New Insights Into the Genetic Variability of Fasciola Hepatica (Trematoda) in Algeria and Relationships with Other Geographic Regions Revealed By Mitochondrial DNA.

This study aims to investigate the level of genetic variability of Fasciola hepatica flukes isolated from cattle in Algeria and to determine the phylogenetic and phylogeographic relationships with sequences isolated worldwide. Mitochondrial (Cytochrome c Oxidase subunit I gene - COI) and nuclear markers (Internal Transcribed Spacers of nuclear ribosomal DNA - ITS) for 24 F. hepatica flukes isolated from 12 cattle in North Algeria were characterised. Only two haplotypes were obtained for the COI gene, resulting in a low level of genetic variation. The analysis of variation among the COI sequences isolated from around the world did not show high levels of genetic divergence, and the phylogenetic analysis revealed a genetic similarity among F. hepatica isolates from different areas of the world. The analysis of the ITS region showed a low level of variability, which prevented obtaining informative phylogenetic and phylogeographic results. The present study also revealed that specimens of F. hepatica are genetically similar in different hosts, indicating that the genetic structure among populations of this parasite is not influenced by the host species. The low levels of genetic variation for COI and ITS regions among fluke isolates from all continents are consistent with a common origin for the flukes' worldwide distribution.

Morphological and genetic variability of the cryptic Gyrodactylus sphinx and Gyrodactylus gerasevi n. sp. (Platyhelminthes: Monogenea) from the Mediterranean Sea and Black Sea: two new members of the cross-ocean distributed Gyrodactylus orecchiae species group.

Gyrodactylus sphinx Dmitrieva & Gerasev, 2000 is the only species of Gyrodactylus originally described from Aidablennius sphynx (Valenciennes) in the Black Sea. In the present study, monogeneans similar to G. sphinx are reported from the same host and from two other species of Blenniidae from the Black Sea, as well as from the Mediterranean Sea. This study aims to verify the taxonomic status of the specimens found in different hosts and localities, other than the type ones of G. sphinx. Twenty-two measurements of the haptoral structures of 169 gyrodactylids were used for the morphological study. Morphometric variability between different samples was analysed using analysis of variance, multivariate analysis of variance (MANOVA) and principal component analysis (PCA). Molecular studies were carried out using the nuclear internal transcribed spacer 2 and 5.8S ribosomal DNA regions. Network, Bayesian phylogenetic and species delimitation analyses were performed to infer the number of taxonomic units and the phylogeographic relationships occurring within and among them. MANOVA revealed a significant dependence of the morphometry of hamuli and marginal hooks on host species and regions, but a clear differentiation between samples was not confirmed by PCA. Moreover, the ranges of all dimensions overlapped between samples. However, molecular analyses suggested the occurrence of at least two taxonomic entities. The most common entity was present in individuals of the Black and Mediterranean seas, and is described here as Gyrodactylus gerasevi n. sp., whereas a second entity recognized as a G. sphinx was found only in individuals from two localities off Crimea. The monophyletic cluster grouping of these two species was placed within a large clade that also included a separate sister cluster with seven other species of the Gyrodactylus orecchiae cross-ocean species group.

Ixodid ticks on wild donkeys in a Mediterranean nature reserve (Asinara National Park): diversity and risk factors.

The Sardinian coloured donkey Equus asinus (Perissodactyla: Equidae) and its albino colour morph represent the wildlife species most typical of the island of Asinara. This Mediterranean island represents a favourable context for ticks and tick-borne diseases; however, knowledge of the tick fauna on Asinara is scarce. A total of 106 Sardinian donkeys were inspected for tick infestation from June to November 2015. All ticks found were collected, classified by stage and sex, and identified to species level. The level of infestation of each donkey was determined; both the overall tick infestation and infestations of each detected species were classified on a scale of 1-3 to give an infestation score (IS). Overall, 256 hard ticks were collected from 60 of 106 donkeys (56.6%). Rhipicephalus bursa, Haemaphysalis punctata and Hyalomma marginatum (all: Ixodida: Ixodidae) infested 26.4%, 28.3% and 6.6% of donkeys, respectively. Different variables affected the IS. With reference to overall tick infestation, a higher IS was observed in donkeys grazing on grassland and Mediterranean shrubland and in albino donkeys compared with coloured donkeys. The collected ticks included species involved in the transmission of pathogens to humans, which highlights the risks for public health in a tourist destination such as Asinara National Park.

