The Evaluation of Gastrointestinal Nematodes in Faecal Samples Using Multiplex Real-Time PCR Assays

Background: The diagnosis of gastrointestinal nematode (GIN) infections in ruminants is routinely based on morphological/morphometric analysis of parasite specimens recovered by coprological methods and followed by larval culture techniques. Such an approach is laborious, time-consuming, requires a skilled expert and moreover suffers from certain limitations. Molecular tools are able to overcome the majority of these issues, providing accurate identication of nematode species and, therefore, may be valuable in sustainable parasite control strategies. Methods: Two multiplex real-time PCR assays for specic detection of six main GIN species, including an internal amplication control to avoid false negative results, were designed targeting SSU rRNA and COI genetic markers, as well as established ITS1/2 sequences. The assays were optimized for analysis of DNA extracted directly from sheep faeces and veried for Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis, Nematodirus battus, Chabertia ovina, and Ashworthius sidemi. Semi ‐ quantitative evaluation of infection intensity was enabled using a plasmid construct and a dilution series of sheep faeces with a known number of nematode eggs. Assays were tested on 44 individually collected faecal samples from three farms and results were compared to those from faecal egg counts (FEC) using the Concentration McMaster technique, and larval cultures (LC). Results: Multiplex real-time PCR assays showed great specicity to target nematodes. During the analysis of faecal samples, the assays proved to have higher sensitivity in strongylid-type egg detection over FEC by revealing 3 false negative samples, while showing moderate agreement in evaluation of infection intensity. The multiplex assays further claried GIN species identication compared to LC, which had confused determination of Teladorsagia spp. for Trichostrongylus spp. Conclusions: Our multiplex assays proved to be a rapid and accurate approach enabling simultaneous and reliable GIN species identication from faeces and semi-quantitative estimation of the number of eggs present. This approach increases diagnostic value and may add a high degree of precision to evaluation of anthelmintic ecacy, where it is important to identify species surviving after treatment. DNA of the nematode species Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis, Nematodirus battus, Ashworthius sidemi, and Chabertia ovina, respectively. We further tested the functionality of both multiplex assays on 44 individual faecal samples from sheep and compared results of a semi-quantitative evaluation of infection intensity to the egg enumerations generated by a conventional faecal otation technique. The ability to reliably identify all targeted GINs present within faeces

PCR-based methods have shown high sensitivity and speci city for the detection of parasite DNA, yet their use for in vivo diagnostics of GIN infections directly from faeces has so far been limited to only three studies by Höglund et al. (2019) [10], Sweeny et al. (2011) [16] and   [17]. The focus of GIN diagnostics using molecular methods is still mainly related to extracted eggs [11,12,13,18,19,20,21,22] and/or cultivated larvae [13,14,22,23]. Faecal DNA extraction is still believed to be unsuitable for usage in most subsequent PCR ampli cations due to the presence of faecal inhibitors [24]. However, developments in DNA extraction procedures and application of inhibitor removal technologies have made such DNA ideal for subsequent PCR-based analyses, especially real-time PCR. This has made this technology a powerful alternative or additional tool to coproscopy [25].
In the present study, we designed two multiplex real-time PCR assays, which included internal ampli cation control, targeting the internal transcribed spacers 1, 2 (ITS1/2), small subunit rRNA (SSU rRNA) and cytochrome c oxidase subunit I (COI) genetic markers to diagnose naturally acquired GIN infections in sheep. We developed six speci c detection systems and describe their use for the detection of genomic DNA of the nematode species Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis, Nematodirus battus, Ashworthius sidemi, and Chabertia ovina, respectively. We further tested the functionality of both multiplex assays on 44 individual faecal samples from sheep and compared results of a semi-quantitative evaluation of infection intensity to the egg enumerations generated by a conventional faecal otation technique. The ability to reliably identify all targeted GINs present within faeces and rank them according to their numerical contribution in a single reaction represents a major advantage over conventional coprological techniques. This approach has clear, practical value, is cost-effective and e cient, and, hence, widely applicable in the control of parasites in ruminant livestock and epidemiological studies.

