Detection of circulating parasite antigens in canine dirofilariasis by counterimmunoelectrophoresis

Circulating Dirofilaria immitis antigen was detected in sera from 24 of 24 infected dogs by counterimmunoelectrophoresis (CIE). Parasite antigen was not detected in sera from uninfected dogs or dogs with Dipetalonema reconditum infection. In experimentally infected dogs, the antigen was first detectable 6.5-8.5 months after infection. Preliminary evidence suggests that the antigen is present in male and female adult worms but not in microfilariae. Sera from dogs with microfilaremic and amicrofilaremic infections contained statistically equivalent amounts of D. immitis antigen. However, a significant correlation was observed between serum parasite antigen content and the number of adult worms present in individual dogs at necropsy. Previous studies from several laboratories have shown that microfilarial counts and serum antibody titers are not related to adult worm counts in canine dirofilariasis or other filarial infections. Thus, CIE detection of D. immitis antigenemia represents a significant improvement over previously available diagnostic techniques because it is more sensitive than microfilarial tests, more specific than antibody tests, and the only test that has been related to infection intensity.     

Anisakis simplex: antigen recognition and antibody production in experimentally infected mice

The kinetics of antibody response to intraperitoneal infection of mice with third stage larvae of Anisakis simplex was investigated by ELISA. Maximum antibody response to excretion-secretion (ES) antigens was reached before maximum response to somatic (SA) antigens. Total immunoglobulin (Ig) production (consisting mainly of IgM and IgG₁ isotypes) was very similar in both cases. Immunoblotting was used to characterize the antigens recognized by the host in the presence or absence of the metabolic products released by the parasite in vivo. Sera from mice infected with live larvae (anti-L₃ L serum) and immunized with dead larvae (anti-L₃ D serum) recognized a similar pattern of bands in immunoblots of ES and SA antigen preparations. In the latter, however, three bands at 14, 17 and 18 kD were only recognized by the anti-L₃ L serum. A possible explanation is that these low molecular weight antigens are ES products released only in vivo. Finally, the immune response in mouse was compared using ELISA and immunoblotting with the response of a human anisakiasis reference serum, and was found to display considerable similarities. This suggests that the mouse may be a useful model for studying the immunobiology of A. simplex in man.     

Quantitative and qualitative changes in the humoral response of dogs through the course of infection with Dirofilaria immitis

The present study compared the humoral response of dogs which developed microfilaremic or occult forms of dirofilariasis following experimental infection with Dirofilaria immitis L3 larvae. Quantitative analysis by ELISA revealed that antibody levels to adult somatic (AS), excretory-secretory (ES), and microfilarial (MF) antigens were highest during the patent phase of infection in dogs with either form of dirofilariasis. Patent sera from dogs destined for occult infections contained anti-AS and anti-MF antibody concentrations of 1,572 and 1,004 micrograms/ml, respectively, while microfilaremic-bound dogs contained 1,044 and 906 micrograms/ml, respectively. Chronic sera (430 days post infection) from occult dogs contained anti-AS and anti-MF antibody levels of 982 and 600 micrograms/ml, respectively, which were higher than in microfilaremic dogs. The antibody response to ES antigen was generally 10-fold less in absolute antibody concentrations at all time points tested. Immunoperoxidase staining of antigens transferred to nitrocellulose revealed the presence of several antigenic proteins which were recognized by occult, and to a lesser extent or not at all, by microfilaremic dogs. Sera drawn from occult-bound dogs 280 days post-infection, a time corresponding to microfilarial clearance (transition phase), contained higher antibody activity to microfilarial proteins weighing 47.5, 42.0, 34.2, and 22.4 kilodaltons compared to the microfilaremic dogs. This difference in antigen recognition became more apparent during the chronic phase of infection.     

Circulating excretory-secretory antigen levels and specific antibody responses in mice infected with Toxocara canis

Circulatory excretory-secretory antigen levels and IgM and IgG responses to larval antigens were monitored in the serum of 20 BALB/c mice that had been given approximately 500 infective eggs of Toxocara canis by stomach tube. Other groups of mice received different doses of infective eggs, ranging from 5 to 1,250 eggs. Excretory-secretory antigens were collected from culture fluid in which mechanically hatched larvae of T. canis were maintained. An indirect enzyme-linked immunosorbent assay was used to monitor specific antibody responses. Circulating antigen levels were monitored using a direct ELISA which incorporated an IgG fraction of a rabbit antiserum to the excretory-secretory antigens as a capture antibody and a biotin-conjugated form of the same rabbit IgG as the second antibody. The antigen-specific IgM response was evident the first week of infection and peaked 3 to 6 weeks post-infection. The antigen-specific IgG response first appeared the second week of infection and peaked at 6 to 8 weeks post-infection. Both isotype levels stayed near their peak values for the remainder of the study. In the untreated sera, circulating antigen was initially evident and highest the first week of infection; the antigen concentrations dropped by the third month of infection to low, but significant, levels that persisted for the duration of the study. The administration of greater than 25 eggs produced antigenemias. There appeared to be a positive linear trend between the number of eggs given and the amount of antigen in the circulation.     

