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infection with Trichuris suis (Li et al., 2012), and the changes in the caprine abomasal microbial composition induced by. H. contortus at 50-days infection (Li et ...
Vet Res Commun DOI 10.1007/s11259-017-9698-5

ORIGINAL ARTICLE

Microbial community and ovine host response varies with early and late stages of Haemonchus contortus infection Saeed El-Ashram 1,2,3,4 & Ibrahim Al Nasr 5,6 & Fathi Abouhajer 7,8 & Maged El-Kemary 4 & Guangping Huang 2 & Güngör Dinçel 9 & Rashid Mehmood 10 & Min Hu 11 & Xun Suo 2,3

Received: 3 April 2017 / Accepted: 17 August 2017 # Springer Science+Business Media B.V. 2017

Abstract The interactions between gastric microbiota, ovine host, and Haemonchus contortus portray the ovine gastric environment as a complex ecosystem, where all factors play a pertinent role in fine-tuning each other and in haemeostasis. We delineated the impact of early and late Haemonchus infection on abomasal and ruminal microbial community, as well as the ovine host. Twelve, parasite-naive lambs were divided into four groups, 7 days post-infection (dpi) and time-matched uninfected-control groups; 50 dpi and time-matched uninfected control groups were used for the experiment. Six sheep were inoculated with 5000 H. contortus infective larvae and followed for 7 or 50 days with their corresponding uninfected-control ones. Ovine abomasal tissues were collected for histological analysis and gastric fluids were collected for PH value measurements, microbial community isolation and Illumina MiSeq platform and

bioinformatic analysis. Our results showed that Haemonchus infection increased the abomasal gastric pH (P = 0.05) and resulted in necrotizing and inflammatory changes that were more severe during acute infection. Furthermore, infection increased the abomasal bacterial load and decreased the ruminal microbiome. A 7-day infection of sheep with H. contortus significantly altered approximately 98% and 94% of genera in the abomasal and ruminal bacterial profile, respectively (P = 0.04– 0.05). However, the approximate altered genera 50 days after infection in the ovine abomasal and ruminal microbiome were about 62% and 69%, correspondingly (P = 0.04–0.05) with increase in some bacteria and decrease in others. Overall, these results indicate that Haemonchus infection plays a crucial role in shaping stomach microbial community composition, and diversity.

Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11259-017-9698-5) contains supplementary material, which is available to authorized users. * Saeed El-Ashram [email protected]

5

College of Science and Arts in Unaizah, Qassim University, Unaizah, Saudi Arabia

* Min Hu [email protected]

6

College of Applied Health Sciences in Ar Rass, Qassim University, Ar Rass 51921, Saudi Arabia

* Xun Suo [email protected]

7

Asmarya University for Islamic Sciences, Zliten, Libya

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College of Animal Sciences and Technology, China Agricultural University (CAU) China, Beijing 100193, China

9

Eskil Vocational School, Aksaray University, Aksaray, Turkey

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Department of Computer Science and Information Technology, University of Management Sciences and Information Technology, Kotli, AJK, Pakistan

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State Key Laboratory of Agricultural Microbiology, Key Laboratory of Development of Veterinary Products, Ministry of Agriculture, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, Hubei, China

1

College of life Science and Engineering, Foshan University, 18 Jiangwan street, Foshan 528231, Guangdong, China

2

National Animal Protozoa Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China

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Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, Beijing 100193, China

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Faculty of Science, Kafrelsheikh University University, Kafr El-Sheikh, Egypt

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Keywords Histological analysis . Haemonchus contortus . Illumina MiSeq platform . Bioinformatic analysis . Abomasal and ruminal microbiome

