Induction of matrix metalloproteinase-9 in mice during Toxocara canis larvae migration

The relationships between inflammation in organs with Toxocara canis larval migration and matrix metalloproteinase-9 (MMP-9) were investigated following the infection of mice with 1,000 infective eggs. Gelatinase activity was defined by gelatin zymography, optimum pH, inhibitor specificity and Western blot analysis. MMP-9 activity was present in the lungs, liver, muscles, and brain during T. canis larval migration. This enzyme had a molecular weight of about 94 kDa and showed maximum activity in the pH range of 6–8. The increased MMP-9 proteinases coincided with larval recovery and the degree of inflammation among the four organs. These results suggest that MMP-9 may be associated with the inflammatory reaction to larval toxocariasis during early migration, and may therefore be a useful marker during T. canis larvae migration.     

Imaging of Toxocara canis larvae labelled by CFSE in BALB/c mice

Mice are used most often as a model for human toxocarosis caused by Toxocara canis larvae. Variety of symptoms developing during the infection reflects behaviour of the larvae, which are able to escape from the intestine and further invade and damage various host organs. In order to find an approach enabling observation on parasite behaviour in mouse in vivo, we used an epifluorescence method and a small animal imaging system (SAIS). Larvae of T. canis were labelled by carboxyfluorescein succinimidyl ester (CFSE) which incorporated on the parasite gastrointestinal tract. Following infection of BALB/c mice by CFSE-labelled larvae it has been observed that staining had no influence on viability and further migratory activity of the parasites through the host organs (the intestine, liver, lungs and brain) where they were detected by SAIS until day 17 p.i. In addition, the dye did not affect larval antigenic activity as well as the development of related immune response. Imaging of parasites labelled by CFSE, therefore, may represent a promising way to study behaviour of T. canis larvae in a paratenic host.     

In vitro translation of mRNA from Toxocara canis larvae

300 micrograms of total RNA was extracted from 1 ml of packed Toxocara canis larvae by centrifugation through a 5.7 M cesium chloride cushion. 60 micrograms of polyadenylated messenger RNA was separated from 300 micrograms of total RNA in an oligothymidylic acid-cellulose gel column. The in vitro translation of the mRNA, isolated from T. canis larvae, was carried out using the rabbit reticulocyte cell-free translation system. Incorporation of [35S]methionine into trichloroacetic acid precipitable material in the lysate containing mRNA was 4-5 times greater than that of control. Translation products were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography. Many polypeptides ranging in molecular weight from 10000 to 100000 were synthesised in the lysate. A T. canis positive human serum was mixed with translation products to form antigen-antibody complexes, which were then absorbed by Staphylococcus aureus Cowan 1 strain and analysed by the autoradiography of SDS-PAGE. Three antigenic polypeptides with molecular weights of 49000, 27000 and 22000 which reacted specifically with IgG antibody in T. canis positive human serum, were demonstrated. The 27000 MW polypeptide reacted particularly strongly with the IgG antibody.     

Cryopreservation and long-term in vitro maintenance of second-stage larvae of Toxocara canis

Second stage larvae of Toxocara canis were isolated from developed eggs, frozen in Eagle's Minimal Essential Medium with 5% dimethyl sulfoxide or 10% glycerol as cryoprotectants according to two cooling schedules and maintained in liquid nitrogen for 1 week. After thawing, the previously frozen larvae (FL) and unfrozen controls (CL) were maintained in a chemically defined medium in vitro for 35 weeks. While CL had motility rates around 95% to 97% throughout the experiment, previously frozen larvae (FL) exhibited rates of 48%–58% at the beginning and of 19%–39% at the end of the 35 week in vitro maintenance period. The surviving FL and CL larvae proved to be infective for mice. Excretory/secretory (ES) antigens isolated from several batches of culture medium in which FL and CL had been maintained reacted in the ELISA with human sera containing antibodies against Toxocara. Antigens from FL and CL separated by SDS-PAGE and silver-stained showed some differences in polypeptide patterns. Western-blot analysis revealed that these differences were not related to antigenic polypeptides but were most likely caused by substances without antigenic properties originating from dead and/or degenerating larvae. It can be concluded that ES antigens produced by previously frozen larvae are essentially the same as those derived from unfrozen controls.The value of cryopreservation of T. canis larvae for routine production of ES antigens will be further evaluated.     

