Dicrocoeliosis in extensive sheep farms: a survey

Background This study investigated the epidemiological and molecular aspects of dicrocoeliosis in extensive sheep farms. Methods From 2013 to 2014, copromicroscopical analyses in 190 dairy sheep farms and anatomo-pathological inspections in six slaughterhouses were carried in Sardinia, Italy. Rectal faecal samples were analyzed using the FLOTAC® method, and anatomo-pathological examinations were based on detecting thickened terminal bile ducts (TTBDs). In addition, genetic analyses were conducted on representative DNA samples of adult Dicrocoelium spp. Results Ninety-seven (51.1%) out of 190 sheep farms were coprologically positive for Dicrocoelium spp. In the liver, on the surface and cut surface, TTBDs were reported in 40.1% (309/770) and 15.3% (118/770) of the animals examined, respectively, with an overall prevalence of 25.5% (196/770). No intraspecific genetic variation was observed among the Dicrocoelium dendriticum isolates. Conclusions Our survey reveals the widespread presence of D. dendriticum in Sardinia, although seasonal, geographical and climatic conditions might be key factors in modulating the infection prevalence. Examining typical lesions due to D. dendriticum in the liver in abattoirs can be used as a marker for tracking chronic dicrocoeliosis infection.

hydatigena, fasciolosis); consequently, veterinarians and farmers may underestimate the importance of this disease [14][15][16]. Infected animals with a parasitic burden of under 1000 individuals of D. dendriticum usually do not show any clinical manifestations [17] and even infections with 4000 parasites can cause mild symptoms [8]. In fact, in previous work we reported that only 33.3% of practitioners diagnose dicrocoeliosis according to clinical symptoms [18].
In addition, sheep with D. dendriticum are often co-infected with other parasites (e.g. gastrointestinal and bronchopulmonary nematodes) making it quite difficult to identify the specific outcomes of each individual parasitosis [5].
Another aspect that may lead to an underestimation of dicrocoeliosis is that this parasitosis is generally not diagnosed with an appropriate coprodiagnostic analysis, thus infected animals are not identified [19]. As a consequence, the infection becomes increasingly persistent, with cumulative effects [19,20]. Moreover, serological techniques do not provide reliable information for diagnosing dicroceliosis, although these tests may be useful when investigating prepatent infections [21]. Sardinia (Italy) has a long-established sheep farming tradition with over 3,200,000 sheep, which represent 45% of the entire stock of the Italian sheep population [22]. Due to the insularity and the high concentration of animals, which all belong to the Sardinian sheep breed, the island is regarded as a unique geographical area for epidemiological studies on parasites [15,16,[23][24][25][26][27][28].
Most of the data on sheep dicrocoeliosis regarding Sardinia have demonstrated that this parasitosis is endemic, though much of the data is not recent [29,30].
In this study, we investigated sheep dicrocoeliosis in Sardinia with particular emphasis on parasitological and molecular aspects in order to provide new insights into its epidemiology in extensive sheep farms.

Copromicroscopical survey in sheep farms
The sample size of the farms studied was estimated considering 15,387 Sardinian sheep farms (National Data Bank of the Italian Ministry of Health; https ://www.vetin fo.it) with an expected Dicrocoelium spp. prevalence of 15%, and confidence level of 95% (http://www.raoso ft.com/sampl esize .html).
A total of 190 dairy sheep farms in Sardinia ( Fig. 1) were investigated from 2013 to 2014. Within each flock, 15 individual rectal faecal samples from sheep older than 3 years of age were collected. These samples were then split into three different faecal pools from five animals, which were then analysed using the FLOTAC ® method with a heavy saturated zinc sulphate solution, specific gravity (SG) 1350) [31,32].
Data were processed for each farm considering the eggs per gram (EPG) mean of the three faecal pools.
The mean intensity (MI) was obtained considering the arithmetic mean of the EPG values in the total number of the infected animals.

