Giovanni Garippa

Molecular characterization of Echinococcus granulosus in sheep and goats of Peloponnesus, Greece

Although cystic echinococcosis (CE) has been a recognized public health problem in Greece, molecular data are lacking regarding the types and prevalences of infecting strains of the etiological agent Echinococcus granulosus. Therefore, we investigated the prevalence of CE and determined the infecting genotypes in sheep and goats in Peloponnesus, a large region of southern Greece. Liver and lung samples were obtained from 210 sheep and 190 goats slaughtered between January and December 2005, and the number, morphology, and fertility of hydatid cysts were determined. Protoscoleces or germinal layers were collected from individual cysts (20 sheep and 20 goats), and DNA was extracted. A polymerase chain reaction (PCR)/seminested PCR system was used to distinguish the G1, G5, and G6/G7 strains, and a specific molecular diagnosis was obtained by sequencing PCR-amplified mitochondrial DNA encoding cytochrome c oxidase subunit 1 and NADH dehydrogenase I genes. The prevalence of CE was 30.4% in sheep and 14.7% in goats; fertile cysts were found in 16.2 and 7.4%, respectively. Overall, 18 of 20 sheep harbored the G1 genotype (common sheep strain), while the remaining two animals had the G3 (buffalo) strain. All 20 goats were infected with the G7 (pig) strain. These results document the prevalence of E. granulosus infection in food animals in this geographical area and reveal for the first time the presence of, at least, three parasite genotypes.

Molecular characterization of Echinococcus granulosus strains in Sardinia

Investigations were undertaken to determine the genotypes of the parasite Echinococcus granulosus that were present in livestock animals on the island of Sardinia. Liver, lung, and spleen samples were obtained from 770 sheep, 229 cattle, and 277 pigs slaughtered in Sardinia between January 2003 and April 2005, and the number and fertility of hydatid cysts were determined. Protoscoleces and/or germinal layer were collected from individual cysts, DNA was extracted from 91 samples, and polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) methods were used for identification of the strain genotype for each sample (G1, G5, G6/G7). Fragments of the mitochondrial cytochrome c oxidase subunit 1 and NADH dehydrogenase I were sequenced. Hydatid disease prevalence of 75.3, 41.5, and 9.4% were found in the organs collected from sheep, cattle, and pigs, respectively. Molecular analysis showed that 89 of 91 ovine, bovine, and swine cysts belonged to the G1 genotype (common sheep strain) of E. granulosus. Parasite isolates from two pigs were identified to belong to the G7 genotype (pig strain). Our results confirm the high prevalence of E. granulosus infection in livestock animals in Sardinia and reveal the presence of at least two parasite genotypes in Sardinia.

Genetic variation within Taenia multiceps in Sardinia, Western Mediterranean (Italy)

Investigations were undertaken on Taenia multiceps to determine if genetic variation was present within the parasites of Sardinia (Italy). Forty samples were obtained from various locations of Sardinia and deoxyribonucleic acid (DNA) was extracted. Polymerase chain reaction (PCR) was performed on NADH dehydrogenase I (ND1) and cytochrome c subunit 1 (CO1) mitochondrial genes and amplicons were then sequenced and aligned with Bioedit software. Pairwise comparison between the ND1 sequences of the T. multiceps isolates showed differences ranging from 1.27 to 2.54% using an isolate obtained from Wales as an outgroup, while COI sequences showed within the samples coming from Sardinia a lesser degree of variability, ranging from 0.22 to 0.67%. Considering the two genes, it was possible to define at least three specific genetic variants in Sardinian samples, which we have termed Tm1, Tm2, and Tm3. This is the first description of genetic variability in T. multiceps. Further investigations will be required to understand to what extent the genetic variability described in this paper would be reflected also in phenotypic differences.

Cystic echinococcosis in equids in Italy

Between March 2003 and February 2007, the livers and the lungs of 2,231 horses from various Italian regions were examined for cystic echinococcosis presence at the time of slaughter. Hydatid cysts were found in six horses, namely four from Sardinia, one from Sicily, and one from Tuscany. The location, number, morphology, and fertility of the cysts found were determined. DNA was extracted from the germinal layers and protoscoleces of the fertile cysts and polymerase chain reactions (PCR) were performed in order to strain type DNA isolates for reduced nicotinamide adenine dinucleotide dehydrogenase subunit 1 (ND1), cytochrome c oxidase subunit 1 (CO1) and 12S partial genes. The PCR products were then purified and sequenced in forward and reverse. Hydatid materials obtained from positive animals were identified as Echinococcus granulosus s.s. (old G1, sheep strain) and Echinococcus equinus (old G4, horse strain) for ND1, CO1, and 12S partial genes. This allowed us to record the presence of the E. equinus in Italy for the first time with molecular tools and also to report new data on the epidemiological situation of this parasite in Italy.

Molecular and epidemiological data on Anisakis spp. (Nematoda: Anisakidae) in commercial fish caught off northern Sardinia (western Mediterranean Sea)

Anisakiasis is a fish-borne zoonosis caused by third stage larvae of the nematode Anisakis sp. present in fish or cephalopods. This is the first contribution to the molecular identification and epidemiology of Anisakis spp. in commercial fish from the Gulf of Asinara (Sardinia, western Mediterranean Sea). Between April 2006 to November 2011, 777 specimens of 10 fish species (Engraulis encrasicolus, Merluccius merluccius, Micromesistius poutassou, Phycis blennoides, Sardina pilchardus, Sardinella aurita, Scomber colias, Sphyraena viridensis, Trachurus mediterraneus, Trachurus trachurus) were examined for Anisakis sp. larvae. A total of 1286 larvae were found in 218 fish. The great majority of larvae were located in the body cavity, and only a small part (60, 4.7%) in the muscle. All the Type I larvae (1272) were identified as Anisakis pegreffii and all the Type II (14) as Anisakis physeteris, confirming that A. pegreffii is the dominant species and the most important agent of human anisakiasis in the western Mediterranean Sea.

