Davey et al.

Parasites & Vectors (2023) 16:19 Parasites & Vectors

https://doi.org/10.1186/s13071-022-05644-6

RESEARCH Open Access

Faecal metabarcoding provides improved

detection and taxonomic resolution
for non‑invasive monitoring of gastrointestinal
nematode parasites in wild moose populations
Marie L. Davey1*, Stefaniya Kamenova2,3, Frode Fossøy1, Erling J. Solberg1, Rebecca Davidson4,
Atle Mysterud1,2 and Christer M. Rolandsen1

Abstract
Background Although wild ungulate populations are heavily monitored throughout Europe, we understand little
of how parasites affect population dynamics, and there is no systematic, long-term monitoring of parasite diversity
and parasite loads. Such monitoring is in part hampered by a lack of time- and cost-effective assay methodologies
with high sensitivity and good taxonomic resolution. DNA metabarcoding has been successfully used to character-
ize the parasitic nemabiome with high taxonomic resolution in a variety of wild and domestic hosts. However, in
order to implement this technique in large-scale, potentially non-invasive monitoring of gastrointestinal parasitic
nematodes (GIN), protocol optimization is required to maximize biodiversity detection, whilst maintaining time- and
cost-effectiveness.
Methods Faecal samples were collected from a wild moose population and GIN communities were characterized
and quantified using both parasitological techniques (egg and larva counting) and DNA metabarcoding of the ITS2
region of rDNA. Three different isolation methods were compared that differed in the volume of starting material and
cell lysis method.
Results Similar nematode faunas were recovered from all samples using both parasitological and metabarcoding
methods, and the approaches were largely congruent. However, metabarcoding assays showed better taxonomic
resolution and slightly higher sensitivity than egg and larvae counts. The metabarcoding was not strictly quantitative,
but the proportion of target nematode sequences recovered was correlated with the parasitologically determined
parasite load. Species detection rates in the metabarcoding assays were maximized using a DNA isolation method
that included mechanical cell disruption and maximized the starting material volume.
Conclusions DNA metabarcoding is a promising technique for the non-invasive, large-scale monitoring of parasitic
GINs in wild ungulate populations, owing to its high taxonomic resolution, increased assay sensitivity, and time- and
cost-effectiveness. Although metabarcoding is not a strictly quantitative method, it may nonetheless be possible
to create a management- and conservation-relevant index for the host parasite load from this data. To optimize the
detection rates and time- and cost-effectiveness of metabarcoding assays, we recommend choosing a DNA isolation
method that involves mechanical cell disruption and maximizes the starting material volume.

*Correspondence:
Marie L. Davey
marie.davey@nina.no
Full list of author information is available at the end of the article

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Davey et al. Parasites & Vectors (2023) 16:19 Page 2 of 15

Keywords Nemabiome, Metabarcoding, ITS2, DNA extraction method, NC1–NC2 primers, Alces alces, Helminth,
Ungulates

Background (superfamily Strongyloidea) in the large intestine [17].