Material And Methods
Biological material. The optimization experiments were carried out with genomic DNA (gDNA) extracted from adult nematodes stored in 70% ethanol, which were recovered from the gastrointestinal tracts of ruminants using standard post-mortem examination. The host animals came from various areas of the Czech Republic and sampling was conducted in 2017-2019. The recovered nematodes were identi ed based on their dominant distinguishing morphological/morphometric characters using a light microscope Olympus BX51 and measurement of their dominant morphological characteristics carried out by QuickPHOTO MICRO 3.0 software (PROMICRA). Adult nematodes were determined as H. contortus, T. circumcincta, T. colubriformis, N. battus, A. sidemi, Ch. ovina, Cooperia curticei, C. pectinata and Oesophagostomum columbianum. One lamb experimentally infected with H. contortus served as a source of eggs for accurate assessment of nematode eggs in faeces.
In total, 44 individual faecal samples for functional veri cation of the multiplex assays were collected rectally on three farms during the autumn of 2019 in the Czech Republic. Approximately 5 g of faeces from the rectum of approximately 10% of the animals in the ock were collected and stored in plastic zipper bags and after transportation frozen at -80 ο C, until further processing for molecular analyses. At the same time, another batch of 5 g of faeces originating from the same animals was collected for immediate quantitative coprological examination using the Concentration McMaster technique with a minimum diagnostic sensitivity of 20 strongyle eggs per gram of faeces (EPG) [26]. Besides strongylid-type egg enumeration, the presence of Nematodirus spp. eggs (includes all species except N. battus, which is distinguished separately) was also recorded for each sample.
In addition to coprological examination, about 1 kg of pooled faecal sample from the whole ock was collected at each farm and incubated under standard conditions to obtain infective L 3 larvae. Faecal pellets were mixed in a beaker with Vermiculite chips, manually homogenized and then kept in moist conditions in an incubator at 28 o C for one week. The developed L 3 s were recovered using Baermannization and identi ed based on their morphological/morphometric features to the genus level according to morphological keys in van Wyk et al. (2004) [15] and van Wyk & Mayhew (2013) [27].
DNA extraction. The gDNA of adult nematodes, which served as reference template DNA during the optimization experiments, was extracted from a single individual of each given nematode species using previously published protocols [28,29] and based on overnight incubation at 55 o C in 50 µl of extraction buffer (100 mM Tris-HCl, 10 mM EDTA, 100 mM NaCl, 1% SDS, 1.5 mM dithiothreitol) containing 0.06 mg proteinase K, followed by alcohol precipitation. Concentration, yield, and purity of extracted gDNA were measured by NanoDrop 8000 Spectrophotometer (Thermo Fisher Scienti c). Extracted gDNA was stored at -20 ο C until processed.
Faecal samples were thawed before the extraction and each zipper bag containing sample was thoroughly manually homogenized. Out of the homogenized faeces, 1 g was taken for further processing and extraction using Quick-DNA Fecal Soil Microbe MiniPrep kit (Zymo Research). Each sample was placed in a sampling container and dissolved by shaking in 800 µl of BashingBead Buffer mixed with 3200 µl of PBS buffer. A negative isolation control (NIC), i.e., a tube without faeces, starting from this step through the whole extraction procedure together with the samples, was included in every extraction procedure. Then, 1200 µl of the sample solution was transferred into ZR BashingBead Lysis Tube and mechanically lysed, ensuring the disruption of eggs and further homogenization of other debris, in a Retsch MM200 mixer mill (RETSCH) for 10 min. Then, lysis tubes were centrifuged, and the supernatant transferred to a Zymo-Spin III-F Filter; the further procedure faithfully followed the kit's manufacturer protocol. After elution, DNA samples were 10× diluted and stored at -20 ο C until use.
Detection systems. Target gene sequences of nematode species of interest were downloaded from the NCBI GenBank database and aligned with the aim to nd conserved genusspeci c regions suitable for oligonucleotide hybridization. Suitable sequence regions were chosen manually and tested in the online tool OligoAnalyzer 3.1 (Integrated DNA Technologies) for melting temperatures and formation of secondary structures. The speci city of each primer probe set was evaluated in silico in Nucleotide BLAST and considered during the design process, following the guidelines by Rodríguez et al. (2015) [30]. Based on accessible sequences and their reaction properties, six detection systems were designed: Haemonchus (ITS2; system was designed to cover species H. contortus and H. placei), Teladorsagia (ITS2; covering T. circumcincta, T. davtiani and T. trifurcata), Trichostrongylus (ITS1; covering T. colubriformis, T. vitrinus and T. rugatus), Nematodirus (SSU rRNA; designed for N. battus), Ashworthius (ITS1; designed for A. sidemi) and Chabertia (COI; designed for Ch. ovina). Duallabeled hydrolysis probes carried a reporter dye (FAM, HEX, TxRd or Cy5) at the 5' end and a compatible black hole quencher (BHQ) at the 3' end. Primers and probes (Table 1) puri ed by HPLC were purchased from SigmaAldrich. Sensitivity was tested in separate reactions for each system at rst and further veri ed in multiplex and on mixed template DNA. Serial 10-fold dilutions (10 ng -10 fg per µl) of stock solutions of gDNA of adult nematodes were used for the tests; similarly, in the case of an experiment with the DNA mixtures, template DNA of three targets was put together (with respect to a certain multiplex assay) and diluted in the same manner. To determine if a non-competitive environmental DNA could interfere with the assay, 10 µg of an excess DNA from sh sperm (SERVA) was added to the samples prior to testing.
