
==== Front
Acta Parasitol
Acta Parasitol
Acta Parasitologica
1230-2821
1896-1851
Springer International Publishing Cham

38951380
861
10.1007/s11686-024-00861-8
Original Paper
First Molecular Identification and Clinical Presentation of Crenosomosis in a Dog from Slovakia
Kaduková Michaela 1
http://orcid.org/0000-0001-6123-8459
Kožár Martin martin.kozar@uvlf.sk

2
Schreiberová Andrea 1
Šišková Barbora 2
Štrkolcová Gabriela 1
1 grid.412971.8 0000 0001 2234 6772 Department of Epizootiology, Parasitology and Protection of One Health, University of Veterinary Medicine and Pharmacy in Košice, Komenského 73, 041 01 Kosice, Slovakia
2 grid.412971.8 0000 0001 2234 6772 Small Animal Clinic, The University of Veterinary Medicine and Pharmacy in Kosice, Komenského 73, 040 01 Kosice, Slovakia
29 6 2024
29 6 2024
2024
69 3 13721381
24 4 2024
18 6 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by/4.0/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
Purpose

Crenosoma vulpis (Dujardin,1845) is a lungworm which has spread worldwide in canines and is associated with upper respiratory infections. In a majority of cases, the infections are accompanied with chronic cough. Diagnosis of lungworms is often underdiagnosed and can be misinterpreted as other respiratory diseases.

Methods

The Small Animal Clinic of the University Veterinary Hospital admitted an 11-month-old dog presented with persistent cough associated with difficulty in breathing and even asphyxia. Based on clinical symptoms, the patient underwent radiological and bronchoscopic examination. Bronchoscopy revealed the presence of lungworms obturating the branches of the tracheobronchial tree. Larvae were collected by bronchoscopic lavage and subjected to parasitological and molecular examination.

Results

Microscopic detection and morphological identification of the worms removed during the bronchoscopy confirmed the presence of female adult worms. The subsequent molecular characterisation of the mitochondrial (cytochrome c oxidase subunit I gene (cox1) and 12S ribosomal DNA (rDNA)), nuclear (18S rDNA) genes, as well as the analysis of the second internal transcribed spacer (ITS-2) region of the ribosomal DNA, confirmed the Crenosoma vulpis species. Faecal samples were processed using the Baermann method, which confirmed the presence of the larval stage 1 of C. vulpis. The therapy with fenbendazole at a dose of 50 mg/kg of live weight once daily for the period of 7 days was initiated for the patient.

Conclusion

This paper presents the first molecularly confirmed clinical case of a Crenosoma vulpis infection in an 11-month-old female dog of the Miniature Schnauzer breed in Slovakia.

Keywords

Crenosoma vulpis
Bronchoscopy
Dog
Foxes
Molecular identification
Baermann technique
http://dx.doi.org/10.13039/501100006109 Vedecká Grantová Agentúra MŠVVaŠ SR a SAV 1/0709/23 Štrkolcová Gabriela University of Veterinary Medicine and PharmacyOpen access funding provided by The Ministry of Education, Science, Research and Sport of the Slovak Republic in cooperation with Centre for Scientific and Technical Information of the Slovak Republic

issue-copyright-statement© Springer Nature Switzerland AG 2024
==== Body
pmcIntroduction

Crenosoma vulpis (Nematoda: Metastrongyloidea), also known as the fox lungworm, is a member of the Crenosomatidae family and it is primarily associated with respiratory infections in canines. It mainly occurs in red foxes (Vulpes vulpes), less frequently in dogs (Canis lupus familiaris), arctic foxes (Alopex lagopus), gray foxes (Urocyon cinereoargentaus), wolves (Canis lupus), coyotes (Canis latrans), and European badgers (Meles meles); it is also present in European pine martens (Martes martes) and beech martens (Martes foina), while from the endemic point of view, it usually lives in the Northern America and Europe [4, 16, 30]. As a result of free movement of foxes in the wild nature without having any natural animal predator, and due to the fact that they stay near the residential areas and tourist locations, foxes are considered to be the main cause of the spread of lungworms to non-endemic regions and as the source of this infection for carnivores [18, 38]. C. vulpis was also reported in foxes in Africa and Algeria [35].

Stunženas and Binkiene (2021) [47] stated in their study that the Crenosoma genus includes 14 validated species, while the most widespread species in Europe is the C. vulpis fox lungworm. A rather frequently occurring Crenosoma striatum species was confirmed mainly in European hedgehogs (Erinaceus europeus) in Portugal and Italy [31]. Other species detected in Europe include Crenosoma melesi–primarily in European badgers (Meles meles), as well as Crenosoma petrowi–identified in European badgers (Meles meles), European pine martens (Martes martes) and beech martens (Martes foina) in Romania [16].

The life cycle of Crenosoma vulpis is indirect. The intermediate hosts include land slugs, such as Arion vulgaris and Limax maximus, as well as common garden snails Cornu aspersum [9, 21]. A definitive host acquires the infection by ingesting an intermediate host whose tissues contain a developing infectious L3 larvae. After the definitive host ingests the infectious larval stage, the larvae migrate through the gastrointestinal tract and via the lymph, and through their subsequent migration through the liver, heart and lungs they reach the locations in the lungs where they transform into sexually mature adults. Females are ovoviviparous; L1 larvae are released from the eggs and after a short period of time they are coughed up and swallowed so they get to the intestine and are eventually released to the external environment with faeces. Adult parasites may live in a definite host as long as 10 months, and their prepatent period is 18–21 days [18, 21, 44]. Depending on the parasite load, clinical symptoms may vary from the asymptomatic form to the nasal discharge, dyspnoea and chronic coughing, which is caused by the irritation of the lungs and bronchi by the parasite [11].

The objective of this paper was to identify the lungworm in a dog with clinical manifestations by applying morphology-molecular analyses.

Material and Methods

Case Presentation/Investigation

In the article, a clinical case of a patient that was referred to the clinic below due to several weeks persisting cough associated with difficulty in breathing and even suffocation is described. The patient–a female dog of the Miniature Schnauzer breed, with the weight of 6.4 kg and the age of 11 months, was treated at a private veterinary clinic, where he was administered two groups of antibiotic therapy and the medication from the group of glucocorticoids. Due to the failure of the conventional therapy, the patient was referred to the Small Animal Clinic of the University Veterinary Hospital at the University of Veterinary Medicine and Pharmacy in Košice, for an endoscopy. The clinical examination did not reveal any significant pathological changes in the patient’s condition (CRT = 2 s; N =  ≤ 2 s), light-pink mucous membranes, in the shock stage, with present normothermia (T = 38.3 °C), the animal was slightly excited, its breathing was shallow and of the costo-abdominal type.

Radiography

Subsequently, an X-ray examination of the chest cavity was performed on the LL and VD projection with the finding of a change in the trachea which was dorsally shifted away, the bronchial lung pattern, significant sharping of the lung field, and a change in the bifurcation region and the heart region (Fig. 1). The summary of all clinical signs indicated.Fig. 1 Radiography

the need for an additional specific diagnostic examination by a direct visualisation of the lower respiratory tract.

