Scanning electron microscopy of viable and calcified Onchocerca gibsoni

SCANNING ELECTRON MICROSCOPY OF VIABLE AND CALCIFIED

~NC~~CE~CA~~~~~N~ P. A. HOLDSWORTH* Department of Parasitology, University of Queensland, St. Lucia, 4067, Queensland, Australia (Received 13 June 1986) P. A. 1987. Scanning electron microscopy of viable and calcified @zchocercff gibsoni. InternationalJoumalfor Parasitology17: 957-964. Scanning electron microscopy (SEM) of adult Onchocerca gibsoni revealed that the cephalic ends of male and female specimens possessed features of little value for differentiation from other species. The marked cuticular ridges of the mid-body region of female worms were ~g~igh~d using SEM, as were the cuticular ~ulations and the body cons~ction at the anal region. Male specimens revealed the uniform annulations of the worm body along with the caudal papillae and the spicules. Microfilariae (mff) possessed cephalic papillae, lateral lines and cuticular stations similar to adult worms. The cephalic hook was the most pro~nent feature at the anterior end of the mff. Calcification of adult worms was found to be mainly intermd, however, one specimen was recorded with external calcification. Energy dispersive X-ray analysis revealed the composition of the calcification to be of calcium phosphate.

&Sk&-HOLDSWORTH

INDEX KEY WORDS: Scanning electron microscopy; Onchocerca gibsoni; microfilariae; cutim.dar ridge; annulations; cephalic hook; papillae; spicules; calcification; energy dispersive X-ray analysis; calcium phosphate.

INTRODUCTION ACCORDINGto Bain & Beveridge (1979), there are few differences in the morphology between @whocerca volvulus and Onchocerca gibsoni nodules and the host response to these parasites in man and cattle, respectively, as remarked on by Cleland & Johnston (191(l), Gilruth & Sweet (1911) and Israel (1959). The similarities of the two species has resulted, in recent years, in the use of 0. gibsoni as a model for the likely action of compounds against 0. voiv~l~ in man (Copeman, D. B., unpublished, Australian Society for Parasitology, Perth, 198 1). This resurgence of interest in 0. gibsoni necessitates a detailed understanding of the worm taxonomically and from a host-parasite viewpoint. In recent years, scting electron microscopy (SEM) has made possible the study of the external morphology of worms at a magnification and resolution not previously at~inable (Hirschm~ 1983). The purpose of this paper is to present detailed information on the microtopographic features of 0. gibsoni, along with some information on the calcification of the worms. MATJZRIALSAND METHODS of worm ~~te~ui~~ its re~~v~l~om

region of freshly killed cattle. Adult worms were dissected in physiological saline, or digested in 4% trypsin at 30°C or collagenase (Sigma Chemical Co., St. Louis, USA) (10 mg in IO0 ml distilled water) at 35’C for 2 days. Microfilariae (mff) were dissected from the uteri of gravid females. S~~n~~ng electron microscopy USES). Worms were washed several times in physiological saline and ultrasonically cleaned in saline plus detergent (Decon 90, Decon Laboratories Ltd, Conway Street, Hove, BN3 5LY, U.K.) 4 : 1 for 5 s. They were rewashed in saline, fixed in formolaceto-afcohol for 12 h, followed by 12 h exposure to 5% glutaraldehyde in 0.1 M cacodylate buffer at a pH of 7.4. Post-fixation took place in 1% osmium tetroxide in deionized water for 2 h, followed by a wash in cacodylate buffer for 5 min. The specimens were then dehydrated in an ascending series of alcohols to amyl acetate, dried in a critical point drier, mounted on stubs and sputter-coated with gold. A Philips 505 and a Cambridge Stereoscan 600SEM were used for viewing. Energy dispersive X-ray analysis (EDAX’). Specimens studied were processed in the same manner as for SEM, except that after study by SEM the gold coating was removed with 1% potassium cvanide solution and then carbon coated. Elements were &lysed using a Joel JSM 35CF SEM with a Tracer Northern TN 2000 Enerav Dispersive Spectrometer. RESULTS

host tissue. Onchocerca gibsoni nodules were collected from the brisket Source

* Present address: Coopers Animal Health Australia Limited, “Birling” (Research Station), The Northern Road, Bringel1yN.S.W. 2171.