Epidemiological survey in slaughterhouses
The sample size of the studied animals was determined considering a total of 3,206,821 heads of Sardinian dairy sheep (National Data Bank of the Italian Ministry of Health; https ://www.vetin fo.it) with an expected Dicrocoelium spp. prevalence of 15%, and confidence level of 95% (http://www.raoso ft.com/sampl esize .html).
From 2013 to 2014, 770 Sarda sheep slaughtered in six different abattoirs in Sardinia, were submitted to anatomo-pathological examination to detect the liver parasites and to evaluate the typical thickened terminal bile duct (TTBD) lesions [33,34], following the EEC Inspection Regulation No. 854 of 2004 (Annex 1, Section IV, Chapter II, point 5) [35] and the methods illustrated by Marcato [36].
Subsequently, according to the guidelines of the World Association for the Advancement of Veterinary Parasitology (WAAVP) [37], an incision on the gall-bladder wall was performed, and the entire liver parenchyma was cut into 0.5-1.0 cm slices, in order to identify and count the adult parasites. The parasitic burdens were classified into five classes, based on the number of parasites found in the organ: (i) ≤ 50; (ii) 50-100; (iii) 100-300; (iv) 300-1000; and (5) > 1000. Five adult Dicrocoelium spp. were taken from each liver in order to confirm the species based on published morphological keys [6].

Genetic analysis
DNA from 15 adult Dicrocoelium spp. representing all four provinces of Sardinia was extracted using a commercial kit, PureLink ® Genomic DNA Mini Kit (Invitrogen, Carlsbad, CA, USA) by following the manufacturer's instructions. DNA samples were amplified by PCR for the regions internal transcribed spacer (ITS2) with the primers and the methods described elsewhere [6,40]. PCR products were purified using a commercial kit (Nucleospin Gel and PCR Clean Up; Macherey-Nagel, Düren, Germany) and sent to an external sequencing service (Eurofins Genomics, Ebersberg, Germany). Sequences were assembled manually with the aid of the CLUSTAL W multiple alignment program [38], and analyzed using the basic local alignment search tool (BLAST) available on the NCBI website (https ://blast .ncbi.nlm.nih.gov/ Blast .cgi.).

Statistical analysis
Data were processed using MINITAB v.12.1 (Minitab Inc., State College, PA, USA) and EpiInfo v.6.04 (CDC, Atlanta, GA, USA). A Chi-square test was performed to compare the prevalence in the four provinces. In order to compare the prevalence rates found in the different seasons, a chi-square trend test was used and odds ratio (OR) values were calculated. Mann-Whitney and Kruskal-Wallis non-parametric tests were used to compare the EPG means. Pearsonʼs correlation test was performed in order to evaluate the correlations between the parasite burden and TTBD score.

Copromicroscopic survey
Ninety-seven out of the 190 examined farms were coprologically positive for Dicrocoelium spp. (51.1%; 95% CI: 43.91-58.07%). Quantitative coprological analysis of Dicrocoelium spp. showed a EPG mean (± standard deviation, SD) of 31.2 ± 68.7 and a MI of 61.1 EPG. Interestingly, dicrocoeliosis prevalence was significantly lower in the summer (37.5%) compared to the winter (90.9%), when the odds ratio (OR) values were four times higher than in other seasons (Table 1). Table 2 shows the prevalence, farm EPG mean, MI values for Dicrocoelium spp. in the faecal pools and OR values in the four provinces. Regarding the Dicrocoelium spp. prevalence, the four provinces showed significant differences (χ 2 = 23.89, df = 3, P < 0.0001); there were also statistically significant differences in EPG means (Kruskal-Wallis H-test: χ 2 = 30.88, P < 0.0001). The province of Nuoro showed the highest prevalence and EPG
TTBD on the surface and the cut surface were reported in 40.1% (309/770) and 15.3% (118/770) of examined livers, respectively (Fig. 2). The hepatic areas most involved were the RL of VF and CL with a prevalence of 24.8% (191/770) and 16.8% (129/770), respectively. TTBD was not observed in the quadrate lobe. Our results did not show any match between the presence of parasites in the examined livers (25.5%) and the TTBD both on the surface (40.1%), and the cut surface (15.3%) (χ 2 = 121.62, df = 2, P < 0.0001). The score values were higher in RL VF    Table 4. There was a significant positive correlation between the parasite burden and the scores for the severity/extension of TTBD as follows: RL of DF (r = 0.538, P < 0.0001), RL VF (r = 0.484, P < 0.0001), LL VF (r = 0.374, P < 0.0001), CL (r = 0.351, P < 0.0001) and the cut surface (r = 0.338, P < 0.0001). According to Colton [39], the correlation, based on reported r-values, between the parasitic burden and RL VF was moderate to good, while the correlations between the parasitic burden and the other hepatic localizations were quite good. Using the morphological examination, all Dicrocoelium spp. were identified as D. dendriticum [6].

