Molecular characterization of Fasciola hepatica from Sardinia based on sequence analysis of genomic and mitochondrial gene markers.

The aim of the present study is to investigate for the first time the genetic diversity of samples identified morphologically as Fasciola hepatica (Platyhelminthes: Trematoda: Digenea) (n=66) from sheep and cattle from two localities of Sardinia and to compare them with available data from other localities by partial sequences of the first (ITS-1), the 5.8S, and second (ITS-2) Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA) genes, the mitochondrial cytochrome c oxidase subunit I (COI), and nicotinamide adenine dinucleotide dehydrogenase subunit I (ND1) genes. Comparison of the sequences from Sardinia with sequences of Fasciola spp. from GenBank confirmed that all samples belong to the species F. hepatica. The nucleotide sequencing of ITS rDNA showed no nucleotide variation in the ITS-1, 5.8S and ITS-2 rDNA sequences among all Sardinian samples, comparing with two ITS-2 haplotypes in standard F. hepatica, showing a substitution C/T in 20 position 859, reported previously from Tunisia, Algeria, Australia, Uruguay and Spain. The present study shows that in Sardinian sheep and cattle there is the most frequent haplotype (FhITS-H1) of F. hepatica species from South Europe. Considering NDI sequences, the phylogenetic trees showed reliable grouping among the haplotypes of F. hepatica from Sardinia and the mitochondrial lineage I, including the main N1 haplotype, observed previously from Europe (Russia, Belarus, Ukraine and Bulgaria), Armenia, West Africa (Nigeria), America (Uruguay and USA), Asia (Turkey, Japan, and China), Georgia, Turkmenistan, Azerbaijan and Australia. Furthermore, common haplotypes FhCOI-H1 and FhCOI-H2 of F. hepatica from Sardinia also corresponded mostly to the first lineage including the main C1 haplotype reported previously from Eastern European and Western Asian populations, they belonged just to a phylogenically distinguishable clade, as F. hepatica from Australia, France, Turkey, Uruguay, Russia, Armenia, Ukraine, Belarus, Turkmenistan, USA, Tunisia and Algeria, indicating that this is the main haplotype involved in the spread of F. hepatica throughout all continents.

Molecular characterization of Fasciola spp. from the endemic area of northern Iran based on nuclear ribosomal DNA sequences.

Fasciolosis caused by Fasciola spp. (Platyhelminthes: Trematoda: Digenea) is considered as the most important helminth infection of ruminants in tropical countries, causing considerable socioeconomic problems. In the endemic regions of the North of Iran, Fasciola hepatica and Fasciola gigantica have been previously characterized on the basis of morphometric differences, but the use of molecular markers is necessary to distinguish exactly between species and intermediate forms. Samples from buffaloes and goats from different localities of northern Iran were identified morphologically and then genetically characterized by sequences of the first (ITS-1) and second (ITS-2) Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA). Comparison of the ITS of the northern Iranian samples with sequences of Fasciola spp. from GenBank showed that the examined specimens had sequences identical to those of the most frequent haplotypes of F. hepatica (n=25, 48.1%) and F. gigantica (n=20, 38.45%), which differed from each other in different variable nucleotide positions of ITS region sequences, and their intermediate forms (n=7, 13.45%), which had nucleotides overlapped between the two Fasciola species in all the positions. The ITS sequences from populations of Fasciola isolates in buffaloes and goats had experienced introgression/hybridization as previously reported in isolates from other ruminants and humans. Based on ITS-1 and ITS-2 sequences, flukes are scattered in pure F. hepatica, F. gigantica and intermediate Fasciola clades, revealing that multiple genotypes of Fasciola are able to infect goats and buffaloes in North of Iran. Furthermore, the phylogenetic trees based upon the ITS-1 and ITS-2 sequences showed a close relationship of the Iranian samples with isolates of F. hepatica and F. gigantica from different localities of Africa and Asia. In the present study, the intergenic transcribed spacers ITS-1 and ITS-2 showed to be reliable approaches for the genetic differentiation of Fasciola spp., providing bases for further studies on F. hepatica, F. gigantica and their intermediate forms in the endemic areas in Asia.

Molecular characterization of Fasciola gigantica from Mauritania based on mitochondrial and nuclear ribosomal DNA sequences.

