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Despite the economic and animal welfare importance of the Poultry Red Mite Dermanyssus gallinae, its genetic structure has been studied in a scattered way so far. The prophylaxis and control of such a globally distributed ectoparasite can be significantly improved by understanding its genetic population structure (composition in species and intraspecific variants). The present study aims to establish a rigorous framework for characterizing the neutral genetic structure of D. gallinae based on a literature review combined with an integrative analysis of the data available in GenBank on population-level nucleotide sequence diversity supplemented by a new dataset. The integrative analysis was conducted on sequence data extracted from GenBank coupled with new sequences of two fragments of the mitochondrial gene encoding Cytochrome Oxidase I (CO1) as well as of an intron of the nuclear gene encoding Tropomyosin (Tpm) from several PRM populations sampled from European poultry farms. Emphasis was placed on using the mitochondrial gene encoding CO1 on which the main universal region of DNA barcoding in animals is located. The species D. gallinae sensu lato is a species complex, encompassing at least two cryptic species, i.e., not distinguishable by morphological characters: D. gallinae sensu stricto and D. gallinae L1. Only D. gallinae s.s. has been recorded among the populations sampled in poultry farms worldwide. Current knowledge suggests they are structured in three mitochondrial groups (haplogroups A, B, and C). Haplogroup A is cosmopolitan, and the other two present slightly contrasted distributions (B rather in the northern part of Europe, C most frequently found in the southern part). Recent data indicate that a dynamic geographic expansion of haplogroup C is underway in Europe. Our results also show that NUMT (nuclear mitochondrial DNA) pseudogenes have generated artifactual groups (haplogroups E and F). It is important to exclude these artifact groups from future analyses to avoid confusion. We provide an operational framework that will promote consistency in the analysis of subsequent results using the CO1 fragment and recommendations for future analyses.

To investigate the presence of Cyclospora cayetanensis, Toxoplasma gondii and Echinococcus spp. in fresh produce sold in Italy, 324 locally produced 'ready-to-eat' (RTE) mixed-salad packages belonging to three brands and 324 berries packages (blueberries and blackberries imported from Peru and Mexico, respectively, and raspberries grown in Italy) were purchased at retail. Nine individual packages from each of the six types of fresh produce were collected monthly for one year, and with the same produce pooled, this resulted in a total of 72 pools for the whole year. Using microscopy (FLOTAC), a Cyclospora-like oocyst was detected in a blueberry sample and a taeniid egg was detected in a RTE-salad sample. Molecular tools confirmed these to be C. cayetanensis and Echinococcus multilocularis, respectively. Toxoplasma gondii was not detected in any of the samples. This study shows for the first time in Europe that imported berries on the Italian market may be contaminated with C. cayetanensis and RTE salads grown in Italy with E. multilocularis. The results indicate a new epidemiological scenario and highlight that current management of fresh produce, locally produced or imported, does not ensure products are free from parasite contamination.

Toxoplasma gondii is a cosmopolitan zoonotic protozoan parasite, and the consumption of raw or undercooked pig meat is one of the most important sources of T. gondii infection. Three predominant lineages, types I, II, and III, are widespread in Europe. Although still poorly understood, a relationship between each type and the severity of illness represents a public health issue. To gain further knowledge of the genotypes in circulation and of the potential risk for consumers, one heart sample and one diaphragm sample (206 total) were taken from each of 103 pig carcasses at an abattoir in Italy. Then, we used 529-bp repetitive element PCR and a B1 real-time PCR high-resolution melting assay coupled with sequencing to detect and genotype T. gondii isolates. T. gondii DNA was detected in 14 pigs (13.6%, 95% confidence interval = 7 to 20.2%), and types I (3.9%), II (5.8%), and III (3.9%) were identified. We found that heart tissue had a significantly higher PCR positivity rate for T. gondii than did diaphragm tissue. This is Europe's largest study on genotyping of T. gondii from pigs, and it demonstrates that all three main lineages are present in carcasses of industrially reared pigs in Italy. There is a potential risk to consumers of infection with any or all of the three lineages, and the related clinical consequences should be taken into account. This study suggests that monitoring of T. gondii types in meat is essential, especially in meat that is traditionally eaten raw or that is minimally processed.

Toxoplasma gondii is a widespread protozoan affecting animals and humans. One of the major routes of human infection is through the consumption of raw or under-cooked meat, particularly of certain animal species, including pigs. Although T. gondii represents an important public health issue, its control at slaughter is not mandatory. Consequently, available information on T. gondii infection in domestic animals destined for human consumption is scarce. Thus, an epidemiologic survey was designed to update information on T. gondii infection in pigs from intensive production. Fattening pigs and sows from conventional farms were sampled. Sera were tested with a commercial ELISA for anti-T. gondii antibodies, whereas molecular analysis by 529 bp repetitive element PCR and B1 real-time PCR with subsequent genotyping was performed on heart samples. Statistical analysis was carried out to detect farm management features and sanitary procedures enhancing the risk of infection. At the farm level, 63.6% (7/11) of farms housing sows and 6.7% (1/15) housing fattening pigs scored positive, with individual prevalences of 8.6% (13/151) in sows and 0.5% (1/219) in fattening pigs. T. gondii DNA was detected in eight sows and one fattening pig, and sequencing revealed the presence of all three genotypes (types I, II, and III). Furthermore, the decrease in the biosecurity level enhanced the risk of infection within a farm. The present survey outcomes confirm the spread of T. gondii infection in pig farms in an area of intensive swine production. The application of stricter sanitary procedures may contribute to a further reduction of infection.

