The partial characterization of proteases present in the excretory/secretory products and exsheathing fluid of the infective (L3) larva of Necator americanus.

Following the observation that live third-stage larvae (L3) could digest gelatin in vitro, gelatinolytic protease activity has been demonstrated at pH 8.5, in both exsheathing fluid (EF) and excretory/secretory (ES) products of infective L3 of Necator americanus. EF resolved as a single band of proteolytic activity, with a mol. wt of 116 kDa, while L3 ES products exhibited multiple bands of proteolysis, at 219, 200, 195, 166, 137, 92, 72 and 62 kDa; weak bands were detectable at 92 and 72 kDa. The EF protease was characterized as cysteine, whereas ES apparently possessed one serine (195 kDa) and seven (219, 200, 166, 137, 92, 72 and 62 kDa) cysteine protease bands and a combination of metallo- and cysteine proteases of approximately the same mol. wts (62, 137 and 219 kDa). Though EF was not able to cleave immunoglobulins, ES was shown to cleave IgG, IgA and IgM, but not IgD or IgE. The activity appeared to be directed toward the Fc portion of the molecule, and was inhibited by PMSF, which is indicative of serine protease activity. The significance of the presence of such apparently diverse proteases in larval products is discussed.     

Towards a suitable antigen for diagnosis of Gnathostoma spinigerum infection.

, , , and 1992. Towards a suitable antigen for diagnosis of Gnathostoma spinigerum infection. International Journal for Parasitology 22: 1151–1156. Advanced third-stage larvae of G. spinigerum were obtained from two separate sources, namely from cysts in the livers of naturally infected eels (L3E) and from experimentally infected mice (L3M). Morphology of the L3E was studied microscopically. The larvae were homogenized in distilled water, 1% Triton X-100 or 1% sodium deoxycholate containing protease inhibitors. Protein compositions of the three crude extracts were compared, on the same weight basis, by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie brilliant blue staining while their antigenicities were studied by Western blot analysis using serum of a patient with parasitologically confirmed gnathostomiasis. Distilled water was found to be the best extraction solution in solubilizing proteins especially the diagnostic antigen, namely the 24,000 (24 kDa) mol. wt component from the larvae. The L3E and L3M contained relatively equal amounts of the 24 kDa antigen. This diagnostic component was anatomically located in the body fluid, oesophagus and intestine of the larva.     

Detection of anti-parelaphostrongylus tenuis antibodies in experimentally infected and free-ranging moose (Alces alces)

Confirming Parelaphostrongylus tenuis infection in moose (Alces alces) and other susceptible hosts is difficult. An enzyme-linked immunosorbent assay (ELISA) was developed using the excretory-secretory (ES) products of third-stage P. tenuis larvae (ES-ELISA) and the test applied to serum samples obtained from seven moose calves (5-9.5 mo old) given infective larvae (L3) in doses approximating those likely to be received in nature (3-30 L3). Anti-P. tenuis immunoglobulin G antibodies were detected in all seven inoculated moose during the course of infection until the termination of experiment 61-243 days post-inoculation (DPI). Five animals tested between 16-25 DPI had significant antibody levels, while a sixth animal did not test positive until 46 DPI. The seventh animal was not tested until 199 DPI. Antibody levels remained elevated in all five animals that harbored adult worms at the termination of the experiment. Whereas, antibody levels showed a gradual decline in the two remaining animals, presumably because of death of worms, and antibodies were undetected in one animal at the time of necropsy. The other animal displayed an anamnestic increase in antibody level following a challenge inoculation of infective larvae. Terminal and peak optical density (OD) values detected by ES-ELISA strongly correlated with inoculation dose (r = 0.98, P = 0.02 and r = 0.95, P = 0.04, respectively) among animals harboring adult worms (n = 4) but not significantly with the number of worms recovered postmortem (peak OD, r = 0.82, P = 0.18; terminal OD, r = 0.93, P = 0.07). Unlike the ES products, use of somatic antigens of the adult worm in ELISA did not provide satisfactory results. Antibodies to P. tenuis were detectable by ES-ELISA in two of 21 free-ranging moose from an enzootic area but not from any of 23 animals from a non-enzootic area. The ES-ELISA appears to be a useful test for assessing exposure of moose to P. tenuis.     