Sandwich ELISA detection of excretory-secretory antigens of Toxocara canis larvae using a specific monoclonal antibody

We produced a new monoclonal antibody (mAb) to the excretory-secretory (ES) antigens of Toxocara canis larvae. The mAb (IgG1) reacts specifically with the 120 kDa protein of many ES molecules and does not have any cross-reactivity with adult T. canis antigens. Sandwich ELISA to detect the ES antigens was performed using the mAb and rabbit polyclonal antiserum. The lower limit for the detection of ES antigen was 4 ng/ml; assay was proportional within a concentration range of 4 ng/ml to 1 microg/ml of ES antigen. This assay system may prove valuable when seeking to quantify parasite burden early in infection and when determining the efficacy of anthelmintic treatment.     

Evaluation of an IgG-ELISA strategy using Taenia solium metacestode somatic and excretory-secretory antigens for diagnosis of neurocysticercosis revealing biological stage of the larvae

Diagnosis of neurocysticercosis (NCC) is complicated because of the variability in clinical presentations and course of the disease where viability of parasite is a major determinant. The present study describes evaluation of ELISAs using Taenia solium metacestode somatic and excretory-secretory (ES) antigens for detection of anti-T. solium metacestode IgG antibodies in serum and cerebrospinal fluid (CSF). And results of the ELISAs in cases with a definitive diagnosis of NCC are correlated with the biological stages of the parasite such as live vesicular or degenerated stage. The sensitivity of the IgG-ELISA using ES antigen is observed to be much higher in serum (88.2%) than in CSF (64.28%) although it is only marginally higher in serum (76.4%) than in CSF (75%) when somatic antigen is used in the ELISA. Whereas, the specificities of the ELISA using either somatic or ES antigen for detection of IgG antibodies in serum (97.97%; 96.96%) and CSF (96.42%; 97.61%) are comparable. A strong association is observed between live stage of the parasite and detection of antibodies in sera and CSF from more number of NCC patients by ELISA using ES antigens. Similarly, detection of antibodies by ELISA using somatic antigens could be associated with the dead or degenerated stage of the parasite in brain. The IgG-ELISA strategy developed in the present study opens up an avenue for diagnosis of NCC in hospitals or in population prevalence studies. The use of crude extracts of ES proteins might improve the serodiagnosis of the cases of NCC carrying live vesicular stage of the parasite larvae.     

Induction of protective immunity in dogs to infection with Dirofilaria immitis using chemically-abbreviated infections

Four dogs were immunized against Dirofilaria immitis infection by a series of 3 larval infections which were each subsequently terminated by ivermectin treatment. Two control dogs received ivermectin treatment alone. Following the final ivermectin treatment, dogs were challenged with infective larvae by subcutaneous inoculation, both free and contained within diffusion chambers. Three weeks after larval challenge the chambers were removed and live larvae were enumerated. Seven months after challenge dogs were killed and necropsied to collect and count adult D. immitis. Chambers recovered from immunized dogs had 63% fewer larvae than chambers from control dogs. At necropsy, control dogs had a mean of 28.5 adult worms whereas the immunized animals had an average of 0.5 worms (range 0-2). Sera collected from immune dogs throughout the study had elevated antibody levels to third- and fourth-larval stage antigens. Significant levels of immune protection were achieved with this immunization regimen. The data suggest that a multiple-stage parasite killing occurs in immune animals. It was not possible to associate immune protection with any of the 5 antigen subsets.     

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。     

Cloning of a cuticular antigen that contains multiple tandem repeats from the filarial parasite Dirofilaria immitis.

