| 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.The control of ovine haemonchosis relies heavily on the employment of anthelmintic drugs.However,the intensive and frequent administration of such anthelmintic drugs has led to the emergence of drug-resistant abomasal parasites and restricted the continued use of these drugs in the future.Furthermore,the vaccines to contro1 H.contortus infection don't achieve the sterile immunity associated with vaccines developed for bacterial and viral disease control.Therefore,a better understanding of the host-parasite interaction is an indispensible to develop a cost effective control strategies as potential options for the control of haemonchosis in sheep and the proper management of sheep in various production systems.In parallel with these,many different parameters have to be taken into consideration:no existence for DNA or RNA extraction method that is suitable for all prokaryotic and eukaryotic organisms with reasonable environment for RNase to have DNA free of RNA and even for DNase to degrade the DNA,and harvesting and purifying parasite oocysts and eggs on a large scale in the shortest duration from diverse volumes of environmental samples.Most of the biochemical and parasitological approaches described in the literature so far do not fulfill the optimal conditions.With this in mind,the aim of the current study is to examine the microbial communities associated with the host and the parasite as well.Furthermore,in vivo and ex vivo systems within the context of Haemonchus infection are essential to further unravel the biology of the parasite at the molecular level with a particular emphasis on improving some parasitological techniques,such as harvesting and purifying parasite eggs on a large-scale in the shortest duration,and biochemical techniques,including nucleic acid extraction.As far as some parasitological and biochemical techniques are concerned,our results showed that DNA and RNA are under triple protection(i.e.EDTA,SDS and NaCl)during lysis step,and this environment is improper for RNase to have DNA liberated of RNA and even for DNase to degrade the DNA.Therefore,the complete removal of RNA under RNase influence is achieved when RNase is added after DNA extraction,which gives optimal quality with any protocols.Similarly,DNA contamination in an isolated RNA is degraded by DNase to obtain high-quality RNA.Furthermore,an alternative way has been described using the combination of electrical cream separator and vacuum filtration for harvesting and purifying eimerian oocysts and haemonchine eggs on large-scale applications with approximately 81%and 92%recovery rates for oocysts and nematode eggs obtained from avian and ovine faeces,correspondingly.The sporulation percentages as a measure of viability in the harvested oocysts and eggs from dry faecal materials are nearly 68%and 74%,respectively,and 12 liters of faecal suspension can be processed in approximately 7.5 min.We hope to encourage other researchers to adapt these very powerful techniques to their needs by making our protocols available to the scientific community at large,and thus make it even more widely applicable.We delineated the impact of early and late Haemonchus infection on abomasal and ruminal microbial community,as well as the ovine host.Ovine abomasal tissues were collected for histological and anatomical analyses and gastric fluids collected to measure PH values,microbial community isolated and subjected to the Illumina Mi Seq platform and bioinformatic analysis.Our results showed that Haemonchus infection increased the abomasal gastric pH(P = 0.04953)and caused a substantial augmentation in anterior blind sac papillae numbers(P = 0.0463),as well as resulted in necrotizing and inflammatory changes that were more severe during acute infection.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 = 0.0369-0.0495).However,the approximate altered genera 50 days after infection in the ovine abomasal and ruminal microbiome were about 62%and 69%,correspondingly(P = 0.0369-0.0495)with increase in some bacteria and decrease in others.Illumina MiSeq platform was exploited to study the microbial community associated with the different stages of the life-cycle of ovine Haemonchus contortus field strains using two distinct amplification primer sets(targeting V3-V4,and V5-V7).Results showed 48(V3-V4 tags)and 28(V5-V7 tags)bacterial genera comprised the microbial flora of H.contortus microbiome.The dominant bacterial genera belonged to Escherichia-Shigella,Pseudomonas and Ochrobactrum,which were shared in all the stages of the parasite life-cycle using V3-V4 and V5-V7 amplicons.Transcriptional profiling of the ovine abomasal tissues has been conducted using the Illumina Hi Seq 4000 platform to segregate early and late H.contortus-infected sheep(7 and 50 days post-infected groups,respectively)from the control naive ones.A total of 548,357 and 7 were substantially induced genes in 7 days post-infection versus uninfected-control group,50 days post-infection versus 7 days post-infection(7 dpi),and 50 days post-infection(50 dpi)versus uninfected-control group,respectively.However,a total of 301,355 and 11 were significantly repressed genes between 7 dpi versus uninfected-control group,50 dpi versus 7 dpi,and 50 dpi versus uninfected-control group,correspondingly.This indicates that H.contortus infection induced a more potent activation of abomasal gene expression in the early stage of infection as compared to the late stage.To study the immediate immune response underlying ovine mucosa invasion by H.contortus larval-stage,we have set up an ex vivo ovine fundic mucosa model which can mimic the multicellular process to explore the early steps in haemonchine nematode infection using the Illumina HiSeq 4000 platform.Ovine abomasal explants were collected for histological and transcriptional analysis and supernatants collected to quantitate LDH enzymes.Key genes observed in response to the addition of L4-inoculum of H.contortus,which included IL-6,IL-8,C1q,atypical chemokine receptor-3,chemokine(C-C motif)ligand-2,manganese superoxide dismutase,integrin alpha-2(ITGA7,8,9),integrin subunit beta-1(ITGB1),integrin subunit beta 6(ITGB6),ICAM-1(intercellular adhesion molecule-1)and actin alpha-1(ACTN1).Overall,these results indicate that Haemonchus infection plays a crucial role in shaping gastric microbial community composition,and diversity,and the parasite microbiome could reflect the external micro-organisms(i.e.micro-and macro-habitats).Moreover,galectins can be viewed as regulatory molecules acting at various stages of infection along the continuum from immediate(Gal-1/6 hours post-inoculation)and early(Gal-4/7 days post-infection)to late(Gal-11/50 days post-infection)Haemonchus infection.These studies are completely widening our understanding of the ovine and haemonchine microbiome,immediate,early and late gene expression profiles in response to Haemonchus infection,improving some parasitological techniques,such as harvesting and purifying parasite eggs on a large-scale in the shortest duration with future application of the fully automatic centrifugal cream separator with mechanical washing and discharge of faecal residue,and biochemical techniques,including nucleic acid extraction,and much remains as important implications for future practice.As galectins are expressed by H.contortus and sheep,it becomes essential to identify,clone,and characterize galectins from many sources.Furthermore,the detection of H.contortus elicitors results in the up-regulation of defense-related genes and the elicitation of inflammatory and immunoregulatory responses.This paucity(the diversity of immune elicitors)should be addressed.Generally,this thesis presents the research already achieved,and anticipates the likely scale of the discoveries to come,with their implications for GIN parasites,including H.comtortus. |
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