Piermarino Milillo

Anthelmintic resistance in cyathostomin populations from horse yards in Italy, United Kingdom and Germany.

BackgroundA large survey was carried out in 2008 in Europe to evaluate the efficacy of fenbendazole (FBZ), pyrantel (PYR), ivermectin (IVM) and moxidectin (MOX), i.e. the major anthelmintic molecules used in current practice against cyathostomins affecting horses. A total of 102 yards and 1704 horses was studied in three countries: 60 yards and 988 horses from Italy, 22 and 396 from the UK, 20 and 320 from Germany. The survey consisted of Faecal Egg Count Reduction Tests (FECRTs) with a faecal egg count reduction (FECR) categorization of (I) resistance present if FECR <90% and the lower 95% confidence limit (LCL) <90%, (II) resistance suspected if FECR ≥ 90% and/or LCL <90% and (III) no resistance if FECR ≥ 90% and LCL >90%. The calculation of FECR data was performed employing bootstrap analysis of group arithmetic means.ResultsThe testing of FBZ on a total of 80 yards resulted in resistance present on more than 80% of the UK and German yards and on significantly fewer in Italy, i.e. in 38% (p < 0.01). PYR, IVM and MOX were tested on a total of 102 yards. For PYR resistance present was found in 25% of the yards with no significant differences between countries. For IVM resistance present was encountered in one Italian and two UK yards (3%), resistance present to MOX was not found in any yard in any country.ConclusionThe results indicate that single and/or multiple drug resistance in equine cyathostomins is present in the three countries, is widespread particularly for FBZ and/or PYR and in one UK yard multiple resistance present was detected to FBZ, PYR and IVM. Macrocylic lactones proved to be the most effective drugs, with some evidence of resistance to IVM and highest activity of MOX, despite a single case of reduced efficacy in Germany. These data call for the development and implementation, among practitioners, owners and managers, of further plans to reduce the expansion of the anthelmintic resistant populations and to use those anthelmintics that remain effective in a manner that preserves their efficacy as long as possible.

Infection by Eucoleus aerophilus in dogs and cats: is another extra-intestinal parasitic nematode of pets emerging in Italy?

The occurrence of the infection by the lungworm Eucoleus aerophilus (syn. Capillaria aerophila) in dogs and cats from Italy has been evaluated with conventional diagnostic procedures. Individual faecal samples from 569 dogs and 200 cats were undertaken to faecal flotation with sugar and zinc sulphate solution. Sixteen dogs (2.8%) and 11 cats (5.5%) scored positive for eggs of E. aerophilus when samples were processed with either of the two flotation solutions. Overall 14 of 16 dogs and eight of 11 cats infected by E. aerophilus showed respiratory symptoms and the most common were general respiratory distress, dry cough, wheezing and sneezing. These results indicate that E. aerophilus is not uncommon and that canine and feline capillariosis is of clinical importance. Given the impact that E. aerophilus infections may have upon animal health and its zoonotic potential, it is strongly advisable to routinely include this disease in the differential diagnosis of (cardio)-respiratory diseases of dogs and cats.

Efficacy of major anthelmintics against horse cyathostomins in France

This paper reports a survey conducted in France during 2011 to evaluate the efficacy of commonly used anthelmintics against horse cyathostomins. A total of 40 farms and 1089 horses were screened for the presence of cyathostomins. All farms but one were positive, with an overall animal infection rate of 53.7%, ranging from 9% to 83% on individual farms. On 445 horses from 30 of these farms, a faecal egg count reduction test (FECRT) was performed to evaluate the efficacy of oral formulations of fenbendazole (FBZ), pyrantel embonate (PYR), ivermectin (IVM) and moxidectin (MOX). Calculation of the mean FECR and 95% confidence intervals (95% CI) around the mean was performed using bootstrap analysis. Resistance to FBZ was found on 17 of 18 farms investigated, with a mean reduction of 57% (95% CI: 38.5-71.2%). Suspected resistance for PYR was found on 6 of 30 farms, and confirmed on another 3 of 30 farms, with a mean reduction for PYR of 94.7% (95% CI: 88.9-98.5%). Reduced efficacy simultaneously of FBZ and PYR was found in 7 farms. Reduced efficacy of IVM was found in one animal on one farm and of MOX in one animal on another farm, and was combined with resistance against FBZ and/or PYR. These results indicate that single and multiple drug resistance and reduced efficacy in equine cyathostomins is present in France. Macrocylic lactones proved to be highly effective compounds against cyathostomins, with reduced efficacy for IVM and MOX in two farms only. These results extend present knowledge on the occurrence of drug resistant cyathostomins in Europe, and illustrate the necessity to use anthelmintics in appropriate worm control programmes.

