Bacterial profiling of Haemonchus contortus gut microbiome infecting Dohne Merino sheep in South Africa

A metagenomic approach was used to study the gut microbiome of Haemonchus contortus field strains and that of its predilection site, the abomasum of Dohne Merino sheep. The abomasum contents and H. contortus were collected from 10 naturally infected Dohne Merino sheep. The H. contortus specimens were classified and sexually differentiated using morphometric characters and was further confirmed through molecular identification. We investigated differences and similarities between the bacterial composition of the adult male and female H. contortus gut microbiomes, which were both dominated by bacteria from the Escherichia, Shigella, Vibrio and Halomonas genera. Major abundance variations were identified between the shared adult male and female H. contortus microbiomes. The results also revealed that Succiniclasticum, Rikenellaceae RC9 gut group and Candidatus Saccharimonas were the predominant genera in the Dohne Merino abomasum. This study provides insight into the highly diverse bacterial composition of the H. contortus gut microbiome and the Dohne Merino abomasum which needs to be studied further to explore the complex interactions of different gastrointestinal nematode microbiomes with the host.

www.nature.com/scientificreports/ of GIN parasites on host microbiome 12 . Currently, there is a lack of scientific information on the microbiome harbored by GIN parasites of livestock and their relationship to the host microbiome. The control of GIN parasites is primarily based on the administration of anthelmintic drugs. The extensive use of anthelmintic drugs leads to the development of drug resistance in GIN parasites, especially the barber's pole worm H. contortus, which is a highly pathogenic and probably the most economically important nematode infecting small ruminants 4,10 . Hence, alternative control measures are needed to effectively control GIN parasites. A novel approach based on biocontrol using symbiotic microbial communities has been suggested to limit the use of chemical based treatments 13 . As such, it is important to understand the dynamics of GIN infections and their respective microbiome in comparison to that of the host which will contribute valuable information towards exploiting the microbiome in an attempt to control GIN in small ruminants 4,10,14 . The current study focused on the characterization of the adult male and female H. contortus microbiome and that of their predilection site, the abomasum of Dohne Merino sheep.

Morphometrics and molecular analysis of H. contortus isolated from Dohne Merino sheep.
A total of 266 GIN, 169 females (63.5%) and 97 males (36.5%) were recovered from 7 out of 10 (70%) abomasum of the Dohne Merino sheep sampled (Table S1 and Fig. S1). A total of 7 pooled adult male H. contortus and 7 pooled female H. contortus samples identified by their morphometric characteristics were confirmed by PCR assay to be H. contortus with an amplification band present at 260 bp (Fig. S3). Care was taken to restrict microbiome contamination (i.e. intact bacteria and their DNA products) originated from the abomasal microbiome (Fig. S2). Table 1, the adult male H. contortus had a higher Operational Taxonomic Unit (OTU) richness than the female (p-value < 0.05). However, when compared to the abomasum, the abomasum microbiome had the highest richness (p-value < 0.05). There was a clear difference between the adult male H. contortus gut microbiome in comparison to the female gut microbiome across all alpha diversity indices except for Faith's Phylogenetic Diversity (PD) which did not show any significant difference (p-value > 0.05) ( Table 1 and Fig. S4). Similarly, in Fig. 1A, the abomasum content was compared to the adult male H. contortus and the female microbiome, and displayed a significant difference in Observed OTUs, Shannon and Simpson indices (p-value < 0.05).

Alpha diversity indices of the adult male and female H. contortus and abomasum microbiome. As shown in
Beta diversity indices of the adult male and female H. contortus and abomasum microbiome. The Principal Coordinates Analyses (PCoA) results indicated that adult male and female H. contortus microbiome clustered together. The abomasum microbiome clustered on its own and was separated from the adult male and female H. contortus microbiome cluster (Fig. 1B). Furthermore, the unweighted (Fig. 1B) and weighted UniFrac (Fig. S5) distance analysis indicated that the microbiome composition of the abomasum was significantly different from that of the adult male and female H. contortus (p-value < 0.05) ( Table S3). The microbiome composition was also significantly different between the abomasum content and the adult female H. contortus (p-value < 0.05). The abomasum and adult male H. contortus microbiome was also found to be significantly different (p-value < 0.05) (Table S3).
Unique and shared microbiome composition. In total, 660 unique bacterial OTUs across the three hosts were identified (Table S4). The abomasum content had higher bacterial OTU abundance followed by the adult female H. contortus whilst the male had the lowest bacterial OTU abundance (Fig. S6). The results showed a total of 57 bacterial OTUs shared between the adult male and female H. contortus. The adult male H. contortus and abomasum shared 113 OTUs with each host having 98 and 169 unique OTUs present, respectively. A total of 16 bacterial OTUs were shared between adult female H. contortus and the abomasum (Fig. 4A).

