distance from vulva to anus (μm)
295 ± 46
Distance from vulva to caudal end (μm)
366 ± 44
Distance from anus to caudal end (μm)
79 ± 3
Vulvar opening (length/width, μm)
Anus (length/width, μm)
Full necropsy and histological examination were carried out on all badgers included in this study. Selected samples from the right atrium, pulmonary arteries, pulmonary parenchyma and tracheobronchial lymph nodes were collected for histological analysis. Samples were fixed in 10 % phosphate-buffered formalin for 24 h, routinely processed, embedded in paraffin wax, cut into 4 μm sections, and stained with hematoxylin and eosin (H&E).
Scanning electron and light microscopy
All adult worms were washed in saline, preserved for 24 h in 0.5 % formalin, dehydrated, cleared in lactophenol, mounted in Canada balsam and analyzed by light microscopy using an Olympus BX 61 microscope (Japan). For scanning electron microscopy, some adult parasites were fixed for 2 h at 4 °C in 2.7 % glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) and washed in PBS. Samples were post-fixed for 1 h with 1 % OsO4. The parasites were dehydrated in an ethanol series (30–100 %), and infiltrated with hexamethyldisilazane, dried, mounted on aluminum stubs coated with a 10 nm gold layer, and examined with a Hitachi SU8230 Scanning Electron Microscope (Japan).
Molecular analyses and species identification
Genomic DNA was extracted from three adult nematodes (one from each positive animal) and 30 L1 stages (a pool of ten from each positive animal) using a commercial kit (Isolate II Genomic DNA Kit, Bioline, London, UK) as stated in the manufacturer’s instructions. PCR amplification of the internal transcribed spacer 2 (ITS2, ∼500 bp) of the rRNA gene was performed using the NC1/NC2 primer pair as previously described . Amplicons were purified using silica membrane spin columns (QIAquick PCR Purification Kit, Qiagen, Halden, Germany) and externally sequenced by Macrogen Europe (Amsterdam). Sequences were compared to those available in the GenBank database by Basic Local Alignment Search Tool (BLAST) analysis. Phylogenetic analyses were conducted using MEGA6 software . The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model . Species identification was based on morphological characteristics, associated with molecular analysis [7, 8].
Morphology and morphometry of Angiostrongylus daskalovi
Larvae were composed of numerous round, basophilic nuclei with scant eosinophilic cytoplasm and a thin amphophilic cuticle (Fig. 8b). Scattered hemorrhagic areas associated with hemosiderin deposits, hemosiderin-laden macrophages (Fig. 8b), and coagulative necrosis were found in the affected parenchyma.
Less affected areas of the lung presented mild congestion, edema, pulmonary atelectasis and alveolar emphysema. The pulmonary arteries exhibited moderate smooth muscle hypertrophy and hyperplasia of the arterial tunica media with mild vacuolar degeneration of the endothelial cells and intimal fibrosis. Multifocal and mild subendocardial fibrosis and minimal mononuclear infiltrates were observed in the right atrium.
The tracheobronchial lymph nodes showed diffuse reactive hyperplasia, multifocal granulomatous reaction centered on parasitic organisms and numerous aggregates of hemosiderin-laden macrophages (Fig. 8c, d). There were no microscopic lesions in the area of pulmonary arteries where adult parasites were located.
This report represents the first comprehensive study of A. daskalovi infection in European badgers. The study identified this species based on morphological, morphometric and molecular analyses. Three species of Angiostrongylus are described in different badger species across the world, namely A. vasorum, A. daskalovi and A. gubernaculatus. Morphometrically, A. vasorum is the smallest species, the length of females ranging between 14.17–17.69 mm and that of males between 11.21–13.91 mm . These values overlap the lower limits of the ranges for length of A. daskalovi: 14.39–31.12 mm in females and 13.36–21.31 mm in males . Our data correspond to specific dimensions of A. daskalovi, and may differentiate these two species even if the recorded variations could be related to the intervals after infection . The third species, A. gubernaculatus is morphometrically very similar to A. daskalovi, but host specificity and geographical range differ amongst the two species. Additionally, another congeneric species, A. chabaudi was recently found and redescribed in wildcats in the same geographical area from Romania, providing a detailed comparison of the morphometric features .