Introduction Among the gastrointestinal parasites that cause losses to the farming industry, for example, Haemonchus, Ostertagia, Trichostrongylus, Nematodirus and Cooperia, the barber’s pole worm, Haemonchus contortus, is the predominant, blood- sucking, highly-pathogenic, and economically-important nematode that infects small ruminants (O'Connor et al., 2006). The economic impact of haemonchosis in sheep and goats has been reviewed in detail elsewhere (Roeber et al., 2013; Haemonchus contortus and Haemonchosis – past, present and future trends, 2016). It has been demonstrated that larvae provoked tiny haemorrhages as early as 3 days post-infection (dpi). Emergence of the larvae into the abomasal lumen commenced between 7 and 11 dpi, and all worms had molted to the 4th stage by 4 dpi. The early 4th stage larva (L4) has a provisional buccal capsule, which facilitates attachment to the abomasal mucosa (Rahman & Collins, 1990). The parasite becomes an L5 stage adult worm after a final molt, and female adult worms commence laying eggs to maintain the H. contortus life cycle (Miller & Horohov, 2006). It is an established fact that shedding of trichostrongylid eggs in faeces is an indication of worm burdens in sheep (Douch et al., 1996). In Saanen goat kids, nematode faecal egg counts peaked at 5–6 weeks of infection with H. contortus (Watson Tg, 1993). The egg excretion peak of H. contortus sheep was observed in the 4th and 5th weeks post-infection in two different breeds (Idris et al., 2011). Egg laying of adult H. contortus is strongly correlated with abomasal pH in lambs (Honde & Bueno, 1982). Existing research recognizes the severe pathogenicity of Campylobacter jejuni associated with concomitant faecal Trichuris suis ova (Shin et al., 2004). A growing body of evidence shows that parasitic infections are associated with elevated abomasal pH and hypergastrinaemia in ruminants and can thus potentially increase anaerobic microbial masses in the abomasum (Simpson et al., 1997; Li et al., 2011; Simcock et al., 1999; Li et al., 2016; Nicholls et al., 1987). All mammals harbor a wide diversity of microbes that live in harmony with their host and colonize the mucosal surfaces, including the digestive, respiratory, and reproductive tracts (Lee & Mazmanian, 2010). As gastrointestinal parasitic helminths and bacteria share the same microhabitat, it is reasonable that these organisms interact with each other and their host. The complex interactions that occur between gastrointestinal parasitic helminths and commensal bacteria are essential for the cross-talk between host and parasite establishment, and mucosal immune system development (Cantacessi et al., 2014; Berrilli et al., 2012). The microbial flora of the large intestine acts synergistically with Trichuris suis to generate the severe clinical syndrome

in conventionally-reared pigs (Rutter & Beer, 1975). Moreover, descriptions of the local and systemic reactions that accompany parasite infection have been reported by (Miller & Horohov, 2006; Adams, 1993; Kringel et al., 2006). Metagenomic tools have been used to examine the alterations of porcine proximal colon microbiota at 21-days of infection with Trichuris suis (Li et al., 2012), and the changes in the caprine abomasal microbial composition induced by H. contortus at 50-days infection (Li et al., 2016). Nevertheless, it is currently unexplored whether similar changes occur during Haemonchus infection in sheep. No investigation has been conducted during early and late H. contortus infection (histrophic larval-stage and persistent adult-stage, respectively) on the ovine microbial community, despite the importance of this parasite and its host. Driven by this need, this work seeks to explore the impact of early and late Haemonchus infection on the abomasal and ruminal microbial community (Illumina MiSeq platform) and the ovine host (histological and anatomical analyses).

Materials and methods Ethics statement Animal experiments were conducted in accordance with the guidelines of Beijing the Municipality on the Review of Welfare and Ethics of Laboratory Animals approved by the Beijing Municipality Administration Office of Laboratory Animals (BAOLA), and under the protocol (CAU-AEC2010–0603) approved by the China Agricultural University Animal Ethics Committee. All experimental procedures were also approved by the Institutional Animal Care and Committee of China Agricultural University (The certificate of Beijing Laboratory Animal employee, ID: 15,883). Animals and parasites Age-related resistance to H. contortus infection is welldocumented (Douch & Morum, 1993; Gruner et al., 2003); therefore, young weaned lambs were used in this study. Twomonth-old lambs (Ovis aries) that had raised under confined living conditions (reared with their mothers on wire-mesh floors, indoors) to avoid worm exposure were moved from a local farmer in Jin Zhan village, Chaoyang, Beijing, China with an average liveweight of 25.02 ± 2.2 kg. Faecal materials and blood were collected for parasitological (faecal egg counts (Gasso et al., 2015)), haemato-biochemical (peripheral eosinophilia, packed cell volume, serum protein and albumin concentrations (Bordoloi et al., 2012; Fausto et al., 2014)), and immunological (Ouchterlony double immunodiffusion (Christomanou & Harzer, 1996; Ouchterlony, 1958)) analyses (unpublished data) to confirm the absence of infection. Thereby, the animals used in the

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experimental infections had no prior exposure to parasitic infections before arriving at the experimental facility. Upon arrival at the China Agricultural University facilities, all sheep were tagged (each sheep was assigned an arbitrary number for sample identification purposes), weighed, and then treated with double doses of ivermectin (Shijiazhuang Fengqiang Animal Pharmaceutical Co., Ltd) and a single dose of levamisole (Hebei New Century Pharmaceutical Co., Ltd). These two, broad-spectrum anthelmintics (Ivermectin and Levamisole) are effective against nematode parasites to assure the removal of any undetected nematode infections. The lambs were acclimatized for four weeks and had reached nearly three months of age before infection experiments were initiated. The animals received a diet containing forage (grass silage): concentrate (barley, maize, wheat, wheat bran and bruised soya) 1:1 ad libitum. Moreover, a mineral animal salt lick block (Qingzhou Zhongyuan Chemical Industry Co., Ltd., China) was included in the facility for each animal group as an essential nutrient for maintaining ovine health. Sheep were exposed to a 12 h/12 h light/darkness regimen at mean internal relative humidity (23.4 ± 2.5) and temperature (19.28 ± 0.94) within the dwelling facility. Freshly prepared third-stage larvae (L3, infective stage) of H. contortus (GenBank accession number: X78803.1) were kindly provided by Professor Dr. Hu from State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430,070, Hubei, China after baermannized and diluted to a concentration of 1000 L3 / mL in sterile phosphate-buffered saline (PBS) (pH 7.4). Larval motility was examined before used.

