Cross-reactions of sera from Toxocara canis-infected mice with Toxascaris leonina and Ascaris suum antigens.

The ELISA method using larval excretory-secretory (E/S) products and homogenized Toxocara canis, Toxascaris leonina and Ascaris suum adult worm extract were used to determine possible cross-reactions in BALB/c and C57BL/10 mice, inoculated with embryonated eggs or adult worm extract of T. canis in single and multiple doses. When we used sera of mice infected with embryonated eggs of T. canis against different heterologous antigens, we observed no cross-reactions in BALB/c mice against A. suum E/S and adult worm extract antigens with a single dose. In multiple doses this was absent too against T. leonina adult worm extract in BALB/c mice, and in both strains against A. suum E/S and adult worm extract. In BALB/c mice inoculated with adult worm extract of T. canis we did not observe cross-reactions with A. suum E/S antigen with both inoculation doses. In the remainder of the experiments, we observed cross-reactions of different intensities.     

Scanning electron microscopy of the eggs of Ascaris lumbricoides, A. suum, Toxocara canis, and T. mystax.

Eggs of Ascaris lumbricoides, A. suum, Toxocara canis, and T. mystax were examined by scanning electron microscopy (SEM). All species under study exhibited pronounced surface ridges. The ridges formed distinctive patterns in T. canis and T. mystax. In the Ascaris species, the ridges are similar except that they are more pronounced in the eggs of A. suum. Operculumlike structures were observed only in Ascaris. Correlation of data from SEM with previously reported transmission electron microscopy suggests that the surface ridges seen in Ascaris eggs are formed by the chitinous layer of the shell.     

Eosinophilia in chimeric mice infected with Toxocara canis

A marked strain variation in eosinophilia following oral infection with Toxocara canis eggs was observed in mice. Mutual radiation chimeras between high and low responder mice in terms of eosinophilia were made and compared with the respective donor and recipient for eosinophilia after the infection. As a result, the degree and time course of eosinophilia in chimeric mice were similar to those in donors. The result suggested that genes which regulate inheritance of the trait, marked eosinophilia in T. canis-infected mice, might be expressed in bone marrow derived cells.     

Decontamination by anaerobic stabilisation of the environment contaminated with enteronematode eggs Toxocara canis and Ascaris suum

Investigations were carried out under operating conditions of Field Composting Factory in Brezno (Slovak Republic) to determine the effect of anaerobic stabilization of organic wastes from public areas on the survival of model helminth Toxocara canis and Ascaris suum eggs. Due to anaerobic conditions, low temperature, low C:N ratio and changes in physical and chemical properties of organic waste, less than 64% of A. suum eggs remained viable after 150 days of stabilisation. The anaerobic stabilisation had a greater effect on the viability of T. canis eggs than on A. suum eggs. The infectivity of T. canis eggs was confirmed by a follow-up experiment in laboratory mice. A small number of T. canis larvae were found in their brain and muscles on day 28 after infection. The results refer to the risks of dissemination, survival and potential spread of endoparasitic developmental stages in the environment through organic wastes subjected to low temperature stabilisation.     

A phosphorylcholine idiotype related to TEPC 15 in mice infected with Ascaris suum.

A serum component, binding antigens having phosphorylcholine (PC) determinants were induced in several strains of mice by infection with Ascaris suum. This component was isolated and demonstrated to be an IgM (K) anti-PC antibody having idiotypic determinants in common with the IgA PC-binding myeloma protein TEPC 15. Rabbit anti-idiotypic antisera prepared with this component had idiotypic specificity for TEPC 15 and cross-idiotypic recognition of MOPC 167 and McPC 603, all IgA PC-binding myeloma proteins. The antisera also recognized determinants not present on TEPC 15. IgM and idiotype levels were quantitated by radial immunodiffusion and PC-specific antibody measured by hemagglutination (HA) with sheep erythrocytes coated with pneumococcal-C-polysaccharide. Mean IgM levels ranged from 2.5 to 8.7 mg/ml, idiotype from 0.54 to 5.3 mg/ml; and HA titers from 1:512 to 1:130,000 in different mouse strains. The high PC-specific antibody response was not duplicated by immunization with dead ascaris larvae or by infection with two other nematode species.     

