The relationships between faecal egg counts and gut microbial composition in UK Thoroughbreds infected by cyathostomins.

A growing body of evidence, particularly in humans and rodents, supports the existence of a complex network of interactions occurring between gastrointestinal (GI) helminth parasites and the gut commensal bacteria, with substantial effects on both host immunity and metabolic potential. However, little is known of the fundamental biology of such interactions in other animal species; nonetheless, given the considerable economic losses associated with GI parasites, particularly in livestock and equines, as well as the global threat of emerging anthelmintic resistance, further explorations of the complexities of host-helminth-microbiota interactions in these species are needed. This study characterises the composition of the equine gut commensal flora associated with the presence, in faecal samples, of low (Clow) and high (Chigh) numbers of eggs of an important group of GI parasites (i.e. the cyathostomins), prior to and following anthelmintic treatment. High-throughput sequencing of bacterial 16S rRNA amplicons and associated bioinformatics and statistical analyses of sequence data revealed strong clustering according to faecal egg counts (P = 0.003). A trend towards increased populations of Methanomicrobia (class) and Dehalobacterium (genus) was observed in Clow in comparison with Chigh. Anthelmintic treatment in Chigh was associated with a significant reduction of the bacterial Phylum TM7 14 days post-ivermectin administration, as well as a transient expansion of Adlercreutzia spp. at 2 days post-treatment. This study provides a first known insight into the discovery of the intimate mechanisms governing host-parasite-microbiota interactions in equines, and sets a basis for the development of novel, biology-based intervention strategies against equine GI helminths based on the manipulation of the commensal gut flora.

Anthelmintics efficacy against intestinal strongyles in horses of Sardinia, Italy.

Intestinal strongyles (IS) are the most important parasites of equids, due to their high prevalence worldwide, pathogenicity and the spread of drug-resistant populations. Despite the large number of horses bred in Sardinia Island, Italy, no data are available on the efficacy of anthelmintic compounds in the control of horse strongylosis. Therefore the aim of the present study was to evaluate the efficacy of five commercial anthelmintic formulations containing fenbendazole (FBZ), pyrantel (PYR), moxidectin (MOX) and two ivermectin formulations (IVM1 and IVM2) against IS in Sardinia by performing a fecal egg count reduction test (FECRT) and investigating the egg reappearance period (ERP) after treatment. In total, 74 horses from 7 farms were examined. Coprocultures performed for individual fecal samples collected at the day of the treatment revealed that cyathostomins were the predominant parasitic species (98.6%). The FECR for all horses belonging to the treatment groups after two weeks was ≥ 95% with a 95% C.I. > 90%. The expected ERP did not decrease in any of the treatment group as FECR values < 90% were found at D60 for FBZ, at D90 for PYR and IVM1, at D150 for IVM2. All horses treated with MOX showed FECRT > 90% for the entire duration of the trial until D150. The results of the present survey indicate that drug-resistant cyathostomin populations are not present in the examined horse population, contrariwise to what observed in other Italian and European regions. The reasons and implications of these results are discussed.

A retrospective and geographical epidemiological survey of traumatic myiasis in southern Italy.

A survey on the prevalence and geographical distribution of traumatic myiasis in sheep, and the risk factors for the disease, was carried out in a region of southern Italy. A total of 138 sheep flocks were selected and visited to acquire data on the presence or absence of traumatic myiasis using both a questionnaire for retrospective analysis and animal inspection. Prevalences registered at the farm and animal levels, respectively, were 8.7% and 6.3% in 2010, and 5.8% and 5.0% in 2011. Records of the occurrence of the parasitic disease in this region are recent: a statistically significant (P < 0.01) progressive increase in the number of farms affected (from 0.7% to 8.7%) has been registered since 2007. Wohlfahrtiosis was found in 11 of 95 (11.6%) geographical units sampled and three significant (P < 0.05) clusters of spatial farm aggregation were identified in the southern part of the study area. A total of 158 presently uninfested farms were considered to be at high risk for transmission as a result of their proximity to infested farms. The spreading of Wohlfahrtia magnifica (Diptera: Sarcophagidae) in southern regions of Italy represents a warning that the risk for infestation may become more significant in other Italian regions, as well as other European countries.

Evidences of increasing risk of dirofilarioses in southern Italy.