Discussion
The barber's pole worm (H. contortus) is a common parasitic nematode infecting small ruminants and one of the most pathogenic GIN of sheep [15][16][17] . Identification of common stomach worms, including H. contortus, in small ruminants has been previously based on recognized morphological characteristics that make it possible to differentiate between Haemonchus species, particularly, H. contortus and H. placei. The spicule lengths and vulvar flap provide the quickest and easiest characteristics to be used for separating populations of H. contortus from H. placei. [18][19][20] . In the present study, the recovered Haemonchus spp. specimens were identified as adult male and female H. contortus based on their morphological characteristics as suggested by Lichtenfels et al. 21 and Gibbons 18 . The adult female H. contortus were recognised mainly by their reddish colour and vulvar flap features. The adult male H. contortus were identified based on the low variability of spicule morphology within species. The morphological classification of the specimens was confirmed by PCR assay using species specific H. contortus primers 22 . Following recovery of the parasites from the abomasum, a rigorous treatment consisting of antibiotic solution (1 mg/ml ampicillin/1 mg/ml gentamicin) and 4% sodium hypochlorite was used to ensure removal of all microbiome contamination on the parasite surface. The PCR assay results with 16S rRNA gene primers further served as an indicator that there was no contamination on the parasite originating from the abomasal microbiome. These results corroborated the previously published results by White 23 and El-Ashram and Suo 10 who reported that sterilisation with this method is sufficient to remove abomasal microbiome contaminants on the surface of the GIN.
The adult male and female H. contortus microbiome were characterised in the absence of external microbiome contaminants on the surface of the parasite. The results indicated that the adult female H. contortus microbiome was dominated by bacterial phyla: Proteobacteria (94%) followed by Firmicutes (3%), Bacteroidetes (1%), Actinobacteria (0.6%) and Cyanobacteria (0.3%). The adult male H. contortus microbiome was dominated by bacterial phyla: Proteobacteria (57%) followed by Firmicutes (24%), Bacteroidetes (8%), Actinobacteria (6%) and Cyanobacteria (2%). These results are similar to those observed in earlier studies conducted on Trichuris muris by White 23 , who reported that the adult T. muris microbiome was dominated by bacterial phyla: Bacteroidetes followed by Firmicutes and Proteobacteria. El-Ashram and Suo 10 and Sinnathamby et al. 4 reported that the H. contortus microbiome was dominated by the bacterial phyla: Proteobacteria followed by Firmicutes, Tenericutes and Actinobacteria, and Firmicutes followed by Proteobacteria and Bacteroidetes respectively. Relatedly, the microbiome composition at phylum level observed in the current study on H. contortus were similar to those observed in previous studies of T. muris, Ascaris suum and H. contortus from United Kingdom, United States of America and China respectively. However, there were variations in abundance of these phyla which are usually influenced by the type of host they inhibit and environmental conditions 10,23 . White 23 , El-Ashram and Suo 10 and Sinnathamby et al. 4 compared the differences and similarity in the microbiome of the different parasite life-cycle stages but not of the different genders of the adult stages and the host. This study therefore provided additional information by comparing the adult male and female H. contortus microbiome as well.
On a genus level, the adult female H. contortus microbiome was dominated by bacterial genera: Escherichia-Shigella (28%) followed by Vibrio (11%), Halomonas (6%), Acinetobacter (2%) and Alteromonadales (2%). The dominant genera in the adult male H. contortus microbiome were Vibrio (15%) followed by Escherichia-Shigella (8%), Halomonas (8%) and Acinetobacter (3%). The predominant bacterial genera in the adult male H. contortus were similar to female but with variation in the genera abundance. The adult female H. contortus showed higher genera abundance than male H. contortus, although the adult male H. contortus had more diversity than female in terms of the overall microbiome. Among the aforementioned genera, Escherichia-Shigella, Vibrio, Halomonas and Acinetobacter were predominantly shared by both adult male and female H. contortus. These results are different from those reported by El-Ashram and Suo 10 who indicated that the predominant bacterial genera of www.nature.com/scientificreports/ www.nature.com/scientificreports/ the H. contortus were Escherichia-Shigella (87.5%), Pseudomonas (5.91%) and Ochrobactrum (5.06%), which we believe is caused by the type of host they infect and environmental conditions of their locations. The microbiome of the adult male and female H. contortus showed similarity and variations in terms of abundances and diversity in the present study. These results indicated that H. contortus harbours a distinct microbiome compared to their host. There was significant difference between adult male and female H. contortus microbiome diversity and abundances. The H. contortus samples in this study were recovered from the same sheep breed, fed under the same diet and under similar environmental conditions. The variations in the microbiome of adult male H. contortus and female may be caused by hormonal changes, immune response or the reproductive needs, including egg development in females, which does not occur in male GINs. The presence of the microbiome in H. contortus suggests a potential nutritional importance of the microbiome to the worms, either providing metabolites that the worm cannot make itself, or aiding the breakdown of ingested blood meal.