Bronchoscopy

The intravenous access was applied (v. cephalica) and venous blood was collected for haematological and biochemical analyses, with the finding of a slight decline in the eosinophil and reticulocyte counts. The patient was then sedated with butorphanol (Butomidor, RP Richter Pharma, Austria) at a dose of 0.2 mg/kg of live weight and medetomidine (Cepetor, CP-Pharma, Germany) at a dose of 0.02 mg/kg of live weight. Subsequently, the patient was put under general anaesthesia with an injection of diazepam (Apaurin, Krka d.d., Slovenia) at a dose of 0.2 mg/kg of live weight and propofol (Propofol, Fresenius Kabi) at a dose of 3 mg/kg of live weight. The patient was maintained under general anaesthesia with hyperoxygenation of the lower respiratory tract.

Following the sedation of the patient, endoscopy of the oral cavity was performed, with the finding of a change in the tonsil region, in particular enlarged tonsils with chronic inflammation. In the rima glottidis region, slight hyperaemia was observed, as well as the finding of prominent polyp formations on the cartilage base of the rima glottidis. After reaching the larynx region, significant swelling and hyperaemia of the mucous membrane was observed in the entire trachea, while the vessels were significantly hyperaemic as well. The bifurcation region showed a typical image of chronic irritation of the lower respiratory tract with hyperaemia and dorsal suppression of the trachea to 30%. Near the branches of the bronchi and the bronchioles, a chronic condition of the mucous membrane was observed, corresponding to the hyperplastic changes in the epithelium with a large amount of foamy exudate. After reaching the caudal regions and exhausting the effusion, the dominant finding was the presence of lungworms obturating the branches of the tracheobronchial tree (Fig. 2). The nostril region did not contain any effusion or depigmentation. After reaching the nasal cavities, hyperaemic changes in the mucosa were observed, as well as the hyperaemic vessels and a swelling with a small amount of viscous phlegm.Fig. 2 Bronchoscopy

Morphological and Molecular Identification of Lungworms

The lungworms collected during the bronchoscopy and the patient’s faeces were subsequently examined at the Department of Epizootiology, Parasitology and Protection of One Health. They were subjected to the microscopic and coprological diagnostics, followed by the molecular identification. All microscopic images and measurements were made using the light microscopy in the PROMICRA Introduces QuickPHOTO 3.0 Microscopy Imaging Software. The faeces of the treated dog, as well as the faeces of the dogs that lived in the same household, were examined by applying the coprological Baermann technique.

Genomic DNA was extracted from 4 adult female lungworms using a commercial kit (DNeasy Blood & Tissue Kit, Qiagen, GmbH, Hilden, Germany) following the manufacturer’s instructions. The molecular identification of the lungworms was carried out by amplification through a polymerase chain reaction (PCR) of 4 different DNA regions – genes of the mitochondrial DNA (cox1 and 12S rDNA) and nuclear DNA (18S rDNA), and based on the analysis of the ITS-2 region of rDNA. The extracted DNA was used as a template for the PCR amplification of an approximately 710 bp region of the mitochondrial cox1 gene with a pair of “universal” primers, widely used for the invertebrate species: LCO1490 forward primer (5’-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 reverse primer (5’-TAAACTTCAGGGTGACCAAAAAATCA-3′) [16, 20, 45] Partial fragments of mitochondrial 12S rRNA (330 bp) and nuclear 18S rRNA (1700 bp) genes were amplified by the conventional PCR using two sets of primers (12SF forward primer: 5′-CGGGAGTAAAGTTTTGTTTAAACCG-3’ and 12SR reverse primer: 5′-CATTGACGGATGGTTTGTACCAC-3′) and (NC18SF1 forward primer: 5′-AAAGATTAAGCCATGCA-3′ and NC5BR reverse primer: 5′-GCAGGTTCACCTACAGAT-3′, respectively) designed by Latrofa et al. 2015. The more variable ITS regions of the ribosomal RNA genes were used to amplify the second internal transcribed spacer ribosomal DNA sequences using the universal direct primers for nematodes (NC16 forward primer: 5′-AGTTCAATCGCAATGGCTT-3′ and NC2 reverse primer: 5′-TTAGTTTCTTTTCCTCCGCT-3′) of 1,250 bp in size [27].

All resulting PCR products were sent to the Microsynth Seqlab (Vienna, Austria) or SEQme (Dobříš, Czech Republic) for purification and sequencing in both strands with the identical primers used for the PCR. The sequencing was performed by the Sanger sequencing method. Resulting sequences were analysed and edited using MEGA X software [29]. and the assemblage of the nucleotide sequences was carried out in Gene Tool Lite 1.0 software (BioTools Inc., Jupiter, FL, USA). The consensus sequences were compared with the sequences deposited in GenBank by applying the nucleotide BLAST algorithm (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The sequences from this study for the cox1, 12S, 18S genes and the ITS-2 region were deposited in GenBank under unique accession numbers (Table 1). For the purpose of a phylogenetic analysis of the cox1 gene, all the sequences of Crenosoma spp. available in the GenBank were selected. The sequences were aligned and the phylogenetic tree of the gene was constructed using the MEGA X software [29]. The phylogenetic analysis was inferred using the statistical method of the Neighbour-Joining algorithm. The optimal tree is shown, with the sum of the branch length of 0.35541990. The percentages of the replicate trees, in which the associated taxa clustered together in the bootstrap test (1,000 replicates), are shown next to the branches. The tree was drawn to a scale, with the branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method, and they are in the units of the number of base substitutions per site. All positions containing gaps and missing data were eliminated (complete deletion option). The analysis involved 29 nucleotide sequences. There were a total of 441 positions in the final dataset (Fig. 5). Table 1 Accession unique numbers for the sequences C. vulpis: 12S rRNA, 18S rRNA, ITS2 region and cox1 gene deposited in the GenBank. (n/d not detected)

Isolates	12S rRNA gene	18S rRNA gene	ITS2 region	cox1 gene	
Cr1Dog1	PP109372 (haplotype I)	n/d	PP106991	PP106438	
Cr2Dog1	PP109373 (haplotype II)	PP107880	PP106992	PP106439	
Cr3Dog1	n/d	PP107881	PP106993	PP106440	
Cr4Dog1	n/d	PP107882	PP106994	PP106441	

Results

Morphological and Coprological Diagnostic

The microscopic diagnostics of the worms collected during bronchoscopy revealed the presence of adult females bearing multiple larvae in their wombs. Based on the morphological features of the parasite (the presence of typical cuticular ridges on the front end; in females, stretched cuticular folds on the posterior end; a visible anus) (Fig. 3), C. vulpis was identified [16].Fig. 3 Morphological diagnosis of Crenosoma vulpis. a (posterior part of C. vulpis), b (anterior part of C. vulpis), c (front part with ring-shaped folds of the cuticle and numerous thorns), d (female of C. vulpis with the uterus filled with the first-stage of larvae)

The faeces of the dog were examined by the coprological Baermann technique, and the presence of stage 1 larvae (L1) was revealed. The size of the larvae ranged from 270 to 290 μm (Fig. 4). The average length of the L1 larvae ranges from 243 μm to 281 μm [12, 34]. In addition, faecal samples of four dogs that lived in the same household were examined by applying the Baermann method; however, the presence of C. vulpis larvae was not confirmed.Fig. 4 Baermann technique–first stage of larvae