Essential oils (EOs) and entomopathogenic fungi such as Beauveria bassiana (Bb) strains have the potential to be used as alternative insecticides and acaricides for controlling ectoparasites as Dermanyssus gallinae. These compounds have some limitations in their use: the acaricidal effect of EOs is rapid, but short-lived, whilst that of Bb is delayed, but long-lived. To evaluate the effect of both compounds combined against D. gallinae, the non-toxic dose of Eucalyptus globulus, Eucalyptus citriodora, Thymus vulgaris and Eugenia caryophyllata essential oils were firstly calculated for "native" strains of Bb. Subsequently, the effects of the combination of selected EOs with Bb against nymph and adult poultry red mites (PRMs) was assessed. EO concentrations ranging from 0.0015 to 8% v/v (i.e., nine double dilutions) were used to evaluate their effect on germination, sporulation and vegetative growth rates of native strains of Bb. A total of 1440 mites (720 nymphs and 720 adults) were divided into three-treated group (TGs) and one control group (CG). In TGs, mites were exposed to Bb in combination with the selected EO (TG1), EO alone (TG2) or Bb (TG3) alone. In the CG, mites were exposed to 0.1% tween 80 plus EO solvent (CG). E. globulus and E. citriodora were toxic for Bb in concentrations higher than 0.2% and 0.003% respectively, whilst E. caryophyllata and T. vulgaris were toxic at all concentrations tested against Bb. Based on the results of the toxicity assays against Bb, E. globulus was chosen to be tested as acaricide resulting non-toxic for Bb at concentration lower than 0.4%. Increased mortality of D. gallinae adults was recorded in TG1 than those in other TGs from 4days post-infection (T+4DPI). A 100% mortality of D. gallinae was recorded in adults at T+9DPI and at T+10DPI in nymphs in TG1 and later than T+11DPI in the other TGs. Used in combination with E. globulus, Bb displayed an earlier acaricidal effect towards both haematophagous D. gallinae stages. The combination of B. bassiana and E. globulus at 0.2% might be used for controlling arthropods of medical and veterinary importance as D. gallinae.

The zoonotic protozoan parasites Toxoplasma gondii, Cryptosporidium parvum and Giardia duodenalis have been recorded worldwide in economically important edible shellfish, and are thus likely to represent a significant public health risk. Therefore, an innovative, user-friendly diagnostic tool is required in order to improve food safety control. The Q3 system is a miniaturized platform whose efficiency and applicability were investigated and compared with results obtained using standard Real-Time PCR. Tanks of saltwater containing acclimated Mytilus galloprovincialis, Ruditapes philippinarum and Ostrea edulis specimens were spiked with purified Cryptosporidium, Giardia and Toxoplasma cysts/oocysts at different concentrations (i.e., 10(3), 10(4) and 10(5)). We then collected 30 specimens for each shellfish species from each group at 24 h and 72 h post-contamination. After DNA extraction, we tested all samples by Real-Time-PCR and Q3, and evaluated the sensitivity, specificity, predictive values, repeatability and concordance between the two systems. Concordance between Real-Time-PCR and Q3 was very good (p

Beauveria bassiana are usually formulated as conidia infection suspensions CIS for field and laboratory applications as biocontrol agent, but the factors affecting CIS shelf-life and virulence, are unexplored. With the aim of investigating the best storage conditions for extending the CIS shelf-life, the potential effects of storage at room 22 ± 1°C and refrigerated temperatures 4 ± 1°C, with and without light and agitation, on viability and virulence of B. bassiana CISs, was assessed. The viability of conidia was evaluated monthly, over a one year period, and the B. bassiana virulence against D. gallinae, five times in one year. B. bassiana had a significantly higher rate of survival at 4 ± 1°C compared to 22 ± 1°C, regardless of the other conditions of storage. The highest mortality rate of D. gallinae was registered for CIS stored at 4 ± 1°C, in dark and without agitation p

In order to establish seawater contamination by emerging protozoan parasites, we used qPCR to molecularly characterize and evaluate the parasitic burden of Giardia duodenalis, Cryptosporidium spp., Toxoplasma gondii, and Cyclospora cayetanensis in 1255 wild bivalve mollusks collected along the Tunisian coasts. T. gondii, G. duodenalis and C. cayetanensis were detected in 6.9% (99% CI=1.6-12.2%) pools of Ruditapes decussatus. None of the samples were found positive to Cryptosporidium spp.; 6.6% pools of R. decussatus were positive for T. gondii Type I, 1.6% for G. duodenalis assemblage A, and 1.6% for the association T. gondii Type I/C. cayetanensis/G. duodenalis assemblage A. R. decussatus harbored up to 77500 oocysts/sample of T. gondii, up to 395 cysts/sample of G. duodenalis, and 526 oocysts/sample of C. cayetanensis. These results provide the first evidence that the Tunisian coasts are contaminated by zoonotic protozoan parasites that can constitute a direct or indirect risk for human health.