Dirofilaria immitis: proteases produced by third- and fourth-stage larvae.

A model of cutaneous extracellular matrix was used to determine if live Dirofilaria immitis larvae secrete proteases which are active at physiological pH and capable of degrading macromolecules found in cutaneous tissue. After 72 hr, 100 third-stage larvae (L3) degraded 24% of the total matrix, while fourth-stage larvae (L4) degraded 10%. A sharp increase in the amount of matrix degraded by L3 corresponded with the onset of the molting process. L3 and L4 degraded comparable amounts of the glycoprotein and elastin components of the matrix, but molting L3 degraded nearly twice the amount of the collagen component (62% vs 35%). Characterization of proteases present in larval-soluble extracts and excretory-secretory products using synthetic substrates and protease inhibitors demonstrated cysteine-protease and metalloprotease activity. Cysteine protease activity was found in whole worm extracts of both L3 and L4. Metalloprotease was secreted at higher levels by molting L3, but was also secreted by L4. Partial separation of the metalloprotease by size-exclusion chromatography indicated that the molecular weight of the native enzyme was in the 49-54 kDa range. The cysteine protease activity was demonstrated in fractions corresponding to 34-39 kDa. The biological function of the D. immitis larval proteases remains to be conclusively determined; however, these data suggest that they are involved in degradation of components of cutaneous tissue and in the molting process.     

Lagochilascaris minor third-stage larvae secrete metalloproteases with specificity for fibrinogen and native collagen

Dissemination of parasitic infections depends on migration through tissues and evasion from both hemostatic processes and immune responses from hosts. Metalloproteases play major roles in these mechanisms of pathogen-host interactions and, thus, are considered drug targets. In this study, we characterized metalloprotease activities in excretory/secretory (ES) products from third stage larvae (L3) of the ascarid Lagochilascaris minor, the causative agent of lagochilascariosis, which demonstrates an impressive migrating capacity across host tissues, including bone. Gel enzymography showed that ES products of L3 display two major gelatinolytic activities. Optimal proteolytic activity was found to occur at neutral/alkaline pH and was associated with two L. minor-secreted metalloproteases of 59 (SM59(Lm)) and 114kDa (SM114(Lm)). We next showed that ES products of L3 were able to hydrolyze fibrinogen and collagen I at neutral pH, but not BSA, in an extensive manner. Furthermore, we demonstrated that ES products of L3 mediate hydrolysis of the triple helical structure of collagen I fibers in mouse mesentery. These results suggest that ES proteases of L3 might facilitate both L. minor migration through host tissues by hydrolyzing collagens of the extracellular matrix and evasion from host hemostatic mechanisms by degrading fibrinogen.     

Brugia pahangi: Immunization with early L3 ES alters parasite migration,and reduces microfilaremia and lymphatic lesion formation in gerbils (Meriones unguiculatus)

Previous studies have shown that intradermally (ID) injected Brugia pahangi L3s migrate through various tissues and into the lymphatics of gerbils in a distinct pattern. Excretory/secretory products (ES) produced at the time of invasion of B. pahangi are likely to be important in this early migration phase of the parasite life cycle in their rodent host. Hence, early L3 ES was collected from 24 h in vitro cultures of B. pahangi L3 larvae and used in immunization experiments to investigate the effect of immunity to early L3 ES on worm migration, survival and development of B. pahangi. Immunization of gerbils with ES in RIBI adjuvant produced antibodies to numerous ES proteins eliciting a strong humoral response to ES and indirect fluorescent antibody (IFA) assay using anti-ES serum recognized the ES proteins on the surface of B. pahangi L3 larvae. Following ES immunization, gerbils were challenged either ID or intraperitoneally (IP) with 100 L3s of B. pahangi and euthanized at 3 or 106 days post inoculation (DPI). Immunization with early ES slowed the migration of ID inoculated L3 at 3 DPI and significantly altered the locations of adult worms at 106 DPI. Immunization did not induce protection in any treatment group. However, immunized animals had significantly fewer microfilariae per female worm suggesting the antigens in ES are important in microfilariae development or survival in the host. The number of lymphatic granulomas was also significantly reduced in ES immunized animals. It is important to note that microfilariae serve as a nidus in these granulomas. Our results shows immunization with early Brugia malayi L3 ES alters the worm migration, affects circulating microfilarial numbers and reduces lymphatic granulomas associated with B. pahangi infection in gerbils.     

