The bacterial community associated with the sheep gastrointestinal nematode parasite Haemonchus contortus
The bacterial community associated with the sheep gastrointestinal nematode parasite Haemonchus contortus
Gajenathirin Sinnathamby 0 1 2
Gemma Henderson 0 2
Saleh Umair 0 2
Peter Janssen 0 2
Ross Bland 0 2
Heather Simpson 0 1 2
0 Wool New Zealand (96MU 25/1.1 Development of alternative methods of parasite control) H.V. Simpson; Massey University Research Fund, H.V. Simpson; C. Alma Baker Trust, H.V. Simpson; E. & C. Thoms Bequest , H.V. Simpson
1 Institute of Veterinary, Animal and Biomedical Sciences, Massey University , Palmerston North , New Zealand , 2 AgResearch Ltd , Palmerston North , New Zealand
2 Editor: Horacio Bach, University of British Columbia , CANADA
Culture-independent methods were used to study the microbiota of adult worms, third-stage larvae and eggs, both in faeces and laid in vitro, of Haemonchus contortus, a nematode parasite of the abomasa of ruminants which is a major cause of production losses and ill-health. Bacteria were identified in eggs, the female reproductive tract and the gut of adult and thirdstage larvae (L3). PCR amplification of 16S rRNA sequences, denaturing gradient gel electrophoresis (DGGE) and clone libraries were used to compare the composition of the microbial communities of the different life-cycle stages of the parasites, as well as parasites and their natural environments. The microbiomes of adult worms and L3 were different from those in the abomasum or faeces respectively. The H. contortus microbiota was mainly comprised of members of the phyla Proteobacteria, Firmicutes and Bacteroidetes. Bacteria were localised in the gut, inside eggs and within the uterus of adult female worms using the universal FISH Eub338 probe, which targets most bacteria, and were also seen in these tissues by light and transmission electron microscopy. Streptococcus/Lactococcus sp. were identified within the distal uterus with the probe Strc493. Sequences from the genera Weissella and Leuconostoc were found in all life-cycle stages, except eggs collected from faeces, in which most sequences belonged to Clostridium sp. Bacteria affiliated with Weissella/Leuconostoc were identified in both PCR-DGGE short sequences and clone libraries of nearly full length 16S rRNA bacterial sequences in all life-cycle stages and subsequently visualised in eggs by fluorescent in situ hybridisation (FISH) with group-specific probes. This strongly suggests they are vertically transmitted endosymbionts. As this study was carried out on a parasite strain which has been maintained in the laboratory, other field isolates will need to be examined to establish whether these bacteria are more widely dispersed and have potential as targets to control H. contortus infections.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Competing interests: The commercial affiliation
with AgResearch Ltd does not alter our adherence
Bacteria have developed symbiotic relationships with multicellular host organisms; these range
from fatal pathogenic infections and parasitism to commensalism (to the advantage of the
symbiont) or mutualism (which benefits both partners) [1±3]. The boundaries between these
associations are not always distinct and may change with the physiology of the bacteria or
host. Symbionts may be located externally or internally (ectosymbionts on the host surface or
endosymbionts living within tissues), be obligate primary endosymbionts or facultative
secondary symbionts and be transmitted vertically or acquired anew by each generation of host.
The most extensively researched symbionts of nematodes are those in filarial and
plant-parasitic nematodes and the entomopathogenic nematodes (EPNs), because of their medical and
agricultural significance, although there are many other unusual bacterial-nematode
symbioses of biological interest.
Most symbionts of nematodes are likely to be associated with either the gut or external
surfaces and to be commensals or mutualists contributing principally to host metabolism. There
is an interesting nutritional symbiosis between bacteria and the gutless stilbonematid marine
worms, which obtain nutrition from external lawns of densely packed ectosymbiotic bacteria,
in many cases a species-specific monoculture [4±7]. The focus of many studies of the
microbiome of Caenorhabditis elegans has been differentiating those species which constitute a food
source or are mutualists from species which are potential pathogens of either the nematode or
other organisms for which the nematode acts as a vector of the bacteria [8±12]. The
microbiomes of the free-living nematodes C. elegans, Caenorhabditis remanei [
] and five grassland soil species [
] were different from those in their environments,
less diverse and dominated by Proteobacteria. As in other multicellular hosts, the microbiome
was not the same in all individuals and dependent on diet, genetics [
] and the presence of
Nematodes symbionts can be exploited to control some important agricultural insect pests.
The EPNs of the genera Steinernematidae and Heterorhabditidae use their γ-proteobacterial
Xenorhabdus sp. symbionts, which colonise the intestine of infective third-stage larvae (L3) to
kill host insects to continue the nematode reproductive cycle [
]. After entry of the L3 into
the haemolymph of an insect, the bacteria are released and induce a fatal septicaemia. The
EPNs reproduce for 2±3 generations within the insect, after which L3 take in symbionts and
emerge into the soil to infect another insect and continue the life-cycle [2,17±19]. In contrast,
the nematodes themselves may be the pests which may be able to be controlled via their
symbionts. This has been proposed for the Verromicrobial endosymbionts of plant-parasitic
nematodes, which are destructive cyst-forming pests of soybeans, potatoes and peas [
Candidatus endosymbionts are specific for each species of nematode and are vertically
transmitted by the females [
Many, but not all, filarial nematode parasites of humans and animals carry the
maternallytransmitted essential endosymbiont Wolbachia pipientis [23±25], which is susceptible to
antibiotic therapy. In these nematodes, the bacteria are necessary for normal worm
embryogenesis, development and adult survival [26±30] and contribute to metabolism [
]. Wolbachia are
transmitted vertically in eggs, thence to the gut of L3, developing lateral cords and adult female
ovary, but not the male testis [
]. Other clades of W. pipientis are essential in many insects,
but there are no confirmed reports of Wolbachia in other nematodes, apart from the
plant-parasitic nematode Radopholus similis [
Gastrointestinal nematodes of farmed livestock cause health and welfare issues and
enormous economic losses in pasture-based grazing systems [34±36]. H. contortus, a blood-sucking
gastric parasite of ruminants, may cause a life-threatening disease in a severe infection [
2 / 25
Anthelmintics are currently the method of choice to control gastrointestinal parasites ,
however, the rapid spread of drench resistance [
] is driving the search for alternatives to
chemical treatment, such as biological control via essential bacterial symbionts. H. contortus
adult female worms lay eggs, which pass out in the faeces and hatch into first-stage larvae (L1)
under favourable warm and moist conditions. L1 develop and moult on the pasture into L2,
both stages feeding on the faecal bacteria, then L2 moult into L3, the infective and non-feeding
stage, which retains the L2 cuticle as a protective sheath. After L3 are consumed by an
appropriate host, they exsheathe (shed the L2 cuticle) in the rumen, move down to the abomasum
and enter the lumen of gastric glands, where they develop and after 2±4 days emerge either as
L4 or immature adult worms. The very fecund female H. contortus lay 5,000±10,000 eggs per
day, beginning after 12±15 days, although this is variable [
The aim of the present study was to investigate the bacteria associated with H. contortus
using DNA fingerprinting. First, the communities in cultured L3, adult worms and eggs (either
recovered from eggs or from adult worms) were compared with those in their environments
by PCR amplification of 16S rRNA sequences and separation of products by denaturing gel
electrophoresis (DGGE). Detailed phylogenetic evolutionary relationships were then
determined to identify the bacteria present in the different life-cycle stages. Bacteria were located
within the parasites by light microscopy (LM), transmission electron microscopy (TEM) and
fluorescence in situ hybridisation (FISH).
