Advances in diagnosis of gastrointestinal nematodes in livestock and companion animals.

Diagnosis of gastrointestinal nematodes in livestock and companion animals has been neglected for years and there has been an historical underinvestment in the development and improvement of diagnostic tools, undermining the undoubted utility of surveillance and control programmes. However, a new impetus by the scientific community and the quickening pace of technological innovations, are promoting a renaissance of interest in developing diagnostic capacity for nematode infections in veterinary parasitology. A cross-cutting priority for diagnostic tools is the development of pen-side tests and associated decision support tools that rapidly inform on the levels of infection and morbidity. This includes development of scalable, parasite detection using artificial intelligence for automated counting of parasitic elements and research towards establishing biomarkers using innovative molecular and proteomic methods. The aim of this review is to assess the state-of-the-art in the diagnosis of helminth infections in livestock and companion animals and presents the current advances of diagnostic methods for intestinal parasites harnessing (i) automated methods for copromicroscopy based on artificial intelligence, (ii) immunodiagnosis, and (iii) molecular- and proteome-based approaches. Regardless of the method used, multiple factors need to be considered before diagnostics test results can be interpreted in terms of control decisions. Guidelines on how to apply diagnostics and how to interpret test results in different animal species are increasingly requested and some were recently made available in veterinary parasitology for the different domestic species.

A five-year retrospective study on ascarid infections in dogs in southern Italy.

INTRODUCTION: A 5-year retrospective analysis of ascarid infections (Toxocara canis and Toxascaris leonina) in dogs from southern Italy was performed to update the epidemiological scenario of these parasites and to identify the risk factors which may favour these infections in animals in this study area. A total of 8,149 dogs, referred to our labs for copromicroscopic analysis using the FLOTAC technique, was considered. A sub-sample of 500 faecal samples were analysed also with the Mini-FLOTAC technique. Of the overall dog samples analysed, 9,2 % (95 % CI = 8,6-9,8) resulted positive for T. canis while 0,5 % (95 % CI = 0,4-0,7) resulted positive for T. leonina. Co-infections with T. canis and T. leonina were found in 0,1 % of dogs (95 % CI = 0,0-0,1). The results obtained by the FLOTAC and Mini-FLOTAC examinations showed a nearly perfect k agreement (k = 0,99, P < 0,001) between these two techniques. Chi-square test showed positivity to T. canis and T. leonina significantly (P < 0,001) associated with dogs housed outdoor (i.e., that lived in garden or in kennel). Moreover, the positivity for T. canis was significantly associated (P < 0,001) also with age (i.e., puppies), as shown by the logistic regression. The decreasing overall prevalence both for T. canis and T. leonina during the years of monitoring, showed that, as suggested by the European Scientific Counsel Companion Animal Parasites, the regular diagnosis could contribute to an efficient control of these parasites.

INTRODUCTION: Une analyse rétrospective sur 5 ans des infections à ascaris (Toxocara canis et Toxascaris leonina) chez les chiens du sud de l'Italie a été réalisée afin de mettre à jour le scénario épidémiologique de ces parasites et d'identifier les facteurs de risque pouvant favoriser ces infections chez les animaux de cette zone d'étude. Au total, 8149 chiens ont été analysés dans notre laboratoire avec une analyse copromicroscopique en utilisant la technique FLOTAC. De plus, un sous-échantillon de 500 échantillons fécaux a été analysé avec la technique Mini-FLOTAC. Sur l'ensemble des échantillons fécaux canins analysés, 9,2 % (IC à 95 % = 8,6 à 9,8) se sont révélés positifs pour T. canis tandis que 0,5 % (IC à 95 % = 0,4 à 0,7) ont été positifs pour T. leonina. Des co-infections avec T. canis et T. leonina ont été trouvées chez 0,1 % des chiens (IC à 95 % = 0,0­0,1). Les résultats obtenus par les examens FLOTAC et Mini-FLOTAC ont montré un coefficient Kappa presque parfait (k = 0,99, p < 0,001) entre ces deux techniques. Le test du chi carré a montré une positivité significative quant aux infections à T. canis et T. leonina (P < 0,001) associées à des chiens hébergés à l'extérieur (jardin ou chenil). De plus, la positivité pour T. canis était également significativement associée (P < 0,001) à l'âge (c'est-à-dire aux chiots), comme le montre la régression logistique. La diminution de la prévalence globale au cours de la période de surveillance a montré que le diagnostic régulier pourrait contribuer à un contrôle efficace de ces parasites à la fois pour T. canis et T. leonina, comme suggéré par le the European Scientific Counsel Companion Animal Parasites.

