Immunity to larval Brugia malayi in BALB/c mice: protective immunity and inhibition of larval development

The objective of this study was to analyze the immune response of mice to the larval stages of Brugia malayi. Male BALB/c mice were inoculated with 3 doses of irradiated third-stage larvae (L-3) of B. malayi and were subsequently challenged with L-3 implanted ip within diffusion chambers. After 3 weeks, larvae were recovered to determine their viability, length, and stage of development. A significant reduction in parasite survival was observed in immunized mice. Furthermore, larvae recovered from immunized mice were significantly shorter than larvae recovered from control mice. All larvae recovered from immunized mice were L-3, whereas 96% of larvae recovered from controls were fourth-stage larvae (L-4). Sera collected from control and immunized mice were tested for the presence of antibodies reactive with L-3 and L-4 antigens using an indirect fluorescent antibody assay employing frozen larval cross-sections as antigen. Sera recovered after challenge of control mice reacted with internal, but not surface, antigens of L-3 and L-4. Alternatively, sera from immunized mice reacted with both internal and external antigens of both L-3 and L-4.     

Protective immunity against Brugia malayi infective larvae in mice. I. Parameters of active and passive immunity

Protective immunity against infective larvae of Brugia malayi was studied in different strains of mice using various sources of antigens. The following strains of mice were susceptible to infective larvae development for 2 weeks after primary ip challenge: BALB/c, C3H/HeJ, C3H/NeN, C3H/HeJms, C57BL/6Jms, and DDD. In comparison to gerbils, BALB/c mice developed stronger resistance to infective larvae after immunization with irradiation attenuated larvae or with killed microfilariae (mf). However, killed mf failed to enhance resistance in C3H/HeJ mice, although C3H/HeN mice were strongly protected and C3H/HeJms mice were protected to a lesser degree by this antigen. Extracts of mf with phosphate buffered saline and sodium dodecyl sulfate both induced high levels of resistance in BALB/c mice. Transfer of resistance from BALB/c mice immunized with attenuated infective larvae to naive mice was accomplished at a high level at protection with nylon wool nonadherent spleen cells (T cells) but not with adherent cells treated with anti-Thy 1.2 serum and complement. In contrast, sera from immunized mice were much less protective.     

日本血吸虫多价DNA疫苗免疫保护作用的研究

目的研究日本血吸虫多价DNA疫苗诱导BALB/c小鼠的免疫保护作用.方法65只BALB/c雌性小鼠随机分为5组,各组小鼠分别肌肉注射纯化的质粒DNA pcDNA3.1(对照组)、pcDNA3.1-TPI(TPI)、pcDNA3.1-(CDR3)6[(CDR3)6]、pcDNA3.1-TPI-linker-(CDR3)6(TLC)和pcDNA3.1-(CDR3)6-linker-TPI(CLT),每鼠100μg.每隔2周加强免疫1次,共3次.末次免疫后4周每鼠经腹部皮肤攻击感染(45±1)条日本血吸虫尾蚴,45 d后剖杀,计数成虫及肝脏虫卵数.首次免疫前2天及感染前2天经尾静脉采血检测IgG抗体水平、抗体亚类IgG1、IgG2a.末次免疫后2周取小鼠脾脏制备单个脾细胞,检测细胞因子IL-2、IL-4、IFN-γ的水平.结果TPI、TLC组和CLT组小鼠血清都检测到特异性IgG抗体,抗体亚类IgG2a/IgG1的比值分别为1.71、2.71和4.70,而其他两组则未检测到特异性IgG抗体;TPI、TLC组和CLT组小鼠的IL-2、IFN-γ较对照组均有不同程度的升高,IL-4则无明显差异.多价DNA疫苗TLC组和CLT组减虫率分别达到37.30%、39.09%,减卵率分别为41.61%、49.54%,均显著高于TPI组和(CDR3)6组(P均＜0.05).结论多价DNA疫苗相对于单价DNA疫苗能诱导小鼠产生较高的免疫保护作用,且诱导宿主产生较高的以Th1为主的免疫应答.     

