The detection and measurement of coproantibodies to Nippostrongylus brasiliensis in rats following a primary infection

Summary Investigations were initiated to study the possible detection and measurement of coproantibodies in animals infected with a gastrointestinal nematode parasite. Faecal extracts, extracts of small intestinal mucosa and sera of rats infected with intestinal nematode Nippostrongylus brasiliensis were examined for total IgA, IgM and IgG levels and haemagglutinating and precipitating antibodies specific to parasite antigens over a 30-day-period following infection. It was found that in both faecal and mucosal extracts immunoglobulin concentrations increased after a primary infection. In faecal extracts there was a seven-fold increase of IgA, a three to six-fold increase of IgG and about a fifty-fold increase of IgM. Haemagglutinins in faecal extracts detected by adult worm excretory-secretory (ES) products and adult worm and infective larvae somatic extracts were observed from 3 days after infection (DAI). Haemagglutinins detected by ES products reached their highest titres on 11–12 DAI while those reacting with adult worm somatic extracts showed the highest level between IS and 19 DAI. A similar pattern of response was found in the antibody levels of the intestinal mucosa. Haemagglutinins detected in faeces during the first 12 DAI reacted with the same antigens as antibodies present in the sera at that time but coproantibodies from 18, 24 and 30 DAI were different from those circulating in sera at that stage of the infection. The results suggest that measurement of coproantibody levels may provide a convenient and useful index of local immune responses to gastrointestinal helminths.     

旋毛虫成虫可溶性抗原和排泄分泌抗原对小鼠免疫保护作用的比较研究

目的　比较旋毛虫成虫可溶性抗原和排泄分泌抗原对小鼠的免疫保护作用。方法　收集人工感染大鼠小肠内的成虫, 经研磨和冻融制备成虫可溶性抗原。采用体外培养的方法从培养液中提取旋毛虫成虫排泄分泌抗原。分别用两种抗原免疫小鼠, 间隔１ 周共免疫３ 次, 末次免疫后１ 周, 每只小鼠攻击感染１００ 条旋毛虫感染性肌肉幼虫。感染后１ 周检查小鼠小肠内成虫数量和雌虫生殖力, 感染后５ 周检查肌肉幼虫负荷。结果　成虫可溶性抗原诱导的成虫减虫率、新生幼虫减虫率和肌肉幼虫减虫率分别为７９５５ ％ 、６２２５ ％ 和６５０ ％ 。成虫排泄分泌抗原诱导的成虫减虫率、新生幼虫减虫率和肌肉幼虫减虫率分别是９７２７ ％ 、８６６０ ％ 和９００ ％ 。结论　实验结果表明旋毛虫成虫可溶性抗原和成虫排泄分泌抗原均能够诱导宿主产生较强的抗攻击感染的免疫力, 但后者的免疫原性更强。     

Importance of immunoglobulin E (IgE) in the protective mechanism against gastrointestinal nematode infection: looking at the intestinal mucosae.

This review discusses experimental evidences that indicate the IgE participation on the effector mechanisms that leads to gastrointestinal nematode elimination. Data discussed here showed that, for most experimental models, the immune response involved in nematode elimination is regulated by Th-2 type cytokines (especially IL-4). However, the mechanism(s) that result in worm elimination is not clear and might be distinct in different nematode species. Parasite specific IgE production, especially the IgE produced by the intestinal mucosae or associated lymphoid organs could participate in the intestinal elimination of Trichinella spiralis from infected rats. Intestinal IgE may also be important to the protective mechanism developed against other gastrointestinal nematodes that penetrate the murine duodenum mucosa tissue, such as Strongyloides venezuelensis and Heligmosomoides polygyrus. At least in Trichinella spiralis infected rats, the results indicated that intestinal IgE might work independently from mast cell degranulation for worm elimination.     