Female worms Tixe cephalic

end appeared disc-like, with the mouth being simple and circular with a t&radiate opening. An inner and outer ring of 4 papillae encircled the mouth. Each of the papillae was spherical or pedunculate in shape. Two amphids were

957

958

P. A. HOLDSWORTH

959

SEM of 0. gibsoni

positioned in the line of the inner ring of papillae (Fig. 1). The anterior end was slightly tapered. The marked cuticular ridges, so characteristic of the genus, did not extend to the anterior extremity of the worm. The cuticular body ridges were most prominent in the mid-body region (Fig. 2). High magnification of the ridges of some specimens revealed a striated pattern running in a horizontal plane with respect to the vertical striation of the surface cuticle (Fig. 2). Whether this was a true surface feature of this species, or an artifact of processing, is difficult to interpret. Those specimens which revealed this feature were exceptionally clean. The characteristic zigzagged sununit of the ridges was present, as were the ramifications at each end. The termini of the ridges in the lateral field were mostly straight, although some were forked and a few uninterrupted ridges crossed the lateral field (Fig. 2). The lateral line was also evident in some specimens. The striation of the cuticular ridges running on a different plane to the striation of the surface cuticle showed a marked contrast in relief on the surface structure of the worms. The anal aperture appeared crescent-shaped. The amtulations and ridges of the caudal end continued to the termini of the worms. The worm body was constricted abruptly at the anus and the tail was a characteristic sausage-like projection (Fig. 3). In some specimens, a pair of rounded prominences could be seen at the distal end of the tail just anterior to the tail lobe (Fig. 4).

surrounding the terminal convex surface of the apparatus, except at the distal extremity where the spicule was bare (Fig. 8). A groove appeared to traverse the length of the right spicule (Fig. 9). The spatulate shape of the right spicule at the distal end resembles, en face, the shape of the vulval opening in the female worm (Fig. 1Q). The adanal and postanal papillae appeared round, pedunculate and similar, except for the size and numbers. Onchocerca gibsoni usually possessed 2-5 pairs of adanal and 1-2 pairs of postanal papillae (Fig. 11). The caudal end appeared slightly tapered and rounded at the base. Microfilariae The cephalic region possessed a circular ‘mouth

(Fig. 12), although on many specimens it appeared elongated (Fig. 13), which may have been an artifact of the fixation process. Surrounding the ‘mouth’ was an inner ring of 4 papillae, with an outer ring of papillae also present (Fig. 12). Such features were essentially the same as those found in adult worms. The cephalic hook in the mff studied here, was the most obvious feature of the cephalic region (Fig. 12). Cuticular annulations were present along the body of the mff, except at the cephalic end which terminated in a smooth dome-like structure (Fig. 12). Lateral lines were also present along the length of the mff (Fig. 13). No excretory or anal pore openings were detected, although these structures may have been hidden due to shrinkage in fixation. The annulations at the caudal end of the mff appeared to carry on to the termini of the worms (Fig. 14).

Male worms The cephalic end resembled

that of the female in structure, being disc-like, with a circular tri-radiate mouth opening and with pairs of oral papillae arranged in two circles. Amphids were present in the line of the inner ring of papillae (Fig. 5). The diameter of the mouth of male worms of all 3 species was approx. 2 pm when measured enface using SEM. The cuticle of the male worm possessed uniform annuli (Fig. 6) however, in some areas the annuli divided. The termini of the annuli in the lateral field appeared mostly straight. The cloaca was bordered with an elevated rim, but the body annuli did not extend onto this (Fig. 7). The right spicule was spatulate at the distal end (Fig. 7), while the left spicule revealed a sheath-lie membranous lamina FIG. 1. Cephalic

region of a female worm showing FIG. 2. Cuticular

External calcification

One nodule extracted from the brisket of a cow, and identified as containing 0. gibsoni (criteria according to Bain & Beveridge, 1979) revealed the anterior end of a female worm protruding from an opening in the nodule. This exposed end was covered, for most of its length, by a deposit of hard material (Fig. 15). SEM showed the deposit to be in a concentric formation but with crystal-like deposits at the proximal end (Fig. 16). EDAX examination of the deposit revealed it to be composed of calcium, with some phosphorus incorporated. EDAX tests, taken from three areas of the exposed part of the worm, showed equal calcium

the mouth (M), cephalic Scale bar = 10 pm.

ridges on the midbody

FIG. 5. Cephalic

(Cep) and amphid

(A).

region of a female worm. Scale bar = 50 pm.