The bacteria associated with H. contortus were identified using PCR amplification of 16S
rRNA sequences from eggs, L3 and adult worms.
Bacterial communities are not identical in parasites and their environment
PCR-DGGE analysis of 16S rRNA sequences showed that the bacterial communities in male
and female adult worms generated very similar DGGE band patterns (S1 Fig). Poor separation
of sequences from abomasal contents did not allow comparison of the communities in adult
worms and contents, however, abomasal bacteria appeared to adhere to the cuticle, as band
patterns from worms set in agar blocks and allowed to migrate out and subsequently cleaned
by washing with 4% sodium hypochlorite differed from bands from manually collected worms
(S2 Fig). Even identical band patterns for two samples are indicative only of similar bacterial
communities, as 16S rRNA sequences which are different in G+C content and/or belong to
different bacterial species can be isolated in one band and, conversely, sequences belonging
to one species can be present in more than one band, due to the presence of multiple rrn
L3 and eggs were subsequently also cleaned with sodium hypochlorite in an attempt to
remove adherent bacteria. The effects of the recovery and cleaning processes on the number of
DGGE bands associated with L3 are shown in S3 Fig. The L3 communities did not simply
reflect those in the faeces in which they were cultivated and the band pattern was altered by a
combination of exsheathing and washing (S3 Fig). Some bacteria present in the adult worm
gut may have persisted throughout the parasite life-cycle from development of L1 to L2, which
feed on faecal bacteria, on to non-feeding L3 and parasitic adult worms.
Eggs would not be expected to have many bacteria associated with them if the eggshells had
been effectively cleaned. This was clearly not the case after sodium hypochlorite washing,
probably because bacteria or DNA were firmly attached to the carbohydrate coat present on
the surface of the eggs [
]. Sheep faecal bacteria appeared to contaminate eggs collected from
the faeces, as there were intense bands in DGGE gels of sequences from eggs extracted from
3 / 25
Fig 1. A representative DGGE gel of PCR amplified products generated from DNA of H. contortus adult worms (HA), L3 (HL) and eggs (HEM: in vitro laid eggs
and HEF: eggs collected from faeces) used for sequencing the bands of interest (left). The phylogenetic affiliations of sequences obtained from the individual DGGE
bands are shown in the table (right).
faeces that were not present in eggs laid in vitro (Fig 1). These bands were subsequently found
to contain sequences of Clostridium sp., which are typical bacteria associated with faeces [43±
46]. The band patterns from in vitro laid eggs and the adult females that laid them were similar
(Fig 1, lanes HA1 and HE2), suggesting these eggs acquired bacteria from gut contents of the
mother during egg-laying.
Preliminary identification of bacteria in adult worms, L3 and eggs
Adult worms, L3 and eggs generally produced similar DGGE bands consisting of 7 major
bands common to all three stages and an eighth not present in eggs (Fig 1). These bands were
excised and ~190bp sequences amplified with the primer set 338f and 518r, which is widely
used for analysing bacterial sequences by the DGGE fingerprinting method [
allowed preliminary identification of bacterial 16S RNA, although the ~190bp sequences
generated were too short for detailed phylogenetic analysis and identification of species. A total of
44 and 21 bacterial sequences were obtained from these 7 bands in 3 separate gels of adult
worms and L3 respectively and 23 sequences from bands 1 and 2 from eggs. An eighth band,
4 / 25
Total lactic acid bacteria (LAB)
Un-cultured rumen bacterium
Flavobacteriaceae or Chryseobacterium
not present in eggs, yielded 45 bacterial sequences from adult worms and 18 from L3; these
were mainly Escherichia (39) and Rhizobia sp. (15). The phylogenetic affiliations of the
sequences are shown in Fig 1 and summarised in Table 1.
About half of the ~190bp sequences were matched to the phylum Firmicutes and the rest
were consistent with sheep gut or ubiquitous environmental bacteria. Lactic acid bacterial and
Proteobacterial sequences dominated those identified from adult worms, L3 and eggs. Alpha-,
beta- and gamma-proteobacteria have been identified in the microbiomes of other nematodes
] and are also ubiquitously present in aquatic environments  and therefore
may be either commensals or contaminants acquired from the parasite environment. These
bacteria are unlikely to have originated from laboratory reagents, which were screened for
Phylogenetic analysis of bacterial sequences from adult worms, L3 and eggs
More detailed phylogenetic evolutionary relationships were determined for longer bacterial
16S rRNA sequences amplified from DNA extracted from adult worms, L3 and eggs. Clone
libraries were constructed using the universal bacterial primer set (27f and 1492r) to amplify
nearly complete length (~1400bp) 16S rRNA sequences and for ~1000bp sequences amplified
by 27f and 1040firmR Firmicutes-specific primers (Table 2).
5 / 25
GCT GGA TTT CTT TCC CAA
Lane et al. (1991)
Muyzer et al. (1993)
Modified from Lane et al.