The effect of Trypanosoma brucei infection on local and systemic antibody responses of rats to Nippostrongylus brasiliensis

Adult Hooded Lister rats were given 5000 Nippostrongylus brasiliensis larvae on day 3 or 7 after infection with Trypanosoma brucei and a second dose of 5000 nematode larvae 28 days later. A similar number of rats was infected only with N. brasiliensis larvae. Comparison of antibody levels in serum and the respiratory and alimentary tracts showed that T. brucei infection influenced both systemic and local antibody responses of rats to N. brasiliensis antigens. After primary infection systemic antibody responses were mainly impaired, the level of suppression depending upon the interval between trypanosome and nematode infections. Anamnestic responses were diminished in both antibody systems. The number of worms reaching the small intestine of T. brucei parasitised rats after primary infection was twice- and after reinfection three-times higher than in rats subjected to nematode infections alone. However, adult nematode expulsion was not delayed. The results suggest that N. brasiliensis infection causes a multiantigenic stimulation of both systemic and local humoral responses of the host. Furthermore, they indicate that depression of systemic antibody responses may enhance worm establishment.     

旋毛虫、卫氏并殖吸虫及华支睾吸虫之间共同抗原的研究

目的鉴定旋毛虫、卫氏并殖吸虫及华支睾吸虫之间的共同抗原,避免血清学诊断这3种寄生虫病时的交叉反应。方法应用十二烷基硫酸钠-聚丙烯酸胺凝胶电泳(SDS-PAGE)和免疫印渍(Western blot)对旋毛虫肌幼虫、卫氏并殖吸虫成虫及华支睾吸虫成虫可溶性抗原中的蛋白组分进行研究。结果旋毛虫肌幼虫、卫氏并殖吸虫成虫及华文睾吸虫成虫3者相同蛋白带的分子量为108、65、53、43、42、31、25、16 kDa。免疫印渍结果表明,旋毛虫和卫氏并殖吸虫抗原中分子量为 65、58、53kDa的蛋白带均与旋毛虫感染的大鼠、小鼠及患者血清、肺吸虫感染的大鼠和患者血清发生反应;旋毛虫和华支睾吸虫抗原中分子量为108kDa的蛋白带均与旋毛虫感染的大鼠、小鼠及患者血清、肝吸虫病患者血清发生反应;而3种可溶性抗原中的53kDa与本实验中所有寄生虫感染的动物和患者均有交叉反应。结论 65、58、53 kDa蛋白为旋毛虫和卫氏并殖吸虫的共同抗原,108kDa蛋白为旋毛虫和华支睾吸虫的共同抗原,而53 kDa蛋白为这3种寄生虫的共同抗原。     

Protective immunity and eosinophilia in IgE-deficient SJA/9 mice infected with Nippostrongylus brasiliensis and Trichinella spiralis.

To elucidate the roles of IgE antibody in host responses to helminth infection, selective IgE-deficient SJA/9 mice were infected with the nematode parasites Nippostrongylus brasiliensis, which elicits remarkable potentiated IgE production, and Trichinella spiralis, which induces strong anti-Trichinella IgE antibody production in normal mice. The kinetics of blood eosinophilia, worm burden after primary infection, and resistance to secondary infection in SJA/9 mice were the same in both infections as those in congenic SJL/J mice used as an IgE-producing control. These results indicate that the host responses examined here operate under IgE-independent mechanisms.     

Immunization of mice with surface antigens from the muscle larvae of Trichinella spiralis

Surface antigens of muscle larvae of Trichinella spiralis were stripped from the cuticle of live worms by the cationic detergent cetyltrimethylammonium bromide (CTAB). Such antigen preparations were shown to contain surface antigens of approximate molecular weights 100,000, 90,000, 69,000, 55,000, 46,000 and 35,000. Immunoprecipitation experiments confirmed that the surface of muscle larvae share antigenic epitopes with antigens contained within and secreted by the stichosome. CTAB antigen preparations were shown to be protective in NIH mice against a challenge infection as assessed by reduction in intestinal worm burden, worm fecundity, worm length and muscle larvae burden. The role of surface antigens in protective immunity to T. spiralis is discussed.     

