A prevalence survey and risk analysis of filariosis in dogs from the Mt. Vesuvius area of southern Italy.

A dog microfilariae prevalence and risk factor survey was conducted in 51 contiguous municipalities of the Mt. Vesuvius area (Campania region, southern Italy) in order to add data to the limited epidemiological information available regarding filarial worms in this zone. Between May 1999 and June 2000, blood samples were collected from 351 asymptomatic dogs. Blood samples were examined using a modified Knott's technique and histochemical staining in order to count and identify microfilariae. The results were subjected to statistical analysis and choroplethic municipal maps (MMs) were drawn by a geographical information system (GIS) software. Microfilariae were detected in 63 of the 351 dogs surveyed, constituting a total filarial prevalence of 17.9%. In particular, 56 dogs (15.9%) showed only microfilariae of Dipetalonema reconditum; three dogs (0.8%) only microfilariae of Dirofilaria repens; two dogs (0.6%) microfilariae of both D. reconditum and D. repens and two dogs (0.6%) microfilariae of both Dirofilaria immitis and D. repens. High D. reconditum prevalence was associated with hunting practice, masculine gender and older dogs. There was also a tendency to find high prevalence in dogs sampled in the afternoon.In conclusion, the presence of microfilariae of D. reconditum in 92% of microfilaraemic dogs indicates that this filarial worm was the predominant filarial species in dogs in the Mt. Vesuvius area.In addition, the general trends of the MMs showed that D. immitis and D. repens were present only in a few municipalities, whereas D. reconditum was widely and homogeneously spread throughout the entire study area.     

A cross-sectional coprological survey of liver flukes in cattle and sheep from an area of the southern Italian Apennines

A cross-sectional coprological survey of liver flukes (Fasciola hepatica and Dicrocoelium dendriticum) was conducted on 81 bovine farms and 197 ovine farms with animals pasturing in an area (3971 km(2)) of the southern Italian Apennines. The farms were selected to be uniformly distributed throughout the study area using geographical information system (GIS) software. Between June 1999 and March 2000, faecal samples were collected from 975 cattle and 3940 sheep and examined using a modified McMaster technique. The results were subjected to statistical analysis and point distribution maps (PDMs) were drawn by GIS.Cattle of 9 of the 81 (11.1%) farms were positive for F. hepatica and of 43 (53.1%) for D. dendriticum. Sheep of 8 of the 197 (4.1%) farms were positive for F. hepatica and of 133 (67.5%) for D. dendriticum. Co-infection was found in cattle of 2 (2.5%) farms, and in sheep of 8 (4.1%) farms.The findings of the present survey show that D. dendriticum was the predominant liver fluke found in cattle and sheep with respect to egg count numbers for both farms and animals. In addition, the general trends of the PDMs show that D. dendriticum was widely and homogeneously spread throughout the study area, whereas F. hepatica was present only in a few concentrated zones of the study area that had both positive bovine and positive ovine farms.     

Use of remote sensing and geographical information systems to identify environmental features that influence the distribution of paramphistomosis in sheep from the southern Italian Apennines

A geographic information system (GIS) was constructed using remote sensing (RS) and landscape feature data together with Calicophoron daubneyi positive survey records from 197 georeferenced ovine farms with animals pasturing in a 3971 km(2) area of the southern Italian Apennines. The objective was to study the spatial distribution of this rumen fluke, identify environmental features that influence its distribution, and develop a preliminary risk assessment model. The GIS for the study area was constructed utilizing the following environmental variables: normalized difference vegetation index (NDVI), land cover, elevation, slope, aspect, and total length of rivers. These variables were then calculated for "buffer zones" consisting of the areas included in a circle of 3 km diameter centered on 197 farms. The environmental data obtained from GIS and RS and from data taken by the veterinarians on the field (stocking rate and presence of streams, springs and brooks on pasture) were analyzed by univariate (Spearman and ANOVA) and multivariate (discriminant) statistical analyses using the farm coprological status (positive/negative) as the dependent variable. Sheep on 32 of the 197 (16.2%) farms, were positive for C. daubneyi, with an average intensity of 52 epg. A multivariate stepwise discriminant analysis model was developed that included moors and heathland, sclerophyllous and coniferous forest vegetation, autumn-winter NDVI and presence of streams, springs and brooks on pasture. The variables entered in the model were also correlated with C. daubneyi positive farms in the univariate tests and are consistent with the environmental requirements of C. daubneyi and its snail intermediate host.     

