Sheep nemabiome diversity and its response to anthelmintic treatment in Swedish sheep herds

doi:10.21203/rs.3.rs-41239/v2

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Sheep nemabiome diversity and its response to anthelmintic treatment in Swedish sheep herds

https://doi.org/10.21203/rs.3.rs-41239/v2

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published 18 Feb, 2021

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Background

A novel way to study the species composition and diversity of nematode parasites in livestock is to perform deep sequencing on composite samples containing a mixture of different species. Herein we describe for the first-time the nematode community structure (nemabiome) inhabiting Swedish sheep and how these are/were affected by host age and recent anthelmintic treatments.

Methods

A total of 158 fecal samples were collected (n=35 in 2007 and n=123 in 2013-2016) and cultured from groups of sheep on 61 commercial farms in the south-central part of country where most animals are grazed. Among the samples, 2 x 44 (56%) were paired collections from the same groups pre- and post-treatment with macrocyclic lactones, benzimidazoles or levamisole. The sequences were analyzed for their nemabiome using the PacBio platform followed by bioinformatic sequence analysis with SCATA. Species richness and diversity were calculated and analyzed in R.

Results

Nematode ITS2 sequences were found in all larval culture samples except two, even though the fecal egg counts were below the McMaster threshold in 20 samples. Sequencing yielded, on average, 1,008 sequences per sample. In total 16 operational taxonomical units (OTU), all had ≥98 % identity to sequences in the NCBI database, were recognized. The OTUs found represented nematode species of which 10 are commonly associated with sheep. Multiple species were identified in all pre anthelmintic treatment larval culture samples. No effects on nematode diversity were found in relation to host age. On the other hand, recent anthelmintic treatment lowered species richness, especially after use of ivermectin and albendazol. Interestingly, despite zero egg counts after use of levamisole, these samples still contained nematode DNA and especially H. contortus.

Conclusions

Our findings provide evidence that nemabiome analysis combined with diversity index analysis provide a sensitive and objective methodology in the study of the efficacy of anthelmintic treatment, as both high and low abundant species were detected.

Parasitology

PacBio

gastrointestinal nematode

livestock

anthelmintics

biodiversity

diversity index

Infection with gastro-intestinal nematode (GIN) parasites is well-known globally as a major veterinary problem which contributes to a decline in the productivity of the global sheep industry [1]. Grazing sheep in Sweden are typically infected by a wide range of GIN with the majority belonging to the superfamily Trichostrongyloidea within the order Strongylida [2], among which some species such as Haemonchus contortus are considered more pathogenic than others [3]. Accordingly, parasite identification is fundamental for the improvement of sustainable parasite control strategies. The relative abundance of the different species present in sheep is driven by a wide range of factors. These include external factors such as climate and managerial factors affecting the exposure to the free living stages [4], and not at least selection reinforced by use of anthelmintic compounds [5].On top of this parasites are influenced by host immunity affected by previous exposure [6] and the presence of other animals that may act as reservoirs [7].

Traditional parasitological diagnostic techniques, based on microscopical examination of fecal eggs counts (FEC) and larval cultures, can provide rough measures about the nematode species or genus composition in sheep [8]. However, a disadvantage with these diagnostic tools is that they rely on trained experts that are rare to find today, but it is clear that they may have major constraints in terms of both sensitivity and specificity [9]. Thus, there is a need to utilize improved diagnostics for the investigation of complex nematode communities. With access to diagnostic instruments that could rapidly rank strongyle nematodes according to their relative contribution to mixed infections would represent a major advantage particularly in terms of the understanding of how selection by anthelmintics shape nematode communities. This need is important given the increasing evidence for anthelmintic resistance (AR) and requests for evidence-based use of anthelmintic drugs in parasite control [10]. Although effective parasite control in general can be achieved through a combination of grazing management strategies and treatment with anthelmintics, this practice is under threat due to the increasing spread of resistance to these veterinary drugs [11].