Metazoan parasites in the head region of the bullet tuna Auxis rochei (Osteichthyes: Scombridae) from the western Mediterranean Sea.

The head region of 72 bullet tuna Auxis rochei from the western Mediterranean Sea (south-east Spain and the Strait of Gibraltar) was examined for parasites. Seven metazoan species were found in the fish from south-east Spain: three monogeneans, two trematodes and two copepods, whereas only three species were isolated in the fish from the Strait of Gibraltar. A comparison of the levels of infection of the parasites according to fish size in south-east Spain showed that the prevalence of Didymozoon auxis and the mean abundance of Allopseudaxine macrova were higher in the larger hosts (range of fork length = 38-44 cm) than in the smaller ones (33-37 cm). A comparison of the parasite infections according to geographical region showed that the mean abundances of Nematobothriinae gen. sp. and Caligus bonito were higher in fish from south-east Spain than in those from the Strait of Gibraltar. A comparison of the parasite fauna of A. rochei from the Mediterranean Sea with the published data on Auxis spp. from the Atlantic, Indian and Pacific Oceans revealed the closest similarity between the Mediterranean A. rochei and the Atlantic A. thazard.

New insights on metastrongyloid lungworms infecting cats of Sardinia, Italy.

In addition to the well-known Aelurostrongylus abstrusus (Strongylida: Angiostrongylidae), Troglostrongylus brevior (Strongylida: Crenosomatidae) has recently been diagnosed as a causative agent of bronco-pulmonary infections of cats in Spain and Italy. However, information concerning the impact of this species of lungworm on feline population is limited to a few case reports. From July 2011 to May 2013 an epidemiological survey was carried out on Sardinia island (Italy), where 107 individual faecal samples were examined by Baermann technique, and first-stage larvae were identified based on their morphology and characterization of molecular markers. The 29.9% (32/107) of cats examined were infested by broncho-pulmonary nematodes and, although A. abstrusus was the most frequently detected (n=27; 25.2%), larvae of T. brevior were also found (n=7; 6.5%). In addition, two cats (1.9%) were co-infested by both species. Overall metastrongyloid infection was higher in female cats (n=22; 38.6%) than in males (n=10; 20%) (χ(2)=4.39; p=0.036). The mean age of positive animals was 21.1 (±29.8) months, being infected animals from 2 months to 10 years of age. Of the 32 animals that scored positive for lungworms only 6 (18.8%) displayed a respiratory condition associated with lungworm infestations. Biomolecular characterization confirmed the morphological diagnosis of A. abstrusus. Positive samples that were identified at genus level as Troglostrongylus spp. were molecularly characterized as T. brevior. This study represents the first epidemiological survey on metastrongyloid lungworms of domestic cats in Sardinia and the first report of T. brevior on this island.

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.

Cercopithifilaria spp. in dogs in Sardinia Island (Italy).

A survey on Cercopithifilaria spp. was carried out on owned and kennelled dogs in Sardinia, Italy. A total of 180 dogs were sampled and tested by microscopic detection or PCR of dermal microfilariae in skin snip sediments. The overall prevalence for Cercopithifilaria spp. at both microscopy and molecular tests was 9.4 % (17/180), while 8.3 % (15/180) of dogs scored positive at microscopic detection of sediments only. Of the 225 microfilariae measured, 212 were identified as Cercopithifilaria bainae and the remaining as Cercopithifilaria sp. II. All samples were molecularly processed for specific amplification of cytochrome oxidase subunit 1 (cox1) and ribosomal 12S gene fragments. The Basic Local Alignment Search Tool analysis of the cox1 and 12S sequences here obtained showed a high nucleotide similarity (99 and 100 %, respectively) with those of C. bainae available in GenBank. In particular, cox1 haplotype I (HI; n=14), haplotype HXVIII (n=2), and a new haplotype, named HXIX (n=1), differing for a single polymorphism from HI, were detected. This study reports data on the occurrence, distribution, and genetic makeup of C. bainae and Cercopithifilaria sp. II infesting dogs in Sardinia, suggesting that these filarioids are spread in areas where Rhipicephalus sanguineus sensu lato ticks occur.