Molecular and Phylogenetic Analysis

The molecular characterisation of the mitochondrial cox1 and 12S rDNA genes and nuclear 18S rDNA genes, and based on the analysis of the ITS-2 region of rDNA, the presence of the Crenosoma vulpis species was confirmed. For the 12S ribosomal RNA gene, 2 high-quality sequences were obtained and compared using the BLAST tool in GenBank; they were a 100% match to the C. vulpis KR920039 sequence (haplotype I; hosts: Vulpes vulpes, Canis lupus familiaris, Meles meles) and the KR920040 sequence (haplotype I; host: Vulpes vulpes), both originated in Italy (Latrofa et al. 2015). Based on that comparison, two haplotypes were determined – for the Cr1Dog1 sample (PP109372) as haplotype I, and for the Cr2Dog1 sample (PP109373) as haplotype II (Table 1). Our sequences for the 18S rRNA gene, deposited under numbers PP107880–PP107882, were compared in GenBank to the C. vulpis KR920038 sequence (hosts: Vulpes vulpes, Canis lupus familiaris, Meles meles from Italy; Latrofa et al., 2015) [30]; and the AJ920367 sequence (host: Vulpes vulpes from Canada; Chilton et al., 2006) [26]; with the identity ranging from 99.60% to 99.73%. Sequences (PP106991–PP106994) for the ITS2 region were compared to OM480716 (host: Canis lupus familiaris from USA; Pohly et al. 2022) [40]; MT808324–MT808325 (host: Vulpes vulpes from United Kingdom; Allen et al. 2020) with the 100% identity, and compared to KF836608 (host: Vulpes vulpes from Germany; Schug et al. 2018) [46] with the identity of 99.41%–99.75%. In the analysis of the cox1 mtDNA gene, four our sequences (PP106438–PP106441) exhibited the 98.10%–99.58% nucleotide identity to the KM216824 sequence of C. vulpis deposited in GenBank (host: Vulpes vulpes from Germany; Schug et al., 2018) [46].

To investigate the phylogenetic relationships among the species of the Crenosoma genus, we selected the PP106438–PP106441 sequences for the cox1 gene of C. vulpes from this study (host: Canis lupus familiaris from Slovakia), as well as all the available reference sequences from GenBank at NCBI for C. vulpes, C. petrowi, C. melesi, C. striatum, and C. goblei from a variety of hosts and different geographic regions. The following sequences of cox1 gene were phylogenetically analysed: C. vulpes KM216824 (host: Vulpes vulpes from Germany; Schug et al. 2018) [46]; ON965049–ON965051 (host: Martes foina from Romania); ON965052–ON965054 (host: Martes martes from Romania) (Latrofa et al. 2015); C. petrowi MZ350755, ON965041–ON965043 (host: Meles meles from Romania); ON965047 and ON965048 (host: Martes martes from Romania); ON965044–ON965046 (host: Martes foina from Romania) (Latrofa et al. 2015); C. melesi ON965036, ON965039, ON965040 and MZ350754 (host: Meles meles from Romania) (Latrofa et al. 2015); C. striatum OQ078756 (host: Atelerix algirus from Mallorca, Spain); KJ579446, KJ579455 and KJ579464 (host: Erinaceus Europeus from Germany); and C. goblei MN207133 (host: Procyon lotor from Washington, USA) [23]. The phylogenetic tree revealed that C. vulpis and C. petrowi constituted a shared clade, even though they divided within the clad into further branches. A majority of the C. petrowi sequences exhibited high homology with a very small number of nucleotide changes; as for C. vulpis, there was a nucleotide variability among the sequences so they split into several smaller branches. C. striatum, C. melesi and C. goblei formed three separate related branches in the phylogenetic tree (Fig. 5).Fig. 5 The tree were constructed using the Neighbour-Joining method (NJ) and depicting the relationships among Crenosoma vulpis, Crenosoma petrowi, Crenosoma goblei, Crenosoma melesi and Crenosoma striatum based on gene cox1 mtDNA sequences data available in theGenBank database

Treatment of Crenosoma vulpis

Based on the findings, the therapy with fenbendazole (Helmigal, PHARMAGAL spol. s r.o., Slovak Republic) at a, oral dose of 50 mg/kg of live weight once daily for the period of 7 days was indicated for the patient, concurrently with the administration of adjuvant preparations for the regeneration of mucous membranes – vitamin A, vitamin D3 (Aquavit ad3, PHARMAGAL spol. s r.o., Slovak Republic) at a dose of 1.5 ml pro toto once daily for the period of 2 weeks; the dosage regimen was q24h during the first week and q48h during the following 7 days.

Two weeks after the completion of the targeted therapy, the coprological examination was repeated by applying the Baermann method. The finding was negative and the X-ray scan of the lung region exhibited a significant improvement compared to the primary condition of the patient.

Discussion

The present study discloses the first clinical case of crenosomosis with the morphological diagnostics and molecular characterisation of the Crenosoma vulpis species in a domestic dog (Canis lupus familiaris) in Slovakia. In a study by Čabanová et al. (2018a) [14], C. vulpis was confirmed by the Baerman method in a dog for the first time in Slovakia, while the presence of C. vulpis in red foxes was detected as early as in 1960s and 1980s in the research conducted by Mituch (1962) [36].

Crenosomosis induced by Crenosoma spp. nematodes may play an important role in chronic respiratory diseases of dogs in Europe [10]. The first such case was reported from the United Kingdom [8]. Since then, the cases of crenosomosis in dogs have been reported from several European countries, such as Ireland, Switzerland, Germany, Italy, Denmark, Belgium, Spain, Austria, Lithuania, France and the Czech Republic [2, 3, 5, 7, 21, 25, 32, 41, 43, 50, 51]. In a majority of those cases, productive cough developed into the chronic form, and dribbled saliva and breathlessness were present. Bronchoscopy revealed the hyperaemic trachea or the presence of mucopurulent exudate.

In the confirmed case of crenosomosis in a dog described herein, the animal was treated with fenbendazole at a dose of 50 mg/kg of live weight once daily per os for the period of 7 days. While the study by Caron et al. (2014) [7] claims that the 7-day therapy with fenbendazole could not cure the infection, and that a better effect would be achieved by a one-time local application of 10% imidacloprid combined with 2.5% moxidectin at a dose of 0.1 ml/kg of live weight, no efficient anthelminthic drugs specific for this parasite are currently marketed. With the use of febantel, fenbendazole, ivermectin and milbemycin oxime, a successful result of the therapy with the absolutely disappeared clinical signs and without the presence of L1 larvae in faeces was confirmed in several studies [4, 8, 39]. In the study by Conboy et al. (2013) [13] with dogs that were experimentally infected with Crenosoma vulpis, the therapy with milbemycin oxime (0.5 mg/kg) and praziquantel (5 mg/kg) was applied with a 98.7% efficiency.