Ungulates are an economically and culturally impor- The taxonomic resolution of egg count surveys is sub-
tant group of species in Europe [1], with a current esti- sequently low, and groups together organisms that can
mated annual harvest of above 7 million individuals [2]. have different or interactive impacts on their hosts [18].
The population ecology of ungulates is well-described in The molecular characterization of GIN parasites
terms of how population density and climate affect vital offers methodological alternatives to traditional parasi-
rates [3, 4], yet we have a limited understanding of the tological approaches. A variety of PCR-based methods
role of parasites in population limitation and regulation. detect, identify, and quantify GIN species in research
There is some evidence of negative impacts of parasites and diagnostic settings [19, 20]. The primary advantage
on host fitness, including body condition, survival, and of molecular approaches in the characterization of GIN
fecundity [5–8], but in a very limited set of species and parasites has been the reliable identification of these
countries. Moreover, the few studies quantifying para- species at any life stage [19, 21]. Recently, both free-liv-
sites in wild ungulates are typically short-term [5, 6, 9, ing and parasitic nemabiome diversity has been investi-
10]. Long-term monitoring of ungulate populations in gated in a variety of environments and hosts using DNA
Europe is extensive and uses either direct estimation of metabarcoding techniques that rely on high-through-
abundances and body condition, or indirect monitor- put sequencing methods (e.g. [22–26]). In particular,
ing of browsing pressure on important forage species DNA metabarcoding of the internal transcribed spacer
[11–13]. However, there is no long-term monitoring of 2 (ITS2) region of ribosomal DNA (rDNA) specifically
parasite diversity and parasite loads in wild ungulates targeting clade V parasitic GIN has been successfully
in Europe, in part due to a lack of suitable methods for applied to adult worms, eggs, and faecal samples (e.g.
estimating parasite diversity and abundance, which are [27–29]). Combined with a curated, well-developed ref-
required for efficient monitoring. erence sequence database (www.​nemab​iome.​ca), this
Parasite monitoring of gastrointestinal nematode provides high quality data with good taxonomic reso-
(GIN) communities in wild ungulate populations is lution for parasitic GIN communities. This method has
methodologically challenging. Traditional parasitologi- been used to successfully characterize the GIN commu-
cal methods for assessing GINs can be labour-intensive nities hosted by a variety of wild ungulates [27, 28, 30,
and not well suited to large-scale, non-invasive, long- 31] and shows substantial promise for allowing non-
term monitoring. Species-level identification of GINs invasive monitoring of GINs in wild populations [31].
typically requires adult specimens. This necessitates In order to implement DNA metabarcoding in large-
harvesting gastrointestinal material from individu- scale, potentially non-invasive monitoring of GIN, pro-
als that have been hunted, culled, lethal sampled, or tocol optimization is required to maximize biodiversity
died of natural causes [6, 14, 15]. This makes it chal- detection, whilst maintaining time- and cost-effective-
lenging to have systematic population representative ness in the protocol. The DNA extraction method has
sampling and non-invasive monitoring in wild popula- been documented to impact the recovery of soil nema-
tions. Faecal egg counts are frequently used to meas- tode biodiversity [32]. More specifically, the detection
ure gastrointestinal helminth burden in livestock (e.g. and sensitivity of polymerase chain reaction (PCR)-
[7, 16]). However, egg counting requires consider- based assays for GIN from faecal samples vary with the
able effort, training, and taxonomic expertise, making type of DNA extraction method used [33, 34]. However,
this task quite demanding for use on large numbers of the impact of the DNA extraction method on parasitic
samples. Gastrointestinal nematodes belonging to the GINs recovery specifically using DNA metabarcoding
order Strongylida produce eggs that are morphologi- of faecal samples has not been previously examined,
cally similar and identification therefore requires either either in terms of biodiversity recovery or in terms of
molecular identification of the eggs or morphological time- and cost-effectiveness. Typically, commercial kits
speciation of hatched larvae, after culture of the eggs for DNA extraction from faecal material are optimized
and larvae. This includes a variety of species, from to retrieve high-quality DNA from a large range of tar-
Ostertagia sp. (superfamily Trichostrongyloidea) in get organisms from both fresh and frozen material.
the abomasum to Bunostomum sp. (superfamily Ancy- However, they rely on small volumes of starting mate-
lostomatoidea) in the small intestine to Chabertia sp. rial, thus potentially limiting the capacity to capture

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Davey et al. Parasites & Vectors (2023) 16:19 Page 3 of 15

sporadic DNA from gastrointestinal parasites, espe- egg (McMaster) and larvae (Baermann) counting, with
cially at periods of low egg-shed. On the other hand, a focus on the detection of GIN diversity and the poten-
DNA extraction kits from soil provide similar purifica- tial for quantification of GIN parasite load, as well as the
tion steps for removal of inhibitory compounds while time- and cost-effectiveness that must be considered for
accommodating large volumes of starting material [35, methods to be effective and practical in large-scale moni-
36]. However, using them is comparatively cost- and toring practices.
time-laborious, potentially negating their advantages in
the context of large-scale monitoring programs.
Here, we assess the impact of the DNA isolation Methods
method from frozen faecal samples on the results of ITS2 Study area
DNA metabarcoding of clade V GIN communities with The study area is located in Trøndelag county in central
the aim of contributing to a robust protocol suitable for Norway within the boreal and alpine vegetation zones
routine studies and long-term parasite monitoring in (Fig. 1). The vegetation is dominated by Scots pine (Pinus
wild ungulate populations. Using faecal samples col- sylvestris), Norway spruce (Picea abies), and downy birch
lected during the capture and global positioning system (Betula pubescens), with grey alder (Alnus incana), aspen
(GPS)-collaring of moose (Alces alces), we compare the (Populus tremula), rowan (Sorbus aucuparia), and goat
results of metabarcoding inventories using commercially willow (Salix caprea) also commonly occurring [37]. The
available DNA isolation kits that differ in the (i) amount study area spans a gentle elevational gradient between
of starting material, (ii) method of cell disruption and approximately 200–700 m above sea level, with active
(iii) labour required. The results were compared with agricultural lands primarily occupying valley bottoms
those from traditional methods, in our case standard and the lower-lying parts of the study area.