Laboratory evidence that dinotefuran, pyriproxyfen and permethrin combination abrogates Leishmania infantum transmissibility by sick dogs.

Dogs are reservoir hosts of leishmaniasis caused by Leishmania infantum and transmitted by phlebotomine vectors. The effect of dinotefuran, pyriproxyfen and permethrin spot-on solution (Vectra®3D, Ceva Santé Animale, Libourne, France) on Leishmania transmissibility by naturally infected dogs via reared Phlebotomus perniciosus, was assessed. Dogs affected by leishmaniasis were submitted to xenodiagnosis and 6 infecting >10% of insects were treated topically on day 0. Antifeeding, insecticidal and anti-transmissibility effects were evaluated through xenodiagnoses performed on days 1, 7 and 28, using individual pre-treatment parameters as control. Feeding and mortality rates were assessed at 24 h, whereas promastigote infection, maturation and burden were assessed up to 96 h post blood meal (potentially infectious rate). On day 1, the anti-feeding efficacy was >95% in 4 dogs, insecticidal efficacy 100% in 4 dogs, and anti-transmissibility effect 100% in 6 dogs. Efficacy rates recorded on day 7 were very similar to day 1. On day 28, anti-feeding and insecticidal efficacy values were much broader, ranging 32.6-100% and 7.7-94.4%, respectively. Potentially infectious insects were recorded from two dogs, with sharp decrease in transmissibility rate as compared with pre-treatment condition. Altogether, Vectra®3D abrogated by >98% the potential Leishmania transmissibility by the examined pool of infected dogs over 1 month.

Ascaris lumbricoides eggs or artefacts? A diagnostic conundrum.

Due to the presence of artefacts in stool samples, the copromicroscopic diagnosis of Ascaris lumbricoides is not always straightforward, particularly in the case of fertilized decorticated eggs. A total of 286 stool samples from 115 schoolchildren in India and 171 adult immigrants in Italy were screened for the presence of A. lumbricoides eggs by both Kato-Katz thick smear and Mini-FLOTAC. If the outer layer of A. lumbricoides eggs was absent, two aliquots of each stool sample were preserved: one for coproculture to identify larvae after development and one to compose a pool of stool for molecular analysis. A total of 64 stool samples (22.4%) were positive for A. lumbricoides using the Kato-Katz thick smear; 36 (56.3%) of these showed mammillated A. lumbricoides eggs, 25 (39.1%) showed elements resembling fertilized decorticated eggs, while three samples (4.7%) showed both mammillated and decorticated eggs. By Mini-FLOTAC, 39 stool samples (13.6%) were positive, while decorticated A. lumbricoides-like eggs were identified as artefacts. These results were confirmed by negative coprocultures and quantitative polymerase chain reaction. Mini-FLOTAC can be used for a reliable diagnosis of A. lumbricoides, thanks to the flotation and translation features which allow a clearer view, resulting in the correct identification of A. lumbricoides eggs.

An integrated approach to control Cystic Echinococcosis in southern Italy.

