Nematodes of the family Crenosomatidae are found in the respiratory tract and sinuses of various mammals (1). The family includes five genera: Paracrenosoma Yun and Kontrimavichus, 1936 (in the respiratory system of insectivores), Troglostrongylus Vevers, 1923 (in the respiratory system of felids), Prestwoodia Anderson, 1978 (in the sinuses of opossums of genus Didelphis), Otostrongylus de Bruyn, 1933 (in the respiratory tract of seals), and Crenosoma Molin, 1861 (in the trachea, bronchi, and bronchioles of carnivores and insectivores) (2). Molinofilaria Vuylsteke 1956 (in the bronchi and veins of pinnipeds) is considered similar to Otostrongylus, but the classification of this genus remains unclear (Anderson, 1978).

The genus Crenosoma is morphologically distinguishable by the presence of a striated and folded cuticle (3). Species belonging to the genus are distributed in Europe, the Americas, and Asia (2, 4–8, 10) (Table 1). The latest review of the genus Crenosoma lists 14 nominal species (43), out of which 9 are found in mustelids. Out of these, three species are found in the New World (C. brasiliense Vieira et al. 2012, C. goblei Dougherty, 1945, C. hermani Anderson, 1962) and six in the Old World [C. melesi Jančev and Genov, 1988, C. petrowi Morozov, 1939, C. schachmatovae Kontrimavichus, 1969, C. schulzi Gagarin, 1958, C. taiga Skrjabin and Petrov, 1928, C. vulpis (Dujardin, 1844)].

There is a relatively large body of literature, often confusing, with reports of various Crenosoma species in mustelids. For instance, Stunženas and Binkiene (43) list C. petrowi as a species distributed in Eurasia but among the hosts, they list two American mustelids. Such a wide distribution over two biogeographical regions is often related to a poor species definition and the absence of genetic data, as most reports are based on morphological identifications. The two most common species of Crenosoma reported in mustelids in Europe are C. melesi and C. petrowi. Surprisingly, prior to the present study, no gene sequences were known for any of the two.

In Romania, Crenosoma vulpis infection in carnivores was documented only in foxes (51, 52), and Crenosoma spp. in bears (53, 54) with limited knowledge regarding the species diversity and distribution range among other carnivores. Wild carnivores are important reservoirs for parasites that can infect domestic animals and humans (55–58). At the same time, badgers are the least studied group in this direction, even though they are reservoirs for Mycobacterium bovis (Infantes-Lorenzo et al., 2019). Romania has a remarkable diversity and abundance of mustelids, with nine extant species recorded in the country (59).

Considering the very limited knowledge and the existence of a wide variety of Crenosoma spp. parasites in mustelids, the present paper aimed to investigate the distribution, prevalence, and diversity of these species among mustelids in Romania. Additionally, identified species were also characterized by partial sequencing of two genes, and the risk factors related to sex, age, and geographical localization were analyzed simultaneously.

The examined mustelids were morphologically identified as Eurasian badger, Meles meles (n = 102), Eurasian otter, Lutra lutra (n = 20), beech marten, Martes foina (n = 36), European pine marten, Martes martes (n = 5), steppe polecat, Mustela eversmanii (n = 1), European mink, Mustela lutreola (n = 1), least weasel, Mustela nivalis (n = 2), European polecat, Mustela putorius (n = 78), and marbled polecat, Vormela peregusna (n = 1). Crenosoma spp. nematodes were detected in the trachea, bronchi and bronchioles (Figure 1) of 26 mustelids (10.6%; 95% CI 7.0–15.1), namely: 16 Eurasian badgers (15.7%; 95% CI 9.2–24.2), 9 beech martens (25%; 95% CI 12.1–42.2), and one European pine marten (20%; 95% CI 0.5–71.6). The nematodes collected from the Eurasian badgers were morphologically identified as C. melesi (n = 13, 12.7%) and C. petrowi (n = 3, 2.9%). In beech martens C. petrowi (n = 6, 16.7%), C. vulpis (n = 1, 2.8%) and Crenosoma spp. (n = 3, 8.3%) were identified, the latter being in a very bad condition, which rendered them unidentifiable to species level. None of the Eurasian badgers, was co-infected with C. melesi and C. petrowi. Co-infections with two Crenosoma species were detected in one beech marten (CJ007577) [C. petrowi + C. vulpis (2.8%)] and the only positive European pine marten (CJ007578) [C. petrowi + C. vulpis (20%)].

Adult C. melesi presented specific circular folds visible along the length of the body (Figure 2a), starting from the middle of the esophagus until after the middle of the body length, with a region in which they were less obvious, followed by their reach back in the region of the anal opening (Figure 2b). The females' body length was between 4.6 and 14.9 mm and their width from 249.9 to 971 μm. From a lateral view, the vulva protruded significantly above the cuticle. Inside the two uteri, eggs in different development stages were observed. The tail has a conic shape with two evident subterminal papillae. The adult males were 4.1 to 5.9 mm in length and 149 to 434.3 μm in width. The cuticular folds were visible until almost half of the body length, after which they disappeared. The copulatory bursa had three lobes, with the lateral ones more developed than the median one. The bursa was sustained by rays which ended in large globular papillae (Figure 2c). The two spicules were almost equal in length, and each was split in two branches in their middle and distal part (Figure 2d).