remove all adhering digesta and worms from the mucosal surface. The contents and washings were collected and poured into a graduated cylinder, and aliquots were withdrawn for worm counts. For microscopic observation, a 1/10 aliquot was fixed in 7% formo1 saline, and the fixed larvae were cleared and stained in a dilute solution of aniline blue in lactophenol. The washed abomasal sections were digested with pepsin-HCI for 6 h at 42 °C. The digest was diluted, and ten two ml aliquots were investigated to estimate the number of larvae present (Dash, 1985; Stear et al., 1995; Blitz & Gibbs, 1971; El-Ashram et al., 2017). Faecal nematode egg counts Faecal egg counts were estimated in a 2 g rectal sample with a modified McMaster’s technique (Vadlejch et al., 2011). Faecal egg counts were conducted on day 0 until the end of the experiment. Abomasal histopathologic examination Small pieces of the abomasal fold were collected in 10% buffered formalin for histopathology. The fixed tissues were washed in running tap water over-night, dehydrated and infiltrated by paraffin wax. Serial paraffin sections (5 μm thickness) were obtained, and the sections were deparaffinized in three, consecutive washings in xylol for 5 min, and rehydrated with five, successive washings with alcohol in descending order of 100, 95, 80, 70, and 50% in deionized water. The histological sections were then subjected to conventional Hematoxylin and Eosin (H and E) staining procedure (Ding et al., 2011).

Parasite infections PH value measurements A total of twelve, 3-month-old sheep were randomly allocated into two groups (uninfected-control and Haemonchus-infected groups) each consisting of six sheep in separate areas within the university facility under the same environmental conditions to avoid cross-contamination. The first group, consisting of 6 sheep, served as control groups. The second group of six sheep after being fasted for 12 h was orally inoculated with a single dose of 5000 H. contortus L3 using a syringe attached to a short rubber tube and maintained for 7- and 50-days after infection. The sheep had food and water withheld for 12 h prior to sacrifice. They were euthanized at 7 (infected and uninfected-control groups, N = 3 for each group) and 50 days post-infection (dpi) (infected and uninfected-control sheep, N = 3 for each group). Worm recovery procedures Immediately after slaughter, the abomasum was tied off at the omaso-abomasal orifice and pylorus. The abomasum then was carefully removed and arranged in its natural position on a tray. It was opened along the greater curvature, and the inner surface was washed with tap water. A fine jet of water was further used to

Within 5 min after euthanasia, the abomasal and rumen digesta were collected apart into clean plastic buckets and thoroughly mixed. Gastric fluid pH was measured separately from the two compartments by a pH meter (Qingdao Tlead International Co., Ltd., China).

Sample collection, DNA extraction, amplification, and processing of samples for high-throughput sequencing Sample collection An aliquot of 50 ml of rumen and abomasum fluids was taken apart from each animal by squeezing the whole abomasum and rumen digesta through four layers of cheesecloth (each gastric compartment was processed separately for each sheep) within 20 min after euthanasia(without the morning feeding). Then the abomasal and ruminal fluids were centrifuged at 5000 rpm for 5 min, and the supernatant was centrifuged at 12,000 rpm for

