Antonio Bosco

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).

Comparison of individual and pooled faecal samples in sheep for the assessment of gastrointestinal strongyle infection intensity and anthelmintic drug efficacy using McMaster and Mini-FLOTAC.

A field study was conducted to validate pooled faecal samples in sheep for the assessment of gastrointestinal (GI) strongyle infection intensity (faecal egg count - FEC) and anthelmintic drug efficacy (FEC reduction - FECR). Ten sheep farms located in the Campania region of southern Italy were selected for the study. In each farm, individual faecal samples from 20 adult sheep (when possible) were collected, before (D0) and after (D14) an anthelmintic treatment with albendazole. For each farm and at each time point (D0 and D14) the faecal samples were examined individually and as pools. Specifically, three different pool sizes (5, 10 and 20 individual sheep samples) and three different analytic sensitivities (namely 10 using Mini-FLOTAC; 15 and 50 using the two variants of McMaster - McM15 and McM50) were compared for FEC and FECR using individual and pooled faecal samples. GI strongyle intensity (eggs per gram of faeces - EPG) of pooled samples correlated positively with mean EPG of individual samples, with very high correlation coefficients (ranging from 0.94 to 0.99) across the 3 different pool sizes and analytic sensitivities. Mini-FLOTAC was more sensitive compared to the two variants of McMaster (McM15 and McM50) for the diagnosis of GI strongyles in sheep (100% vs 88.5% vs 75.9%) and resulted in significant higher FEC compared to both McM15 and McM50, with a mean difference in egg counts of approximately 90 EPG (p<0.001). The drug efficacy results showed that FECR was higher than 98% at most farms independently of the pool size and analytic sensitivity. With the exception of two farms, FECR was 100% when calculated for individual animals and across the different pool size and analytic sensitivities. In conclusion, the present study highlighted that pooling ovine faecal samples is a rapid procedure that holds promise as a valid strategy for assessing GI strongyles FEC and FECR in sheep.

A comparison of the FECPAK and Mini-FLOTAC faecal egg counting techniques.

Faecal egg counts (FECs) are used for detecting and quantifying nematode infections and are the basis for determining drug efficacy and anthelmintic resistance in faecal egg count reduction tests (FECRTs). Currently, several FEC techniques are available for detecting and quantifying eggs of sheep nematodes. A comparison of the egg counts from the FECPAK (with a minimum detection limit of 30 eggs per gram (epg)) and Mini-FLOTAC (with a minimum detection limit of 5 epg) showed better diagnostic performance with Mini-FLOTAC in terms of measurement error (level of over- or under-estimation of FEC) and precision (variability in FEC). A tendency to under-estimate FEC was observed with the FECPAK particularly at egg densities of less than 500 epg. It is concluded that Mini-FLOTAC is a reliable diagnostic tool offering reduced measurement error and a higher level of precision.

Haemonchus contortus: spatial risk distribution for infection in sheep in Europe.

Haemonchus contortus is a species of gastrointestinal strongyles of primary concern for sheep. This highly pathogenic, blood-feeding helminth negatively influences animal health, welfare and productivity. In order to elucidate the current scenario in terms of prevalence and intensity of H. contortus infection in sheep farms across Europe, a standardized crosssectional survey was conducted in three pilot areas in Ireland, Switzerland and Italy, all part of the EU funded GLOWORM project. Two consecutive field surveys (in 2012 and 2013) were conducted in the three countries in the same period (August-October) in 259 sheep farms in total. Harmonized, diagnostic procedures (from farm to laboratory) based on pooled samples, the FLOTAC technique and coproculture were used. The georeferenced parasitological results were modelled (at the pilot area level) following a Bayesian geostatistical approach with correction for preferential sampling and accounting for climatic and environmental covariates. The observed H. contortus prevalence rates did vary between the countries showing high values in Switzerland (77%) and Italy (73%) compared to Ireland (4%). Spatial patterns of H. contortus distribution were detected in Switzerland and Italy with a north-south gradient. The latent factor analysis highlighted the importance of seasonality and annual cyclicity within country (particularly in southern Italy), while mean temperature and rainfall dominated between country variations in the prevalence of H. contortus infection.

The maintenance of anthelmintic efficacy in sheep in a Mediterranean climate

To determine how far management practices on small ruminant dairy flocks in southern Italy have influenced the development of anthelmintic resistance (AR), faecal egg count (FEC) reduction tests were run on 54 sheep farms using the FLOTAC technique with a sensitivity of 2 eggs per gram of faeces. Tests were run on groups of sheep (12-20 animals per group) using six anthelmintics administered orally: levamisole on 8 farms, ivermectin (half and full dose) on 8 farms, moxidectin on 3 farms, monepantel on 8 farms, netobimin on 22 farms (pooled samples) and albendazole on 5 farms (pooled samples). Different formulae and statistics were used to calculate FEC reductions and confidence intervals based on the presence of a control group and/or the use of pooled samples. A very high average efficacy was obtained with all anthelmintics tested as follows: levamisole 99.3% (range across farms 98-100%), ivermectin half dose 99.5% (98.0-100%), ivermectin full dose 99.9% (99.3-100%), moxidectin 100% (99.9-100%), monepantel 99.4% (97-100%), netobimin 99.1% (92-100%) and albendazole 100%. The results suggest that the development of AR in small ruminants could be limited in countries with a Mediterranean type of climate provided that refugia of the nematode populations are maintained, anthelmintic use is restricted and movement of animals is not permitted to spread resistance.

