Parasitology Research

, Volume 110, Issue 2, pp 971–978

Morphological characterization of adult Fascioloides magna (Trematoda: Fasciolidae): first SEM report

Authors

    • Department of Pathobiology, Faculty of Veterinary MedicineUrmia University
  • Christine M. Budke
    • Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical SciencesTexas A&M University
  • Thomas M. Craig
    • Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical SciencesTexas A&M University
Original Paper

DOI: 10.1007/s00436-011-2582-2

Cite this article as:
Naem, S., Budke, C.M. & Craig, T.M. Parasitol Res (2012) 110: 971. doi:10.1007/s00436-011-2582-2

Abstract

Five adult Fascioloides magna specimens were recovered from the livers of naturally infected calves from Texas, USA. Scanning electron microscopy was used to study the morphological characteristics of the trematodes. These mature flukes measured 35–100 mm in length by 15–25 mm in width and had an oval dorsoventrally flattened body, with no anterior cone. The tegument was armed with sharp spines. Around the oral and ventral suckers, some of the spines were small, with a sharp point, while others had serrated edges with 15–22 sharp points. The surface of the oral sucker was covered by an interesting pattern of tegument, small dome-shaped and ciliated papillae. The ventral sucker showed a smooth surface and two unknown spine-like structures. There were fewer spines at the base of the genital pore than on other parts of the anterior end of the worm. At the anterior end of the ventral side, well-developed spines were observed, while at the posterior end of the ventral side, the spines were small, mostly with one or three points and blunted edges. At the posterior end of the dorsal side, the spines became progressively fewer, smaller, and shorter. Around the excretory pore, the tegument was folded, with no spines, and small groups of dome-shaped and ciliated papillae were present. The cirrus organ showed a smooth surface, with small pores on the dorsal side and small groups of tiny spines between the folds. The eggs measured 168 × 101 μm and had a protoplasmic appendage at the pole opposite the operculum. At the posterior end of the dorsal side, and toward the right, a pore with a very thin rim was present, which could be the terminus of Laurer’s canal.

Introduction

Fascioloides magna (Bassi 1875) is known as the giant liver fluke, the large American liver fluke, or the deer fluke and is an important parasite of a variety of wild and domestic ruminants in North America and Europe. A total of ten lymnaeid snails have been reported as intermediate hosts of F. magna (Foreyt 1990). In the snail, the miracidium–cercaria phase lasts 7 to 8 weeks. Cercaria exit the snail and encyst as metacercaria on vegetation which is eaten by the final host. After infection of the final host, the young flukes migrate from the gut to the liver, and their further development differs with the host species. In cattle, the flukes are immobilized by a heavy capsule and most infections are clinically inapparent. In sheep, the fluke is of pathogenic importance due to the failure of the host to limit the activity of the fluke by encapsulation. In sheep, wide tunnels are made through the liver parenchyma, and infection with only two or three flukes can result in fatal injury (Soulsby 1986). Apart from the epidemiological, genetic, and molecular findings that have already been demonstrated by other researchers (Ursprung et al. 2006; Novobilsky et al. 2007; Kralova-Hromadova et al. 2008; Bazsalovicsova et al. 2010; Reblanova et al. 2010; Radvansky et al. 2011), there is a paucity of information on F. magna using scanning electron microscopy (SEM) (Coil 1977, 1981; Fried et al. 1986; Qureshi et al. 1989). The present study reports the findings of SEM evaluation of adult F. magna removed from naturally infected calves.

Materials and methods

During a period of 6 months, from June to November 2009, five adult F. magna were removed from naturally infected calves located in Texas, USA, and sent to the Department of Nematology, University of California, Davis, California, in AFA solution (alcohol, formaldehyde, acetic acid, distilled water, and glycerine). The worms were washed in 2% sodium cacodylate buffer (pH 7.2) and then placed in 4% glutaraldehyde. The specimens were then transferred to the Electron Microscopy Facility at the Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada, for SEM study.