From Lane et al. (1991) Meier et al. (1999) Amman et al. (1990) Wallner et al. (1993)
Harmsen et al. (2002) Collins et al. (1993) Jang et al. (2002) Franks et al. (1998)
Manz et al. (1992)
Ashelford et al. (2002)
Piccini et al. (2006)
These sequences have been deposited in the GenBank database: Weissella sp, MF148162,
MF148163, MF148164, MF148165, MF148166, MF148167, MF148168, MF148169, MF148170,
MF148171, MF148172, MF148173; Leuconostoc sp. MF148174, MF148175, MF148176,
MF148177, MF148178, MF148179; Staphylococcus sp. MF148180, MF148181, MF148182,
MF148183; Lactococcus sp. MF148184, MF148185, MF148186, MF148187; MF148195,
MF148196, MF148197, MF148198, MF148199, MF148200 Streptococcus sp. MF148188,
MF148189, MF148190, MF148191, MF148192, MF148193, MF148194; Lactococcus sp. rumen
bacterium clone, MF148201, MF148202, MF148203, MF148204; uncultured bacterial clone,
MF148205, MF148206, MF148207, MF148208, MF148209, MF148210, MF148211, MF148212,
MF148213, MF148214, MF148215, MF148216, MF148217, MF148218, MF148219, MF148220,
MF148221, MF148222, MF148223, MF148224, MF148225, MF148226, MF148227. The initial
taxonomic identification of ~1400bp bacterial 16S rRNA sequences (amplified by 27f and
1492r) is shown in Table 3.
Sequences belonging to the phylum Bacteroidetes were identified only in adult worms and
the majority were identified as uncultured rumen bacteria. Only a single Spiroplasma sequence
was identified, in eggs collected from faeces.
Amongst the phylum Proteobacteria, the dominant genera were Mesorhizobium,
Stenotrophomonas, Pseudomonas, Comamonas and Rhizobium and no sequence was common to adult
worms, L3 and eggs. Mesorhizobium were found only in adult worms and L3, the majority of
the Stenotrophomonas in eggs laid in vitro and Pseudomonas in eggs collected from faeces. As
Proteobacteria are ubiquitous in the environment [
], they may be acquired on to the surface
of parasites from their environment, whereas Mesorhizobium, Stenotrophomonas and
Pseudomonas may be gut residents. Sequences of the phylum Proteobacteria were not subjected to
detailed analysis because of the high sequence variation identified by the universal bacterial
16S rRNA primer pair. Further phylogenetic analysis was carried out only for phylum
6 / 25
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Fig 2. Phylogenetic tree (based upon the Maximum Likelihood method) of phylum Firmicutes 16S rRNA gene sequences from H. contortus using the
universal primer set 27f and 1492r and reference 16S rRNA gene sequences. The 13 groups were identified from closest type strain sequences. Sequences which
have been compressed are represented as triangles. Bootstrap values are shown at each node (percent of 500 replicates). The scale bar indicates 0.02 nucleotide
substitutions per nucleotide position.
Firmicutes sequences, which were the only ones identified in all three H. contortus life-cycle
stages (adult worms, L3 and eggs).
Phylogenetic analysis of phylum Firmicutes ~1400bp and ~1100bp 16S
Phylum Firmicutes sequences had the same phylogenetic affiliations and the phylogenetic
trees had similar topology and robustness for the Maximum Likelihood (ML), Neighbour
Joining (NJ) and Maximum Parsimony methods. Fig 2 shows the tree generated by the ML method
for the ~ 1400bp sequences, and detailed trees for ~ 1400bp and ~ 1000bp sequences are
shown in S4±S6 Figs. Sequences grouped into 13 clusters in the phylogenetic analysis of ~
1400bp sequences (using the universal primer set 27f and 1492r) (Table 4) and 4 clusters in the
analysis of ~1000bp sequences (using the Firmicutes-specific set 27f and 1040firmR) (Table 5).
Detailed phylogenetic analysis of the relatively small number of sequences of the order
Clostridiales suggested that many may belong to yet to be described genera or species, as these
sequences had very low sequence similarity with type strain sequences and their groups also
had low bootstrap values. Most of the clone sequences within the CCT cluster grouped
separately from the known type strain Clostridium thermocellum and the cultured species
8 / 25
Closest cultured and type strain (GenBank accession)
Lactococcus plantarum DSM
Lactococcus raffinolactis DSM
subsp. coli NCDO964T
Lactococcus lactis subsp. lactis
NCDO_604T (DSM 20481)
Lactococcus fujiensis strain:
Lactococcus lactis subsp. lactis
NCDO_604T (DSM 20481)
Leuconostoc citreum ATCC
Leuconostoc citreum strain B/
Weissella confusa JCM1093T
DSM 1237 T (L09173)
Clostridium sufflavum strain
strain: JCM 17853T
Veillonella parvula ATCC
Eubacterium tenue DSM
Clostridium sordellii strain
Robinsoniella peoriensis strain
9 / 25
Genus Closest cultured and type strain (GenBank accession)
Leuconostoc Leuconostoc citreum strain
Weissella Weissella confusa gene JCM1093T
Lactobacillus Lactobacillus ingluviei strain KR3T
Lactobacillus fermentum strain KN02
Streptococcus Streptococcus infantarius strain
Streptococcus equinus strain: BP1-7
Clostridium sufflavum. These clone sequences had a low bootstrap value (55) with their type
strain and cultured strain sequences in the detailed phylogenetic tree (S5 Fig). Sequences
belonging to the genera Clostridium and Lactococcus were present in adult worms and faecal
eggs, but no sequences belonging to Clostridium sp. were identified in L3, probably due to
their removal by exsheathing. Clostridium sp. are mammalian gut residents [43±46], suggesting
sheep gut bacteria are present on the surface of the parasites or in the worm gut.
Sequences (~1400bp) belonging to the lactic acid bacteria Lactococcus, Streptococcus,
Leuconostoc and Weissella were identified in adult worms, L3 and eggs, similar to the findings from
the short sequences retrieved from DGGE bands (Table 2). These sequences had over 99%
sequence similarity with those from type strains identified as Weissella confusa, Leuconostoc
citreum, Lactococcus plantarum, Lactococcus raffinolactis, Lactococcus lactis, and Streptococcus
infantarius. Although sequences (~1400bp) from the genera Weissella and Leuconostoc were
not found in eggs collected from faeces, partial 16S rRNA bacterial sequences (~1000bp) using
the phylum Firmicutes-specific primer confirmed that Weissella and Leuconostoc sequences
can be obtained from all life-cycle stages, including eggs collected from faeces. Notably, those
sequences amplified from in vitro laid eggs using the phylum Fimicutes-specific primer were
all Leuconostoc or Weissella. The failure to find lactic acid bacterial sequences from faecal eggs
is not surprising, as most belonged to Clostridium sp. and the most abundant species would
dominate the clones used for sequencing. The reason for the large number of sequences of
Clostridium sp. could be either primer bias or the presence of large numbers faecal bacteria
adhering to the eggs.