The influence of flotation solution, sample dilution and the choice of McMaster slide area (volume) on the reliability of the McMaster technique in estimating the faecal egg counts of gastrointestinal strongyles and Dicrocoelium dendriticum in sheep

The present study was aimed to evaluate the influence of flotation solution, sample dilution, and the choice of McMaster slide area (volume) on the reliability of the McMaster technique in estimating the faecal egg counts of gastrointestinal (GI) strongyles and Dicrocoelium dendriticum in a composite sample of faeces from naturally infected sheep. Fourteen flotation solutions having densities between 1.200 and 1.450, and six sample dilutions, 1:10, 1:15, 1:20, 1:30, 1:40 and 1:50 were used. Each of the six dilutions was divided into 70 aliquots in order to have five replicates of each of the 14 flotation solutions at each of the six dilutions. For each McMaster slide, the GI strongyle and D. dendriticum egg counts were performed under one grid (McM 0.15 ml), two grids (McM 0.3 ml), one chamber (McM 0.5 ml), and both chambers (McM 1.0 ml). Mean eggs per gram (EPG) of faeces of GI strongyles and D. dendriticum were calculated and statistical analyses were performed on the resulting data. The type of flotation solution used significantly influenced the EPG in the GI strongyles and in the D. dendriticum egg counts. All the sucrose-based solutions at density between 1.200 and 1.350 floated more GI strongyle eggs than the others. With respect to D. dendriticum, only six solutions were capable of floating eggs and the potassium iodomercurate solution (density 1.440) floated more eggs than the others. The reliability of the McMaster technique regarding sample dilution was high for both GI strongyle and D. dendriticum EPG at 1:10 and 1:15, and then progressively decreased with increasing dilution. The reliability of the McMaster technique regarding the choice of the McMaster slide area (volume) was high for both GI strongyle and D. dendriticum EPG at the McMaster slide area (volume) of 1.0 ml, i.e. the total area of the McMaster slide. The EPG counts resulting from choosing any of the other three McMaster slide areas (volumes), i.e. McM 0.15 ml, McM 0.3 ml, or McM 0.5 ml, produced unreliable over-estimates. The findings of the present study show that the highest reliability of the McMaster technique for estimating GI strongyle and D. dendriticum egg counts in faeces from pastured sheep is obtained when using flotation solutions based on sucrose for GI strongyles, and potassium iodomercurate for D. dendriticum, dilutions which do not exceed 1:15, and the McMaster slide area (volume) of 1.0 ml.     

Evaluation of the pharmacokinetic profile of artesunate, artemether and their metabolites in sheep naturally infected with Fasciola hepatica

The pharmacokinetic (PK) parameters of artesunate, artemether and their metabolites dihydroartemisinin (DHA) and dihydroartemisinin-glucuronide (DHA-glucuronide) were determined in sheep naturally infected with Fasciola hepatica. Sheep were treated either with artesunate (intramuscular (i.m.): 40 and 60 mg/kg) or artemether (i.m.: 40 and 160 mg/kg; oral: 80 mg/kg). Blood samples were withdrawn at selected time points post treatment and the artemisinins were quantified in plasma by liquid chromatography and tandem mass spectrometry (LC-MS/MS). The in vitro effect of the metabolites against F. hepatica was investigated using a phenotype-based assay and scanning electron microscopy (SEM). Following artesunate applications (40 and 60 mg/kg), comparable C(max) (maximal plasma concentration) and AUCs (area under the plasma concentration-time curve) were observed for artesunate (C(max): 8.4×10(3) and 9.4×10(3)ng/ml; AUC: 6.9×10(5) and 9.7×10(5) ng min/ml), DHA (C(max): both 2.4×10(3)ng/ml; AUC: 3.7×10(5) and 5.0×10(5) ng min/ml), and DHA-glucuronide (C(max): 1.7×10(4) and 1.6×10(4)ng/ml; AUC: 2.6×10(6) and 3.3×10(6) ng min/ml). Mean elimination half-lifes (t(1/2)) of artesunate, DHA and DHA-glucuronide ranged between 58 and 63 min, 94 and 113min, and 89 and 98 min, respectively. The i.m. oil-based drug formulation liberated artemether slowly and constant levels of artemether and its metabolites were observed during the entire sampling period (24 h). The AUCs of all analytes were significantly higher for the i.m. 160 mg/kg dose compared to i.m. 40 and oral 80 mg/kg doses (P=0.018). Mean C(max) of artemether (2126 and 426 ng/ml) and DHA-glucuronide (3477 and 1587 ng/ml) were higher following oral compared to i.m. (160 mg/kg) treatments (P>0.068), whereas C(max) of DHA was significantly higher following i.m. applications (P=0.0062). DHA rapidly reduced the viability of F. hepatica in vitro, whereas DHA-glucuronide showed no activity. SEM observations revealed only minor and focal tegumental alterations in few of the DHA treated worms. The calculated PK parameters reflect the anthelmintic activity of artesunate and artemether following different routes of application and will aid in the design of future studies with these drugs.     

