肝片吸虫免疫显性抗原基因的筛选及鉴定

为筛选出特异的肝片吸虫诊断候选抗原,拓展诊断靶标,利用肝片吸虫阳性血清筛选肝片吸虫cDNA表达文库,获得肝片吸虫特异性抗原基因,采用RT-PCR技术扩增目的基因,连接表达载体pET-32a(+),构建重组质粒,转化入感受态细胞BL21(DE3)中,利用IPTG对重组蛋白进行诱导表达,通过SDS-PAGE及Western blot技术对蛋白表达情况进行鉴定。结果显示:经筛选获得了17个肝片吸虫免疫显性抗原基因,其中假定蛋白Fh010935为肝片吸虫特异性抗原基因;扩增得到的Fh010935核苷酸序列大小为144 bp,编码48个氨基酸,A+T含量为55.56%;Fh010935蛋白由1个α-螺旋,2个β-折叠和3个β-转角构成,具有3个抗原表位,推测该蛋白可能具有较好的抗原性;重组蛋白主要以包涵体形式表达,分子量大小约24 ku,可以被肝片吸虫感染阳性血清特异性识别,具有较好的反应原性。提示:假定蛋白Fh010935可作为肝片吸虫病诊断候选抗原,为疾病诊断制剂的开发提供前期基础。     

肝片吸虫免疫显性抗原基因的筛选及鉴定

为筛选出特异的肝片吸虫诊断候选抗原,拓展诊断靶标,利用肝片吸虫阳性血清筛选肝片吸虫cDNA表达文库,获得肝片吸虫特异性抗原基因,采用RT-PCR技术扩增目的基因,连接表达载体pET-32a(+),构建重组质粒,转化入感受态细胞BL21(DE3)中,利用IPTG对重组蛋白进行诱导表达,通过SDS-PAGE及Western blot技术对蛋白表达情况进行鉴定。结果显示:经筛选获得了17个肝片吸虫免疫显性抗原基因,其中假定蛋白Fh010935为肝片吸虫特异性抗原基因;扩增得到的Fh010935核苷酸序列大小为144 bp,编码48个氨基酸,A+T含量为55.56%;Fh010935蛋白由1个α-螺旋,2个β-折叠和3个β-转角构成,具有3个抗原表位,推测该蛋白可能具有较好的抗原性;重组蛋白主要以包涵体形式表达,分子量大小约24 ku,可以被肝片吸虫感染阳性血清特异性识别,具有较好的反应原性。提示:假定蛋白Fh010935可作为肝片吸虫病诊断候选抗原,为疾病诊断制剂的开发提供前期基础。     

Use of immunosorbent-purified antigens of Fasciola hepatica in enzyme immunoassays

A monoclonal antibody specific for the T1 tegumental antigen of Fasciola hepatica was used as a solid-phase immunosorbent for the purification of T1 antigen from homogenised mature F hepatica. Material fractionated by this technique was successfully used in enzyme-linked immunoassays to detect antibodies to F hepatica in sera from sheep and cattle. Species differences in response to infection by F hepatica were demonstrated.     

Progress in the development of Fasciola hepatica vaccine using recombinant fatty acid binding protein with the adjuvant adaptation system ADAD

Fatty acid binding proteins (FABP) have been designed as a potential vaccine against fasciolosis. In this work, the immunoprophylaxis of the recombinant Fh15 FABP from F. hepatica (Fh15) in adjuvant/immunomodulator ADAD system was evaluated using mice and sheep challenged with F. hepatica. The ADAD system combines the Fh15 antigen with an immunomodulator (hydroalcoholic extract of Polypodium leucotomos; PAL) and/or an adjuvant (saponins of Quillaja saponaria; Qs) in a water/oil emulsion (30/70) with a non-mineral oil (Montanide). All the infected control mice died by 41-48 days post-infection. The mice vaccinated with ADAD only with PAL+Fh15 present a survival rate of 40-50% and those vaccinated with ADAD containing PAL+Qs+Fh15 had a survival rate of 50-62.5%. IgG1 antibodies were lower in surviving mice in comparison with non-surviving mice. The sheep vaccinated with ADAD PAL+Qs+Fh15 showed lower fluke recovery (43%), less hepatic lesions and higher post-infection daily weight gain than F. hepatica infected control animals. Thus, the ADAD system using recombinant fatty acid binding proteins from F. hepatica could be a good option to develop vaccines against F. hepatica.     