The trematodes were placed into fresh 4% glutaraldehyde for 2 h, post-fixed in 2% osmium tetraoxide in cacodylate buffer (pH 7.2) at 4°C for 2 h, and dehydrated through a series of increasing ethanol concentrations (50%, 75%, 96%, and 100%). They were then dried with liquid CO2 in a critical point drier (Critical Point Drier, model no. 2800, Ladd Research Industries, Burlington, VT, USA). Dried specimens were cut into three pieces; each piece was placed on a numbered aluminum stab, which was coated with gold in an ion-coating sputter (Sputter Coater, model no. E5100, Polaron Instruments, USA) and viewed with a JEOL JSM-840 scanning electron microscope operated at 20 kV. Photographs were taken with a digital acquisition system for analogue SEMs (GW Electronics, Norcross, GA, USA).

Results

Mature flukes were large and flat, measuring 35–100 mm in length by 15–25 mm in width. The flukes were reddish brown in color, covered by spiny tegument, and had no anterior cone. Ventral and oral suckers were prominent and easily observed. In addition to oral and ventral suckers, genital and excretory pores were clearly visible (Fig. 1). The surface of the oral sucker was covered by a patterned tegument and small, dome-shaped, and ciliated papillae (Figs. 2 and 3). Different types of spines were present around the oral sucker (Figs. 4, 5, and 6). Some of the spines were small, with a sharp point, and some had serrated edges with 15–22 sharp points.
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Figs. 1–6

Scanning electron micrographs of the anterior end of F. magna. 1 Oral sucker (OS), genital pore (GP), ventral sucker (VS), and excretory pore (EP). Bar, 0.02 μm 2 Oral sucker (OS) and dome-shaped papillae (arrows). Bar, 0.32 μm 3 An interesting pattern of tegument around the oral sucker (T), small dome-shaped papilla (DP), and ciliated papillae (arrows). Bar, 2.5 μm 4 Pattern of tegument around the oral sucker (T), different types of spines (S), small dome-shaped papillae (DP), and ciliated papilla (arrow). Bar, 2.5 μm 5 Around the oral sucker and different types of spines including spines with eight sharp points (white S), spines with two or three points (black S), and spines with one sharp point (arrows). Bar, 1.25 μm 6 An interesting pattern of tegument around the oral sucker (arrows) and different types of spines (S). Bar, 1.25 μm

The ventral sucker was smaller than the oral sucker (Figs. 1 and 7) and possessed a smooth surface, with two spine-like structures (Fig. 8). In addition, the ventral sucker was surrounded by a variety of spines and small groups of ciliated papillae (Figs. 9 and 10).
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Figs. 7–10

Scanning electron micrographs of the anterior end of F. magna. 7 Ventral sucker (VS) and tegument around the ventral sucker (T). Bar, 0.08 μm 8 Smooth surface of ventral sucker and two unknown spine-like structures (asterisks). Bar, 2.5 μm 9 Around the ventral sucker, different types of spines (arrows and arrowheads) and small groups of ciliated papillae (asterisks). Bar, 0.64 μm 10 Higher magnification of tegument around the ventral sucker (T) and small groups of ciliated papillae (asterisk). Bar, 2.5 μm

The genital pore was located between the two suckers (Fig. 1), and the cirrus organ had a smooth surface with small pores on the dorsal side (Figs. 11, 12, and 13). In some areas, between the folds of the cirrus organ, small pores and groups of tiny spines could be seen (Fig. 14). There were fewer spines at the base of the genital pore than on other parts of the anterior end of the worm. In some cases, eggs were observed under the cirrus organ (Fig. 12). The eggs measured 168 × 101 μm and had a protoplasmic appendage at the pole opposite the operculum (Figs. 15 and 16).
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Figs. 11–16

Scanning electron micrographs of the anterior end of F. magna. 11 Tegument around the genital pore (T), cirrus organ (C), base of cirrus organ (asterisk), and dorsal side of cirrus organ including some small pores (arrow). Bar, 0.16 μm 12 Cirrus organ (C), base of cirrus organ (asterisk), and three eggs (E). Bar, 0.2 μm 13 Surface of cirrus organ (C). Bar, 1.25 μm 14 Dorsal side of cirrus organ, some small pores (arrows), and groups of tiny spines. Bar, 2.5 μm 15 Eggs (E) and operculum (O). Bar, 0.64 μm 16 Egg (Egg) and a protoplasmic appendage at the pole opposite the operculum (A). Bar, 2.5 μm