Fig. 1 Map of study area. Maps showing (a) the location of the study area in central Norway and (b) an overview of the study area with points
representing the locations where faecal samples were collected from 29 GPS-collared moose

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Davey et al. Parasites & Vectors (2023) 16:19 Page 4 of 15

Sample collection animal had a mono- or mixed infections with protostron-

All faecal samples were collected fresh directly from gylid larvae. The number of larvae per gram faeces (LPG)
the rectum of moose that had been captured and anes- was estimated from the subsample count (number of
thetized to equip them with GPS collars to study their larvae detected in 100 μl × 10/the weight of the faeces in
space use (Rolandsen et al., unpublished data). Moose the faecal sample). A second 100-μl subsample was taken
were darted from a helicopter during winter, and all pro- from the unhomogenized sediment if no larvae were
cedures were approved by the Norwegian Environment detected in the first subsample. If no larvae were detected
Agency and the Norwegian Food Safety Authority, which in the second subsample, then the results were recorded
is the animal research authority in Norway. Twenty- as no larvae detected.
nine individuals were sampled, and no recaptures were
included in the analyses. Five to 10 faecal pellets were Molecular detection of intestinal parasites
selected and placed in individual clean plastic contain- DNA extraction and sequencing
ers. Faecal samples were sent at ambient temperatures Faecal samples were defrosted overnight at 4 °C and each
for parasitological analyses (McMaster and Baermann sample thoroughly homogenized in a clean zip-lock bag.
counts), and thereafter stored at −20 °C until DNA DNA was isolated from subsamples of each faecal sample
extraction for metabarcoding analysis. using one of three DNA isolation protocols (Fig. 2):

(1) QIAmp Fast DNA Stool Mini Kit

Nematode counts and identification
The abundance of endoparasitic eggs and oocysts was From each faecal sample, 220 mg of wet weight was
estimated using a modified McMasters method and withdrawn using a disposable lab spatula (Chem-
zinc–chloride/sodium chloride flotation fluid (with glass, UK). Subsamples were stored in sterile 2-ml
a specific gravity of 1.3) [17, 38] with a 3 g faecal sam- microcentrifuge tubes and DNA was extracted
ple mixed with 57 ml tap water. A total of 1 ml flotation immediately after sub-sampling. DNA extractions
fluid was examined for eggs giving a theoretical detection were carried out using the QIAamp Fast DNA
limit of 20 eggs per gram (EPG)/oocysts per gram (OPG). Stool Mini Kit (Qiagen, Germany) according to the
Eggs and oocysts were identified to genus level (Monie- manufacturer’s instructions. Two blank extractions
zia sp., Trichuris sp., Nematodirus sp., and Eimeria sp.) (ultra-pure Milli-Q water instead of DNA) were
and, where possible, species level (Strongyloides papil- included to monitor for possible contamination.
(2) MP FastDNA™ Spin Kit for Soil (50 ml volume)
losus, Nematodirus battus), based on morphological
characteristics. Several GIN eggs can only be identified
to order, given morphological similarities and size over-
lap. Therefore, Chabertia sp., Cooperia sp., Haemonchus Between 1.5 and 4 g of faecal wet weight was
sp., Oesophagostomum sp., Ostertagia sp., Spiculoptera- placed in a 50-ml centrifuge tube containing Lysing
gia sp., Teladorsagia sp. and Trichostrongylus sp. were Matrix E (MP Biomedicals), which comprises 1.4
grouped as strongyle-type eggs. The Baermann technique mm ceramic spheres, 0.1 mm silica spheres and 25
was used to isolate, quantify and identify parasitic first- 4-mm glass beads. Samples were homogenized by

the faecal material using the MP FastDNA™ Spin

stage (L1) larvae in the faeces [38]. A 10-g faecal sam- shaking at 6 m/s for 40 s. DNA was isolated from
ple, wrapped in gauze, was suspended in tepid water in a
conical glass for a minimum of 12 h at room temperature. Kit for Soil (50 ml) according to the manufactur-
The fluid above the 10 ml mark was aspirated and dis- er’s directions, but excluding the initial three steps
carded, whilst the bottom 10 ml, including the sediment, intended to remove humus and litter from soil sam-
was transferred to a 15 ml conical tube and centrifuged ples.
at 1500×g for 5 min. The supernatant was then aspirated (3) MP FastDNA™ Spin Kit for Soil (2 ml volume)
to the 1 ml mark and a 100-μl subsample of remaining
homogenized sediment examined at ×100 magnifica- Two millilitres of the homogenized, lysed faecal
tion for larvae. Larvae were identified and counted. Lar- suspension prepared in protocol 2 was transferred
vae were recorded as hatched GIN larvae, the lungworm
lated from it using the MP FastDNA™ Spin Kit for
to a 2-ml microcentrifuge tube and DNA was iso-
Dictyocaulus sp. or dorsal spine larvae (DSL, protostron-
gylid larvae) based on the morphological appearance of Soil (2 ml) according to the manufacturer’s direc-
the tail (straight tail/s-shaped tail with spine) as well as tions, beginning the protocol with the addition of
larval length. Only the first 10 protostrongylid larvae protein precipitation solution (PPS) according to
in each sample were measured to evaluate whether the the 2-ml protocol.

