Toxocara canis larvae viability after disinfectant—exposition

The effect of three routinely used disinfectants on the embryonary development of Toxocara canis eggs was evaluated both in vivo and in vitro. In the in vitro experiment, T. canis eggs were treated with the ethanol, sodium hypochlorite, and one commercial mix of benzalconium chloride and formaldehyde, and the embryonary development was assessed. After a period of 24 days incubation, ethanol was the best disinfectant because it prevented the development of the T. canis larvae 2 in the eggs, and sodium hypochlorite caused degeneration in 50% eggs. By using the commercial mix, 25% T. canis eggs developed to 2nd stage larvae. In the in vivo experiment, the embryonated eggs treated with the disinfectants were inoculated to mice, and their brain tissues were examined for larval presence on the 24th day postinfection. In addition, a control group was set up for comparison with the infected groups. No injury or T. canis larvae were observed in mice infected with sodium hypochlorite-treated eggs, opposite to that recorded in the animals infected with the commercial disinfectant-treated eggs. These results showed that both ethanol and sodium hypochlorite are very appropriate because of their full efficacy against infective T. canis eggs. Disinfection of kennels, animal shelters, cages, and veterinary clinics with one of these products to eliminate T. canis eggs and to avoid contamination is strongly recommended.     

Visceral larva migrans—an immunofluorescent examination of rabbit and human sera for antibodies to the ES antigens of the second stage larvae of Toxocara canis, Toxocara cati and Toxascaris leonina (Nematoda)

Precipitates were demonstrated in vitro at the orifices of the second stage larvae of Toxocara canis and T. cati when placed in sera derived from rabbits infected with these nematodes. Cross-reactions occurred between these two species.

Furthermore, these precipitates occurring at the oral, excretory pore and the anal orifices of these larvae were shown to be of a specific antibody nature by the use of the direct fluorescent antibody (Coons) staining technique.

The second stage larvae of Toxascaris leonina did not react in this way when examined in the above-mentioned experimental system, or in sera or globulins derived from rabbits infected with T. leonina.

Human sera, taken from clinically suspect cases of visceral larva migrans, were examined in this manner (q.v.). Comparable results were obtained, and it was possible to determine whether fluorescent antibodies were present, and to use this information as an aid to the clinical diagnosis of this disease.

The significance of these findings in relation to the aetiology, pathogenesis and immuno-diagnosis of visceral larva migrans is discussed.

     

Fungi predatory activity on embryonated <Emphasis Type="Italic">Toxocara canis</Emphasis> eggs inoculated in domestic chickens (<Emphasis Type="Italic">Gallus gallus domesticus</Emphasis>) and destruction of second stage larvae

The objective of this study was to evaluate the infectivity of Toxocara canis eggs after interacting with isolated nematophagous fungi of the species Duddingtonia flagrans (AC001) and Pochonia chlamydosporia (VC4), and test the predatory activity of the isolated AC001 on T. canis second stage larvae after 7 days of interaction. In assay A, 5000 embryonated T. canis eggs previously in contact with the AC001 and VC4 isolated for 10 days were inoculated into domestic chickens (Gallus gallus domesticus), and then these animals were necropsied to collect material (digested liver, intestine, muscles and lungs) at 3-, 7-, 14-, and 21-day intervals after inoculation. In assay A, the results demonstrated that the prior interaction of the eggs with isolated AC001 and VC4 decreases the amount of larvae found in the collected organs. Difference (p?<?0.01) was observed in the medium larvae counts recovered from liver, lung, intestine, and muscle of animals in the treated groups when compared to the animals in the control group. At the end of assay A, a percentage reduction of 87.1 % (AC001) and 84.5 % (VC4) respectively was recorded. In the result of assay B, the isolated AC001 showed differences (p?<?0.01) compared to the control group, with a reduction of 53.4 % in the recovery of L₂. Through these results, it is justified to mention that prior interaction of embryonated T. canis eggs with the tested fungal isolates were efficient in reducing the development and migration of this parasite, in addition to the first report of proven predatory activity on L₂.     

Schistosoma japonicum soluble egg antigens: Separation by con a chromatography and immunoaffinity purification

A crude soluble egg antigen (SEA) preparation from Schistosoma japonicum eggs was used for immunoabsorbent fractionation of anti-SEA antibody. Anti-SEA antibodies were isolated from serum pooled from mice infected with S. japonicum for 7 weeks and from mice infected for 12 weeks. These anti-SEA immunoglobulins were then used for immunoabsorbent fractionation of specific SEA antigens. Crude SEA was also partially purified by Con A chromatography. Crude SEA, the Con A fraction, an antigen isolated with 7 week infection serum, and antigens isolated with 12 week infection serum were analysed by immunoprecipitation and SDS polyacrylamide gel electrophoresis.     

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.     