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10 min. The supernatant was decanted, and the pellet of each sample was shipped to Beijing Allwegene Technology Co., Ltd., China on dry ice for sequencing of the abomasal and ruminal microbiota employing the Illumina MiSeq® platform. Microbial DNA extraction Microbial genomic DNA from samples was extracted using the E.Z.N.A.® Bacterial DNA Kit (Omega Bio-tek, Norcross, GA, U.S.) according to manufacturer’s instructions and (ElAshram et al., 2016). DNA products were assessed by electrophoresis using a 1% agarose gel run at 100 V for 30 min, and the sizes of the products were validated by comparison with a molecular size standard. The quantity and quality of the DNA were evaluated by measuring the absorbance employing an ultraviolet spectrophotometer (DU1 640, Beckman Instruments, Inc., CA, USA) at 260 and 280 nm. All microbial genomic DNA was stored at −20 °C before further analysis. Amplicon generation and high-throughput sequencing The 16S ribosomal RNA (rRNA) gene contains nine hypervariable regions enclosed by regions of more conserved sequences. A region of approximately 468 bp encircling the V3V4 hypervariable region within the 16S rRNA gene was subjected to high-throughput sequencing to maximize the effective length of sequencing reads (Mizrahi-Man et al., 2013). Sequencing was done on the Illumina MiSeq® platform at Beijing Allwegene Technology Co., Ltd., China. The V3-V4 region of the bacterial 16S rRNA gene sequences was amplified by employing the universal primers 338F (5′ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGAC TACHVGGGTWTCTAAT -3′) (Wu et al., 2016; El-Ashram & Suo, 2017). Briefly, each 50 μL of polymerase chain reaction (PCR) reaction contains 10 ng of abomasal or ruminal microbial genomic DNA as the template, 0.25 μL Pyrobest™ DNA Polymerase (Takara Biotechnology, Dalian CO., LTD), and 1 μL of 10 μM of each primer. PCR reactions were carried out using the following protocol: (1) an initial denaturation step performed at 95 °C for 5 min followed by 30 cycles of denaturation (95 °C, 30 s), annealing (56 °C, 30 s) and extension (72 °C, 40 s), and a final elongation of 10 min at 72 °C. The PCR products were separated by 1% agarose gel electrophoresis, and the 468 bp fragment was purified by using the E.Z.N.A.® Gel Extraction kit (Omega Bio-tek, GA, US). Sequencing libraries were generated using NEBNext ® Ultra DNA Library Prep Kit for Illumina ® (Illumina, USA) following manufacturer’s recommendations, and index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. Then, the library was sequenced on an Illumina MiSeq platform (Beijing Allwegene Technology Co., Ltd., China), and 300 bp pairedend reads were generated.

Statistical and bioinformatics analysis After trimming the adaptor and primer sequences from Illumina reads, the raw sequences were assembled for each sample according to the unique barcode using quantitative insights into microbial ecology (Qiime) (V1.7.0,http://qiime. org/scripts/split_libraries_fastq.html). Paired-end reads from the original DNA fragments were merged using FLASH (V1.2.7, http://ccb.jhu.edu/software/FLASH/). Quality filtering was performed under specific filtering conditions to obtain the high-quality clean tags according to the pipeline tool Qiime. The clean tags were compared against the reference database (Gold database,http://drive5.com/uchime/ uchime_download.html) employing UCHIME algorithm (UCHIME Algorithm, http://www.drive5.com/usearch/manual/ uchime_algo.html) to detect and remove chimeric sequences. Paired-end reads were assigned to each sample according to the unique barcodes. Sequence analysis was performed by UPARSE software package (Uparse v7.0.1001,http://drive5.com/uparse/) using the UPARSE-OTU ref. algorithms. Alpha (within samples) and beta (among samples) diversities were analyzed employing in-house Perl scripts. Sequences with ≥97% identities were assigned to the same OTUs. Representative sequences for each OTU were picked up to be used in the ribosomal database project (RDP) classifier to annotate the taxonomic information for each representative sequence. In order to estimate alpha diversity, we rarified the operational taxonomic units (OTUs) and calculated five metrics: Phylogenetic diversity (PD) Whole tree, Good’s coverage, Chao1, Shannon and Observed species. Rarefaction curves were created based on these five metrics. The Kruskal-Wallis test (Qi et al., 2014) was used to compare diversity metrics, such as genus evenness, Simpson and Shannon-Wiener species diversity indices. The level of significance was determined at P < 0.05. The Principal Coordinates Analysis (PCoA) was conducted to explore the differences in the bacterial community structures and was displayed with the WGCNA, stat and ggplot2 packages in the R software (Version 2.15.3) (Wu et al., 2012). Weighted and un-weighted unifrac distances were calculated employing QIIME.

Results Parasitological data Worm recovery rate, as a percentage of the number of infective larvae administered are listed in S1 Table. It remained approximately constant from 7 to 50 days after infection (p = 0.67; a twotailed t-test using Instat software [Graphpad Software, San Diego, Calif]). Eggs first appeared in the faeces at day 16 postinfection, peaked on day 34 post-infection, and then declined in subsequent days in lambs infected with H. contortus (S2 Table).