In the neighbouring Czech Republic, Husník et al. (2011) [25] confirmed C. vulpis in a 1-year-old female dog of the Shetland Sheepdog breed. The patient was presented with tachypnoea and moist cough, and bronchoscopy revealed the hyperaemic trachea and phlegm-purulent exudate. Adults were collected with the use of bronchoalveolar lavage and after the microscopic analysis, the larvae were identified as Crenosoma vulpis. The application of the Baermann sedimentation method confirmed the presence of stage 1 larvae (L1). Similarly to the present study, the therapy indicated for the patient included fenbendazole (50 mg/kg) once daily for the period of 3 days, but it was combined with doxycycline (5 mg/kg) per os twice daily for the period of two weeks. That therapy too has been proven efficient.

At present, there is no evidence that gender or age represent a potential predisposition to this parasitic disease. It is assumed that dogs usually acquire the infection at the age of approximately 1 year [4, 33]. This assumption was confirmed by the present study, since the infection was detected in an 11-year-old dog. One of the risk factors that affect the outbreak of the disease is the living environment. The dogs that live in rural regions are exposed to a higher risk of infection than the dogs in urban regions due to the potential presence of foxes and a higher concentration of intermediate hosts [48].

Foxes as the most frequent reservoirs of cardiopulmonary parasites, including C. vulpis, are responsible for the extension of the geographical distribution of C. vulpis to urban regions in Europe [18, 30, 49]. Čabanová et al. (2018b) [13] confirmed C. vulpis in foxes in 17.51% of cases, and according to the existing data, foxes inhabit as much as 93.5% of the territory in Slovakia [28]. Out of all European countries, the highest prevalence of crenosomosis was reported from Norway 58.2%; Lithuania 53.8%; Bosnia and Herzegovina 45.3%; Pyrenees 44.8%; Portugal 39.29%; Germany 32.6%; Romania 32.0% and Italy 28.4% [6, 15, 17, 19, 22, 24, 37, 46].

In Europe, Latrofa et al. (2015) was the first team to confirm by a molecular analysis the presence of C. vulpis in dogs in Italy; later in 2022, it was confirmed by Remesar et al. in Spain. At present, there is only a very little available data in GenBank about the C. vulpis species, not only with regard to dogs as hosts, but also general data about the species. Molecular detection based on the database confirmed C. vulpis in the European countries and in the Northern America, while the most frequent definite hosts were Vulpes vulpes (Italy, Canada, United Kingdom, Germany, Bosnia and Herzegovina); Canis lupus familiaris (Italy, Spain, USA); Meles meles (Italy, Romania); and Martes foina and Martes martes (Romania) [16, 24, 26, 30, 40, 42, 46].

Species identification was carried out using the "universal" DNA primers – LCO 1490 and HCO 2198, which were originally intended for the amplification of highly-conservated regions of mitochondrial genes cytochrome c oxidase subunit I (cox1) in several taxons of invertebrates [16, 20]. The phylogenetic tree of the cox1 gene was compiled out of all sequences of Crenosoma spp. available in GenBank. The phylogenetic analysis for this gene showed a nucleotide variability among the sequences obtained from C. vulpis, and divided them into several smaller branches. One branch represents our sequences from Slovakia, which together with the sequences from Germany (host: Vulpes vulpes) constitute a homologue group, unlike other sequences obtained from Romania. It is assumed that this variability may be associated with the geographical spread of this parasite and with the diversity of its hosts. The C. vulpis and C. petrowi species are morphologically and genetically related. A comparison of sequences of the cox1 gene of those two species in GenBank revealed a high degree of percentual identity; moreover, a phylogenetic analysis showed a high degree of the relationship between C. petrowi and C. vulpis [16]. Since the GenBank does not contain any sequences of 12S, 18S or ITS-2 for C. petrowi, it is impossible to subject them to a phylogenetic analysis together with our sequences for C. vulpis. However, there is currently only a very little available data on the occurrence of C. petrowi in canines, since it was mostly detected in Eurasia and America (Addison et al. 1994) [1, 16]. Since the two species exhibited a high degree of relationship, C. vulpis was confirmed in our study by using also other genes (12S and 18S genes, ITS-2 region,). In the study by Latrofa et al. (2015) [30] conducted in Italy with Vulpes vulpes, Canis lupus familiaris and Meles meles, four haplotypes (I–IV) were identified based on the 12S rRNA target gene for C. vulpis, while our two C. vulpis lungworms were categorised as haplotypes I and II. An interesting fact is that both haplotypes I and II had the same host, in our case a dog, whereas in the aforementioned Italian study, as much as 3 haplotypes were detected in a single Vulpes vulpes individual. According to Latrofa et al. (2015) [30] haplotype I ranks among the most frequently occurring haplotypes in various hosts in Romania. Hodžić et al. (2016) [24] confirmed a new haplotype V for C. vulpis in the Vulpes vulpes foxes population in Bosnia and Herzegovina.

The most frequently used detection method is the Baermann technique, which is regarded as the most efficient method for the diagnostics of C. vulpis. It is cost-effective and easy to perform, but it is rarely used in veterinary clinics [48]. Rinaldi et al. (2007) [43] performed the detection of C. vulpis by applying the FLOTAC technique, and compared their results with the standard copro-microscopic methods: Baermann technique, McMaster technique, faecal flotation, and the Wisconsin method. The results showed that the FLOTAC method confirmed a larger number of larvae per gram of faeces when compared to the other methods. The findings obtained in the study indicate a potential improvement in the exact diagnostics of the lungworm infection in dogs.

Conclusion

An increase in the population of foxes in Europe, as well as their more and more frequent migration across the urbanised regions, may result in the elevated numbers of infections in domestic dogs. Infection caused by the Crenosoma vulpis species in domestic dogs is generally regarded as rare, but it may often be overlooked. The spread of this parasite across Slovakia or even Europe may therefore be actually much more extensive than currently assumed. In cases where persisting cough and lung lesions are present, especially in young dogs, veterinary doctors should consider a potential presence of C. vulpis, as well as other lungworms, and include the Baermann technique in the routine examination methods intended for lungworms.

For the first time, the clinical presence of C. vulpis is confirmed in dogs in Slovakia via molecular analyses.

Author's Contributions

Department of Epizootiology, Parasitology and Protection of One Health, University of veterinary medicine and pharmacy in Košice, Komenského 73, 041 01, Kosice, Slovakia. Michaela Kaduková, DVM., Andrea Schreiberová, RNDr., PhD., Gabriela Štrkolcová Assoc. prof., DVM., PhD. Small Animal Clinic, The University of Veterinary Medicine and Pharmacy in Kosice, Komenského 73, 040 01 Kosice, Slovakia. Martin Kožár, DVM., PhD., Barbora Šišková, DVM.

Funding

Open access funding provided by The Ministry of Education, Science, Research and Sport of the Slovak Republic in cooperation with Centre for Scientific and Technical Information of the Slovak Republic. This research was funded by the projects of the Scientific Grant Agency of the Ministry of Education of the SR and the Slovak Academy of Sciences, VEGA 1/0709/23.

Data Availability

The data supporting the findings of this study are available within the article.

Declarations

Conflict of Interests

The authors declare no competing interests.