To evaluate the occurrence of Toxoplasma gondii and to genetically characterize its isolates in carcasses of industrial fattening pigs, blood, diaphragm, and heart samples were collected from 375 carcasses of pigs slaughtered to be processed for Parma ham production. Pigs had been bred on approved farms (n = 75) located in the so-called Food Valley in Italy. Sera were examined for immunoglobulin G antibodies to T. gondii by modified agglutination test (MAT). Both heart and diaphragm samples from seropositive carcasses were processed for the presence of T. gondii DNA (B1 locus) by real-time PCR and high resolution melting (HRM) assay. Anti-Toxoplasma antibodies were detected in 2.1% of pig carcasses, with titers from 1:10 to 1:320. T. gondii DNA was detected in all (eight) seropositive carcasses and in 11 (5 heart and 6 diaphragm samples) of 16 samples; that is, it was detected in heart tissue in two subjects, in diaphragm tissue in three subjects, and in both muscle tissues in three subjects. Toxoplasma genotypes were determined in seven of eight carcasses: type III was identified in four carcasses, type II in two, and both III and II in one carcass. The serological findings and the molecular detection of T. gondii strains suggest that cured meat products obtained from industrially bred pigs may be potential sources of toxoplasmosis for humans. Our results provide novel, important information regarding the seroprevalence and molecular prevalence of T. gondii in intensively reared pigs within this specific region of Italy, particularly because Parma ham from this region is known and consumed worldwide. On-farm preventive measures combined with slaughterhouse monitoring of carcasses of pigs bred for cured meat production should never be overlooked to prevent the introduction of T. gondii into the food chain and to ensure safety for consumers of these products.

Tap water samples from the toilets of an Italian national railway train were collected over a period of 10 months and tested for the presence of Cyclospora cayetanensis (C. cayetanensis) using EvaGreen® real-time polymerase chain reaction (RT-PCR) assay coupled with high resolution melting (HRM) analysis for protozoan detection and oocyst quantification. C. cayetanensis positive samples were detected in March, April, and May 2013, with the number of oocysts of 4, 5, and 11 per liter, respectively. This is the first finding of C. cayetanensis in water samples in Italy. The findings call for an improvement of hygiene and water safety by the Italian national railway company.

Giardia duodenalis is considered a potentially zoonotic protozoan. Some animal species, including infected dogs, may play an important role in the spread of Giardia cysts through environmental contamination with their feces. In the present study, a commercial enzyme-linked immunosorbent assay (ELISA) was used to examine 143 samples of dog feces collected in urban areas as an indicator of the risk of field contamination. Using a Bayesian statistical approach, the ELISA showed a sensitivity of 88.9% and a specificity of 95.8% with positive and negative predictive values of 89.6% and 95.4%, respectively. The test affords the advantage of rapid processing of fecal samples without a complex technical structure and extensive costly labor. Moreover, the present results show that the assay provides public health veterinarians with a practical tool that can be used in screening programs, as a valid alternative or as an adjunct to other tests, in order to assess the biological risk of exposure to G. duodenalis cysts from dogs in human settlements. However, the test may not be completely accurate for human health risk evaluation, as it does not discriminate between zoonotic and non-zoonotic isolates.

Molecular characterization of Giardia duodenalis cysts from humans and animals living in well-defined contexts is useful to study the circulation of isolates and represents a tool to evaluate zoonotic infection risk. The presence of giardiasis in children living in a disadvantaged and socially deprived small Rom community, as well in dogs roaming freely in the same context was carried out by microscopic analysis and beta-giardin gene amplification. Five out of 14 children were found positive at microscopic examination for G. duodenalis and six positive at PCR, while eight out of 14 dogs tested both microscopically and molecularly positive for G. duodenalis. Moreover, most of the children and dogs were symptomatic. Molecular characterization of Giardia positive samples from children and dogs showed 99.5% identity with Giardia Assemblage A1. The dog-specific genotypes C and D were not found. The findings of this survey provide the first European evidence to support the possible role of dogs in zoonotic transmission involving children and stray dogs in a closed context with very low standards of hygiene (i.e. Rom community), and these results show the need to monitor the health of marginal populations to safeguard ethnic minority groups.

In the present survey, 276 horses bred on 16 farms located in central and southern Italy were investigated for the presence of drug resistant cyathostomes by a Fecal Egg Count Reduction Test (FECRT). Sixteen to 20 animals were selected on each farm and randomly assigned to one of four equally sized treatment groups. Groups were treated with fenbendazole, pyrantel pamoate, ivermectin or moxidectin. Resistance to fenbendazole was declared on six farms (37.5%) and suspected in two farms (12.5%), with FECR values ranging from 41% to 88.3%. Resistance to pyrantel was found in two farms (12.5%) and was suspected in one case (6.2%), with FECR values ranging from 43% to 85.4%. Macrocyclic lactones remained effective on all farms. Only cyathostome third stage larvae (L3) were found in fecal cultures after treatment. This paper reports the first wide survey conducted in Italy for anthelmintic resistance in equine cyathostomes. The results indicate that multiple drug resistant equine cyathostomes are present in the central and southern regions of Italy. These data call for a geographically and numerically broader investigation of horse farms in all regions and for the development and implementation -among veterinarians, owners and managers of a plan to reduce the expansion of these anthelmintic resistant populations and control these important parasites.

Two hundred hair/skin samples were collected from 2002 to 2004 from two groups of cats (privately owned and stray cats from a shelter) and 165 samples were obtained during the same period from persons in whom dermatophyte infection was highly suspected. The epidemiological data were statistically evaluated. Thirteen of the 100 privately owned cats (13%) and 100% of the stray cats were positive; of the 165 human samples examined 109 (66%) were positive for dermatophytes. Microsporum canis was the most common dermatophyte isolated in both cat groups while Trichophyton mentagrophytes was the most common in humans. Interestingly, a geophylic dermatophyte species (Microsporum gypseum) was found to be present and associated with clinical signs. Living in the countryside proved to be a risk factor for dermatophytoses in privately owned cats while in humans the main risk factor for M. canis was contact with animals followed by young age. None of the variables considered was associated with positivity for T. mentagrophytes while positivity for other fungi was correlated with life in the countryside.