Genetic analysis
No intraspecific variations were observed for the ITS2 gene sequence (GenBank: MG004688) among the D. dendriticum isolates. In addition, the same isolates showed a homology of 99%, with the Iranian ITS-B haplotype isolate (GenBank: JQ966973) [40], and a homology of 99% and 98% with the Italian isolates DQ379986.2 [41] and EF547132.1 [6], respectively. The sequence alignment of ITS showed a T/A substitution at the 153 codon with an index diversity of 0.002, compared with the sequence of the above mentioned Iranian isolate.

Discussion
The present survey provides an update of various epidemiological aspects of sheep dicrocoeliosis in Sardinia. Our copromicroscopic survey on the farms revealed the widespread presence of D. denditricum. However, the distribution of this parasite does not appear to be homogeneous across the island, with significantly more farms affected in the province of Nuoro, which is located in the central part of the island. It should also be highlighted that this area is characterised by the highest altitude with an average altitude of 496 meters) (http://www.comun i-itali ani.it/20/clima .html) and the lowest average temperature compared with the other provinces (http://www.sar.sarde gna.it/pubbl icazi oni/ riepi loghi mensi li/mensi li.asp).
The EPG means and OR values appear to be statistically higher in the winter in other regions of Italy [33] and also in other countries [42][43][44]. In Spain it has been reported that the mountainous pastures located over 600 meters and with temperatures of < 11.8 °C present the highest risk of infection with D. dendriticum [44]. These findings suggest that in some geographical areas and, especially during the winter, it is important to monitor and carry out anthelmintic treatments against Dicrocoelium spp. in sheep. We also found a different prevalence and EPG means values between the two years studied, thus suggesting that the epidemiology of the dicrocoeliosis could also be influenced by annual climatic conditions.
Our survey demonstrated that inspections at slaughterhouses can detect the presence of the typical lesions due to D. dendriticum in the liver, and can thus be used to monitor the presence of chronic infections in a given flock. Underestimating the numbers of infected sheep is thus leading to the spread of parasitosis in Sardinia, which probably explains the high prevalence among sheep flocks on the island.
According to Ambrosi [33], infections with threshold values of under 100 adult parasites are not easily detected by copromicroscopical analysis. The same author [33] reported that approximately 7% of farms with EPG means values over 100 EPG could incur production losses. However, we found that only 3% of the examined livers in slaughterhouses showed a burden of over 1000 D. dendriticum. At the same time, the mild clinical signs might contribute to chronic infection and potentially to a loss of productive performance, which could be an interesting research line for further studies on this parasite. Although previous papers have reported a high variability within D. dendriticum [48], both in terms of genetic and morphological parameters, no intraspecific variation was observed within our isolates and our results were consistent with findings in other surveys carried out in Italy [6,41] and in Iran [40].

Conclusions
This present study show the widespread presence of D. dendriticum in Sardinia and highlights the key role of abattoirs and of the coprological analysis in the monitoring of parasitic diseases, through which farmers and practitioners can be given the data needed for diagnosing D. dendriticum and thus for setting up specific anthelmintic treatments.