Given the spread of Aedes albopictus from northern to southern Italy, and the lack of updated data on Dirofilaria infections, this study was carried out to assess the infection risk for dogs and cats in Apulia region. During a 2-year study, 175 A. albopictus female specimens and samples of blood from 427 dogs (309 privately owned dogs and 118 shelter dogs) and 12 cats were collected. All blood samples were subjected to a modified Knott method, to a test for the detection of circulating Dirofilaria immitis antigen, and to a Dirofilaria species-specific real-time PCR for the simultaneous detection of D. immitis and Dirofilaria repens, targeting on partial cytochrome oxidase subunit 1 and internal transcribed spacer-2, respectively. Two abdomen and one thorax pools from A. albopictus were positive for D. immitis, with minimum infection rates of 1.14 and 0.51, respectively, and a probability of a single positive specimen to be infected of P = 0.6 % (95 % confidence interval (CI) = 0.12-1.73). Out of 439 examined subjects, 22 (5.0 %) tested positive for Dirofilaria spp. in at least one diagnostic test. A specific D. immitis infestation rate of 3.5 % was found among the privately owned dogs, while shelter dogs tested positive only for D. repens with a prevalence of 3.4 %; one cat tested molecularly positive for D. immitis. There was a significantly higher rate of positivity among guard dogs for D. immitis (odds ratio, 6.24, 95 % CI, 1.26-25.28; P < 0.05). The increasing risk of D. immitis infection in southern Italy is supported by the noteworthy positivity of A. albopictus populations and the cat. Our data highlight the usefulness to include filarioid infestation in routine diagnosis.

Occurrence of Giardia duodenalis infection in chinchillas (Chincilla lanigera) from Italian breeding facilities.

The present work investigated the occurrence of Giardia infection in Chinchilla lanigera reared in three Italian breeding facilities and determined their role as potential zoonotic reservoir. One hundred and four fecal samples were tested for the presence of Giardia spp. cysts using a Direct Fluorescent Assay (DFA). A high positivity rate (39.4%) was found despite all animals were asymptomatic at the time of sampling. Thirty-one positive samples were genetically characterized by sequence analysis of the ITS1-5.8S-ITS2 region of the Giardia ribosomal DNA. Assemblages B (29 isolates) and C (two isolates) were identified. These results showed that Giardia infection can be common in chinchillas, thus spurring further molecular epizootiological studies of the infection to assess the zoonotic potential or host specificity of their isolates, to determine the source of infections, to identify the routes of transmission, and to control the infection among animal populations.

Detection of specific antibodies in dogs infected with Angiostrongylus vasorum.

Canine angiostrongylosis, caused by the nematode Angiostrongylus vasorum, is an emerging cardiopulmonary disease in Europe which can be fatal if left untreated. We determined the diagnostic value of the specific detection of antibodies against A. vasorum adult somatic antigen, adult excretory/secretory (E/S) antigen and first stage larvae (L1) somatic antigen in ELISAs. Also, A. vasorum adult somatic antigen purified by monoclonal antibodies (mAb) was evaluated in a sandwich-ELISA. Among the crude antigens, the best sensitivities when testing 21 naturally infected dogs were obtained using adult E/S and somatic antigen (85.7% and 76.2%, respectively), which were comparable with the results of the sandwich-ELISA based on mAb-purified antigens (81%). The ELISA performed with L1 antigen had the lowest sensitivity (42.9%). In experimentally inoculated dogs, the sensitivities ranged from 97.7% to 100% with all test settings. The specificity was 98.8% (92.5-99.9%, 95% CI) with all ELISAs using sera of 82 randomly selected dogs. Cross-reactions using adult somatic, adult E/S and L1 somatic antigen were observed in sera of dogs infected with Crenosoma vulpis, Dirofilaria immitis, Dirofilaria repens, and Eucoleus aerophilus. In contrast, using the mAb-purified antigens, the cross-reactions were minimal. Depending on the antigens used, specific antibodies were detected starting between 13 and 21 days post experimental inoculation (dpi), and at latest between 35 and 48 dpi, thus before or around the onset of patency. The serological follow-up of four A. vasorum-infected dogs after anthelmintic treatment at 88 dpi showed a decrease of antibody levels after drug administration, and the animals became seronegative 2-9 weeks later. Two untreated dogs remained seropositive. In four dogs treated 4 dpi, virtually no antibody-reaction was detectable, with the exception of the ELISA performed with L1 antigen. The early detection of specific antibodies against A. vasorum by ELISA represents a valid alternative for a reliable diagnosis and for follow-up investigations after anthelmintic treatment.