In order to evaluate the temporal occurrence of Cryptosporidium oocysts in Ruditapes philippinarum clams bred along the northeastern Italian Adriatic coast and molecularly characterize the isolates, 2,160 specimens (180 clams per month) were collected from three clam farms from January to December 2004. Two farms (sites A and B) were located in Venice (Chioggia, Veneto region) and one (site C) in the Marano Lagoons (Friuli Venezia Giulia region). Clams from 36 pools (i.e., one pool of 60 clams per month per site) were subjected to a high-sensitivity seminested PCR assay specific for a 360-bp diagnostic region internal to the Cryptosporidium spp. outer wall protein gene. Positive amplicons were sequenced and analyzed. Cryptosporidium DNA was found in clams from seven pools (sites A and B) during 1 month of sampling at site A and 6 months of sampling at site B, with Cryptosporidium hominis and Cryptosporidium parvum being detected. The expected infection rate of the clams was 0.36%. Site B showed a significantly higher expected infection rate (1.15%) than did the other sites (A = 0.14% and C = 0%). Given its high sensitivity and specificity, this seminested PCR assay can be considered a reliable tool for detecting and distinguishing species within the Cryptosporidium genus. The seasonal pattern of contamination and the related public health risks are of particular concern.

To date, in Europe, there is scant information on the occurrence of Cyclospora in water from treatment plants and in humans, and no data are available on soil or fresh plant products. Here, we undertook the first molecular survey of Cyclospora in multiple biological matrices collected from the Apulia region of southern Italy. Samples of irrigation water from four municipal treatment plants, eight different types of vegetables or fruit (cucumber, lettuce, fennel, celery, tomato, melon, endive and chicory) and soil from the same farms on which these plants were grown, as well as faecal samples from humans living in the same region were tested by qPCR-coupled single-strand conformation polymorphism (SSCP) analysis and DNA sequencing. Cyclospora was detected in 15.5% of all 213 samples tested. Specifically, this protist was detected in (i) treated water (21.3% of 94 samples), well water (6.2% of 16), but not drinking water (0% of 3); (ii) soil (11.8% of 51 samples) and vegetables (12.2% of 49), with the highest prevalence (18.7%) on fennel; and (iii) human stools (27.5% of 40 samples). In environmental and food samples, Cyclospora was detected mainly in autumn and was significantly more prevalent in the faeces from humans of 40-50 years of age. This is the first comprehensive molecular survey of Cyclospora in environmental, food and human faecal samples in Europe. These data suggest that irrigation water, soil and vegetables might be contaminated by Cyclospora cayetanensis, which might represent a source of infection to humans in the study area and calls for monitoring by health authorities.

PCR Reverse Line Blot (RLB) hybridization and sequencing were used to determine the dynamics of infection with tick-borne pathogens in one hundred apparently healthy sheep in Italy. Blood samples were tested once prior to the onset of the grazing season (June 2010) and once after the end of the grazing season (August 2010). Ticks collected from sheep and from the vegetation were also tested by PCR/RLB. Before grazing, 56% of the sheep harbored several tick-borne pathogens: Anaplasma ovis was the most prevalent (41%), followed by A. ovis co-infected with Theileria sp. OT3 (14%). After grazing, 87% of sheep were positive for A. ovis alone (41%), co-infected with Theileria sp. OT3 (8%) or co-infected with Babesia motasi (5%). Other sheep were infected with Anaplasma phagocytophilum alone (20%), co-infected with B. motasi (7%) or with Theileria sp. OT3 (5%) (p0.001). After grazing, sheep were significantly more infected with tick-borne pathogens than before grazing. Ticks collected were all Haemaphysalis punctata (n-89) and 36% were positive for A. ovis, Ehrlichia ovina and A. ovis combined with A. phagocytophilum. Phylogenetic analysis including isolates from countries in the Mediterranean Basin show circulation of the same variants of Theileria sp. OT3, whereas two different geographical origins for the isolates of A. ovis and A. phagocytophilum were identified. This is the first report from Italy of Theileria sp. OT3 in sheep, whereas the detection of Ehrlichia ovina in ticks is worth noting, and the presence of A. phagocytophilum in sheep and in ticks poses a potential public health risk.

The purpose of this investigation was to analyze the association between different social determinants of health at the local scale and Giardia duodenalis infection in diverse settlements in southern Cote d’Ivoire. Stool samples from 306 individuals aged 1–16 years were collected from six rural villages and a small town. Five variables were categorized to classify the increasing risk levels of acquiring intestinal parasites. Giardia prevalences (%) and odds ratios (ORs) were evaluated. Correlation and regression analyses were conducted to determine the correlation coefficients and to propose predictive models based on social determinants to forecast the risk of giardiasis. The overall observed prevalence of Giardia was 21.6 %. When the analysis was conducted at the local level, the percentage of infected people varied from a minimum of 12.7 up to 36.4 %. A significant association (p

To evaluate the presence of Toxoplasma gondii and Cyclospora cayetanensis in edible shellfish, a total of 795 specimens of Mytilus galloprovincialis from 8 different sites on the west coast of Turkey (Bays of Izmir and Mersin) were examined. Fifty-three pooled samples were created and subjected to EvaGreen (R) RealTime PCR assay and HRM analysis for protozoans detection and oocyst quantification. Seven out of 8 sites were contaminated with at least one protozoan species. Twenty-one out of 53 (39.6%) samples tested positive for T. gondii, C cayetanensis or both pathogens: 26.4% samples positive for C cayetanensis; 9.4% for T. gondii, and 3.8% for both protozoans. The number of oocysts in positive samples ranged from 6 to 30 for T gondii and from 3 to 48 for C cayetanensis. This is the first report of T. gondii Type 1, and C. cayetanensis in M. galloprovincialis, the most consumed shellfish species in Turkey. (C) 2014 Elsevier Ltd. All rights reserved.