Morphological and molecular characterization of bovine coenurosis in Sardinia, Italy.

Coenurosis is a central nervous system disease of wild and domestic ruminants caused by Coenurus cerebralis, a bladder worm stage of Taenia multiceps). Even in Sardinia island, this metacestode seems to be widespread in sheep (Scala et al. Vet Parasitol 143(3-4):294-298, 2007) where coenurosis is an important health problem (Varcasia et al. Parasitol Res 99(5):622-626, 2006) the last and unique report of coenurosis in cattle was in 1990 (Cubeddu et al. 1990). In the present paper, a case of bovine coenurosis in Sardinia was described 22 years after the first report with a morphological a biomolecular characterization. A 2-year-old Limousine bull was euthanized in the Bolotana (NU) municipality (Central Sardinia). The remote anamnesis achieved from the farmer reporting that the bull showed neurological symptoms from 1 year of age previously classified as nutritional problems by the farm's veterinary. The breeder also says that the bull have by self-produced the skull fracture by hitting a gaff in the farm. The skull was opened and the brain removed and carefully examined showing two coenurus cysts containing clear fluid with numerous scoleces both in the right hemisphere. Morphological features of the cysts and mt-DNA sequencing confirm that the parasites were T. multiceps Coenuri.

Molecular characterization of subcutaneous and muscular coenurosis of goats in United Arab Emirates.

Coenurosis is a parasitic disease caused by the larval stage of Taenia multiceps, which affects various ruminants species, particularly sheep and goats, and occasionally humans. In this note, non-cerebral coenurosis in goats with the goal to compare morphological and biomolecular characteristics is focused in order to determine if the non-cerebral specimens should be considered a new specie or a strain of T. multiceps. Three hundred goats slaughtered with macroscopic lesions due to the presence of metacestodes were examined in an abattoir in Dubai, United Arab Emirates in order to evaluate the presence of extra-cerebral coenuri. Forty-eight coenuri were found under the skin, between fasciae of the skeletal muscles, diaphragm and in the abdominal cavities. Morphologically, the examined non-cerebral coenuri recovered showed the same features reported by other authors for Coenurus cerebralis, but their location outside the central nervous system suggests that they may be a different strain or genetic variants of T. multiceps. Nine caprine coenuri were then processed for sequencing of mitochondrial partial COI (396 bp) and ND1 (471 bp) genes indicating that they had a pairwise distance of 1.0-1.3% and 2.4-4.1% compared with parasite' COI sequences from Italy (Tm1-Tm3 strains) and Erzurum strains of ovine origin, respectively; whereas it had 0.6-1.3% and 0.4-1.1% pairwise distance for ND1. Phylogenetic trees of their ND1 and COI sequences using the maximum composite likelihood method with MEGA showed that these nine parasites recovered in Emirates abattoir could be grouped into a new strain. In conclusion, morphological and molecular characterization of caprine coenurosis from Dubai, United Arab Emirates, shows that the specimens are significantly different from other strains or genotypes in COI sequence, which suggests that they should belong to different genotypes or strains of T. multiceps.

Cystic echinococcosis in Sardinia: farmers' knowledge and dog infection in sheep farms.