The Mini-FLOTAC technique for the diagnosis of helminth and protozoan infections in humans and animals

This protocol is an extension to: Nat. Protoc. 5, 503–515 (2010); doi: 10.1038/nprot.2009.235; published online 25 February 2010The FLOTAC is a sensitive, accurate, and precise technique for the diagnosis of protozoan and helminth infections in humans and animals. However, it requires centrifugation, and hence might be out of reach in resource-constrained settings. As an extension of the original FLOTAC protocol, this protocol describes the Mini-FLOTAC technique, a logical evolution of FLOTAC conceived to perform multivalent, qualitative, and quantitative diagnosis of helminth and protozoan infections in human and animal feces, and urine. This has been found to be of most use in the processing of large numbers of samples with rapid laboratory workup, and for veterinary applications directly on-farm. In addition to the Mini-FLOTAC apparatus, we describe the use of the Fill-FLOTAC, a closed system used to facilitate the performance of the first four consecutive steps of the Mini-FLOTAC technique: fecal sample collection and weighing, homogenization, filtration, and filling of the Mini-FLOTAC chambers. Processing of an individual sample using this protocol requires ∼12 min.

Outbreak of acute fasciolosis in sheep farms in a Mediterranean area arising as a possible consequence of climate change.

The objective of the present study was to investigate whether climate change in recent years have influenced the onset of acute outbreaks of Fasciola hepatica in ovine farms in southern Italy. In May-June 2014, a severe outbreak of F. hepatica occurred in three sheep farms in the Campania region. Clinical, coprological and necroscopic examinations were performed. Morbidity and mortality due to F. hepatica were 3-67% and 3-50%, respectively. Coprological examinations showed high values of F. hepatica eggs per gram (EPG) of faeces (860-1,240). Similarly, high adult parasitic burdens were found in animals that had sucombed (124-426 flukes). The study area was georeferenced and climatic data (temperature, humidity, days of rain and total amount of rainfall) were recorded at four georeferenced meterological stations in the study area. Montly data were processed and analyzed for the period 2000-2013 to evaluate the change of the climatic parameters during these years. The results show that there was a significant increase (P<0.001) of temperature, increased rainfall and increase in the number of rainy days compared to previous years. In addition to the outbreak reported here, we discuss the potential effects of climate change on the epidemiology of F. hepatica and the implications for sheep farming in the Mediterranean area.

The basic reproduction quotient (Q0) as a potential spatial predictor of the seasonality of ovine haemonchosis.

Haemonchus contortus is a gastrointestinal nematode parasite of small ruminants, which feeds on blood and causes significant disease and production loss in sheep and goats, especially in warmer parts of the world. The life cycle includes free-living immature stages, which are subject to climatic influences on development, survival and availability, and this species therefore exhibits spatio-temporal heterogeneity in its infection pressure based on the prevailing climate. Models that better explain this heterogeneity could predict future epidemiological changes. The basic reproduction quotient (Q0) was used as a simple process-based model to predict climate-driven changes in the potential transmission of H. contortus across widely different geo-climatic zones, and showed good agreement with the observed frequency of this species in the gastrointestinal nematode fauna of sheep (r = 0.81, P <0.01). Averaged monthly Q0 output was further used within a geographical information system (GIS) to produce preliminary haemonchosis risk maps for the United Kingdom (UK) over a four-year historical span and under future climate change scenarios. Prolonged transmission seasons throughout the UK are predicted, especially in the south although with restricted transmission in peak summer due to rainfall limitation. Additional predictive ability might be achieved if information such as host density and distribution, grazing pattern and edaphic conditions were included as risk layers in the GIS-based risk map. However, validation of such risk maps presents a significant challenge, with georeferenced observed data of sufficient spatial and temporal resolution rarely available and difficult to obtain.

Sheep and Fasciola hepatica in Europe: the GLOWORM experience.

Fasciola hepatica infection challenges health, welfare and productivity of small ruminants throughout the world. The distribution of F. hepatica in sheep in Europe is usually scattered and studies are generally concerned with a single area making it difficult to compare results from different environments, climates and management regimes. In order to elucidate the current scenario in terms of prevalence and intensity of F. hepatica infection in sheep farms across Europe, a standardized cross-sectional survey was conducted in three pilot areas in Ireland, Switzerland and Italy, all part of the EU funded GLOWORM project. Two consecutive field surveys (in 2012 and 2013) were conducted in the three countries in the same period (August-October) in 361 sheep farms in total. Harmonized procedures (from farm to laboratory) based on pooled samples and the highly sensitive and accurate, diagnostic FLOTAC technique were used. The georeferenced parasitological results were modelled (at the pilot area level) following a Bayesian geostatistical approach with correction for preferential sampling and accounting for climatic and environmental covariates. The observed F. hepatica prevalence rates did not differ between the two study years in any of the three pilot areas, but they did vary between the countries showing high values in Ireland (61.6%) compared to Italy (7.9%) and Switzerland (4.0%). Spatial patterns of F. hepatica distribution were detected by the Bayesian geostatistical approach in Ireland with a high risk of infection in the south-western part of the pilot area there. The latent factor analysis highlighted the importance of year-to-year variation of mean temperature, rainfall and seasonality within a country, while long-term trends of temperature and rainfall dominated between countries with respect to prevalence of infection.

The comparison of FLOTAC, FECPAK and McMaster techniques for nematode egg counts in cattle.

Three methods, FLOTAC, FECPAK and McMaster were compared for accuracy and sensitivity for counting numbers of nematode eggs in faeces of naturally infected cattle with high or low nematode egg counts. Only FLOTAC gave positive results for 12 replicates from pooled samples with low egg counts making it more sensitive than FECPAK (67%) and McMaster (41.7%). FLOTAC resulted in generally higher egg counts and lower coefficients of variation than the other two methods used. The reliability of FECPAK and McMaster is depended on the area under the slide counted. All three methods can be used for making decisions whether to treat but FLOTAC or Mini-FLOTAC should be used for faecal egg count reduction tests when lower egg counts are present.