Molecular Detection of Capillaria aerophila, an Agent of Canine and Feline Pulmonary Capillariosis

ABSTRACT Capillaria aerophila, a trichuroid nematode causing pulmonary infections in wild and domestic carnivores, is occasionally and potentially poorly recognized in infections of humans due to clinicopathological mimicry and a lack of accurate, robust laboratory diagnostics. The present work evaluated the efficiency of a DNA-based assay amplifying a partial cytochrome c oxidase subunit 1 (cox1) gene of C. aerophila in the diagnosis of lung capillariosis. Fecal samples from 34 dogs and 10 cats positive at parasitological examination for C. aerophila and other endoparasites (i.e., other lungworms, whipworms, roundworms, hookworms, tapeworms, and/or coccidia) and from 44 animals negative for C. aerophila but positive for other endoparasites were molecularly examined. Of the 44 samples positive for C. aerophila at copromicroscopy, 43 scored positive (i.e., 33/34 dogs and 10/10 cats) in seminested PCR, resulting in a sensitivity of 97 to 100%. Samples that were copromicroscopy negative for C. aerophila although positive for other endoparasites never produced a PCR product or nonspecific amplicons. The specific PCR amplification of C. aerophila (i.e., specificity of 100%) was confirmed by a nucleotide sequence analysis of the cox1 amplicons. The potential implications of the molecular diagnosis of lung capillariosis are discussed.

Effects of worm control practices examined by a combined faecal egg count and questionnaire survey on horse farms in Germany, Italy and the UK.

BackgroundFor the control of worm infections, the strategic use of anthelmintics, often accompanied by additional farm and/or pasture management procedures, is currently applied on most horse farms in industrialized countries. However, the particular effects of the specific worm control procedures are often unclear and have only been investigated to a limited extent. We examined faecal egg count (FEC), faecal egg count reduction (FECR) and questionnaire data on farm and pasture management procedures. The aim of this study was to determine whether specific worm control practices reported to be applied in European horse farms affect worm prevalence.ResultsThis study involved 20 German, 26 Italian and 16 UK horse farms for each of which FEC were performed on a minimum of 16 horses. In total, 2029 horse faecal samples were quantitatively analysed for helminth eggs, resulting in 56.3% of the faecal samples being positive for strongylid eggs. The prevalence in the 742 German horse samples (48.1%) was significantly lower than that in the 737 Italian (61.1%) and the 550 UK (60.9%) samples. As expected, a significant effect of horse age on the infection prevalence was observed, with adult horses showing lower prevalences and lower mean FEC than foals and yearlings. The majority of the participating farms were stud farms (n = 29), followed by riding stables (n = 27) and racehorse stables (n = 6). The prevalence of strongyle infection by farm type differed between countries. While in Germany, horses on riding farms were significantly less often strongyle positive, in the UK horses on stud farms showed the lowest strongyle prevalences, whereas in Italy, no significant difference between farm types were seen. On all farms, horses received routine/preventive anthelmintic treatment. An effect of treatment frequency on strongyle prevalence was only encountered with adult horses. On farms performing more than one annual treatment, faecal samples were significantly less often positive. Furthermore, by comparing the FECR results of individual horses with their pre-treatment FEC, it was found that high pre-treatment FEC were associated with a significantly higher probability for a FECR below 90%.ConclusionOverall, age-dependent strongyle infection patterns and general worm control approaches were found to be similar on horse farms in the three countries. Also, a negative association of pre-treatment FEC and treatment efficacy was consistently found in all countries. However, mean strongyle prevalences and frequencies of anthelmintic treatments were considerably different. In addition to the age-dependent prevalence patterns, the finding of a possible negative association between high FEC and reduced FECR might argue for a focus on horses showing high pre-treatment FEC when monitoring anthelmintic treatment efficacy in the field.

Distribution and species-specific occurrence of cyathostomins (Nematoda, Strongylida) in naturally infected horses from Italy, United Kingdom and Germany

