Identification of the Egyptian species of Fasciola
W.M. Lotfy
a,∗, H.N. El-Morshedy
b, M. Abou El-Hoda
a, M.M. El-Tawila
c, E.A. Omar
b, H.F. Farag
aaParasitology Department, Medical Research Institute, Alexandria University, 165 El-Horreya Avenue, Alexandria, Egypt
bTropical Health Department, High Institute of Public Health, Alexandria University, Alexandria, Egypt
cNutrition Department, High Institute of Public Health, Alexandria University, Alexandria, Egypt Received 11 May 2001; accepted 22 October 2001
Abstract
Reports on the species of Fasciola present in the Nile Delta, Egypt, appear controversial. Some authors reported the presence of both Fasciola gigantica and Fasciola hepatica, others reported F.
gigantica only and mentioned that F. hepatica was found only in imported animals.
This study was an attempt to identify the species of Fasciola flukes collected from locally bred animals. Morphologic, morphoanatomic, morphometric, and chemotaxonomic criteria of the fluke isolates were studied. Speciation based on morphologic and morphometric data was not decisive due to overlap in the values of most measurements. Morphoanatomic data proved the presence of both the species, and isoelectric focusing (IEF) of fluke soluble protein confirmed the presence of both F. gigantica and F. hepatica in Egypt. © 2002 Elsevier Science B.V. All rights reserved.
Keywords: Fasciola gigantica; Fasciola hepatica; Taxonomy; Speciation; Isoelectric focusing
1. Introduction
Animal fasciolosis is highly enzootic in Egypt. Fasciola gigantica was considered the en- dogenous species of Fasciola found in the Nile Delta (Halawani and Gindy, 1957), while Fas- ciola hepatica was thought to be present only in imported animals (Soliman and Farid, 1960;
El-Azazy and Schillhorn Van Veen, 1983; Abd-Rabo and Abou-Rawash, 1998). Malacolog- ical data supported this assumption, where Lymnaea truncatula, the snail intermediate host of F. hepatica, was not detected in most of the snail surveys conducted in the Nile Delta, and Lymnaea cailliaudi was the incriminated snail intermediate host (Allam, 1992; Farag, 1998).
∗Corresponding author. Tel.:+20-3-428-2331; fax:+20-3-428-3719.
E-mail address: [email protected] (W.M. Lotfy).
0304-4017/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 0 1 7 ( 0 1 ) 0 0 6 1 3 - 6
Since late 1970s, human fasciolosis has received an increasing attention (Ragab and Farag, 1978; Farag et al., 1979), and the flow of cases to the Medical Research Institute is continuous especially from Abis rural area in the vicinity of Alexandria (Farag, 1998).
Based on egg measurements F. hepatica was the species incriminated (Farag et al., 1979;
Allam, 1992). However, this parameter is not satisfactory, due to the presence of an overlap in the egg size of both the species.
Soluble protein isoelectric focusing (IEF) a highly sensitive and specific technique was used efficiently for the differentiation of several parasite species, namely Nematodirus (Rickard et al., 1997), Diphyllobothrium (Bylund and Djupsund, 1977), Hymenolepis (Dixon and Arai, 1985), Moniezia (Johnson and Hoberg, 1989), and Fasciola (Lee and Zimmerman, 1993). Moreover, IEF could be successfully used for identification of the different strains of Echinococcus granulosus (Kumaratilake et al., 1979; Kumaratilake and Thompson, 1984;
Baldock et al., 1985).
The present study aimed at identification of the Egyptian species of Fasciola depending on morphologic, morphoanatomic and morphometric differences. IEF was also used in attempt to confirm the findings.
2. Material and methods
2.1. Collection of worms
Fasciola worms were collected from the livers of locally bred buffalo and cattle, slaugh- tered at the abattoir of the Faculty of Agriculture, Alexandria University. Only adult flukes were collected, these were identified by the presence of numerous eggs in the uterus. The flukes were washed in PBS, then incubated in PBS at 37◦C for at least 4 h to allow them to expel their gut contents (Lee et al., 1992).
