Elaphostrongylus spp . from Scandinavian cervidae-a scanning electron microscope study ( SEM )

Nematodes of the genus Elaphostrongylus collected from moose (Alces alces L.), reindeer (Rangifer tarandus tarandus L.), and red deer (Cervus elaphus L.), respectively, were studied by means of scanning electron microscopy. Morphological differences in the ribs of the genital bursa were demonstrated. The Elaphostrongylus species from reindeer and red deer differed from each other in four ribs of the genital bursa. These results agree with the morphological characters of Exervi and E.rangiferi described by Cameron (1931) and Mitskevitch (1960). The genital bursa of Elaphostrongylus sp. from moose, in accordance with the description of E.alces by Steen et al. (1989) showed characteristics differing from those found in Elaphostrongylus spp. from reindeer and red deer respectively. These results support the hypothesis that there are three separate species of Elaphostrongylus present in Scandinavian Cervidae.


Introduction
The genus Elaphostrongylus was erected by Cameron (1931) for a protostrongylid species which he described and named E.cervi.Boev and Shul'ts (1950) proposed Elaphostrongylus to be the type genus of the subfamily Elaphostrongylinae.Four species of the genus Elaphostrongylus have been described on the basis of light microscopy (LM): E.cervi Cameron, 1931, E.panticola Lyubimov, 1945, E.rangiferi Mitskevich, 1960, and E.alces Steen et al., 1989.The taxonomy is based mainly on the structure of the male bursa.However the differentiation of species has been questioned by several authors.Kutzer and Prosl (1975) considered E.rangiferi to be synonymous with E.cervi.Pryadko and Boev (1971) stated that E.rangiferi and E.panticola are subspecies of E.cervi.Kontrimavichus et al. (1976) expressed the opinion that the differences in morphology are due to adaptation to different host species.
The aim of this study was to compare the males of the genus Elaphostrongylus spp.found in moose (Alces alces L.), reindeer (Rangifer tarandus tarandus L.), and red deer (Cervus elaphus L.), respectively, by means of scanning electron microscopy (SEM) in order to elucidate the taxonomy of the genus.

Materials and methods
Nematodes were collected from Swedish moose found dead or shot during the hunting season in counties in which reindeer were or were not present.The sampling of nematodes from Swedish reindeer was performed on slaughtered animals from counties also inhabited by moose.Specimens from red deer were collected in Denmark from game animals, both free living and Five.The dorsal rib is divided into two dorsodorsal, two medio-dorsal and one externo-dorsal rib.
Four.Two ribs dx. and two ribs sin.One rib sin. is short, while one rib sin.and the two ribs dx. are slightly longer than the externodorsal rib dx.
Four.Two ribs dx. and two ribs sin., ending close to the margin of the bursa. Oval.
One.The dorsal rib is divided into two buckles with two branches each.
Two.One rib dx. and one rib sin.with two branches, ending at 5/10 (n= 12) of the bursa.
Four.Two ribs dx. and two ribs sin.well developed, ending close to the margin of the bursa.
One.The dorsal rib is split into two branches, with complementary branches.
Four.Two ribs dx. and two ribs sin.They are thin, ending at the margin of the bursa.
Two.One rib dx. and Two.One rib dx. and one one rib sin.ending at rib sin.ending at 7/10 7/10 (n = 12) of the bursa (n = 6) of the bursa.farmed where neither moose nor reindeer excist.
Fourteen Elaphostrongylus males from each of moose and reindeer, and eight from red deer were examined.Seven males of Elaphostrongylus from moose originated from counties also inhabited by reindeer whereas the remaining seven did not.
The nematodes were immersion fixed for at least 24 h in a mixture of 3 % glutaraldehyde and 3 % paraformaldehyde in 0.1 M phosphate buffert (pH 7.4).The specimens were then postfixed in phosphate buffered 1 % Os04 at 4°C for 1 h, rinsed, dehydrated in an ethanol series ending in absolute ethanol.The ethanol was then replaced stepwise by Freon TF, which served as an intermediate fluid.The specimens were critical-point dried using liquid CO2, mo-unted on stubs with double adhesive tape, and sputter coated with palladium.The specimens were examined and photographed in a JEOL JSM 820 at 5-15 kv.
The lengths of the ribs were measured in proportion to the distance from the base (    A phasmidlike structure is seen in lateral view (Fig. V arrow B).Elaphostrongylus sp. in moose from different localities, irrespectively if reindeer was present, showed the same morphological structure of the bursa.

Elaphostrongylus sp. from reindeer (Fig. VI-VIII) (Table 1)
The dorsal rib has a broad base divided into two buckles (Fig. VI;  together on the inner surface, close to the margin of the bursa.The shape of the bursa is circular.The genital cone is well developed with a papilla located medially on the dorsal side (Fig.