Location of bacteria in H. contortus
Symbionts were identified by a combination of light microscopy (LM), transmission electron
microscopy (TEM) and fluorescence in situ hybridisation (FISH). Bacteria were apparent in
three locations in histological sections of adult worms: in the gut lumen of male and female
worms, and in females also within the uterus and eggs. The group-, class- and species-specific
FISH probes (Table 2) used to identify the morphology and locations of bacteria in H.
contortus were selected based on the phylogenetic analyses. The bacteria visible in a female worm by
LM and TEM are summarised in Fig 3.
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Fig 3. Location and morphology of bacteria in the gut (top), the uterus (centre) and eggs (bottom) of an adult
female H. contortus. Left: LM image of a whole unstained worm showing the sites of collection of tissues; middle: LM
images of H & E stained tissues; right: TEM images. Bacteria are shown at successively higher magnifications. Those in
the eggs were not seen in LM sections. In H & E stained sections, bacteria are shown in boxes and in TEM sections are
indicated by arrows.
PLOS ONE | https://doi.org/10.1371/journal.pone.0192164
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Bacteria could not be seen on the surface of the worms, using either LM or FISH. Live
helminths actively remove cells [
], chemicals , antibodies [55±57] and lectins [
attached to their surface by continuously replacing their cuticles. Therefore, it is unlikely that
there are permanent bacterial communities on the surface of H. contortus, whereas some free
living marine nematodes carry large permanent populations of sulphur-oxidising
ectosymbionts which provide nutrients to their host [
Gut bacteria. Bacteria in the gut lumen of female worms in sections stained with H & E
were a mixed population of gram-positive and gram-negative bacteria. In TEM sections, there
were diverse morphotypes in the gut in a single worm and these differed amongst worms,
although many worm sections contained no visible bacteria. Bacteria were seen in the gut
lumen and none was attached to microvilli or within any specialised structures. Gut bacteria in
adult worms hybridised with the eubacterial probe (EUB338) and in a few sections also with
the Strc493 probe, which hybridises with most Streptococcus sp. and some Lactococcus sp.
Neither the lactic acid bacterial group- nor the Weissella species-specific probes targeted any
bacteria in the gut. Although Proteobacterial sequences were identified in clone libraries, these
bacteria were not visualised using class-specific Proteobacterial FISH probes.
The H. contortus microbiota was mainly comprised of members of the phyla Proteobacteria,
Firmicutes and Bacteroidetes (Tables 1 and 3). A large proportion of sequences were consistent
with the vertically-transmitted Weissella/Leuconostoc endosymbionts and the Lactococcus/
Streptococcus sp. seen in the uterus of female worms. The remainder, which are likely to be
attached to the cuticle or in transit or resident in the intestine of L3 or adult worms, were
dominated by Proteobacteria, Clostridium sp. and Lactococcus sp. Sequences belonging to
Bacteroidetes, typical ruminant foregut bacteria [60±63], were identified in adult worms, but not L3 or
eggs (Fig 1 and Table 1). Rumen bacteria can remain alive in abomasal contents, particularly
when the pH is raised by the presence of abomasal parasites [
] and either dead or live rumen
bacterial cells could contribute to the DNA and the sequences subsequently identified in the
adult worm samples. In adult worms and L3, the most frequently identified 16S rRNA
sequences belonging to the phylum Proteobacteria were from Mesorhizobium sp., which are
agriculturally important soil and rhizosphere bacteria [
]. Some of these may have been
become associated with the free living stages (L1 or L2) of the parasite, which feed on bacteria,
and remained in the dormant, non-feeding L3 stage and subsequently survived in L4 and adult
worms. Proteobacteria also dominated the microbiomes of the plant-parasitic nematode
Meloidogyne incognita [
] and free-living species [13±15].
The gut in many of the worm sections contained no bacteria, which could either be the
result of the delay in recovery using the agar method and preparation for FISH or from
expulsion of bacteria by agar taken in through the mouth and moving along the intestine. Although
not detected by FISH probes, even a small amount of DNA from these bacteria could have
been detected by PCR, especially if the primer pair preferentially amplified bacterial sequences
from the phylum Proteobacteria over the phylum Firmicutes. The rapid transit time of
contents through the nematode gut, estimated to be less than 2 min in C. elegans [
than for a comprehensive study of the H. contortus gut microbiome, the worms should be
manually collected from digesta and externally cleaned very rapidly to prevent loss of gut
Bacteria in the uterus. In most female worms, there were numerous gram-positive
bacteria in the distal uterus, but not in the proximal uterus near the ovaries. In TEM sections, they
were densely grouped near the wall of the uterus between the wall and the egg shells. Bacteria
were of a single morphotype, 300±500 nm in diameter, smaller than those in either the eggs or
in the gut, but none had a thick cell wall characteristic of gram-positive bacteria. Sperm were
also present within the uterus adjacent to fully formed eggs. These coccoid or diplococcoid
12 / 25
Fig 4. Bacteria inside an egg, near the ovipositor in a female worm. Bacteria were targeted by EUB338 (FITC-labelled, left) and Wgp (Cy3-labelled, right)
probes. The EUB338 probe targets all bacteria and the Wgp probe targets Weissella sp. Not all bacteria were targeted by the Wgp probe. E: egg; ES: egg shell.
bacteria are close relatives of either Lactococcus sp. or Streptococcus sp., as they were targeted
by the Strc493 FISH probe. The Strc493 probe was not able to be combined with other
speciesspecific probes because of different optimal hybridisation stringency conditions. The bacteria
detected in the uterus were not the same as those present in eggs, as in separate FISH
experiments, none of the probes Lab158, Wgp and S-G-Wei-0121-a-S-20 hybridised with bacteria
within the uterus, unlike those in eggs.
Despite their large numbers, these bacteria appeared not to be pathogens for the nematodes,
which were the usual size for the species, fully active and contained eggs of normal
morphology. This nematode culture is fully pathogenic to sheep and has the pre-patent period and high
egg production usual for H. contortus. The possible routes of entry of those bacteria into the
uterus could either from the environment (abomasum) or transmitted by male worms during
mating, after which they become resident in the uterus. The worms could be bacterial vectors,
as some Streptococcus sp. are opportunistic pathogens, while others are commensal bacteria in
animals and humans [
]. Their location in the distal, rather than proximal, uterus suggests
they are acquired at each generation and not vertically transmitted.