The Effects of Maternal Ethanol Consumption on Lactational Transfer of Immunity to Trichinella spiralis in Rats

Transient immunity to the intestinal parasite Trichinella spiralis can be transferred from the mother to the neonate during lactation. The goal of this study was to determine whether maternal ingestion of ethanol during pregnancy and lactation inhibited expression of anti- T. spiralis immunity in nursing pups. Groups of female rats were infected with 1000 T. spiralis L1 larva, mated, and fed either ethanol-containing or isocaloric liquid diets and maintained on diets through pregnancy and lactation or were fed the liquid diets for 30 days before T. spiralis infection, mated, and maintained on diets through pregnancy and lactation. Pups were challenged orally with 200 T. spiralis larva at 14 days postdelivery (preweaning period) or 21 days postdelivery (postweaning period) and were sacrificed either 3 or 8 days after respective challenge. Intestinal worm counts and serum titers of anti-T. spiralis IgG antibodies were determined for each pup. No difference in the number of intestinal worms between pups of ethanol-treated and pair-fed dams that received ethanol diet after T. spiralis infection was observed in the preweaning period. This was also true of pups from the dams sacrificed at 3 days after challenge in the postweaning period. However, similar pups sacrificed at 8 days after challenge showed significantly higher worm counts (decreased immunity) relative to their pair-fed controls. Pups of dams that received ethanol containing diet 30 days prior to T. spiralis-infection showed significantly higher numbers of intestinal worms relative to pair-fed pups at both the preweaning and postweaning periods.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of Trichinella spiralis antigens in urine of men and animals

The practical inability to diagnose Trichinella spiralis antibodies in man before day 20 post infection (dpi) has stimulated interest in the development of immunodiagnostic test to detect circulating antigens. Our previous experience showed that soon after infection immune complexes as well as uncomplexed parasite antigens in sera of infected rats could be detected. To diagnose the presence of antigen in urine, double sandwich-capture ELISA was applied using a peroxidase-conjugated rabbit immunoglobulin to T. spiralis larval antigens. The plates were coated with metabolic (AES) or somatic (AS) larval antigens. Mice were infected with 500 T. spiralis larvae. The urine samples from experimentally infected mice taken from 1 to 41 dpi. and the urine samples from patients of the Clinical Hospital in Bia?ystok taken from 3 to 120 dpi were examined. Before testing, the urine samples were heated for 6 min. at 100 degrees C and centrifuged for 6 min. at 5000 g, supernatants were used in ELISA. The presence of T. spiralis antigens in mice urine samples was detected between 6-26 days post infection (dpi) using double sandwich-capture ELISA. All samples taken later were negative as samples taken from uninfected mice. 3 from 9 human urine samples taken 3-10 dpi were positive, the remaining samples taken 3-10 and 10-30 dpi showed values near to "cut-off". In both mice and human urine samples the higher level of antigens was detected in ELISA when somatic larval antigen was used. The T. spiralis antigens were present in urine of infected men and mice in the first phase of infection.     

旋毛虫成虫抗原的免疫保护性研究

目的 比较旋毛虫成虫虫体抗原、排泄分泌抗原和表面抗原对小鼠产生的免疫保护作用。 方法 检查免疫鼠和对照鼠肠道成虫、肌幼虫和血液中的嗜酸性粒细胞数;用ELISA测血清中抗旋毛虫肌幼虫IgG抗体滴度。 结果旋毛虫成虫虫体抗原、排泄分泌抗原和表面抗原免疫组的成虫减虫率分别为84.48％、89.98％和85.16％;肌幼虫减虫率分别为69.82％、78.80％和73.94％。3种抗原免疫组小鼠血中的嗜酸性粒细胞(EOS)数明显增多,血清中IgG抗体滴度明显升高,IgG抗体的几何平均倒数滴度(GMRT)分别是未免疫组的6.96、7.99和6.06倍。 结论 旋毛虫成虫虫体抗原、排泄分泌抗原和表面抗原均能诱导宿主产生较强的抗攻击感染保护力,且可激发特异性体液免疫和细胞免疫。成虫的排泄分泌抗原显示出更强的免疫原性。     