An unusual antigen composed of tandemly repeated protein units was cloned from the filarial parasite Dirofilaria immitis. The antigen was initially identified by screening a lambda gt11 cDNA library with serum from dogs immunized with irradiated D. immitis third-stage larvae. DNA sequence analysis of the cDNA clone, Di5, revealed a continuous open reading frame composed of two 399-base-pair repeats arranged in tandem. Southern blot analysis of genomic D. immitis DNA showed that the gene coding for Di5 is composed of a tandem array of 25-50 copies of this same 399-base-pair repeat. Antiserum raised against recombinant Di5 protein detected a protein "ladder," from about 14 to greater than 200 kDa with steps approximately 15 kDa apart, on immunoblots of D. immitis extract. Metabolic labeling of adult parasites with [35S]methionine showed that Di5 is synthesized as a large precursor that is subsequently cleaved to produce the ladder-like array. These results suggest that the characteristic ladder is created by proteolytic cleavage of the precursor at the same site in each monomer. The Di5 antigen was localized to the cuticle and hypodermis of adult D. immitis by immunoelectron microscopy. Both male and female parasites were found to release Di5 when cultured in vitro. DNA hybridization analysis demonstrated that Di5 is a member of a gene family present in many filarial parasites that infect both animal and human populations.     

Neurocysticercosis immunodiagnosis using Taenia solium cysticerci crude soluble extract, excretory secretory and lower molecular mass antigens in serum and urine samples of Indian children

Neurocysticercosis (NCC), the most common neurological disorder of parasite etiology, results from lodgment of Taenia solium cysticerci in the central nervous system and is now increasingly being recognized in children. The confirmed diagnosis is based collectively on radiological findings and serodiagnostic techniques. The serodiagnostic techniques have variable sensitivity and specificity depending upon the technique, antigens used, location and number of cysts. Crude soluble extract (CSE), excretory secretory (ES) and lower molecular mass (LMM) (10-30 kDa) antigenic fraction of T. solium cysticerci were evaluated for antibody detection in serum and urine samples by ELISA. Serum and urine samples were collected each from 125 clinically suspected and radiologically proven NCC (111 with single Computed Tomography (CT) lesions and 14 with multiple CT lesions) and 125 control subjects (60 with neurological disorders other than NCC, 40 with other parasitic diseases and 25 apparently healthy subjects). The sensitivity of the ELISA with the use of CSE, ES and LMM antigenic fractions was 38.4%, 63.2% and 30.4% with serum (cut off dilution 400), 46.4%, 44% and 47.2% with neat urine and the specificity was 88%, 76.8% and 85.6% with serum (cut off dilution 400), 66.4%, 65.2% and 58.4% with neat urine samples, respectively. The study suggests that detection of antibody to ES antigen in serum samples may serve useful purpose for the serodiagnosis of human NCC.     

A case of Ascaris suum visceral larva migrans diagnosed by using A. suum larval excretory-secretory (ES) antigen

A 42-y-old female presented with common cold-like symptoms. Laboratory data showed mild liver dysfunction together with peripheral blood eosinophilia. She was suspected of having a helminthic infection, however parasite eggs or larvae were not detected by repeated stool examinations. Eventually she transpired to have a high IgG antibody titer against excretory-secretary (ES) antigen of Ascaris suum larvae, but not of Toxocara canis larvae, suggesting that she had been suffering from visceral larva migrans (VLM) caused by A. suum. Her sickness improved without any treatment. Current results clearly highlight the usefulness of ES antigens derived from larvae of A. suum for the fine discrimination of VLM caused by A. suum and by T. canis. Application of A. suum derived ES antigens as a diagnostic tool may reveal the distinctive clinical features of VLM caused by A. suum.     

Immunodiagnosis of human fascioliasis by enzyme-linked immunosorbent assay using excretory-secretory products

In sera from patients with fascioliasis the enzyme-linked immunosorbent assay (ELISA) was used to detect antibody using excretory-secretory products (ES) from Fasciola hepatica adult worms. The specificity of ES-ELISA (with OD values greater than 0.38) allowed the differentiation among fascioliasis, schistosomiasis, clonorchiasis, and other human parasite infections.     

Clinicopathologic and histologic evaluation of Dirofilaria immitis-induced nephropathy in dogs

Twelve beagles were infected with 200 Dirofilaria immitis infective larvae to study glomerular lesions associated with filariasis. All developed high serum levels of antibodies to dirofilarial antigens and became persistently microfilaremic. The dogs were killed at various times between 398 and 562 days post-infection and renal lesions were examined by light, electron, and immunofluorescent microscopy and antibody elution techniques. A membranoproliferative glomerulonephritis was observed in all dogs. Immunofluorescence was positive in all; predominantly in a fine granular pattern along the glomerular capillary wall. Ultrastructural examination showed intramembranous globular electron-dense deposits and a linear band of fine electron-dense particles in all dogs. Antibody elution studies demonstrated antibody reactive to dirofilarial antigens. In a subsequent experiment, an aqueous-soluble antigen prepared from adult female D. immitis was infused into the renal arteries of 5 heartworm-naive dogs. Immunofluorescent examination of the infused kidneys showed dirofilarial antigen present on the glomerular capillary wall in a fine granular pattern indicating there was adherence of the antigen to the capillary wall. These observations support the hypothesis of in situ immune complex formation as part of the pathogenesis of glomerulonephritis associated with dirofilariasis.     