Expression of interleukin 4, interleukin 4 splice variants and interferon gamma mRNA in calves experimentally infected with Fasciola hepatica

Interleukin 4 (IL-4) is expected to play a dominant role in the development of T helper (Th) 2 cells. Th2 immune responses with expression of relatively large amounts of interleukin 4 (IL-4) but little interferon gamma (IFN-gamma) are characteristic for chronic helminth infections. But no information is available about IL4 expression during early Fasciola hepatica (F. hepatica) infections in cattle. Therefore, we investigated F. hepatica specific IL-4 and IFN-gamma mRNA expression in peripheral blood mononuclear cells (PBMCs) from calves experimentally infected with F. hepatica. Cells were collected prior to infection and on post-inoculation days (PIDs) 10, 28 and 70. Interestingly, PBMCs responded to stimulation with F. hepatica secretory-excretory products (FhSEP) already on PID 10 and expressed high amounts of IL-4 but not of IFN-gamma mRNA suggesting that F. hepatica induced a Th2 biased early immune response which was not restricted to the site of infection. Later in infection IL-4 mRNA expression decreased whereas IFN-gamma mRNA expression increased slightly. Isolated lymph node cells (LNCs) stimulated with FhSEP and, even more importantly, non-stimulated LN tissue samples indicated highly polarized Th2 type immune responses in the draining (hepatic) lymph node, but not in the retropharyngeal lymph node. During preliminary experiments, two splice variants of bovine IL-4 mRNA, boIL-4delta2 and boIL-4delta3, were detected. Since a human IL-4delta2 was assumed to act as competitive inhibitor of IL-4, it was important to know whether expression of these splice variants of bovine IL-4 have a regulatory function during an immune response to infection with F. hepatica. Indeed, IL-4 splice variants could be detected in a number of samples, but quantitative analysis did not yield any clue to their function. Therefore, the significance of bovine IL-4 splice variants remains to be determined.     

Antigenic relationship among field isolates of Tritrichomonas foetus from cattle

Analysis of protein and antigen profiles of Tritrichomonas foetus isolates from cattle from 5 western states was accomplished by sodium dodecyl sulfate polyacrylamide-gel electrophoresis, immunoblot, immunoprecipitation, and fluorography techniques. Total protein profiles of all isolates were compared by Coomassie brilliant blue staining of T foetus protein samples prepared by 4 protein-extraction methods. Antigenic tritrichomonas proteins were identified by immunoblot assay with polyclonal bovine or rabbit anti-T foetus serum. Additionally, [14C]glucosamine-labeled T foetus was used for total and antigenic glycoprotein analyses. Detectable differences in the composition of total proteins or antigenic tritrichomonal proteins were not observed among all isolates. However, intensity differences in some antigenic protein bands were apparent. Bovine and rabbit sera from immunized animals possessed antibodies to the same antigenic tritrichomonal proteins. Each T foetus isolate contained 4 to 7 molecular weight size classes of glycoprotein, which were labeled by [14C]glucosamine; however, only 3 to 4 glycoproteins were identified as antigens by bovine or rabbit antiserum.     

Alternative activation of ruminant macrophages by Fasciola hepatica

The helminth parasite, Fasciola hepatica, has a worldwide distribution and infects a wide variety of mammalian hosts, including ruminants and man. In response to infection, these hosts mount a type 2 helper (Th2) response that is highly polarized and results in the downregulation of type 1 helper (Th1) mechanisms. In a murine macrophage model F. hepatica induces alternative activation of macrophages. These macrophages differ from classically activated cells in that they preferentially use arginase instead of inducible nitric oxide synthase (iNOS) for metabolism of nitrogen. In this study we sought to characterize macrophage phenotype following stimulation of the ovine cell line MOCL7 with recombinant F. hepatica enzymes and crude parasite extracts. An in vitro model using the MOCL7 cell line was established and arginase levels in cells were used to determine the activation status of cells. Stimulation of this cell-line in vitro with F. hepatica products induces alternative activation. We have also found a chitinase-like protein in supernatants which is capable of differentiating alternatively activated from classically activated macrophages.     