The excretory pore was present at the posterior end of the dorsal side of the fluke (Figs. 1 and 17) and was surrounded by small groups of dome-shaped and ciliated papillae in some areas (Fig. 18). Spines differed in number, size, and shape (Fig. 19). The tegument was folded, with an absence of spines around the excretory pore (Fig. 20).
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Figs. 17–20

Scanning electron micrographs of the posterior end of F. magna. 17 Excretory pore (EP) surrounded by groups of small spines (asterisks). Bar, 0.32 μm 18 Tegument around the excretory pore (T) including small groups of dome-shaped (DP) and ciliated papillae (asterisks). Bar, 2.5 μm 19 Tegument around the excretory pore (T) including different types of spines (S). Bar, 2.5 μm 20 Tegument around the excretory pore (T) was folded (arrows) and showed no spines. Bar, 1.25 μm

The tegument pattern on the ventral side was slightly different from the dorsal side. At the anterior end of the ventral side, well-developed spines were observed. Some of the spines had one or two points while others had more (Fig. 21). A cluster of small, ciliated papillae could be seen between the spines at the anterior end of the fluke (Fig. 22). At the mid-region on the ventral side, small, dome-shaped papillae and different types of spines were present (Figs. 23 and 24). At the posterior end of the ventral side, the tegument was covered with smaller spines, mostly with one or two points and blunted edges (Figs. 25 and 26).
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Figs. 21–26

Scanning electron micrographs of F. magna: pattern of the tegument of ventral side. 21 Anterior end of the ventral side including well-developed spines. Some of spines had one or two points (arrow) and some had more (S). Clusters of small ciliated papillae were seen between the spines (asterisks). Bar, 0.64 μm 22 Tegument of the anterior end of the ventral side (T) including different types of spines (S) and cluster of small ciliated papillae (asterisk). Bar, 2.5 μm 23 Mid-region of the ventral side, different types of spines (S), and a small dome-shaped papilla (arrowhead). Bar, 2.5 μm 24 Tegument of mid-region of the ventral side (T) including different types of spines (S). Bar, 2.5 μm 25 Tegument of the posterior end of the ventral side (T) which was covered with smaller-sized spines. Bar, 0.32 μm 26 Tegument of the posterior end of the ventral side (T) with smaller-sized spines, mostly with one or two points, and blunted edges (S). Bar, 0.64 μm

At the anterior end of the dorsal side, the tegument revealed similar features to that of the anterior end of the ventral side, with very small dome-shaped papillae and small ciliated papillae (Figs. 27 and 28). At the mid-region of the dorsal side and toward the lateral part of the body, the spines were more prominent than the papillae (Fig. 29). At the posterior end of the dorsal side, and toward the right, a pore with a very thin rim could be seen (Fig. 30), which may be the terminus of Laurer’s canal. Furthermore, the spines were small, with one sharp point, and became progressively smaller and shorter at the posterior end of the dorsal side (Figs. 31 and 32).
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Figs. 27–32

Scanning electron micrographs of F. magna: pattern of the tegument of dorsal side. 27 Tegument of the anterior end of the dorsal side (T), different types of spines (black and white S), and some small dome-shaped papillae (asterisk). Bar, 1.25 μm 28 Tegument of the anterior end of the dorsal side (T) with very small sized spine (S), and small ciliated papilla (arrow). Bar, 2.5 μm 29 Tegument of mid-region of the dorsal side, towards the lateral part of the body (T). Spines are more prominent than papillae (S). Bar, 1.25 μm 30 Posterior end of the dorsal side, on the right part of the body, a pore with a very thin rim was seen (asterisk). It could be Laurer’s canal pore. Around this pore, there were few small spines with one sharp point (arrows). Bar, 0.16 μm 31 Higher magnification of the posterior end of the dorsal side. There were few small spines with one sharp point (arrows). Bar, 0.64 μm 32 Tegument around the excretory pore (T) with very small spines (arrowheads). Bar, 1.25 μm

Discussion

This paper describes, for the first time using SEM, most of the surface features of adult F. magna (Trematoda: Fasciolida) collected from naturally infected calves. Penetration of the miracidium of F. magna into the snail host Fossaria bulimoides was first described using SEM by Coil (1981). The same technique has also been used to study encysted metacercariae and newly excysted F. magna juveniles (Fried et al. 1986). In another study, the efficacy of triclabendazole against fascioliasis was investigated by Qureshi et al. (1989), and SEM images were used to show extensive damage to the tegumental surface in the treated group.