Real-time PCR standard curve and internal control. A plasmid DNA construct serving as a positive control in the reaction was prepared based on ITS2 sequence of H. contortus. The same ampli ed sequence as targeted in the relevant multiplex assay by speci c primers to H. contortus was cloned via Clone Jet (Thermo Fisher Scienti c) into vector pJET1.2/blunt and transformed into DH10B competent E. coli cells. The plasmid was puri ed with NucleoBond Xtra kit (Macherey-Nagel), and its DNA concentration determined by NanoDrop. The plasmid construct was sequenced to con rm the proper insertion of intended sequence. The copy number was calculated using the formula: copies/µl = X ng * 6.022 × 10 23 molecules/mole / (N * 660 g/mole) * 1 × 10 9 ng/g; where X is the concentration of the plasmid [ng/µl] and N is the length of dsDNA [plasmid with insert]. The calculated copy number was used for preparation of the plasmid stock solution 1 × 10 10 in 50 µg/ml of carrier DNA from sh sperm and the standard curve was generated by serial dilutions of the plasmid stock to give: 5 × 10 7 , 1 × 10 7 , 5 × 10 6 , 1 × 10 6 , 5 × 10 5 , 1 × 10 5 , 5 × 10 4 , and 1 × 10 4 copies per µl. This series was analysed in duplicate by real-time PCR using Multiplex1 assay in three replications (three separate plates) to test the e cacy and reproducibility of the reaction. To relate the technical calibration curves of plasmid control to the number of eggs in sheep faeces, we spiked 5 g of parasite-naïve (trichostrongyle egg-free) sheep faeces with a given number of H. contortus eggs. Two independently diluted faecal concentration series ranging from 7,500 to 1 EPG were processed following the DNA extraction for faecal samples described above. We analysed the samples in duplicates and in three replications using Multiplex1 assay simultaneously with plasmid control.
The sequence of the internal ampli cation control (IAC) was created by Mikel et al. 2016 [31] from ancient mitochondrial DNA sequences of two extinct species and synthesized de novo (Sigma-Aldrich). This non-competitive synthetic sequence was cloned into a plasmid, puri ed and diluted using the same protocol as with H. contortus plasmid control. Plasmid together with IAC speci c primers and hydrolysis probe (Table 1) was incorporated into reaction premix of both multiplex assays to differentiate between truly negative and false negative (inhibited) samples [32]. Plasmid standard, no-template control (NTC) and NICs (in case of real sample analysis) were included on each analysed plate, while IAC was incorporated in every sample, inclusive of controls. The raw data were analysed using the Bio-Rad CFX Manager 3.0 (Bio-Rad Laboratories). The quanti cation cycle (Cq) values of the samples were read after adjustment of the relative uorescence units (RFU) threshold line to 140 for FAM, 120 for HEX and 210 for TxRd channel. The sample was considered to be positive only when both replicates provided a positive result and conversely, the sample was considered to be negative only when both replicates gave a negative result, but the IAC gave a positive signal; if this condition was not met, the real-time PCR and/or the DNA extraction was repeated.