Cystic echinococcosis (CE) is a severe zoonosis, caused by the larval stage of the tapeworm Echinococcus granulosus. This helminth infection is of increasing public health and socio-economic concern due to the considerable morbidity rates that cause economic losses both in the public health sector and in the livestock industry. Control programmes against E. granulosus are considered long-term actions which require an integrated approach and high expenditure of time and financial resources. Since 2010, an integrated approach to control CE has been implemented in a highly endemic area of continental southern Italy (Campania region). Innovative procedures and tools have been developed and exploited during the control programme based on the following strategies: i) active and passive surveillance in livestock (using geospatial tools for georeferencing), ii) diagnosis in dogs (using the FLOTAC techniques and molecular analysis), iii) targeted treatment of farm dogs (using purpose-built confinement cages), iv) early diagnosis in livestock (by ultrasonography), v) surveillance in humans (through hospital discharge records analysis), vi) monitoring the food chain (analysing raw vegetables), vii) outreach activities to the general public (through dissemination material, e.g. brochures, gadgets, videos, virtual reality). Over eight years, the integrated approach and the new strategies developed have resulted in a noteworthy reduction of the parasite infection rates in livestock (e.g. up to 30 % in sheep). The results obtained so far highlight that using a one health multidisciplinary and multi-institution effort is of pivotal importance in preparing CE control programmes at regional level and could be extended to other endemic Mediterranean areas.

Assessment of anthelmintic efficacy against cattle gastrointestinal nematodes in western France and southern Italy.

Our objective was to measure the efficacy of ivermectin (IVM) and benzimidazoles (BZ, i.e. fenbendazole and albendazole) in 15 cattle farms in western France and southern Italy. A total of 11 groups were treated with IVM and 11 with BZ. Efficacy was assessed by calculating the percentage of faecal egg count reduction (%FECR) using the pre- and post-treatment arithmetic means. Anthelmintic resistance was considered to be present when the %FECR was <95% and the lower limit of the 95% confidence interval <90%. For IVM, the percentages of FECR ranged from 73% to 100%. Lack of efficacy to IVM was detected in two farms out of four in France, but was not detected in any of the seven farms in Italy. For BZ, the percentages of FECR ranged from 95% to 100%. No case of BZ resistance was detected in the five farms in France and the six farms in Italy.

Survey of Baylisascaris spp. in captive striped skunks (Mephitis mephitis) in some European areas.

Skunks are popular carnivore species kept both in zoological institutions and in households where they are hand raised as exotic pets. These small carnivores are considered the main definitive hosts of the roundworm Baylisascaris columnaris. The purpose of this survey was to investigate the occurrence of Baylisascaris spp. in striped skunks kept as pets or in private zoo collections in some European areas. Copromicroscopic data from two laboratories, one in Italy and one in Germany, were used. A total of 60 animals were selected. Samples came from Germany (n = 30), Italy (n = 23), United Kingdom (n = 5), Austria (n = 1), and the Netherlands (n = 1). Twenty-eight animals were certainly kept as pets in private households in Italy and the UK. Fifteen out of 60 animals (25%) were positive for Baylisascaris spp. Molecular identification of adult parasites was performed in ten of those animals, revealing B. columnaris in all cases. To the authors' knowledge, this is the first survey of Baylisascaris spp. in captive skunks in Europe.

Heat treatment of serum samples from stray dogs naturally exposed to Dirofilaria immitis and Dirofilaria repens in Romania.

Pre-heating of serum samples has been shown to reverse false negative antigen tests for Dirofilaria immitis infection in dogs. Here the authors report the results of serum sampling in a population of dogs naturally exposed to D. immitis and Dirofilaria repens infection by testing in ELISA before and after heat treatment. Of 194 dogs sampled from four cities in Romania, D. immitis circulating antigens were found in 16 (8.2%) non heated samples and in 52 (26.8%) heated samples. Of the 108 dogs examined by Knott test, 24 dogs (22.2%) were positive for circulating mf. Subsequent PCR identification showed six dogs had D. immitis mf only, 12 dogs, had only D. repens mf, and 5 were positive for both. Fifty% of dogs with circulating D. immitis mf had positive antigen tests before and after heating, while the other 50% reverted to positive only after heat treatment. Sixty% of dogs with mixed D. immitis/D. repens infection were antigen positive before and after heating, while the other 40% converted to positive after heating. Antigen testing for D. immitis in the 12 dogs with only D. repens mf gave conflicting results. Only two dogs (16%) were antigen negative both before and after heat treatment. Six dogs (50%) became antigen positive after heating and four dogs (30%) were antigen positive both before and after heat treatment. Results would suggest that: false negative result for antigen testing can be reverted by heating of the serum sample; dogs infected with D. repens may have also an occult infection with D. immitis; heat treatment of serum from D. repens-infected dogs can reveal an occult infection with D. immitis.