For some time, the molecular identification of parasitic nematodes largely relies on amplification of the internal transcribed spacer two (ITS2) region located between the 5.8S and 28S subunits of the ribosome encoding genes [12]. Today there are several DNA-based tests available for genotyping of GIN that offers the potential to detect, identify and quantify especially strongyle nematode parasites in ruminants (Hunt and Lello, 2012; Elmahalawy et al., 2018; Roeber et al., 2011). However, these different technologies in general suffer from limitations in terms of the number genera and/or species that can be detected since they are restricted to the few parasites that are considered to be of particular interest and to which primer probe sets have been designed. Clearly the advent of deep amplicon sequencing using next-generation sequencing (NGS) platforms has generated new prospects and simplified the sequencing of gene amplicons in the study of microbial communities that usually exist as mixed infections within their hosts. Like for any microbe, NGS allows for the characterization of complex nematode communities and it open up new possibilities to identify community components even at low relative abundances at an unprecedented depth with minimal cost and labor. Recently the term “nemabiome” was created to describe the community structure of nematodes in ruminant livestock [16]. By using an Illumina based deep amplicon next generation pooled sequencing approach targeting the nematode ITS2 rRNA gene, a pipeline was developed which so far has been used to study the entire nemabiome in beef cattle [16], bison [17] and dairy cattle [18] in Canada and the US, as well as in UK sheep [19]. Similarly, the nemabiome of equines were recently studied by a slightly different methodology [20]. Combined, these studies have revealed detailed insights into the nematode diversity in each of these hosts using a truly non-invasive diagnostic approach.

In this paper we; describe the species composition and diversity of GIN by studying the nemabiome in 158 sheep larval cultures collected on 61 commercial farms distributed across Sweden. The results were analyzed by investigate how community structure was affected; i) by host age, ii) in response to anthelmintic treatments, and iii) its long term temporal effects. This was conducted by using data generated on the Pacific Biosciences (PacBio) platform, which for other microbial communities has shown to produce less bias compared to other deep sequencing technologies [21]. The OTUs we refer to were distinguished using SCATA (https://scata.mykopat.slu.se/), which is a specifically designed analysis framework for the evaluation of sequenced tagged amplicons derived from eukaryotic communities.

Sample collection

The sampling was carried out either as part of random investigation or from commercial sheep farms suffering from recurrent problems with GIN. In total, 158 fecal samples were obtained from 19 farms during 2007 (n=35 lambs), and on 42 farms between 2013 and 2016 (n=123, of which 74 from adults and 49 from lambs), (Fig. 1). On each sampling occasion approximately two tablespoons of fresh faeces were collected per rectum from each of 10 or 15 randomly selected animals (either ewes or lambs) in the flock. This was done by the farmer or a veterinarian at the Farm and Animal Health in Sweden. After treatment, the sheep were kept in their respective herd. Individual fecal samples were placed in marked zip-locked plastic bags where after the air was pressed out before sealing and then sent over night by national post to the diagnostic laboratory (Vidilab AB).

On 44 of the sampling occasions, paired pre- and post-treatment samples were obtained, 24 paired fecal samples from adults and 35 from lambs, i.e. 88 samples. Among these 26 groups were treated according to the recommended dose with the macrocylic lactone ivermectin (IVM), 13 with the benzimidazole albendazole (ABZ), and 5 with the imidazothiazole derivative levamisole (LEV). On 10 farms the efficacy of several anthelmintics were tested in parallel using different groups of animals in the flock. The post-treatment collection was done from the same animals between 7 to 10 days after deworming in accordance with [22]. These samples originated from routine monitoring program and collected by veterinarians and thus, they reflect the actual use of anthelmintics on sheep farms in Sweden.

Parasitological investigation and DNA extraction

Upon arrival to the laboratory faeces were investigated for gastrointestinal nematode (GIN) strongyle eggs. The number of nematode eggs were first counted using a modified McMaster technology based on 3 g of faeces and with a minimum diagnostic sensitivity of 50 nematode eggs per gram faeces (EPG) as described previously [23].