Bacteria in eggs. Eggs collected from faeces and also in female worms contained a small
number of spherical bacteria, 800±1000 nm in diameter, which were very close relatives of W.
confusa. They were clearly seen within eggs in 2 of 28 TEM sections of female worms, but
could not be recognised in LM sections. Although clearly defined, the cell walls were not as
thick as those in bacteria in other locations. Bacteria within eggs in females were hybridised by
the lactic acid bacterial group-specific probe Lab158, as well as the Weissella species-specific
probes (Wgp and S-G-Wei-0121-a-S-20) (Fig 4). Lab158 also hybridised with eggs in faeces.
There may be closely related lactic acid bacteria in the eggs, as not all could be targeted by
Weissella sp-specific probes, because there were more EUB338 signals than that from Wgp and
S-G-Wei-0121-a-S-20. The bacteria were either coccoid or diplococcoid, consistent with TEM
images, and scattered throughout the H. contortus egg as individual cells or in small clusters
when viewed at different focal planes in confocal microscopic images.
Other bacterial sequences were also present in clone libraries constructed from eggs,
although no bacteria were seen either by FISH or TEM on the egg surface. This is consistent with
13 / 25
RNA/DNA of faecal bacteria contaminating eggs in faeces or carried by female worms attached
to eggs laid in vitro. Clostridium sp. are mammalian gut residents [43±46] and sequences
belonging to this genus were prominent in eggs collected from faeces. Similarly, sequences belonging to
Sternotrophomonas sp. were dominant in the clone library of eggs laid in vitro.
Maternal transmission of the Weissella/Leuconostoc endosymbionts is strongly supported
by their visualisation in eggs, both in the female and after laying, and identification in all three
life-cycle stages of nearly full length (~1400bp and ~1000bp) 16S rRNA gene sequences and
short sequences (~190bp) from DGGE bands. Despite identifying similar bacterial sequences
in L3 and male and female worms, these bacteria were not able to be visualised in L3 and male
worms by FISH, perhaps due to dormancy of the bacteria. In L3 sections, background
fluorescence was so strong that true FISH signals could not be distinguished from false positives.
Therefore, the location of bacteria in sections of L3 and male worms and the details of the
route of vertical transmission remain unknown.
The most likely source of the endosymbionts is rumen fluid, in which they form a minor,
but variable, component of the microbiome [
], however, they also have been detected in
plants, fermented foods, meat products and in human and animal gut contents, milk and saliva
]. Weissella are also rare opportunistic pathogens of humans and animals causing infections
of the heart and artificial joints, abscesses and bacteraemia, probably after entry from the
intestinal mucosa [
]. They do not appear to be pathogenic for H. contortus, although
experimental feeding to C. elegans caused a moderately extended life span, compared with worms fed
Escherichia coli, due to dietary restriction and stress induction . Weissella sp. may use H.
contortus as a vector, similar to the situation involving vertical transmission of endosymbiotic
Neorickettsia in parasitic trematodes (flatworms) and horizontal transmission to trematode
vertebrate hosts, which then develop serious diseases [73±75].
The Weissella/Leuconostoc endosymbionts in H. contortus could be evolving from
free-living to mutualistic endosymbiotic bacteria. This process has been induced experimentally in
Rhizobia in legumes through acquiring essential genes and genome re-modelling [76±78],
leading ultimately to a reduced symbiotic genome as non-essential genes are lost [
Horizontal transfer of genes from associated bacteria or spontaneous mutations similarly may be
shaping the development of free-living Weissella into endosymbionts of H. contortus. They
may be at the transitional stage between free-living and endosymbionts, with gene sequences
evolving in only some of the bacteria in the population; this could explain why fewer
symbionts in eggs were targeted by Weissella species-specific probes than by the Lab158 FISH probe,
resulting in an erroneously interpretation of multiple species. The specific probes may
hybridise with the highly variable regions, which may be evolving, whereas Lab158 targets a
conserved region in the 16S rRNA gene of most of the lactic acid bacterial group; this region may
not be evolving at this time.
The best known vertically transmitted symbionts are the Wolbachia and Verrucomicrobia,
which manipulate respectively the biology of filarial [
] and plant parasitic nematodes
]. More recently, a Comomonas sp. has identified in eggs, the gut cells of L3 and adult
Spirocerca lupi, a parasite principally of canids . Although this symbiont was present in the
strain of S. lupi prevalent in Israel, it did not appear to be in the parasites in South Africa [
A similar situation could exist in H. contortus, in which the symbiont is currently known only
in a laboratory strain of the nematode and its distribution in the field is unknown.
The microbial communities of H. contortus were shown by PCR-DGGE and constructing
clone libraries of sequences to differ from the communities in the natural environments of
14 / 25
adult parasites in the abomasum or developing L3 in faeces. Detailed phylogenetic
evolutionary relationships showed that members of the phyla Proteobacteria, Firmicutes and
Bacteroidetes were associated with adult worms, larvae and eggs in faeces and laid in vitro. Bacteria
were identified in eggs, the female reproductive tract and the gut of adult and L3 larvae using
the universal FISH Eub338 probe, which targets most bacteria, and were also seen by light and
transmission electron microscopy. Those in the reproductive tract were of two different
morphotypes and their sequences matched to unrelated type species in the phylum Firmicutes.
Streptococcus or Lactococcus sp. were targeted within the distal uterus with the probe Strc493,
whereas sequences from the genera Weissella and Leuconostoc were found in all life-cycle
stages, except eggs collected from faeces, which were dominated by sequences belonging to
Clostridium sp. Bacteria closely related to W. confusa were identified both by PCR-DGGE
short sequences and in clone libraries of nearly full length 16S rRNA bacterial sequences in all
life-cycle stages; they were subsequently visualised only in eggs by fluorescent in situ
hybridisation (FISH) with group-specific probes despite detection of their DNA in L3, female and male
worms. This strongly suggests they are vertically transmitted endosymbionts of a laboratory
strain of H. contortus.
Materials and methods
Maintenance in the laboratory for 10 years of a pure culture of H. contortus, originally collected
from the field, by regular passage through sheep and recovery of adult worms from euthanased
sheep were carried out in accordance with the requirements of the Massey University Animal
Ethics Committee approval #09/11 for this project.
Abomasal fluid and adult worms were recovered from infected sheep on Day 21 post
]. Briefly, the abomasum was removed and abomasal contents and saline washings
were mixed 2:1 with warmed 3% agar, allowed to set and the solidified blocks incubated at
37ÊC in a saline bath. Clumps of parasites were collected from the saline soon after emergence
and placed in medium appropriate for microscopy or molecular biology. Some samples of
adult worms were also manually collected from abomasal contents. Male and female
populations were separated under a dissecting microscope, based on morphological differences.