The bias, accuracy and precision of faecal egg count reduction test results in cattle using McMaster, Cornell-Wisconsin and FLOTAC egg counting methods.

The faecal egg count reduction test (FECRT) is the recommended method to monitor anthelmintic drug efficacy in cattle. There is a large variation in faecal egg count (FEC) methods applied to determine FECRT. However, it remains unclear whether FEC methods with an equal analytic sensitivity, but with different methodologies, result in equal FECRT results. We therefore, compared the bias, accuracy and precision of FECRT results for Cornell-Wisconsin (analytic sensitivity = 1 egg per gram faeces (EPG)), FLOTAC (analytic sensitivity = 1 EPG) and McMaster method (analytic sensitivity = 10 EPG) across four levels of egg excretion (1-49 EPG; 50-149 EPG; 150-299 EPG; 300-600 EPG). Finally, we assessed the sensitivity of the FEC methods to detect a truly reduced efficacy. To this end, two different criteria were used to define reduced efficacy based on FECR, including those described in the WAAVP guidelines (FECRT <95% and lower limit of 95%CI <90%) (Coles et al., 1992) and those proposed by El-Abdellati et al. (2010) (upper limit of 95%CI <95%). There was no significant difference in bias and accuracy of FECRT results across the three methods. FLOTAC provided the most precise FECRT results. Cornell-Wisconsin and McMaster gave similar imprecise results. FECRT were significantly underestimated when baseline FEC were low and drugs were more efficacious. For all FEC methods, precision and accuracy of the FECRT improved as egg excretion increased, this effect was greatest for McMaster and least for Cornell-Wisconsin. The sensitivity of the three methods to detect a truly reduced efficacy was high (>90%). Yet, the sensitivity of McMaster and Cornell-Wisconsin may drop when drugs only show sub-optimal efficacy. Overall, the study indicates that the precision of FECRT is affected by the methodology of FEC, and that the level of egg excretion should be considered in the final interpretation of the FECRT. However, more comprehensive studies are required to provide more insights into the complex interplay of factors inherent to study design (sample size and FEC method) and host-parasite interactions (level of egg excretion and aggregation across the host population).

FLOTAC can detect parasitic and pseudoparasitic elements in reptiles.

Reptiles have increased in popularity worldwide; snakes and lizards are frequently used as pets. As a consequence of their popularity, the interest of the scientific community in these animals has increased. In order to acquire epidemiological data on the parasitic infections affecting reptiles in Italy a survey was carried out in 125 snakes and 25 lizards bred in the Campania region of southern Italy. Individual fecal samples were collected and FLOTAC was used for copromicroscopic diagnosis. Eimeriidae, oxyurids, strongylids, other gastro-intestinal nematodes and pulmonary nematodes were the most representative parasites found. Eggs of pseudoparasites (mites, oxyurids and trichurids affecting rodents) were also found. The use of FLOTAC for diagnosis of parasitic infections in reptiles has demonstrated to be a rapid and sensitive test to improve diagnosis and acquire new information on the parasitological fauna of reptiles.

Calibration and diagnostic accuracy of simple flotation, McMaster and FLOTAC for parasite egg counts in sheep.