FIG. 3. The caudal region of a female worm showing the cuticular FIG. 4. The caudal

papillae

annulations

(Ca) and the anus (An). Scale bar = 10 pm.

region of a female worm revealing a pair of rounded prominences (TI). Scale bar = 4 pm.

(Rp), just anterior

to the terminal

region of a male worm showing the inner and outer ring of papillae (Cep), the t&radiate is shown at higher magnification in the insert, and the amphids (A). Scale bar = 4 ,um. FIG. 6. Cuticular

ammlations

FIG. 7. The right spicule (Rs) protruding FIG. 8. The left spicule (Ls) protruding

at the midbody

lobe

mouth (M), which

region of a male worm. Scale bar = 50 pm.

from the cloaca, which is surrounded from the cloaca, which is surrounded

by caudal papillae by caudal papillae

(Cp). Scale bar = 10 m. (Cp). Scale bar = 10 F.

960

P. A. HOLDSWORTH

SEM of 0. gibsoni

levels along the length of the deposit (Figs. 18 & 19). The base of the exposed area, which had no’deposit, showed insignificant levels of calcium (Fig. 20). Phosphorus levels at the same three test areas revealed the first two to have low levels (Figs. 18 & 19) and the third, insignificant levels (Fig. 29). Internal calcification

EDAX revealed the composition of the calcification (Fig. 17) to be mainly of calcium and phosphorus elements (Fig. 21). The internal calcification resulted in marked distortion of the cephalic features of female worms. The calcium deposits appear lobulated, resulting in a pitting effect of the cuticle of female worms (Fig. 22). The cuticle had an atypical, thin, double-ridged . appearance (Fig. 23). Internal calcification of the caudal regions caused marked enlargement of the body ammlations along with that of the lateral line appearances (Fig. 2.4). Male 0. gibsoni were also found internally calcified. DISCUSSION This

study confirmed that the cephalic ends of adult worms possessed features of little value for species differentiation from other Onchocerca species, as suggested previously by other workers using light microscopy. The ornamentation of the female cuticle is considered to be an important criterion for species differentiation in bovine Onchocerca spp. (Ottley & Moorhouse, 1979); the present study illustrated the marked cuticular ridges of 0. gibsoni. The pair of rounded prominences found on the tails of some female specimens in this study have been recorded by other workers. Chauhan & Pande (1978) described these structures as two ‘nipple-like papillae’ in 0. gutturosa, from buffalo. Eberhard (1979) called them semi-subterminal papillae in 0. gutturosa of cattle, while Ottley & Moorhouse (1979) described in 0. gibsoni a trilobulate projection 5 pm long, which was apparently the terminal attachment of the body musculature. The structures viewed under SEM appeared secretory in function due to the debris adhering to their surface. Various functions have been attributed to spicules. It is suspected that the groove of the right spicule

961

forms a channel for sperm transfer when aligned with the left spicule. The spatulate shape of the right spicule at the distal end may function as a ‘holdfast’ for both spicules when inside the female reproductive tract. The functional roles of various surface structures of mff are at present unclear. McLaren (1972) reported that the cephalic hook in mff of various filarial genera was merely a cuticular extension of the ventral rim of the apparatus of the amphidial channel, which turns obliquely. Simpson & Laurence (1972) reported that the hook was hardened by deposition of a keratinous scleroprotein. No amphidial openings were detected on the cephalic surface as were reported in 0. volvulus mff by Martinez-Palomo & Martinez-Baez (1977). The pentagonal shape of the cephalic ends of 0. volvulus mff reported by Martinez-Palomo & Martinez-BQez (1977) was not seen in any of the 0. gibsoni mff studied here. It is suspected that the shape they observed was a result of distortion caused by processing. The lateral lines of the mff became obscure at the cephalic ends of the mff and their anterior termination could not be seen. This may have been the reason why no amphids were detected. If the lateral lines terminate anteriorly at the amphids, then both structures may have been hidden at the terminal end of the mff, due probably to the fixation process. The observation of ammli to the terminus of the tail was at variance with that of Martinez-Palomo & Martinez-Baez (1977) who reported that the annulations of 0. volvulus terminated before the end of the tail. Their electron micrographs however show the flattened armulations, which may have resulted from fixation shrinkage or collapse in the specimens. The observation of the anterior end of a female 0. gibsoni worm protruding from a nodule, and the ensuing external calcification of it, has not been reported before in this species. Schulz-Key (1975) reported that Onchocerca flexuosa females in red deer extrude their anterior ends from the nodule to release mff. However, the dense fibrous capsule of 0. gibsoni nodules and the poorly developed body muscle in the female worms make such activity unlikely (Beveridge, Kummerow &Wilkinson, 1980). It is more probable that the anterior end of the worm, in this case, remained outside the nodule instead of being drawn inside during formation as suggested by Mohammed (1931).