Cystic Echinococcosis (CE) is one of the most widespread parasitic diseases in Sardinia, the second largest Mediterranean island where almost 3,558,000 milk sheep were raised extensively. The aim of this survey was to evaluate the level of farmers' knowledge on CE transmission, focusing on the role of human to facilitate the persistence of this zoonosis in Sardinia after 14 years after the last campaign against CE. The other goal of the survey is to update on presence of Echinococcus granulosus in its definitive hosts through three ELISA coproantigen tests. An interview was carried out with 172 farmers. The questionnaire was designed to include possible factors associated with the transmission of Echinococcosis: ownership and number of dogs, the use of anthelmintic drugs against dog cestode, frequency of anthelmintic treatment in dogs, home slaughtering and offal disposal. Individual faecal samples were retrieved from 300 dogs, and after a preliminary macroscopic examination to discover adult worms and/or proglottids, was submitted to copromicroscopic examination. Coproantigens were then extracted according to the protocol described by Allan et al. (1992), and subsequently stored at -20°C until use. Faecal soluble antigens from E. granulosus were detected using three different ELISA coproantigen assays: (a) the commercially produced Chekit Echinotest (Bommeli, Bern, CH) based on polyclonal antibodies against adult excretory/secretory (E/S) antigens; (b) a sandwich ELISA that uses rabbit polyclonal antibodies against adult E/S antigens and biotinylated monoclonal antibody EmA9 produced against adult Echinococcus multilocularis somatic extract (Malgor et al., 1997); and (c) a sandwich assay that uses monoclonal antibody EgC3 produced by immunization with adult E. granulosus E/S products (Casaravilla et al., 2005). Questionnaire results reveal that on all farms home-slaughtering was done, and offal was used as dog meal raw (17%) or after boiling (37%), discarded in the trash (23%), or buried superficially (15%). Most farmers (69%) declared to deworm their dogs, but only 10% used cestodicidal drugs. The coprological survey of 300 farm dogs using sedimentation, flotation and three different coproantigen (CA) ELISAs resulted in a faecal prevalence of 8.3% for taeniid eggs, while the CA tests gave prevalences of 3% (Chekit Echinotest, Bommeli), 6% (EmA9 sandwich ELISA) and 10% (EgC3 sandwich ELISA). Our results show that this is not only an educational problem, but also an economic one, stressing the need that future control plans should follow an integrative approach including veterinary and medical services, farmers, breeders' associations and the Government.

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.

Preliminary field trial of a vaccine against coenurosis caused by Taenia multiceps.

Taenia multiceps is a taeniid cestode that in its adult stage lives in the small intestine of dogs and other canids. In the intermediate hosts, the larval stage of T. multiceps causes coenurosis, a common disease in the CNS of ruminants, which typically leads to the death of the infected animals. Recent research into new methods for control of coenurosis and other taeniid cestode infections such as hydatidosis has identified vaccination as a potentially valuable new tool. In order to test the applicability of vaccination as an approach for control of T. multiceps infection in sheep, a field trial was carried out against natural infection in Sardinian farms (Italy) with recombinant proteins of T. multiceps. The recombinant proteins with Quil A as adjuvant were injected subcutaneously, the first administered to lambs at 10-12 weeks of age and a booster dose given after 2-4 weeks. A total of 632 sheep were selected, belonging to the "replacement quota" of six different farms, of which 424 were used as controls (unvaccinated) and 208 were vaccinated. After a period of more than 40 months from the beginning of the field trial, 33 episodes of cerebral coenurosis occurred in the monitored farms, including 32 cases in control sheep and l case in a vaccinated animal. Statistical analysis revealed a significant reduction in the number of coenurosis cases in the vaccinated animals (chi(2)=14.08, P<0.001). This is the first successful field test of a practical vaccine against T. multiceps and, considering the high degree of effectiveness achieved, could be a prelude to routine application in field situations of particular risk, such as Sardinia.

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.

Genetic variation within and between G1 and G3 genotypes of Echinococcus granulosus in Italy revealed by multilocus DNA sequencing.

Numerous studies have provided evidence that Echinococcus granulosus exists as a complex of different strains, that differ in a wide variety of criteria that have an impact on the epidemiology, pathology and control of cystic hydatid disease (CHD) and, to date, 10 distinct genotypes (G1-G10) have been identified. In Italy, sequence analysis of the mitochondrial cox1 and nad1 genes showed the occurrence of the G1 genotype, the common sheep strain, the G3 genotype, the buffalo strain and of one isolate identified as G2 genotype, the Tasmanian sheep strain. In the present work, we have analysed E. granulosus strains in Italy, by genotyping a large sample of isolates and by checking out the genetic differentiation within and among the G1 and G3 genotypes using an additional mitochondrial gene as marker, the rrnS gene. Sequencing of the rrnS gene revealed a significant genetic differentiation between isolates identified as belonging to the G1 and G3 genotypes, with fixed nucleotide substitutions. This study provides further evidence of the occurrence of the E. granulosus G3 buffalo strain in Italy, a strain previously thought to be confined to the Indian region.

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.

A survey of Taenia multiceps coenurosis in Sardinian sheep.