Thereafter subsamples of approximately 2 g faeces were collected from each animals in all groups and pooled in separate plastic containers. After blending the pooled fresh faeces with Vermiculite, the eggs were cultured for approximately 10 days in a moistened chamber at ≈20 °C [22]. Infective third-stage larvae (L3) were harvested using the Petri-dish method, concentrated in a Falcon tube and collected into an Eppendorf-tube before storage in a freezer at approximately -18 °C [14]. DNA was then extracted from the larval cultures (one per group and sampling occasion) using the Nucleospin® DNA tissue kit (Macherey-Nagel, Düren, Germany) following the manufacturers guidelines.

Library preparations and sequencing

Nematode parasite ITS-2 rDNA fragments were amplified using the NC1-NC2 primer pair [24] tagged with an 8 bp long barcode [25] on the 5’ side of each primer. PCR was conducted in 50µl reactions consisting of 0.2 µM dNTPs, 0.75 µM MgCl2, 1µM of each of NC1-NC2 primer pair, 2.5µl DNA template and 1.25 U DreamTaq DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA). The PCR cycling consisted of an initial denaturation for 3 min at 95 °C followed by 20-34 cycles of 30 s at 94 °C, 30 s at 55 °C and 30 s at 72 °C and finally 7 min at 72 °C where the number of PCR cycles for each sample was determined depending on template concentration. Each sample was purified using Agencourt AMPure beads (Beckman Coulter, IN, USA) and the purified PCR product was measured fluorometrically (Qubit Fluorometer). The same amount of PCR product from each sample was pooled into four libraries. Sequence ligation to each library and sequencing on PacBio SMRT cell V3 RSII were performed by SciLifeLab, Uppsala, Sweden.

Bioinformatics

The raw sequence reads were processed using the SCATA bioinformatics pipeline (http://scata.mykopat.slu.se/). Sequences with an average quality score below 10 or 20 at any single position were removed. The primer and tag sequences were then removed from the remaining reads. SCATA uses blast as a search engine to cluster sequences based on similarity. During the clustering procedure, sequences were compared parwise for similarity using USEARCH [26] with a minimum length set to 85% of the longest sequence. Pairwise alignments were scored using a scoring function with 1 in penalty for mismatch, 0 for gap opening and 1 for gap extension. Homopolymers were collapsed to 3 bp when OTUs cannot be delimited based on differences in homopolymer regions [27]. Sequences were clustered into operational taxonomic units (OTUs) by single linkage clustering with a 98.5% sequence similarity required to be assigned to OTUs [28]. OTU sequences were manually searched for similarity with reported sequences using NCBI BLAST and OTUs not representing nematodes were omitted. OTUs containing less than 1% of the total reads per sample were filtered to reduce the background noise and background contamination. For transparency, a scatterplot with number of reads per sample (Fig. S1), unfiltered data and analyses can be found in the supplementary data file (Tables S1-S2 and Fig. S2-S4).

Statistical analysis

All statistical analyses on community structure were performed in R v4.0.2 [29]. Species richness was calculated by summing up the number of helminth species per individual. To investigate helminth community diversity and if it is dominated by a few species, the inverse Simpson’s diversity index was calculated using Vegan package [30]. The value of Simpson's D ranges from 0 to 1, with 0 representing infinite diversity and 1 representing no diversity, so the larger the value of D, the lower the diversity. For this reason, Simpson's index is expressed as its inverse (1/D). We used linear models (LM) for Fig2-3 and generalized mixed models (GLMM), with a repeated measure design, was used to assess the effects on the nemabiome composition after anthelmintic treatment (Fig. 4G-L), using respective diversity indices as the response variable, treatment as a fixed factor and farm as a random structure. GLMM was calculated using the MCMCglmm package [31]. Using a bayesian framework is robust for analyzing data with unbalanced sample size groups. Run parameters are presented in Table 2. Post-treatment larval culture samples for two IVM farms were omitted due to successful treatment (Fig. 4J), hence no nematode DNA was to be analyzed. Data was visualized with ggplot2 [32].