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Davey et al. Parasites & Vectors (2023) 16:19 Page 5 of 15

Fig. 2 Schematic representation of the methodological comparisons investigated in this study. Faecal samples were subjected to both
parasitological egg and larva counts, and subjected to three types of DNA isolation protocols and the GIN communities characterized using
metabarcoding of the ITS2 region of rDNA

The NC1–NC2 primer set targeting the clade V group amounts, and sequenced in one paired-end 300 bp run
of parasitic GINs [39] was used to amplify the ITS2 on the Illumina MiSeq sequencing platform with v3
region of rDNA from the DNA isolated from the fae- chemistry at the Genomics Core Facility (GCF), Nor-
cal samples, isolation negative controls, and from three wegian University of Science and Technology (NTNU),
PCR-negative controls containing water instead of tem- Trondheim, Norway.
plate DNA. PCR reactions contained 1× KAPA HiFi
HotStart ReadyMix (Roche, Switzerland), 0.2 µM of the Bioinformatics and statistical analyses
forward and reverse primers, and 25 ng template DNA The MiSeq Reporter on the Illumina MiSeq sequencing
with a final volume of 25 µl. PCR conditions consisted platform was used to demultiplex samples and remove
of an initial denaturing step of 5 min at 95 °C, followed adapters. Primer sequences were identified and removed
by 35 cycles of 1 min at 95 °C, 1 min at 54 °C and 1 min from both the 5′ and 3′ ends of forward and reverse reads
at 72 °C with a final elongation step of 5 min at 72 °C. using cutadapt v.1.9.1 [40], allowing up to 15% mismatch
PCR products were quantified using an Agilent 4200 across the length of the primer. Quality filtering, error
TapeStation and cleaned of excess primers and nucleo- correction, and chimera detection were all conducted
tides using magnetic beads (Mag-Bind RxnPure Plus) to using the DADA2 v.1.12 package for R [41]. Reads were
select fragments between 200 and 600 base pairs (bp) quality filtered to remove all sequences with ambiguous
in length. The size-selected amplicons were used as a bases, > 2 expected errors in the forward direction and
template for a second, indexing PCR using the Nex- reverse directions, and length < 50 bp after truncation at
tera XT Index Kit (Illumina, USA) according to the the first instance of a base with a quality score < 15. Error
manufacturer’s instructions. The indexed samples were rates were estimated for forward and reverse sequences
again cleaned as described above, pooled in equimolar and forward and reverse reads were merged with a

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Davey et al. Parasites & Vectors (2023) 16:19 Page 6 of 15

minimum overlap of 30 bp, and amplicon sequence vari- effect and biological sample included as a random effect
ants (ASVs) were inferred for each sample. Chimeric in both cases. A general linear model with a quasibino-
sequence variants were assessed on a per-sample basis, mial distribution was used to assess the relationship
as chimeric events occur at the individual PCR level. If between the proportion of target nematode reads recov-
a sequence variant was flagged as chimeric in more than ered and the total GIN egg and larvae count per gram of
90% of the samples it occurred in, it was removed. Taxon- faeces. The DNA isolation method was included in the
omy was assigned to ASVs using the naïve Bayesian clas- model as a fixed effect.
sifier [42] implemented in DADA2 and a custom version
of the Nematode ITS2 v.1.0.0 database ([24, 43], www.​ Results
nemab​iome.​ca), including additional reference sequences Parasitological assays
of Nematodirus, Nematodirella, Spiculopteragia, and Visual counts of eggs and larvae found moose hosted an
Dictyocaulus species retrieved from GenBank (Accession average of 1.6 (± standard deviation [SD]: 1.09) nema-
No: MW837830-MW837840, KT438069, AY168865). tode taxa per individual, although 7% (2/29) of individu-
Minimum confidence estimates of 80% were required als had no detectable parasites in their faeces (Tables 1;
for a successful assignment against the custom database Additional file 2: Tables S1–S4). Strongylid-type nema-
at any given taxonomic level. Each ASV was also sub- todes were the most frequently detected taxon, occur-
jected to a BLAST search against the NCBI nucleotide ring in all but three individuals. All other taxa (Trichuris,
non-redundant database. Any ASV with the best BLAST Capillaria, Nematodirus, Elaphostrongylus, Varestron-
match to a lineage outside the order Strongylida, or that gylus) were only detected sporadically across individuals
could not be assigned with confidence > 80% at the order (Table 1). Most eggs and larvae could only be identified to
level was designated a non-target amplification and the order level, with the exception of Nematodirus, Tri-
excluded from further analyses. ASVs that could not be churis, and Capillaris eggs, and larvae of Varestrongylus
successfully assigned to the species level were clustered alces and Elaphostrongylus alces (Fig. 3B; Additional File
using VSEARCH [44] at 97% sequence similarity to cre- 1: Figs. S1–S2). Overall, parasitological assays detected
ate species-unit proxies that were subsequently assigned fewer unique taxonomic units than metabarcoding assays
taxonomy at the genus or family level. at the family, genus, and species levels (Fig. 3A). The
All statistical analyses were conducted in the R statisti- average parasitic GIN egg load was 43.9 eggs per gram of
cal environment [45]. To examine variation in the propor- faeces but varied up to two orders of magnitude between
tional abundance of the individual taxa recovered from individuals (SD: 36.8, range: 0.140). The average parasitic
a sample by each method, log twofold changes between GIN larval load was also highly variable, with a mean of
methods were calculated per pairwise sample:method 5.6 larvae per gram of faeces (SD: 16.6, range: 0–82.14).
combination for each taxon recovered. General linear
models were used to assess differences in ASV and spe- Metabarcoding assays
cies recovery between DNA isolation protocols, with the Amplicon sequencing generated a total of 6,053,739
log-transformed sequencing depth included as a fixed high-quality sequences, with a mean of 65,775 per

