Introduction

The filariid nematodes are viviparous and produce their larval stages (microfilariae) in the bloodstream (Keymer 1982). While microfilariae of Eufilaria delicata Supperer, 1958 and Ornithofilaria mavis Leiper, 1909 (Nematoda: Filarioidea) readily occur in birds in Europe, occurrence of the adult stages of these nematodes is rare (Sonin 1966; Kučera 1982; Baruš 1992; Hauptmanová 2003; Votýpka et al. 2003; Hauptmanová et al. 2004; Palinauskas et al. 2005). They are known to be transmitted by dipterans including genera Simulium and Culicoides (Anderson 2000).

Adult filariids of the genus Eufilaria Seurat, 1921 (Lemdaniinae) are mostly found in subcutaneous tissue around the larynx, esophagus, and crop, and also in subcutaneous tissue of the leg joints (Anderson 2000; Bartlett 2008). E. delicata adults and microfilariae have been recorded in passerine birds (Passeriformes) of the genus Turdus (Turdidae) in Austria and France (Supperer 1958; Bain 1980), in Garrulus glandarius (Passeriformes, Corvidae) in Austria (Supperer 1958), and Corvus frugilegus (Corvidae) in Moldavia (Sonin 1966). Findings of microfilariae have also been recorded in Spain in Turdidae (López-Caballero 1978a).

The adult filariids of the genus Ornithofilaria Gönnert, 1937 (Splendidofilariinae) have been found in birds’ leg joint tissues, subcutaneous tissue, and vessel walls (Anderson 2000). Adult specimens of O. mavis have been found in the birds of the genus Turdus in England, Germany, France, Austria, Spain, and Poland (Leiper 1909; Gönnert 1937; Chabaud and Golvan 1956; Supperer 1958; López-Caballero 1978a; Okulewicz 1981, 1997), while microfilariae of this species were found repeatedly in the blood of the same host in Spain (Jimenéz-Millan and López-Caballero 1975; López-Caballero 1978b; Cano-Martil et al. 1989; López-Caballero et al. 1992). This species also has been recorded in Coccothraustes coccothraustes (Passeriformes, Fringillidae) in the microfilariae stage in the Czech Republic (Hauptmanová et al. 2004).

The aim of our study was to determine the prevalence and intensity of microfilarial infection in birds during post-nesting and pre-nesting periods in the Czech Republic and to detect any adult specimens of the species E. delicata in Turdus philomelos.

Materials and methods

The birds were captured at the locality Čerťák (49° 34′ N, 17° 59′ E), near Nový Jičín in the northeastern part of the Czech Republic. This site is situated in mixed forest at the altitude of 370–400 m a.s.l. The first phase of examination was carried out in the post-nesting period during August–September 2005, and the second in the pre-nesting period during spring migration in April 2007. The birds were captured with ornithological mist nets, identified, ringed, scanned for the presence of subcutaneous cysts, and after blood collection released back into the wild.

One drop of blood was taken from the vena ulnaris cutanea and the blood smear was made by standard procedure. The smears were air-dried and then fixed by methanol. In the laboratory, the smears were stained with a combination of May-Grünwald and Giemsa-Romanowski stains, using a method according to Pappenheim (Lukas and Jamroz 1961). The stained smears were examined microscopically for the presence of microfilariae under ×200 magnification. If microfilariae were present, their morphology was studied under ×1,000 magnification and measurements were carried out using the QuickPHOTOMICRO 2.2 software. In microfilariae, total body length, head space, tail length, maximum width of body, head width, tail width, and distance of fixed points from anterior body end were measured. Fixed point values were expressed as percentages of the total body length. In infected birds, the total number of microfilariae in a whole blood smear was counted. Infection intensity was categorized into three levels: low (1–10 microfilariae per slide), medium (11–20 microfilariae per slide), and high (>20 microfilariae per slide).

In one individual T. philomelos, a cyst was recorded on the left heel joint. The cyst was slit with a scalpel at a length of about 2 mm and the contents were removed by forceps. The incision was then disinfected with ethanol and the bird was released. No cysts were found on other parts of the body. The cyst contained nematodes, which were fixed in 96% ethanol. The nematodes were cleared with glycerin, after which they were examined using a light microscope (Olympus BX 61) equipped with differential interference contrast optics, a digital image analysis system (analySIS auto 5.0), and a drawing attachment. After examination, the specimens were stored in vials with 70% ethanol. Measurements are given in micrometers (μm), unless otherwise stated, with the means in parentheses.

