First phenotypic description of Fasciola hepatica/Fasciola gigantica intermediate forms from the human endemic area of the Nile Delta, Egypt

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Abstract

Fasciola gigantica is the main fasciolid species in Africa; however, F. hepatica and F. gigantica overlap in some countries. Egypt deserves mentioning because of the emerging situation of human fascioliasis in the Nile Delta area. The morphometric characteristics of fasciolid adults infecting the main livestock species present in the Nile Delta human endemic area are analyzed through a computer image analysis system (CIAS) on the basis of standardized measurements known to be useful for the differentiation of both fasciolid species. This is the first time that such a study is performed in an African country and, therefore, the results are compared to (i) F. hepatica (European Mediterranean area) and F. gigantica (Burkina Faso) standard populations, i.e. geographical areas where both species do not co-exist, and (ii) F. hepatica and F. gigantica populations from geographical areas where both species do co-exist, including the presence of intermediate forms (Iran). Results indicate the presence of F. hepatica, F. gigantica and intermediate forms (Fasciola sp.) in Egypt for the first time, and demonstrate the usefulness of CIAS for the phenotypic characterization of liver fluke adults from a concrete fascioliasis endemic area. Body roundness, body length over body width, and distance between the ventral sucker and the posterior end of the body provide useful tools for studying inter- and intraspecific morphological diversity in Fasciola adults. The application of these markers to specimens from geographical areas where F. hepatica and F. gigantica co-exist, such as in Egypt and Iran, suggest a strong population-level variation in Fasciola adult morphology.

Introduction

Fascioliasis is an important human disease caused by Fasciola hepatica and F. gigantica, transmitted by freshwater lymnaeid snails and with many livestock species acting as reservoirs (Mas-Coma, 2004). The geographical distribution of both causal agents overlaps in many African and Asian countries (Mas-Coma et al., 2005). Despite the importance to differentiate between the infection by either fasciolid species, due to their distinct epidemiological, pathological and control characteristics, there is, unfortunately, neither a direct coprological nor an indirect immunological test available for their diagnosis. The specific differentiation can only be made by either a morphological study of adult flukes (Periago et al., 2006) or by molecular tools (Marcilla et al., 2002). Hence, subjects diagnosed are currently referred to as infected by Fasciola sp. (Mas-Coma et al., 2005).

The overlapping distribution of F. hepatica and F. gigantica has also led to a long ranging controversy on the taxonomic identity of the Fasciola species found in Far East countries, in which some resemble F. hepatica, whereas others resemble F. gigantica, with intermediate forms also being present and involving phenomena such as abnormal gametogenesis, diploidy, triploidy and mixoploidy, parthenogenesis, and hybridization events between different genotypes (see review in Mas-Coma and Bargues, 1997). The existence of hybrid forms was confirmed when it was shown that Japanese fasciolids from animals presented ribosomal DNA sequences almost identical to those of F. hepatica and mitochondrial DNA sequences almost identical to those of F. gigantica (Itagaki and Tsutsumi, 1998, Itagaki et al., 1998).

Such peculiar phenomena have only been described in animal endemic areas of Asia. Nevertheless, intermediate forms have also been described in the province of Gilan, Iran, where fascioliasis poses a serious health problem, including large human epidemics (Ashrafi et al., 2006).

Fasciola gigantica is the main fasciolid species in Africa, but both fasciolids overlap in some countries. Among these Egypt deserves mentioning because of the emerging situation of human fascioliasis in the Nile Delta area (Curtale et al., 2000). In the mid-90s, 830,000 individuals were estimated to be infected and 27.7 million people were considered at risk (WHO, 1995). Surveys performed a few years later showed that the level of human endemicity was higher than initially estimated (Esteban et al., 2003), and appropriate global diagnostic and selective treatment campaigns by administration of triclabendazole for human use (Egaten®) were implemented (Curtale et al., 2005).

Additional studies about the epidemiology and transmission of the disease to humans are needed to obtain the baseline on which to establish appropriate control measures. This study represents a step further in this endeavour, by analyzing the morphometric characteristics of fasciolid adults infecting the main livestock species present in the Nile Delta human endemic area. For this reason, a computer image analysis system (CIAS) was applied (Valero et al., 2005) on the basis of standardized measurements known to be useful for the differentiation of both fasciolid species (Periago et al., 2006). Since it is the first time that such a study is performed in an African country, the results are compared to (i) F. hepatica (European Mediterranean area) and F. gigantica (Burkina Faso) standard populations, i.e. geographical areas where both species do not co-exist (data published by Periago et al., 2006), and (ii) F. hepatica and F. gigantica populations from geographical areas where both species do co-exist, including the presence of intermediate forms (Iran).

Section snippets

Parasites

Only adult flukes found in livers of naturally infected bovines (cattle and buffaloes) were used, since previous studies revealed that the definitive host species decisively influences the size of fasciolid adults and eggs, mainly because of the size of the liver duct microhabitat (Valero et al., 2001a, Valero et al., 2002). All fasciolid specimens included in the study were gravid adult flukes. The samples analyzed included the largest possible worm variability (different stages of maturity,

Results

The specimens from Egypt were first grouped according to BR, BL/BW and VS-P criteria into F. hepatica-like, F. gigantica-like or Fasciola sp.-like. The application of these criteria shows intermediate forms (or Fasciola sp.-like) in Egypt. In the same way, the specimens from Iran were grouped into F. hepatica-like, F. gigantica-like or Fasciola sp.-like.

The comparison of the range of values obtained in Egypt (Table 1) shows that besides the measurements used as criteria (BR, BL/BW and VS-P),

Discussion

According to the criteria proposed by Periago et al. (2006) for the differentiation of liver fluke adults of both fasciolid species, the results of this study indicate the presence of F. hepatica, F. gigantica and intermediate forms (Fasciola sp.) in Egypt. The existence of morphological intermediate forms of Fasciola in countries from the eastern Mediterranean region has long been suspected (Halawani and Gindy, 1957, Farag et al., 1979), but intermediate forms in Africa have not been described

Acknowledgements

Field and laboratory work of authors from the Parasitology Department of Valencia funded by Project FIS No. PI030545 of the Ministry of Health, Madrid, Projects No. BOS2002-01978 and No. SAF2006-09278 of the Ministry of Education and Science, Madrid, and the Red de Investigación de Centros de Enfermedades Tropicales—RICET (Projects No. C03/04, No. PI030545 and No. ISCIII-RETIC RD06/0021/0017 of the Programme of Redes Temáticas de Investigación Cooperativa) of the Fondo de Investigación

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