New insights into the morphology, molecular characterization and identification of Baylisascaris transfuga (Ascaridida, Ascarididae).

Species ranked within the genus Baylisascaris (Ascaridida, Ascarididae) have been implicated in clinical and subclinical intestinal diseases in their natural hosts (e.g., raccoons and bears) as well as in life-threatening larva migrans syndromes in a number of incidental hosts, including humans. Following the diagnosis of Baylisascaris transfuga infestation in two captive polar bears, living in the zoo park of Pistoia (Tuscany, Italy), nematodes (n=300; both sexes) have been characterized by morphological and molecular methods by sequencing and analysing ribosomal (large ribosomal DNA (28S) and internal transcribed spacer region 1 and 2 (ITSs)) and mitochondrial (cytochrome c oxidase subunit 1 (cox1) and cytochrome c oxidase subunit 2 (cox2)) target regions. In addition, seven faecal samples were collected from the animal enclosure and submitted to copromicroscopic and molecular examination. All nematodes were morphologically identified as B. transfuga and their main distinctive features are here presented. No variation in size and nucleotide polymorphisms was detected within each target sequence among all samples analysed. These data contribute to facilitate an accurate diagnosis of this little known nematode infestation in order to apply appropriate anthelmintic strategies.

The mitochondrial genome of the common cattle grub, Hypoderma lineatum.

The mitochondrial DNA of the cattle grub Hypoderma lineatum (de Villers) (Diptera: Oestridae) was completely sequenced. The entire molecule was 16,354 bp long and presented a heavy bias towards A + T, which accounted for 77.8% of the whole genome. Hypoderma lineatum genes were organized in the same order and orientation as in the mitochondrial genomes available for other species belonging to the Oestroidea superfamily and compared in this study [Chrysomya putoria (Wiedemann), Cochliomyia hominivorax (Coquerel), Lucilia sericata (Meigen) and Dermatobia hominis (L.)], except for the occurrence of a 102-bp non-coding region partially present in other species. The complete sequence of H. lineatum will represent a useful dataset to evaluate the evolutionary pattern of mtDNA within Oestroidea by using molecular information in diagnostic, taxonomic and evolutionary studies.

Detection of Bonamia ostreae and B. exitiosa (Haplosporidia) in Ostrea edulis from the Adriatic Sea (Italy).

The flat oyster Ostrea edulis L. is widespread along the Italian coasts. In particular, the Manfredonia Gulf (Adriatic Sea) represents an important site where natural beds subsist. Previous monitoring conducted in 1990 by light microscopy and ultrastructural studies revealed the presence of Bonamia-like microcell parasites in some flat oysters: following this observation, a new sampling of O. edulis was carried out at this location in 2007. Of 750 oysters collected, 3 showed the presence of uninucleated microcells (2 to 3 microm diameter) free or inside the haemocyte cytoplasm by cytology and histopathology. Molecular analysis confirmed that the microcells in 2 oysters were B. exitiosa, whereas in the third oyster the microcells were B. ostreae. Moreover, molecular studies were carried out to confirm the existence of Bonamia sp. in archived samples, confirming the presence of B. ostreae in the Manfredonia Gulf since 1990.

Expression of the micro-opioid receptor on Malassezia pachydermatis and its effect in modulating phospholipase production.

Malassezia spp. may act as opportunistic skin pathogens in humans and animals. Malassezia pachydermatis proliferation and phospholipase production may play a pathogenic role in the occurrence of skin lesions in dogs. This study investigates the presence of mu-opioid receptor (MOR) in M. pachydermatis strains isolated from healthy dogs and dogs with skin lesions and its effects on phospholipase activity (p.a.). P.a. of 64 M. pachydermatis isolates was evaluated using different concentrations of naloxone (Nx), a MOR antagonist. Isolates were divided into Group A (i.e., 40 isolates from 26 dogs with dermatitis) and Group B (i.e., 24 isolates from 12 healthy dogs). The MOR expression was analyzed by Western blot and immunofluorescence. A statistically higher p.a. than that of the controls was found with isolates in Group A at a Nx concentration of 10(-6) M (P<0.05). No isolate in Group B displayed p.a. in either control samples or in the presence of any Nx concentration. Immunoblotting revealed two positive MOR immunoreactive bands of approximately 65 and 98 kDa. MOR expression and localization was also demonstrated by immunofluorescence in isolates from Groups A and B. This study provides the first evidence of MOR expression on M. pachydermatis cell membranes pointing to its possible role in modulating p.a. production in isolates from dogs with skin lesions.