Immunological genomics of Brugia malayi: filarial genes implicated in immune evasion and protective immunity

Filarial nematodes are metazoan parasites with genome sizes of> 100 million base pairs, probably encoding 15 000-20 000 genes. Within this considerable gene complement, it seems likely that filariae have evolved a spectrum of immune evasion products which underpin their ability to live for many years within the human host. Moreover, no suitable vaccine currently exists for human filarial diseases, and few markers have yet been established for diagnostic use. In this review, we bring together biochemical and immunological data on prominent filarial proteins with the exciting new information provided by the Filarial Genome Project's expressed sequence tag (EST) database. In this discussion, we focus on those genes with the highest immunological profile, such as inhibitors of host enzymes, cytokine homologues and stage-specific surface proteins, as well as products associated with the mosquito-borne infective larva which offer the best opportunity for an anti-filarial vaccine. These gene products provide a fascinating glimpse of the molecular repertoire which helminth parasites have evolved to manipulate and evade the mammalian immune response.     

小鼠巨细胞病毒性心肌炎模型的建立

目的　用小鼠巨细胞病毒(murinecytomegalovirus, MCMV)K181株建立BALB/c小鼠巨细胞病毒性心肌炎模型。方法　25只健康纯系5周龄BALB/c小鼠腹腔接种MCMVK181病毒悬液(1×104PFU/只),观察小鼠血清cTnI浓度变化和心肌组织MCMV病毒滴度及病理改变。结果小鼠腹腔接种病毒悬液后第3天即可在心肌组织中检测到低滴度MCMV,第5 ~7天达高峰,随后迅速下降,第10天时已不能检测到MCMV;小鼠心肌组织病理改变出现在MCMV感染后第3天, 7~10天达高峰,随后逐渐减轻,病变可持续到病毒感染后3 ~4个月;血清cTnI水平在MCMV感染后第3天即有升高, 7~10天达高峰, 14天仍维持较高水平。结论　成功建立小鼠巨细胞病毒性心肌炎模型,可为巨细胞病毒性心肌炎的治疗、药物筛选、疾病发病机制以及疾病预后评估提供一个良好研究平台。     

Stage–specific antigens of Hymenolepis microstoma recognized in BALB/c mice

Antigenicity of eggs (oncospheres), cysticercoids and adults (with immature segments only) of the bile duct tapeworm Hymenolepsis microstoma was analysed using immunoblotting techniques and indirect immunofluorescent antibody (IFA) techniques with immune sera of BALB/c mice (i) infected with different doses of cysticercoids, (ii) during patent or prepatent infection with the lumen phase of the parasite or (iii) sensitized with live or dead eggs. Antibody responses detected by IFA test and immunoblotting showed that antigenicity of eggs (oncospheres) differed from that of cysticercoids and adults. Single worm infections were sufficient to stimulate antibody responses. Mice which had patent infection showed strong antibody responses to all three (egg (oncosphere), cysticercoid, adult) antigens, while mice given two prepatent infections showed some antibody responses to cysticercoid and adult antigens only. Although the normal intermediate hosts of this parasite are arthropods, antibodies to some major egg (oncosphere) antigens were produced in mice given eggs of this parasite orally, either through inoculation of eggs or ingestion of faeces contaminated with eggs. Antibodies were not produced in mice dosed with non-viable eggs. The results are consistent with the hypothesis that cestode parasites express phase- (or stage-) specific antigens.     

A simple technique for the in vitro cultivation of nocturnally subperiodic Brugia malayi infective larvae

A simple system for the in vitro cultivation of nocturnally subperiodic Brugia malayi was developed. The manner of cultivation consisted of a 1:1 (v/v) mixture of Iscove's Modified Dulbecco's medium and NCTC-135 medium supplemented with 20% fetal bovine serum by using candle jar incubation at 37 degrees C instead of CO2 incubator. Changing the media: every 2 days, 3 days and changing media on day 7, then every 2 days produced a larval survival rate of 50% (70/140) on day 10, 49% (82/166) on day 6, and 53% (105/200) on day 9. With this technique, up to 50% of the infective stage larvae (L3) survived for up to 10 days and had long life for at least 27 days in all experiments with low larval survival rate in the fourth week. In addition, the culture system promoted molting L3 to fourth stage larvae (L4) after 7 days, as shown by light microscope.     