Results
Optimizations. In the speci city tests, no nematode DNAs other than those targeted were ampli ed and, in all samples, there was a clear signal for the IAC. The only exception was found in the detection system for Nematodirus where 1 ng of T. circumcincta DNA was non-speci cally ampli ed with Cq 34.73 (Table 2). However, this was not observed in the testing of real samples, even though uorescent signal for Teladorsagia has been reported frequently. The limit of detection (LOD) was the same or better when the mixed samples were run in multiplex mode than when single template DNA was run in either in singleplex and multiplex (data not shown). The LOD of detection systems in multiplex assays was determined to be at least 500 fg of target DNA with the exception of the Ashworthius system, where the LOD was 5 pg (Table 3). An addition of non-competitive excess DNA from sh sperm had no impact on assay performance (data not shown).  (Figure 1), the PCR e ciency was established to be 99.63%, correlation coe cient 0.9997, slope -3.3308 and y intercept 40.074. The standard deviation (SD) of the Cq values within the plasmid DNA replicates between plates was < 0.11 and the coe cient of variation (CV) < 0.76% (Table 4). Semi-quantitative data evaluation. Based on median values of faecal series calibration curve ( Figure 2A) the PCR e ciency was established to be 83.37%, correlation coe cient 0.9869, slope -3.7974 and y intercept 32.181. The SD of the replicates between plates was < 0.59 and CV < 2.11%.
The Cq values of faecal concentration series ranging from 7,500 to 1 EPG were correlated to the corresponding values of H. contortus ITS2 plasmid standard copies enabling a semi-quantitative estimation ( Figure 2B) of the number of eggs in unknown samples.
Since not all GIN detection systems are using the same target marker gene, "correction factors" were calculated to overcome their different representation in the respective genome (an issue for all ITS sequences [13] To enable evaluation, the Cq value of each detected target sequence in the analysed sample was rstly assigned to plasmid copy number counting with the calibration curve of plasmid control on the plate, sample dilution and respective correction factor. Plasmid copy number was then used to estimate the EPG value of the target, based on the formula for semi-quantitative evaluation ( Figure 2B). EPG values of individual targets of the sample were summed to obtain the estimation of overall infection intensity (total EPG), whereas the limit values for interpretation were set to < 100 EPG in low infection, 100-1000 EPG medium infection and > 1000 EPG high infection; the same cut-off values were used for evaluation of faecal egg counts (FEC) results. It was found that 1 EPG corresponds to 234 copies of plasmid standard, which was considered a minimum limit for a positive detection in the faecal samples.    Discussion PGE in ruminants is usually caused by a mixture of GINs occurring simultaneously [3,33]. Therefore, the potential implications for studying these parasites (population biology, epidemiology, treatment e cacy etc.) offered by quantitative and multiplex PCR assays seem highly valuable. The advantages are manifold and besides simultaneous detection of multiple parasites present in a sample (use of three different dyes for the six targets means that only one third of the number of reactions must be performed to diagnose all six GINs) include high-throughput capacity, wide dynamic range and avoidance of inaccuracies related to end-point analysis. High-throughput sequencing devices are being increasingly applied to the challenge of GIN identi cation [34] and complex nematode community studies [35,36,37]. This 'nemabiome'-type technology is still relatively expensive and requires expertise and complicated bioinformatic analysis, so is still the preserve of specialist laboratories. In contrast, real-time PCR technology is very widespread today and became common equipment of larger laboratories, allowing diagnostic procedures based on these platforms to be used relatively routinely. This fact, together with time and laboratory simplicity ensuring immediate results without bioinformatic pipelines, bringing our present multiplex real-time PCR assays for speci c detection and semi-quantitative evaluation of the most prevalent GIN infections directly from faeces of domestic livestock closer to the wider scienti c community.   [17] developed an automated multiplexed-tandem PCR (MT-PCR), with properties very similar to our approach, with the possible exception that the individual species real-time components are carried out in parallel, in separate reactions, rather than simultaneously in the same reaction. The platform, targeting ITS2 regions of six GINs (Haemonchus spp., T. circumcincta, Trichostrongylus spp., Oesophagostomum spp., Ch. ovina, and C. curticei) was validated for European applications [22] and showed promise for routine use in practice. Although they tested direct extraction of DNA out of 0.25 g of faeces using the PowerSoil DNA puri cation kit (MoBio) as one of the possible methods in the MTPCR study [17], the results showed decreased detection sensitivity which led to decision of the continued use of previously extracted eggs/L 3 larvae only. In comparison, we see the advantage of our approach in The parasites for which the multiplex real-time PCR assays are described in this study represent, apart from A. sidemi, the most important GIN of sheep. The oligonucleotides designed for this study show sequence identity with the currently available NCBI GenBank database gene sequences of individual targets (data not shown). Therefore, even though we did not investigate the speci c detection of further targeted species of the same genera (namely H. placei, T. davtiani, T. trifurcata, T. vitrinus, and T. rugatus) due to the unavailability of DNA material, it can be assumed that the respective probes will detect even these closely related species for which the present detection systems were intended [14].