VIII arrow).
Elaphostrongylus sp. in reindeer from localities with or without moose showed similar morphological structures of the genital bursa, which differed markedly from those found in Elaphostrongylus sp. from moose.

Elaphostrongylus sp. from red deer (Fig. IX-XI) (Table 1)
The dorsal rib is split into two branches visible as indentations on the inner surface of the bur-sa.Each branch is split into complementary branches (Fig. IX;arrows), visible as convexities on the inner surface of the bursa.
The externo-dorsal ribs ad dextram et ad sinistram (Fig. IX;3,3) are separated from the dorsal rib and slightly shorter than the dorsal rib's complementary branches.Both end at the middle (n = 5) of the bursa as pronounced knobs (Fig. IX;3,3) on its outer surface.
There are two lateral ribs on each side (Fig. X;4,5,5).These are thin, ending close together as knobs (Fig. X;4,5,5) at the inner margin of the bursa.The externo-lateral ribs ad sinistram (Fig. X;6) et ad dextram are shorter than the lateral ribs and end as pronounced knobs (Fig. X;6) at seven-tenths (n = 6) on the outer surface of the bursa.The two ventral ribs on each side are (Fig. X;7,7;8,8) thinner and longer than the externo-lateral ribs and end close together on the inner surface near the margin of the bursa.One is shaped as a knob (Fig. X;7,7) and the other as a pit (Fig. X;8,8).The shape of the bursa is circular (Fig. X).The genital cone is well developed, supported by a pair of lateral bulges on the dorsal side (Fig. XI arrows).

Discussion
The Elaphostrongylus spp.investigated from three different Scandinavian Cervidae species reveal pronounced and constant differences in the shape of the genital bursa, the bursal ribs, branches, and the genital cone (Table 1).
The bursae of Elaphostrongylus spp.from reindeer and red deer are circular, and the dorsal ribs are seen as buckles, indentations and convexities, often split into branches.

Elaphostrongylus
sp. from moose differs strongly from those of the other two Elaphostrongylus spp.The bursa and the ribs have characteristic shapes i.e the bursa is oval and has well developed long and slender ribs diverging from the base.
It is noticeable that the differences in the appearance of the bursae in Elaphostrongylus sp. in moose and reindeer are constant even if the hosts share the same habitat.Kontrimavichus et al. (1976), however, have suggested that the differences between the species in the genus Elaphostrongylus are results of their parasitism in different hosts.They demand a cross-infection study of hosts by the different elaphostrongyles to be essential for cla-   The lateral ribs (4,5) end close to the inner margin of the bursa with two knobs (4' : ",5' : ").The ventral ribs (7,8) are shorter than the lateral one and end as a knob (7) and a pit (8), respectively.The genital cone has a papilla (arrow) located medially on the dorsal side.The externo-dorsal ribs (3,3) are shorter than the complementary branches of the dorsal rib.They end in a pronounced knob (3,3) on the outer surface of the bursa.

Fig. X:
Caudal view.Ventral side (arrow).The lateral ribs (4;5,5) end as knobs (4;5,5) at the inner margin of the bursa.The externo-lateral ribs (6) are shorter than the lateral ribs (4;5,5) and end as knobs (6) on the outer surface of the bursa.The ventral ribs (7,7,8,8) end close together at the inner margin of the bursa, one as a knob (7,7) and one as a pit (8,8).The shape of the bursa is circular.rifying the differentatiation of the species of the genus.
In 1964, Enigk and Hildebrandt performed a cross-infection study with the genus Dictyocaulus in order to investigate the species of D.filaria, D.viviparus and D.eckerti.D.filaria turned out to be a specific species with other hosts than D.viviparus.D.eckerti was estimated to be synonymous of D.viviparus.
Later D.viviparus has been considered to have different strains with different patogenicity for Cervidae and Bovidae (Corrigal et al 1988).
These circumstances should as well be considered when evaluating the taxonomy of the genus Elaphostrongylus .
However, the detailed morphology of the genital bursae as elucidated by SEM in Elaphostrongylus spp.from red deer, reindeer, and moose strengthens earlier descriptions based on light microscopy of E.cervi by Cameron (1931), E.rangiferi by Mitskevich (1960) and E.alces by Steen et al. (1989).
The results therefore support the current taxonomic designation of these parasites as being three distinct species.

