Introduction

Seven species have been assigned to overall protostrongylid fauna of leporids (Kontrimavichus et al. 1976). These are Protostrongylus boughtoni Goble & Dougherty, 1943; Protostrongylus cuniculorum (Joyeux & Gaud, 1946) Schulz & Kadenazii, 1949; Protostrongylus kamenskyi Schulz, 1930; Protostrongylus oryctolagi Baboš, 1955; Protostrongylus pulmonalis (Frölich, 1802) Goble & Dougherty, 1943; Protostrongylus tauricus Schulz & Kadenazii, 1949 and Protostrongylus terminals (Passerini, 1884) Schulz, Orlow & Kutass, 1933.

According to the available literature the following species are known from the European brown hare: P. cuniculorum reported from Italy (Barbiera 1960) and Hungary (Babos 1961), P. terminalis reported from Belarus (Merkusheva 1960), Poland (Czaplinska et al. 1965) and Bulgaria (Yanchev 1970); P. tauricus reported from Crimea (Babos 1961), Georgia (Rodonaia 1967), Bulgaria (Genov 1970; Panayotova-Pencheva et al. 2014), Austria (Kutzer and Frey 1976), Azerbaijan (Fataliyev 2013) and Russia (Movsesyan et al. 2016); P. pulmonalis reported from Slovakia (Sitko et al. 1970), Sweden (Burgaz 1970), Germany (Gottschalk 1973; Nickel and Gottwald 1979; Forstner and Ilg 1982; Allgower 1992), Finland (Soveri and Valtonern 1983), Italy (Costantini et al. 1990), Austria, the Czech Republic (Chroust et al. 2012), Poland (Kornas et al. 2014) and France (Lesage et al. 2014); and P. oryctolagi reported from France (Lesage et al. 2014).

Protostrongylids cause serious pathological changes in the lungs and are considered by many authors as parasites of health importance for their hosts. Frolich et al. (2003) have pointed out that lungworm infections caused by protostrongylids are among the major parasitic diseases especially in young hares. These parasites cause pleuropneumonia and may lead to a general loss of condition especially if secondary bacterial infections occur (Frolich et al. 2003). According to Ilic et al. (2014) protostrongylids are very important endoparasites for hares and wild rabbits. These authors assume that the reduction in the hare population, which is recorded in whole Europe is caused by a number of reasons including protostrongyloses. Chroust et al. (2012) have found that these infections influence health and decrease the body weight of hares in Austrian and Czech hunting grounds. Other authors (Lesage et al. 2014) consider that protostrongylidoses are among the most dangerous diseases in hares that under definite conditions can be the reason in influencing their number.

Two protostrongylid species have been previously found in the European brown hare in our country—P. tauricus which has been recorded and described by Genov (1970) and Panayotova-Pencheva et al. (2014) and P. terminalis, recorded by Yanchev (1970). The objective of this study was to assess for the presence of other protostrongylid nematode species infecting the European brown hare in Bulgaria. Two protostrongylid species were found during our present investigations. One of them, no doubt, was P. tauricus, but the other one was unknown to us. In relation to the fact that there is confusion in defining the morphological characteristics among the protostrongylid species in leporids (Lesage et al. 2014), we decided after the identification of the second species to supply some morphometrical data about it in the present materials.

Materials and methods

Lungs of 790 brown hares from 190 areas of Bulgaria (Fig. 1) were investigated for presence of parasites. A standard technique for helminthological necropsy of the lungs was used, in which the trachea, the large, the middle and the small bronchi were consecutively cut. To find the small, hardly visible worms located in the bronchioles and alveoli, the abnormal lung tissues were examined as it has been previously described (Panayotova-Pencheva 2011). Nematode specimens were collected in a physiological solution and stored in 70% ethanol. The specimens used for identification were cleared with lactophenol. Pictures were taken using a light microscope “Leica DM5000 B”, supplied with a camera and software (Leica Application Suite LAS v. 3.1) as well as with a microscope “Amplival” supplied with a Web camera Logitech 4000. The measurements were made with the classical parasitological methods or through analysing computer programme Image-Pro Plus—Version 6 as it has been described by Panayotova-Pencheva and Alexandrov (2008). Representative specimens were deposited in the collection of the Institute of experimental morphology, pathology and anthropology with museum, Bulgarian Academy of Sciences, Sofia, Bulgaria.