Ethical Approval

Not applicable.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
==== Refs
References

1. Addison EM Fraser GA Life cycle of Crenosoma petrowi (Nematoda:Metastrongyloidea) from black bears (Ursus americanus) Can J Zool 1994 72 2 300 302 10.1139/z94-04
Addison EM, Fraser GA (1994) Life cycle of Crenosoma petrowi (Nematoda:Metastrongyloidea) from black bears (Ursus americanus). Can J Zool 72(2):300–302. 10.1139/z94-0410.1139/z94-04
2. Al-Sabi MNS Kapel CHMO Johansson A Espersen MC Koch H Willesen JL A coprological investigation of gastrointestinal and cardiopulmonary parasites in hunting dogs in Denmark Vet Parasitol 2013 196 4 366 372 10.1016/j.vetpar.2013.03.027 23602361
Al-Sabi MNS, Kapel CHMO, Johansson A, Espersen MC, Koch H, Willesen JL (2013) A coprological investigation of gastrointestinal and cardiopulmonary parasites in hunting dogs in Denmark. Vet Parasitol 196(4):366–372. 10.1016/j.vetpar.2013.03.02723602361 10.1016/j.vetpar.2013.03.027
3. Barutzki D Schaper R Natural infections of Angiostrongylus vasorum and Crenosoma vulpis in dogs in Germany Parasito Res 2009 105 34 48 10.1007/s00436-009-1494-x
Barutzki D, Schaper R (2009) Natural infections of Angiostrongylus vasorum and Crenosoma vulpis in dogs in Germany. Parasito Res 105:34–48. 10.1007/s00436-009-1494-x10.1007/s00436-009-1494-x
4. Bihr T Conboy GA Lungworm (Crenosoma vulpis) infection in dogs on prince Edward Island Can Vet J 1999 40 555 12001335
Bihr T, Conboy GA (1999) Lungworm (Crenosoma vulpis) infection in dogs on prince Edward Island. Can Vet J 40:55512001335
5. Bourgoin G Callait-Cardinal MP Bouhsira E Polack B Ariza CR Carassou L Lienard E Drake J Prevalence of major digestive and respiratory helminths in dogs and cats in France: results of a multicenter study Parasit Vect 2022 15 314 10.1186/s13071-022-05368-7
Bourgoin G, Callait-Cardinal MP, Bouhsira E, Polack B, Ariza CR, Carassou L, Lienard E, Drake J (2022) Prevalence of major digestive and respiratory helminths in dogs and cats in France: results of a multicenter study. Parasit Vect 15:314. 10.1186/s13071-022-05368-710.1186/s13071-022-05368-7
6. Bružinskaite-Schmidhalter R Šarkunas M Malakauskas A Mathis A Torgerson PR Deplazes P Helminths of red foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides) in Lithuania Parasitol 2012 139 120 127 10.1017/S0031182011001715
Bružinskaite-Schmidhalter R, Šarkunas M, Malakauskas A, Mathis A, Torgerson PR, Deplazes P (2012) Helminths of red foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides) in Lithuania. Parasitol 139:120–127. 10.1017/S003118201100171510.1017/S0031182011001715
7. Caron Y Merveille ACH Losson B Billen F Crenosoma vulpis infection in two young dogs in Belgium Vet Rec Case Rep 2014 10.1136/vetreccr-2014-000098
Caron Y, Merveille ACH, Losson B, Billen F (2014) Crenosoma vulpis infection in two young dogs in Belgium. Vet Rec Case Rep. 10.1136/vetreccr-2014-00009810.1136/vetreccr-2014-000098
8. Cobb MA Fisher MA Crenosoma vulpis infection in a dog Vet Rec 1992 130 452 10.1136/vr.130.20.452 1621347
Cobb MA, Fisher MA (1992) Crenosoma vulpis infection in a dog. Vet Rec 130:452. 10.1136/vr.130.20.4521621347 10.1136/vr.130.20.452
9. Colella V Mutafchiev Y Cavalera MA Giannelli A Lia RP Dantas-Torres F Otranto D Development of Crenosoma vulpis in the common garden snail Cornu aspersum: implications for epidemiological studies Parasit Vectors 2016 9 208 10.1186/s13071-016-1483-8 27079792
Colella V, Mutafchiev Y, Cavalera MA, Giannelli A, Lia RP, Dantas-Torres F, Otranto D (2016) Development of Crenosoma vulpis in the common garden snail Cornu aspersum: implications for epidemiological studies. Parasit Vectors 9:208. 10.1186/s13071-016-1483-827079792 10.1186/s13071-016-1483-8
10. Conboy G Natural infections of Crenosoma vulpis and Angiostrongylus vasorum in dogs in Atlantic Canada and their treatment with milbemycin oxime Vet Rec 2004 155 16 18 10.1136/vr.155.1.16 15264484
Conboy G (2004) Natural infections of Crenosoma vulpis and Angiostrongylus vasorum in dogs in Atlantic Canada and their treatment with milbemycin oxime. Vet Rec 155:16–18. 10.1136/vr.155.1.1615264484 10.1136/vr.155.1.16
11. Conboy G Bourque A Miller L Seewald W Schenker R Efficacy of Milbemax (milbemycin oxime+ praziquantel) in the treatment of dogs experimentally infected with Crenosoma vulpis Vet Parasitol 2013 198 319 324 10.1016/j.vetpar.2013.09.016 24144516
Conboy G, Bourque A, Miller L, Seewald W, Schenker R (2013) Efficacy of Milbemax (milbemycin oxime+ praziquantel) in the treatment of dogs experimentally infected with Crenosoma vulpis. Vet Parasitol 198:319–324. 10.1016/j.vetpar.2013.09.01624144516 10.1016/j.vetpar.2013.09.016
12. Craig RE Anderson RC The genus Crenosoma (Nematoda: Metastrongyloidea) in new world mammals Can J Zool 1972 50 1555 1561 10.1139/z72-204 4656823
Craig RE, Anderson RC (1972) The genus Crenosoma (Nematoda: Metastrongyloidea) in new world mammals. Can J Zool 50:1555–15614656823 10.1139/z72-204
13. Čabanova V Miterpakova M Druga M Hurnikova Z Valentova D GIS-based environmental analysis of fox and canine lungworm distribution: an epidemiological study of Angiostrongylus vasorum and Crenosoma vulpis in red foxes from Slovakia Parasitol Res 2018 117 521 530 10.1007/s00436-017-5728-z 29297093
Čabanova V, Miterpakova M, Druga M, Hurnikova Z, Valentova D (2018) GIS-based environmental analysis of fox and canine lungworm distribution: an epidemiological study of Angiostrongylus vasorum and Crenosoma vulpis in red foxes from Slovakia. Parasitol Res 117:521–530. 10.1007/s00436-017-5728-z29297093 10.1007/s00436-017-5728-z
14. Čabanova V Hurnikova Z Miterpakova M Dirbakova K Bendova A Kocak P Lungworm infections in dogs from central Europe Vet Med Czech 2018 63 367 372 10.17221/24/2018-VETMED