Four
Fig. I: Elaphostrongylus sp. from reindeer.Male genital bursa.Ventral view.Base of the bursa (arrow A) margin of the bursa (arrow B).
Fig. I B) to the margin (Fig. I A) of the genital bursa (Fig. I)-Results Common morphological traits The bursas lack distinct divisions into lobes.The bursal ribs are well developed.The genital cones are pronounced.Specific morphological traits Elaphostrongylus sp. from moose (Fig. II-IV), (Table 1).The dorsal rib is divided into two dorso-dorsal (Fig. II; 1,1), two medio-dorsal (Fig. II;2,2), and one externo-dorsal rib ad dextram (Fig. II;3), all split to the base of the dorsal rib.The dorsodorsal and the medio-dorsal ribs end as knobs on the inner and the outer surface (Fig. II;2,2) of the bursa.The externo-dorsal rib ad dextram ends at eight -tenths (Fig. II;3) (n = 7) of the bursa on the outer surface as a pronounced knob (Fig. IV;3).The slightly longer externodorsal rib ad sinistram (Fig. III;3) is situated close to the lateral ribs (Fig. III;4,5), ending as a knob on the inner surface of the bursa.The two lateral ribs ad dextram (Fig. IV;4,5) are as long as the left externo-dorsal rib (Fig. III;3) and end as two knobs on the inner surface of the bursa.The posterio-lateral rib ad sinistram (Fig. III;4) was the same length as the two right lateral ribs and like them ends as a knob on the inner surface of the bursa (Fig. IV;arrow 4).The medio-lateral rib ad sinistram (Fig. Ill;5) is shorter than the posterio-lateral rib ad sinistram ending as a knob (Fig. III;5) on the outer surface of the bursa.The externo-lateral rib (Fig. IV;6) is visible only at the right lateral view, well separated from the two lateral ribs, ending at seven-tenths (n = 8) of the bursa as a pronounced knob (Fig. IV;6) on its outer surface.The two ventral ribs (Fig. IV;7,8) end together close to the margin of the bursa as knobs (Fig. IV;7*,8*) on its inner surface, on both the right and left sides.The shape of the bursa is oval (Fig. IV,V).The genital cone has a papilla on the ventral side (Fig. V arrow A).

Fig. V :
Fig. V: Ventro-lateral view.The genital cone has a papilla (arrow A) on the ventral side.Note the phasmid-like structure (arrow B), and the oval shape of the bursa.
1,1) with two branches each.One branch is short (Fig. VI arrows) ending on the inner surface of the bursa, the other is thinn (Fig. VI;2,2) and visible as an indentation on the inner surface of the bursa.The externo-dorsal ribs ad sinistram (Fig. VI;3, arrow a) et ad dextram are separated from both the dorsal rib and the lateral ribs (Fig. VI;4,5).The externo-dorsal ribs are split into two branches: one short (Fig. VI;3) ending at fifth-tenths (n=12) of the bursa with a pronounced knob (Fig. VII;3) on its outer surface: one thin, ending diffusely on the bursa (Fig. VI,VII;arrow There are two lateral ribs on each side (Fig. VI, VIII; 4,4*,5,5*).These are well developed, longer than the other ribs, ending together as knobs (Fig. VIII;4*,5*) close to the inner margin of the bursa.The externo-lateral rib (Fig. VI,VII,VIII;6) and the lateral ribs on both sides seem to be connected at the base.The externolateral ribs are half the length of the lateral ribs ending at seven-tenths (n=12) of the bursa in pronounced knobs (Fig. VI,VII,VIII;6) on its outer surface.The two ventral ribs (Fig. VIII;7,8) on each side are longer than the externo-lateral ribs but shorter than the two lateral ribs.One of the ventral ribs ends as a knob (Fig. VIII;7) and the other as a pit (Fig. VIII;8)

Fig
Fig. VI: Dorsal view.The dorsal rib is divided into two buckles (1,1)with two branches each.One branch is short (arrows), ending on the inner surface, the other (2,2) is thinner and visible as an indentation of the inner surface of the bursa.The externo-dorsal ribs are split into two branches, one short (3) and one thin (arrow a).The externo-lateral ribs (6) are half the length of the lateral ribs (4,5).

Fig
Fig. VII:Lateral view sin.The short branch of the externo-dorsal rib ends a a pronounced knob (3) on the outer surface, while the thin branch (arrow a) ends diffusely of the bursa.The externo-lateral rib (6) ends in a knob (6) on the outer surface of the bursa.

Fig
Fig. VIII:Dorso-lateral view.The lateral ribs (4,5) end close to the inner margin of the bursa with two knobs (4' : ",5' : ").The ventral ribs (7,8) are shorter than the lateral one and end as a knob (7) and a pit (8), respectively.The genital cone has a papilla (arrow) located medially on the dorsal side.

Fig
Fig. IX: Dorsal view.The dorsal rib is split into two branches.Each branch is split into complementary branches (arrows).The externo-dorsal ribs (3,3) are shorter than the complementary branches of the dorsal rib.They end in a pronounced knob (3,3) on the outer surface of the bursa.

Fig
Fig. XI: Caudal view.The genital cone has two pairs of lateral bulges on the dorsal side (arrows).Note the circular shape of the bursa.