Translation products of mRNA from infective larvae of Trichinella spiralis include an antigenic polypeptide

Total RNA was extracted from packed infective larvae of Trichinella spiralis by centrifugation through a 5.7 M caesium chloride cushion. Polyadenylated messenger RNA was separated from total RNA in an oligothymidylic acid-cellulose gel column. The in vitro translation of the mRNA, isolated from infective larvae of T. spiralis, was carried out using the rabbit reticulocyte cell-free translation system. Incorporation of 35S-methionine into the trichloroacetic acid precipitates in the lysate containing mRNA was 5 times greater than that in control. The translation products were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography. Many polypeptides with molecular weights of less than 100,000 were synthesized in the lysate. A T. spiralis positive mouse serum was mixed with translation products to form antigen-antibody complexes, which were then absorbed by Staphylococcus aureus Cowan 1 strain and analysed by autoradiography of SDS-PAGE. An antigenic polypeptide with a molecular weight of 48,000 was demonstrated to react specifically with IgG antibody in T. spiralis positive mouse serum. T. spiralis larvae were cultured in methionine-free medium containing 35S-methionine, and antigenic polypeptides in somatic extracts and ES products were compared with those in translation products by autoradiography of SDS-PAGE. Several polypeptides in ES products and somatic extracts reacted specifically with IgG antibodies in positive serum. Especially the polypeptide with a molecular weight of 48,000 in ES products strongly reacted with IgG antibody in positive serum.     

Characterisation of stage-specific proteins of Oesophagostomum dentatum by preparative isoelectric focusing and lectin blotting

The nodular worm of pigs, Oesophagostomum dentatum, has previously been shown to undergo distinct biochemical changes during its life cycle. This phenomenon was studied in more detail for the early parasitic stages. Differences between infective third-stage larvae (L3), parasitic fourth-stage larvae cultivated in vitro (L4c), and pre-adult larvae recovered from the intestinal contents of pigs (L4p) were compared with respect to their protein and glycoprotein patterns by solubility-based protein fractionation and preparative isoelectric focusing followed by SDS-PAGE or by Western blotting with various lectins. While differences between the L4 were only minor (only three bands were specific for either L4c or L4p), L3 displayed distinctly different protein patterns with four L3-specific and nine L4-specific bands. Concanavalin A bound to a variety of glycoproteins, partly in a stage-specific manner, while Ricinus communis Agglutinin 120, Wheat Germ Agglutinin, Peanut Agglutinin and Soybean Agglutinin bound to fewer, partly stage-specific, molecules.     

Immunodiagnosis of angiostrongyliasis with monoclonal antibodies recognizing a circulating antigen of mol. wt 91,000 from Angiostrongylus cantonensis

In the analysis of excretory-secretory (ES) antigens from infective third-stage larvae (L3) of Angiostrongylus cantonensis, one major component of mol.wt 91,000 was not precipitated by pooled sera of patients with eosinophilic meningoencephalitis. Monoclonal antibodies (Mc Ab) secreted from two hybridoma cell lines, established against somatic antigens of L3, recognized this molecule but with different epitope specificities indicated by an additivity index (A.I.) of 83%. The 2 Mc Ab (TD2 and 3A5) belonged to IgG2a and IgM classes, respectively. Combinations of TD2 and 3A5 were used in a sensitive enzyme-linked fluorescent assay (ELFA) for the immunodiagnosis of human angiostrongyliasis. The double-antibody sandwich ELFA method was applied firstly to sera from experimentally infected rats using either TD2 or 3A5 to coat the assay plates. Two fluorescence unit (F.U.) peaks appeared in sera from infected rats collected 18 and 44 days after infection. Specimens from 35 patients were tested, all cerebrospinal fluids (CSF) and most sera (88%) showed positive reactions and the average F.U. of CSF was greater than that of serum.     