Cross-reactivity between sera from dogs experimentally infected with Dirofilaria immitis and crude extract of Toxocara canis

This study was performed to investigate whethere there is cross-reactivity between Dirofilaria immitis and three intestinal nematodes of dogs. In ELISA, D. immitis-infected dog sera obtained at the 4th molting stage (9-11 weeks) and microfilaremic stage (25-30 weeks) were shown to be highly reactive with crude extract of T. canis. In immunoblotting, some antigenic fractions, 44, 57, 88, 100 kDa of crude extract of T. canis, were found to be positive reaction with sera of dogs infected with D. immitis. However, little or no cross-reaction were observed between sera of D. immitis-infected dogs and crude extract antigen of T. vulpis or A. caninum. These result suggest that there are partial cross reaction between sera of D. immitis-infected dogs and the antigen of T. canis.     

Demonstration of carboxyl and thiol protease activities in adult Schistosoma mansoni, Dirofilaria immitis, Angiostrongylus cantonensis and Ascaris suum

Evidence has been presented showing two kinds of acidic protease activities in adult Schistosoma mansoni, Dirofilaria immitis, Angiostrongylus cantonensis and Ascaris suum. A haemoglobinolytic activity without adding any SH-containing compounds was maximal at pH 3.5, 2.5, 3.0 and 3.5 in S. mansoni, D. immitis, Angiostrongylus cantonensis and Ascaris suum respectively. The inhibitor studies demonstrated that this activity is ascribable to carboxyl protease(s). In the presence of dithiothreitol, activity on Azocoll (azo-dye coupled hide powder) was maximal at pH 4.6, 4.6, 3.5 and 5.6 in S. mansoni, D. immitis, Angiostrongylus cantonensis and Ascaris suum respectively. The effects of inhibitors demonstrated that this activity belongs to the thiol protease class. The intraspecific distribution of the protease activities was studied in some of the nematodes from which the organs could be anatomically separated. The distribution patterns of the haemoglobinolytic and azocollytic activities were quite different in An. cantonensis and much the same in As. suum. Based on the present results, acidic haemoglobinolytic activities reported in adult S. mansoni by other authors are thought to be due to carboxyl and thiol protease(s) respectively.     

An improved method for recovery of muscle-stage larvae from mice infected with Toxocara canis

We report a modified digestion method that improves the recovery of Toxocara canis larvae from skeletal muscle. Minced muscle tissue from infected mice was incubated in artificial gastric juice for 48 hr at 37 C, and ethanol was added for the second 24 hr. This procedure allowed the larvae to be identified and counted more quickly than with the standard digestion method. This method allows measurement of the total number of larvae present in muscle tissue following oral intubation of embryonated eggs, although it does not permit counting of live larvae.     

Prolactin evokes lactational transmission of larvae in mice infected with Toxocara canis

We investigated the trans-lactational maternal–neonatal transmission of Toxocara canis larvae in mice, with particular interest in the role of prolactin in their migration to the mammary gland. Two female mice were infected with 300 T. canis eggs soon after delivery of 27 offspring. After 1 week of breast-feeding, seven larvae were recovered from 4 of 13 offspring. After 2 weeks of lactation, 101 larvae were recovered from all the remaining offspring. Daily prolactin administration (5 μg) was performed 2 weeks before T. canis infection and continued until 2 weeks after infection in six non-pregnant female mice, which resulted in larval accumulation in the mammary gland. Furthermore, prolactin administration in female mice that had been infected with T. canis 4 weeks prior to prolactin treatment induced migration of larvae into the mammary gland. These findings suggest that prolactin is a promoting factor contributing to lactational transmission of T. canis larvae in mice.     