In the present study, both oral and ventral suckers in mature flukes were covered with transverse folds and had no spines. An investigation on Fasciola gigantica indicated that adult flukes were leaf-shaped with tapered anterior and posterior ends. Oral and ventral suckers had thick rims in F. hepatica (Dangprasert et al. 2001). In F. magna, different types of spines were seen around the oral sucker and ventral suckers. Some of these spines were small with a sharp point, while some had 15–22 sharp points. At the anterior part of the ventral surface of F. hepatica, the spines were small and closely spaced. Each spine had a serrated edge with 16–20 sharp points (Dangprasert et al. 2001).

The surface of the cirrus organ of F. magna showed small groups of tiny spines in some areas. In contrast, the cirrus organ of F. hepatica is covered by spines with a sharp point, primarily on the dorsal side of the cirrus organ (S. Naem, unpublished observation). A study of F. gigantica indicated that the cirrus was sausage-shaped with dorsal spines, was curved slightly ventrally, and was folded markedly anteriorly. Also, the cirrus was shown to have pseudostriations dorsally and was ornamented with variable sizes of sharp spines, while no spines were observed on the ventral area (Srimuzipo et al. 2000). In some specimens, a few eggs were seen under the cirrus organ of F. magna. The eggs had a smooth surface with an operculum and a protoplasmic appendage at the opposite pole. The latter characteristic is also used in the differentiation of eggs of F. magna from F. hepatica and F. gigantica.

Around the excretory pore of F. magna, spine-free areas were observed. Another SEM study carried out on F. gigantica showed an area of spineless tegument, posterior to the excretory pore, while ciliated papillae were observed in the same area (Mahmoud and Hegazi 2007).

In the present study, the pattern of the tegument on the ventral side was slightly different from the dorsal side. A study on the morphological features and surface topography of adult F. gigantica described the number, size, and shape of spines as well as both clusters of and solitary sensory papillae surrounding the oral sucker (Srimuzipo et al. 2000). At the mid-region on the ventral side of F. magna, spines with one or two points were more predominant than spines with three or more points. Spines on the middle region of F. gigantica were large and elongate in shape, with serrations that included 11–15 sharp points. In addition, the tegument among the spines appeared corrugated, with transverse folds alternating with grooves (Srimuzipo et al. 2000). At the posterior end on the ventral side of F. magna, the tegument was covered by smaller-sized papillae, most of which had one or two points, blunted edges, and few papillae. On the posterior quarter of F. gigantica, the spines were much smaller in size and without serrated edges (Srimuzipo et al. 2000).

At the posterior end of the oral sucker, the tegument showed similar features to that of the anterior end of the ventral side. Srimuzipo et al. (2000) found similar surface topography on both the ventral and dorsal sides of F. gigantica, except that the dorsal spines appeared fewer in number and smaller in size than the ventral spines. Interestingly, a pore with a very thin rim was observed at the posterior end on the dorsal right side of the body of F. magna and is most likely Laurer’s canal pore. Around this pore, there were a few small spines with one sharp point and no papillae.

Acknowledgments

We heartily thank members of the Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, for providing the specimens of F. magna. Also, we would like to express our gratitude to Professor Larry A. Arsenault, head of the electron microscope facility, Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada, for his assistance in providing access to SEM facilities during the study. In addition, we would like to acknowledge the valuable help of Mr. Ernie Spitzer, Chief Technician, and all of the technicians in the electron microscope facility. Lastly, we appreciate our colleague Dr. Saeed Maslak for his great assistance in editing the SEM images.

Copyright information

© Springer-Verlag 2011