In vitro culture of Parascaris equorum larvae and initial investigation of parasite excretory-secretory products

Currently, diagnosis of Parascaris equorum infection in equids is limited to patent infections. The goals of this study were to culture P. equorum larvae in vitro and identify excretory-secretory (ES) products for prepatent diagnostic testing. Parascaris equorum L2/L3 larvae were hatched and cultured for up to 3 weeks for ES product collection. Fifth stage (L5) P. equorum were also cultured for ES product collection. Examination of ES fractions by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and silver stain revealed L2/L3 products ranging from 12–94 kDa and L5 products ranging from 12–189 kDa. Western blot analyses were conducted using polyclonal antibodies produced against P. equorum or Baylisascaris procyonis L2/L3 ES products, sera from rabbits inoculated with B. procyonis or Toxocara canis eggs, and sera from animals naturally infected with P. equorum or T. canis. Western blot results indicated parasite antigens migrating at 19 and 34 kDa may be useful for specifically detecting P. equorum infections.     

Cross-reactivity of Toxocariasis with Crude Antigen of Toxascaris leonina Larvae by ELISA

Roundworms of Toxocara canis and Toxascaris leonina are common gastrointestinal helminths of canids over the world. Humans are infected with T. canis larvae through ingestion of infective eggs in contaminated environments or larvae by consumption of raw or uncooked meat or livers. Recently, patients of clinically diagnosed toxocariasis are increasing and require correct diagnosis in Korea. The present study investigated serological cross-reactivity between crude antigens of T. canis (TCLA) and T. leonina (TLLA) larvae. We collected serum specimens from 177 toxocariasis patients who were clinically suspected in the Seoul National University Hospital and 115 healthy controls. An ELISA method for toxocariasis was used to evaluate diagnostic efficacy of TLLA for serodiagnosis of human toxocariasis. The IgG ELISA using TLLA gave 14 (14.3%) positives of 98 TCLA positive specimens among 177 suspected toxocariasis patients. Most of them showed high absorbances with TCLA. In conclusion, there is a partial cross reaction between serum specimens of toxocariasis and TLLA.

Graphical Abstract

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Effect of various doses of infectiveToxocara canis andToxocara cati eggs on the humoral response and distribution of larvae in mice

The effect of 5–2,500 infectiveToxocara canis and 5–1,000T. cati eggs on the humoral immune response and on the distribution of larvae in the organism was studied in paratenic hosts — inbred C57BL6/J mice. With each dose ofT. canis eggs the maximal antibody level was recorded on day 56 post infection and was followed by a moderate decline that lasted until day 154 of the experiment. A correlation between the antibody level and the egg count was observed only with the infective dose of 5–50 eggs. A more rapid occurrence of antibodies was recorded in mice infected with a high dose of eggs. In those given 5 and 7T. cati eggs the antibody level exceeded the extinction threshold value only from day 21 to day 84. Low doses ofT. canis (n=5) andT. cati (n=7) eggs caused a comparable distribution of larvae in mice, and the larval recoveries on day 70 post infection ranged between 10.00% and 25.74%. Following a dose of 500T. cati eggs, 22.28% of the larvae were recovered, although only 1.08% were localized in the brain. A dose of 1,000T. canis eggs yielded, 36.37% of the larvae, with as much as 28.13% being found in the brain.     

Temperature and the development and survival of infective <Emphasis Type="Italic">Toxocara canis</Emphasis> larvae

The influence of temperature on the development and survival of Toxocara canis larvae was investigated under laboratory conditions, in water at 15, 20, 25, 30 and 35°C and at room temperature 22°C ± 1°C. T. canis eggs were able to develop to the larvated stage at all the tested temperatures. Development rate increased with temperature. Linear regression of development rate against temperature predicted a lower development threshold of 11.8°C. Eggs survived cooling to 1 and −2°C for 6 weeks, and could develop to the infective, larvated stage when transferred to higher temperatures, but their development rates were then retarded compared with non-chilled eggs. Larvated eggs remained viable after 7 weeks of incubation across the tested temperature range, with the highest percentage viability (47%) obtained at 25°C. Development of eggs to the infective larval stage required, on average, 121 degree days between 20°C and 30°C. Results provide a basis for predicting variation in the infectivity of eggs in the environment over time in different climates.     

Establishment and migration pattern of<Emphasis Type="Italic"> Toxocara canis</Emphasis> larvae in chickens

Migrations of Toxocara canis larvae were observed in experimentally infected chickens. Three groups of three chickens were inoculated orally with T. canis eggs. Within each group, individual chickens received either 5,000, 10,000, or 20,000 eggs. A group of infected chickens was then necropsied at either 1, 3 or 6 days post infection (dpi). The entire duodenum, spleen, liver, heart, lungs, right inner pectoral muscle, and brain were subjected to pepsin digestion for larval recovery. Larvae were predominately (>87%) recovered from the liver and lungs, and only a few larvae were seen in other organs or tissues in all chickens, with the exception of the duodenum at 1 dpi of chickens inoculated with 20,000 eggs. The percentage of total larval recovery varied widely among chickens (range: 0.4–16.7%). Similar numbers of larvae were distributed in the liver and lungs at 1 dpi. Subsequently, more larvae were found in the lungs than the liver at 3 dpi, whereas the larval distributions in the liver and lungs were reversed at 6 dpi. These observations suggest that T. canis larvae can migrate by a hepatopulmonary route in the chicken, and reinforces the possibility that chickens harboring migrating T. canis larvae may pose a zoonotic risk, especially if the liver is consumed.     