Identification of the intermediate hosts of Habronema microstoma and Habronema muscae under field conditions.

A polymerase chain reaction (PCR)-based assay was used for the specific detection of Habronema microstoma and Habronema muscae (Nematoda, Spirurida) in order to identify the intermediate hosts of both nematode species under field conditions. A total of 1087 netted and 165 laboratory-bred flies were tested. Flies were identified as Musca domestica Linnaeus 1758, Musca autumnalis De Geer 1776, Haematobia irritans (Linnaeus 1758), Haematobia titillans (De Geer 1907) and Stomoxys calcitrans (Linnaeus 1758) (Muscidae). Genomic DNA was extracted from pools of fly heads, thoraces and abdomens, and 703 samples were subjected to a duplex two-step semi-nested PCR assay to specifically detect diagnostic regions within the ribosomal ITS2 sequence of both H. microstoma and H. muscae. Stomoxys calcitrans specimens were positive for H. microstoma DNA and M. domestica specimens were positive for H. muscae DNA. In particular, PCR-positive samples derived from both farm-netted and laboratory-bred flies. The present study represents the first evidence of the vectorial competence of different fly species as intermediate hosts of Habronema stomachworms under field conditions. We discuss the roles of S. calcitrans and M. domestica in transmitting H. microstoma and H. muscae.

First description of the endogenous life cycle of Hypoderma sinense affecting yaks and cattle in China.

Larvae belonging to five species of Hypoderma spp. (Diptera, Oestridae) cause myiasis in wild and domestic ruminants that is characterized by migrations within deep tissues. In China hypodermosis is one of the most important arthropod diseases affecting ruminants and, moreover, represents a significant zoonosis, with numerous reports of Hypoderma spp. affecting farmers. Recently, a sixth species, Hypoderma sinense Pleske, has been rediscovered but the endogenous migration pathway within the host body is completely unknown and it represents a major constraint for the control of larval infection. In December 2003 a total of 165 larval stages of Hypoderma spp. were collected from different anatomical sites of 40 yaks slaughtered at an abattoir in the province of Gansu, China. The morphological characters and size of the recovered larvae were used to infer migratory routes and 45 specimens were also subjected to a polymerase chain reaction (PCR) assay of cox1 mtDNA and amplicons sequenced. All the larvae molecularly processed were identified as H. sinense and sequence identity was confirmed by a PCR-restriction fragment length polymorphism (PCR-RFLP) tool carried out using BfaI and HinfI endonucleases. The finding of H. sinense larvae only in the oesophagus or both in oesophagus and subcutaneous tissue of 12 and 15 animals, respectively, indicates that H. sinense larvae migrate through the oesophagus similarly to Hypoderma lineatum (De Villers). The description of the endogenous life cycle of H. sinense will help to determine the timing of specific treatment programmes to guarantee the improvement of animal welfare and health, thus resulting in an increase in livestock production in China.

Morphological variability and genetic identity in Rhinoestrus spp. causing horse nasal myiasis.

Larvae of Rhinoestrus purpureus (Brauer) and Rhinoestrus usbekistanicus Gan (Diptera: Oestridae) cause nasal myiases of equids. During a recent epidemiological survey in southern Italy some morphological and taxonomical doubts arose concerning the identification of Rhinoestrus third stage larvae on the basis of the features of the posterior spiracles and the distribution of dorsal spines on the third segment. Four different morphotypes were retrieved: R. usbekistanicus-like, R. purpureus-like and two morphotypes with shared features. The genes encoding for the mitochondrial cytochrome oxidase I (COI) and for the ribosomal subunits 16S and 28S of the four morphotypes of Rhinoestrus were investigated to determine whether they belonged to a single taxon or they displayed genetic differences indicative of more than one species. The three genes showed a very low level of sequence variation (COI 0-0.43%, 16S 0-1.45%, 28S 0-0.23%) falling within the intraspecific ranges previously described for Oestridae species. Finally, the peritreme features and the spinulation of the third segment of the four morphotypes examined could not be used to differentiate the two species.

A third species of Hypoderma (Diptera: Oestridae) affecting cattle and yaks in China: molecular and morphological evidence.