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.

In the poultry industry, control of the red mite D. gallinae primarily relies worldwide on acaricides registered for use in agriculture or for livestock, and those most widely used are carbamates, followed by amidines, pyrethroids and organophosphates. Due to the repeated use of acaricides - sometimes in high concentrations - to control infestation, red mites may become resistant, and acaricides may accumulate in chicken organs and tissues, and also in eggs. To highlight some situations of misuse/abuse of chemicals and of risk to human health, we investigated laying hens, destined to the slaughterhouse, for the presence of acaricide residues in their organs and tissues. We used 45 hens from which we collected a total of 225 samples from the following tissues and organs: skin, fat, liver, muscle, hearth, and kidney. In these samples we analyzed the residual contents of carbaryl and permethrin by LC-MS/MS. Ninety-one (40.4%) samples were positive to carbaryl and four samples (1.7%) were positive to permethrin. Concentrations of carbaryl exceeding the detection limit (0.005 ppm) were registered in the skin and fat of birds from two farms (p

Faecal samples were individually collected from pet (n=63) and zoo (n=83) birds representing 14 orders and 63 species. All the samples were examined by faecal flotation technique. In a subgroup of samples (n=75), molecular assays were also used to detectCryptosporidiumoocysts andGiardia duodenaliscysts. Overall, 35.6% of the birds harboured parasites (42.2% of zoo birds and 27% of pet birds), including Strongyles-Capillarids (8.9%),Ascaridia(6.8%), Strongyles (5.5%),G. duodenalisAssemblage A (5.3%), Coccidia (4.1%),Cryptosporidium(4%),Porrocaecum(2.7%),Porrocaecum-Capillarids (2%), andSyngamus-Capillarids (0.7%). The zoonoticG. duodenalisAssemblage A andCryptosporidiumwere exclusively found in Psittaciformes, with prevalences of 10.3% and 7.7% within this bird group. Zoo birds were more likely to harbor mixed infections (OR = 14.81) and symptomatic birds to be parasitized (OR = 4.72). Clinicians should be aware of the public health implications posed by zoonoticG. duodenalisAssemblages andCryptosporidiumspecies in captive birds.

To evaluate the presence of Toxoplasma gondii in edible farmed shellfish, 1734 shellfish specimens i.e., 109 Crassostrea gigas (6 pools), 660 Mytilus galloprovincialis (22 pools), 804 Tapes decussatus (28 pools) and 161 Tapes philippinarum (6 pools), were collected from the Varano Lagoon (Apulia, Italy). Shellfish from 62 pools were subjected to two molecular techniques: a nested-PCR assay, and a fluorescent amplicon generation (FLAG) real-time PCR assay, both based on the multi-copy B1 target, were performed. One pooled sample of gills from C. gigas and one pooled sample of haemolymphs from T. decussatus were assessed as positive for T. gondii DNA by both techniques. The results demonstrated the presence of T. gondii in edible farmed C. gigas and T. decussatus and indicate that there may be a considerable health threat involved in eating contaminated raw shellfish.

Parasites are a problem wherever poultry are raised, either in large poultry infrastructures or small backyard flocks, and are responsible for economic losses that can be significant. This chapter will discuss the most economically important parasites, all having a negative impact on egg production and egg quality. However, it will mostly focus on the red mite, Dermanyssus gallinae, because of its outstanding negative impact on animal health, welfare and production. The authors will review different aspects including biology of red mites and new insights of its control.

Seven farms, two in Central Italy and five in Southern Italy, were visited for the presence of larvae-infested wounds. Ninety-six (3%) out of 3129 sheep examined had a traumatic myiasis with a very high percentage of infested animals found only in a farm (10.5%). Wounds were mainly localized on the vulva and prepuce. Infested animals were restless, anxious, and reluctant to graze. None of the 10 goats in one infested flock had wounds, whereas a shepherd dog from another flock presented a wound on the ear. All the larvae and adults reared from them in the laboratory were identified as Wohlfahrtia magnifica (Schiner, 1862) (Diptera, Sarcophagidae). The cases presented here are the first clinical cases of wohlfahrtiosis in sheep and dogs reported in Italy, and indicate that this myiasis is endemic in this country. A wider epidemiological investigation on the prevalence, incidence and seasonality of W. magnifica infestation of Italian W. magnifica population/s would be useful to understand its geographical provenience and the likely risk for its spreading throughout Italy and other non-endemic areas.

A survey on the prevalence of Thelazia spp. in Abruzzo region (Italy) in slaughtered native horses was conducted from August 29, 1997 to August 28, 1998. Both eyes from 128 eight-month to 11 year-old native animals were examined. 50 horses (39.06 %) were found parasitized by Thelazia lacrymalis . 502 specimens (371 females, 88 males and 12 larvae) were collected. In the infected horses the numbers of T. lacrymalis ranged from 1 to 48, with a mean count burden of 3.92 per head (SD = 7.79). T. lacrymalis specimens were mainly in the excretory ducts of the Harderian gland, and also in the ducts of the lacrimal glands, free in the conjunctiva and behind the nictitancte. Gross examination showed a conjunctivitis, more frequently a follicular conjunctivitis, in the 58 % of the infected horses.