Cross-reactions between crude antigens of larval Taenia solium (Cysticercus cellulosae) and other helminths of pigs

A crude antigen extract of larval Taenia solium was shown by immunodiffusion (ID) and immunoelectrophoresis (IEP) to cross-react with rabbit antisera against pig serum proteins and larval T. hydatigena, and by enzyme-linked immunosorbent assay (ELISA) with antisera against pig serum proteins, Fasciolopsis buski, larval T. hydatigena, hydatid cyst, Hymenolepis diminuta and Dipylidium caninum. Immunoblotting demonstrated that the crude antigens extract contained epitopes of pig serum proteins of 48 and 66 kDa. The crude extract also contained a subunit of antigen B (95 kDa) which was also found in T. hydatigena and H. diminuta. Immunoperoxidase and indirect immunofluorescence studies showed that cross-reacting antigens were distributed mainly on the tegument of T. solium.     

Immunization of rats against Fasciola hepatica using crude antigens conjugated with Freund's adjuvant or oligodeoxynucleotides

Chronic Fasciola hepatica infection is correlated with the development of a T helper (Th2)-predominant immune response. To determine whether immunostimulatory CpG-containing oligodeoxynucleotides (CpG-ODN) or Freund's complete adjuvant (FCA), known to promote a Th1 (T helper 1) immune responses, could provide protection from F. hepatica infection, total homogenate (TH) of F. hepatica mixed with CpG-ODN or FCA were injected subcutaneously (s.c.) into Wistar rats. A F. hepatica-specific Th1-predominant immune response was induced with CpG-ODN or FCA in lymph nodes of immunized animals. Lymph node cells from TH-CpG-ODN or TH-FCA immunized rats showed increased antigen-specific proliferation with high levels of INFgamma, compared to lymphocytes from rats injected with TH alone. In contrast, these two groups of immunized animals did not modify IL-4 release by draining lymph node cells, when they were subsequently stimulated with TH in vitro. However, a significant reduction in the burden of flukes (76.7%) was only observed in rats immunized with TH-FCA. Conversely, immunization of rats with TH-CpG-ODN did not promote protection against the parasite. Therefore, even though CpG-ODNs and FCA induced Th1 type responses, only FCA provided a significant protection to rats infected with F. hepatica.     

Identification of bovine herpesvirus-1 polypeptides involved in serum neutralization

Bovine herpesvirus (infectious bovine rhinotracheitis virus)-infected cell antigens were solubilized with Nonidet P-40. The crude antigen extract was separated by reaction with bovine hyperimmune serum in line immunoelectrophoresis; individual immunoprecipitates were used to immunize rabbits. Rabbit sera possessing serum neutralizing activity were analyzed by reaction with crude antigen extract in immunoprecipitation sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Four virus-specified glycopeptides, with molecular weights of 69-75K, 77-81K, 82-92K and 108-115K, appeared to be involved in inducing serum neutralizing antibody.     

Vaccine potential of inclusion bodies containing cysteine proteinase of Fasciola hepatica in calves and lambs experimentally challenged with metacercariae of the fluke