Ashworthius detection was included in the presented multiplex assay for the following reasons. This hematophagous abomasal nematode is phylogenetically related and morphologically/morphometrically almost indistinguishable in immature stages from H. contortus, and poses a threat to become one of the most widespread pathogenic GINs of autochthonous European ruminants [29]. This invasive parasite originally endemic in Asiatic deer was probably introduced to Europe by sika deer in the late 19th and early 20th century and since then it has successfully spread among new hosts (such as red deer, roe deer, fallow deer, or moose) and is highly pathogenic in some e.g. European bison [38]. The susceptibility of domestic sheep to ashworthiosis has so far only been con rmed experimentally [39], whereas the rst cases of natural infection in cattle were recorded in Poland by Moskwa et al. (2015) [40]. Thus, horizontal transmission of this parasite from wild ruminants to domestic livestock is probably just a matter of time in all regions where these hosts share the same pastures. Thus, having access to a reliable diagnostic tool that can detect A. sidemi in livestock was the main purpose of including this parasite in our multiplex assay.
Detection systems for members in the genus Nematodirus are typically not included in similar studies devoted to strongyle nematodes of livestock due to their distinguishability based on the eggs, which are much larger and contain distinctive dark cells. However, N. battus is an especially important pathogen in temperate climates causing signi cant health problems in grazing lambs in spring. For this reason, we decided to include this species in the multiplex assay aimed at faecal samples, which could be used as an alternative tool to coproscopy. During the assay's speci city tests, we noticed a weaker non-speci c ampli cation of T. circumcincta DNA (Table 2) with the Nematodirus system. However, this phenomenon was not recorded during tests on faecal samples, even in those with a high EPG in the Teladorsagia system (Table 6). This anomaly could be caused either by the presence of other Teladorsagia species than T. circumcincta, to which the N. battus probe did not bind, due to sequence variations or it was more likely due to the presence of an excess amount of nonspeci c DNAs in a complex sample, such as faeces, that might block nonspeci c hybridization during the PCR reaction the same way as sheared DNAs [32,41,42]; such enhancement of speci city has previously been demonstrated by the usage of competing primers [43] or presence of tRNA [44]. In the present study, Nematodirus spp. was detected only in six faecal samples out of 44 examined by McMaster (Table 5). In ve of these samples (No. 32, 41-44) the Nematodirus spp. eggs were identi ed, while in one (No. 8) N. battus was also present but only judged on a single egg. Sample No. 8 was re-extracted and tested repeatedly by multiplex real-time PCR but always with negative results. This could be as a result either of the misidenti cation of Nematodirus species based on a single egg (since other species than N. battus would not be detected by our assay, e.g., N. licollis, N. helvetianus), sampling error (no eggs were present in the faecal sample used for molecular analysis), or because the amount of DNA was below the LOD for this system. The use and suitability of this detection system needs to be further evaluated during a broader study on eld samples from sheep ocks.
The LOD determined by assays sensitivity tests on adult nematodes gDNAs (Table 3) cover the range of 0.5-5 pg which equates to a proportion of gDNA extracted from a single egg [11]. These results are consistent with previous studies based on other singleplex [11,21] and multiplex real-time PCR assays [25].
To re ne the data interpretation for faecal samples, the limit of 1 EPG corresponding to 234 plasmid copies was established. In the present study, this values of individual targets. The reason for this consideration was that semi-quantitative evaluation together with knowledge of species/genus composition is more valuable than absolute quanti cation, given that the intensity of nematode infection does not have to re ect the number of eggs excreted per gram of faeces; the exception to this are nematodes with a high biotic potential (e.g., H. contortus and Oesophagostominae) [11]. Based on this knowledge and experience, we conclude that a semi-quantitative enumeration of different GIN eggs in faeces based just on ITS2 H. contortus plasmid control is feasible by our multiplex real-time PCR assays. The results of individual faecal samples (Table 6)  . Nematode composition based on LC proportionally agreed with those resulting from multiplex assays, however, a slight bias can be observed in the example of F1, where Trichostrongylus DNA was not detected but microscopically determined at 8% according to LC results. Since Teladorsagia was the second most abundant genus on this farm, this indicates a confusion of Trichostrongylus and Teladorsagia larvae during microscopical identi cation of L 3 . Usually these two genera are grouped together even with an experienced observer [27]. The high sensitivity of the present multiplex realtime PCR, together with the approach of individual animal testing, preventing bias of the result due to animals with aggregated parasites, are the required features for reliable GIN diagnostics.