2.2. Morphologic, morphoanatomic and morphometric identification
• One hundred seventy three adult Fasciola flukes were stained with alcoholic carmine hydrochloride to study the morphological details and the branching pattern of intestine, ovaries, and testes (Gradwohl, 1970). According to the different morphologic and mor- phoanatomic characters, worms were affiliated to two different groups.
• Viable worms were subjected to morphometric examination. They were measured after they have lost vigorous contractions. The following measurements were assessed: fluke length, fluke breadth, cephalic cone length, cephalic cone breadth, testes length and length of the area behind the testes.
• Transverse sections of the fluke acetabular region of six worms of group I and six worms of group II were stained by haematoxylin and eosin to study the dorsal cuticular scales.
Worms were chosen among the larger specimens of each group.
2.3. Isoelectric focusing
Preparation of protein samples. Typical and atypical worms of each group, obtained from different buffalo and cattle hosts, were studied after the IEF technique. The preparation of
the protein samples was performed according to Lee et al. (1992). Briefly, flukes were stored at−70◦C, then they were thawed, homogenized individually and centrifuged at 11 000×g for 12 min. The clear layer was obtained and protein concentrations of the samples were estimated according to Lowry et al. (1951). The protein concentration was adjusted to 1–1.5 g/dl.
2.4. Marker proteins
Marker protein kit for isoelectric points (pI’s) measurement was obtained from Pharmacia Fine Chemicals AB, Uppsala, Sweden. It contained nine different proteins with pI’s in the 3.6–10.0 pH range. An aqueous solution of protein mixture (2 g/dl) was prepared as described by Radola (1980a,b).
2.5. Polyacrylamide gel
It was prepared as described by Radola (1980a,b), and the flap technique was adopted:
ultrathin-layer polyacrylamide gels (100m) were prepared on commercially available gel-fix. After polymerization, the gels were covered, protected and stored in a refrigerator until used.
2.6. The IEF technique
Ultrathin-layer IEF was carried out using the horizontal technique in a flat-bed cham- ber. After sample application IEF was operated at 4◦C under standard running conditions (prefocusing at 450 V for 10 min, focusing at 450 V for 15 min, 800 V for 10 min, 1000 V for 5 min, 1200 V for 5 min, 1500 V for 5 min, and finally 1800 V for 5 min) and separation distance of 7 cm (Radola, 1980a,b). The gel was then stained and destained, and was left to dry at room temperature (Radola, 1980a; Frey and Radola, 1982).
The soluble protein banding patterns of F. gigantica and F. hepatica were studied and compared.
3. Results
3.1. Morphometric and morphoanatomic data
Fig. 1 demonstrates whole mounts of Fasciola flukes obtained from buffalo and cat- tle. Considering the general shape some worms resembled F. hepatica, others F. gigan- tica, while intermediate forms could not be classified. Accordingly, a detailed study of the morphoanatomy of all worms was undertaken for a preliminary identification. The morphoanatomic study focused on intestinal branching, on the shape of the ovary and the testes and on the study of cuticular scales. Morphoanatomic differences appear in Fig. 2.
Median intestinal branches in some worms revealed few branches resembling small pouches (Fig. 2A), in others numerous secondary and tertiary branches were observed (Fig. 2B). The ovarian branches were smaller and club-shaped in some worms (Fig. 2C), while in others
Fig. 1. Whole mounts of Fasciola adult flukes (scale bar: 10 mm). 1, 2 and 3 identified as group I by detailed morphoanatomy and 4, 5 and 6 identified as group II by detailed morphoanatomy.
they were larger, more numerous and gave a more complicated appearance (Fig. 2D). Also, in relation to the testicular branches, they were simple in some warms (Fig. 2E), extremely sinuous and tightly coiled in others (Fig. 2F).