Species diversity

The 159,224 high quality reads were assembled into OTUs, with on average 1,008 reads per sample (range 9-1885 prior to filtering). After filtering, we identified 16 nematode species, based on OTU identification, from 158 sheep, of which 13 had a NCBI BLAST query cover of more than 99% (Table 1). Of the 158 larval culture samples, all except two collected post-treatment produced nematode OTUs. Of these, the five most common species consisted of roughly 97% of the reads for both adult sheep and lambs (Fig. 2; Table 1). The species in the stacked bar plots in Fig. 2A, 3A and 4A-C are calculated using fractions of the total number of reads per sample. Thus, each sample have equal representation in the figures. On average adults were infected by fewer species compared to lambs (Species richness, Fig. 2B.) (LM, F_(1.112)=2.9019, P=0.01704). However, the species composition showed that the nematode community was dominated by a few species, among ewes compared to lambs (Inverse Simpson’s diversity index, Fig. 2C.) (LM, F_(1.112)=8.3942, P=0.004529).

Table 1. Identification of species from OTUs using BLAST (basic local alignment search tool). The top five species represent 97% of the reads. Nematodes were identified in 156 (99%) out of 158 larval culture samples. The most common species was Haemonchus contortus, which was identified in 127 (80%) of the positive samples and was also represented by most of the sequence reads. The ITS2 fragments for all OTUs varied in length from 266 to 455 bp.

Best species match from BLAST search	Query cover	Identity	Number of samples	OTU Sequence length	Number of reads
Chabertia ovina	100	100	37	283	8418
Haemonchus contortus	100	100	127	281	102110
Teladorsagia circumcincta	100	99	80	296	28293
Trichostrongylus vitrinus	100	100	40	288	10311
Oesophagostomum venulosum	99	100	20	308	3288
Bunostomum trigonocephalum	100	100	3	281	199
Cooperia curticei	100	99	1	292	800
Cooperia oncophora	100	100	5	291	1290
Coronocyclus coronatus	100	99	1	281	735
Cylicocyclus nassatus	100	98	10	370	1294
Cylicostephanus minutus	99	100	3	266	200
Cystocaulus ocreatus	100	99	3	418	85
Muellerius capillaris	100	100	2	455	35
Ostertagia leptospicularis	100	99	1	289	66
Ostertagia ostertagi	100	100	3	288	364
Varestrongylus eleguneniensis	94	100	2	411	23

Nemabiome composition

For lambs, we tested larval culture samples from two time periods, 2007 and 2013–2016 while samples for adults were only collected between 2013–2016. Among the lambs, Chabertia ovina decreased and H. contortus increased in frequency in comparison with adults. The species richness in lambs between the two time periods differed between the time periods (LM, F₍_1.60)=6.942, p=0.0107). It was also a higher dominance among a few species in 2013–2016 than in 2007, based on the difference in inverse Simpson’s diversity index between the years according to a linear model (LM, F₍_1.60)=5.8339, p=0.01878) (Fig. 3).

Influence of anthelmintic treatment

Treatment with IVM was most effective against Oesophagostomum venulosum, ABZ was effective against C. ovina and Trichostrongylus vitrinus, while LEV showed high efficacy against Teladorsagia circumcincta and also had good effect against H. contortus (Fig. 4A-C). Furthermore, the post-treatment EPG was lower for all anthelmintic substances (Fig. 4 D-F), and especially for LEV where no nematode eggs were found after treatment. In contrast, eggs remained in 17/26 (65%) and 7/13 (54%) of the groups treated with IVM and ABZ, respectively. Post-treatment lowered the species richness on IVM or ABZ treated sheep, (Fig. 4D-E) while no change in species dominance was detected (Fig. 4J-K). For LEV, the treatment was highly effective and the EPG in post-treated sheep was lower than 50 (the detection limit of the McMaster) for all farms (n=5). Still, nemabiome data was generated and the low number parasites remaining notably changed in species composition although it did not notably affect species richness or species dominance (Fig. 4I-L) See Table 2 for detailed summary statistics.