Crenosoma petrowi adults had a transparent cuticle that formed evident folds that protrude in their posterior part (Figure 3e). The rings formed by the cuticle were visible until half of the body length for female worms and only in the anterior third in males, where the folds stretch and become unapparent. The females detected in Eurasian badger hosts were 6.7–9.2 mm in length and in 402.2–406 μm in width. The ones detected in the two Martes species were 3.6 to 5.8 mm in length and 283.3–384.1 μm in width. The vulva was lacking the appendage above the cuticle, and it was localized in the anterior third of the worm. Eggs and larvae were visible inside the uteri. The anal opening was very close to the posterior extremity of the nematode (110–210 μm) (Figure 3d). Male nematodes collected from badgers were 3.4–4 mm in length and 220.3–262.3 μm in width. The male specimens collected from both species of martens were slightly smaller, 2.4–3.2 mm in length and 154.8–224.8 μm in width. The copulatory bursa had three well distinctive parts, and it is sustained by rays. The spicules were almost equal in length and slightly curved at their caudal end (Figures 3a, b). On the dorsal side of the spicules there was a thin protrusion visible in the second third of the length (Figures 3a, c). The gubernaculum had the shape of a barque from a lateral view (Figure 3b).

Crenosoma vulpis adults have a cuticular sheath that formed evident folds visible in the anterior part and were stretched in the posterior extremity. The females detected in Martes foina were 9.4–10.3 mm in length and 348.6–427 μm in width. The ones collected from Martes martes were 4.6 to 6.7 mm in length and 357.3–455.4 μm in width. The vulva was positioned almost in the middle of the body, closer to the posterior extremity and the anus was at 117.3–189.3 μm from the caudal extremity (Figure 4a). Two papillae (phasmids) are visible on the lateral parts of the tail. The eggs containing a larva were visible and measured only in one specimen collected from Martes foina and had a size of 75.2–76 × 39.7–42.7 μm (Figure 4b). No male specimens of C. vulpis were found.

The measurements of all the nematodes that were morphologically characterized are available in the Supplementary material 2.

High-quality sequences were obtained for all of the three markers for a total of 21 Crenosoma specimens, belonging to 14 hosts: ten Eurasian badgers, three beech martens, and one pine marten. The LSU sequences were highly conserved and insufficient for a clear differentiation among species (Supplementary material 3). However, the differences and distances between the cox 1 isolates were in agreement with the morphological identification of the species. The phylogenetic analysis revealed that C. melesi formed a separate clade, while C. petrowi clustered with C. vulpis and is more closely related to C. goblei (Figure 5).

According to the bioregion, overall, the differences in the prevalence of infection were significant (X² = 13.18; d.f. = 4; p = 0.01).

In the Eurasian badgers, juveniles were significantly more frequently infected as compared to adults (X² = 8.95; d.f. = 1; p = 0.002), while the differences between sex and bioregion were not significant. For the beech martens, no significant differences were identified.

Thirteen (12.75%) of the Eurasian badgers were infected with C. melesi, while the other three (2.94%) harbored C. petrowi. Among beech martens, C. petrowi was identified in six (16.67%) individuals, of which one (2.78%) was co-infected with C. vulpis. The remaining three (8.33%) were positive for Crenosoma spp. The statistical data is available in Supplementary material 4. The distribution map of the Crenosoma species and the positive hosts is shown in Figure 6.

The genus Crenosoma is widely distributed and its species infect a wide variety of mammal hosts. Crenosoma vulpis, infecting mainly canid hosts, is the most studied species of the genus, followed by C. striatum, parasitic in hedgehogs (Table 1). Although there are numerous studies which report the infection with Crenosoma, the identification was based only on the morphological characteristics, correlated with the assumed host specificity. The morphological identification of species is mainly based on the number and aspect of the anterior cuticular folds, the aspect of the female tales and the dimensions and aspects of the copulatory bursa and rays in male nematodes. Nowadays, when genetic tools are largely available, the lack of molecular data could be considered a limitation of the published studies, which could have affected the knowledge on parasite-host associations. Interestingly, up to now, there is very little information about the genetic sequences of Crenosoma species. Partial cox1 gene sequences are available for only three species in the GenBank (C. striatum, C. vulpis, and C. goblei), while SSU and/or LSU sequences are known for four species (C. striatum, C. vulpis, C. goblei, and C. mephitidis).