Immunity to Heligmosomoides polygyrus induced by subcutaneous vaccination with post-infection larvae

The objective of this study was to investigate, using the Heligmosomoides polygyrus (= Nematospiroides dubius)-mouse model, whether live post-infection trichostrongylid larvae recovered from the intestinal wall of donor animals and placed subcutaneously would serve as vaccine protecting against oral challenge by third-stage (infective) larvae (L3). Experiments were conducted to determine the effect of number and age of post-infective larvae as well as age and sex of host on vaccination. Vaccinated BALB/cByJ mice were challenged with 30 L3 and total adult worm burdens compared between vaccinated groups and sham-treated controls (greater than 90% infection rates). All mice subcutaneously vaccinated with either five or 10 larvae harbored significantly fewer challenge parasites in their intestines than did sham-treated controls (P less than 0.001). Both young and mature mice were significantly protected against challenge by the subcutaneous larval vaccine. Adult female mice had significantly (P less than 0.05) fewer parasites than adult male mice. The age of the larvae (indicated as the days between infection and harvesting of the larvae) was important in that day-4 or day-6 larvae (L4) were significantly more protective (P less than 0.001) than day-2 (L3) or day-8 larvae (L5-preadult). Reduction in worm burden for young vaccinated animals ranged from 31 to 39% (P less than 0.001) and for mature animals from 88 to 100% (P less than 0.001). Passive transfer to serum resulted in the reduction of worm burdens by 26-40% (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and preliminary characterization of a novel cuticular antigen from the filarial parasite Dirofilaria immitis

We have described here the cloning and partial characterization of a cDNA encoding a cuticular antigen of Dirofilaria immitis. A 48-h third-stage larval D. immitis cDNA library was immunoscreened with sera raised in mice against third-stage larval cuticles (mouse anti-L3 cuticle antisera). A strongly immunoreactive clone (L3MC4) was isolated. Sequence analysis of L3MC4 showed that it was a partial length cDNA. The missing 5′ end of the clone was amplified by PCR from D. immitis adult female first-strand cDNA using the nematode 22-base splice leader sequence and a L3MC4-specific antisense primer. The composite cDNA sequence comprised 616 bases (nDiL3MC4) encoding a full-length protein of 146 amino acids (DiL3MC4). GenBank analysis showed that DiL3MC4 shared some homology to an unknown C. elegans gene product (31%) at the amino acid level. However, there were no related filarial expressed sequence tags in the current GenBank™ database. Antibodies to recombinant DiL3MC4 (rDiL3MC4) identified a 19-kDa native antigen in the adults and in the L3 and L4 larval stages of D. immitis. In addition, the antibodies bound to the cortical layers of the L3 cuticle, as revealed by immuno-gold electron microscopy. The native protein was not detected in larval and adult excretory–secretory products. Immunoblot analysis showed that serum from a rabbit that was repeatedly injected with a small number of D. immitis third stage larvae reacted with rDiL3MC4. Thus, DiL3MC4 is a novel cuticular antigen of a filarial parasite.     

Effect of flavan-3-ols on in vitro egg hatching, larval development and viability of infective larvae of Trichostrongylus colubriformis