Although, emphasis must be placed on proper homogenization and mechanical cell disruption of the faeces, there are several advantages of using multiplex real-time PCR assays compared to LC. The most noticeable advantage is the signi cant shortening in number of steps included in the procedure resulting in reduction of the whole procedure duration from 7 days on cultured L 3 [46], 2 days with L 1 [14], to only a few hours in the case of the present multiplex assays.
Detection of speci c DNA also allows GIN diagnostics on frozen faecal samples, where the vast majority of eggs rupture or are otherwise damaged causing gDNA release.
Another indisputable but little known and underestimated advantage of using biological samples directly is the presence of parasite cell-free DNA (cfDNA). CfDNA is released during disintegration of different life stages (cellular apoptosis/necrosis) and active secretion from cells and its presence (and other parasite components) within the faeces cannot be identi ed by microscopy [47]. The detection of cfDNA is being increasingly applied as an e cient biomarker for the accurate diagnosis of parasites occupying tissues and the circulatory system. The presence of extracellular cfDNA helps to overcome problems connected with intermittent egg shedding and thus greatly increases the sensitivity and accuracy of the semi-quantitative diagnostic assay. Its presence can also reveal the prepatent stage or the hypobiotic larvae (arrested larval development) [11], since cfDNA is released by the larvae present in the wall of the gastrointestinal tract. The theory of cfDNA and its potential to increase assay accuracy might be supported by individual faecal sample results that tested negative by microscopical FEC; in the case of sample No. 25, we assume that a low intensity of infection detected by multiplex assay (total EPG = 44) could adversely affect faecal otation, which may be subject to a dilution error, but this was presumably not the case in the other two samples. Samples No.
31 and 34 tested negative by faecal otation technique but highly positive by multiplex real-time PCR [25], with rankings of severe infections. In both samples, high levels of Haemonchus, Teladorsagia and Trichostrongylus were detected. A possible explanation for these results could be a prepatent period of infection with a high representation of developing larval stages. Although less likely scenarios such as some form of hypobiosis or sampling error given, e.g., by intermittent egg shedding, cannot be ruled out; the faeces for both tests were collected at the same time of the day, and we reckon that in such a heavy infection would be at least some eggs present anyway.
No molecular approach for speci c DNA detection of GINs in sheep using fresh faeces has yet been implemented in standard diagnosis practice. In our opinion, the two multiplex real-time PCR assays presented in this study may allow reliable evaluation of GIN genus/species richness within European sheep ocks and thus improve diagnosis of GIN infections in ruminant livestock. Presented preliminary evidence of the assays' ability to rapidly identify and rank nematodes according to their numerical contribution to observed FEC in mixed infections may also suggest the potential to become a powerful alternative or practical adjunct to conventional faecal egg count reduction tests (FECRT) to enable the rapid inference of which strongyle species or genera are susceptible or resistant to particular anthelmintic drugs [12,16]. However, further evaluation and re nements of these assays needs to be carried out during further testing on real samples to re ne its precision; then it could usefully supplement or replace the conventional coprological techniques for the purpose of routine diagnosis.

Conclusions
Two multiplex real-time PCR assays designed in this study, containing among other things an internal ampli cation control to avoid false negative results, targeting the main GINs of sheep and were successfully tested for detection of H. contortus, T. circumcincta, T. colubriformis, N. battus, Ch. ovina, and, atypically, A. sidemi as an invasive threat to sheep ocks. These assays were optimized to be performed using DNA extracted directly from faeces and tests of individually collected faecal samples from three farms showed greater sensitivity of molecular screening in comparison to coproscopy. The knowledge of species proportion within the sample, together with semi-quantitative evaluation of FEC for each genus/species adds value to the diagnostic readout and, in our opinion, has the potential to improve current diagnostic approaches. The assays will allow the performance of extensive epidemiological/demographics studies on the major gastrointestinal parasites of sheep and also add a high degree of precision to anthelmintic e cacy testing protocols.

Declarations
Ethics approval and consent to participate The farmers gave permission to collect faecal samples from animals and provided gastrointestinal tract of sheep slaughtered for human consumption.

Consent for publication
Not applicable.

Availability of data and materials
All data generated or analysed during this study are included in this published article. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

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