Scanning electron microscopy revealed similar structures of the anterior and posterior ends as described in adult A. vasorum , but also identified particular structures. At the anterior extremity, the mouth opening is surrounded by six papillae and two amphidal pores, but the small tooth observed in the mouth cavity of A. daskalovi is not known in A. vasorum. Comparison of the copulatory bursa of A. vasorum with that of A. daskalovi reveals only a slight difference in the median dorsal ray whose digitations are sometimes separated by a papilla, but none of the males of A. daskalovi exhibited this structure.
Despite the morphometric differences, these three species may have a common origin. Angiocaulus gubernaculatus which resembles Angiostrongylus sp., currently accepted as a synonym , may represent a common ancestor for the Brazilian and European populations of A. vasorum. Some authors consider that A. vasorum is the ancestral species that subsequently spread globally with its carnivore hosts, and evolved into genetically distinct populations in various host species . The phylogenetic analysis performed herein is also supportive of this hypothesis, as A. daskalovi clustered with A. vasorum.
To our knowledge, this is the first description of the first-stage larva (L1) of A. daskalovi. The differentiation of first-stage larvae of different Angiostrongylus species is difficult due to several common characters such as the transparent body, sigmoid tail, the presence of a dorsal spine and a visible notch. Their lengths partly overlap from one species to another: 310–400 μm for A. vasorum and 336–412 μm for A. daskalovi in the present study (L1s of A. gubernaculatus are not described). This does not allow a clear differentiation based on morphological and morphometric criteria. However, the adults and larvae of A. daskalovi identified in this study were 100 % similar to adults of Angiostrongylus sp. recovered from a badger in Spain and tentatively identified, based on morphology and morphometry, as A. daskalovi .
The presence of molting larvae in tracheobronchial lymph nodes and eggs in the lung parenchyma provides evidence that the life-cycle of A. daskalovi is probably similar to that of A. vasorum, following the type II of the known development in the definitive hosts . During their migration, larvae may exert a significant pathogenic action. Hypertrophy and hyperplasia of the arterial tunica media are explainable by pulmonary hypertension due to the presence of nematodes in the lung arteries, being similar to those lesions recorded in dogs naturally infected with A. vasorum . The presence of hemosiderin deposits and hemosiderin-laden macrophages, edema, pulmonary atelectasis, alveolar emphysema, diffuse congestion of the lungs and the presence of the nodules randomly distributed within all lung lobes are similar to those produced by A. vasorum in dogs . All these pathological alterations of the lungs and the pulmonary arteries confirm that A. daskalovi might play an important pathogenic role in infected badgers.
Although the species of Angiostrongylus infecting carnivores seem to show a relatively well-defined host specificity, some studies report certain overlaps. As badgers can occasionally be infected with A. vasorum, dogs might also be infected occasionally with A. daskalovi. Our new molecular data and larval morphology can partly solve possible misdiagnosis problems in European carnivores. Still to be solved is the life-cycle of A. daskalovi.
The current study confirms the existence of A. daskalovi patent infection in badgers from Romania and provides the first description of the larvae, its pathological effect, and its phylogenetic relationships with other congeneric species.
BLAST, Basic local alignment search tool analysis; DNA, deoxyribonucleic acid; H&E, Hematoxylin and eosin; L1, First larval stage; PCR, polymerase chain reaction; SEM, Scanning electron microscopy
We are indebted to Cristian Domșa for preparing the map and to all the people and hunters which provided the dead badgers.
This study was supported by UEFISCDI Grant PCE 236/2011.
Availability of data and materials
The data supporting the conclusions of this article are included within the article.
CMG, ADM and VC conceived and designed the study. GD and GD’A performed the necropsy. IAM and AMI performed molecular analysis of the parasites. MT conducted the histopathological study. LBT conducted the SEM analysis. AS collected the carcasses of the badgers. CMG wrote the paper. All authors contributed to improve the manuscript and read and approved the final version.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
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