The present study was aimed at carrying out a calibration and a comparison of diagnostic accuracy of three faecal egg counts (FEC) techniques, simple flotation, McMaster and FLOTAC, in order to find the best flotation solution (FS) for Dicrocoelium dendriticum, Moniezia expansa and gastrointestinal (GI) strongyle eggs, and to evaluate the influence of faecal preservation methods combined with FS on egg counts. Simple flotation failed to give satisfactory results with any samples. Overall, FLOTAC resulted in similar or higher eggs per gram of faeces (EPG) and lower coefficient of variation (CV) than McMaster. The "gold standard" for D. dendriticum was obtained with FLOTAC when using FS7 (EPG=219, CV=3.9%) and FS8 (EPG=226, CV=5.2%) on fresh faeces. The "gold standard" for M. expansa was obtained with FLOTAC, using FS3 (EPG=122, CV=4.1%) on fresh faeces. The "gold standard" for GI strongyles was obtained with FLOTAC when using FS5 (EPG=320, CV=4%) and FS2 (EPG=298, CV=5%). As regard to faecal preservation methods, formalin 5% and 10% or freezing showed performance similar to fresh faeces for eggs of D. dendriticum and M. expansa. However, these methods of preservation were not as successful with GI strongyle eggs. Vacuum packing with storage at +4°C permitted storage of GI strongyle eggs for up to 21 days prior to counting. Where accurate egg counts are required in ovine samples the optimum method of counting is the use of FLOTAC. In addition, we suggest the use of two solutions that are easy and cheap to purchase and prepare, saturated sodium chloride (FS2) for nematoda and cestoda eggs and saturated zinc sulphate (FS7) for trematoda eggs and nematoda larvae.

Intestinal parasites in immigrants in the city of Naples (southern Italy).

The present study was aimed at carrying out a cross-sectional copromicroscopic survey of helminths and intestinal protozoa in immigrants in Naples (southern Italy). Between October 2008 and November 2009, a total of 514 immigrants were tested comparing the FLOTAC dual technique and the ethyl acetate concentration technique. Combined results of the two techniques served as a diagnostic 'gold' standard and revealed an overall prevalence of parasitic infections of 61.9% (318/514). The ethyl acetate concentration technique detected a low number of positive results (49.0%) and this was confirmed for each helminth/protozoa species detected. Among helminths, Trichuris trichiura (3.9%), hookworms (3.7%) and Ascaris lumbricoides (1.4%) were the most prevalent. Strongyloides stercoralis (0.4%), Enterobius vermicularis (0.4%), Schistosoma mansoni (1.0%), Hymenolepis nana (1.6%) and Taenia spp. (0.2%) were also found, as well as zoonotic helminths, as Trichostrongylus spp. (0.8%) and Dicrocoelium dendriticum (0.8%). As regard to pathogenic protozoa, Blastocystis hominis was the most commonly detected (52.7%), followed by Entamoeba histolytica/Entamoeba dispar/Entamoeba moshkovskii (11.9%) and Giardia duodenalis (4.5%). Several issues concerning diagnosis, epidemiology and public health impact of parasitic infections in immigrants are offered for discussion. In conclusion, the present paper pointed out the need of better diagnosis and cure of the immigrant population in order to improve access to health care of this neglected and marginalised population group, for its own protection and care.

FLOTAC: an improved method for diagnosis of lungworm infections in sheep.

The FLOTAC techniques involve the spinning of faecal samples onto the surface of counting chambers to permit enumeration of parasitic elements (eggs, larvae, oocysts and cysts) to an accuracy of one parasitic element per gram of faeces. In the present study it is demonstrated that FLOTAC provides a rapid and very sensitive method for counting of lungworm larvae of sheep. The optimum flotation solution for lungworm larvae is zinc sulphate and mercury II iodide (s.g. 1.45) although zinc sulphate (s.g. 1.20 or 1.35) on its own also gave good results. Samples preserved in 5% formalin gave the highest counts but fresh, frozen and samples in 10% formalin also gave higher counts than McMaster and simple flotation. Larval counts of 307 field samples gave up to 1.27x more positives samples than use of Baermann funnels and up to 4.18x more larvae per sample. As FLOTAC is faster than Baermannisation of samples it offers a better method of counting larvae in ruminant faecal samples.

The role of cattle in the epidemiology of Echinococcus granulosus in an endemic area of southern Italy.