Worms were placed on a sterile filter and washed alternately five times with 4% sodium
hypochlorite for 5±10 sec and rinsed with approximately 200 ml ultrapure water. Ultrapure water
was prepared from MilliQ water by filter sterilisation, autoclaving and ultraviolet irradiation
for 48 h.
Approximately 500±600 eggs were collected either from faeces or after laying by female
worms in vitro. After recovery from agar, 15 adult females/tube were placed in 1.5 ml
phosphate buffered saline (PBS) in each microcentrifuge tube to lay eggs during an overnight
incubation at 37ÊC. After manual removal of the worms, the egg suspensions were pooled and the
eggs separated and washed on a sterile filter, as described above for adult worms. Eggs were
separated from faeces by sequentially passing through sieves down to 20 μm mesh, which
retained the eggs along with some particulate matter. This suspension was placed on top of a
saturated NaCl solution for 5 min and eggs allowed to stick to a glass surface placed on top.
The eggs were washed on to a sterile filter and treated as described above for laid eggs in vitro.
L3 were cultured in faeces collected into faecal bags on infected sheep. Faeces were placed
in trays, moistened, covered and cultured for 10 days at 22±24ÊC. L3 were separated from
faeces by their movement through paper tissues (Baermannisation), washed and stored in reverse
osmosis (RO) water at 10ÊC. Before use, L3 were exsheathed by incubating at 37ÊC in 0.05%
sodium hypochlorite solution (Clark Product Ltd, Rotorua, New Zealand) for 15±20 min.
15 / 25
After confirming microscopically that 95±100% had exsheathed, they were again
Baermannised in ultrapure water for 12 h.
Molecular fingerprinting of bacterial communities in H. contortus
DNA was extracted from parasites and samples from their environment, bacterial 16S rRNA
genes were amplified and bacterial sequences were subjected to phylogenetic analysis.
Extraction of DNA
DNA was extracted from 0.5 g samples of 4% sodium hypochlorite washed adult worms,
exsheathed L3 and eggs, as well as abomasal fluid and faeces. Samples were homogenised using
a sterile micro-centrifuge pestle, incubated in lysis solution, plus 20 μl of 20 mg/ml proteinase
K (Life Technologies) solution for 2.5 h at 37ÊC. DNA was extracted from 200 μl samples, as
described in the manufacturer's manual of the QIAamp DNA stool-kit (Qiagen, Hilden,
Germany). The quantity and the purity of the DNA were determined using a NanoDrop ND-1000
UV-Vis spectrophotometer (NanoDrop Technologies) or using the Qubit ™ ds DNA HS Assay
Kits on a Qubit fluorometer (Invitrogen) according to the manufacturer's instructions.
Samples were stored at -20ÊC for downstream applications.
PCR-DGGE. Bacterial 16S rRNA genes were amplified in a 50 μl reaction mixture
containing: 5.1 μl 10X PCR buffer with Mg2+, 5 μl of 2 mM dNTP, 0.5 μl of 10 μM forward
primer and 0.5 μl of 10 μM reverse primer, 2.5 U (0.5 μl) of native Taq DNA polymerase
(Roche Diagnostics, Mannheim, Germany), 5 μl of template and 33.4 μl of ultrapure water.
The following touchdown PCR was used: stage I: 3 min initial denaturation at 95ÊC, followed
by stage II: 10 cycles of denaturation (30 sec at 95ÊC), initial annealing (30 sec at 62ÊC) and
elongation (30 sec at 72ÊC), -0.5ÊC/cycle, followed by stage III: 26 cycles of denaturation (30
sec at 95ÊC), annealing (30 sec 57ÊC) and elongation (30 sec at 72ÊC) with final elongation at
72ÊC for 10 min. PCR amplifications were carried out with the universal bacterial primers,
338f and 518r (Table 1); the forward primer (338f) had a 40-nucleotide GC-clamp added to
Amplified DNA was analysed by agarose gel electrophoresis. Gels were stained with SYBR
safe DNA stain (Invitrogen), visualised using UV trans-illumination and photographed using
a BIO-RAD Molecular Imager1 Gel Doc™ XR (BIO-RAD Laboratories, Milan, Italy). The
concentration of the extracted DNA was measured and the purity was determined using a
NanoDrop ND-1000 UV-Vis spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA)
according to the manufacturer's instructions. PCR products were purified with the Wizard1
SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA).
Purified PCR products (300 ng) were mixed with equal volumes of DGGE loading dye
(0.05% [w/v] bromophenol blue, 0.05% [w/v] xylene cyanol, 70% [w/v] glycerol in water, pH
8.0) and loaded into the DGGE gel wells. A 1 kb-plus marker (Invitrogen) was also run on
each gel. The gels were electrophoresed with 1X TAE buffer which contained 40 mM tris
(hydroxymethyl) aminomethane, 65 mM acetic acid and 10 mM EDTA, adjusted to pH 8 with
5 M NaOH. The electrophoresis was performed at 60ÊC for 5 h at 200 V. After the
electrophoresis, gels were stained with 3 μl of SYBR Gold (Invitrogen) in 600 ml of MQ water for 20 min
on a shaker and then destained overnight. The gel was visualised using UV trans-illumination
Sequences from DGGE bands
Bands of interest in adult, L3 and egg samples were excised and aseptically transferred into
sterile centrifuge tubes. 100 μl ultrapure water was added to the tube containing the excised
16 / 25
band and vortexed for 1min, then the water was removed. Gel slices were finely broken in
50 μl of ultrapure water using sterile pipette tips. Tubes were incubated overnight at 4ÊC and
the following day were vortexed for 5 sec and centrifuged at 28,000 g for 1 min. The
supernatants were transferred to sterile centrifuge tubes and the DNA fragments in the supernatant
were re-amplified with the primers 338f and 518r, using the touchdown PCR protocol with an
initial annealing temperature of 62ÊC. Purified PCR products were cloned by ligation into a
plasmid vector (pCR 2.1TOPO-TA cloning vector, Invitrogen) and transformed into
chemically competent E.coli TOP-10 cells, using a TOPO-TA cloning system (Invitrogen) according
to the manufacturer's instructions. Amplified PCR products were sequenced using the primer
M13f by Macrogen Inc. (Seoul, Republic of Korea).