旋毛虫排泄分泌抗原对小鼠的保护性免疫

目的比较旋毛虫成虫排泄分泌抗原(ES抗原)、肌幼虫ES抗原、成虫和肌幼虫ES混合抗原对小鼠的免疫保护作用。方法用生理盐水培养法从培养液中提取成虫ES抗原、肌幼虫ES抗原,分别用成虫ES抗原、肌幼虫ES抗原、成虫和肌幼虫ES混合抗原免疫小鼠,同时设佐剂组和对照组,间隔7d共免疫3次。末次免疫后7天,每只小鼠用200条旋毛虫感染期幼虫经口进行攻击感染。感染后7天和30天检查各组小鼠肠道成虫数和肌幼虫数。结果旋毛虫成虫ES抗原组、肌幼虫ES抗原组、成虫和肌幼虫ES混合抗原组的成虫减虫率分别为87.95%、69.48%、84.34%,肌幼虫减虫率分别为74.79%、87.97%、86.87%。成虫ES抗原组、成虫与肌幼虫ES抗原混合组的成虫减虫率均高于肌幼虫ES抗原组(P均<0.05)。肌幼虫ES抗原组、成虫与肌幼虫ES抗原混合组的肌幼虫减虫率均高于成虫ES抗原组(P均<0.01)。结论旋毛虫成虫和肌幼虫ES混合抗原均能诱导小鼠产生抗成虫及肌幼虫较强的免疫力。     

Establishment of T-Helper-2 immune response based gerbil model of enteric infection

BACKGROUND: The reciprocal antagonism of T-helper-1 (Th-1) and Th-2 type immune responses suggests that helminth parasitic infection may ameliorate disease where a Th-1 type response dominates. The Mongolian gerbil has been useful in the investigation of the pathogenesis of gastric cancer, since long-term infection of gerbils with Helicobacter pylori induces adenocarcinoma. In this study the kinetics of worm expulsion and associated immune responses in gerbils infected with Trichinella spiralis were investigated in an attempt to establish an animal model of parasitic infection that could be helpful when investigating the effect of a Th-2 type response on Th-1-based intestinal disorders. METHODS: Gerbils were infected with various doses of infective T. spiralis larvae and were euthanized on different days after infection to investigate the intestinal worm recovery, goblet cell population, eosinophil response and serum IgG1 responses. RESULTS: The number of worms recovered from the intestine was dependent on the number of larvae used for the infection. Almost all worms were expelled spontaneously by day 26 post-infection, when the gerbils had been infected with 375 or 750 larvae. The number of intestinal goblet cells, eosinophils and the serum IgG1 level significantly increased following infection compared with the control. CONCLUSION: This is the first comprehensive report on the time-course of T. spiralis infection in gerbils. The data indicate that the T. spiralis-infected gerbil could be used as a model of the Th-2-based response to investigate the effect of a parasite-induced Th-2 response on various Th-1-mediated intestinal disorders such as H. pylori-induced gastritis and gastric carcinoma.     

旋毛虫成虫可溶性抗原免疫小鼠T淋巴细胞亚群的动态变化

目的　观察旋毛虫成虫可溶性抗原免疫小鼠T淋巴细胞亚群的动态变化。方法采用流式细胞术分别检测旋毛虫感染后 7、14、2 1、2 8、35天小鼠外周血CD+ 4 、CD+ 8T淋巴细胞的百分率。结果　免疫小鼠CD+ 4 、CD+ 8细胞增加 ,CD+ 4 /CD+ 8细胞比值与正常组比较无明显差异。结论　旋毛虫成虫可溶性抗原免疫小鼠未出现免疫抑制现象     

旋毛虫抗原基因Ts88原核表达及重组蛋白鉴定

目的　原核表达旋毛虫抗原基因Ts88,并对重组蛋白的抗原性进行鉴定。　方法　免疫筛选cDNA文库得到旋毛虫抗原新基因Ts88,克隆入原核表达载体pET28c(+),异丙基βD硫代半乳糖苷诱导表达。重组蛋白免疫小鼠制备免疫血清,ELISA检测抗体滴度。蛋白质印迹法检测重组蛋白的抗原性。免疫荧光试验确定Ts88蛋白在虫体的分布。　结果　经原核表达的Ts88重组蛋白,用其免疫小鼠可得到高滴度的抗体血清。蛋白质印迹法结果表明该重组蛋白能与旋毛虫病患者血清、旋毛虫病猪血清、人工感染及人工免疫兔血清反应,不与其他蠕虫病患者血清发生交叉反应 ,并能识别旋毛虫虫体天然抗原成分。免疫荧光试验显示Ts88蛋白主要分布于虫体体壁。　结论　成功制备Ts88基因的重组蛋白,证明该蛋白具有较好的抗原性     