Genetic characterization of Fasciola hepatica from Tunisia and Algeria based on mitochondrial and nuclear DNA sequences

Fasciolosis caused by Fasciola spp. (Platyhelminthes: Trematoda: Digenea) is considered the most important helminth infection of ruminants in tropical countries, causing considerable socioeconomic problems. In the present study, samples identified morphologically as Fasciola hepatica from sheep and cattle from different geographical locations of Tunisia and Algeria 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) and mitochondrial Cytochrome c Oxidase subunit I (COI) gene. Comparison of the ITS and COI sequences of the North African samples with sequences of Fasciola spp. from GenBank confirmed that all samples from Tunisia and Algeria samples belong to a single species, namely F. hepatica. Several specimens from Tunisia and Algeria showed a substitution C/T in position 859 in the ITS-2 sequences, previously reported from Spain, suggesting that the above mentioned variant may have a common origin and spread recently throughout the three countries because of movement of infected animals. This is the first molecular characterization of F. hepatica in North Africa which provides a foundation for further studies on Fasciola spp. in Tunisia and Algeria.

Outbreak of Sciaenacotyle panceri (Monogenea) on cage-reared meagre Argyrosomus regius (Osteichthyes) from the western Mediterranean Sea

Two different batches of meagre Argyrosomus regius were stocked in the same floating cage located in north-eastern Sardinia (western Mediterranean Sea). After 4 mo, the fish started showing non-specific disease signs, such as lethargy, emaciation, gill anaemia and mortality. In total 65 specimens of meagre of both stocks were sampled for parasitological and microbiological analysis, and only 1 species of parasite, the microcotylid monogenean Sciaenacotyle panceri, was found on the gills of caged meagre. No protozoan or other metazoan parasites were found and no bacteria or viruses were isolated. After the outbreak, a total mortality rate of 5 to 10% was estimated for the older fish stock, while no noteworthy mortality was recorded in younger fish (<2%). This is the first account of S. panceri on the gills of cultured meagre as causative agent of disease and mortality. The effects of the presence of this parasite emphasise the need for disease control strategies of cultured meagre, and investigating the transfer of microcotylids from wild to caged fish.

Cystic echinococcosis in Europe and in Italy

Cystic Echinococcosis remains a public health problem especially in the Mediterranean Region and the economic impact of Echinococcosis is great despite efforts to control it. In this review, an attempt is made to assess the present epidemiological situation of Echinococcosis in Europe and Italy.

Besnoitia besnoiti among cattle in insular and northwestern Italy: endemic infection or isolated outbreaks?

BackgroundBovine besnoitiosis, caused by the apicomplexan Besnoitia besnoiti, is a chronic and debilitating disease considered as emerging in Europe. In Spain, Portugal and France it is endemic and foci of infection were recorded in Germany, Switzerland, Hungary, Greece and Italy. In Italy, cases of bovine besnoitiosis were registered both in imported and autochthonous cattle, and mostly in central regions; high seroprevalence was also revealed by an epidemiological survey performed in the southern part of the country. Aiming to update information on the disease in northwestern and insular areas of Italy, where data on bovine besnoitiosis were missing, a serosurvey was designed for the present study.MethodsThree thousand one hundred and forty bovine blood samples from both dairy and beef farms (n = 126) were collected in northwestern regions (Lombardy, Piedmont and Liguria) and in the island of Sardinia. Samples were analyzed by a standardized in-house ELISA and those resulted positive were re-tested by Western Blot (WB) for confirmation. On results obtained by both ELISA and WB, apparent (AP) and true prevalence (TP) were calculated at individual and herd levels. Further, a panel of sera resulted positive to ELISA was analyzed by IFAT.ResultsA total of 712 animals (AP = 22.7%; TP = 18.8%) and 109 farms (AP = 86.5%; TP = 88.2%) showed a positive reaction in ELISA. Only ten (AP = 0.3%; TP = 0%) specimens proceeding from five farms (AP = 3.9%; TP = 1.7%) from Lombardy were confirmed positive to the WB, corresponding to two Holstein Friesian cows and eight beef cattle. IFAT showed a low sensitivity (44.4%) scoring positive in only four samples out of 9 positive to WB.ConclusionsThe survey demonstrated that bovine besnoitiosis cannot still be considered endemic in whole Italy. In fact, independent foci of infection were registered only in Lombardy region. Therefore, a sanitary strategy aimed to increase control measures and to organize monitoring plans, by adequate diagnostic tools is necessary to avoid overestimation of B. besnoiti in Italy.

Reinstatement of Philometra jordanoi (López-Neyra, 1951) (Nematoda:Philometridae): a parasite of the Mediterranean dusky grouper Epinephelus marginatus (Lowe) (Osteichthyes, Serranidae)

Morphological examination of a single male philometrid nematode, recovered from the ovary of a dusky grouper Epinephelus marginatus caught off the north-east Mallorca (western Mediterranean), revealed morphological differences between this specimen and philometrid males from the gonads of other marine fish hosts. These differences, together with discrepancies between the descriptions of philometrid females found in the gonads of Mediterranean E. marginatus and those described to date in various hosts and/or seas, lead us to propose that Philometra jordanoi (López-Neyra, 1951) should be reinstated as a species parasitic in the gonads of the Mediterranean dusky grouper. The male of P. jordanoi is described for the first time.