Fig. 1
figure 1

Areas of Bulgaria from which brown hares have been obtained

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Results

Protostrongylid nematodes were found in 60 (7.6%) of the 790 necropsied lungs. Of these, 59 (7.5%) were identified as P. tauricus. Only one individual hare captured in the village of Borino (Rhodope Mountains, Smolyan district, 41.690° N, 24.309° W) harboured specimens identified as P. cuniculorum. Three intact male specimens, three posterior and one anterior ends of females and some fragments of P. cuniculorum were found in the middle bronchi. The following morphometric description of the species was made based on these. Thin nematodes with a thread form. The males are whitish and the females—pale-brown. Poorly developed labia and some papillae are visible around the mouth opening (Fig. 2). The oesophagus is cylindrical, weakly dilated in its distal part.

Fig. 2
figure 2

Anterior end of P. cuniculorum from a brown hare from Bulgaria: 1—papilla, 2—labia (original picture)

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Males

The copulatory bursa consists of one dorsal and two symmetrical lateral parts. The dorsal ray of the copulatory bursa has a half-spherical form and some well pronounced papillae (Fig. 3a). The exterodorsal rays are wide but short and do not reach the edge of the copulatory bursa (Fig. 3b). The lateral rays have a common trunk. The anterolateral ones are separated from the base of the trunk, they do not reach the edge of the copulatory bursa (Fig. 3b, c). The middle-lateral and posterolateral rays are separated a little lower of the trunk, they remain united to each other to middle of their length and almost touch the edge of the bursa (Fig. 3b,c). The ventral rays start also with a common trunk, and are separated in the middle of their length (Fig. 3b, c). The spicules (Fig. 4a) have a spongiform-comb structure. The proximal ends of the spicule stems are widened and the distal ones are more narrow and rounded. The spicule wings are transparent in their distal parts, and they are longer than the spicule stems (Fig. 4b). This peculiarity however, is visible only when the spicules are outside the body of the helminth. The gubernaculum has a capitulum, corpus and crura. The capitulum displays two pairs of spikes with pointed ends (Fig. 4a, c). The corpus is built of two thin, filiform parts that connect the capitulum with the crura (Fig. 4c). In some cases, the parts of the corpus are hardly visible (Fig. 4a). The crura of gubernaculum are well developed and their lateral surfaces are toothed (Fig. 4a). The telamon is clamp-shaped, massive and greatly chitinized (Fig. 4a).

Fig. 3
figure 3

Male P. cuniculorum from a brown hare from Bulgaria. a Dorsal ray of copulatory bursa. b Dorsolateral view of the copulatory bursa: 1—ventral rays, 2—anterolateral ray, 3—middle and posterolateral rays, 4—exterodorsal ray, 5—dorsal ray. c Ventrolateral view of the copulatory bursa: 1—ventral rays, 2—anterolateral ray, 3—middle and posterolateral rays (original pictures)

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Fig. 4
figure 4

Male P. cuniculorum from a brown hare from Bulgaria: a) Dorsal view: 1-spicules, 2-gubernaculum’s capitulum, 3-gubernaculum’s crura, 4-telamon; b) Lateral view: 1-spicule stem, 2-spicule wing; c) Dorsolateral view: 1-gubernaculum’s capitulum, 2-gubernaculum’s corpus, 3-gubernaculum’s crura (original pictures)

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Females

The posterior end of the body has a conical form and the anus is situated near to its tip (Fig. 5a). The vulva is also opened to the posterior end of the body (Fig. 5a). A small dilation of the cuticle is observed around its opening (Fig. 5b). The eggs in the uterus are oval-shaped and embryonated (Fig. 5a). Measurements of the different structures are presented in Table 1.

Fig. 5
figure 5

Female P. cuniculorum from a brown hare from Bulgaria. a Posterior end: 1—anus, 2—vulva. b Region of the vulva (original pictures)

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Table 1 Metric data (in μm) on P. terminalis and P. cuniculorum from different sources
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Discussion

Taking into account, the key for identification of protostrongylids (Kontrimavichus et al. 1976) and the morphometric descriptions of the different protostrongylid species (Boev 1975), we assumed that there were two possibilities for identification of the specimens found by us—P. terminalis and P. cuniculorum. Examining the available literature on these two species, the data provided below were found.

For the first time, P. terminalis has been described in materials from the Mountain hare (Lepus timidus L.) from Tuscany, Italy (Passerini 1884). Czaplinska et al. (1965) have found it in European brown hares from Poland. In their work, these authors have pointed out that the species (under the synonym Protostrongylus commutatus) has been previously described in materials from Poland. However, clear deviations in the morphometric data by the different authors have been observed. Czaplinska et al. (1965) provide their own results, describing the species under the name Protostrongylus terminalis (Passerini 1884) Kamensky, 1095 = P. commutatus (Diesing 1851). Unfortunately, the data of these authors are only metric (Table 1), the morphological peculiarities are not described, and the presence of a gubernaculum is not mentioned.