FIG. 9. The distal end of the right spicule. A groove (G) is seen to traverse the spicule and it is suggested in sperm transfer. Scale bar = 4 pm. FIG. 10. The vulval opening FIG. 11. The caudal

of the female revealing

a shape similar to that of the distal end of the right spicule of the male. Scale bar = 4 pm.

region of a mate worm revealing the cloaca (Cl); caudal Scale bar = 10 pm.

FIG. 12. Cephalic region of a microfilaria at ‘birth’. The cephalic the ‘mouth’ (M) surrounded by papillae (Cep). Cuticular FIG. 13. Cephalic FIG. 14. Caudal

that this is functional

papillae

(Cp) and cuticular

annulations

(Ca).

hook (Ch) is the most prominent surface feature, along with ammlations (Ca) arc also visible. Scale bar = 1 pm.

end of in ufero microfilariae. The ‘mouth here appears elongated compared with that of Fig. 12. The cuticular annulations (Ca) and the lateral line (L) are also visible. Scale bar = 5 pm. end of an in utero microfilaria

showing the cuticular annulations worm. Scale bar = 5 p.

(Ca) continuing

to the terminus

of the

962

P. A. HOLDSWORTH

963

SEM of 0. gibsoni

FIG. 22. The cephalic

region

FIG. 23. The midbody FIG. 24. The caudal

of a female

worm

showing the distortion bar= 1Opm.

effect of internal

region of a female worm showing the pitting effect of internal

calcification.

region of a female worm showing the distortion and marked enlargement due to internal calcification. Scale bar = 0.5 mm.

Because external calcification is unusual and since knowledge of the actual process involved in calcification is so limited, interpretation of this is difficult. Histology of 0. volvuZus suggests that normally calcification commences within the worm (Kershaw, Ross & Webber, 1955). In this present case, however, no gross signs of internal calcification were seen in the exposed section of the worm. Although the EDAX reading suggested the calcification to be calcium phosphate, it must be mentioned that calcium carbonate may also be significantly present, as reported in 0. volvulus by Albiez (198 S), however, since EDAX only registers elements above 11 on the periodic table, carbon does not appear on the graph reading. As carbon is a fundamental component of most biological organisms its presence can be assumed. FIG. 15. The anterior

and pitting

end of a female

worm

FIG. 16. Higher magnification

of Fig. 15 showing the crystalline

FIGS. l&19

calcification

Scale

Scale bar = 50 pm.

of the cuticular

annulations

Nitisuwirjo & Ladds (1980) suggested that calcification of 0. gibsoni in nodules may occur more frequently than that in 0. volvulus nodules in man. Albiez, Biittner & Schulz-Key (1984) reported 8% of female worms calcified in 0. voZvulus nodules, while Nitisuwirjo & Ladds (1980) recorded a 22% occurrence of focal mineralization in 0. gibsoni nodules, and also reported that some mineralization was not associated with obvious parasites and was located within the fibrous capsule. These areas may represent the calcified remnants of male worms incorporated within the fibrous tissue. The difference between external calcification of the worm outside the nodule and internal calcification inside the nodule may suggest that the nodule influences, to some extent, the form of calcification. This is not supported by 0. flexuosa females, where

protruding from the nodule showing elements. Scale bar = 0.1 mm.

FIG. 17. Section exhibiting

calcification.

a deposit

of calicum

and phosphorus

deposit. Scale bar = 0.025 mm.

in a female worm. Scale bar = 100 pm.

AND 20. EDAX of the three probe sites indicated

in Fig. 15.

FIG. 2 1. EDAX of the calcified worm shown in Fig. 17.