The effects of flavan-3-ols (the monomer units of condensed tannins (CT)) and their galloyl derivatives on the viability of eggs, the development of first stage (L1) larvae, and the viability of the infective larvae of Trichostrongylus colubriformis were investigated under in vitro conditions. Each of the flavan-3-ol gallates showed some inhibition of egg hatching at 100 μg/ml, and 100% inhibition at 1000 μg/ml, with epigallocatechin gallate being the most effective in the egg hatch (EH) assay. In contrast, none of the flavan-3-ols were able to completely inhibit egg hatching. The flavan-3-ols and galloyl derivatives dose-dependently inhibited the development of infective larvae as assessed by the larval development (LD) assay. A larval migration inhibition (LMI) assay was used to assess the effect of flavan-3-ols and their galloyl derivatives on the motility of the infective third-stage (L3) larvae of T. colubriformis. In general, the flavan-3-ol gallates were more effective than the flavan-3-ols at immobilising the infective larvae as evidenced by their ability to inhibit more (P<0.05–0.01) larvae from passing through the LMI sieves. At 500 μg/ml, epigallocatechin gallate inhibited significantly more (P<0.1) larvae from passing through the sieves than did catechin gallate, epicatechin gallate, or gallocatechin gallate. Comparisons were made between the flavan-3-ols and their galloyl derivatives with the in vitro effects of CT extracts from several forage legumes, which have exhibited effects on parasites in vivo. The forage legumes tested at 200–500 μg/ml reduced the proportion of eggs that hatch, with comparable results to those obtained using the flavan-3-ols. The activities may be influenced by the prodelphinidin: procyanidin (PD:PC) ratios: CT extracts from Lotus pendunculatus and sainfoin have PD:PC ratios of 70:30 and 77:23, respectively, whereas the less active CT extract from Lotus corniculatus has a PD:PC ratio of 27:73. The active CT extracts from forage legumes have epigallocatechin as the dominant flavan-3-ol extender unit, and epigallocatechin is the most active flavan-3-ol in both the EH and LD assays.     

薇甘菊甲醇提取物对二疣犀甲生长发育的影响

【目的】探讨入侵杂草薇甘菊Mikania micrantha的利用价值, 及其在棕榈害虫二疣犀甲Oryctes rhinoceros生态防控中的应用前景。【方法】采用浸渍法和室内饲喂法, 研究了薇甘菊甲醇提取物对二疣犀甲取食量、 卵孵化、 化蛹、 羽化及幼虫发育等的影响。【结果】薇甘菊提取物对二疣犀甲具有很好的生长发育调节活性。拒食活性研究结果表明, 在不同供试浓度下, 其对二疣犀甲3龄幼虫均表现拒食活性, 且拒食率与处理浓度呈正相关。薇甘菊提取物处理二疣犀甲的卵后, 孵化率明显降低, 且孵化期延长, 10 mg/mL提取物处理后孵化率仅达66.66%, 孵化期比对照延迟3 d, 同时初孵幼虫死亡率也高达40.43%。采用添加薇甘菊提取物的饲料饲喂1龄幼虫后, 幼虫体重增长减缓, 在处理浓度为10和5 mg/g时, 处理后90 d体重增加量分别为3.83 g和4.53 g, 而对照组体重增加量达到6.87 g。薇甘菊提取物处理老熟幼虫后, 对其化蛹具有抑制作用, 造成化蛹率降低, 化蛹时间延长及蛹的畸形。经薇甘菊提取物处理二疣犀甲蛹后, 成虫羽化率降低, 羽化时间明显延长, 且畸形成虫数量增加, 主要表现为翅无法正常伸展、 虫体瘦小、 无法正常爬行等。【结论】薇甘菊具有开发为生物源昆虫生长调节剂、 并在二疣犀甲防控中应用的潜力。     

Daily rhythms of clock gene expression and feeding behavior during the larval development in gilthead seabream,Sparus aurata