Morphometric data from Leiper (1909), Gönnert (1937), Chabaud and Golvan (1956), Supperer (1958), Bain (1980), and Okulewicz (1981) were used in identifying adult nematodes. Indications from Supperer (1958), López-Caballero (1978a, b), Cano-Martil et al. (1989), López-Caballero et al. (1992), and Hauptmanová et al. (2004) were used in morphometric determination of microfilariae. Reference material (voucher specimens) of the E. delicata was deposited in the Helminthological Collection of the Institute of Parasitology, Academy of Sciences of the Czech Republic, České Budějovice, Czech Republic (Cat. No. N-946).

Morphometric differences between microfilariae of different host species and seasons were tested by Mann–Whitney U test statistics.

Results

Prevalence and intensity of microfilarial infection

During the 2005 post-nesting period, 677 birds including 31 species were examined (Table 1). Microfilariae were found in three birds (prevalence 0.4%). Three individuals of T. philomelos were infected with E. delicata.

Table 1 The list of all investigated and positive birds in the years 2005 and 2007
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During the 2007 pre-nesting period, 438 birds of 31 species were examined (Table 1). Microfilarial infection was recorded in 11 birds (prevalence 2.5%). Infected individuals belonged to species: Turdus merula (three infections with E. delicata, one mixed infection with E. delicata and O. mavis), T. philomelos (two infections with E. delicata, one mixed infection with E. delicata and O. mavis, one infection with undetermined microfilariae) Erithacus rubecula (one mixed infection with E. delicata and O. mavis, one infection with undetermined microfilariae), and Poecile montanus (one infection with undetermined microfilariae). Prevalences and intesity of infections are given in Table 2.

Table 2 Intensity of infection and prevalence in positive birds
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Morphometric characteristics of microfilariae

Microfilariae E. delicata (Fig. 1a, b) were found without cuticle cover, and the cuticle appears not to be transversally striated. Anterior body end (head space) is clearly rounded, pale, with slight prominence. The cephalic space is about as long as the body width. Interruption of the cellular stem indicates the location of the neural ganglion, not equally well visible in all specimens. The nerve ring is visible as a short brightening over the whole width of the body. The excretory pore is recognizable as a short and pale lateral spot. The inner body is visible as a distinct elongated homogeneous space, filling the entire inner space of the microfilarial body. The anal pore, like excretory pore, is visible only as a pale lateral spot, opening with the anal cell. The posterior end of the body is conical and ends with a sharply pointed tapering tail. The morphometric characteristics of the microfilariae are given in Table 3, and comparison of the fixed point values from our measurements with those of other authors are given in Table 4.

Fig. 1
figure 1

a, b Microfilaria E. delicata from host T. philomelos. 1 head space, 2 nerve ring, 3 excretory pore, 4 inner body, 5 anal pore. Scale bar = 10 μm

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Table 3 Morphometric characteristics of microfilariae Eufilaria delicata in various bird hosts (n = number of all microfilariae in all infected birds in one host species)
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Table 4 Values of fixed points for Eufilaria delicata and Eufilaria sp. according to previous references and our findings. In our measurements were indicated statistical significant differences in head space (n = number of all microfilariae in all infected birds in one host species)
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Statistically significant difference in the mean head space morphological parameters of E. delicata between host species T. philomelos and T. merula was found (Mann–Whitney U = 159.0; p = 0.005). Differences at other fixed points were not found. The difference in the mean body length of microfilariae E. delicata between T. philomelos and T. merula was statistically not significant. Statistically significant difference in the body length of E. delicata was found between post-nesting (2005) and pre-nesting (2007) period (Mann–Whitney U = 109.0; p < 0.001).

Microfilariae O. mavis (Fig. 2a, b) were covered with smooth cuticle, which appears not to be transversally striated. Front and rear end of the body were clearly rounded. The cephalic space is about as wide as its length. The caudal part is tapering, but narrowing is not very sharp and creating a broad, rounded tail. Nerve ring is visible as a short brightening over the whole width of the body. The excretory pore is visible as a short and pale lateral spot in the microfilaria. The inner body is a relatively clear space, varying in size, located closer to the posterior body end. The anal pore is structured like excretory pore and is located close to the tail part. The morphometric characteristics of the microfilariae are given in Table 5, and comparison of the fixed point values from our measurements with those of other authors are in Table 6.