Čabanova V, Hurnikova Z, Miterpakova M, Dirbakova K, Bendova A, Kocak P (2018) Lungworm infections in dogs from central Europe. Vet Med Czech 63:367–372. 10.17221/24/2018-VETMED10.17221/24/2018-VETMED
15. Davidson RK Gjerde B Vikøren T Lilehaug A Handeland K Prevalence of Trichinella larvae and extra-intestinal nematodes in Norwegian red foxes (Vulpes vulpes) Vet Parasitol 2006 136 30 316 10.1016/j.vetpar.2005.11.015
Davidson RK, Gjerde B, Vikøren T, Lilehaug A, Handeland K (2006) Prevalence of Trichinella larvae and extra-intestinal nematodes in Norwegian red foxes (Vulpes vulpes). Vet Parasitol 136:30–316. 10.1016/j.vetpar.2005.11.01510.1016/j.vetpar.2005.11.015
16. Deak G Ionică AM Gherman CM Mihalca AD Diversity of Crenosoma species in mustelids with the first molecular characterization of C. melesi and C. petrowi Front Vet Sci 2023 17 1094554 10.3389/fvets.2023.1094554
Deak G, Ionică AM, Gherman CM, Mihalca AD (2023) Diversity of Crenosoma species in mustelids with the first molecular characterization of C. melesi and C. petrowi. Front Vet Sci 17:1094554. 10.3389/fvets.2023.1094554. (PMID: 37138924; PMCID: PMC10150068.)10.3389/fvets.2023.1094554
17. Deak G Gherman CM Ionică M Péter Á Sándor A Mihalca AD Biotic and abiotic factors influencing the prevalence, intensity and distribution of Eucoleus aerophilus and Crenosoma vulpis in red foxes, Vulpes vulpes from Romania IJP-PAW 2020 12 121 125 10.1016/j.ijppaw.2020.05.009 32547917
Deak G, Gherman CM, Ionică M, Péter Á, Sándor A, Mihalca AD (2020) Biotic and abiotic factors influencing the prevalence, intensity and distribution of Eucoleus aerophilus and Crenosoma vulpis in red foxes, Vulpes vulpes from Romania. IJP-PAW 12:121–125. 10.1016/j.ijppaw.2020.05.00932547917 10.1016/j.ijppaw.2020.05.009
18. Deplazes P Eckert J Mathis A Samson-Himmelstjerna G Zahner H Parasitology in veterinary medicine First edition, Wageningen Academic 2016 10.1016/j.vprsr.2022.100714
Deplazes P, Eckert J, Mathis A, Samson-Himmelstjerna G, Zahner H (2016) Parasitology in veterinary medicine. First edition, Wageningen Academic. 10.1016/j.vprsr.2022.10071410.1016/j.vprsr.2022.100714
19. Figueiredo A Oliveira L Madeira de Carvalho L Fonseca C Torres RT Parasite species of the endangered Iberian wolf (Canis lupus signatus) and a sympatric widespread carnivore IJP-PAW 2016 5 164 167 10.1016/j.ijppaw.2016.04.002 27358768
Figueiredo A, Oliveira L, Madeira de Carvalho L, Fonseca C, Torres RT (2016) Parasite species of the endangered Iberian wolf (Canis lupus signatus) and a sympatric widespread carnivore. IJP-PAW 5:164–167. 10.1016/j.ijppaw.2016.04.00227358768 10.1016/j.ijppaw.2016.04.002
20. Folmer O Black M Hoeh W Lutz R Vrijenhoek R DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates Mol Mar Biol Biotechnol 1994 3 294 299 7881515
Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol 3:294–2997881515
21. Fuehrer HP Morelli S Bleiche J Detection of Crenosoma spp., Angiostrongylus vasorum and Aelurostrongylus abstrusus in Gastropods in Eastern Austria Pathog Dis 2020 9 12 1046 10.3390/pathogens9121046
Fuehrer HP, Morelli S, Bleiche J et al (2020) Detection of Crenosoma spp., Angiostrongylus vasorum and Aelurostrongylus abstrusus in Gastropods in Eastern Austria. Pathog Dis 9(12):1046. 10.3390/pathogens912104610.3390/pathogens9121046
22. Garrido-castañé I Ortuño A Marco J Castellà J Cardiopulmonary helminths in foxes from the Pyrenees Acta Parasitol 2015 60 4 712 715 10.1515/ap-2015-0101 26408595
Garrido-castañé I, Ortuño A, Marco J, Castellà J (2015) Cardiopulmonary helminths in foxes from the Pyrenees. Acta Parasitol 60(4):712–715. 10.1515/ap-2015-010126408595 10.1515/ap-2015-0101
23. Groves BA Yabsley MJ Swanepoel L Garner MM Lungworm (Crenosoma goblei) infection in unweaned free-ranging Raccoons (Procyon lotor) in Washington State, USA J Wildl Dis 2020 56 419 423 10.7589/2019-03-060 31596677
Groves BA, Yabsley MJ, Swanepoel L, Garner MM (2020) Lungworm (Crenosoma goblei) infection in unweaned free-ranging Raccoons (Procyon lotor) in Washington State, USA. J Wildl Dis 56:419–42331596677 10.7589/2019-03-060
24. Hodžić A Alić A Klebić I Kadrić M Brianti E Duscher GG Red fox (Vulpes vulpes) as a potential reservoir host of cardiorespiratory parasites in Bosnia and Herzegovina Vet Parasitol 2016 223 63 70 10.1016/j.vetpar.2016.04.016.10.1136/vetreccr-2014-000098 27198779
Hodžić A, Alić A, Klebić I, Kadrić M, Brianti E, Duscher GG (2016) Red fox (Vulpes vulpes) as a potential reservoir host of cardiorespiratory parasites in Bosnia and Herzegovina. Vet Parasitol 223:63–70. 10.1016/j.vetpar.2016.04.016.10.1136/vetreccr-2014-00009827198779 10.1016/j.vetpar.2016.04.016.10.1136/vetreccr-2014-000098
25. Husník R Sloboda M Kovaříková S Koudela B Infection with Crenosoma vulpis lungworm in a dog in the Czech Republic Helminthologia 2011 48 56 58 10.2478/s11687-011-0010-x
Husník R, Sloboda M, Kovaříková S, Koudela B (2011) Infection with Crenosoma vulpis lungworm in a dog in the Czech Republic. Helminthologia 48:56–58. 10.2478/s11687-011-0010-x10.2478/s11687-011-0010-x
26. Chilton N Huby-Chilton F Gasser R Beveridge I The evolutionary origins of nematodes within the order Strongylida are related to predilection sites within hosts Mol Phylogenethis evol. 2006 40 118 128 10.1016/j.ympev.2006.01.003
Chilton N, Huby-Chilton F, Gasser R, Beveridge I (2006) The evolutionary origins of nematodes within the order Strongylida are related to predilection sites within hosts. Mol Phylogenethis evol. 40:118–128. 10.1016/j.ympev.2006.01.00310.1016/j.ympev.2006.01.003