The Dohne Merino abomasum microbiome naturally infected with H. Contortus field strains were characterised to gain insight into the impact that GIN parasites infection has on the host microbiome and how these interactions influence H. contortus microbiome. The abomasum microbiome was characterised and found to be dominated by the following bacterial phyla: Firmicutes (45%), Bacteroidetes (26%), Actinobacteria (7%), Patescibacteria (4%) and Planctomycetes (3%). Similar observations were reported in previous studies on small ruminants which indicated that the abomasum was dominated by Bacteroidetes, Firmicutes, Actinobacteria and Proteobacteria. 24,25 . With regard to the abundance of these predominant phyla, there were variations found across the studies. Interestingly, the phyla Patescibacteria and Planctomycetes were found in abundance in the abomasum content in the present study with none of these phyla reported in other previously published studies [23][24][25][26] . These variations between the present study and previously published studies could be due to known factors that affect microbiome composition such as parasite burden, breed type, diets and different environmental condition. www.nature.com/scientificreports/ The predominant assigned bacterial genera observed in the abomasum content were Succiniclasticum (5%), Rikenellaceae RC9 gut group (4%), Candidatus Saccharimonas (4%), Ruminococcaceae UCG-011 3%, p-1088-a5 gut group (3%) and Prevotella 1 (3%). These predominant genera are known to specialize in complex polysaccharides degradation, such as starch and cellulose, and contribute in the metabolism of dietary fiber in ruminants 27 . These results agree with observations reported by Zeng et al. 25 , Tanca et al. 28 and Zhang et al. 27 whereby predominant genera in the GIT depend on the parasite burden, diet and environmental condition. Hence, there were variations in the microbiome observed in the present study and previously published studies 25,27,28 .
The results further indicated that all abomasum content microbiome clustered together, suggesting that the sheep abomasum harbour similar microbiome. The adult male and female H. contortus microbiome clustered together, indicating that they harbour similar microbiome as well, which could be due to similar living environmental conditions and diet. El-Ashram and Suo 10 reported similar results which shows the H. contortus eggs and adult stage microbiome cluster together because they share similar environmental conditions inside the host. When compared, the adult male H. contortus and female appeared to have lower bacterial diversity than the abomasum content. Hence, the H. contortus microbiome does not simply reflect their host abomasum content microbiome, although they spend majority of their life-cycle in the host abomasum. A previous study by Sinnathamby et al. 4 reported that the different parasite life-cycle stages microbiomes are different from that of their predilection site. Moreover, El-Ashram and Suo 10 ,Sinnathamby et al. 4 also reported that during the H. contortus life cycle development stages, the egg stage, L1, and L2 and L3 interact with different microbiome from their respective environment, thus each stage might have distinct microbiome that they pick up from their environment and the host.
Furthermore, we found that the adult male H. contortus harbour a higher number of unique bacterial OTUs compared to the female and shared a higher number of bacterial OTUs with the abomasum content than the female. These results suggest that there are various selective measures at play within the H. contortus digestive tract, different to that of the host gastrointestinal tract. Therefore, some bacterial species are unable to persist and others thrive. The metabolic needs of the H. contortus might vary to that of its host and microbiome selection will depend on what GIN parasite require for its own fitness and survival within the host. These results correspond with Sinnathamby et al. 4 observations whereby they reported that the H. contortus harbour distinct/unique microbiome from its host, which are important for their survival. Furthermore, the present study indicated that the abomasum content harbour higher OTUs abundance compared to H. contortus, which would be due to its higher metabolic needs. This observation is similar to previous studies on T. muris and H. contortus by White et al. 29 and Sinnathamby et al. 4 which reported that the host harbour more diverse microbiome than the parasite.
Previous studies by Lu et al. 30 ,Gómez-Garzón et al. 31 and Abdel-Salam et al. 32 reported that bacterial strains, Pseudoalteromonas marina, Vibrio attantica strain 5-16, Pseudomonas aeruginosa and beteli, and Lysinibacillus can produce nematicidal volatile compounds with activities against the root-knot nematode Meloidogyne incognita and pine wood nematodes. Interestingly, similar bacterial genera were observed in the unique OTUs in the present study as well. As result, these bacteria can be manipulated in livestock production system due to their nematicidal activity to target pathogenic GIN in livestock such as H. contortus and potentially become biological control agents for GIN parasites while reducing the use of chemical based treatments 31,32 . The presence of these bacteria gives hope that it is possible to use symbiotic microbiome to control GIN parasites in livestock.
In conclusion, the adult male H. contortus appears to harbour more diverse microbiome as compared to the female. The abomasum has more diversity compared to both adult male and female H. contortus. Data obtained in this study has shown presence of bacterial genera (Vibrio, Lysinibacillus and Anaplasma) in the H. contortus and abomasum that can produce nematicidal volatile compounds that are active against nematodes, with possibility to be manipulated to control populations of GIN parasites within the sheep abomasum. To determine the exact nature of microbiome-H. contortus and host-H. Contortus microbiome interaction, additional and complementary analyses are necessary to uncover specific molecular mechanisms underlying the nematodemicrobiome as a method of controlling nematodes.