Fasciolosis caused by Fasciola hepatica and Fasciola gigantica (Platyhelminthes: Trematoda: Digenea) is considered the most important helminth infection of ruminants in tropical countries, causing considerable socioeconomic problems. From Africa, F. gigantica has been previously characterized from Burkina Faso, Senegal, Kenya, Zambia and Mali, while F. hepatica has been reported from Morocco and Tunisia, and both species have been observed from Ethiopia and Egypt on the basis of morphometric differences, while the use of molecular markers is necessary to distinguish exactly between species. Samples identified morphologically as F. gigantica (n=60) from sheep and cattle from different geographical localities of Mauritania were genetically characterized by sequences of the first (ITS-1), the 5.8S, and second (ITS-2) Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA) genes and the mitochondrial Cytochrome c Oxidase I (COI) gene. Comparison of the sequences of the Mauritanian samples with sequences of Fasciola spp. from GenBank confirmed that all samples belong to the species F. gigantica. The nucleotide sequencing of ITS rDNA of F. gigantica showed no nucleotide variation in the ITS-1, 5.8S, and ITS-2 rDNA sequences among all samples examined and those from Burkina Faso, Kenya, Egypt and Iran. The phylogenetic trees based on the ITS-1 and ITS-2 sequences showed a close relationship of the Mauritanian samples with isolates of F. gigantica from different localities of Africa and Asia. The COI genotypes of the Mauritanian specimens of F. gigantica had a high level of diversity, and they belonged to the F. gigantica phylogenically distinguishable clade. The present study is the first molecular characterization of F. gigantica in sheep and cattle from Mauritania, allowing a reliable approach for the genetic differentiation of Fasciola spp. and providing basis for further studies on liver flukes in the African countries.

Molecular characterization of Hysterothylacium aduncum (Nematoda: Raphidascaridae) from different fish caught off the Tunisian coast based on nuclear ribosomal DNA sequences.

Larval forms of the genus Hysterothylacium have been previously reported in teleost fish from the North African coasts of central Mediterranean Sea by morphological analysis. In the present study, samples identified morphologically as Hysterothylacium aduncum (n = 62), from Merluccius merluccius, Trachurus mediterraneus and Pagellus erythrinus from different geographical locations of the Tunisian coasts, were genetically characterised by sequences of the first (ITS-1), the 5.8S and second (ITS-2) Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA). Comparison of the sequences obtained with those available in public gene databases confirmed that all the samples from the Tunisian coasts belong to a single species, namely H. aduncum. All specimens from the Tunisian coasts showed one indel in position 787 in ITS-2 sequences not reported by any of the previously published sequences from the Atlantic and Pacific Oceans, Adriatic Sea (Mediterranean Sea) and the East Greenland Sea, suggesting the existence of a population-specific pattern exhibiting a low differentiation of this parasite in this area. This is the first molecular characterization of H. aduncum from the Tunisian coasts using ITS rDNA sequences which allows the definition of genetic markers for their unequivocal identification, and provides further biological data on these nematodes in marine fish off the Tunisian coasts, improving the picture of the occurrence of these taxa in the North African coasts of central Mediterranean Sea.

Prevalence of the gastro-intestinal parasites of domestic chicken Gallus domesticus Linnaeus, 1758 in Tunisia according to the agro-ecological zones.

Helminthosis is a very important disease affecting the poultry industry, especially the traditionally reared free ranging chickens. In Tunisia, the poultry production is considered as the most important source of protein in as much as chickens provide 53 % of animal protein production. The traditionally reared poultry farming system exposes chickens to many types of parasites, however, very little work has been done to establish the extend of helminth infection in Tunisia. The aim of this work is to investigate various aspects of helminth infections. A significant difference (p < 0.01) was found between the prevalence rates of helminth parasites in the different agro-ecological zones. The highest prevalence was observed in lowland areas of northern Tunisia (Siliana district). This suggests that agro-ecology has a major influence on the distribution of helminth parasites. Recovered nematodes included Heterakis spp. (100 %), Ascaridia galli (53.33 %) and Acuaria hamulosa (37 %). The principal cestode species encountered were Hymenolepis spp. (73.33 %) and Raillietina spp. (33.33 %).

Anisakid parasites of two forkbeards (Phycis blennoides and Phycis phycis) from the eastern Mediterranean coasts in Tunisia.

Two gadiform species with a successive bathymetric and an ecological and economical importance in the Mediterranean fishing industry, Phycis blennoides and Phycis phycis, were selected for the present study. A total of 592 fresh specimens belonging to the Gadiformes genus were obtained from local commercial fisheries. The investigation was centred on anisakid parasites of 272 specimens of the greater forkbeard (P. blennoides) and 320 of the forkbeard (P. phycis) captured off the Mediterranean coasts of Tunisia (eastern Mediterranean Sea). Four species of nematodes were identified: Anisakis simplex s.1., Anisakis physeteris, Hysterothylacium aduncum and Hysterothylacium fabri. The total prevalence was 53.75% in the forkbeard and 51.47% in the greater forkbeard. The highest values of prevalence (38.75%, 2-14), mean intensity (6.74+/-3.4) and mean abundance (2.61) were all obtained for H. fabri L4 in the forkbeard. The most frequent parasite in the greater forkbeard was H. aduncum L3 with 32.35% (1-3) prevalence and values of 1.21+/-0.58 and 0.39 for mean intensity and mean abundance, respectively. The infestation parameters were also analysed according to the host length, and prevalence was highest in P. blennoides longer than 35 cm in respect of all anisakid species. Whilst in P. phycis, the highest prevalence, conditioned by H. fabri parasitisation, was found in fish with length reaching a maximum of more than 40 cm. When the data were grouped seasonally, clear patterns were observed for P. blennoides and P. phycis species, with prevalence and mean intensity of all the anisakid species peaking in spring and summer.