27. Chilton NB Huby-Chilton F Gasser RB First complete large subunit ribosomal RNA sequence and secondary structure for a parasitic nematode: phylogenetic and diagnostic implications Mol Cell Probes 2003 17 33 39 10.1016/S0890-8508(02)00107-X 12628592
Chilton NB, Huby-Chilton F, Gasser RB (2003) First complete large subunit ribosomal RNA sequence and secondary structure for a parasitic nematode: phylogenetic and diagnostic implications. Mol Cell Probes 17:33–39. 10.1016/S0890-8508(02)00107-X12628592 10.1016/S0890-8508(02)00107-X
28. Krištofík J, Danko Š (2012) The mammals of Slovakia: distribution, bionomy and protection. Vvydavateľstvo Slovenskej Akadémie Vied, Bratislava
29. Kumar S Stecher G Li M Knyaz C Tamura K MEGA X: molecular evolutionary genetics analysis across computing platforms Mol Biol Evol 2018 35 1547 1549 10.1093/molbev/msy096 29722887
Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35:1547–154929722887 10.1093/molbev/msy096
30. Latrofa MS Riccardo PL Giannelli A Colella V SantoroD´Alessio MN Campbell BE Parisi A Dantas-Torres F Mutafchiev Y Veneziano V Otranto D Crenosoma vulpis in wild and domestic carnivores from Italy: a morphological and molecular study Parasitol Res 2015 114 3611 3617 10.1007/s00436-015-4583-z 26103959
Latrofa MS, Riccardo PL, Giannelli A, Colella V, SantoroD´Alessio MN, Campbell BE, Parisi A, Dantas-Torres F, Mutafchiev Y, Veneziano V, Otranto D (2015) Crenosoma vulpis in wild and domestic carnivores from Italy: a morphological and molecular study. Parasitol Res 114:3611–3617. 10.1007/s00436-015-4583-z26103959 10.1007/s00436-015-4583-z
31. Mariacher A Santini A Del Lesto I Tonon S Cardini E Barone A Eleni C Fichi G Perrucci S Endoparasite infections of the European Hedgehog (Erinaceus europaeus) in Central Italy Animals 2021 11 3171 10.3390/ani11113171 34827903
Mariacher A, Santini A, Del Lesto I, Tonon S, Cardini E, Barone A, Eleni C, Fichi G, Perrucci S (2021) Endoparasite infections of the European Hedgehog (Erinaceus europaeus) in Central Italy. Animals 11:3171. 10.3390/ani1111317134827903 10.3390/ani11113171
32. Martínez-Carrasco C Berriatua E Garijo M Martínez J Alonso FD Ruiz de Ybáñez R Epidemiological study of non-systemic parasitism in dogs in southeast Mediterranean Spain assessed by coprological and post-mortem examination Zoonoses publ health 2007 54 5 195 203 10.1111/j.1863-2378.2007.01047.x
Martínez-Carrasco C, Berriatua E, Garijo M, Martínez J, Alonso FD, Ruiz de Ybáñez R (2007) Epidemiological study of non-systemic parasitism in dogs in southeast Mediterranean Spain assessed by coprological and post-mortem examination. Zoonoses publ health 54(5):195–203. 10.1111/j.1863-2378.2007.01047.x10.1111/j.1863-2378.2007.01047.x
33. Matos B Colella V Alho AM Otranto D Doyle R Madeira de Carvalho L Crenosoma vulpis infection in a four-month old puppy Helminthologia 2016 53 3 276 280 10.1515/helmin-2016-0027
Matos B, Colella V, Alho AM, Otranto D, Doyle R, Madeira de Carvalho L (2016) Crenosoma vulpis infection in a four-month old puppy. Helminthologia 53(3):276–280. 10.1515/helmin-2016-002710.1515/helmin-2016-0027
34. McGarry JW Morgan ER Identification of first-stage larvae of matastrogyles from drog Vet rec 2009 165 9 258 261 10.1136/vr.165.9.258 19717830
McGarry JW, Morgan ER (2009) Identification of first-stage larvae of matastrogyles from drog. Vet rec 165(9):258–261. 10.1136/vr.165.9.25819717830 10.1136/vr.165.9.258
35. Mechouk N Deak G Ionică AM Toma CG Bouslama Z Mihalca AD First report of Crenosoma vulpis in Africa and Eucoleus aerophilus in Algeria IJP PAW 2023 20 187 191 10.1016/j.ijppaw.2023.03.003 36941973
Mechouk N, Deak G, Ionică AM, Toma CG, Bouslama Z, Mihalca AD (2023) First report of Crenosoma vulpis in Africa and Eucoleus aerophilus in Algeria. IJP PAW 20:187–191. 10.1016/j.ijppaw.2023.03.00336941973 10.1016/j.ijppaw.2023.03.003
36. Mituch J Contribution to the knowledge of the helminthofauna in the red fox (Vulpes vulpes crucigera L.) in Slovakia Vet Med 1962 7 227 238
Mituch J (1962) Contribution to the knowledge of the helminthofauna in the red fox (Vulpes vulpes crucigera L.) in Slovakia. Vet Med 7:227–238
37. Morandi B Bertaso S Conboy G Gustinelli A Galuppi R Tosi G Poglayen G Crenosoma vulpis in red foxes (Vulpes vulpes) in Northern Italy Parasitol Res 2019 118 1981 1985 10.1007/s00436-019-06272-3 30972572
Morandi B, Bertaso S, Conboy G, Gustinelli A, Galuppi R, Tosi G, Poglayen G (2019) Crenosoma vulpis in red foxes (Vulpes vulpes) in Northern Italy. Parasitol Res 118:1981–1985. 10.1007/s00436-019-06272-330972572 10.1007/s00436-019-06272-3
38. Otranto D Cantacessi C Dantas-Torres F Brianti E Pfeffer M Genchi C Guberti V Capelli G Deplazes P The role of wild canids and felids in spreading parasites to dogs and cats in Europe. Part II: Helminths and arthropods Vet Parasitol 2015 213 2 24 37 10.1016/j.vetpar.2015.04.020 26049678
Otranto D, Cantacessi C, Dantas-Torres F, Brianti E, Pfeffer M, Genchi C, Guberti V, Capelli G, Deplazes P (2015) The role of wild canids and felids in spreading parasites to dogs and cats in Europe. Part II: Helminths and arthropods. Vet Parasitol 213(2):24–37. 10.1016/j.vetpar.2015.04.02026049678 10.1016/j.vetpar.2015.04.020
39. Peterson EN Barr SC Gould WJ Beck KA Bowman DD Use of fenbendazole for treatment of Crenosoma vulpis infection in a dog J Am Vet Med Assoc 1993 202 1483 10.2460/javma.1993.202.09.1483 8496106
Peterson EN, Barr SC, Gould WJ, Beck KA, Bowman DD (1993) Use of fenbendazole for treatment of Crenosoma vulpis infection in a dog. J Am Vet Med Assoc 202:14838496106 10.2460/javma.1993.202.09.1483
40. Pohly AG Nijveldt EA Stone MS Walden HDS Ossiboff RJ Conrado FO Infection with the fox lungworm (Crenosoma vulpis) in two dogs from New England - Two clinical reports and updated geographic distribution in North America Vet Parasitol Reg Stud Reports 2022 30 100714 10.1016/j.vprsr.2022.100714 35431072