Fig. 2
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a, b Microfilaria O. mavis from host T. philomelos. 1 head space, 2 nerve ring, 3 excretory pore, 4 inner body, 5 anal pore. Scale bar = 10 μm

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Table 5 Morphometric characteristics of microfilariae Ornithofilaria mavis according to a previous reference and our findings (n = number of all microfilariae in all infected birds in one host species)
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Table 6 Values of fixed points for Ornithofilaria mavis and O. böhmi according to previous references and our findings (n = number of all microfilariae in all infected birds in one host species)
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Microfilariae sp. from P. montanus (Fig. 3) had a total body length of 69–98 μm (average 83 μm). The average width was 5.3 μm (5–6 μm). The head space was rounded and as long as wide (4 μm). Nerve ring was visible only in single individual of microfilaria and appeared as a narrow brightening across the whole width of the microfilaria, with its location 23 μm from the anterior end of the body. Excretory pore was not visible in any of the measured microfilariae. Inner body was visible only in two microfilariae as a homogeneous space filling the whole inner space of the microfilaria body (44–54 μm, average 49 μm). Anal pore is visible as a brightening opening to the lateral side of the body (59–68 μm, average 63.5 μm) near to the posterior body end. The tail part is tapering to a cone shape with sharp peak. Length of tail is 11–12 μm (11.5 μm), width of tail end is 1–2 μm (1.7 μm). The fixed point values in our measurements and those for microfilaria from the blood of Parus major according to Hauptmanová (2003) and comparable measurements for Eufilaria bartlettae are shown in Table 7.

Fig. 3
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Microfilaria sp. from the host P. montanus. Scale bar = 10 μm

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Table 7 Values of fixed points for Eufilaria bartlettae according to previous references and values of fixed points for microfilariae from Poecile montanus (our findings)
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Description of the adult E. delicata nematodes from T. philomelos

Seven specimens of adult nematodes (three males, two females, two without sex determination, because they were not prepared and remain in the collection of the authors) were located in the subcutaneous cyst in the heel joint in one T. philomelos captured July 30, 2005. A high-intensity infection with E. delicata microfilariae was found in the blood of the same bird. A total of 62 and 34 birds from genus Turdus were examined in 2005 and 2007, respectively, but no other parasitic cysts were found on these or other birds.

The anterior end of the nematodes was widely rounded; posterior end was slightly narrower, but also rounded. The oral orifice was small and rounded without lips. The cephalic papillae were rudimentary or completely lost. The oral cavity was small, extending into a thin esophagus, with unmarked transition to the gut. The cuticle was thin with faint longitudinal stripes.

Male (Table 8, Fig. 4, a–f): Male body length reached approximately half of the female body length. Maximum body width at the anterior ganglion was 83–104 μm, at the level of cloaca 67–90 μm. Maximum width of esophagus was 20–21 μm. Two weakly sclerotized spicules had a slightly different length (3–22 μm difference). The width of the proximal part of the longer spicule was 18–19 μm, shorter spicule 16–18 μm. The distal ends of the spicules were pointed. Small bulges were present laterally to the cloaca, supported by rudimentary papillae. A less pronounced and similarly rounded bulge is present above the upper rim of the cloacal orifice (other measurements in Table 8.)

Table 8 Size of E. delicata or E. bartlettae males according to previous references and our findings
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Fig. 4
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Adults of E. delicata. a–f (male), g–i (female). a anterior part of male, b detail of cephalic region, c, d detail of caudal part, lateral view, e spicules, f testis around esophagus, g detail of cephalic region, h vulva region, i caudal part, lateral view. Scale bars: A (200 μm), B–H (50 μm), I (100 μm)

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Female (Table 9, Fig. 4, g–i): Body width at the anterior ganglion 154 μm, at vulva 274 μm. The gut forms a broadened ampula approximately 290–660 μm from the posterior end. Both uterus branches are filled with microfilariae.

Table 9 Size of E. delicata females according to previous references and our findings
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Discussion

The prevalence of microfilariae in birds is dependent upon many factors, including species, sex, age of host, ethology of host, locality, and sampling period (Kučera 1981). We found the prevalence of microfilarial infection to be generally low in the Czech Republic, although in the 2007 pre-nesting period, it was higher than in the 2005 post-nesting period. In hematozoan parasites, the higher prevalence in spring was interpreted to be caused by the weakening of the organism by increased load during breeding and increased number of suitable vectors, and lower number of parasites reflecting the relative abundance of juveniles in the population (Deviche et al. 2001). Another aspect may be the migration of birds and the fact that migrants move through more terrain, which increases the likelihood of encountering a higher diversity of parasite vectors (Møller and Erritzøe 1998, Smith et al. 2004, Valkiūnas 2005).