Despite intensive research efforts, progress in the development of effective anti-Fasciola hepatica vaccine has not been satisfactory. However, it has been found that cysteine proteinases of F. hepatica are very important candidates for a vaccine antigen because of their role in fluke biology and in the host-parasite relationship. In our previous experiments we found that recombinant cysteine proteinase which we have cloned from adult F. hepatica (CPFhW) can protect rats against the liver fluke infection when administered intramuscularly or when given intranasally in the form of cDNA. In the present experiments we aimed to evaluate the protectivity of the mucosal vaccination in calves and lambs with inclusion bodies containing recombinant CPFhW using different vaccination doses and various sites of antigen delivery. Female calves vaccinated intranasally with two doses of 300 microg of the recombinant CPFhW showed 54.2% protection against the subsequent challenge of 400 metacercariae (mc). Flukes which developed in vaccinated calves showed a reduction of reproductive potential. Male Corriedale lambs vaccinated at the age of 4 months demanded three doses of the antigen to gain 56.5% of protection to a challenge with 250 mc of F. hepatica. Vaccinated animals showed significantly lower blood eosinophil counts. No correlation was found between serum and mucosal IgG or IgA reacting with F. hepatica ES antigens and the protection level.     

The shedding of the outer glycocalyx of juvenile Fasciola hepatica

Freshly excysted Fasciola hepatica possess an outer glycocalyx which on incubation at 37 degrees C is rapidly shed. Using an ELISA technique the release of this parasite antigen was shown to be temperature-dependent and to occur in both normal bovine serum as well as in serum free conditions. The ELISA failed to detect the antibody--antigen complexes that occurred when flukes were incubated in immune serum. Release of specific parasite antigen fell slowly with in vitro cultured flukes, but increased with in vivo cultured flukes. Using a fluorescence inhibition assay, antigens with high ELISA titres inhibited surface fluorescence of the parasite suggesting that the ELISA was detecting surface antigens as well as other parasite metabolic products. Several metabolic blocking agents and commercial antihelminthics were titrated against juvenile F. hepatica to screen for inhibition of the surface--tegument shedding: there was little selective inhibition of surface shedding without a significant loss of motility.     

Requirement for MHC class II positive accessory cells in an antigen specific bovine T cell response

Purified populations of bovine antigen presenting cells (APCs) and T cells have been isolated from peripheral blood and characterised using various monoclonal antibodies (mAbs) for cell surface markers. Bovine APCs were found in an adherent cell fraction and were non-specific esterase positive, phagocytic and expressed bovine major histocompatibility complex (MHC) class II determinants, all of which are typical macrophage characteristics. T cells were rigorously depleted of accessory cell function before being used in an antigen presenting cell assay. The generation of T helper cells in response to the soluble antigen, ovalbumin, was entirely dependent upon a critical number of APCs. Further the proliferative response was inhibited by several mAbs to bovine MHC class II molecules. Thus the interaction between bovine APCs and helper/inducer T lymphocytes (TH/I) appears to be similar to that in other species.     

Acquired immunity in schistosomiasis with purified Fasciola hepatica cross-reactive antigens

We have previously demonstrated that a Fasciola hepatica-derived adult worm antigen, which is cross-reactive with Schistosoma mansoni and designated FhSmIII(M), induces resistance to challenge infection with S. mansoni in mice. The current review concerns the methods developed to isolate and partially characterize a major component of FhSmIII(M), a 12-kDa polypeptide, as well as immunity studies involving this antigen. Utilizing conventional gel filtration, followed by diethylaminoethyl (DEAE) Sephadex A-120 and monitoring the fractions by polyacrylamide gel electrophoresis (PAGE) and enzyme-linked immunoelectrotransfer blot techniques (EITB), we were able to isolate the 12-kDa antigenic polypeptide to homogeneity. Conventional gel filtration chromatography was followed by high-pressure, liquid anion, exchange chromatography, when highly purified material was needed, although the effective yields diminished drastically with the latter. Mice, rabbits and calves with a primary infection of F. hepatica developed antibodies (detectable in enzyme linked immunosorbent assay (ELISA) to the F. hepatica 12-kDa polypeptide within 2 weeks of infection. Mice with a primary infection of S. mansoni developed significant, but low, levels of anti-12-kDa antibodies by 7 weeks post-infection. Immunization of mice with microgram amounts of this 12-kDa polypeptide in Freunds' adjuvant resulted in the development of up to 77% less S. mansoni worms than the controls. Treatment with either endoglycosidase H, neuraminidase or dithiothreitol had no effect on the protein's mobility on sodium dodecyl sulfate (SDS)-PAGE or in its recognition by antibodies, suggesting the absence of carbohydrate moieties or disulphide bonds in relation to its antigenic determinants. Degradation by proteinase K further confirmed its polypeptide nature and points to recombinant DNA technology for the large-scale manufacture of this potential vaccine. Further use of this antigen in immunity studies should greatly contribute to the clarification of the mechanisms involved in cross-resistance against schistosomiasis.     