The Effects of Ethanol Consumption on the Expression of Immunity to Trichinella spiralis in Rats

The effects of ethanol ingestion on immune responses of female rats against Trichinella spiralis (T. spiralis) infections were investigated. Female rats were pair-fed either ethanol-containing or isocaloric control liquid diets for 68 days, during which time they underwent one pregnancy cycle. For some studies, animals were fed ethanol diets for 68 days beginning prior to pregnancy and continuing through lactation and involution. They were infected on Day 7 of involution with 1000 L1 larvae of T. spiralis and tested for a primary rejection response 10 days later. To test for a secondary immune response, rats were infected with T. spiralis, placed on ethanol-containing liquid diet 15 days later, and after 68 days on diets, challenged with 1000 T. spiralis larvae and killed 3 days later. For primary immunized studies, ethanol-treated animals demonstrated significantly lower levels of anti-T. spiralis serum antibodies in ELISA, reduced rates of H3 thymidine incorporation by lymph node cells stimulated with T. spiralis antigen and significantly higher numbers of intestinal worm burdens (decreased immunity) compared with pair-fed controls. For animals sensitized to T. spiralis prior to pregnancy and given a secondary challenge during involution, no differences were found between ethanol and pair-fed animals in their ability to reject their worm burdens or in anti-T. spiralis serum antibody levels; however, ethanol-treated animals showed reduced rates of thymidine incorporation by lymph node cells when stimulated with T. spiralis antigen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stem cell factor contributes to intestinal mucosal mast cell hyperplasia in rats infected with Nippostrongylus brasiliensis or Trichinella spiralis, but anti-stem cell factor treatment decreases parasite egg production during N brasiliensis infection

We assessed the effects of the c-kit ligand, stem cell factor (SCF), in the jejunal mucosal mast cell hyperplasia that occurs during infection with the intestinal nematodes, Nippostrongylus brasiliensis or Trichinella spiralis in rats. Compared with vehicle-treated rats, rats treated with SCF (25 micrograms/kg/d, intravenous [i.v.] for 14 days) during N brasiliensis infection exhibited significantly higher levels of the rat mucosal mast cell (MMC)-associated protease, rat mast cell protease II (RMCP II) in the jejunum and serum on day 8 of infection, but not on days 10 or 15 of infection. By contrast, in comparison to rats treated with normal sheep IgG, rats treated with a polyclonal sheep antirat SCF antibody exhibited markedly decreased numbers of jejunal MMCs, levels of jejunal RMCP II, and serum concentrations of RMCP II during infection with either nematode, particularly at the earlier intervals of infection (< or = day 10). Taken together, these findings indicate that SCF importantly contributes to MMC hyperplasia and/or survival during N brasiliensis or T spiralis infection in rats, but that levels of endogenous SCF are adequate to sustain near maximal MMC hyperplasia during infection with these nematodes. Notably, treatment of rats with SCF somewhat increased, and treatment with anti- SCF significantly decreased, parasite egg production during N brasiliensis infection. This finding raises the interesting possibility that certain activities of intestinal MMCs may contribute to parasite fecundity during infection with this nematode.     

Immunoglobulin class specific responses to biochemically defined antigens of Trichinella spiralis

A comparison of the humoral response to resistant (NIH) and susceptible (C3H) strains of mice, which reject adult worms at different rates during a primary infection, was made following infection with Trichinella spiralis. The serum concentration of immunoglobulins of the heavy chain classes IgM, IgG1, IgG2, IgG3 and IgA were determined by single radial immunodiffusion. Antibodies of the same immunoglobulin isotypes to biochemically defined, stage specific surface and secreted components of three stages of parasite development were also determined using an isotype specific immuno-coprecipitation assay. Independent variation of the responses of each immunoglobulin isotype was observed. The specific anti-parasite response did not reflect total serum immunoglobulin levels in all immunoglobulin classes, and this is discussed in relation to basic mechanisms of immunoglobulin class switching. Finally a close correlation was observed in resistant (NIH) mice between the production of IgA antibody to surface components of adult worms and accelerated expulsion of this stage of the worm from the gastrointestinal tract. The possible relevance of this IgA response is further indicated by the failure of susceptible mice to synthesise IgA antibodies to the same surface antigens.     