An improved Graves' disease model established by using in vivo electroporation exhibited long-term immunity to hyperthyroidism in BALB/c mice

In Graves' disease, the overstimulation of the thyroid gland and hyperthyroidism are caused by autoantibodies directed against the TSH receptor (TSHR) that mimics the action of TSH. The establishment of an animal model is an important step to study the pathophysiology of autoimmune hyperthyroidism and for immunological analysis. In this study, we adopted the technique of electroporation (EP) for genetic immunization to achieve considerable enhancement of in vivo human TSHR (hTSHR) expression and efficient induction of hyperthyroidism in mice. In a preliminary study using beta-galactosidase (beta-gal) expression vectors, beta-gal introduced into the muscle by EP showed over 40-fold higher enzymatic activity than that introduced via previous direct gene transfer methods. The sustained hTSHR mRNA expression derived from cDNA transferred by EP was detectable in muscle tissue for at least 2 wk by RT-PCR. Based on these results, we induced hyperthyroidism via two expression vectors inserted with hTSHR or hTSHR289His cDNA. Consequently, 12.0-31.8% BALB/c mice immunized with hTSHR and 79.2-95.7% immunized with hTSHR289His showed high total T(4) levels due to the TSHR-stimulating antibody after three to four times repeated immunization by EP, and thyroid follicles of which were hyperplastic and had highly irregular epithelium. Moreover, TSHR-stimulating antibody surprisingly persisted more than 8 months after the last immunization. These results demonstrate that genetic immunization by in vivo EP is more efficient than previous procedures, and that it is useful for delineating the pathophysiology of Graves' disease.     

The analysis of the humoral response of the BALB/c mouse immunized with radiation attenuated third stage larvae of Brugia pahangi

BALB/c mice immunized with L3 of Brugia pahangi, irradiated at 45kRad from a ¹³⁷Caesium source, are strongly immune to challenge infection (75–100% reduction in the recovery of challenge infection larvae on day 6 post-challenge). The target of immunity appears to be the post-infective L3, as challenge infection larvae are killed within 5–6 days of infection. By immunoblot analysis, serum from immune animals recognizes a limited set of somatic antigens, the majority of which are shared between different life cycle stages. Serum from immune mice also strongly recognizes larval surface antigens by immunofluorescence, some of which may be stage specific. The larval surface determinants do not appear to be protein or glycoprotein by standard immunochemical analysis. A proportion of the antibody response of the BALB/c mouse is directed towards phosphorylcholine epitopes on filarial antigens, but the limited antigen recognition cannot be explained on the basis of the mouse strain used, as CBA/Ca mice recognize a similar limited set of antigens.     

Antioxidant responsiveness in BALB/c mice exposed to ozone

BACKGROUND: A single, acute exposure to ozone has been shown to modify the antioxidant defense mechanism in the respiratory tract. OBJECTIVE: The aim of this study was to evaluate the effects of ozone exposure on antioxidant response in BALB/c mice. METHODS: We measured enhanced pause of breathing (Penh) as a marker of airway obstruction using barometric whole-body plethysmography before and after ozone exposure [groups (n = 6): filtered air, 0.12 ppm, 0.5 ppm, 1 ppm, 2 ppm] for 3 h. Antioxidant levels were measured using high-performance liquid chromatography with electrochemical detection in bronchoalveolar lavage (BAL) fluid and lung tissue homogenates. RESULTS: Malondialdehyde concentrations in lung tissue homogenates were significantly increased in the group exposed to 2-ppm ozone compared to the filtered air group. Uric acid and gamma-tocopherol concentrations in BAL fluid were significantly increased in the ozone exposure group compared to the filtered air group (p < 0.01). Uric acid concentrations were increased in a concentration-dependent manner according to ozone concentration to which the animals were exposed. Increases in Penh after ozone exposure were significantly higher in an ozone concentration-dependent manner. The proportion of neutrophils in BAL fluid was significantly higher in the group exposed to 2 ppm than in the filtered air and the group exposed to 0.12 ppm (p < 0.01, respectively). The level of ascorbate correlated with the level of gamma-tocopherol. CONCLUSION: These findings suggest that antioxidant responses may serve as a protective mechanism against a range of oxidants in BALB/c mice exposed to ozone.     

Differential Susceptibility of BALB/c and BALB/cBy mice to Graves' hyperthyroidism.