Bovine lineage-specific gamma delta (gammadelta) T cell GD3.5 antibody cross-reacts with cell surface antigens on ovine and caprine lymphocytes

A specifically defined function for gammadelta T cells is still a topic of intense debate. Thus, there is great importance in the identification of lineage-specific markers that characterize these cells. Early studies describe a family of lineage-specific cell-surface molecules on ruminant gammadelta T cells called WC1. More recently, a 220-240 kDa lineage-specific bovine gammadelta T cell surface marker, GD3.5Ag, was reported. Here, we used anti-bovine GD3.5Ab to study its cross-reactivity with caprine and ovine lymphocytes. Flow cytometric analysis demonstrated that GD3.5Ab binds ovine and caprine lymphocytes. In addition, immunoprecipitation experiments with GD3.5Ab demonstrate an ovine and caprine cross-reactive antigen that is similar in size to the bovine antigen. These results suggest that GD3.5Ag is well conserved among ruminant species and GD3.5Ag may play an important role in gammadelta T cell biology.     

Serum antibody isotype responses of Fasciola-infected sheep and cattle to excretory and secretory products of Fasciola species

This study investigated the immunoglobulin isotype responses of sheep and cattle chronically infected with Fasciola hepatica and Fasciola gigantica to adult F. hepatica excretory/secretory products (Fh-ES) or F. gigantica excretory/secretory products (Fg-ES), respectively. An antibody enzyme-linked immunosorbent assay (Ab-ELISA) was used to determine serum antibody (total Ig, IgG(1), IgM, IgG(2) and IgA) responses. At necropsy, the mean number of flukes recovered was lower in cattle than in sheep. All F. hepatica and F. gigantica infected sheep and cattle showed an increased total Ig levels from 3 to 4 weeks post-infection (wpi). Among isotypes IgG(1) was most dominant while IgM was the earliest (2 wpi) to be detected in both sheep and cattle infected with both F. hepatica and F. gigantica animals. IgG(2) response was early (2 wpi) in sheep infected by F. hepatica but there was no response in sheep infected with F. gigantica. There was a late and strong IgG(2) response in cattle infected with both flukes. The IgA isotype showed an early and a clear biphasic response in sheep with F. hepatica but was less pronounced in F. gigantica infected sheep. While IgA response to Fh-ES was noticed 5 wpi in F. hepatica infected cattle, it appeared much later (21 wpi) in those infected with F. gigantica. The dominance of IgG(1) isotype in infected sheep and cattle suggest an associated Th2 response. This early response to adult Fasciola spp. ES antigen suggests an early exposure to the antigen presumably through the cross-reacting ES products of juvenile flukes. There is clearly difference in IgG(2) isotype response in cattle (resistant) compared to sheep (susceptible). The late IgG(2) response in cattle may suggest late Th1 involvement in bovine cellular responses to adult Fh-ES/Fg-ES.     

Humoral immune response in calves to single-dose, trickle and challenge infections with Fasciola hepatica