Characterization of excretory-secretory products of adult Dictyocaulus viviparus and the antibody response to them in infection and vaccination

In vitro released products of the adult stage of the bovine lungworm, Dictyocaulus viviparus, were characterized according to their SDS-PAGE profile, glycosylation pattern, in vitro synthesis and antigenicity in the context of infection and vaccination with irradiated larvae. Biosynthetic labelling experiments with ³⁵S-methionine indicated active synthesis of ES throughout this time. There was, however, little incorporation of ³H-glucosamine into ES products, and lectin affinity chromatography and glycopeptidase F digestion identified only one glycosylated component. Immunoprecipitation of ¹²⁵l-labelled ES products with sera from calves patently infected with D. viviparus demonstrated that all of these, with the exception of two components, are antigenic to the bovine host. One of those not immunoprecipitated was shown to be host serum albumin carried over into culture. A limited degree of cross-reactivity between nematode species was observed, with a D. viviparus female-specific antigen of 290 kDa being recognized by serum antibody from calves infected with the gastrointestinal nematodes Cooperia oncophora and Ostertagia ostertagi. Calves vaccinated with irradiated larvae of D. viviparus, despite not being exposed to the adult stage of the parasite, also showed some recognition of adult ES products. This might suggest that vaccination with irradiated larvae operates against both pre-pulmonary and pulmonary stages of the infection.     

抗旋毛虫肌幼虫ES抗原IgY的制备及鉴定

目的制备抗旋毛虫肌幼虫排泄-分泌(excretory-secretory,ES)抗原的鸡卵黄免疫球蛋白(IgY),测定其效价及用于检测抗原的敏感性。方法 4只24w龄罗曼母鸡用旋毛虫肌幼虫ES抗原经大腿外侧与胸部肌肉免疫4次(首次剂量为500μg/只,加强剂量为250μg/只),每次间隔10d。取免疫前和首次免疫后42d的鸡蛋卵黄,用水稀释法提取IgY,考马斯亮蓝法测定蛋白含量,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、蛋白质印迹(Western blot)及间接荧光抗体试验(IFAT)对IgY进行分析,ELISA检测纯化后IgY的效价及检测抗原的敏感性。结果罗曼鸡经ES抗原免疫后,每枚鸡蛋经提纯后均可得到约70mg抗体,SDS-PAGE表明纯化的IgY有2条主要蛋白带,分子量为67kDa、23kDa,Western blot与IFAT发现提纯的IgY可识别肌幼虫虫体与ES抗原。IgY的抗体效价为1∶107,IgY-夹心ELISA检测旋毛虫抗原的敏感性为1.17ng/mL。结论制备的抗旋毛虫ES抗原的IgY具有较高的效价与敏感性。     

Dynamics of immune responses related to clinical status in Brugia pahangi-infected dogs

Clinical signs of lymph node enlargement, limb edema, lymph duct fibrosis, and microfilaremia were monitored in dogs with chronic Brugia pahangi infections. During the study a single rear limb of each dog was reinfected with multiple low doses of infective larvae. The changing immune responses to parasite antigens prepared from three sources--Brugia pahangi adult worm homogenate extract, adult worm excretory-secretory products, and microfilaria excretory-secretory products--were monitored by Western blot ELISA of antigens fractionated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and by microtiter plate ELISA. Assays were used to detect antibodies in both the the IgG and IgE classes. A wide range of clinical manifestations was demonstrated in response to reinfection: asymptomatic, amicrofilaremic; asymptomatic, microfilaremic; acute short duration node enlargement and/or limb edema with microfilaremia; and chronic limb edema, amicrofilaremic. On microtiter plate ELISA, the dogs demonstrating the highest anti-adult worm homogenate titers were amicrofilaremic and were asymptomatic or developed chronic limb edema, dogs with high anti-mf ES titers were persistently amicrofilaremic, and the most marked increases against all three antigen sources upon reinfection occurred in low or amicrofilaremic dogs. Quantitative changes in antibody levels against the three crude antigen sources following reinfection were often paralleled by distinct changes in recognition of specific bands of antigens fractionated by SDS-PAGE.     