Vector sequences were removed using MEGA version 5 (Molecular Evolutionary Genetics
]. The sequences were checked for quality of the chromatogram (evenly-spaced
peaks) and miscalled nucleotides. Good quality sequences were analysed with known
sequences available in the GenBank database. The BLASTn search option of the National
Center for Biotechnology Information (NCBI) web site (http://www.ncbi.nlm.nih.gov) was used to
compare sequences of close evolutionary relatives with sequences obtained from the DGGE
Phylogenetic analysis of bacterial sequences from adult worms, L3 and eggs
Nearly complete length (~1400bp) and ~1000bp 16S rRNA gene sequences were amplified
respectively with the universal bacterial primer set 27f and 1492r or 27f and the phylum
Firmicutes-specific primer 1040firmR (Table 1). The PCR mixture (25 μl) contained 2.6 μl of 10X
reaction buffer with MgCl2, 2.5 μl of dNTP, 0.5 μl of 0.5 μM of each primer, 0.5 μl of 2.5 U of
Taq DNA polymerase (Roche), and 15.9 μl DNA-free ultrapure water. The PCR was carried
out using the touchdown PCR programme with an initial annealing temperature of 62ÊC.
For the detailed phylogenetic analysis, the ~1400bp and ~1000bp bacterial sequences were
matched with those in the GenBank database [
] using the BLASTn option [
] to obtain the
closest sequences of uncultured bacteria, cultured isolates and type strains. Additionally, the
sequence match option of Ribosomal Database Project (RDP) was used to obtain information
on the closest type strain. For phylogenetic tree building, sequences from the GenBank
database from uncultured bacteria with similarity >99%, the closest cultured isolate and type strain
sequences which had the highest similarity to the sequence were combined with those from H.
contortus. Sequences were globally aligned with ClustalW using the MEGA 5.0 software
]. The alignment was manually corrected by comparison with previously identified
The online chimeric detection programme Bellerophon [
] (Huber et al., 2004) was used
to identify chimeric sequences. The phylogenetic affiliations of sequences were created with
the phylogeny option of MEGA 5.0 [
] (Tamura et al., 2011), using the default settings for the
NJ, ML and parsimony methods. Phylogenetic trees of ~1400bp sequences obtained from NJ,
ML and parsimony were compared with each other to verify the robustness of tree topology.
Additionally, two NJ trees were created from the first and last ~400bp of aligned ~1400bp
sequences and compared with each other for further detection of chimeric sequences. Potential
chimeric sequences were excluded from further tree building analysis. Distance matrices of
aligned sequences were also made using the Geneious software package [
]. The same
procedure was carried out for ~1000bp sequences. The final dendrograms of sequences of phylum
Firmicutes (~1400bp and ~1000bp) were inferred using the NJ, ML and parsimony methods.
Each analysis included sequences from the closest cultured and type strains and bacterial
sequences identified in H. contortus.
17 / 25
Light microscopy (LM). Adult worms were fixed in 10% [v/v] neutral buffered formalin
overnight, then routinely processed in a Leica TP1050 Tissue processor (Global Science and
Technology, Auckland, New Zealand) and paraffin embedded (Leica Histo Embedder,
Germany). Sections 5 μm thick were cut on a Leica RM 2235 manual rotary ultramicrotome
(Wetzlar, Germany), using a S35 Feather disposable microtome blade (Osaka, Japan). Sections
were de-waxed and stained with hematoxylin and eosin (H & E) using a Leica Autostainer XL
(Global Science and Technology, Auckland, New Zealand) or by the Gram Twort method.
Slides were washed, dried and a drop of Entellan immersion oil (Merck New Zealand Ltd,
Auckland, and NZ) was added as a mounting solution. Sections were covered with cover slips
and viewed under with an OLYMPUS BX61 microscope (OLYMPUS, Tokyo, Japan).
Transmission electron microscopy. Adult female worms were sliced into pieces 6±8 mm
long, fixed for 2±3 days in 3% glutaraldehyde and 2% formaldehyde in 0.1 M phosphate buffer
(Na2HPO4.12H2O and KH2PO4), pH 7.2 and post-fixed in 1% OsO4 in phosphate buffer.
Tissues were embedded in resin and 1 μm thick sections cut on an Ultra-microtome (Leica
Microsytems, Wetzlar, Germany). Sections were stained with 0.05% toluidine blue and areas
of interest were chosen by LM. Sections 100 nm were cut and double stained with uranyl
acetate and lead citrate and observed by electron microscopy (Philips CM10 Transmission
Electron Microscope with SIS Morada high-resolution digital imaging) at 60 kV at the Manawatu
Microscopy and Imaging Centre, Massey University (MMIC).
Fluorescence in situ hybridisation (FISH)
Eggs from faeces and laid in vitro, exsheathed L3 and male and female worms were collected as
described above. To reduce gut emptying, female worms were also collected from abomasal
contents after euthanasia of the sheep and fixed immediately. Adult worms and L3 were
straightened by incubating for approximately 12 h at 4ÊC in PBS, then all lifecycle stages were
fixed overnight at 4ÊC in 4% paraformaldehyde in PBS (PFA). After residual PFA had been
removed by washing twice with PBS, adult worms were transferred to 70% ethanol and stored
at -20ÊC until automated, routine histological processing through graded alcohol solutions
and 100% xylene (Leica TP1050 tissue processor, Wetzlar, Germany) and paraffin embedding
(Leica Histo Embedder, Wetzlar, Germany). Eggs and L3 were processed in microcentrifuge
tubes and centrifuged at 17,100 g for 1 min between dehydrating and washing steps. This was
followed by serial immersion of the samples in ethanol-xylene solutions of 3:1, 1:1 and 1:3 [v/
v] and finally in 100% xylene for 10 min. After removing the xylene, the samples were
embedded in paraffin blocks (Leica Histo Embedder).
Sections 3 μm thick were cut as for LM and 2 sections, each containing 3 adult worms, 100±
200 eggs or L3, were placed on each slide (Menzel-Glaser Superfrost, Lomb Scientific Pty Ltd,
Sydney, Australia). Sections were de-parafinised by heating for 3±5 sec at 100ÊC, immersed in
100% xylene for 15 min and then in 100% ethanol for 15 min. These two steps were repeated
twice and then the slides were washed in MQ water. The slides were thoroughly air-dried
and treated with 1 mg/ml lysozyme (Sigma) for 10 min for the Lab158, Strc493, Wgp and
S-G-Wei-0121-a-S-20 probes, but not Proteobacterial class-specific probes. Lysozyme was
removed under running tap water and the slides were air-dried thoroughly.