Materials and methods
Ethical approval. Ethical approval was obtained from the Onderstepoort Veterinary Institute Animal Ethics Committee (AEC) (AEC No: 36.18) and the scientific committee of the Integrated Pest Management subprogram, North-West University. All methods in this study were approved by AEC as well as the scientific committee of the Integrated Pest Management subprogram, North-West University, and performed in accordance with the relevant guidelines and regulations.
Animals and parasites. The study was conducted using 10 Dohne Merino sheep (Table S1), collected from Wauldby farm (E27° 37′ 32.7"'' S32° 35′ 07′') which consist of a flock of over 450 ewes situated in the Stutterheim district in the Eastern Cape Province, South Africa. This farm has a well-documented history of natural H. contortus infection.

Recovery and morphological characterization of gastrointestinal parasites. Naturally infected
Dohne Merino sheep were slaughtered under humane conditions. The abomasum was detached from the rest of the GIT and the contents of the abomasum including adult worms were collected 4,33 . The worms were recovered from 7 of the 10 sampled Dohne Merino sheep. The worms were first examined in detail with the assistance of an experienced helminthologist to identify and sexually differentiate worms based on their morphological characters as described by Lichtenfels et al. 20 and Jacquiet et al. 34 (Fig. S1). The morphological identification was further confirmed by PCR assay using species specific primers (Table S2). Amplicon library preparation and sequencing. A region of approximately 470 bp to 500 bp encircling the V3-V4 hypervariable region within the 16S rRNA gene was amplified using a set of commonly used primers for the analysis of the gut microbiome (Table S2). The region of 260 bp was also amplified using species specific primers for confirmation of H. contortus (Table S2)  Pre-processing and data analysis. De-multiplexed, paired-end fastq reads and a mapping file or metadata file were imported into the Quantitative Insights Into Microbial Ecology 2 program (QIIME2, ver. 2018.6.0, https ://qiime 2.org/). Sequencing reads were pre-processed, quality filtered and analysed using the Divisive Amplicon Denoising Algorithm 2 (DADA2) software package, wrapped in QIIME2 version 2018.6.0. Resultant feature sequences were summarized and then annotated using the QIIME2 q2-feature-classifier plugin and Silva 132 99% OTUs (full-length, seven-level taxonomy) classifier pre-trained to the full-length Silva database to assign taxonomy to all ribosomal sequence variants.

Statistical analysis.
Alpha and beta-diversity analyses were performed with the q2-diversity plugin wrapped in QIIME2 at a sampling depth of 15,000 sequences. Alpha diversity metrics (Shannon diversity index, Simpson diversity index, Pielou's measure of species evenness and Faith's Phylogenetic Diversity) were calculated and statistical significance determined using Kruskal-Wallis tests. The Beta diversity was calculated by Bray-Curtis and Jaccard distances, unweighted and weighted UniFrac metrics. Principal Coordinate analyses (PCoA) was performed based on unweighted and weighted UniFrac distances and visualized with QIIME2 emperor visualizer (ver. 2018.6.0) and the statistical significances were calculated using Permutational Multivariate Analysis Of Variance (PERMANOVA). Additional analysis were performed with in-house python scripts (Python version 3.6. 1, 2017-03-21), KronaTools and RStudio software using phyloseq package (R version 3.5.0, 2018-04-23). www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Data availability
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.