Table 2. Output from MCMCglmm testing of the impact of each of the three anthelmintic drug treatments (IVM=ivermectin, ABZ=albendazole, LEV=levamisole) on species richness and inverse Simpson’s diversity index (see also Fig. 4G-4L) for farms sampled before and after anthelmintic treatment. As an example, untreated sheep were infected with on average 0.77 species more compared to ivermectin treated sheep. With LEV, no such effect was found for species richness, nor for any of the Inverse Simpsons’ indices.

Post- and pre treatment	Posterior mean	Lower 95% CI	Upper 95%Ci	Effective sample size	pMCMC¹
Species richness IVM	0.7701	0.2814	1.2294	10000	0.0024**	Fig 4G
Species richness VBZ	0.97106	0.07297	1.91494	10691	0.041*	Fig 4H
Species richness LEV	-0.07496	-1.68539	1.70185	10000	0.8738	Fig 4I
Inv Simpson IVM	0.20156	-0.07581	0.47853	9062	0.153	Fig 4J
Inv Simpson VBZ	0.2319	-0.108	0.5962	10000	0.186	Fig 4K
Inv Simpson LEV	-0.2686	-0.9549	0.4001	10000	0.3822	Fig 4L
¹Statistically significant fixed effects are marked with * (p<0.05) and ** (p<0.01)
Prior settings:	R (V=2, nu =0.2); G (V=2, nu = 0.02)
Run settings:	Burn-in = 50,000; Iterations = 100,000; Thinning interval = 10

In this study we have for the first time investigated nemabiome in 158 larval culture samples from 61 commercial sheep farms in Sweden. The aims were to find out how nematode community structure (species composition and diversity) were influenced; i) by host age (in ewes and lambs), ii) in the short-term following recent anthelmintic treatment with ABZ, IVM or LEV, and iii) long-term in samples collected several years apart. For this we used DNA extracted from composite larval cultures which then were analyzed using a PacBio NGS pipeline generating sequence data (amplicons) that were clustered into OTUs with a specifically designed analysis framework developed for eukaryotes (SCATA) [33–35]. We identified five common OTUs representing 97% of the reads, among which H. contortus and T. circumcincta were the two dominant species. There was, in general, no significant differences in nemabiome post-treatment between ewes and lambs. In contrast, use of anthelmintics had a short-term dramatic effect, but not on nemabiome in post-treatment larval culture samples collected several years apart indicating there was no long-term effect.

In total, 16 OTUs were identified, five of which are well-known and globally distributed strongyle nematodes associated with sheep; i.e. C. ovina, H. contortus, T. circumcincta, Trichostrongylus vitrinus and Oesophagostomum venulosum. This group of big-five constituted the majority of reads (97%). All of these had a match of ≥99 to 100 in query cover and identity according to BLAST searches and were therefore considered as valid species. We also recognized five other species reported from sheep with the same confidence in most cases (Table 1). These were represented by two strongylids; Bunostomum trigonocephalum and Cooperia curticei, plus three metastrongylids (small lungworms); Cystocaulus ocreatus, Muellerius capillaris and and a species related to Varestrongylus eleguneniensis. All had an identity of ≥99 and with the exception of the match to Varestrongylus eleguneniensis and a query cover of 100%. However, combined these five represented less than 1% of the total reads and they were only found in a few samples. This indicates that the epidemiological consequences and clinical impact of these parasites are probably of less importance for sheep raised under Swedish conditions. With the exception of T. colubriformis, which was absent in our data set, we recognized all eight major species found in UK sheep based on data generated with a similar approach [19]. However, the relative abundance of the distinctive species differed. In our study five species dominated and with H. contortus being more prevalent in Swedish compared to UK sheep. From this, it follows that there is a difference in nemabiome composition in sheep between different climatic conditions. Thus, microclimatic influences would be worthwhile to investigate especially in the light of the possible impact of climate change.