Table 1 Gastrointestinal nematode parasite loads estimated from moose faecal samples (n = 29)
Taxon No. positive individuals EPGb ± SD (range) LPGc ± SD (range)

Strongyle-type eggs 26 47.5 ± 35.8 (1:140) NA

Nematodirus spp. eggs 3 13.5 ± 10.8 (1:20) NA
Trichuris sp. 8 46.7 ± 44.9 (20–155.1) NA
Capillaria sp. 2 19.7 ± 0.43 (19.4–20) NA
Strongylid-type larvae 3 NA 38.51 ± 37.8 (16.3:82.1)
Protostrongylid-type ­larvaed 4 NA 11.7 ± 10.0 (3.16:25)
3V/4Ea
Twenty-nine moose faecal samples were analysed using the McMaster and Baermann techniques. The total number of individual moose testing positive for a given
group is reported, as well as the eggs or larvae per gram of faeces
NA not applicable
a
V/E: number of individuals infected with Varestrongylus alces/Elaphostrongylus alces
b
EPG: eggs per gram
c
LPG: larvae per gram
d
The presence of Elaphostrongylus alces and Varestrongylus alces larvae are reported, but larval counts are combined as protostrongylid-type larvae due to the
presence of ambiguous individuals

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Davey et al. Parasites & Vectors (2023) 16:19 Page 7 of 15

Fig. 3 Taxonomic resolution of parasitological and molecular methods. Comparisons of the (a) total taxonomic diversity recovered by the different
parasitological and molecular methods at the family, genus, and species levels and (b) the taxonomic resolution achieved for the occurrences
detected by each method. In (a) the taxonomic units on the y axis represent the detected number of families, genera, and species for each of the
three groupings from left to right, respectively. Morph morphological, MP2 MP soil kit 2 ml, MP50 MP soil kit 50 ml, QS Qiagen stool kit

sample (range: 1796–1,234,331), of which 5,011,920 (Additional file 1: Fig. S3, Additional file 2: Table S5). The
were assigned to the phylum Nematoda (mean: 57,608 most frequently occurring species belonged to Osterta-
sequences per sample, range: 16–276,841). Diverse GIN gia, Trichostrongylus, and Nematodirus (Additional file 1:
species assemblages were recovered from all moose faecal Figs. S1–S2). Compared with traditional parasitologi-
samples, including 10 genera from six strongylid families cal investigations of the faecal samples, metabarcoding
(Chabertia, Cooperia, Elaphostrongylus, Haemonchus, recovered a greater diversity of parasitic GIN families
Nematodirus, Ostertagia, Spiculopteragia, Teladorsagia, and genera (Fig. 3a) and provided higher taxonomic reso-
Trichostrongylus, and Varestrongylus) (Fig. 3; Additional lution for more of the occurrences detected (Fig. 3b).
file 1: Figs. S1–S2). Nematode sequences were not recov- The three methods of DNA isolation tested recovered
ered from the isolation and PCR-negative control sam- highly similar GIN communities from each individual
ples. An average of 18.5 ASVs (sd: 6.7, range: 1–37) were with regards to composition (Additional file 1: Figs. S1–
recovered per moose individual, representing an average S2). The proportional abundances of individual taxa were
of 6.7 species (sd: 3.17, range: 1–13). Although the num- consistent across methods for highly abundant taxa like
ber of ASVs recovered was significantly correlated with Ostertagia sp. 1 and Trichostrongylus sp. 1, and more
sequencing depth, the number of species recovered was variable among low abundance taxa like Nematodirus sp.
not, indicating that the sequencing depth was sufficient 1 and Trichostrongylus axei (Fig. 4). Proportional abun-
to recover all of the GIN species present in the samples dances were more consistent between the MP soil kit

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Davey et al. Parasites & Vectors (2023) 16:19 Page 8 of 15