Light is the main environmental time cue which synchronizes daily rhythms and the molecular clock of vertebrates. Indeed, alterations in photoperiod have profound physiological effects in fish (e.g. reproduction and early development). In order to identify the changes in clock genes expression in gilthead seabream larvae during ontogeny, three different photoperiods were tested: a regular 12L:12D cycle (LD), a continuous light 24L:0D (LL) and a two-phases photoperiod (LL?+?LD) in which the photoperiod changed from LL to LD on day 15 after hatching (dph). Larvae were sampled on 10, 18, 30 and 60 days post-hatch (dph) during a 24?h cycle. In addition to the expression of clock genes (clock, bmal1, cry1 and per3), food intake was measured. Under LD photoperiod, larvae feed intake and clock genes expression showed a rhythmic pattern with a strong light synchronization, with the acrophases occurring at the same hour in all tested ages. Under LL photoperiod, the larvae also showed a rhythmic pattern but the acrophases occurred at different times depending on the age, although at the end of the experiment (60 dph) clock genes expression and feed intake rhythms were similar to those larvae exposed to LD photoperiod. Moreover, the expression levels of bmal1 and cry1 were much lower than in LD photoperiod. Under the LL?+?LD photoperiod, the 10 dph larvae showed the same patterns as LL treatment while 18 and 30 dph larvae showed the same patterns as LD treatment. These results revealed the presence of internal factors driving rhythmic physiological responses during larvae development under constant environmental conditions. The LL?+?LD treatment demonstrates the plasticity of the clock genes expression and the strong effect of light as synchronizer in developing fish larvae.     

Passive transfer of protective immunity to larval Dirofilaria immitis from dogs to BALB/c mice

Protective immunity to larval Dirofilaria immitis has been demonstrated in both the natural host, the dog, and in an experimental host, the mouse. In the present study, sera were collected and pooled from dogs that had been shown to have protective immunity to larval D. immitis. The pooled serum was inoculated into normal BALB/cByJ mice that then were challenged with third-stage larvae (L3) implanted in diffusion chambers. Two weeks postchallenge no significant difference was seen in either parasite survival or growth. Three weeks postchallenge, there was a significant decrease in parasite survival in mice receiving serum from immune dogs. Living larvae recovered at 3 wk postchallenge were significantly shorter than cohorts recovered from control mice. Antibody responses to L3 and forth-stage larvae (L4) surface antigens, to L3 and L4 aqueous soluble antigens, and to an excretory-secretory antigen fraction were measured. Only antibody responses to L3 surface antigens were elevated in the immune serum as compared to controls, thus suggesting a possible role for antibodies with specificity for surface antigens in protective immunity.     

The distribution and localization of the stage-specific GP31 antigen from infective Ostertagia circumcincta larvae.

The 31,000 mol. wt glycoprotein (GP31) antigen of infective third-stage (L3) Ostertagia circumcincta larvae was shown, by surface labelling experiments and immunofluorescent antibody staining of whole larvae and larval sections, to be distributed internally. When transverse sections of L3 O. circumcincta, taken from the anterior pharyngeal region, were further examined by electron microscopy, after immunogold staining with rabbit anti-GP31 antiserum, the GP31 antigen was found to be specifically located in 'secretory organelles' within the cells of the oesophageal glands. By in vitro culturing L3 O. circumcincta in medium supplemented with 35S-methionine and then analysing the excretory-secretory material released by the larvae, it was found that the GP31 molecule was one of the major components of the excretory-secretory complex. The purified GP31 molecule had no detectable proteolytic activity in protein degradation assays. On examination of Triton X-100 extracts of infective larvae from other nematode parasite species, a predominant antigen similar to GP31 was found in Trichostrongylus colubriformis and Haemonchus contortus, but in Toxocara canis a minor component corresponding in mol. wt to GP31 was also detected. Based on these results the possible role of GP31 as a candidate antigen for a broad spectrum molecular vaccine against gastrointestinal nematode parasites in sheep is discussed.     

Arrested development and the histotropic phase of Ascaridia galli in the chicken

Studies on 300 worm-free chickens infected with Ascaridia galli indicated that the histotropic phase is a normal part of the life cycle and that it involves both second- and third-stage larvae. The duration of the histotropic phase was dose-dependent. At low dose rates (50 eggs) it occurred from days 3 to 16 and was terminated abruptly by the third ecdysis. At high dose rates (2000 eggs) it was prolonged until at least day 54, because third-stage larvae became arrested in their development and the third ecdysis was delayed. Arrested development of Ascaridia galli was significantly suppressed following treatment of birds with cyclophosphamide, but expulsion of worms was not prevented. This result suggests the involvement of host antibodies in the induction of the arrested state.