BALB/c mice are susceptible to the induction of Graves' hyperthyroidism. To investigate the susceptibility of BALB/c substrains of mice to the induction of hyperthyroidism, we immunized BALB/cJ and BALB/cByJ mice with an adenovirus expressing amino acid residues 1-289 of thyrotropin receptor (TSHR). The data presented in this article showed that 17 of 26 (65%) BALB/c and only 4 of 30 (13%) BALB/cBy mice developed hyperthyroidism. Hyperthyroid mice displayed characteristics of Graves' disease, such as thyroid-stimulating antibodies and enlarged thyroid glands. To explore the differences in the susceptibility of these substrains for hyperthyroidism, we examined the TSHR antibodies in three different assays. The TSHR antibodies determined in a radioreceptor assay (TSH binding inhibitory immunoglobulins) were similar in both of these BALB/c substrains. The TSHR antibody titers of total IgG, IgG1, and IgG2a were measured by an enzyme-linked immunosorbent assay and were found to be similar in these mice. There were no significant differences between these two groups of mice in the thyroid-stimulating antibody activity. However, BALB/cBy mice had significantly higher TSH-blocking antibody activity compared to BALB/c mice. TSHR-specific proliferation of splenocytes and secretion of cytokines interferon-gamma and interleukin-4 by spleen cells were comparable in both the groups. BALB/cJ and BALB/cByJ mice both belong to same MHC haplotype, H-2(d), but differ in the Qa-2 region of class Ib molecule. This report shows the importance of other genes, such as Qa-2 region of class Ib molecule in addition to MHC class II, in the susceptibility of Graves' hyperthyroidism.     

Effects of serum from treated patients on antibody-dependent cell adherence to the infective larvae of Brugia malayi

Adherence assays were used to demonstrate the in vitro effect of serum-dependent cellular adherence of human buffy coat cells to infective larvae of Brugia malayi in filariasis patients treated with antifilarial drugs. In this study, microfilaraemic patients were treated with either diethylcarbamazine citrate (DEC), mebendazole or levamisole hydrochloride. It was found that DEC and mebendazole decreased the motility of infective larvae due to a direct action of the drugs. Sera of levamisole-treated patients caused increased adherence of human buffy coat cells to infective larvae, leading to a decrease in motility and cuticular damage as confirmed by scanning electron microscopic studies. However, serum of levamisole-treated patients alone could cause a similar lethal effect on infective larvae. Studies with the indirect fluorescent antibody test suggested that IgM was involved in this phenomenon. Complement did not appear to be important.     

Protective immunity in the rat model of congenital toxoplasmosis and the potential of excreted-secreted antigens as vaccine components

Toxoplasma infection is a major cause of severe foetal pathology both in humans and in domestic animals, particularly sheep. We have previously reported the development of an experimental model to study congenital toxoplasmosis in the rat. Here we demonstrate that, as in humans, total protection against congenital toxoplasmosis can be achieved regardless of the strain of Toxoplasma gondii used to infect rats, or when initial and challenge infections were carried out with different strains. Chronic infection is associated with a highly specific immunity that involves both B-and T-cell responses beginning at day 10 postinfection. The antibody isotype analysis revealed that whereas immunoglobulin (Ig)G2b is the major elicited isotype, no IgG1 antibodies are detected. T cell proliferation was assayed using crude Toxoplasma extracts or excretory-secretory antigens (ESA). The analysis of T cell supernatants showed the specific secretion of both interleukin-2 and interferon-gamma by activated T cells. Immunization of rats before pregnancy with either crude Toxoplasma extracts or with ESA elicited a B cell response that included antibodies of the IgG1 isotype and conferred on the newborns high levels of protection. Preliminary experiments of immunization using two HPLC-purified ESA, GRA2 and GRA5, conferred, a significant protection although to a lesser extent. This experimental model represents an attractive model for the identification of future vaccine candidates against congenital toxoplasmosis.     

Cryopreservation of microfilariae and third-stage larvae of Brugia malayi and subsequent development in the hosts

Methods are studied for the cryopreservation of microfilariae (mf) and third-stage larvae (L3) of periodic Brugia malayi. RPMI-1640 tissue culture medium containing 6% dimethyl sulfoxide (DMSO) and 15% newborn calf serum were used as cryoprotectant. The larvae survived best when specimens were frozen at the rate of -0.5 degrees C to -1.0 degrees C per minute using the vapor phase of liquid nitrogen, when the temperature reached -70 degrees C to -90 degrees C the specimens were placed directly into the liquid nitrogen (-196 degrees C). After the thawing of the mf which had been stored for 6,212 and 375 days in cryogen, 96.2% of the mf were shown to be viable and developed in Aedes togoi. It was also shown that the survival rate of L3 cryopreserved for 28-321 days was also 96.2% and that, when 107 L3 frozen for 321 days were inoculated after being thawed into one jird, one live female adult was recovered at autopsy 71 days after inoculation, its morphology being the same as the unfrozen specimens. There was no correlation between the time of cryopreservation and the survival rate of the larvae.     