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Haemonchus infection-associated histopathologic changes of the abomasum Microscopic evaluation of the abomasal samples showed histopathologic lesions for all infected sheep. The early Haemonchus-infected abomasal tissues displayed haemorrhages and degeneration and/or necrosis of the mucosa. Furthermore, the mucosal changes were characterized by cystic enlargements with infiltration by mononuclear cells, especially lymphocytes. However, the late (50 dpi) infected abomasum was represented by milder hemorrhage, severe congestion of blood vessels in the lamina propria, and infiltration by mononuclear cells, notably lymphocytes, in conjunction with mild degeneration and/or necrosis of the mucosa. No histopathologic lesions of the abomasal mucosa were observed for the uninfected-control sheep (Fig. 1a-f). Fig. 1 Haemonchus infectionassociated histopathological changes of the abomasum. No significant lesions were noted in the abomasal tissue of the uninfected-control sheep. H&E 20X (a, b). Section of the early infected abomasum characterized by mild degeneration and/or necrosis, and infiltration by mononuclear cells, especially lymphocytes (arrow). H&E 10X (c). Section of the early infected abomasum characterized by hemorrhage and cystic enlargements (arrow). H&E 10X (d). Section of the late infected abomasum characterized by mild hemorrhage and infiltration of mononuclear cells, especially lymphocytes (arrow). H&E 10X (e). Section of the late infected abomasum characterized by severe congestion (arrow), H&E 10X (f)

Haemonchus infection-associated microhabitat changes In infected groups, abomasal and ruminal pH values were significantly increased during the early stage of infection (i.e. 7 dpi) as compared to uninfected-control sheep. Moreover, the infection significantly augmented the abomasal pH value at 50 dpi. No significant differences in the ruminal pH value at 50 dpi were found compared to the uninfected-control group (S3 Table). Haemonchus infection-associated abomasal and ruminal microbial changes The 16S rRNA amplicon metagenomic analysis of abomasal/ruminal bacteria Four groups of sheep, 7 dpi and time-matched uninfected-control groups; 50 dpi and time-matched uninfected-control groups, were used for the experiment; however, only three groups were

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presented for all the data. No significant differences were observed between the uninfected-control groups of the two time points. Thereby, one group was presented as a control group. Rarefaction analysis (S4 Table; S5 Table) of abomasal and ruminal bacteria revealed that the number of OTUs for 16S rRNA gene sequences tended to plateau after 67,618 and 38,035 sequences respectively at 97% identity. This suggested that the sequencing result could relatively reflect the microbial diversity and the OTU richness as a function of sequencing of these abomasal and ruminal samples precisely. General analyses of the sequencing data indicated that a total average of 95,916/100282, 91,042/47756 and 104,282/102143 reads for abomasal/ruminal 16S rRNA gene sequences of uninfected-control, 7 dpi and 50 dpi groups, respectively, could be paired successfully. After removing short and low-quality reads, singletons, replicates and chimeras, the average of 92,539/99894, 86,230/45655 and 100,369/101649 clean tags were retained for 16S rRNA gene sequences of uninfected-control, 7 dpi and 50 dpi groups of abomasal and ruminal microbial communities, respectively. Based on 97% identity, a total of 3342.99992 ± 4.58235/ 3369 ± 24.9045, 4187.9986 ± 8.8916/2238.99767 ± 13.0035, and 3351.33178 ± 2.3094/2658.99267 ± 25.9895 operational taxonomic units (OTUs) for uninfected control, 7 dpi and 50 dpi groups were obtained for abomasal and ruminal 16S rRNA gene sequences (S6 Table) in corresponding order. Therefore, infection increased the abomasal bacterial load and decreased the ruminal bacterial load. The body sites with the greatest number of core OTUs, defined as OTUs shared among the three groups (100%) sampled, were the abomasal site followed by the rumen. All three groups of abomasal and ruminal microbiomes (i.e. uninfected-control, 7 dpi and 50 dpi groups) shared 978 and 570 OTUs, correspondingly (S7 Table; S8 Table). The Venn Diagrams showed that 28.36%/ 51.795, 45.95%/46.18 and 25.76%/42.87 of the OTUs were unique to the three groups of the abomasum and rumen, respectively (Figs. 2Ai and 3Bi). We then identified the set of core OTUs present in all sheep (infected and uninfected-control) across the abomasum and rumen. As shown in S9 Table, there were 495 OTUs existing in all ovine samples (abomasum and rumen), the most abundant of which belonged to genus Prevotella, Butyrivibrio, RC9_gut_group and Treponema. Diversity index analysis of microbial community Samples were evaluated for alpha diversity (microbial diversity within samples) and beta diversity (community diversity between samples) analysis. Alpha rarefaction curves The alpha diversity indices represent species richness/number of OTUs per group that was present in the abomasal and ruminal samples at different time intervals compared to the uninfected-