In cattle experimentally infected with Fasciola hepatica, parasite specific IgG1 and IgG2 responses were studied. Additionally parasite specific IgE production was assessed by the Passive Cutaneous Anaphylaxis reaction. The primary infection was administered either as a single-dose or as a trickle infection over a 4-week period. Animals were challenged 4 months later. Titres of IgG1 and IgG2 against excretory-secretory parasite products (FhESAg), and against a whole-worm extract (FhSomAg) were measured by enzyme-linked immunosorbent assay (ELISA) in relation to weight gain, serum hepatic enzyme levels, and fluke infection rate. At necropsy, the mean number of flukes recovered was similar in both infected groups. The two ELISAs specific for bovine IgG1 showed analogous sensitivity and specificity (92% and 94%). Cross-reactivity was observed towards Echinococcus granulosus, Cysticercus tenuicollis, and C. ovis but not towards C. bovis, Cooperia spp., and Ostertagia spp. FhESAg gave rise to apparently more stable specific IgG1 titres as compared to FhSomAg. Mean IgG1 titres were significantly higher in the single-dose-infected group than in the trickle-infected group during the early migratory phase of the infection (week 2 to week 4 (FhSomAg) or week 6 (FhESAg)). IgG2 values were consistently lower than IgG1 levels. The kinetic response of both isotypes yielded a similar pattern. Specific IgE antibodies were detected in cattle of both infected groups from week 2 post-primary infection (PPI) onwards. The mean serum glutamate dehydrogenase (GLDH) and gamma-glutamyl transferase (gammaGT) activities were significantly higher in the single-dose-infected group for 3 weeks around peak levels (12-14 weeks PPI and 14-16 weeks PPI for GLDH and gammaGT respectively). Western blotting revealed a major antigenic fraction in FhESAg (26-30 kDa) recognized specifically by sera from F. hepatica infected calves as early as 6-8 weeks PPI. Experimental challenge caused no statistically significant modification of any parameter (IgG1 and IgG2 titres, enzymatic activities, immunoblotting) used to monitor the course of the infection. No correlation was found between fluke size and number, and antibody titres, suggesting that IgG1 production has little protective effect against F. hepatica infection.     

Fasciola hepatica: studies on vaccination of rats and mice with a surface antigen prepared from fluke homogenate by means of a monoclonal antibody

T1 tegumental antigen was isolated from a homogenate of eight- to 10-week-old Fasciola hepatica using a T1-specific monoclonal antibody bound to sepharose in an antibody-affinity column. Rats and mice were vaccinated with T1 antigen in Freund's complete adjuvant, and control groups received equivalent amounts of non-T1 antigen (eluted from the antibody-affinity column) or ovalbumin. On completion of the immunisation programme, serum samples were collected for ELISA and IFA testing. The animals were challenged by oral infection with F hepatica metacercariae or, for several vaccinated rats, by intraperitoneal transplantation of live adult flukes. At autopsy, worm-burden and liver damage was assessed for each animal and the condition of transplanted flukes was examined. Comparison of test and control groups of animals showed that neither T1 nor non-T1 antigens provided significant protection against challenge, although specific antibody responses against the appropriate sensitising antigen were engendered. Flukes transplanted to the peritoneal cavity of immunised rats survived without damage, although they became encased in hollow fibrous capsules of host origin. The results lend support to the pre-existing concept that glycocalyx turnover by discharge of T1 secretory bodies at the apical surface of migrating flukes provides an efficient means of protection for the parasite against host immunity.     

Differentiation of parapoxviruses by application of orf virus-specific monoclonal antibodies against cell surface proteins.

Monoclonal antibodies were produced against orf virus-specified cell surface proteins in an attempt to develop reagents capable of differentiating between members of the Parapoxviridae. Two immunization protocols were used to induce an anti-orf response in BALB/c mice, one of which resulted in virus replication in the recipient. The monoclonal antibodies produced were tested for crossreactivity with bovine papular stomatitis virus (BPS) and milker's node virus (MNV) by indirect immunofluorescence assay (IFA) and immunoblotting. The results indicate that significant antigenic overlap exists between isolates of orf, MNV and BPS, even at the level of specificity provided by monoclonal antibodies. One monoclonal antibody reacted strongly in IFA with orf virus isolates, very weakly with MNV, and not at all with BPS. On immunoblots this same antibody recognized a 40-43 kDa protein in orf virus-infected cells, and also a 45-48 kDa protein in cells infected with MNV or BPS virus. The data suggest that it may be possible to define parapoxvirus strains on the basis of small variations in specific virus-directed cell surface proteins.