弓形虫DNA疫苗联合诱导BALB/c小鼠保护性免疫力的研究

研究弓形虫表面抗原P30和P22 DNA疫苗联合诱导BALB/c小鼠的保护性免疫作用。48只BALB/c小鼠随机分成4组:A组(NS对照组),肌注生理盐水100μl/鼠;B组(空质粒对照组),肌注pBK-CMV 100μl/鼠(1mg/ml);C组(pBK-P30免疫组)肌注pBK-P30 100μl/鼠(1mg/ml);D组(pBK-P30&pBK-P22联合免疫组)分别肌注pBK-P30和pBK-P22各100μl/鼠(1mg/ml),免疫后5周,通过ConA刺激,MTT法测定免疫鼠脾脏T淋巴细胞转化率,使用间接免疫荧光法,用流式细胞仪对CD4+、CD8+T细胞群进行测定。免疫后10周,免疫鼠腹腔注射弓形虫速殖子攻击感染。结果表明,ConA刺激小鼠脾T淋巴细胞均发生增殖反应,疫苗免疫组略高于两对照组,但4组之间的差异均无显著性(P>0.05)。T淋巴细胞亚群CD4+、CD8+动态分析,CD4+T细胞在各组的增殖均不明显(P>0.05),但两免疫组CD8+T细胞数量升高,CD4+/CD8+比值降低,与空质粒pBK-CMV对照组和NS对照组之间差异均有显著性(P<0.05,P<0.01),pBK-CMV对照组与NS对照组之间差异也有显著性(P<0.01),两免疫组之间差异无显著性(P>0.05)。弓形虫速殖子攻击感染,联合免疫组平均存活时间较两对照组均延长(P<0.05)。但两免疫组间、pBK-P30免疫组与两对照组之间差异无显著性(P>0.05)。弓形虫DNA疫苗能诱导BALB/c鼠产生特异性细胞免疫应答及部分抗虫免疫保护作用,此两种DNA疫苗联合免疫有一定的免疫保护效果。     

The role of CD4 cells in protective immunity to Brugia pahangi

The BALB/c mouse immunized sub-cutaneously (s.c.) with 45 kRad attenuated third stage larvae (L₃) of the lymphatic filarial nematode Brugia pahangi is strongly immune to a challenge infection (75–100% reduction in recovery at day six post challenge). Analysis of spleen cell supernatants from immunized mice re-stimulated in vitro, with parasite antigen or the non specific T cell mitogen Con-A reveals a cytokine profile (IL-4, IL-5 and IL-9) which indicates that the Th2 subset of CD4 cells has been expanded. In an attempt to formally prove a critical role for CD4 cells in immunity in this model system, immunized mice were given either anti-CD4 or anti-CD8 neutralizing antibodies. Administration of anti-CD4 antibody had a significant and detrimental effect on the immune response whereas anti-CD8 antibody had a negligible effect on immunity. The efficacy of antibody in neutralizing their target cells was determined by fluorescence activated cell sorting analysis (FACS). Spleen cells from anti-CD4 treated immunized mice, when re-stimulated with parasite antigen had a significantly reduced potential to secrete IL-4, IL-5 and IL-9 in vitro and serum from these mice had reduced levels of parasite specific IgG and IgE. These results demonstrate a critical role for CD4 T cells in host protective immunity to B. pahangi in vivo and strongly suggest that some component of the Th2 response plays an important role in the immune response elicited in this model system.     

A monoclonal antibody to surface antigens on microfilariae of Brugia malayi reduces microfilaremia in infected jirds

A monoclonal antibody to surface antigens of Brugia malayi microfilariae promotes the adherence of peripheral blood cells to microfilariae in vitro and reduces microfilaremia in vivo. The antibody reacts with epitopes present on two stage-specific antigens with estimated molecular weights of 70,000 and 75,000 daltons.     