control group using various metrics (Fig. 2Ai-Avi, S2 Table, Fig. 3Bii-Bvi, S5 Table). Alpha rarefaction curves of the abomasal and ruminal bacterial communities (7 dpi and 50 dpi) were computed exploiting the alpha_rarefaction.py in Qiime. Alpha diversity metrics, such as Phylogenetic diversity (PD) whole tree, Good’s coverage, Chao1, Shannon and Observed species were plotted. They illustrated the number of observed species in a random pool of sequences in different depths (number of sequences). The common microbial diversity indices, such as Simpson’s index, Shannon-Wiener Species diversity index (H) and genus richness (S6 Table), were evaluated. Our results suggest that the abomasal and ruminal microbial community of the early H. contortus-infected sheep had been more taxon-rich compared to that in the late infection sheep. Additionally, it is apparent from our data that the early and late Haemonchus infection groups had abrogated archaeal DNA. The abomasal and ruminal microbial communities of the early and late H. contortus -infected sheep displayed significant differences employing genus evenness, Simpson and Shannon-Wiener species diversity indices at the genus-level in comparison with the uninfectedcontrol group. Beta diversity Computing differences between microbial communities were calculated exploiting the default beta diversity metrics of weighted and unweighted UniFrac (Lozupone & Knight, 2005)(beta_diversity_through_plots.py). PCoA was plotted using the result of UniFrac distance matrices to determine the similarity between groups of samples/time-points. The three-dimensional PCoA plots were visualized employing the Emperor tool (Vazquez-Baeza et al., 2013). The beta diversity analysis showed three, very distinct clusters separating Haemonchus- infected (early and late stages of infection) and uninfected-control sheep (Fig. 4). The analysis of microbial community structure and dominant taxa The abomasal and ruminal microbial communities were studied to identify H. contortus-induced shifts using the Illumina MiSeq platform in an experiment where the animals were fed a fixedformula diet containing forage and concentrate in a ratio of 1 to 1. Throughout Haemonchus infection, significant changes in the abundance and composition of abomasal and ruminal microbiota were observed. Sixteen phyla for uninfected control group, sixteen phyla for 7 dpi group, and fifteen phyla for 50 dpi group from the abomasal microbial community were identified. For a better survey, taxonomic abundances of all taxa can be visualized in a Krona plot (Ondov et al., 2011)that is based on the average relative abundance of the abomasal and ruminal microbial community associated with the different stages of Haemonchus- infected sheep (Fig. 5).

Vet Res Commun Fig. 2 Venn diagram (Ai) and alpha diversity analysis (Aii-Avi) of Haemonchus- infected (7 and 50 dpi) and uninfected-control sheep based on 16S rRNA gene sequence of abomasal microbiota and 97% identity at genus level. Rarefraction curves for (Aii) observed species (Aiii) Shannon, (Aiv) Chao1, (Av) Good’s coverage, and (Avi) PD whole tree. Data represent mean values from 3 separate samples per group. The rarefaction curves for abomasal microbiota reached the near plateau phase depicting satisfying sampling depth

For all groups, including the uninfected-control, 7 dpi and 50 dpi groups, the luminal abomasum microbiota was highly dominated (i.e. had a relative abundance greater than 1.0%) by microbes of the following bacterial phyla: Bacteroidetes (65.25%), Firmicutes (24.96%), Fibrobacteres (2.79%), Lentisphaerae (2.18%), Proteobacteria (1.41%), Spirochaetae (1.07%), and Tenericutes (1.05%) for the uninfected control group, Bacteroidetes (71.23%), Firmicutes (18.43%), Proteobacteria (5.6%), and Lentisphaerae (1.23%) for the 7 dpi group, Bacteroidetes (57.7%), Firmicutes (35.42%), Proteobacteria (2.86%), and Fibrobacteres (1.14%) for the 50 dpi group. Among the classes collectively detected, Bacteroidia and Clostridia accounted for closely (88.11%), (84.48%) and (87.63%) for the uninfected-control, 7 dpi and 50 dpi groups respectively. The most abundant genus in the ovine abomasum was Prevetella, which accounted for approximately (38.56%), (55.4%) and (42.23%) of all sequences for the uninfected-

control, 7 dpi and 50 dpi groups in corresponding order. However, the highly dominant ruminal OTUs belonged to B a c t e ro i d e t e s ( 7 9 . 8 2 % ) , F i r m i c u t e s ( 1 4 . 3 3 % ) , Proteobacteria (1.43%) and Spirochaetae (1.3%) for control group, Bacteroidetes (61.26%), Firmicutes (31.25%), Proteobacteria (2.23%), Lentisphaerae (1.36%) and Spirochaetae (1.28%) for 7 dpi group, Bacteroidetes (76.16%), Firmicutes (5.99%), Proteobacteria (15.75%) and Spirochaetae (1.44%) for the 50 dpi group. Among the classes collectively detected, Bacteroidia and Clostridia accounted for approximately 90.44%, 86.23%, and 79.79% for the uninfected control, 7 dpi and 50 dpi groups, respectively. The most highly abundant genus in the ovine rumen was Prevetella, which accounted for nearly 60.1%, 49.07% and 68.97% of all sequences for the uninfected-control, 7 dpi and 50 dpi groups, respectively. Infection seemed to induce significant changes in the microbial composition of the abomasal phyla, including