Fig. 3 demonstrates the cuticular scales of the dorsal side of the acetabular region. In group I they were small and thin, while in group II they were stout long and broad at their root and were striated. Depending on these findings, worms were divided to two groups:
group I similar to F. hepatica and group II similar to F. gigantica.
3.2. Morphometric data
They are presented in Table 1. In spite of the presence of significant difference between the mean values of all parameters measured (except the breadth), range values denoted a wide overlap between the two species. The overlap was not marked in the fluke length, testes length and length of the area behind the testes.
3.3. Morphometric indices
They are presented in Table 2. Although there were significant differences between the mean values of all indices, yet the range values still showed overlap, when flukes were considered individually. The smallest overlap was observed in the index FL/FB.
Table 1
Morphometric characterization of adult Fasciola flukes isolated from animal hostsa
Group I(N=37) Group II(N=42) t-Test (p-value) Fluke length
Range 9–25 19–41 0.000
Mean (±S.D.) 16.1 (±4) 30 (±6)
Fluke breadth
Range 5–15 6–13 0.699
Mean (±S.D.) 9 (±2.2) 8.9 (±1.7)
Cephalic cone length
Range 1–4 1–4 0.002
Mean (±S.D.) 1.9 (±0.8) 2.4 (±0.6)
Cephalic cone breadth
Range 1–3 1–3 0.000
Mean (±S.D.) 1.9 (±0.7) 2.4 (±0.5)
Testes length
Range 5–14 8–19 0.000
Mean (±S.D.) 8.1 (±2.3) 13.6 (±2.8)
Length of the area behind the testes
Range 2–7 5–19 0.000
Mean (±S.D.) 4.5 (±1.3) 11.2 (±3.1)
aAll measurements are in millimeter.
Table 2
Different morphometric indices used for characterization of adult Fasciola flukes isolated from animal hosts Group I(N=37) Group II(N=42) t-Test (p-value) FL/FBa
Range 1.2–2.6 2.1–5.4 0.000
Mean (±S.D.) 1.8 (±0.4) 3.5 (±0.7)
FL/CCLb
Range 5.7–16 6.3–30 0.000
Mean (±S.D.) 9.4 (±2.6) 13.5 (±4.4)
FB/CCBc
Range 2.7–11 2.3–7 0.000
Mean (±S.D.) 5.3 (±1.9) 3.9 (±1.1)
FL/Td
Range 1.5–2.5 1.7–3 0.001
Mean (±S.D.) 2.0 (±0.2) 2.2 (±0.3)
FL/Pe
Range 2.4–6 2.1–4 0.000
Mean (±S.D.) 3.7 (±0.8) 2.8 (±0.4)
aFluke length over fluke breadth.
bFluke length over cephalic cone length.
cFluke breadth over cephalic cone breadth.
dFluke length over testes length.
eFluke length over length of the area behind the testes.
Fig. 2. Morphoanatomic differences between group I (A, C and E) and group II (B, D and F), scale bar: 1 mm. (A) and (B): median intestinal branches; (C) and (D): ovarian branches; (E) and (F): testicular branches.
3.4. Electrophoretic data
Figs. 4 and 5 show the soluble protein banding patterns of worms of the two groups, obtained from buffalo and cattle, by focusing in the pH range 5–8. Protein banding pattern of group I from the same or different hosts revealed minimal differences in the various samples. Similarly, in group II slight differences were observed between the samples. Many bands were common between the two groups, yet obvious differences were observed in the pH range 5.14–7.4.
4. Discussion
Nowadays the species of Fasciola present in the Nile Delta is not well defined. Previously, F. gigantica was the only species reported in animals. When human fasciolosis emerged, it was reported to be due to F. hepatica and importation of infected animals was considered
Fig. 3. Cuticular scales of the dorsal side of the acetabular region, the lower series represents group I, while the upper series represents group II (scale bar: 0.1 mm).
the source of propagation of infection. However, the absence of L. truncatula in an area endemic for human fasciolosis, posed a question for the possibility of local transmission.