In addition to the above species, we identified five species usually not found in sheep. Among these, three species are mainly linked to cattle; Cooperia oncophora, Ostertagia leptospicularis, and O. ostertagi [2]. However, we also identified several cyathostomins with best BLAST matches to Coronocyclus coronatus and Cylicocyclus nassatus, that are normally associated with equids. To the best of our knowledge it is unknown if transmission of nematodes from equids to sheep can occur. Still, at least the matches both for C.coronatus and C. ocreatus showed high query cover and identity (100 and 99), indicating that these findings are valid. Although both C. nassatus and C. minutus had an identity of 100%, the query cover was low (81-86).

In contrast, opportunities for cross-infections between cattle and sheep nematodes have been studied through experimental cross-infections [36]. It was shown that both Cooperia spp. and Ostertagia spp. can infect lambs, however there are varying degrees of host specificity in these genera, with C. oncophora and O. ostertagi being more adapted to cattle than to sheep. However, as stated by Herlich (1971), it cannot be precluded that small numbers of O. ostertagi may attain sexual maturity in sheep under natural grazing conditions, which is in line with our findings.

Interestingly, we recognized no species primarily associated with wildlife., However, there are generalist known to infect a wide range of wildlife ungulates in Europe [7]. Not at least roe-deer in Spain [38], Italy [39], Turkey [40], and France [41], is known to be infected with species identified herein. Among these, particularly H. contortus is the most pathogenic nematode of sheep in Sweden [3] and is therefore of major interest. Although, roe-deer is abundant in Sweden, knowledge about its nemabiome and its role as a reservoir of livestock parasites is presently unknown. Thus, this needs to be studied in the future, especially with focus on H. contortus, not at least in face of climatic change and its propensity to develop resistance to anthelmintic drugs and the possibility of roe-deer acting as a reservoir and spreading it widely. Since it is known that there are cryptic species that appear identical but are genetically distinct, particularly among members of the superfamily Trichostrongyloidea that hybridize [42], we are convinced that nemabiome analysis is well suited for this.

Clearly, the culturing conditions for nematode eggs can have an impact the nemabiome composition [43]. For example, it has been shown that fewer eggs of T. circumcincta develop to the third stage than for T. colubriformis. Also, the development of larvae of other strongyles are heavily influenced by refrigeration [43]. Extracting DNA from fecal matter or from larval cultures both had advantages and disadvantages. 1) DNA from fecal samples would contain DNA from all eggs and might contain inhibitors while fecal samples containing few parasite eggs might lead to an underestimation of species diversity and contain inhibitors, 2) Larval culture samples will contain more parasite larvae, while the hatchability might be impared due to storage. Besides from the input material, the number of recognized OTUs are also influenced; i) by primer design and PCR conditions, ii) the choice of NGS platform and bioinformatics pipeline for OTU clustering and recognition, and not at least iii) available sequence information in the sequence databases. In this study we took advantage of the commonly used primers NC1 and NC2 and compared the obtained sequence with information in NCBI. In line with previous studies, these universal primers spanning the second (ITS2) internal transcribed spacer in the nuclear ribosomal DNA array not only amplify a wide range of livestock genera of nematodes of veterinary interest (i.e. Bunostomum, Chabertia, Cooperia, Dictyocaulus, Haemonchus, Nematodirus, Oesophagostomum, Ostertagia, Varestrongylus, Teladorsagia, and Trichostrongylus), but also provide suitable genetic markers for species delimitation (for a review see Gasser et al., 2008). In our study, most of these genera were found as well as five additional genera (Table 1.). Still, it is unclear where to define the cut-off for discriminating between nematode species, as genetic isolation is generally used to define species boundaries rather than DNA differences [44]. Nevertheless, in agreement with previously studies on nemabiome in livestock and horses [16–20], a cut-off identity threshold of ≥99 seems reasonable. However, as shown in our study, the query cover also needs to be considered. Still, when targeting the ITS region it has been shown that PacBio reflects the composition better than Illumina MiSeq for metabarcoding of fungal communities due to sequence length variation [35]. In this study we have shown that ITS2 for the different OTUs varied between 266 and 455 bp (Table 1) and we thereby detected metastrongyles. However, to what extent nemabiome composition is affected by sequence length variation needs to be explored in more detail. Further investigations also need to address factors that may introduce sequencing bias.