Eosinophil peroxidase levels in hearts and lungs of mice infected with Toxocara canis

Toxocara canis infection of abnormal hosts results in a condition in which infective larvae migrate through the soft tissues of the body, exclusive of the skin. This condition is known as visceral larva migrans (VLM) and causes a syndrome characterized by hepatosplenomegaly, hyperglobulinemia, hypereosinophilia, and transient pulmonary infiltrates. Because of the known association between hypereosinophilia and eosinophilic heart disease, we have been studying the hearts of mice infected with T. canis for evidence of myocardial damage and have previously described a severe eosinophilic myocarditis that leads to a marked myocardial fibrosis. We have measured eosinophil peroxidase (EPO) levels (a marker enzyme for specific granules of eosinophils) in homogenized lungs, homogenized hearts, and eosinophils recovered from the lungs of mice infected with T. canis over a 6-wk period. A marked accumulation of EPO was observed in the lungs of infected mice from day 14 postinfection (PI) to at least 6 wk of infection. Most of the EPO was associated with eosinophils that comprise the bulk of the pulmonary infiltrates associated with the VLM syndrome. However, following bronchoalveolar lavage, cytochemical localization of EPO activity in lungs from infected mice suggested that eosinophil degranulation had resulted in this marker enzyme being deposited within the pulmonary parenchyma. Peak levels of EPO were found in the myocardium by day 14 PI and declined over the 6-wk period. These levels equaled about 1/3 of the levels seen in the lungs of the same mice. These studies suggest that in mice infected with T. canis, the presence of increased numbers of eosinophils may lead to marked peroxidatic cardiopulmonary damage.     

Follow-up of antibody avidity in BALB/c mice infected with Toxocara canis

In human Toxocara canis infection, an association has been shown between high IgG avidity in the chronic phase and low IgG avidity in recently acquired toxocarosis. The evolution of the antibody response in terms of avidity has been carried out through a T. canis infection in BALB/c mice. Infection with T. canis embryonated eggs (EE) was carried out with single doses (SD) of 6, 12, 50, 100, 200 or 1000 EE/mouse and with multiple doses (MD) of 200 and 1000 EE. Specific antibodies against T. canis (IgM+G, IgG, IgG1 and IgM) were detected by ELISA and Western Blot (WB) techniques in the presence and absence of urea. With the ELISA method, an increase in the avidity index (AI) of around 50% was detected from days 40-80 p.i. to the end of the study, with all the doses studied. The WB method showed the presence of high avidity antibodies bound to 100 kDa and 75 kDa T. canis proteins in all the cases when the IgM+G and the IgG1 antibodies were investigated. Antibodies of variable avidity were observed in those sera that recognized the group of low molecular weight proteins, between 37 kDa and 25 kDa.     

Eosinophils in the cerebrospinal fluid of mice infected with Angiostrongylus cantonensis are resistant to apoptosis.

Interleukin-5 (IL-5) transgenic mice were used to assess the immunological features of CSF eosinophils from mice infected with Angiostrongylus cantonensis. CSF eosinophils were hypodense by day 14 post infection (p.i.). CSF eosinophils survived longer in vitro than peritoneal eosinophils collected from cadmium sulphate (CdSO₄) -treated normal IL-5 transgenic mice. Apoptosis was measured by Annexin V binding and the presence of a distinct laddering pattern of DNA fragmentation on agarose electrophoresis. Regardless of the presence or absence of Actinomycin D, CSF eosinophils collected from IL-5 transgenic mice from days 15–36 p.i. exhibited less apoptosis than peritoneal eosinophils collected from uninfected IL-5 transgenic mice. CSF eosinophils collected from A. cantonensis infected C57BL/6 mice at days 15–34 p.i. showed elongation of survival time and less apoptosis during in vitro cultivation. Reduced apoptosis was noted only in CSF eosinophils, but not in peritoneal eosinophils recovered from the same infected IL-5 transgenic mice. CPP32/Caspase 3 activity of cultured peritoneal eosinophils from both infected and uninfected IL-5 transgenic mice was higher than that of cultured CSF eosinophils. Stimulation with A23187 readily induced apoptosis of peritoneal eosinophils, but not CSF eosinophils or peritoneal eosinophils cultured with mouse recombinant IL-5. The latter cells were morphologically identical to hypodense eosinophils. RT-PCR analysis indicated that bcl-2 and bcl-x_L mRNA expression was higher in CSF eosinophils compared with peritoneal eosinophils and this expression in the latter cells was upregulated after culture with mouse recombinant IL-5. These results suggest that CSF eosinophils, shifting to hypodense status through an accumulation from peripheral blood, are resistant to apoptosis. These changes may explain the long-lasting, helminthotoxic and neurotoxic actions of CSF eosinophils in A. cantonensis infection.     