In Bulgaria, Yanchev (1970) has found protostrongylids in the trachea and bronchi in brown hares from the region of Godech, which is located in Northwestern Bulgaria, about 50 km northwest of Sofia. He has identified them as P. terminalis. Yanchev (1970) has provided his data about the dimensions of different helminth structures as well as those of other authors. However, there is considerable variation in the metric range for some structures. For example, the length of the male specimens is between 18 and 40 mm, of the females—between 28 and 90 mm, the length of the oesophagus of males—between 114 and 665 μm and the spicule length—between 160 and 180 μm. All authors quoted by Yanchev (1970) have not provided data about the gubernaculum. Furthermore, his data are also only metric (Table 1). There is no description of the parasite’s morphology, which does not give us enough grounds to be sure in the identification of the species.

Boev (1975) has re-described P. terminalis based on the original drawings by Passerini (1884). According to this re-description the spicules display the usual structure, the gubernaculum has a capitulum, corpus and crura. The capitulum is in the shape of a transversely lying bracket, with lateral branches, slightly pointed distally. The gubernaculum’s corpus is in the form of a thick, colourless spindle, longer than the crura. The crura are dark-coloured in the distal half and colourless in the proximal, the character of their distal ends is depicted unclearly. This description, however, is mainly schematic, and there are no taxonomic features about the differentiation of the species, such as morphology and size of the spicules, gubernaculum size, structure of the telamon, dorsal rays of copulatory bursa and the morphological features of the female specimens.

Protostrongylus cuniculorum has been originally reported by Joyeux and Gaud (1946) as a variety of Protostrongylus rufescens, known as P. rufescens var. cuniculorum. Shults and Kadenazii (1949) have elevated this variety to the species level—P. cuniculorum. The validity of the species has been later confirmed by Boev (1950). Barbiera (1960) describing the species has pointed out that the distal ends of the spicule stems were slightly enlarged and rounded, without wings, and the dorsal ray of the copulatory bursa was quadrangular in shape with six papillae.

Babos (1961) has pointed out that P. cuniculorum looked like P. terminalis (Passerini, 1884) but has left the name P. cuniculorum. He has described its features as follows: The spicule wings may overlap the ends of the stems. The capitulum of the gubernaculum has two pairs of branches very often some of the dorsal ones being hardly visible. The body of the gubernaculum is clearly visible only when it is separated from the helminth, and in a native preparation, even after colouring the body remains homogeneous with the surrounding tissues. The gubernaculum crura are not smooth, but have protrusions in both the distal and proximal parts. There is no provagina in the females.

According to Boev (1975), the morphology of the posterior end of the male specimens described by Barbiera (1960) completely corresponds to the one in the drawings of Joyeux and Gaud (1946). Probably because of this he has included the species in his monograph under the name Protostrongylus cuniculorum (Joyeux & Gaud, 1946) Schulz & Kadenazii, 1949. Taking into account, the close morphological resemblance between P. cuniculorum and P. terminalis mentioned in the literary sources, the author commented on the possible unification of the two species. According to him, however, only the equal number of the branches of gubernaculum capitulum and the equal length of the larvae supported the synonymization of the two species. But these characteristics are not that specific and can also apply to other species. Therefore, for assisting in the differential diagnosis of these two species, these characteristics should be considered together with other ones such as the spicule length, the character of their distal ends, and the morphology of gubernaculum’s crura. Boev (1975) has considered that this was not the suitable moment for synonymization of P. cuniculorum and P. terminalis due to the scanty description of P. terminalis according to the above mentioned criteria. According to him, this issue could be solved after the study on a large quantity of pulmonary helminths from hares from Tuscany, Italy, type location of P. terminalis. Until then, it would be appropriate to consider P. cuniculorum as an independent species. We fully agree with this view, and in our case we had to relate the obtained helminths to one of the two species. Having in mind the morphological features that we have observed in them, such as spicule wings that protrude over the stem ends, toothed lateral surfaces of the gubernaculum’s crura, a bigger length of gubernaculum’s crura than the gubernaculum’s corpus and the metric data about the two species (Table 1) we consider that at this stage, we should determine the nematodes found by us as P. cuniculorum. Further morphometric studies on new materials from the country and other regions, as well as the application of molecular-biological studies, would contribute to a better understanding of the validity of this species.

Conclusion

According to the available literature (Boev 1975; Babos 1961; Blasco et al. 1996) P. cuniculorum is a parasite commonly found in the European rabbit and the European brown hare in areas of southwestern and central Europe. Herein, we provide the first record for P. cuniculorum in the brown hare in Bulgaria and southeastern Europe.