Cryptosporidiosis is a worldwide-diffused protozoan disease causing important economic losses to animal husbandry and livestock production. Additionally, several species/genotypes of Cryptosporidium have a relevant zoonotic potential and ruminants may be important sources of infection for human beings. Nonetheless, in Europe, little is known of the presence of Cryptosporidium in sheep nor on the species/genotypes involved. To obtain information on the occurrence of cryptosporidiosis in lambs and the potential zoonotic role of the Cryptosporidium isolates, one hundred and forty-nine faecal samples individually collected from lambs in central Italy have been examined for the presence of Cryptosporidium. All faecal specimens were processed with a commercial ELISA kit immunoassay and all ELISA-positive samples were further analyzed genetically. Twenty-six ELISA-positive samples scored positive at the PCR and the sequences obtained displayed 100% identity with the zoonotic Cryptosporidum parvum. This work suggests for the first time that lambs in Italy may shed C. parvum, thus representing a potential public health hazard.

Cyathostome populations in horses on two farms located in central Italy with a history of fenbendazole (FBZ) resistance were investigated with the Faecal Egg Count Reduction Test to evaluate the susceptibility to oxibendazole and moxidectin. Faecal eggs were collected pre- and post-treatment on each farm and molecularly examined with a Reverse Line Blot (RLB) assay able to unequivocally detect and identify 13 cyathostome species. Resistance to FBZ was confirmed on both farms, while oxibendazole and moxidectin demonstrated 97% and 100% efficacy, respectively. Overall eight species of cyathostomes (Coronocyclus labiatus, Cylicocyclus ashworthi, Cylicocyclus nassatus, Cyathostomum catinatum, Cylicostephanus longibursatus, Cylicostephanus goldi, Cylicostephanus calicatus and Cylicocyclus insigne) were identified in pre-treatment samples. Coronocyclus labiatus and C. goldi were identified after treatment with FBZ while C. calicatus and C. labiatus were shown to be

Giardia and Cryptosporidium spp. are important enteric protozoan pathogens for humans and animals, and have been found to contaminate water as well as edible shellfish all over the world. This is the first study to simultaneously investigate the presence of Giardia and Cryptosporidium in inflowing water and harvested shellfish in a geographically closed environment (Varano Lagoon, Southern Italy). Samples of treated wastewater were collected each month - at the outlet from the treatment plant, and downstream at the inlet into the lagoon - from the channels flowing into the Lagoon, together with specimens of Ruditapes decussatus and Mytilus galloprovincialis from shellfish-farms on the same lagoon. Giardia cysts were found by immunofluorescence (IF) microscopy in 16 out of 21 samples of treated wastewater and in 7 out of 21 samples from downstream water channels, and viable cysts were also detected by a beta-giardin RT-PCR. G. duodenalis Assemblages A and B were identified by small ribosomal subunit (18S-rDNA) and triosephosphate isomerase (tpi)-PCR, followed by sequencing. Cryptosporidium oocysts were found by IF in 5 out of 21 wastewater samples, and in 8 out of 21 samples from water channels. Molecular analysis identified the zoonotic species Cryptosporidium parvum by oocyst wall protein (COWP)-PCR and sequencing. Higher concentrations of Giardia cysts than Cryptosporidium oocysts were registered in almost all wastewater and water samples. IF and molecular testing of shellfish gave negative results for both protozoa. Wastewaters carrying Giardia and Cryptosporidium (oo)cysts are discharged into the Lagoon; however, the shellfish harvested in the same environment were found to be unaffected, thus suggesting that physical, ecological and climatic conditions may prevent contamination of harvested shellfish.

To investigate the prevalence and cyst burden of Giardia duodenalis as well as the genetic identity and possible zoonotic potential of isolates in canine faeces left on soil, a total of 143 freshly passed faecal specimens were collected in four different green urban areas and examined by Real-time PCR. Overall, 30.8% of faeces from different dogs that defecated in urban areas were found to harbour Giardia cysts, with single area positivity proportions ranging from 4.2% to 47.8%; significant (P0.05) and highly significant (P0.01) differences were found in the prevalence among different areas investigated. The number of cysts ranged from 2 to 1428 per gram of faeces. Results emphasize that a high risk of dog-to-dog Giardia transmission may occur in public areas where high quantities of faeces from infected dogs are daily left to cumulate on the ground. Results suggest there is a low risk for zoonotic transmission of Giardia: only 0.6% of canine faeces contaminating urban areas were carrying subgenotype A1, which is considered as the most potentially transmissible to humans.

In order to update and implement the data on the epidemiological situation of giardiosis in the Italian arena, stool samples from 240 dogs living in the Abruzzo region (central Italy) were examined for prevalence, risk factors, and genotypes. Giardia duodenalis cysts were detected in 26.6% of the dogs and kennel dogs tested. Dogs younger than 12 months (38%) and with diarrhea (46%) were statistically more affected. Species-specific G. duodenalis assemblages (C and D) were identified in kennel and in privately owned dogs, while the zoonotic assemblage A was identified in privately owned dogs. In light of these results, giardiosis in dogs, and mainly in well-cared dogs, is still a problem of concern and may pose a public health risk.

Giardia and Cryptosporidium spp. are protozoa that cause human and animal disease worldwide and often exhibit zoonotic transmission. This review gives ample information concerning the epidemiology of these parasites in Italy, i.e. prevalence data in humans, farm and pet animals, shellfish and aquatic environment. Moreover, it reports genotyping results obtained from different isolates, with particular emphasis on the spread of host-specific and zoonotic species/genotypes of various origin, and on molecular data that make the Italian situation different from that of other countries. Finally, possible explanations are given for the infrequent reports of Giardia and Cryptosporidium spp. outbreaks, despite widespread faecal contamination by these parasites.