The protective effect of a DNA vaccine encoding the Toxoplasma gondii cyclophilin gene in BALB/c mice

Toxoplasmosis is a world‐wide zoonosis that causes significant public health and veterinary problems. The study of vaccines remains the most promising method for the future prevention and control of toxoplasmosis. Recombinant Toxoplasma gondii cyclophilin has been shown to have potent PPIase and IL‐12‐inducing activities, thus promoting the stabilization of T. gondii's life cycle and maintaining the survival of its host during evolution. In this study, the T. gondii cyclophilin gene was used to construct a DNA vaccine (pVAX1‐TgCyP). The immune response and protective efficacy of the vaccine against T. gondii infection in BALB/c mice were evaluated. All BALB/c mice that were vaccinated with pVAX1‐TgCyP developed a high response with TgCyP‐specific antibodies, and significant splenocyte proliferation (P < 0·05) compared with pVAX1 vector and PBS groups. pVAX1‐TgCyP also induced a significant Th1 type immune response, indicated by the higher production of IL‐2 and IFN‐γ (P < 0·05). The survival rate of BALB/c mice increased significantly after vaccination with pVAX1‐TgCyP (37·5%) (P < 0·05). These results indicate that TgCyP is a highly efficacious vaccine candidate that can generate protective immunity against T. gondii infection in BALB/c mice.     

Neutrophils have a protective role during early stages of Leishmania amazonensis infection in BALB/c mice

Neutrophils are involved in the early stages of immune responses to pathogens. Here, we investigated the role of neutrophils during the establishment of Leishmania amazonensis infection in BALB/c and C57BL/6 mice. First, we showed an accumulation of neutrophils between 6 and 24 h post‐infection, followed by a reduction in neutrophil numbers after 72 h. Next, we depleted neutrophils prior to infection using RB6‐8C5 or 1A8 mAb. Neutrophil depletion led to faster lesion development, increased parasite numbers and higher arginase activity during the first week of infection in BALB/c mice, but not in C57BL/6 mice. Increased susceptibility was accompanied by augmented levels of anti‐L. amazonensis IgG and increased production of IL‐10 and IL‐17. Because IL‐10 is a mediator of susceptibility to Leishmania infection, we blocked IL‐10 signalling in neutrophil‐depleted mice using anti‐IL‐10R. Interestingly, inhibition of IL‐10 signalling abrogated the increase in parasite loads observed in neutrophil‐depleted mice, suggesting that parasite proliferation is at least partially mediated by IL‐10. Additionally, we tested the effect of IL‐17 in inflammatory macrophages and observed that IL‐17 increased arginase activity and favoured parasite growth. Taken together, our data indicate that neutrophils control parasite numbers and limit lesion development during the first week of infection in BALB/c mice.     

Excretory/secretory proteases and mechanical movement of Anisakis pegreffii infective larvae in the penetration of BALB/c mice gastrointestine

Anisakiasis is a human parasitic disease caused by infection with the infective larvae of Anisakis. Accidental infection in humans causes the gastrointestinal pathophysiological effects of mechanical tissue damage by migrating larvae. The mechanism of the infective larval invasion and migration is suspected to involve larval excretory/secretory proteases and motility. This study demonstrates the penetration rate of the infective larvae of Anisakis pegreffii in mouse gastrointestine depends on the time after infection, and that only 15% of larvae remain in the gastrointestinal tract 3 h after infection. Strong activities of matrix metalloproteinases (MMPs) and serine proteases, especially plasmin, were found in the excretory/secretory products of A. pegreffii; these can be inhibited by ONO-4817 and phenylmethylsulfonyl fluoride, respectively. The protease activity was also significantly decreased in another 1 h of cultivation of larvae in fresh 0.9% normal saline (NS) after previous cultivation for 48 h in NS. The motility scores of larvae were significantly lower after 48 h of cultivation in NS. The penetration rate of A. pegreffii larvae in the gastrointestine of infected mice sequentially were 90% in the freshly prepared, 68% in serine protease inhibited, 55% in MMPs inhibited larvae, and 16% in larvae cultivated in NS for 48 h. Therefore, this study demonstrates that MMPs and serine proteases excreted and secreted by A. pegreffii and the mechanical movement of infective larvae participate in the penetration of the gastrointestine of mice after infection.