Fig. 4 Differences in proportional abundance estimates across taxa. Each panel represents log fold change comparisons calculated using the
method indicated in the panel label as a reference value and represented by the dashed vertical line. The number of faecal samples for which the
log fold changes were calculated is indicated in parentheses after the taxon name. Samples with non-detection of a taxon by one or more methods
were excluded from the analysis. MP2 MP soil kit 2 ml, MP50 MP soil kit 50 ml, QS Qiagen stool kit

extractions than between either the MP soil kit or the between the proportional abundances of species recov-
QIAamp stool kit (Fig. 4). Among taxa with > 10 com- ered from the bulk isolation and the 2 ml aliquot for any
parisons, we did not observe consistent, systematic over given sample (df = 1536, t = 762.564, P < 2e−16, Fig. 6),
or under estimation by any of the methods (Fig. 4). The highlighting the consistency of results when using either
Qiagen stool kit-based protocol (220 mg starting mate- of the MP soil kit protocols. Although ASV recovery was
rial) yielded significantly fewer ASVs (degrees of free- significantly correlated with sequencing depth, species
dom [df] = 86, t = −6.228, P < 0.001) and species (df = 86, recovery was not, and there was no significant interac-
t = −10.026, P < 0.001) than the MP soil kit-based proto- tion between isolation protocol and sequencing depth
cols (2–4 g starting material, Fig. 5). There were no sig- (Additional file 1: Fig. S2, Additional file 2: Table S5).
nificant differences in ASV and species recovery between The GIN communities recovered by metabarcoding
the 2 ml and 50 ml MP soil kit protocols (ASVs: df = 86, from the faeces were largely consistent with those recov-
t = 0.468, P = 0.641; species: df = 86, t = 0.036, P = 0.971) ered using traditional parasitological investigations, albeit
(Fig. 5). In addition, there was a strong correlation with higher taxonomic resolution (Fig. 3; Additional File

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Davey et al. Parasites & Vectors (2023) 16:19 Page 9 of 15

Fig. 5 ASV and species recovery using different DNA isolation methods. Comparison of ASV (a) and species (b) recovery using different DNA
isolation methods. Values represent the proportion of the total ASVs or species recovered per individual. MP2: MP Biomedicals FastDNA™ Spin Kit for
Soil (2 ml), MP50: MP Biomedicals FastDNA™ Spin Kit for Soil (50 ml), QS: Qiagen QIAamp Fast DNA Stool Mini Kit

1: Fig. S1). However, the results were not entirely con- parasite loads. Such monitoring is in part hampered by
gruent, as metabarcoding detected E. alces and V. alces lack of time- and cost- effective assay methodologies with
in only two of the four samples where they were recov- high sensitivity and good taxonomic resolution. DNA-
ered by parasitological investigations (Additional file 1: based methods are increasingly used for the characteriza-
Fig. S1). Furthermore, metabarcoding recovered GIN tion of biodiversity in a variety of contexts [46], and here
species from two samples where no eggs or larvae were we explore the suitability of DNA metabarcoding for par-
observed during the parasitological investigations (Addi- asite monitoring and attempt to optimize the DNA isola-
tional file 1: Fig. S1). The proportion of metabarcoding tion step of this method.
reads that could be assigned to target nematode taxa var-
ied greatly between samples (range: 0.08–99.8%) and was Effects of DNA isolation method
significantly correlated with the individual’s total egg and Both ASV and species recovery was higher when DNA
larval load (df = 86, t = 2.445, P = 0.026) (Fig. 7). was isolated with the MP soil kit as compared with the
Qiagen stool kit, indicating that GIN assay sensitiv-
Discussion ity and resolution can be substantially impacted by the
Although wild ungulate populations are heavily moni- DNA isolation method. The importance of DNA isola-
tored throughout Europe, we understand little of how tion in the detection of parasitic nematode species has
parasites affect population dynamics, and there is no sys- been highlighted during the development of diagnostic
tematic, long-term monitoring of parasite diversity and quantitative PCR (qPCR) tests for commercially relevant

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Davey et al. Parasites & Vectors (2023) 16:19 Page 10 of 15

Fig. 6 Comparison of bulk and sub-sampled DNA isolations. Correlation between the proportional abundance of GIN species when isolated from
bulk faecal samples (MP Soil 50 ml) or from an aliquot of the same homogenized faecal sample (MP Soil 2 ml) (P < 2e−16, R2 = 0.997, t = 762.564). A
1:1 relationship is indicated by the dotted line, while the fitted correlation is indicated by a solid blue line

Fig. 7 Parasite load in 29 moose estimated by molecular and parasitological assays of faecal samples. The parasite load of moose was estimated
from metabarcoding data as the proportion of nematode to non-target sequences recovered and then compared with parasitological egg and
larvae counts (df = 86, t = 2.445, P = 0.026). Results are shown for each of the three different DNA extraction methods tested