The number of microfilariae in the smear can be generally very low and does not accurately reflect their density in blood (Kučera 1982), which can be influenced by the circadian rhythm of their occurrence (they occur regularly at night, while sampling is done predominantly during the day) (Kučera 1982; Anderson 2000). Irregular occurrence of microfilariae also negatively impacts the evaluated intensity of infection, and therefore, low levels of infection estimated in blood smears may not adequately reflect the current state of the microfilarial presence in the blood stream.

In comparison with the reports for the genus Turdus (López-Caballero 1978a), our findings identically confirm a higher occurrence of microfilariae in the species T. philomelos than in the species T. merula, but in contrast, our study showed more infections caused by the species E. delicata than O. mavis. This can be due to the geographic location of our trapping sites, as well as to the trapping dates. The comparable research in Spain was done between November and March (López-Caballero 1978a).

Our findings complement the description regarding the occurrence of microfilariae E. delicata in T. philomelos and T. merula (Supperer 1958; López-Caballero 1978a, b; Bain 1980; Cano-Martil et al. 1989), confirm the presence of this species in central Europe, and expand the known distribution of E. delicata northward.

We documented the first record of O. mavis in E. rubecula. In the Czech Republic, the nematode O. mavis had been recorded in Coccothraustes coccothraustes (Hauptmanová et al. 2004). Findigs of O. mavis in T. philomelos and T. merula are new host records for the Czech Republic. We found mixed infection with E. delicata and O. mavis microfilariae in three birds (T. merula, T. philomelos, and E. rubecula). Mixed infections of this type have previously been recorded in T. philomelos and T. iliacus in Spain (López-Caballero 1978a).

Findings of microfilariae have been rare in tits (Passeriformes: Paridae). Unspecified microfilariae have been reported in Sweden for P. major (Allander and Bennett 1994) and in Russia for P. montanus (Palinauskas et al. 2005). Hauptmanová (2003) described a single microfilaria in P. major and determined it as E. bartlettae Bain, 1980. Comparison of fixed points from that individual (Hauptmanová 2003) and our results (see Table 5) showed a high level of similarity. However, the difference in body length (our material is shorter; average length 83 μm), supports the suggestion that this may not be the same species. According to the shape of the tail (conical with sharp peak), which is an important morphological feature, we placed the microfilariae from P. montanus into the genus Eufilaria.

The finding of adult nematodes E. delicata completes previously described findings in the birds of genus Turdus (Supperer 1958; Bain 1980). Values of measurable characteristics in adult individuals of E. delicata by other authors (Supperer 1958; Bain 1980) show significant differences (maximum body width, esophagus length, location of testes and cloaca, ampullary gut enlargement, vaginal length, and location of the posterior ovary). Body size measurements are not useful for species identification if the size variation of the available specimens is so extensive. In such cases, characteristic morphological traits are to be preferred for species identification. Unclear borders between inner organs may lead to inaccuracies of the measurements. Departures from the real values can be also caused by a small number of specimens measured or inherently high variability of body proportions, compression of the specimen (affects mostly the maximum body and gut width) during the preparation of the slide. The comparison of the morphological characteristics of adult males and females found in our material with the data in the descriptions from already named authors demonstrates that adult nematodes from the host T. philomelos are conspecific with E. delicata (Supperer 1958). It was also possible to identify microfilariae present in the blood of this bird.

In France, Bain (1980) described a new species of filariae named E. bartlettae only on the basis of the material from male specimens collected from T. merula.This taxon is morphologically very close to E. delicata, but it differs slightly in morphology and topography of the pericloacal bulges, as well as in its morphometrics. Considering the intraspecific variability of morphometric characteristics of males and females, we suggest the taxon E. bartlettae to be valid, probably as a sibling species to E. delicata.

Sites in the avian hosts occupied by adult Eufilaria nematodes are generally subcutaneous connective tissues of the head and neck, connective tissue around the trachea, esophagus and crop, and occasionally subcutaneous connective tissue of the groin or legs (Anderson 2000; Bartlett 2008). Subcutaneous connective tissues in birds have been reported as parasite locations for the nematode E. delicata (Supperer 1958; Bain 1980). We have observed the leg joint to be an occasional location of the nematode E. delicata in T. philomelos. Such localization is common in birds of the family Turdidae for the filarids O. mavis and O. böhmi Supperer, 1958, from which E. delicata morpholocically is very different. The species O. böhmi was considered by Sonin (1966) to be a synonym of the previous.

The adults, as well as microfilariae of E. delicate in T. philomelos, represent the first documented finding of this nematode in the Czech Republic.