Disseminated granulomatous disease in a cat caused by larvae of Toxocara canis

The gross and histological findings of a case of disseminated granulomatous disease caused by larval nematode infection in a cat are presented. The larvae were identified as those of Toxocara canis and the lesions associated with infection of cats with various ascaridoid larvae are discussed.     

Biological activity of Paecilomyces genus against Toxocara canis eggs

Saprophytic soil fungi can exert ovicidal and ovistatic effects on helminths with differing degrees of efficiency. The representatives of such fungi from temperate regions, Paecilomyces lilacinus (Thom) Samson and P. marquandii (Masse) Hughes, exhibit recognized ovicidal activity on some nematodes. We evaluated the action in vitro of P. lilacinus and P. marquandii on the zoonotic canine roundworm eggs of Toxocara canis. Eggs exposed and unexposed to fungal samples were observed by both light and scanning electron microscopy on days 4, 7 and 14 post-inoculation. Ovicidal activity of P. lilacinus on T. canis eggs was considered to be high and that of P. marquandii to be intermediate. Received: 14 February 2000 / Accepted: 16 March 2000     

The impact of single versus repeated exposure of mice to Toxocara canis

Infection with T. canis can alter dramatically the brain and behavior of the host. Previous results suggest that if the mammalian host is exposed either simultaneously to lead, or has a history of prior exposure to that toxic substance, the magnitude of the behavioral reaction to T. canis may be modified or even reduced. The present data suggest that the magnitude of both the behavioral and tissue/immune reactions may be less if the organism has multiple, instead of a single exposure, to T. canis. Lead, and perhaps other environmental toxicants may alter neurotropic products of the parasite, the behavior of the parasite, and/or reactivity of the host in the presence of the parasite. Such considerations may help explain, in part, the relative rarity of reported toxocariasis in humans, despite the fact that serological indices suggest that exposure to T. canis may be as high as 7% of the world population.     

The progression of behavioral and pathological effects of the parasite Toxocara canis in the mouse

Toxocara canis, the parasitic roundworm of the dog may infect aberrant hosts including mice and humans. The present study examined the behavioral and pathological changes at each of three postintubation periods (Period 1: 8-10 days, 2: 49-51 days, and 3: 84-86 days postintubation, respectively) in independent groups of mice intubated with 1000 eggs of T. canis. Eight-ten days after intubation Toxocara infected animals typically showed depressed levels of activity relative to saline-intubated controls. The scope and severity of behavioral changes were attenuated when different mice were tested 49-51 days after infection, and then became more severe when the third set of animals was tested 84-86 days after intubation. While brain pathology increased over the three periods, visceral organs showed marked pathology 8-10 days after intubation followed by a decrease in severity. These data suggest that Toxocara associated pathological changes in visceral organs and in the brain have behavioral consequences in mice. Given the similarity in migratory pathways of this parasite in rodents and humans, and the findings of T. canis larvae in human brain tissue, the results of this animal study may have implications concerning the possible etiology of behavioral disorders for children who have a known history of pica for dirt.     

Purification and characterization of excretory and secretory antigen of Toxocara canis larvae.

Sialoglycoprotein (GP) of human erythrocytes was incorporated into liposomes and its effect on the Fc receptor-mediated phagocytic reaction of human PMN cells was examined. Whereas liposomes carrying 2,4-dinitrophenylated lipid were, upon opsonization with rabbit anti-DNP, readily ingested by PMN cells and induced the NBT-reducing reaction, these reactions were markedly suppressed when GP was incorporated into the target liposomes. The inhibitory activity was found in the glycophorin A and B fractions, but the latter was more active than the former on a weight basis. It was estimated that incorporation of only a single molecule of GP per vesicle of 6000 lipid molecules may be sufficient to protect the particle from phagocytosis, but there was an apparent antagonism between the suppressive GP and opsonizing antibody as, with more antibody, more GP became necessary to inhibit phagocytosis. The effect of GP was largely abolished by trypsin treatment of GP-bearing liposomes or by the addition of F(ab')2 of anti-GP.     

