Gastrointestinal parasite assemblages from the wild rodent capybara (Hydrochoerus hydrochaeris) inhabiting a natural protected area from Argentina.

Knowledge about parasitic diseases of wildlife will help us to understand the dynamics of parasites and their effects on host populations. The capybara (Hydrochoerus hydrochaeris) is the largest living rodent in the world, and its distribution is associated with the presence of tropical and subtropical wetlands in South America. The Los Padres Lake Integral Reserve (LPLIR) is an important conservation zone in the pampean region of Argentina. One of the emblematic species found within the reserve is the capybara. The objective of this study was to determine the gastrointestinal parasites present in wild capybaras of the LPLIR and to compare different coprological methodologies. Free-ranging capybara fresh feces from 57 individuals were randomly collected from the area of LPLIR in the summer of 2022. Three different techniques were applied: spontaneous sedimentation technique (SS), INTA modified McMaster technique (MM), and Mini-FLOTAC (MF) technique. Fifty-six samples from all samples analysed (56/57, 98%) were found to be positive for gastrointestinal parasites. Two species of Strongylida, Protozoophaga obesa, Echinocoleus hydrochaeris, one unidentified nematode, one unidentified spirurid, and at least two morphotypes of Eimeria spp. oocysts were recorded. There were found significant differences in the proportion of positive samples and in richness by technique, but no significant differences were found in parasite counting. In conclusion, the choice of methodology depends on the specific objectives of the study. This is the first parasitological study of capybaras from the LPLIR and represents an exploration of parasite communities present in these wild rodents at their southernmost distribution.

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.

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.

Species diversity and spatial distribution of ixodid ticks on small ruminants in Greece.

A cross sectional field study on the distribution of Ixodidae ticks was carried out over two consecutive tick seasons (2012 and 2013) in small ruminants (309 farms) located in mainland and five islands of Greece. A total of 2108 ticks (1199 females, 908 males, and 1 nymph) were collected from sheep and goats. Two species of Rhipicephalus, two of Ixodes, five of Hyalomma, three of Haemaphysalis, and one of Dermacentor were detected. The species with the widest distribution were Rhipicephalus sanguineus s.l. (64.8 %), R. bursa (25.9 %), and Dermacentor marginatus (4.1 %), whereas the least frequently collected species were Ixodes ricinus, I. gibbosus, Haemaphysalis parva, H. sulcata, H. punctata, Hyalomma marginatum, H. excavatum, H. dromedarii, H. rufipes, and H. impeltatum representing together less than 5.3 % of the collections. R. sanguineus s.l. was the predominant species, while D. marginatus and I. gibbosus were restricted to the northeastern Aegean Sea islands. This is the first survey on tick fauna from mainland and islands in Greece.

Survey of Hymenolepis spp. in pet rodents in Italy.

We carried out the first survey of Hymenolepis spp. infection in pet rodents in Italy. Fresh fecal samples were collected from 172 pet rodents as follows: guinea pigs (Cavia porcellus; n = 60), squirrels (Callosciurus finlaysonii, Callosciurus prevosti, Tamias striatus, Tamias sibiricus, Sciurus calorinensis; n = 52), hamsters (Phodopus campbelli, Mesocricetus auratus; n = 30), chinchillas (Chinchilla lanigera; n = 13), rats (Rattus norvegicus; n = 10), and mice (Mus minutoides; n = 7). These animals were housed either in pet shops or in private houses. All fecal samples were processed using the FLOTAC pellet technique to assess the number of eggs per gram (EPG) of feces. Eggs of Hymenolepis nana were found in 24 out of 172 (13.9 %; 95 % confidence interval = 9.3-20.2 %) pet rodents. Of those rodents, 41.6 % (10/24) were rats (mean EPG = 55.7; range = 2-200), 29.2 % (7/24) mice (mean EPG = 64.5; range = 32-120), 25.0 % (6/24) were chinchillas (mean EPG = 25.5; range = 10-50), and 4.2 % (1/24) hamsters (P. campbelli) (EPG = 86.0). In addition, Hymenolepis diminuta eggs were found in 2 out of 172 (1.2 %; 95 % confidence interval = 0.2-4.6 %) rodents examined, both of which (100 %; 2/2) were pet squirrels (C. prevosti) (mean EPG = 10; range = 4-16). To the authors' knowledge, this is the first report of a natural infection of H. diminuta in pet squirrels.

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.

Prevalence and molecular identification of Cryptosporidium isolates from pet lizards and snakes in Italy.

In order to acquire prevalence and genetic data on Cryptosporidium infections in captive lizards and snakes kept as pets, a survey was conducted on 150 individual reptiles from southern Italy. Fecal samples were preserved in 5% formalin and analyzed using a commercial immunofluorescence assay (IFA) for the detection of Cryptosporidium oocysts and Giardia cysts. IFA revealed the presence of Cryptosporidium oocysts in nine of the 150 samples examined (6.0%), precisely in 6/125 snakes (4.8%) and in 3/25 lizards (12.0%); all fecal samples tested negative for the presence of Giardia cysts. Molecular characterization based on nested PCR amplification and sequencing of the SSU-rRNA gene, revealed the presence of Cryptosporidium serpentis in three samples from snakes (Boa constrictor constrictor, Elapheguttata guttata guttata and Python molurus).