Isolation of specific antigens from Angiostrongylus cantonensis. 2. By affinity chromatography

The crude adult worm extract of Angiostrongylus cantonensis was subjected to a series of affinity chromatography for selective removal of host antigens as well as cross-reactive components with other helminths. The purified fraction designated as AC(p) with molecular weights ranging from 10,000-42,000 was found to contain antigenic components specific to A. cantonensis as determined by immunoelectrophoresis and enzyme-linked immunosorbent assay.     

Isolation of specific antigens from Angiostrongylus cantonensis. 1. Preparative flatbed isoelectric focusing

Electrophoresis on SDS gel and analytical isoelectric focusing showed that a crude extract of Angiostrongylus cantonensis consisted of at least 40 protein components with molecular weights ranging from 13 000-70 000 and isoelectric points of pI values ranging from 3.7-10.0. Crossed-immunoelectrophoresis with a hyperimmune antiserum to A. cantonensis showed at least 40 different antigenic components in the crude worm extract which were cross-reactive with those of Ascaris suum, Metastrongylus apri and Toxocara canis. Using preparative isoelectric focusing, the somatic worm preparation was divided into 13 equal fractions, of which 3, 4 and 5, with pI values of 3.7, 4.0 and 4.45 respectively, were later shown by immunoelectrophoretic techniques and enzyme-linked immunosorbent assay to contain antigens specific to A. cantonensis.     

Alteration in density of eosinophils in the cerebrospinal fluid of mice infected with Angiostrongylus cantonensis

The density of eosinophils in the cerebrospinal fluid (CSF) and blood of male ddY mice infected with Angiostrongylus cantonensis was examined on days 14, 20 and 27 post-infection (p.i.) with discontinuous Percoll gradient centrifugation. Normal blood eosinophils had a density of between 1.070 and 1.080 g ml-1. No significant changes in density in blood eosinophils were noted during the course of the observations. CSF eosinophils began to become hypodense (defined as density less than 1.070 g ml-1) on day 20 p.i., and 88% of eosinophils were hypodense on day 27. Our results suggest therefore that eosinophils probably become hypodense in the CSF and brain tissues, but not in the blood.     

Experimental infection routes of Angiostrongylus cantonensis in mice.

Stomach intubation is the most common method used in the experimental infection of animals with Angiostrongylus cantonensis. In order to compare the effectiveness of other possible transmission methods, groups of BALB/c mice were given infective third-stage larvae of A. cantonensis by different routes including intraperitoneal or subcutaneous injections, and penetration of anal mucosa, vaginal mucosa, conjunctival mucosa, lacerated skin, unabraded skin, foot pad and tail skin, while stomach intubation was used as control. Recovery of fifth-stage larvae was higher in mice inoculated with third-stage larvae subcutaneously. Successful infections were established through all experimental transmission routes except tail skin penetration. This study suggests that oral infection may not be the only route for the transmission of human angiostrongyliasis, and subcutaneous infection may be a better method for experimental infection.