An epidemiological and molecular survey was conducted to investigate the role of cattle in the transmission chain of cystic echinococcosis (CE) in the Campania region of southern Italy. Out of a total of 434 cattle examined for CE, 45 (10.4%) were found infected. A total of 363 cysts were collected from the infected animals: 239 in the liver and 124 in the lungs. The cysts were either sterile (42.7%) or calcified/caseous (57.3%); no fertile cysts were found. Most of the cysts had sizes <3 cm (77.1%) and were unilocular (78.8%). The results of the linear regression model did not show any significant correlation between the age of infected cattle and the number of cysts. The sequencing of the mitochondrial cytochrome C oxidase subunit 1 (CO1) gene of 40 hydatid cysts produced sequences of 419 bp for each sample analyzed. Alignment of the obtained sequences with those present in GenBank showed 100% identity with the common sheep G1 (n=21 cysts), the Tasmanian sheep G2 (n=2 cysts), and the buffalo G3 (n=17 cysts) strains, which constitute the species Echinococcus granulosus sensu stricto. The findings reported in the present study show that CE is widespread in cattle bred in the Campania region of southern Italy. However, the absence of fertile cysts and of the cattle strain (G5, E. ortleppi) suggests that cattle would not have any role in the persistence of this important zoonosis but rather a role as indicators of CE infection in this endemic area.

Molecular update on cystic echinococcosis in cattle and water buffaloes of southern Italy.

Cystic echinococcosis (CE)--caused by the larval stage (hydatid cyst) of the cestode Echinococcus granulosus--is one of the most widespread zoonoses of veterinary and medical importance. Molecular techniques have allowed the identification of 10 different genotypes (G1-G10) of the parasite. The present paper is an update regarding the E. granulosus genotypes infecting water buffaloes and cattle bred in the Campania region of southern Italy. The molecular study was performed on 30 hydatid cysts (11 from water buffaloes and 19 from cattle). Two different mitochondrial DNA genes, namely the cytochrome c oxidase subunits 1 and the 12S ribosomal DNA (12S rDNA) were used as genetic markers. Three different genotypes of E. granulosus were unequivocally identified, i.e. the G1 (common sheep), G2 (Tasmanian sheep) and G3 (buffalo) genotypes, as well as some G1 and G2 variants. It should be noted that the present study demonstrated for the first time: (i) the presence of the G2 genotype in water buffaloes from a Mediterranean area; and (ii) the fact that the analysed portion of the 12S rDNA gene can not discriminate between the G2 and G3 genotypes of E. granulosus. The finding of the G1, G2 and G3 genotypes in large ruminants from southern Italy is of epidemiological relevance and immediate public health importance because of their recognized infectivity in humans.

Giardia and Cryptosporidium in canine faecal samples contaminating an urban area.

The aim of the present paper was to continue the study on the presence of parasitic elements in the canine faeces contaminating the urban environment of Naples (southern Italy), focussing on the protozoa Giardia and Cryptosporidium. The total number of sub-areas studied was 143, and the total number of canine faecal samples collected and examined was 415. Each faecal sample was tested for the presence of copro-antigens of Giardia and Cryptosporidium using two commercially available enzyme-linked immunosorbent assays. Giardia antigens were found in 19.6% (28/143) of the sub-areas and in 7.7% (32/415) of the canine faeces collected. Cryptosporidium antigens were found in 4.2% (6/143) of the sub-areas and in 1.7% (7/415) of the canine faeces collected. Co-infection was not found in any sample. The results of the logistic regression models did not show any association between the positivity to Giardia or Cryptosporidium and the independent demographic variables (human population density, male and female population density) taken into consideration. In conclusion, the findings of the present study revealed the presence of Giardia and Cryptosporidium in canine faecal samples from the urban environment of Naples; however, the zoonotic potential of these findings was not assessed due to the lack of information on species/genotypes detected.

Characterization of the 28S and the second internal transcribed spacer of ribosomal DNA of Dicrocoelium dendriticum and Dicrocoelium hospes.