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Davey et al. Parasites & Vectors (2023) 16:19 Page 11 of 15

species [34, 47, 48]. The eggs of GIN species are known morphological assays in two of the samples. These appar-
to be recalcitrant and difficult to break open [47], which ent detection failures by the metabarcoding method
can prevent effective DNA isolation. The MP soil kit could be a result of primer-related bias, although this
includes a mechanical grinding step intended to physi- seems unlikely given that both species were successfully
cally disrupt cells, while the Qiagen stool kit does not detected in other samples. Instead, the differences in the
and instead depends only on chemical lysis to free cellu- detection of E. alces and V. alces between the methods
lar DNA within the sample. It would appear the grind- may be attributed to stochastic differences in the faecal
ing step in the MP soil kit successfully ruptured more subsamples subjected to parasitological and metabarcod-
nematode eggs in the samples and a physical homogeni- ing analyses, as different volumes of faecal matter were
zation step is important for optimizing the sensitivity of analysed, and eggs and larvae can be unevenly distrib-
metabarcoding assays for GIN communities. However, it uted between faecal pellets. These stochastic differences
must also be noted that the total starting faecal biomass in the occurrence of eggs and larvae in the faecal mate-
used in the Qiagen stool kit was approximately 220 mg, rial may also be driving the increased GIN detection rate
while the MP soil kit used an order of magnitude more observed with the metabarcoding approach. Alterna-
starting biomass (2–4 g). The increased starting material tively, the increased detection rate could be due to con-
effectively increases the sampling effort, which, as would tamination or false positives using the metabarcoding
be expected, yields greater sensitivity in the assays. The method, although we argue this is unlikely, as there was
lack of significant differences in ASV and species recov- no systematic contamination observed in the sequenced
ery and strong correlation in species proportional abun- PCR and extraction negative controls, and multiple spe-
dances between the isolation from a 2-ml aliquot of the cies were detected in each of the samples. Given that no
homogenized faecal material and the entire biomass with GIN taxa were detected solely by parasitological methods
the MP soil kit suggests that the time- and cost-saving and not concurrently by metabarcoding, we instead argue
advantages of a 2 ml-based extraction protocol can be that metabarcoding of GIN DNA isolated from frozen
retained without sacrificing metabarcoding assay sensi- faecal samples has increased sensitivity when compared
tivity, as long as there is a preliminary homogenization with egg and larval counts from the same samples when
step with larger amounts of faecal biomass. Use of a 2 ml- they are fresh, most likely in cases with low egg and larval
based extraction kit allows simultaneous treatment of abundance. Other PCR-based methods have been dem-
24–96 samples at all steps of the isolation protocol, while onstrated to have increased sensitivity over traditional
50-ml bulk extractions are restricted to simultaneous microscopy-based methods for GIN detection [50–53],
handling of eight samples in some steps of the isolation but to our knowledge, this has not been previously dem-
protocol. In addition, there was a 60% cost saving per onstrated for DNA metabarcoding. We hypothesize that
sample in using the described sub-sampling method with this increased sensitivity can be attributed to the meta-
a 2-ml kit as opposed to doing bulk isolations. Simplifi- barcoding method also detecting extracellular DNA
cation and streamlining of laboratory protocols for DNA derived from adult worms [54] in the gastrointestinal
extraction and metabarcoding contribute to reducing tract that may be shedding few or no eggs at the time of
costs and increasing time efficiency, further increasing sampling. While other species-specific PCR-based meth-
the utility of non-invasive metabarcoding for large scale ods may have similar detection sensitivity with better
monitoring of GIN communities in wild populations. cost-effectivity, DNA metabarcoding-based approaches
do not require a priori knowledge of the GIN community
Metabarcoding for characterizing GIN communities in wild and have the potential to detect unexpected and/or atypi-
ungulates cal GIN infections.
DNA-based methods are increasingly used for the char- The metabarcoding approach consistently recovered
acterization of biodiversity in a variety of contexts [46], more GIN genera and families, providing improved taxo-
and in general, have proven to be both more sensitive nomic resolution as compared with traditional morpho-
and provide better taxonomic resolution for the taxa logical assays. This is primarily driven by the capacity for
detected [49]. In this paper, we demonstrate that DNA metabarcoding methods to distinguish between strongyle-
metabarcoding is a highly valuable approach for the char- type eggs that cannot be identified to species based on
acterization of GIN parasites in wild ungulates such as morphology [17]. Only three GIN genera were detected
moose. Using a molecular-based approach, we detected with traditional methods as compared with 10 using DNA
GIN species in all samples investigated, while egg and metabarcoding. Such improvement in taxonomic resolu-
larval counts detected GIN in 93% of samples. Never- tion allows for better estimation of the diversity and the
theless, E. alces and V. alces were detected exclusively by range of species infecting a given individual. Although the

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Davey et al. Parasites & Vectors (2023) 16:19 Page 12 of 15