Pohly AG, Nijveldt EA, Stone MS, Walden HDS, Ossiboff RJ, Conrado FO (2022) Infection with the fox lungworm (Crenosoma vulpis) in two dogs from New England - Two clinical reports and updated geographic distribution in North America. Vet Parasitol Reg Stud Reports 30:100714. 10.1016/j.vprsr.2022.10071435431072 10.1016/j.vprsr.2022.100714
41. Reilly GAC McGarry JW Martin M Belford C Crenosoma vulpis, the fox lungworm, in a dog in Ireland Vet Rec 2000 146 26 764 765 10.1136/vr.146.26.764 10909912
Reilly GAC, McGarry JW, Martin M, Belford C (2000) Crenosoma vulpis, the fox lungworm, in a dog in Ireland. Vet Rec 146(26):764–765. 10.1136/vr.146.26.76410909912 10.1136/vr.146.26.764
42. Remesar S García-Dios D Calabuig N Prieto A Díaz-Cao JM López-Lorenzo G López C Fernández G Morrondo P Panadero R Díaz P Cardiorespiratory nematodes and co-infections with gastrointestinal parasites in new arrivals at dog and cat shelters in north-western Spain Transbound Emerg Dis 2022 69 e3141 e3153 10.1111/tbed.14670 35880814
Remesar S, García-Dios D, Calabuig N, Prieto A, Díaz-Cao JM, López-Lorenzo G, López C, Fernández G, Morrondo P, Panadero R, Díaz P (2022) Cardiorespiratory nematodes and co-infections with gastrointestinal parasites in new arrivals at dog and cat shelters in north-western Spain. Transbound Emerg Dis 69:e3141–e3153. 10.1111/tbed.14670. (Epub 2022 Aug 9. PMID: 35880814; PMCID: PMC9804432)35880814 10.1111/tbed.14670
43. Rinaldi L Calabria G Carbone S Carrela A Cringoli G Crenosoma vulpis in dog: first case report in Italy and use of the FLOTAC technique for copromicroscopic diagnosis Parasitol Res 2007 101 1681 1684 10.1007/s00436-007-0713-6 17805573
Rinaldi L, Calabria G, Carbone S, Carrela A, Cringoli G (2007) Crenosoma vulpis in dog: first case report in Italy and use of the FLOTAC technique for copromicroscopic diagnosis. Parasitol Res 101:1681–1684. 10.1007/s00436-007-0713-617805573 10.1007/s00436-007-0713-6
44. Saari S, Näreaho A, Nikander S (2018) Canine parasites and parasitic diseases. Academic press
45. Sharma P Kobayashi T Are universal DNA primers really universal? J Appl Genet 2014 55 485 496 10.1007/s13353-014-0218-9 24839163
Sharma P, Kobayashi T (2014) Are universal DNA primers really universal? J Appl Genet 55:485–496. 10.1007/s13353-014-0218-9. (Epub 2014 May 17 PMID: 24839163)24839163 10.1007/s13353-014-0218-9
46. Schug K Krämer F Schaper R Hirzmann J Failing K Hermosilla C Taubert A Prevalence survey on lungworm (Angiostrongylus vasorum, Crenosoma vulpis, Eucoleus aerophilus) infections of wild red foxes (Vulpes vulpes) in central Germany Parasit vectors 2018 11 1 11 12 10.1186/s13071-018-2672-4 29301570
Schug K, Krämer F, Schaper R, Hirzmann J, Failing K, Hermosilla C, Taubert A (2018) Prevalence survey on lungworm (Angiostrongylus vasorum, Crenosoma vulpis, Eucoleus aerophilus) infections of wild red foxes (Vulpes vulpes) in central Germany. Parasit vectors 11(1):11–12. 10.1186/s13071-018-2672-429301570 10.1186/s13071-018-2672-4
47. Stunženas V Binkiene R Descriptiion of Crenosoma vismani n. sp. Parastic int the lungs of Lynx lynx (Carnivora: Felidae) with identification key to the species of the genus Crenosoma molin, 1861 (Nematoda: Crenosomatidae) Syst Parasitol 2021 98 73 83 10.1007/s11230-020-09961-1 33184731
Stunženas V, Binkiene R (2021) Descriptiion of Crenosoma vismani n. sp. Parastic int the lungs of Lynx lynx (Carnivora: Felidae) with identification key to the species of the genus Crenosoma molin, 1861 (Nematoda: Crenosomatidae). Syst Parasitol 98:73–83. 10.1007/s11230-020-09961-133184731 10.1007/s11230-020-09961-1
48. Taubert A Pantchev N Vrhovec MG Bauer Ch Hermosilla C Lungworm infections (Angiostrongylus vasorum, Crenosoma vulpis, Aelurostrongylus abstrusus) in dogs and cats in Germany and Denmark in 2003–2007 Vet Parasitol 2009 159 2 175 180 10.1016/j.vetpar.2008.10.005 19019555
Taubert A, Pantchev N, Vrhovec MG, Bauer Ch, Hermosilla C (2009) Lungworm infections (Angiostrongylus vasorum, Crenosoma vulpis, Aelurostrongylus abstrusus) in dogs and cats in Germany and Denmark in 2003–2007. Vet Parasitol 159(2):175–180. 10.1016/j.vetpar.2008.10.00519019555 10.1016/j.vetpar.2008.10.005
49. Tolnai Z Széll Z Sréter T Environmental determinants of the spatial distribution of Angiostrongylus vasorum, Crenosoma vulpis and Eucoleus aerophilus in Hungary Vet Parasitol 2015 207 3–4 355 358 10.1016/j.vetpar.2014.12.008 25547643
Tolnai Z, Széll Z, Sréter T (2015) Environmental determinants of the spatial distribution of Angiostrongylus vasorum, Crenosoma vulpis and Eucoleus aerophilus in Hungary. Vet Parasitol 207(3–4):355–358. 10.1016/j.vetpar.2014.12.00825547643 10.1016/j.vetpar.2014.12.008
50. Unterer S Deplazes P Arnold P Flückiger M Reusch CE Glaus TM Spontaneous Crenosoma vulpis infection in 10 dogs: laboratory, radiographic and endoscopic findings Schweiz Arch Tierheilkd 2002 144 4 174 179 10.1024/0036-7281.144.4.174 12038215
Unterer S, Deplazes P, Arnold P, Flückiger M, Reusch CE, Glaus TM (2002) Spontaneous Crenosoma vulpis infection in 10 dogs: laboratory, radiographic and endoscopic findings. Schweiz Arch Tierheilkd 144(4):174–179. 10.1024/0036-7281.144.4.17412038215 10.1024/0036-7281.144.4.174
51. Vekšins A Ponomarjova O Sandersen CH Klavina A Crenosoma vulpis associated eosinophilic bronchopneumopathy in a young dog in Latvia Vet Rec Case Rep 2021 9 4 176 10.1002/vrc2.176
Vekšins A, Ponomarjova O, Sandersen CH, Klavina A (2021) Crenosoma vulpis associated eosinophilic bronchopneumopathy in a young dog in Latvia. Vet Rec Case Rep 9(4):176. 10.1002/vrc2.17610.1002/vrc2.176