Tear IgA-ELISA: a novel and sensitive method for diagnosis of ophthalmic cysticercosis

For the first time, presence of locally secreted specific IgA antibodies in tear specimen from human with ophthalmic cysticercosis is documented in the present study. The ELISA using Taenia solium metacestode excretory secretory (ES) antigen demonstrated a diagnostic level of IgA antibodies in tears with 100% sensitivity (6 out of 6 confirmed cases of ophthalmic cysticercosis) whereas, 25 of 34 (73.52%) clinically suspected cases were diagnosed positive. The ELISA using T. solium metacestode somatic antigen detected a diagnostic titre of IgA antibody in tears with a sensitivity of 50% (3 out of 6 confirmed cases). The specificity of the tear IgAELISA using T. solium metacestode somatic and ES antigens is observed to be 94.87% and 92.3%, respectively. Overall in tears, the ELISA using T. solium metacestode ES antigens for detection of IgA antibodies shows a higher diagnostic efficiency (93.33%) compared to that using T. solium metacestode somatic antigen (88.88%). The sensitivities of the ELISA for detection of IgA antibodies in tears is observed to be higher than that for detection of IgG antibodies in serum using either somatic or ES antigens of the parasite.     

一株抗泡球蚴包囊角皮层单克隆抗体杂交瘤细胞的制备

在抗泡球蚴单克隆抗体的制备过程中, 成功地获得了一株被命名５－５Ａ７ （ＩｇＧ１） 的特异性与泡球蚴包囊角皮层结合反应的单克隆抗体杂交瘤细胞株。与７ 种异源性蠕虫抗原及ＢＡＬＢ／ｃ 小鼠肾脏可溶性抗原的ＥＬＩＳＡ反应结果, 显示了高度的特异性。与旋毛虫包囊、人蛔虫成虫组织切片以及砂鼠和ＢＡＬＢ／ｃ 小鼠的正常脏器组织切片的免疫组化研究, 亦均未显示任何非特异性染色。通过胰蛋白酶、蛋白水解酶的蛋白溶解和过氧化酸氧化反应实验证实, 单克隆抗体５－５Ａ７ 所认识的泡球蚴抗原决定簇的化学性质为碳水化合物。我们期望, 单克隆抗体５－５Ａ７ 的制备, 将为泡球蚴病的免疫治疗实验研究提供一条可能的途径。     

Elution of cytomegalovirus antibodies from adenocarcinoma of the colon.

Eluates were prepared by high salt extraction from normal colonic mucosa and adenocarcinomatous tissue from 28 patients, eight more from unmatched colonic tissue and five from patients with other gastrointestinal disease. Immunoglobulins were detected by ELISA: IgG was present in 24% eluates from normal colon and 21% from carcinomas; IgA in 55% eluates from normal colon and 39% from carcinomas; IgM in 55% from normal colon and 37% from carcinomas. Cytomegalovirus-specific antibody was found in 15% eluates from normal colon and in 18% carcinomas. Out of the 28 matched specimens, cytomegalovirus-specific IgG was detected in one normal and four tumour eluates, specific IgA in two normal and four tumour eluates, and specific IgM in two normal and two tumour eluates. In two instances cytomegalovirus-specific antibody was present in the eluates prepared from the normal and tumour tissue of the same patient. Of those eluates which contained cytomegalovirus-specific antibodies by ELISA, two were positive by anti-complement immunofluorescence of human embryo fibroblasts infected with cytomegalovirus strain AD-169. It seems possible, therefore, that cytomegalovirus antigens on colonic cells may be masked by complexing with anti-cytomegalovirus antibodies, and may not therefore be detected by techniques such as immunofluorescence.     

Stage-specific surface antigens of the cattle lungworm Dictyocaulus viviparus

Immunofluorescence on live Dictyocaulus viviparus parasites revealed a significant antibody response by vaccinated and patently infected bovine hosts to the sheath of infective larvae (L3), a structure which is generally thought to be shed from the parasite surface prior to invasion of host tissue. In contrast, surface-exposed antigens of the adult, egg and pulmonary L1 stages were recognized only by serum antibody from calves exposed to a patent lungworm infection. Radioiodination of sheathed L3 identified a restricted set of components while a more complex pattern of labelled material was observed with adult parasites. Many more components of adult worms were labelled by the Bolton-Hunter than by the lodogen reagent, probably reflecting the more penetrative labelling propensities of the former. Stage-specificity of surface-associated antigens of adult parasites was demonstrated by their immunoprecipitation by antibody from patently-infected, but not from vaccinated, calves. There was no in vitro release of the major iodinatable surface-associated antigens of adult parasites nor any binding of antibody raised against adult excretory-secretory (ES) products to the surface of living adult worms, suggesting that surface components do not contribute to adult ES products in this species. Antibody responses to the surface of adults. L1 and eggs were specific to patently-infected animals and may provide a useful indicator of exposure to patent infection.