We observed an effect in relation to host age and long-term effects were observed when we compared the larval culture samples collected in 2007 and 2013-2016 (Fig.2, 3). The observation that adults had less diversity than lambs is expected as adults can build up resistance to some parasites. It is also illustrated by that some species dominated the nemabiomes in adults. It is clear that use of anthelmintics drastically influenced the nemabiome composition (Fig. 4). In this context it is important to consider that the use of the tested drugs has changed during the last decades. In Sweden, the drug of first choice whenever H. contortus is present has, from the 1960’s until recently, changed from ABZ to IVM [45]. However, with emerging evidence for double resistant H. contortus strains, this practice has changed. Today, LEV is increasingly used, but currently only on farms that neither respond adequately to IVM nor ABZ. Interestingly our data show that the identified nematode species responded differently to these drugs. Of particular practical interest is that both IVM and ABZ were unable to control H. contortus on several farms, whereas LEV still has a reasonable efficacy. However, despite there was always zero-egg counts post-treatment with LEV it is evident that in particular H. contortus survived at a low level. Likewise, T. circumcincta, which is the second most important nematode in Swedish sheep, survived treatment with either ivermectin or ABZ, but then to a lesser extent than H. contortus, whereas not at all after LEV treatment. This is partly in contrast to the situation reported several years ago in a global context, when resistance to LEV was widely prevalent among several trichostrongylid nematodes of sheep [46]. Overall, we believe the observed patterns reflect the current anthelmintic resistance situation in GIN of sheep in Sweden. Of major practical concern is whether LEV resistant H. contortus will appear with increasing use of this drug. In countries with more intense sheep production (e.g. New Zealand), the use of LEV has decreased in favor of other drugs or drug combinations with increased reports of resistance [47]. However, in the past when LEV was used as a single compound in sheep, high levels of resistance emerged, like for most other commonly used anthelmintics [48]. In light of this, our observations showing low levels of surviving H. contortus is of great concern.

In our study, the nemabiome approach proved to be a powerful method of studying nematode community diversity in sheep and how it is influenced by factors such as host age and the most recent use of anthelmintic treatment. Clearly, by using another molecular approach (droplet digital PCR), estimates of sheep nematode diversity was underestimated [14]. From this study it is clear that the effects of recent anthelmintic treatment of gastrointestinal parasites in sheep can be investigated with higher precision by using high throughput sequencing.

Ethics approval and consent to participate

No ethical permissions were necessary for this study as the parasites were collected from samples sent in for routine veterinary diagnostics.

Consent for publication

Not applicable

Availability of data and material

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. The raw ITS2 reads are stored at the European Nucleotide Archive (ENA) under accession number XXXXXX.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by the Swedish Farmers’ Foundation for Agricultural Research grant O-16-20-742 and Formas 2016-1744.

Authors' contributions

Both authors designed the study. PH analyzed the data. JH and PH wrote the manuscript, reviewed it and have approved the final version. Funding was provided to JH.

Acknowledgements

The authors are grateful to DVM Katarina Gustafsson and Vidilab AB for their help with sample collection. We would also acknowledge SLU Metabarcoding Laboratory – UMBLA for PacBio library preparation and SNP&SEQ Technology Platform at SciLifeLab in Uppsala, Sweden for PacBio sequencing.

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Additional files Additional file: Table S1. Species identification without 1% cut-off. Table 2. MCMCglmm analysis without 1% cut-off. Figure S1. Scatterplot with number of sequences per sample. Figure S2. Nemabiome composition for adults and lambs without 1% cut-off. Figure S3. Nemabiome composition for lambs for two periods using data without 1% cut-off. Figure S4. Nemabiome composition and diversity indices without 1% cut-off.

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Sheep nemabiome diversity and its response to anthelmintic treatment in Swedish sheep herds

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