Eosinophilic myocarditis in CBA/J mice infected with Toxocara canis.

In humans, chronic eosinophilia has been associated clinically with endomyocardial fibrosis and myocardial damage. Mice infected with Toxocara canis have a marked eosinophilia, and develop eosinophil-rich granulomatous lesions in the soft tissues of the body, especially the lungs, liver, brain, and skeletal muscle. Few reports have described myocardial lesions associated with T. canis infections in mice. We examined the hearts of CBA/J mice killed at weekly intervals over an 8-week period for evidence of myocardial damage that might be attributable to eosinophils. Total white blood cell counts and eosinophil counts were obtained during this period, and revealed a peak white blood cell count of approximately 28,000 cells/mm3 at day 7 after infection and a peak eosinophil count of approximately 4,000 cells/mm3 at day 14 after infection. Myocardial lesions in the ventricular wall began as focal infiltrates of eosinophils and histiocytes, then progressed into granulomata containing necrotic debris. Collagen deposition was noted by day 21 after infection. By day 42 after infection, the lesions had contracted greatly because of a loss of cellularity, and consisted mainly of fibroblasts and hemosiderin-laden macrophages. Myocyte damage, characterized by increased eosinophilia and necrosis, was observed. T. canis-infected CBA/J mice thus offer a useful model to study eosinophil-dependent myocardial damage.     

In vitro studies on the effects of flubendazole against Toxocara canis and Ascaris suum

Adult Toxocara canis and Ascaris suum were incubated in vitro in media containing 0.1, 1, 10 or 100 µg/ml flubendazole in order to study drug-derived effects. This incubation was done for 8 h and repeated (in some groups) after 24 h for another 8 h. The onset and intensity of flubendazole-derived effects were dosage-dependent and time-dependent, i.e. the same grade of damage was reached when incubating for a longer period at a low dosage or for a shorter period in medium containing a high amount (10 or 100 µg/ml) of flubendazole. A repeated incubation in drug-containing medium was superior to a single exposure. Flubendazole is apparently able to penetrate into the worm's interior via the cuticle. This became evident in worms with sealed orifices, which showed identical damage to worms which were not sealed. The type of tissue damage due to flubendazole was identical in both worm species when exposed to any of the drug dosages used. The principal mode of action of flubendazole was based on the complete reduction of microtubuli-polymerisation inside the parasite's cells. This apparently led to the complete destruction of the hypodermis, muscle layer and intestine. Flubendazole also stopped the formation of gametes. Summarising, even low concentrations of flubendazole (0.1 µg/ml) led to significant and irreversible damage in all worms studied.     

In vitro studies on the effects of flubendazole against Toxocara canis and Ascaris suum

Adult Toxocara canis and Ascaris suum were incubated in vitro in media containing 0.1, 1, 10 or 100 micro g/ml flubendazole in order to study drug-derived effects. This incubation was done for 8 h and repeated (in some groups) after 24 h for another 8 h. The onset and intensity of flubendazole-derived effects were dosage-dependent and time-dependent, i.e. the same grade of damage was reached when incubating for a longer period at a low dosage or for a shorter period in medium containing a high amount (10 or 100 micro g/ml) of flubendazole. A repeated incubation in drug-containing medium was superior to a single exposure. Flubendazole is apparently able to penetrate into the worm's interior via the cuticle. This became evident in worms with sealed orifices, which showed identical damage to worms which were not sealed. The type of tissue damage due to flubendazole was identical in both worm species when exposed to any of the drug dosages used. The principal mode of action of flubendazole was based on the complete reduction of microtubuli-polymerisation inside the parasite's cells. This apparently led to the complete destruction of the hypodermis, muscle layer and intestine. Flubendazole also stopped the formation of gametes. Summarising, even low concentrations of flubendazole (0.1 micro g/ml) led to significant and irreversible damage in all worms studied.     