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.

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.

New insights into the biology, diagnosis and immune response to Dirofilaria repens in the canine host.

Dogs are the primary host for Dirofilaria repens, therefore it is mandatory to accurately diagnose the canine infection and to expand our current knowledge on parasite biology and the immune response of the infected host for a better prevention.Thus, the aim of the present study was to provide new insights from experimental infections of dogs with D. repens, focusing on the evaluation of: 1) the pre-patent period and 2) the antibody response against D. repens somatic antigens and against the Wolbachia endosymbiont. Briefly, on Day 0, twenty purpose-bred Beagle dogs were experimentally infected with 50 infective larvae (L3) of D. repens. Starting from Day 58 until the last day of the study (Day 281), blood samples were collected on a monthly basis for detection of antibodies against D. repens (Dr) and recombinant Wolbachia surface protein (rWSP) by non-commercial IgG-ELISAs. Additional samples were collected on Days 220, 245 and 281 for the detection of microfilariae (mff) using the modified Knott's test and biomolecular analysis, following two PCR protocols: Gioia et al. (2010; protocol A) and Rishniw et al. (2006- protocol B). The results were analysed by univariate statistical analyses using 2 × 2 contingency tables and K Cohen was calculated to assess the agreement among all the diagnostic techniques. Overall, the outcome of the study revealed that out of the 20 dogs experimentally infected with D. repens, 16 (80 %) were microfilaraemic, 17 (85 %) were positive at DNA detection in the blood, 18 (90 %) had D. repens antibodies and 16 (80 %) had Wolbachia antibodies on the last day of the study. The overall k agreement between Knott's and PCR protocol B was 0.442 (P = 0.0001) and increased throughout the study, reaching 0.828 (P = 0.0001) on Day 281. To the authors knowledge, this is only the second study reporting antibody response to D. repens somatic antigen in experimentally infected dogs. ELISA results showed that an antibody response develops before the onset of patency, and steadily increases with time. Results would suggest that the development of an immunological response to infection could lead to application in epidemiological studies, risk assessment and as an aid in the diagnostic approach in dogs, in particular for early infections without mff.

New insights into the biology, diagnosis and immune response to Dirofilaria repens in the canine host.

Dogs are the primary host for Dirofilaria repens, therefore it is mandatory to accurately diagnose the canine infection and to expand our current knowledge on parasite biology and the immune response of the infected host for a better prevention.Thus, the aim of the present study was to provide new insights from experimental infections of dogs with D. repens, focusing on the evaluation of: 1) the pre-patent period and 2) the antibody response against D. repens somatic antigens and against the Wolbachia endosymbiont. Briefly, on Day 0, twenty purpose-bred Beagle dogs were experimentally infected with 50 infective larvae (L3) of D. repens. Starting from Day 58 until the last day of the study (Day 281), blood samples were collected on a monthly basis for detection of antibodies against D. repens (Dr) and recombinant Wolbachia surface protein (rWSP) by non-commercial IgG-ELISAs. Additional samples were collected on Days 220, 245 and 281 for the detection of microfilariae (mff) using the modified Knott's test and biomolecular analysis, following two PCR protocols: Gioia et al. (2010; protocol A) and Rishniw et al. (2006- protocol B). The results were analysed by univariate statistical analyses using 2×2 contingency tables and K Cohen was calculated to assess the agreement among all the diagnostic techniques. Overall, the outcome of the study revealed that out of the 20 dogs experimentally infected with D. repens, 16 (80 %) were microfilaraemic, 17 (85 %) were positive at DNA detection in the blood, 18 (90 %) had D. repens antibodies and 16 (80 %) had Wolbachia antibodies on the last day of the study. The overall k agreement between Knott's and PCR protocol B was 0.442 (P=0.0001) and increased throughout the study, reaching 0.828 (P=0.0001) on Day 281. To the authors knowledge, this is only the second study reporting antibody response to D. repens somatic antigen in experimentally infected dogs. ELISA results showed that an antibody response develops before the onset of patency, and steadily increases with time. Results would suggest that the development of an immunological response to infection could lead to application in epidemiological studies, risk assessment and as an aid in the diagnostic approach in dogs, in particular for early infections without mff.

Is gastrointestinal strongyle faecal egg count influenced by hour of sample collection and worm burden in goats?