Some researchers denied the presence of F. hepatica among local breeds of animals and pointed to the great difficulties in species differentiation (Abd-Rabo and Abou-Rawash, 1998). Proper identification of Fasciola species present in Egypt is important for control, as
Fig. 4. Soluble protein banding patterns of worms of group I. Focusing: 5–8 pH gradient. Samples: (1–3) from buffalo, (4–6) from cattle and (M) marker proteins.
Fig. 5. Soluble protein banding patterns of worms of group II. Focusing: 5–8 pH gradient. Samples: (1–4) from buffalo, (5–6) from cattle and (M) marker proteins.
identification followed by biological and ecological characterization is important to obtain the basic information necessary for the field control of each of F. hepatica and F. gigantica.
In the present work, worms were obtained from locally infected animals. Morphologic and morphoanatomic studies were performed. At the extremes of the morphologic range some flukes resembled typical F. gigantica and F. hepatica, yet intermediate forms were difficult to classify.
Depending on previous studies the branching of the intestinal caeca was an important aid for differentiation (Sahba et al., 1972; Kimura et al., 1984). Ovarian branching was reported to reveal a more complicated appearance in F. gigantica than F. hepatica (Sahba et al., 1972).
The testicular branching could as well be used for differentiation, it was extremely sinuous in F. gigantica (Varma, 1953; Bergeon and Laurent, 1970). As to the cuticular armature, the shape and size of the scales were different in the two species (Varma, 1953). Accordingly, in the present study worms of group I could be preliminarily specified as F. hepatica, those of group II as F. gigantica.
Regarding the morphometric data, there was a wide range of values for each parameter in the same group. The allometric differences of body parts and organs of trematodes can be influenced by (1) host species (Fischthal et al., 1980), (2) intensity of infection, competitive inhibition among flukes and crowding effect (Fischthal et al., 1982; Thorpe, 1965), and (3) age resistance and host reactions in previously infected hosts (Hayes et al., 1974).
From the findings of the present study, it could be concluded that when any morphometric measurement was considered alone a great overlap between the species was observed. The fluke length, testes length and length of the area behind the testes were the most important measurements.
Considering the morphometric indices, significant differences in mean values were observed between the two groups; yet there was still overlap in the range values. FL/FB revealed the smallest overlap and could be considered as an important index in
differentiation, particularly when the worms were measured in warm isotonic saline immediately after their removal from the body, as recommended by Kendall (1965).
IFF was applied to flukes of the two groups. Soluble protein banding patterns of group I from the same or different hosts (buffalo and cattle) revealed minimal differences in the various samples. Similarly, in group II slight differences were observed between the samples. According to Lee and Zimmerman (1993), the differences between flukes of the same species and from the same host may be due to intraspecific variations.
By comparing the banding of the two groups most bands appeared common; few were specific to each group. Specific differences were observed in the area of the pH range 5.14–7.4. According to Lee and Zimmerman (1993), the soluble body proteins of F. hepatica have 15 dominant peaks in the pH range 4.6–9.3, whereas those of F. gigantica have 18 dominant peaks in the same pH range; peak number 10 of F. hepatica(pI =7.12±0.012), was not present in F. gigantica, and in F. gigantica peaks 6(pI =5.61±0.05), 7(pI = 5.91±0.03), and 8(pI =5.97±0.02)were unique. These results are comparable to the present results, accordingly worms of group I could be considered as F. hepatica, those of group II as F. gigantica.
In conclusion, in the present study, by comparing the electrophoretic results with the morphologic and morphometric findings, the results were matching, so that the presence of both the Fasciola species in Egyptian animals could be confirmed. No intermediate forms resulting from hybridization between the two species were detected.
Due to the presence of allometric differences and the great overlap between the two species, morphometric measurements alone are not recommended for speciation. Mor- phoanatomical studies and IEF clearly differentiate between the two species and either can be used for species identification. Soluble protein IEF can be considered a powerful tool for differentiation between the two species of Fasciola even when the classical morphologic methods fail.
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