Effects of Toxocara canis infection on hemopoietic stem cells and hemopoietic factors in mice

The effects of Toxocara canis infection on hemopoietic stem cells and hemopoietic factors were examined in mice. Severe eosinophilia was observed with a peak 14 days after infection. When the numbers of hemopoietic stem cells in peripheral blood, spleen, and bone marrow were examined by spleen colony assay (CFU-S), those in peripheral blood and spleen increased in parallel with peripheral blood eosinophilia. On the other hand, CFU-S in bone marrow did not alter significantly throughout the course of infection. Interleukin (IL)-3, which is known as multi-colony-stimulating factor and is involved in the growth/differentiation of various blood cells including stem cells, was produced by spleen cells of infected mice. The time course study showed that concanavalin A stimulated IL-3 production peaked on day 7 after infection, whereas that with excretory secretory antigen peaked on day 14. Even without stimulation, spleen cells obtained on day 21 after infection produced IL-3 spontaneously. IL-5, which is known to have eosinophil differentiation factor activity, was also produced by spleen cells obtained on day 13 after infection. These results suggest that in response to increased demand for eosinophils, hemopoietic stem cells migrate into various extramedullar hemopoietic organs where they grow/differentiate into mature eosinophils, depending on the hemopoietic factors.     

The behavior and pathogenicity ofToxacara canis larvae in mice of different strains

In the present study the behavior and pathogenicity of second-stage larvae ofToxocara canis were examined in different mouse strains with special emphasis on the major histocompatibility complex (MHC). Mice of the inbred strains BALB, C3H, C57BL, and DBA and the outbred strain NMRI were infected orally with 1000 second-stage larvae ofT. canis. The clinical behavior of the animals: the numbers of larvae detected in the liver, lungs, brain and musculature; the hematological and serological parameters; and histological sections were examined. In mice of the BALB strain, no death occurred during the entire period of the investigation and the pattern of body-weight development of infected and uninfected animals was almost identical. The highest larval counts in the brain of all strains were found in BALB mice. The percentage of eosinophils in the blood of BALB mice increased after the 8th week postinfection, whereas it decreased in the other strains. Histological and pathophysiological changes developed to a lesser extent in this strain than in the other strains. In mice of the strains C3H, C57BL, DBA, and NMRI, deaths occurred from the 4th week postinfection onward. The infected animals lost weight in comparison with the uninfected controls; the numbers of larvae found in the brains of infected mice of the above-mentioned strains were lower than those detected in the BALB strain. There is no evidence that mechanical damage caused by migrating larvae in the brain tissue is mainly responsible for symptoms of central nervous toxocariasis. Likewise, the assumption that the MHC is involved in the allergicinflammatory response in the brain could not be proven: infected mice of the BALB and DBA strains reacted completely differently, although both are equipped with the same MHC haplotype.     

Ultrasonography for early diagnosis of Toxocara canis infection in puppies

Parasitology Research - Toxocara canis is one of the most common intestinal parasites in dogs and represents a highly infectious zoonotic parasite worldwide. Adult worms live in the bowel of dogs,...     

Immunoglobulin class of antibodies in monkeys infected with Toxocara canis

Sera of monkeys (Macaca sinica), experimentally infected with multiple doses of eggs of Toxocara canis, were subjected to Sephadex G-200 gel filtration and DEAE cellulose ion exchange chromatography. The distribution of antibodies in the various serum fractions was determined by the conglutinating complement absorption test (CCAT) and the agar-gel diffusion precipitin test using antigens prepared from the adult and the embryonated eggs of T. canis and by the formation of precipitates at the oral orifice of artificially hatched living 2nd stage larvae of T. canis.In serum samples taken 1 and 2 weeks after initial infection, CCAT antibodies were located mainly in the macroglobulin fractions of serum. By 1 to 4 weeks after a third dose of eggs such antibodies were located mainly in the 7S fraction of immunoglobulins.Antibodies detected by gel precipitation were located in the macroglobulin fraction of serum, while those that produced oral precipitates on 2nd stage larvae occurred in the 7S fractions.     

Lung manifestation of visceral larva migration syndrome due to Toxocara canis infection

32 year-old patient was hospitalized because of disseminated lung lesions. 2 years earlier he manifested chorioditis. Exact disease history suggested suspicion of toxocare infection. The diagnosis was confirmed by serological tests with anti-Toxocara canis antibodies, bronchial lavage and chest CT scan. Administration of 450 mg of dietylokarbamasin (Hetrazan) resulted in complete resolution of pulmonary lesions.