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.

Two hundred and forty kennel and privately owned dogs were tested for a molecular epidemiological study on Cryptosporidium infection. Genomic DNA was extracted from individual faecal samples. All the DNA extracts were analysed with a PCR assay specific for a approximately 400-bp fragment internal to the gene encoding for the Cryptosporidium oocyst wall protein. The prevalence was 3.3% and it was higher in kennel dogs and in dogs with gastrointestinal symptoms. Cryptosporidium parvum was detected by sequencing analysis in six kennel dogs and one privately owned dog, and Cryptosporidium canis was detected in one kennel dog. This is the first investigation on the prevalence of Cryptosporidium in Italian dogs and has pointed to the existence of genotypes that may be of public health significance.

Gastric habronemosis of horses caused by Habronema microstoma and Habronema muscae (Nematoda, Spirurida) is characterized by catarrhal gastritis, diarrhoea, progressive weight loss and ulcers. Despite its importance in the equine industry and in clinical practice, knowledge of the epidemiology of this infection is still incomplete as diagnosis in live animals is challenging. A two-step semi-nested PCR assay using ribosomal DNA (rDNA) markers has recently been used for the molecular diagnosis in vivo of gastric habronemosis based on the detection of H. microstoma and/or H. muscae DNA in equine faeces. To evaluate the field efficacy of this assay, a molecular epidemiological survey was carried out on equid gastric habronemosis in central Italy. One hundred and fifty-three individual faecal samples were collected from live native horses and subjected to both coprological examination and the two-step semi-nested PCR. When flotation procedures were performed no horse tested positive for Habronema spp. larvated eggs while 96 animals (61.2%) were positive for other endoparasites (i.e. strongyles, oxyurids, ascarids). Two-step semi-nested PCR detected 86 samples (53.6%) that were positive for H. microstoma and/or H. muscae DNA. H. microstoma prevalences showed statistically significant differences; the highest prevalence was observed in horses infected by other gastrointestinal parasites and concomitantly by H. muscae. No statistical differences were found between the prevalence of Habronema spp. infection and sex, age, breeding management, and antiparasitic treatments. This field survey provided further information on habronemosis and its epidemiology.

Tortoises of the genus Testudo living in Italy are Testudo hermanni, Testudo graeca and Testudo marginata. Although a great deal of information has been acquired on the internal medicine and surgery on these animals, little is known of their parasitological fauna. A survey on the presence of gastro-intestinal parasitic nematodes in tortoises bred in Italy was carried out to acquire extensive epidemiological data. Stool samples of 62 tortoises (37 T. hermanni hermanni, 13 T. graeca, 6 T. hermanni boettgeri and 6 T. marginata) were macroscopically examined and subjected to qualitative (flotation and Baermann techniques) and quantitative (McMaster technique) microscopic examinations. Adult nematodes were identified using morphological keys. Eggs of oxyurids (species indistinguishable) and adults of Alaeuris numidica, Mehdiella microstoma, Mehdiella uncinata, Tachygonetria longicollis, Tachygonetria conica and Tachygonetria palearcticus (Oxyurida, Pharyngodonidae); larvae and adults of Atractis dactyluris (Ascaridida, Atractidae); and eggs and adults of Angusticaecum holopterum (Ascaridida, Ascarididae) were identified in all animals >1 year, whereas all tortoises

Giardiosis in domestic ruminants is an important parasitic disease and it has been shown to impair growth in lambs, thus constituting a disease of economic concern. In Europe, surveys on the prevalence of giardiosis in sheep are limited. In order to obtain additional information on the presence of giardiosis and on the potential zoonotic role of Giardia duodenalis affecting sheep in central Italy, faecal samples of 325 native sheep from 20 farms in Abruzzo region (Italy) were examined for the presence of Giardia and the isolates were genotyped and sequenced. G. duodenalis cysts were detected in five of the 325 sheep (1.5%) (mean of 450 cysts/g) coming from two farms. The 770-bp fragment of the glutamate dehydrogenase gene and the 753-bp fragment of the beta-giardin gene showed 100% homology with the Assemblage AI. This work suggests for the first time in Italy that sheep carry a Giardia genotype, which can be a potential public health hazard.

Bivalves filter large volumes of water and can concentrate organisms which are pathogenic for humans and animals. Our aim was to evaluate the presence of Cryptosporidium spp. in clams from the Adriatic coast (Abruzzo region) and genetically characterize the oocysts isolated from the clams. From March to July 2003, 960 specimens of clams (Chamelea gallina) present in nature were collected at 500 m from the Tordino, Tronto, Vibrata and Vomano river mouths on the Adriatic sea. The haemolymph and tissues were extracted from the specimens (240 per river mouth) after the specimens had been identified, measured and weighed (live weight). Immunofluorescence tests (IFA) were performed on pools (n = 32) of samples and oocysts of Cryptosporidium spp. were detected in 23 pools of C. gallina. To identify the Cryptosporidium species, all the pools IFA-positive were tested by a PCR assay specific for the Cryptosporidium outer wall protein (COWP) gene. Positive amplicons then were sequenced and analysed. Two pools of clams were positive for Cryptosporidium parvum Genotype 2 (the "bovine" i.e. zoonotic genotype). This is the first time that C. parvum was found in clams from the Adriatic sea in Italy and the case might be of public health importance.

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.