Isolates of Dicrocoelium dendriticum (n=150) from sheep and cattle bred in southern Italy and isolates (n=5) of D. hospes from a Bos indicus from Senegal were characterized genetically. The 28S region and the second internal transcribed spacer (ITS-2) plus flanking 5.8S and 28S sequences (ITS-2+) of ribosomal DNA (rDNA) were amplified by polymerase chain reaction and sequenced from individual flukes. Regarding the 28S rDNA, sequences of 568 and 581 bp were obtained for D. dendriticum and D. hospes, respectively. No intraspecific variation was observed between the 28S rDNA of all the D. dendriticum specimens studied and the D. dendriticum 28S rDNA sequence present in GenBank. However, intraspecific variation was observed in the 28S rDNA of the D. hospes specimens compared to the sequence present in GenBank. Regarding the ITS2+ rDNA, sequences of 402 and 428 bp were obtained for D. dendriticum and D. hospes, respectively; both sequences were deposited in GenBank. Variations intra- and interpopulation were observed for D. dendriticum, whereas 100% identity was observed in all the ITS2+ sequences of D. hospes. With respect to the interspecific variations, the ITS-2+ of D. dendriticum and D. hospes differed in 33 positions. The findings of the present study showed an ITS2+ sequence variability (8.2-8.5%) between D. dendriticum and D. hospes, thus demonstrating the utility of this sequence to discriminate the two species.

A comparison of five methods for DNA isolation from liver and rumen flukes to perform ITS-2+ amplification.

Five different DNA isolation methods (4 commercial kits and a modification of phenol-chloroform method) were compared for the discrimination of adults of Fasciola hepatica and Dicrocoelium dendriticum (liver flukes), and Calicophoron daubneyi (rumen fluke) collected from sheep in southern Italy. The second internal transcribed spacer (ITS-2) of ribosomal DNA (rDNA) plus flanking 5.8S and 28S sequence (ITS-2+) was amplified by polymerase chain reaction (PCR) from serial diluted DNA templates (6 ng - 60 fg) of each fluke species. Overall, in terms of efficiency in detection limit, the best results were obtained either with phenol-chloroform purification or with QIAamp DNA Mini Kit (Qiagen), but using this latter method, rapid, safe and not expensive, an increased level of sensitivity sufficient to detect small amounts of target-DNA was achieved. In addition, electrophoresis analysis following PCR also showed that ITS-2+ could be useful as a genetic marker for the molecular identification of F. hepatica, D. dendriticum and C. daubneyi in definitive and intermediate hosts. Furthermore, for the first time, the ITS-2 sequence of D. dendriticum was defined.

Cystic echinococcosis in water buffaloes: epidemiological survey and molecular evidence of ovine (G1) and buffalo (G3) strains.

A survey of cystic echinococcosis (CE) in the water buffalo (Bubalus bubalis) of the Italian Mediterranean breed was carried out in Campania, a region of southern Italy. In addition, a molecular study was performed on 48 hydatid cysts coming from 48 water buffaloes in order to determine the Echinococcus granulosus strain(s) present in this host. Out of a total of 722 water buffaloes examined for CE, 76 (10.5%) were found infected. The average number of cysts per buffalo was 4.3 (minimum 1, maximum 45). Seventeen buffaloes had hydatid cysts only in the liver (with an average of 5 cysts/liver), 34 only in the lungs (with an average of 1.8 cysts/lungs), and 25 buffaloes had cysts both in the liver and in the lungs. Fertile cysts were found in 10 (13.2%) out of the 76 positive buffaloes. The sequencing of the mitochondrial cytochrome C oxidase subunit 1 (CO1) gene of the 48 hydatid cysts produced sequences of 419 bp for each sample analysed. For 33 samples, alignment of the obtained sequences with those present in GenBank showed a total homology with the common domestic sheep strain G1; for 15 samples, sequences obtained showed 100% homology with buffalo strain G3. The findings of the present survey represent the first epidemiological and molecular comprehensive studies on CE in water buffalo from an endemic area for E. granulosus.