Biological Roles Of Galectins Of Haemonchus Contortus

Haemonchus contortus is an important gastrointestinal nematode parasite ofruminants (notably goats and sheep) in many parts of the world, which belongs toHaemonchus genus of Trichostrongylidae family. It is a blood-feeding nematodeparasite that infects the abomasums (fourth or true stomach) and small intestine ofruminants. Infection can lead to anaemia, loss of condition and the death of the host(especially lambs) in severe cases, which cause significant economic loss ofhusbandry industry.Lectins are proteins that bind to specific carbohydrate structures, which existvastly in nature. They are not derived from immune systems and absent of enzymeactivities, but can cause the agglutination of cells. The functions of lectins areinvolved in a series of physiological and pathological processes, such as fecundation,development, cell proliferation, differentiation, homeostasis, immune response,infection, inflammation, cancer cell metastasis and cardioencephalo-angiologicaldiseases associated with thrombus formation and so on. Galectins consisted of afamily of lectins, that specifically bindβ-galactoside sugar structures in a metalindependent manner. The functions of these molecules are extremely diverse, asgalectins have been shown to be involved in processes such as cell adhesion,apoptosis and immune responses.Although there have been more than one hundred years history in scientificresearch on lectins of plants, it is very late to commence the research on the lectins ofnematode parasites. As to the research on galectins of H. contortus parasite, it wasonly started in recent ten years. Substantial protection against H. contortus has beenachieved by immunizing sheep with a glycoprotein fraction isolated from the intestinal membranes of this parasite (Smith et al. 1994). This fraction was termedHaemonchus galactose-containing glycoprotein (H-gal-GP) complex. Vaccinationwith H-gal-GP gives a＞70% reduction in worm burden with a concomitant reductionin faecal egg output of＞90% when compared with unvaccinated, control animals.Since several reports demonstrated that galectin was a part of the H-gal-GP complex,a number of galectins of H. contortus have been cloned and characterized. Galectinshave been focused on as one of the most promising antigens of vaccine. Previously,Hco-gal-m and Hco-gal-f galectins were cloned from male and female H. contortusand were expressed by our laboratory. The research work was sequentially carried asfollows:1. Recombinant galectins of Hemonchus contortus inhibited goat cytokine mRNAtranscription of peripheral blood lymphocyte cells in vitroThe effects of recombinant galectins of male and female H. contortus(rHco-gal-m/f) on the mRNA levels of IL-1β, IL-2, IL-4, IL-6, interferon-γ(IFN-γ)and tumor necrosis factor-α(TNF-α) of goat peripheral blood lymphoccyte cells(PBLCs) were observed in this study. Blood samples were collected from five,randomly selected two year old healthy goats. PBLCs were separated and cultured invitro with ConA or LPS, at varying rHco-gal-m/f protein concentrations of 0μg/ml,10μg/ml, 20μg/ml and 40μg/ml. Semi-quantitative reverse transcription (RT)-PCRwas employed to test the synthesis of mRNA. The results showed that the mRNAtranscriptions of IL-1β, IL-2, IL-4, IL-6, IFN-γand TNF-αin the PBLCs wereinhibited by rHco-gal-m in a dose-dependent manner, rHco-gal-f inhibited mRNAsynthesis of IL-1β, IL-4, IFN-γ, and TNF-αof the same cells in similar fashion. Thefindings suggested that the recombinant galectin proteins of H. contortus coulddecrease the transcription of cytokines in vitro.2. Recombinant galectins of Hemonchus contortus induced goat peripheral bloodlymphocytes apoptosisThe effect of H. contortus galectin peptides rHco-gal-m/f to induce apoptosis inthe peripheral blood lymphocyte cells (PBLCs) of goats was investigated. Analysis ofapoptosis was carded out with agarose gel eleetrophoresis, flow