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 ,i s 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 Hypodermaspecies. 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. Larvae of Hypoderma spp. (Diptera, Oestridae) cause myia- sis to develop in warbles under the skin of domestic and wild ruminants (Zumpt, 1965). The most common species are Hy- poderma bovis (Linnaeus, 1758) and Hypoderma lineatum(De Villers, 1789), mainly affecting cattle, Hypoderma diana Brauer, 1858, Hypoderma actaeonBrauer, 1858, and Hypoder- ma tarandi (Linnaeus, 1758), affecting cervids (roe deer, red deer, and reindeer, respectively). Myiasis caused by larvae of Hypoderma spp. greatly impairs livestock production and wild ruminant welfare by inducing mechanical damage to internal organs and skin and by down- regulating the host's immune system (Boulard, 1989; Chabaudie and Boulard, 1992; Scholl, 1993; Nicolas-Gaulard et al., 1995; Boulard, 2002). In addition to the 5 species noted above, 2 other species have been described by Zumpt (1965), i.e., Hypoderma capreola Rubtzov, 1939, which has also been considered as synonymous of H. diana (see Grunin, 1962), and Hypoderma moschiferi Brauer, 1863. An eighth species, H. sinense, was described by Pleske (1926), but the author did not make comparisons between the adult flies and those of the other species (Grunin, 1969) and did not deposit holotypes. Furthermore, this species has long been considered a synonym of H. lineatum (see Zumpt, 1965), thought to affect animals living in mountainous regions (Grun- in, 1969). Identification of third larval stages (L3) of Hypoderma spp. is currently performed using 3 morphological keys (James, 1947; Zumpt, 1965; Sugar, 1976) on the basis of the presence or absence of the spines on the ventral surface of the 10th segment or on the structure of the posterior spiracles. A com- parative description of H. bovis, H. lineatum, H. diana, H. ac- taeon, and H. tarandi has been made using scanning electron

The prophylactic efficacy of microdoses of injectable and pour-on ivermectin formulations against larval stages of Przhevalskiana silenus was assessed in naturally infected goats in the region of Calabria (southern Italy).Sixty-eight goats from two goat farms were divided into five groups: one group remained untreated, while the other four groups were treated with microdoses of ivermectin (5 and 10 microg/kg injectable formulation and 10 and 20 microg/kg pour-on formulation). The microdoses of ivermectin were fully effective in the treatment of goat warble fly infestation (GWFI) as no larvae emerged from the warbles in the treated groups, while all the larvae emerged in the control groups. Irrespective of the type of formulation used, the difference between the treated groups and the control group was statistically significant (P< 0.001). By contrast, no statistical differences were found between the goats treated with the injectable formulation and those receiving the pour-on applications, and between the two doses of the injectable and pour-on formulations used. Given the plasma concentrations it attains at its lowest dose (0.052 - 0.042 ng/ml for the injectable formulation and 0.030 ng/ml for the pour-on) the injectable formulation seems to offer the most reliable route for the administration of ivermectin microdoses and it is acceptable for milk consumption. The introduction of ivermectin in the early eighties and the use of microdoses in some cases have made it possible to control cattle hypodermosis in large areas of Europe. As with cattle hypodermosis, the administration of ivermectin microdoses in goats is particularly interesting because of the low costs involved and the low levels of residues found in goat milk; it may thus constitute the basis for GWFI control campaigns in areas where the disease is prevalent.

The occurrence of Thelazia gulosa and T. skrjabini in cattle in Italy, together with an update of the prevalence of Thelazia spp. in the southern Italy, is reported. On 764 slaughtered native cattle, fifty-five (7.2 %) bovines were infected by Thelazia spp: T. rhodesi was found in 44 (80 %) animals, T. gulosa was found in 19 (34.5 %) cattle and only one (1.8 %) cattle harboured T. skrjabini . This is the first report of T. gulosa and T. skrjabini in Southern Europe.

Despite the public health importance and the possible zoonotic role of Giardia, epidemiological survey on the prevalence of giardiosis in Italy are lacking and fragmentary, compared to other countries. A cross-sectional coprological study on giardiosis was carried out in central and northern Italy in dogs and humans with the aim to providing data about prevalence and potential risk factors. A total of 616 dogs (436 from central and 180 from northern Italy) and 300 humans (central Italy) were examined. The overall prevalence was 21.3% in dogs and 2% in humans. No differences were noted between dog’s prevalence in central and northern Italy (22.2% and 19.4% respectively). The major risk factors for giardiosis in dogs were represented by density of dogs and age, i.e. dogs kept in kennel (PR=2.13, APe=53%) and younger than 1 year (PR=1.99, APe=50%) showed higher prevalence than others. Higher prevalence was noted also in dogs with gastrointestinal symptoms (PR=1.86%, APe=46%). Looking forward to obtain information on these Giardia genotypes, all the measures for the prevention of giardiosis, screening of dogs for giardiosis, particularly puppies, even those with no symptoms, and their treatment to prevent eventual human infection is highly recommended.

The red mite, Dermanyssus gallinae (Acarina: Mesostigmata), is a temporary blood-sucking ectoparasite of poultry and other avian species, with a worldwide distribution. It can occasionally bite mammals, including humans, and cause dermatitis. Medical textbooks frequently cite itching and cutaneous lesions resulting from red-mite bites as a normal occurrence in subjects working in close conjunction with poultry. In fact, D. gallinae is one of the most common pests and a major cause of economic loss in the poultry industry, with a farm prevalence of up to 90%.1 In the literature, red-mite dermatitis (RMD) is generally regarded as an urban hygiene issue. Not only …