A broad scale study was carried out in 2008 to evaluate the distribution and species-specific occurrence of cyathostomin populations in horse yards from Europe. In total 102 properties and 3123 horses were included in Italy (60 yards and 1646 animals), United Kingdom (22 yards and 737 animals) and Germany (20 yards and 740 animals). Individual faecal samples were examined with a McMaster technique while pooled samples were subjected to the microscopic examination of in vitro cultured larvae and to a Reverse Line Blot (RLB) assay able to molecularly identify the most diffused 13 species of cyathostomins. All yards were positive for the presence of cyathostomins both at the McMaster technique and at the microscopic examination of cultured larvae. One thousand and nine hundred thirty-one horses (61.8%) showed a positive faecal egg count, i.e. 1110 (67.4%), 463 (62.8%) and 358 (48.3%) from Italy, UK and Germany respectively. Out of the 1931 positive animals 1133 (36.3%) showed a faecal egg count per gram >150, specifically 694 (42.2%) from Italy, 237 (32.2%) from UK and 202 (27.3%) from Germany. The molecular results showed that all 13 species that can be detected by the RLB were found in each of the three countries, with a range of 3-13 species present in individual yards. The five most prevalent were Cylicocyclus nassatus, Cylicostephanus longibursatus, Cyathostomum catinatum, Cylicocyclus goldi and Cyathostomum pateratum. The relevance of these results and related biological and epidemiological features are discussed, together with their significance for both future studies of cyathostomins and further intervention programs aiming to control the spread of anthelmintic-resistant populations.

Application of a Reverse Line Blot hybridisation assay for the species-specific identification of cyathostomins (Nematoda, Strongylida) from benzimidazole-treated horses in the Slovak Republic.

Five horse farms located in eastern Slovakia were investigated for the presence of benzimidazole-resistant strongyles by faecal egg count reduction test and egg hatch assay. Coprocultures were prepared for each farm from faecal samples taken pre- and post-treatment and harvested larvae were molecularly examined with a Reverse Line Blot assay. Faecal egg count reduction values ranged from 0 to 52.5% and all farms were positive for benzimidazole-resistant cyathostomins. Seven benzimidazole-resistant cyathostomin species were molecularly identified on farms before and also after treatment. These data demonstrate that resistance to benzimidazoles is well established in cyathostomin populations from horse farms in the Slovak Republic and that the molecular assay was able to determine the species-specific distribution of resistant cyathostomins under field conditions.

Giardia duodenalis sub-Assemblage of animal and human origin in horses

In order to evaluate infection occurrence and the potential zoonotic role of horse isolates of Giardia duodenalis, 431 individual fecal samples were genetically characterized by PCR tests -coupled sequencing and phylogenetic analysis. Thirty-seven (8.6%) animals resulted infected by different Assemblage. The presence of sub-Assemblage was assessed by characterizing the β-giardin gene for 16 of the 37 positive horses. Ten isolates showed 99.6% to 100% homology with the sub-Assemblage described as B1-2 and B1-6, three Assemblage A showed 99.8% homology with sub-Assemblage A1, while one Assemblage E displayed 98.8% homology with sub-Assemblage E3. Furthermore, one isolate characterized as Assemblage A showed 99.6% homology with the sub-Assemblage B1-2 and one characterized as E was 100% identical with sub-Assemblage B1-6. These results demonstrate the presence of both animal and human sub-Assemblage of G. duodenalis in horses from Italy. Epidemiological and sanitary implications are discussed.

Faecal Cyathostomin Egg Count distribution and efficacy of anthelmintics against cyathostomins in Italy: a matter of geography?

BackgroundIn the framework of a trial carried out in 2008 in Europe to evaluate the efficacy of major parasiticides against horse cyathostomins, pre- and/or post-treatment Faecal Egg Counts (FEC) were evaluated in a total of 84 yards and 2105 horses from nine different regions from the South, the Center, the North-Center and North-East of Italy. Specifically, on the basis of FECs of the horses present in each property, 60 out of the 84 yards were enrolled for a Faecal Egg Count Reduction Test (FECRT) using fenbendazole, pyrantel, ivermectin and moxidectin.ResultsOf the 1646 horses bred in the 60 recruited yards, 416 animals had a FEC between 50 and 150 Eggs Per Gram (EPG) of faeces and 694 a FEC >150 EPG (i.e. with total of 1110 positive animals). Of the 1110 positive animals, those with the highest FECs (i.e. 988) were included in the FECRT. The FECRT for four anthelmintic compounds showed remarkable differences in terms of prevalence of reduced and equivocal efficacy against cyathostomins in the different areas of Italy. Administration of fenbendazole and pyrantel resulted in resistance present or suspected in about half of the yards examined while resistance to ivermectin was found in one yard from central Italy and suspected resistance was detected in three more yards, one in each the North, the Center and the South. Treatment with moxidectin was 100% effective in all yards examined.ConclusionCyathostomin populations in the South and the Center of Italy were more susceptible to fenbendazole and pyrantel than the populations present in the Center-North and North-Eastern areas of Italy. Fenbendazole and/or pyrantel were ineffective in almost all properties from the North of Italy. The reasons for such a difference among the Italian regions in terms of FECs and efficacy of antiparasitic drugs are discussed, together with the role that veterinarians, and horse owners and managers should have for effective worm control programs in this country.