Cattle and yak hypodermosis in China is caused by Hypoderma bovis and H. lineatum, with a prevalence reaching up to 98-100% of the animals and maximum intensities exceeding 400 warbles for each animal. A third species, H. sinense, is also considered by Chinese researchers to affect livestock. The molecular characterization of the most variable region of the mitochondrial cytochrome oxidase I gene and of the ribosomal 28S gene has been performed for the third-stage larvae collected from cattle and yaks in China and identified (on the basis of the spinulation on the ventral side of the 10th segment) as H. bovis, H. lineatum, and H. sinense. Amplicons were digested with the HinfI and BfaI restriction enzymes, which provided diagnostic profiles to simultaneously differentiate the 3 Hypoderma species. Third-stage larvae of H. sinense were also examined by scanning electron microscopy, which revealed proper morphological characteristics different from those of H. bovis and H. lineatum. The molecular and morphological evidence herein reported support the existence of a third species of Hypoderma affecting cattle and yaks in China, and the results provide new tools for unequivocal identification of this species and present key components for the evaluation of its endogenous cycle and pathogenicity in animals and humans.

Biology of Thelazia callipaeda (Spirurida, Thelaziidae) eyeworms in naturally infected definitive hosts.

Thelazia callipaeda (Spirurida, Thelaziidae) eyeworm causes ocular infection in carnivores and humans in the Far East; this infection has been recently reported also in Europe--northern and southern Italy--in dogs, cats and foxes. The natural vector/s of T. callipaeda is/are unknown and the development of the nematode in its definitive hosts is limited to an experimental trial on dogs. To contribute new insights into the development of T. callipaeda in the definitive host in field conditions, eyeworms were collected from naturally infected dogs from an area with a high prevalence of infection (up to 60.14%) in the Basilicata region of southern Italy, from January 2002 to December 2003. Conjunctival secretions were also collected and examined for the presence of immature stages. The presence of blastomerized eggs throughout the period--except for the months from May to November--indicates a seasonality in the reproductive activity of T. callipaeda, coinciding with the presence/absence of the vector. In fact, 1st-stage larvae were found in the lachrymal secretions of dogs in summer (June--July 2002 and 2003), ready to be ingested by flies feeding about the eyes. The evidence of 4th-stage larvae in March 2002 and April, July and October 2003 may be accounted for by the presence of flies that act as intermediate hosts of T. callipaeda from early spring to early autumn. The presence of immature stages in October indicates an overlapping generation of nematodes and a 2nd cycle of vector infection. This basic knowledge of the development of T. callipaeda will hopefully help future epidemiological studies to identify the intermediate hosts and define the likely risk for vectors in field conditions.

[Myiasis caused by Oestridae: serological and molecular diagnosis]. / Le miasi da Oestridae: diagnosi sierologica e molecolare.

Myiasis-causing Oestridae (bot flies) infect several animal species world-wide, from palaearctic to subtropical/tropical areas. Oestrids affect livestock production causing abortion, reduced milk production, losses in weight and fertility, poor hide quality and an impairment of the host's immune system. In the last few years much research has been carried out on the immunology of these infestations, in order to acquire efficient and reliable diagnostic serological tools; the genome of the different species of Oestridae has been studied to further their molecular identification, taxonomy and phylogenesis. The immunodiagnostic methods for many myiasis causing Oestrids have proven to be a viable alternative to the clinical parasitological examination or the post-mortem examination. Numerous serological tests have been developed for the diagnosis of bovine hypodermosis caused by Hypoderma bovis and Hypoderma lineatum, and ELISAs using larval hypodermin C as the antigen are currently used on serum, individual and pooled milk samples to detect the presence of circulating anti-Hypoderma antibodies. In Italy the best period to sample the animals is November-January, since it is in this period that the antibody kinetics of the animals reaches a peak. Recently the efficacy of the ELISA test on pasteurized milk samples has been demonstrated, allowing the diagnosis of bovine hypodermosis also in areas where there is no information on the presence of the disease and the sampling of the animals is laborious. The cross-reactivity between Przhevalskiana silenus antigens and anti-Hypoderma antibodies led to assessing the usefulness of a simple and cost-effective ELISA for the diagnosis of goat warble fly infection. In particular, it has been demonstrated that infected goats display an antibody peak in November-December in blood and milk, thus making this period suitable for sampling. Although no extensive data is available on the immunology of sheep and goat oestrosis caused by Oestrus ovis, the efficacy of ELISA has been demonstrated by correlating serological results with clinical post-mortem examinations. No immunological techniques are currently used to diagnose gasterophilosis of equids and only one study reports the efficacy of ELISA for detecting anti-Gasterophilus antibodies in infected equids. Several studies have been conducted into the molecular characterization of the mitochondrial DNA (mtDNA)--in particular of the gene encoding for the cytochrome oxidase I (COI)--for many free-living and parasitic arthropods for diagnostic, taxonomic and phylogenetic purposes. As regards Oestridae causing myiasis, the first study features a PCR-RFLP assay of the most common Italian species (i.e. H. bovis, H. lineatum, Gasterophilus intestinalis, P. silenus, O. ovis), which showed clear genetic differences among the genera examined, but no inter-specific variation between the two species of Hypoderma considered. The molecular characterization of the most variable region of the COI gene (encoding for the region from E4 to the terminal COOH) was able to clearly differentiate H. bovis and H. lineatum. The E4-COOH region of the COI gene has been characterized for 18 oestrid species and from a taxonomical point of view, molecular data confirm the morphological classification, with the examined species divided into four subfamilies. New insights have also been gained on the molecular differentiation of the most common species of Hypoderma (i.e. H. bovis, H. lineatum, Hypoderma actaeon, Hypoderma diana and Hypoderma tarandi) and, in particular, the restriction enzyme BfaI, provides a diagnostic profile that can be used to simultaneously differentiate all the species examined. The characterization of the E4-COOH COI gene and the hypervariable region of the gene encoding for the ribosomal Isu revealed the identity of Hypoderma sinense as a new species, infecting cattle and yaks in China. Finally, the molecular analysis of the same mitochondrial and ribosomal regions showed that P. silenus, Przhevalskiana aegagri and Przhevalskiana crossii are morphotypes of the same species.

