| Haemonchus contortus is an important ruminant gastrointestinal nematode, which is also able to infect human and the rodent. The prevalence of Haemonchusis relies on the climate, geography, grazing conditions and host conditions. Drug resistance and the lack of effective vaccines bring difficulties to the control of the disease. The development of immunological protective method is in urgent need, in which exploring the rule of host immune response to H. contortus infection is a vital research domain. In this study, the prevalence of sheep and goat gastrointestinal parasitic disease and the phylogenetic evolution of H. contortus in some parts of China were analyzed. Microarray was used to compare gene expression to obtain a systematic transcriptional profiling of the T lymphocytes in primary H. contortus infected sheep. Quantitative RT-PCR was utilized to analyze the dynamic expression of cytokines in T lymphocytes, which contributed to determine the type of T lymphocytes immune response. Nematode-cell co-culture system and gerbil infection model were built to mimic the host early immune response to H. contortus infection. The immunity index in co-culture system and gerbil model was detected to state the early immune response. The results would lay a foundation for researches on the host immune response to H. contortus infection.1. Epidemiology of sheep and goat gastrointestinal parasitic disease and the phylogenetic analysis of H. contortus in some parts of ChinaFour hundred and ninety fecal samples from nine provinces of China, including Shandong, Jilin, Heilongjiang, Neimenggu, Liaoning, Hebei, Jiangsu, Tianjin and Qinghai, were collected in winter and autumn to examine eggs of sheep and goat parasites. Fifteen adult samples from Jiaxing, Zhejiang were gathered to analyze the phylogenetic evolution of H. contortus during the decade by ITS-2 and ND4 sequence analysis. Severe parasite infections all over the country were observed in the results. The infection rate of protozoon, nematode and tapeworm were respectively 83.27%, 64.29% and 49.80%. The most three severe provinces were Tianjin, Qinghai and Neimenggu. The least three severe provinces were Jiangsu, Heilongjiang and Shandong. The phylogenetic analysis of Jiaxing strain of H. contortus showed that ITS-2 and ND4 varied very little and relatively randomly. The ranges of genetic variation of ITS-2 and ND4 were 0.0-3.9% and 0.4-4.8%. This survey would provide a basis for making a suitable control strategy.2. Profiling of differentially expressed genes in sheep T lymphocytes response to an artificial primary H. contortus infectionTo study the host T lymphocytes response, a primary H. contortus infection model in sheep was built, and microarrays were used to compare gene expression between 0 days post infection (dpi),3 dpi,30 dpi and 60 dpi infected sheep to obtain a systematic transcriptional profiling of the T lymphocytes in H. contortus artificial-infected sheep. H. contortus began to lay eggs at 25 dpi and peaked by 30 dpi. A spawning cycle was finished by 43dpi. T lymphocytes proliferated from the beginning of infection. Serum level of total IgG significantly increased since the infection and reached the top at 30 dpi. A higher level was remained until 60dpi. In microarrays,853,242,42,1058,805 and 102 differentially expressed genes were obtained in the 3d vs. 0d,30d vs. 0d,60d vs. 0d,30d vs.3d,60d vs.3d and 60d vs.30d comparisons, respectively. Gene Ontology and KEGG pathway analysis indicated that these genes were involved in metabolism, signaling, cell growth and immune system processes. Functional analysis of significant differentially expressed genes including SLC9A3R2, ABCB9, COMMD4, SUGT1, FCER1Q GSK3A, PAK4 and FCER2 revealed a crucial association with cellular homeostasis maintainence and immune response. The increased expression level of IL-13 at 60 dpi was possibly related to the clearance of nematodes at late infection. The rule of sheep immune response to H. contortus infection was clarified to some extent.3. Sheep T lymphocytes immune response to H. contortus infectionBased on the sheep infection model, the expression level of thirteen cytokines in sheep T lymphocytes at 0 dpi,3 dpi,30 dpi and 60 dpi were detected by qRT-PCR. At 3 dpi, the expression of regulative cytokines IL-10 and TGF-β expression was increased, which indicated a leading role of Tregs in early infections. At 30 dpi, the expression levels of Thl cytokines IL-2, IFN-y, TNF-β and GM-CSF were elevated, as well as Th2 cytokines IL-13, IL-15 and IL-33, showing a Th1-and Th2-type immune response. At 60 dpi, only Thl cytokines raised their expression levels, demonstrating a strong Thl-type immune response. This experiment was a supplement to the description of sheep immune response to H. contortus infection.4. Research on the early immune response utilizing a nematode-cell co-culture systemTo study the host early immune response, an air-liquid nematode-cell co-culture system was built using tranwell inserts. Microscopic examination revealed two locomotive behaviours of H. contortus, either a sinusoidal motion or a circular burrowing one. The larvae ingested cells based on the Far-Red labeled cells and the filling of intestinal tract with labeled proteins from cells. The length of larvae at 0,4 and 7 days post co-culture (dpc) were 981.5±24.3 μm,1113.9±24.4μm and 1157.0 ±47.6 μm respectively, which at 7 dpc was similar to that in the host at 4 dpi. Reduced vitality of larvae was observed after co-colture, and larvae presented a state of curled up. Cells were dislodged from the collagen pad by larvae at the same time, and necrotic. The necrosis rate of MGC at 4 dpc increased from 12.91% to 53.25% compared to 0 dpc, and that of BGC increased from 2.63% to 24.81%. IL-33 gene expression in cells was significantly increased after co-culture, consistent with a high expression in the host. The expression level of IL-33 in MGC at 4 and 7 dpc were 2.520-fold and 4.180-fold higher than that at 0 dpc. The relative expression of IL-33 in BGC at 4 and 7 dpc raised to 1.732 and 5.150 compares to that at 0 dpc as well. Tissue damage and early immune response were studied by the nematode-cell co-culture system in vitro.5. Primary exploration of a gerbil model infected with H. contortusTo obtain a convenient and useful lab animal infection model for research on early immune response, a gerbil model infected with H. contortus was built. Larvae collected from gerbil stomach were at the early stage of L4. The lengths of larvae at 4, 7 and 18 dpi were 1099.8±169.0μm,1273.0±121.7μm and 1553.4±132.8μm. Larvae recovered at 4 dpi were separated into males and females. Some differentiation into functional entities, such as the vulva ovejector primordium appeared in the genital primordium at 7 dpi. Growth of larvae in gerbils was much slower than that in sheep. Rod-like crystalline inclusions were observed in the intestinal cells of larvae. Blood routine examination of gerbils displayed unobvious changes after infection. Histological examination of gerbil stomach sections showed that larvae were located in the lumens or at the mucosal surfaces with few evident inflammatory changes. These results demonstrated that gerbil model needed to be further studied, although H. contortus succeeded to parasitize gerbils.In conclusion, the investigation of the prevalence of sheep and goat gastrointestinal parasitic diseases and the phylogenetic analysis of H. contortus in some parts of China were performed. Differentially expressed genes and immune response type in sheep T lymphocytes response to an artificial primary H. contortus infection were analyzed. The host early immune response to H. contortus infection was studied by building the nematode-cell co-culture system and gerbil infection model. This study would pave for researches on the host immune response to H. contortus infection, and provide a basis for making effective immunological control strategies. |
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