964

P. A. HOI.DSWORTH

the cephalic end can be extruded from the nodule with no response. Further studies on the structure and formation of the tunnels and envelopes of Onchocerca spp. may give some insight into what initiates calcification within these worms, and why, in some cases, worms can degenerate or even be calcified in parts, whereas the other regions are well preserved. Acknowledgements-I

wish to thank Mr J. Hardy for technical assistance in processin worm specimens and Mrs W. Gardiner for typing tI!e manuscript. This work was funded by the Australian Meat Research ~~~ (Grant UQ 15) to whom I express my

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ALBIEZ E. J. 1985. Calcification

ALB~EZE. J., B~TTNERD. W. & SCHULZ-KEYH. 1984. Studies onnodulesand adult Onchocerea voivtdus during a noduiectomy trial in hyperendemic villages in Liberia and Upper Volta. II. Comparison of the macrofilaria population in adult nodule carriers. Tropenmedizin und Parasitologie 35: 163-166. BAIN0. & BEVERIDGE I. 1979. Redescription d’Onchocerca gibsoni C.&J. 1910. Annalesde Parasitologic Humaine et

65: 329-388.

GILRUTHJ. A. & SWEETG. 1911. Onchocerca gibsoni: the cause of worm nod&s in Australian cattle. Department of Trade and Customs, Commonwealth of Australia, Sydney, pp. l-34. HIRSCHMANNH. 1983. SEM as a tool in nematode taxonomy. In: Concepts in Nematode Systematics (Edited by STONE A. R., PLATTH. I. & KHALILL. F.), Vol. 22, pp. 95-l 11. Academic Press, London. ISRAELM. S. 1959. The nodule in onchocerciasis. Transactions of the Royal Society of Tropical Medicine and Hygiene 53: 142-147. KERSHAWW.E., Ross J.A. & WEBBERW. A. F. 1955. Calcification in nodules containing Onchocerca volvulus. Transactions of the Royal Society of Tropical Medicine and Hygiene 49: 300.

MCLAREND. J. 1972. Ultrastructural studies on micro~~~~32.(Nematoda:Filarioidea). Parus~toZo# 65: MARTINEZ-PALO~AO A. & MARTINEZ-B~EZM. 1977. Ultrastructure of the microfilaria of Onchocerca voivuius from Mexico. Journal OfParasitology 63: 1007-1018. MOHAMMED A. S. 1931. Contribution to the study of the pathology and morbid histology of human and bovine onchocerciasis. Annals of Tropical Medicine and FarasitoIogy 25: 21 S-298.

Comparee 54: 69-80.

BEVERIDGEI., KUMMEROWE. L. & WILKINSONP. 1980. Experimental infection of laboratory rodents and calves with microfilariae of Onchocerca gibsoni. Tropenmedizin und Parasitologic 31: 82-86.

-

CHAUHANP.P. S. & PANDE B. P. 1978. variations within the species Onchocerca 0. gutturosa in buffaloes and cattle reference to the male tail and cuticular Journal of He~minthoio#

(Nematoda:Filarioidea) found in cattle in the United States. I. Systematics of 0. gutturosa and 0. lienalis with a description of 0. stilesi n.sp. Journal of Parasitology

Moroholoeical arrmllata~and

in India, with ornamen~tion.

52: 343-353.

NITISUWIRJOS. & LADDS P. W. 1980. A quantitative histopathological study of Onchocerca gibsoni noduies in cattle. Tropenmedizin-und Parasitoiogie 31: 467-474. OTTLEY M. L. & MOORHOUSED. E. 1979. Onchocerca (Nematoda:FiIarioidea) from Queensland cattle: a redescription of Onchdcercagibsoni Cleland &Johnston 19 10 and 0. lienahs (Stiles 1892). Zoologisher Anzeiger 203: 369-377.

CLE~AND J.B. & JOHNSTONH. 1910. Worm nests in Australian cattle due to Filaria (Onchocerca) gibsoni. With notes on similar structures in camels. Journal of the

SCHULZ-KEYH. 1975. Untersuchungen uber die Filarien der Cerviden in Siiddeutschland. I. Knotenbildung, Geschlechterfindung und Mikrotilarienausschuttung bei Onchocerca flexuosa (Wedl, 1856) in Rothirsch (C&us

Proceedings of the Royal Society of New South Wales 44: 156-171. EBERHAXDM. L. 1979. Studies on the Onchocerca

%&ON M. G.-& LAURENCEB. R. 1972”. Histochemical studies on microfilariae. Parasito~o~ 64: 61-88.

elaahusl. Tropenmedizin und Parasitolouie 26: 60-69.