Climate and Dirofilaria infection in Europe

Climatic changes, together with an increase in the movement of cats and dogs across Europe, have caused an increase in the geographical range of several vector borne parasites like Dirofilaria, and in the risk of infection for animals and humans. The present paper reviews the effects of climate and other global drivers on Dirofilaria immitis and Dirofilaria repens infections in Europe and the possible implications on the transmission and control of these mosquito-borne nematodes. In the last several years, growing degree day (GDD)-based forecast models, which use wide or local scale temperature data, have been developed to predict the occurrence and seasonality of Dirofilaria in different parts of the world. All these models are based on the fact that: there is a threshold of 14 °C below which Dirofilaria development will not proceed; and there is a requirement of 130 GDD for larvae to reach infectivity and a maximum life expectancy of 30 days for a vector mosquito. The output of these models predicts that the summer temperatures (with peaks in July) are sufficient to facilitate extrinsic incubation of Dirofilaria even at high latitudes. The global warming projected by the Intergovernmental Panel on Climate Change suggests that warm summers suitable for Dirofilaria transmission in Europe will be the rule in the future decades and if the actual trend of temperature increase continues, filarial infection should spread into previously infection-free areas. These factors not only favour incubation of Dirofilaria, but also impact on mosquito species. Recent findings have also demonstrated that Aedes albopictus is now considered to be an important, competent vector of Dirofilaria infections. This mosquito species could spread from southern to northern European countries in the near future, changing the epidemiological patterns of dirofilariosis both in humans and animals.     

Serological survey of Encephalitozoon cuniculi in farm rabbits in Italy

Rabbit sera (n = 1600) from 40 commercial farms were submitted to a serological screening for Encephalitozoon cuniculi by an enzyme-linked immunosorbent assay (ELISA) and a carbon immunoassay (CIA test). Antibodies anti-Encephalitozoon cuniculi were found in 505/1600 (31.6%) sera analysed, and all the farms (100%) resulted positive. Rabbits older than 4 months showed a significantly higher seropositivity for E. cuniculi (chi-squared test: p < 0.0001) than rabbits under 4 months, E. cuniculi sero-prevalence showed an increasing trend in rabbits within the farm along with the increase in the “number of rabbits on the farm”; however, this trend was not significant (Spearman’s correlation: p = 0.073).The findings of the present study confirm that rabbit is the main reservoir of E. cuniculi; they are of epidemiological relevance and immediate public health importance because of the recognized infectivity in humans by the microsporidium.     

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.     

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.     

Neospora caninum in pastured cattle: determination of climatic, environmental, farm management and individual animal risk factors using remote sensing and geographical information systems

A cross-sectional serological survey was conducted on cattle pasturing in an area of the southern Italian Apennines to evaluate the seroprevalence to Neospora caninum, and to investigate the climatic, environmental, farm management, and individual animal factors that influence the distribution of this protozoan. Blood samples were collected from 864 pastured cattle raised on 81 farms. Serum samples were tested for antibodies to N. caninum using an ELISA assay (HerdCheck), IDEXX). A geographical information system (GIS) for the study area was constructed using the following remote sensing (RS) and landscape feature data: autumn-winter, spring and summer normalized difference vegetation index (NDVI), land cover, elevation, slope, aspect, mean rainfall and minimum, mean, and maximum temperature in spring, summer, autumn and winter. Data on each of these features were then extracted for "buffer zones" consisting of the area included in a circle of 3 km diameter centered on the 81 geo-referenced centroids of the main cattle pastures. Climatic and environmental data obtained from RS and GIS and individual animal characteristics and farm management data obtained from a questionnaire were analyzed in relation to N. caninum seropositivity and antibody titres both by linear and logistic regression models. Out of the 81 farms sampled, 63 (77.8%) had at least one tested animal positive for N. caninum. Out of the 864 bovine sera samples, 266 (30.8%) were found to have antibodies to N. caninum. The results of the logistic regression model show that significantly high seroprevalence to N. caninum was found in heifers/steers and adults, in cattle raised on farms having a large number of dogs, and in cattle raised in buffer zones having high minimum temperatures in the spring, and a narrow extension of summer NDVI. In addition, positive linear correlations were found between N. caninum antibody titres and the number of dogs on farm, and the minimum temperature in spring. All the above determined risk factors for N. caninum seroprevalence indicate that horizontal infection resulting from the ingestion of oocysts shed by dogs is the most probable route of N. caninum infection in pastured cattle of the southern Italian Apennines.