cytometry andtransmission electron microscopy. The results indicated that there were visibleapoptosis bodies and typical DNA ladders by genomic DNA fragmentation. Thequantitative analysis of apoptosis by flow cytometry indicated that rHco-gal-m/f induced apoptosis was time and dose dependent. Ultrastructural studies of the PBLCsrevealed that a large number of apoptotic cells were present in galectin-treated cells,which had the typical morphologic changes of apoptosis such as reduction of thecytoplasmic volume, loss of cell surface microvilli, chromatin condensation andfragmentation of the apoptotic ceils into small apoptotic bodies.3. Anticoagulant effects of recombinant galectins of Hemonchus contortusAnticoagulant effects of rHco-gal-m/f were examined. Eight goats were selectedto determine the coagulant time of rHco-gal-m/f utilizing the tube test. It was foundthat rHco-gal-m/f possessed anticoagulant effects and prolonged the coagulant time ofgoat blood (P＜0.01, P＜0.01). These anticoagulant effects were enhanced byincreasing protein concentration.4. Protection studies with recombinant galectins of Haemonchus contortusThe efficacy in terms of Hemonchus contortus infection, of two recombinantproteins of H. contortus, rHco-gal-m and rHco-gal-f, were studied in about 9-10month old goats. 16 animals were allocated into four experimental groups of fourgoats each. Experimental groups were treated as follows: 10mg group wasadministered with 10mg of rHco-gal-m/f(5mg rHco-gal-m and 5mg rHco-gal-f). 5mggroup was administered with 5mg of rHco-gal-m/f (2.5mg rHco-gal-m and 2.5mgrHco-gal-f). The proteins were emulsified with complete Freund's adjuvant, andadministered subcutaneously. A booster dose was repeated with the protein suspendedin the incomplete Freund's adjuvant after two weeks period. Unchallenged groupserved as the unvaccinated control animals that were not challenged with L₃, butsham-vaccinated with 1ml of PBS. Challenged group were the unvaccinated control,challenged with L₃ infection and sham-vaccinated with 1ml of PBS. Vaccinatedanimals received two vaccinations at 2 week intervals. 5000 infective H. contortus L₃were given two weeks after the final injection. Faecal egg counts were performedonce every two days from day 22 after challenge until the end of the experiment.Goats were killed 35 days alter challenge. The abomasum was opened immediately,and worm counts were performed. The results showed that the faecal egg output andworm burdens were reduced by 37.25% (P＜0.05) and 41.1% (P＜0.05), respectivelyin the 10mg vaccinated group at the end of the experiment. Corresponding faecal eggoutput and worm burdens reductions in the 5mg vaccinated group, were 48.03%(P＜0.01) and 46.19% (P＜0.01), respectively. These findings suggested thatrHco-gal-m/f protein against adult H. contortus offers partial immuno-protective effects against homologous infection in the goat.5. Humoral and local immune responses of goats vaccinated with recombinantgalectins of Haemonchus contortusThe efficacy in terms of Hemonchus contortus infection, of two recombinantproteins of H. contortus, rHco-gal-m and rHco-gal-f, were studied in about 9-10month old goats. In all goats, rHco-gal-m/f-specific immunoglobulin IgG titres, serumIgA, mucosal surface (MS) IgA and mucosal homogenate (MH) IgA concentrationswere determined with ELISA. Before L₃ challenge infection, the 10mg and 5mgvaccinated groups had significantly higher rHco-gal-m/f-specific IgG titres than theunchallenged or challenged control goats (P＜0.01, P＜0.01). The serum IgA levels ofthe 10mg and 5mg vaccinated groups increased as the experiment progressed.Following L₃ challenge, the 10mg and 5mg vaccinated groups had significantly higherrHco-gal-m/f-specific IgG titres than the unchallenged or challenged control groups(P＜0.01, P＜0.01). The two vaccinated and challenged control groups had significantlyhigher serum IgA compared to the unchallenged controls (P＜0.05, P＜0.05,P＜0.05).The MS IgA level in the two vaccinated and challenged control groups also differedsignificantly compared to the unchallenged control at necropsy (P＜0.01, P＜0.01,P＜0.01). There was a significant difference in the MH IgA levels between the twovaccinated groups and challenged controls (P＜0.05, P＜0.05). No significantdifference was detected in the rHco-gal-m/f-specific IgG titres, serum IgA, MS IgAand MH IgA levels between the 10mg and 5mg vaccinated groups. A significantlynegative correlation was found between the specific anti-rHco-gal-m/f IgG titres andworm burdens (r=-0.657; P＜0.05), and between MH IgA level and worm burdens (r=-0.692, P＜0.05). These findings suggested that the rHco-gal-m/f-specific IgG and MHIgA could play a role in protection against H. contortus parasite.6. The CD4⁺T, CD8⁺T and B lymphocyte immune responses of goats vaccinatedwith recombinant galectins of Haemonchus contortusIn order to investigate protective mechanism of acquired immunity in goat, theperipheral CD4⁺, CD8⁺T and B lymphocytes were determined by flow cytometry. Itwas found that prior to L₃ challenge infection, the %CD4⁺ and %CD8⁺ T lymphocyteswere significantly higher in the 10mg vaccinated group as compared to theunchallenged control at (P＜0.05 and P＜0.01) respectively. The % B lymphocyteswere also significantly higher between the 10mg or 5mg vaccinated groups andunchallenged control group before L₃ challenge (P＜0.05, P＜0.01). After L₃ challenge infection, the two vaccinated and challenged control groups had significantly higher%CD4⁺ T lymphocytes than the unchallenged control group (P＜0.05, P＜0.05, P＜0.05).There was significant difference in %CD8⁺ T lymphocyte between the challengedcontrol group and 10mg vaccinated group(P＜0.01). The challenged control group hadsignificantly higher %B lymphocytes than the two vaccinated groups (P＜0.01,P＜0.01). No significant difference was detected in the CD4⁺, CD8⁺T and Blymphocyte levels between the 10mg and 5mg vaccinated groups. However, asignificantly positive correlation was found between %B lymphocytes and wormburdens (r=0.587; P＜0.05).7. The granulocyte and monocyte immune responses of goats vaccinated withrecombinant galectins of Haemonchus contortusThe peripheral blood eosinophil, basophil, neutrophil, monocyte counts andhemoglobin concentration were determined in goats vaccinated with recombinantgalectin proteins. It was found that eosinophil levels between the two vaccinatedgroups were significantly higher than the unchallenged control group before L₃challenge infection (P＜0.01, P＜0.01). The 10mg vaccinated group also hadsignificantly more neutrophils than the unchallenged control group during this period(P＜0.01). The monocytes in the two vaccinated groups increased as the experimentadvanced. However, there was no significant difference in the amount of hemoglobinbetween the four groups prior to L₃ challenge. After L₃ challenge infection, theeosinophils were significantly higher in the two vaccinated groups compared to theunchallenged control (P＜0.01, P＜0.01, P＜0.01). The neutrophils in the 10mgvaccinated group were significantly higher than the challenged control (P＜0.05). Themonocyte cells were significantly higher between the 5mg vaccinated or challengedgroups compared to the unchallenged control group (P＜0.05, P＜0.01). Thehemoglobin level was significantly higher between the two vaccinated groups orunchallenged group compared to the challenged control group (P＜0.05, P＜0.05,P＜0.01). No significant difference was found in the eosinophil, basophil, neutrophil,monocyte and hemoglobin levels between the 10mg and 5mg vaccinated groups.Notably, there was a positive correlation between eosinophil cells and cumulativeEPG (r=0.537), although this was not significant. A positive correlation was detectedbetween monocytes and worm burdens (r=0.636; P＜0.05), and a negative correlationbetween hemoglobin level and worm burdens (r=-0.744; P＜0.01).