The group-, class- and species-specific probes Lab158, Wgp, S-G-Wei-0121-a-S-20, Strc493,
Alf73a, Beta1 and SteMal_439 were selected from the literature to target the bacterial species
identified from ~190bp and ~1400bp sequences from H. contortus (Table 1) and analysed using
the probeBase website [
] (Loy et al., 2007). In the absence of a suitable 16S rRNA probe for
Alphaproteobacteria, a 23S rRNA targeted probe was selected. Labelled (Cy3 or FITC) probes
18 / 25
were purchased from Eurofins MWG Operon (Ebersberg, Germany) and Sigma Aldrich
(Auckland, New Zealand). The specificity was determined for 10 reference cultures (S1 Table)
and the optimum formamide concentration and hybridisation stringency for each probe were
determined (S2 Table) as the formamide concentration immediately below that in which
specific signals decreased and there were no non-specific signals from non-target species.
FISH was carried at 46ÊC for 2 h in humidified containers on multiple slides, each
containing two consecutive serial sections of either 3 adult worms, 100±200 eggs or 100±200 L3,
isolated by drawing a hydrophobic barrier around each nematode section. Hybridisation buffer
(20 mM tris-HCl, 0.9 M NaCl, 0.01% sodium dodecyl sulphate, pH 7.2), containing 50 ng/μl
probe and formamide was added to cover each section on the slide. Each experiment included
a control for autofluorescence (hybridisation buffer without probes) and a negative control
(Non-EUB338 labelled with FITC or Cy3) and the universal bacterial EUB338. Following
hybridisation, each slide was rinsed immediately using a pipette containing the appropriate
washing buffer (pre-heated to 48ÊC) and then placed in a tube containing washing buffer at
48ÊC for 10±15 min. After removal from the washing buffer, the slides were immediately
rinsed briefly in a beaker of ice-cold distilled water and then thoroughly air-dried.
Sections were mounted, viewed under both phase contrast and at appropriate wave lengths
under a confocal laser scanning microscope (Leica TCS SP5 DM 6000B Leica Microsystems,
Germany) and photographed at 100x magnification. The appropriate excitation and emission
wave lengths to avoid non-specific signals were determined using EUB338 Cy3 and FITC
labelled reference bacteria as 470±495 and 510±550 nm for FITC and 535±555 and 570±625
nm for Cy3. Images were analysed using the Leica LAS AF and Leica LAS AF lite (Leica
Microsystems CMS GmbH, Wetzlar, Germany) imaging software packages.
S1 Fig. A DGGE gel (6% acrylamide) of PCR amplified products of DNA extracted from
sodium hypochlorite washed male (HAM) and a female (HAF) H. contortus from each of
three sheep. Samples were amplified using the universal bacterial 16S rRNA primers 338f
(GC-clamp) and 518r. The gel was a portion of a 30±45% denaturing gradient.
S2 Fig. A DGGE gel (6% acrylamide) of PCR amplified products generated from the DNA
extracted from manually collected worms from the abomasal mucosa and sodium
hypochlorite washed worms from three sheep. The PCR was carried out using the universal
bacterial 16S rRNA primers 338f (40bp GC clamp) and 518r. The gel was a portion of a 30±45%
S3 Fig. DGGE gels (6% acrylamide) of PCR amplified products of DNA extracted from
samples collected during H. contortus larval culture from faeces. The DNA was amplified
using the universal bacterial 16S rRNA primers 338f (40bp GC clamp) and 518r. DGGE gel
30±55% denaturing gradient: Lanes: A-fresh faecal sample, B-11 day old faecal sample mixed
with vermiculite, C-larvae in water, D-larvae collected on a sieve, E-larvae collected at the
bottom of a tube connected to the funnel, F-sheathed larvae washed with absolute ethanol, G-RO
water, M-1 kb plus DNA ladder.
S4 Fig. Phylogenetic tree (ML method) of phylum Firmicutes ~1400bp 16S rRNA genes
sequences from H. contortus using the primer set 27f and 1492r and reference gene
sequences. Sequences belonging to order Clostridiales were compressed and represented as a
19 / 25
triangle in the dendrogram. GenBank accession numbers of reference sequences are given
before the reference cultures; (T) designates a type strain. Bootstrap values are shown at each
node (percent of 500 replicates). HA: adult worms; HL: L3; HEF: eggs collected from faeces;
HEM: eggs laid in vitro. The scale bar indicates 0.02 nucleotide substitutions per nucleotide
S5 Fig. Phylogenetic tree (ML method) of phylum Firmicutes ~1400bp bacterial 16S rRNA
genes sequences from H. contortus using the primer set 27f and 1492r and reference gene
sequences. Sequences belonging to families Leuconostocaceae, Streptococcaceae and
Staphylococcaceae were compressed and represented as triangles in this dendrogram. GenBank
accession numbers of reference sequences are given before the reference cultures; (T)
designates a type strain. Bootstrap values are shown at each node (percent of 500 replicates). HA:
adult worms; HL: L3; HEF: eggs collected from faeces; HEM: eggs laid in vitro. The scale bar
indicates 0.02 nucleotide substitutions per nucleotide position.
S6 Fig. Phylogenetic tree (ML method) of phylum Firmicutes ~1000bp bacterial 16S rRNA
genes sequences from H. contortus using the primer set 27f and 1040firmR and reference
16S rRNA gene sequences. GenBank accession numbers of reference sequences are given
before the reference cultures; (T) designates a type strain. Bootstrap values are shown at each
node (percent of 500 replicates). A: adult worms; L: L3; FE: eggs collected from faeces; ME:
eggs laid in vitro. The scale bar indicates 0.02 nucleotide substitutions per nucleotide position.
S1 Table. Specificity, target and non-target species used to optimise bacterial probes used
for fluorescence in situ hybridisation (FISH).
S2 Table. Formamide concentrations for optimal hybridisation stringency of the bacterial
species-, group- and class-specific fluorochrome-labelled probes used to identify bacteria
in H. contortus by FISH.
Conceptualization: Gajenathirin Sinnathamby, Gemma Henderson, Peter Janssen, Ross
Bland, Heather Simpson.
Data curation: Gajenathirin Sinnathamby, Saleh Umair.
Formal analysis: Gajenathirin Sinnathamby, Saleh Umair.
Funding acquisition: Heather Simpson.
Investigation: Gajenathirin Sinnathamby.
Methodology: Gajenathirin Sinnathamby, Gemma Henderson, Peter Janssen, Ross Bland,
Project administration: Heather Simpson.
Supervision: Gemma Henderson, Peter Janssen, Ross Bland, Heather Simpson.
Visualization: Gajenathirin Sinnathamby.
20 / 25
Writing ± original draft: Gajenathirin Sinnathamby, Gemma Henderson, Heather Simpson.
Writing ± review & editing: Heather Simpson.
21 / 25
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