Vet Res Commun Fig. 3 Venn diagram (Bi) and alpha diversity analysis (Bii-Bvi) of Haemonchus- infected (7 and 50 dpi) and uninfected-control sheep based on 16S rRNA gene sequence of ruminal microbiota and 97% identity at genus level. Rarefraction curves for (Bii) observed species (Biii) Shannon, (Biv) Chao1, (Bv) Good’s coverage, and (Bvi) PD whole tree. Data represent mean values from 3 separate samples per group. The rarefaction curves for ruminal microbiota reached the near plateau phase depicting satisfying sampling depth

L e n t i s p h a e r a e , B a c t e r o i d e t e s , Te n e r i c u t e s , Candidate_division_TM7, Synergistetes, Firmicutes, E l u s i m i c ro b i a , F i b ro b a c t e re s , P ro t e o b a c t e r i a , Cyanobacteria, Actinobacteria and Spirochaetae, and ruminal microbiota phyla, such as Tenericutes, Lentisphaerae, F i b ro b a c t e r e s , P ro t e o b a c t e r i a , S p i ro c h a e t a e , Actinobacteria, Candidate_division_TM7, Firmicutes, and Bacteroidetes compared to those of uninfected-control animals (S10 Table; S11 Table).

Discussion The current study was conducted to investigate Haemonchus infection-associated abomasal histopathological, abomasal and ruminal microhabitat, and abomasal and ruminal microbial changes.

The more extreme histopathological changes observed in the abomasal mucosa at 7 dpi were necrosis of the mucosa, severe cystic enlargements, and infiltration by mononuclear cells, especially lymphocytes compared to the mild changes observed in the 50 dpi group. Similar changes have been reported previously by (Salman & Duncan, 1984; Javanbakht et al., 2014). With respect to pH values, the present study showed an association between the histopathological changes of the abomasum and its luminal pH value. The abomasal pH was significantly increased during the early (7 dpi) and late (50 dpi) stages of infection compared to the uninfected-control group (P = 0.05). These results are in line with those of previous studies (Li et al., 2016; Rahman & Collins, 1991). Moreover, the Kruskal-Wallis test showed that ruminal fluid pH values of the healthy and infected sheep were statistically augmented at 7 dpi (P = 0.05); however, the ruminal pH values remained largely within normal limits at 50 dpi (P = 0.13). These findings

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Fig. 4 Principal coordinates analysis (PCoA) of abomasal (a) and ruminal (b) microbial community (between sample diversity). The plot displayed three very distinct clusters of gastric microbial communities delineating Haemonchus- infected (7 dpi and 50 dpi) and uninfected-control sheep

corroborate the ideas of (Hertzberg et al., 2000), who suggested that Ostertagia leptospicularis mediated inhibition of abomasal acid secretion in sheep. Lowered numbers of parietal cells due to the severe abomasal histopathological changes associated

with the histotrophic larval phase are the most likely causes of the abomasal pH increase. This study has been unable to demonstrate any significant effect on the live weight of lambs infected with H. contortus

Fig. 5 Graphical representation of the average relative abundance of abomasal (a-c) and ruminal bacterial (d-f) diversity from phylum to genus level based on 16S rRNA gene sequence (V3-V4 region)

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Fig. 5 (continued)

Fig. 5 (continued)

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Fig. 5 (continued)

Fig. 5 (continued)

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Fig. 5 (continued)

(i.e. ovine yield). However, these findings do not support previous research (Li et al., 2016). This inconsistency may be because of the different food regimens employed and the genetic background of hosts (goats versus sheep). These results further support the idea that lambs on diets sufficient in protein and trace elements have an increased capacity to withstand parasitic pathogenesis and harbor a less severe parasitic infection (Strain & Stear, 2001; Louvandini et al., 2006; Coop & Field, 1983). Additionally, compared to sheep, the goat immune response against parasitic nematodes was less effective (Hoste et al., 2008). Furthermore, the experimental infection by a gastrointestinal ovine parasite increased the abomasal bacterial load and decreased the ruminal microbiome. The abomasal microbiome showed significant differences in various microbial diversity indices, including Simpson’s index, Shannon-Wiener Species diversity index, and genus richness at the genus level in the 7 and 50 dpi groups compared to the uninfected-control (P = 0.04–0.05). Although these results differ from some published studies (Li et al., 2016; Cantacessi et al., 2014), they are consistent with those of (Lee et al., 2014), who reported soil-transmitted helminth colonization was associated with increased gut microbial diversity. Relatedly, the ruminal microbiome displayed significant differences in various microbial diversity indices, such as Simpson’s index, Shannon-Wiener Species diversity index