两种新型免疫佐剂CT-B、Saponin制备的旋毛虫疫苗对小鼠免疫保护作用的比较研究

目的　比较两种新型免疫佐剂CT -B(霍乱毒素B亚单位 )、saponin(皂素 )制备的旋毛虫疫苗对小鼠的免疫保护作用。方法　将旋毛虫肌幼虫可溶性抗原分别与CT -B、saponin混合 ,以口服或皮下注射途径免疫NIH小鼠 ,间隔 1周共免疫 3次 ,末次免疫后 1周 ,与对照组同时予 2 0 0条旋毛虫感染期肌幼虫攻击 ,比较三组小鼠肠道成虫数、雌虫生殖力及肌幼虫数。结果　与对照组比较 ,CT -B组成虫减虫率、新生幼虫减虫率及肌幼虫减虫率分别为 91 5 9%、6 1 74 %和 90 32 % ,saponin组成虫减虫率、新生幼虫减虫率及肌幼虫减虫率分别为 79 2 1%、6 7 4 4 %和 88 39%。 结论　CT -B和saponin均能有效提高机体对旋毛虫的保护性免疫力 ,CT -B对肠道成虫的影响更显著     

Infection of inbred and nude (athymic) rats with Brugia spp.

Summary Infective larvae of Brugia pahangi were injected subcutaneously into inbred PVG (-RIT) rats, and ‘nude’ (PVG -rnu/rnu) (athymic) rats. Adult worms or circulating microfilariae were recovered from 20/34 (59%) of PVG -RTI^C rats and from 30/30 (100%) of ‘nude’ rats. Fertile worms were regularly found in the lumbar lymphatics and hearts of both strains of rat. Blood eosinophilia first developed in PVG -RTI^c rats about 17 days, and in all such animals by 6 weeks. High circulating eosinophil counts persisted only in patent animals, proving a useful hallmark for the presence of microfilariae. Nude rats despite patency, developed eosinophilia only latterly and then to a lesser extent. Specific anti-2?. pahangi IgG antibody was first detected at 7 days in all infected PVG -RTT rats, with levels rising until 8 weeks and remaining high only in microfilaraemic animals; total IgE showed a similar response. Specific IgE rose in all the eight patent rats inconsistently and only to low levels in eight non-patent infected rats. IgG and IgE were undetectable in nude rats. Other strains of inbred rats of different RTI haplotype were also successfully infected with B. pahangi and the human parasite B. malayi, a total of 10/23 (43%) and 5/15 (33%) becoming patent respectively. In the small numbers tested no major influence of RTI haplotype was detected. Infection by the intraperitoneal route did not result in the development of microfilariae. The difference in patency rates between ‘nude’ and normal PVG rats supports the contention that the development of filarial infections is T lymphocyte dependent. Inbred and ‘nude’ rats provide a valuable model of human filariasis, in which many features of filarial immunopathology can be studied.     

Free radical generation during primary infections with Nippostrongylus brasiliensis

The production of free radicals during infection of the rat with Nippostrongylus brasiliensis was investigated. Lipid peroxidation, which is the best documented effect of free radicals, was monitored in the small intestines of infected rats by measurement of malonyldialdehyde and was found to be increased at the time of worm rejection. The capacity of peritoneal leucocytes to produce free radicals, as measured by chemiluminescence, was monitored in rats infected with different doses of N. brasiliensis. Rejection of N. brasiliensis from rats infected with 6000 third-stage larvae (L3) began 2 days earlier than in rats infected with only 600 L3. Maximal free radical generation also occurred 2 days earlier and was quantitatively greater in rats infected with 6000 L3. Free radical generation by leucocytes in response to live adult N. brasiliensis was enhanced by plasma from infected rats indicating the existence of a plasma-borne factor responsible for the initiation of free radical generation in response to N. brasiliensis.