A survey was carried out to assess the occurrence of Coenurus cerebralis infection in Sardinian sheep. A prevalence of 0.35% was observed when 566 regularly slaughtered sheep were examined. However, in 120 sheep with suspected symptoms of coenurosis examined from November 2001 to October 2002, a total of 299 cerebral coenurosis lesions were observed with an incidence of 1% per year. Lesions were classified as migratory, cystic and secondary. Most migratory lesions were found in sheep aged 3-6 months. Cavitary lesions containing cysts in different developing stages were found with high incidence per year in sheep aged 7-12 months. Secondary lesions due to the development of Coenurus were most frequent in sheep aged 19-36 months. Most sheep were found infected in spring and in early summer, between March and June. Most lesions were located in the cortex. The mean number of protoscolices per cyst was 149 (range 10-370).

Sero-epidemiological update on sheep toxoplasmosis in Sardinia, Italy.

In order to better understand the epidemiology of ovine toxoplasmosis in Sardinia, a serological survey was carried out on 22 flocks with no fertility problems. In total 1043 sera (9% of the 11,382 sheep raised in the flocks) were examined by means of a commercial ELISA kit. To verify the performance of ELISA test, 160 selected sera were tested again with a gold standard test (IFAT). Performance of the commercial ELISA kit was summarised in terms of Sensitivity (SE), Specificity (SP), positive and negative Likelihood Ratios (LR+; LR-). The overall seroprevalence with ELISA test was recorded as 51.3%. It was generally increasing according to age and was significantly lower in animals younger than one year (with the exception of < 1 month old lambs). This survey provided data on the current Toxoplasma gondii sheep seroprevalence in Sardinia, confirmed a still high parasite pressure and pointed out that consumption of raw or undercooked ovine meat can be considered a potential risk factor for humans.

[Environmental control of gastrointestinal strongylosis in sheep and goats]. / Profilassi ambientale delle strongilosi gastrointestinali degli ovini e dei caprini.

Gastrointestinal strongylosis are the dominant parasitic infections of sheep and goats. The successful control of these parasites cannot be done exclusively with anthelmintics, but the first step is an integrated program for environmental prophylaxis. The correct planning of the prophylaxis program has to be preceded by the analysis of the related problems: (1) the parasitological status of farm livestock; (2) knowledge of the farm management; (3) hydrogeological, pedological and climatic-environmental aspects. The environmental control strategies can be resumed as follows: avoiding animals from different farms to share the same pasture; avoiding animals of different age classes to graze together; parcel the pastures to permit a rational rotation; rotational grazing of pastures according to the seasonal development of parasites; stocking rate; young animals grazing ahead of the older animal ones; crop management practices (draining, ploughing, harrowing, scrub clearing, fertilizing, etc.). These measures make the habitat less suitable for the free-living stages of gastrointestinal strongyles, reducing the potential of infection of the same pastures.

Updates on cystic echinococcosis (CE) in Italy.

An update on Cystic Echinococcosis (CE) diffusion in Italy during 2003-2005 is reported. CE seems to have a sporadic diffusion in the northern part of the country where this disease plays a minor role (prevalence < 1%). Recent investigations have shown the occurrence of CE cases in humans from the mountains between Reggio Emilia and Modena, with an average year incidence between 9.4 and 5.6/100,000. In Abruzzo prevalences in sheep and cattle are 20.2% and 15.3%, with a fertility of 4.6% and 1.3%, respectively. In the same region, G1 and G3 strains were identified and a prevalence of 31% in dogs was found with CaELISA. In Campania, CE prevalence was 14.8% in cattle, with no viable cysts recovered, and 10.5% in water buffaloes, with a fertility of 1.4%. Biotechnologies allowed to find G1 and G3 strains in water buffaloes. In Sicily, CE was found in 67.1% of cattle, with a fertility of 4%, and in 57.6% of sheep, with 9.2% of viable cysts. Biomolecular investigations have found G1 strain in sheep and cattle. In dogs, a prevalence of 5.6% for Echinococcus granulosus was reported. In Sardinia CE prevalence was 75.3% in sheep and 41.5% in cattle, with a fertility of 10.3% and 2.6%, respectively. CE was found also in 9.4% of pigs, with fertility of 6.5%. The G1 strain was recovered in sheep and cattle while the G7 in pigs.

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.