[Ecology of Thelazia spp. in cattle and their vectors in Italy]. / Ecologia di Thelazia spp. e dei vettori in Italia.

The genus Thelazia (Spirurida, Thelaziidae) includes a cosmopolitan group of eyeworm spirurids responsible for ocular infections in domestic and wild animals and transmitted by different species of muscids. Bovine thelaziosis is caused by Thelazia rhodesi Desmarest 1828, Thelazia gulosa Railliet & Henry 1910, and Thelazia skrjabini Erschow 1928, which occur in many countries; T. gulosa and T. skrjabini have been reported mainly in the New World, while T. rhodesi is particularly common in the Old World. In Italy, T. rhodesi was reported in southern regions a long time ago and, recently, T. gulosa and T. skrjabini have been identified in autochthonous cattle first in Apulia and then in Sardinia. Thirteen species of Musca are listed as intermediate hosts of eyeworms, but only Musca autumnalis and Musca larvipara have been demonstrated to act as vectors of Thelazia in the ex-URSS, North America, ex-Czechoslovakia and more recently in Sweden. In Italy, after the reports of T. gulosa and T. skrjabini in southern regions, the intermediate hosts of bovine eyeworms were initially only suspected as the predominant secretophagous Muscidae collected from the periocular region of cattle with thelaziosis were the face flies, M. autumnalis and M. larvipara, followed by Musca osiris, Musca tempestiva and Musca domestica. The well-known constraints in the identification of immature eyeworms to species by fly dissection and also the time-consuming techniques used constitute important obstacles to epidemiological field studies (i.e. vector identification and/or role, prevalence and pattern of infection in flies, etc.). Molecular studies have recently permitted to further investigations into this area. A PCR-RFLP analysis of the ribosomal ITS-1 sequence was developed to differentiate the 3 species of Thelazia (i.e. T. gulosa, T. rhodesi and T. skrjabini) found in Italy, then a molecular epidemiological survey has recently been carried out in field conditions throughout five seasons of fly activity and has identified the role of M. autumnalis, M. larvipara, M. osiris and M. domestica as vectors of T. gulosa and of M. autumnalis and M. larvipara of T. rhodesi. Moreover, M. osiris was described, for the first time, to act as a vector of T. gulosa and M. larvipara of T. gulosa and T. rhodesi. The mean prevalence in the fly population examined was found to be 2.86%. The molecular techniques have opened new perspectives for further research on the ecology and epidemiology not only of Thelazia in cattle but also of other autochthonous species of Thelazia which have been also recorded in Italy, such as Thelazia callipaeda, which is responsible for human and canid ocular infection and Thelazia lacrymalis, the horse eyeworm whose epidemiological molecular studies are in progress.