and genus richness at the early and late stage of infection compared to the uninfected-control sheep (P = 0.05). These results further confirm the association between Haemonchus infection, and bacterial community structure and diversity. Strikingly, several studies have documented that infection with helminth parasites elicited a significant change within the gut microbiome structure and function (Li et al., 2011; Li et al., 2012; Plieskatt et al., 2013). A 7-day infection of sheep with H. contortus significantly altered nearly 98% and 94% of genera in the abomasal and ruminal microbiome, in a corresponding manner (P = 0.04–0.05). In this context, the approximate altered genera 50 days after infection in the ovine abomasal and ruminal microbiome were around 62% and 69%, respectively (P = 0.04–0.05). Strikingly, Haemonchus infection may have pronounced effect on the abomasal and ruminal microbiome at 7 dpi; however, only slight differences at 50 dpi have been found in the abomasal and ruminal microbiome. Relatedly, infection altered the abundance of nearly 13% of genera in the proximal colon microbiome in a 21-day infection of pigs with the whipworm Trichuris suis (Li et al., 2012). A 50-day infection of goats with H. contortus significantly altered the abundance of approximately 19% of the 432 species-level OTUs detected per sample in the abomasal microbiome (Li et al., 2016). Similar effects on the gastrointestinal microbiome have been reported in hamsters infected with the liver fluke, Opisthorchis viverrini, and in pigs

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infected with the whipworm Trichuris suis (Lee et al., 2014; Plieskatt et al., 2013). Furthermore, our results did show that the abomasal microbiome of Haemonchus - infected sheep had greater genus richness at 7 dpi compared to 50 dpi and control-uninfected groups, respectively (P = 0.04). Similarly, the genus richness of the 7 dpi ruminal microbial group tends to greater than in control-uninfected and 50 dpi groups, correspondingly (P = 0.05). The most striking result to emerge from the abomasal and ruminal microbial data is that the genus, Prevotella was significantly different in the 7 dpi group compared to the controluninfected and 50 dpi groups (P = 0.05). It has been demonstrated that the dominance of Prevotella was associated with higher carbohydrates, especially fiber-rich diets in humans (Wu et al., 2011). In ruminants, Prevotella possesses a ratelimiting dipeptidyl peptidase type IV (DPP-IV) activity, which plays a vital role in ruminal protein degradation (Li et al., 2016). These results corroborate the ideas of (Li et al., 2016), who suggested that the increased Prevotella richness in Haemonchus-infected goats would have functional significance in host protein metabolism. This view is supported by (Alauzet et al., 2010), who reported that changes in the abundance and diversity of Prevotella were observed during dysbacteriosis -associated diseases. Helminths possess potent immuno-modulatory activity. Manipulation of the gut butyrate biosynthesis underlies antiinflammatory responses induced by altering butyrateproducing bacteria abundance (Forbes et al., 2016; Zaiss & Harris, 2016). In 2000, (Saemann et al., 2000) published a paper in which they described the anti-inflammatory property of butyrate (Saemann et al., 2000). Intriguingly, our data show significant differences in the relative abundance of the genus Butyrivibrio in the early and late Haemonchus - infected sheep in the abomasal and ruminal microbiome (P = 0.05) compared to the uninfected-control group. These results do support the previous research of (Li et al., 2016), who reported that Haemonchus infection could significantly alter the abundance of butyrate-producing bacteria of the abomasum at day 50 of the post-infection period. The ruminal exsheathment of the third-stage larvae (L3) of H. contortus (Hertzberg et al., 2002), the severe interaction between the histotrophic larvae and the ovine abomasum, and the mucosal and systemic immunity to the histotrophic stage and adult stage of H. contortus (Miller & Horohov, 2006; Adams, 1993; Kringel et al., 2006) could be the reason for the global changes in the gastric microbiota. Therefore, the abomasal parasite interacts with the ovine abomasal and ruminal microbiome modifying the balance between host and gastric microbiota. In general, therefore, it seems that H. contortus manipulates its microhabitat, which facilitates their survival, increases their reproduction, and maintains their niche restriction. Future research should thereby concentrate on the investigation of

prebiotic and probiotic supplements under the influence of helminths, bacteria, and the host for the long-term maintenance of sheep health. Acknowledgments This research was supported by the National Key Basic Research Program (973 program) of China (Grant No. 2015CB150300), and the Start-up Research Grant Program provided by Foshan University, Foshan city, Guangdong province for distinguished researchers. Additionally, the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author Contributions Revising the final version. Compliance with ethical standards Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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