Gastrointestinal (GI) strongyle infections remain one of the main constraints to goat production worldwide. Although several indicators of GI strongyle infections have been proposed, faecal egg count (FEC) techniques are the most common approaches for the estimation of prevalence and intensity of these parasites. However, FEC may be subjected to a within-individual variation due to factors such as weather, season, random day-to-day variation and phase of the parasitic infection. A longitudinal study of GI strongyle FEC was conducted on 63 dairy goats to evaluate the effects of hour of sample collection on FEC and the relationship between FEC and worm burden. The goats were naturally infected with Teladorsagia circumcincta, Haemonchus contortus, Trichostrongylus colubriformis and Oesophagostomum venulosum. Every 3 weeks for 14 months faeces were collected every 2 h for 24 h from three individually caged goats. The faeces were those passed by the goats during the 2 h preceding each collection. For each goat a sample from this material was analyzed using the Flotac double technique. To explore relationships between FECs and adult parasite counts, on the day following sampling, the three goats were euthanized and the nematodes present in the abomasa and intestines were recovered, identified and counted. The results of the general linear model did not show any significant effect of the hour of faecal sample collection on GI strongyle FEC, whereas a significant effect of the month of faecal sample collection was found. The results of the Pearson correlation showed a significant (P=0.000) positive relationship between FEC and worm burden (r=0.6), in particular regarding H. contortus (r=0.9). In conclusion, the present study showed that the hour of sample collection does not influence the GI strongyle FEC and that there is a good relationship between FEC and total GI strongyle worm burden in goats.

Effects of strategic anthelmintic treatments on the milk production of dairy sheep naturally infected by gastrointestinal strongyles.

The present paper was aimed at assessing the benefit of strategic anthelmintic treatments on milk production in four commercial dairy sheep farms in Southern Italy whose animals were naturally infected by gastrointestinal (GI) strongyles and Dicrocoelium dendriticum. The scheme was based on two treatments timed in relationship to parturition, i.e. the first with moxidectin in the periparturient period and the second with netobimin at the mid/end of lactation. On each farm, two similar groups (20 animals each) were formed, one untreated control group and one group treated according to the above-mentioned scheme. Faecal egg counts (FEC) were performed on each study animal at the start of the trial and then monthly until the end of the study; in addition, milk production was recorded for each animal fortnightly in each farm for the lactation period. The results showed a significant increase in milk yield across all four farms that ranged from 19% to 44% improvement in milk yield. The benefit in milk yield in addition to considerably reduced egg output of the treated animals provide clear evidence that the two main aims of prophylactic parasite control, i.e. to maintain or improve animal performance and to reduce pasture contamination, can be achieved using strategic anthelmintic treatments.

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.

Economic efficacy of anthelmintic treatments in dairy sheep naturally infected by gastrointestinal strongyles.

The aim of the present paper was to assess benefit of strategic anthelmintic treatments on milk production in six commercial dairy sheep farms, located in southern Italy, whose animals were naturally infected with gastrointestinal strongyles. On each farm, two similar groups were formed, one untreated control group and one treated group. In all the treated groups, the strategic anthelmintic schemes were based on: (i) only one treatment with moxidectin in the periparturient period (February, Farm No. 6), or; (ii) two treatments, i.e. the first with moxidectin performed in the periparturient period (February, Farms Nos. 1, 2, 3 and 4) or in the postparturient period (April, Farm No. 5), and the second with netobimin at the mid/end of lactation (June, Farms Nos. 1, 2, 3, 4 and 5). Faecal egg count reduction (FECR) tests were performed on each farm in order to asses the anthelmintic efficacy of the drugs used. In addition, milk yield measurements for each animal fortnightly in each farm for the lactation period were performed. In terms of FECR, both moxidectin and netobimin were effective in all the 6 studied farms. Regarding milk production, overall in the 6 study farms the mean daily milk productions of the treated groups were higher than those of the control group. However, there were important differences between the 6 farms, i.e. the increase of milk production in the treated groups versus the control groups was as follows: +18.9% (Farm 1), +30.4% (Farm 2), +4.0% (Farm 3), +37.0% (Farm 4), +5.5% (Farm 5) and +40.8% (Farm 6). The results of the study showed that the economic efficacy of an anthelmintic treatment is not a cause-effect issue, but is a multifactorial issue which depends upon the quali-quantitative parasitological status of the animals, the pathogenesis of the species of parasites, the virulence of the strains of parasites, the local epidemiology, the timing of treatment, the breed of animal in terms of genetics and production types, nutrient supply.

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.

Global Distribution of Alveolar and Cystic Echinococcosis.

Alveolar echinococcosis (AE) and cystic echinococcosis (CE) are severe helminthic zoonoses. Echinococcus multilocularis (causative agent of AE) is widely distributed in the northern hemisphere where it is typically maintained in a wild animal cycle including canids as definitive hosts and rodents as intermediate hosts. The species Echinococcus granulosus, Echinococcus ortleppi, Echinococcus canadensis and Echinococcus intermedius are the causative agents of CE with a worldwide distribution and a highly variable human disease burden in the different endemic areas depending upon human behavioural risk factors, the diversity and ecology of animal host assemblages and the genetic diversity within Echinococcus species which differ in their zoonotic potential and pathogenicity. Both AE and CE are regarded as neglected zoonoses, with a higher overall burden of disease for CE due to its global distribution and high regional prevalence, but a higher pathogenicity and case fatality rate for AE, especially in Asia. Over the past two decades, numerous studies have addressed the epidemiology and distribution of these Echinococcus species worldwide, resulting in better-defined boundaries of the endemic areas. This chapter presents the global distribution of Echinococcus species and human AE and CE in maps and summarizes the global data on host assemblages, transmission, prevalence in animal definitive hosts, incidence in people and molecular epidemiology.

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.