metabarcoding approach improved taxonomic resolution Host specificity of parasites and spillover among host
over the morphological assays, it must be noted that the species
primer combination used (NC1–NC2) is limited to clade V Wild ungulates can act as infection reservoirs for domes-
GIN, and as such will not detect other parasite groups that tic hosts [30, 59]. With evidence that parasite loads in
are typically included in Baermann and McMaster assays wild ungulate populations are affected by land use (spe-
(e.g. Moniezia, Eimeria, Trichuris, Capillaria). Moreo- cifically livestock rearing) and climate change [60, 61],
ver, several of the GIN sequence variants recovered could a better understanding of the dynamics of host-parasite
only be identified with high confidence to the genus, fam- interactions and the ensuing effects on host population
ily, or order level. Of the 10 most abundant ASVs identi- dynamics is urgent. A major benefit of the metabarcod-
fied to these higher taxonomic levels, six had 98% identity ing approach is increased taxonomic resolution. Such
or less to a reference sequence in the database, suggesting insight is required to understand the host specificity of
that there is a lack of reference sequences for GIN parasites the parasite community and to predict the parasite spillo-
of wild ungulates. For example, Spiculopteragia alcis and ver in host communities of wild and domestic ungulates.
Ostertagia kolchida are two known GIN parasites of moose A number of the GIN species detected in the moose fae-
that were not included in the identification database. Fur- cal samples are commonly known from domestic animals
ther reference database development will be needed to (e.g. Chabertia ovina, Cooperia oncophora, and Tela-
support the implementation of DNA metabarcoding in dorsagia circumcincta) where they cause host morbid-
large-scale monitoring of GIN infections in these wild ity [17]. This is consistent with earlier observations that
populations. GIN taxa in co-occurring wild cervids and domestic ani-
Finally, measures of parasite load are of particular mals frequently overlap [62–65] and further supports the
interest for monitoring GIN infections, as they correlate theory that wild ungulate populations can act as reser-
with host body condition, fecundity, and survival in pop- voirs for GIN parasites of domestic animals with recip-
ulations of wild ungulates [55, 56]. Traditional egg and rocal infections occurring between species [59, 62]. The
larval count methods from faecal samples provide a non- high taxonomic resolution of DNA metabarcoding-based
invasive method for estimating parasite load, but involve GIN monitoring in wild ungulate populations has the
laborious isolation procedures that make the method potential to provide not only valuable data for conserva-
suboptimal for large-scale monitoring programs where tion and management decisions, but also provide insight
high throughput of many samples is required. In the cur- into the parasite spillover between co-occurring wild and
rent study, we observe a significant relationship between domestic species and their impact on each other’s health.
the proportion of nematode sequences recovered from
the samples and the parasite load as determined by egg
Conclusions
and larval counting. On the individual species level, it
DNA metabarcoding is a promising technique for the
is well documented that DNA metabarcoding sequence
non-invasive, large-scale monitoring of parasitic GINs in
abundance is at best, semi-quantitative [57] in rela-
wild ungulate populations. Metabarcoding assays provide
tion to the number of individuals or biomass, although
increased sensitivity and taxonomic resolution compared
the method provides robust estimates of proportional
with traditional egg and larva isolation and identification
abundances within GIN communities in a single host
methods. While not strictly a quantitative method, our
[58]. The correlation between total parasite load and the
results indicate that with further research, it may none-
ratio of nematode sequences to non-target sequences
theless be possible to create a management- and conser-
has not previously been reported. While DNA metabar-
vation-relevant index for host parasite load. The DNA
coding may be unreliable for estimating individual spe-
isolation method significantly impacted species recovery,
cies abundances, this result suggests it may nevertheless
and for monitoring of GIN species from faecal samples,
provide a very coarse estimate of the total parasite load.
we recommend the use of a DNA isolation protocol that
However, this result must be interpreted with extreme
(1) includes a mechanical cell disruption step and (2)
caution given the small number of samples (n = 29), and
maximizes starting material volume.
the small number of samples with high parasite load
(> 100 eggs and larvae per gram: three samples). Further
research is required to determine whether this relation- Abbreviations
ship can provide a meaningful index for parasite loads at DNA Deoxyribonucleic acid
GIN Gastrointestinal nematode
levels affecting host condition, which would be relevant ITS Internal transcribed spacer
for management and conservation in wild populations. PCR Polymerase chain reaction

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Davey et al. Parasites & Vectors (2023) 16:19 Page 13 of 15

Consent for publication

Supplementary Information Not applicable.
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s13071-​022-​05644-6. Competing interests
The authors declare no competing interests.
Additional file 1: Figure S1. Comparison of 29 moose individuals’
parasitic nematode communities detected using parasitological and Author details
1
metabarcoding assays of faecal samples. Parasitological surveys included Norwegian Institute for Nature Research (NINA), Trondheim, Norway. 2 Univer-
counting of eggs and larvae. Metabarcoding of faeces samples was con- sity of Oslo, Oslo, Norway. 3 Norwegian University of Life Sciences, Ås, Norway.
4
ducted using three different DNA isolation protocols. Point type indicates Norwegian Veterinary Institute, Tromsø, Norway.
the lowest taxonomic level a method successfully identified the taxon at.
Figure S2. Taxonomic summary of the gastrointestinal nematode com- Received: 6 July 2022 Accepted: 28 December 2022
munity recovered by DNA metabarcoding. Faecal samples of 29 moose
were analysed. Sequences that could not be identified to the species level
are grouped at the lowest level of taxonomy possible, and the number of
operational taxonomic units (OTUs) recovered in the group is indicated
in parentheses following the taxon name. Figure S3. The relationship References
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