The present study brings important details regarding the diversity of Crenosoma species in mustelids and host-parasite association. Moreover, two species, namely C. melesi and C. petrowi were reported for the first time in Romania. Crenosoma melesi is a respiratory strongyle typically infecting Eurasian badgers, which was initially described in Bulgaria, followed by few reports in other European countries. Crenosoma melesi was identified also in other mustelid species (Table 1). Although in our study we found a higher prevalence of infection in juvenile Eurasian badgers, as no data is available from other studies across the range of this host, we cannot conclude that there is an age risk. Generally, higher prevalence in young animals has been documented for other nematode species and it is believed that this could be due to their curious behavior, increased immunity gained with the age, or differences in their food habits (52, 65).

Crenosoma petrowi was identified in three mustelid species: Meles meles, Martes foina, and Martes martes. Additionally, this species is much more similar to C. vulpis in regard to its wide range of parasitized animals, including canids and Ursus americanus, and also to its wide distribution range (Table 1). Moreover, the present results are in accordance with this statement as based on the phylogenetic analysis, C. petrowi is closely related to C. vulpis (Supplementary material 3), and genetically distant to C. melesi. However, the reports of C. petrowi in canid hosts are questionable, as artificial infections of red foxes were unsuccessful (17).

Previously, only C. vulpis was morphologically identified in red foxes from Romania (52), and is now morphologically and molecularly confirmed in two mustelid species, M. martes and M. foina. In red foxes, the abundance of C. vulpis has a strong positive relationship with the presence of wetlands, and environmental factors mostly act on the intermediate hosts, regulating the distribution pattern (52). However, animals examined in the present study were grouped based on bioregions, and both animals infected with C. vulpis originated in a continental bioregion, located in the same county (Maramureş). This infection could be associated with an endemic area of C. vulpis rather than a specific parasite-host association, with mustelids being only accidental hosts.

The absence of C. vulpis in Eurasian badgers could be related to a previous misidentification of this species in badgers' hosts, or to a negative parasite-host association. Until more information is available on this topic, we can hypothesize that badgers are unsuitable hosts that may accidentally get infected in endemic areas. The only positive M. martes was found to be co-infected with C. vulpis and C. petrowi, which to the best of our knowledge is the first report of a co-infection with two species of the Crenosoma genus in the same host. Similarly, in one M. foina we detected the same species association. These findings underline the importance of complementary identification methods, such as molecular typing of specimens, especially for genera that are known to have more than one species parasitic in a particular host. Ideally, morphological identification should be followed by molecular confirmation.

In addition, C. vulpis was detected for the first time in a European pine marten. The morphological descriptions of Crenosoma specimens from the present study are in accordance with data previously reported (8, 16), with slight differences in morphometrics.

Besides the two species of martens and the badgers, all the other mustelids were negative for infection with Crenosoma spp. For M. nivalis, M. lutreola, M. eversmanii and V. peregusna the lack of infection could be attributed to the low number of examined animals. However, 31 L. lutra and 78 M. putorius were examined with no Crenosoma nematodes detected. Both mustelids are suitable hosts and were previously found to be infected with Crenosoma spp. (Petrov, 1940; Kontrimaviciius et al., 1976; Nugaraitė et al., 2014; Kretschmar, 2016). There is only one report of infection with C. vulpis in one otter, in the Asian part of the former USSR (16). Most likely, due to its habitat and food preferences consisting mainly in crustaceans and fish (66), this host is not ecologically exposed to infective Crenosoma larvae. Infection with C. taiga was identified only once in one European polecat from a zoo in Moscow in 1940 (15) and since then, no other reports are available. More recently, infection with C. schachmatovae was reported in polecats from Lithuania (18, 19). In the present study, all the examined polecats were negative, and we could speculate that C. schachmatovae is absent in Romania (Table 1).

The present paper presents the first complex study on Crenosoma parasitic in mustelids and points up the need of respecting specific identification protocols when dealing with multiple-species parasites. Moreover, the study highlights the need for further studies in order to elucidate the host-parasite associations, pathological implications on mustelid hosts, as well as the exploration of other gene sequences and their suitability for phylogenetic taxonomy.

There are three species of the genus Crenosoma infecting Romanian mustelids. Co-infections with two species of the genus in the same animal host are possible. We report a new host-parasite association for M. martes and C. vulpis. Sequences of C. melesi and C. petrowi were described for the first time. The cox1 difference between the identified species is in accordance with the morphological identification. Further studies are needed in order to determine the host-parasite associations and to improve the understanding of the epidemiology of Crenosoma nematodes.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

The animal study was reviewed and approved by the ethical decision nr. 232 23.11.2020, approved by Bioethics Committee of USAMV Cluj-Napoca.

GD helped with the collection of the carcasses, performed the necropsies, morphologically identified the nematodes, analyzed the data, and wrote the manuscript. AI performed necropsies, the molecular work, and the statistical analysis. CG performed the necropsies and revised the manuscript. AM coordinated the study, financially supported the molecular work, and critically revised the manuscript. All authors contributed to the article and approved the submitted version.