Matrix metalloproteinases activation in Toxocara canis induced pulmonary pathogenesis

BackgroundToxocara canis, a source of visceral larva migrans, causes toxocariasis and induces respiratory symptoms. The reasons by which the pulmonary pathological alteration in the lungs infected with T. canis remain unclear.MethodsThe involvement of the pulmonary pathological alteration by histology, enzyme activity, and Western blot analysis in the lungs of BALB/c mice after the infection of 2000 embryonated eggs.ResultsThe pathological effects gradually increased after the infection culminated in severe leukocyte infiltration and hemorrhage from days 4–14 post-inoculation. Gelatin zymography using substrate showed that the relative activity of matrix metalloproteinase (MMP) −9 and MMP-2 significantly increased in T. canis-infected mice. Western blot analysis indicated that the MMPs protein level of fibronectin monomer significantly increased in T. canis-infected mice compared with that in uninfected control. T. canis larvae mainly initiated leukocyte infiltration and hemorrhage in the lungs.ConclusionThese phenomena subsequently induced the activities of MMPs in parallel with the pathological changes in early stage pulmonary inflammation. In conclusion, T. canis larval migration activated the MMPs and caused pulmonary pathogenesis.     

Blocking anti- Trichinella spiralis antibodies in chronically infected rats

 Specific anti-newborn larva antibodies present in the serum of rats chronically infected with Trichinella spiralis were incapable of inducing killing of newborn larvae by activation of normal peritoneal cells. These late antibodies blocked the cytotoxic reaction induced by early antibodies produced a few weeks after infection. Passive transference of late serum to normal mice failed to induce protective immunity against infection by newborn T. spiralis larvae. When late immunoserum was fractionated by gel filtration, blocking activity was found only in the fraction containing IgG subclasses. By indirect immunofluorescence assay and cytotoxic reaction it was shown that blocking antibodies were specific for newborn larvae and could not be adsorbed with muscle larvae. It is concluded that the synthesis of anti-newborn larva antibodies is modulated in the course of a chronic infection: early antibodies developing shortly after infection are cytotoxic, whereas blocking antibodies predominate in the late population. Furthermore, the results suggest that during a chronic infection, resistance to reinfection may be modified. Received: 10 March 1995 / Accepted: 20 May 1995     

Seroprevalence of Ehrlichia canis and of Canine Granulocytic Ehrlichia Infection in Dogs in Switzerland

Serum samples from 996 dogs in Switzerland were examined for antibodies to Ehrlichia canis and to the agent causing canine granulocytic ehrlichiosis (CGE). Ehrlichiosis, borreliosis, and systemic illness not associated with ticks were suspected in 75, 122, and 157 of these dogs, respectively. The remainder of the serum samples were obtained from clinically healthy dogs which resided north (n = 235) or south (n = 407) of the Alps. The serum samples were tested by an indirect immunofluorescence technique for antibodies to the two agents incriminated, E. canis and Ehrlichia phagocytophila, a surrogate marker of the agent of CGE. Twenty-two of 996 (2.2%) serum samples had antibodies to E. canis and were distributed as follows: 20 of 75 (26.7%) samples from dogs suspected of having ehrlichiosis, 1 of 122 (0.8%) from dogs suspected of having borreliosis, and 1 of 407 (0.2%) from healthy dogs which resided south of the Alps. Of the 75 (7.5%) serum samples that had antibodies to E. phagocytophila, significantly more samples were from ill dogs than from healthy dogs. Among the sera from healthy dogs, antibodies to E. phagocytophila were significantly more prevalent in the north. Because seropositive dogs had a history of travel outside Switzerland and because Rhipicephalus sanguineus is found exclusively south of the Alps, it was presumed that, in contrast to the agent of CGE, E. canis is not indigenous to Switzerland.     

Observations on the transmission and development ofToxocara pteropodis (Ascaridoidea: Nematoda) in the Australian Grey-Headed Flying-Fox,Pteropus poliocephalus (Pteropodidae: Megachiroptera)

Findings in the Australian Grey-Headed Flying-Fox,Pteropus poliocephalus, have elucidated the life-cycle ofToxocara pteropodis. In adult bats, other than parturient females, larvae were found only in the livers. Following parturition, larvae were recovered only from mammary glands up to 2 weeks post-partum. Developing larvae were found only in the intestine of young bats from the age of two days onwards; there was no evidence of pulmonary migration. The evidence indicates that juvenile bats commence passingToxocara eggs in their faeces at about 2 months of age and expel the worms spontaneously following weaning at about 5 months. The eggs passed in the faeces of the young bat and its mother are disseminated through-out their environment and embryonate rapidly, being infective to mice after 10 days. Under natural conditions the eggs remain viable for 6 weeks or less and are infective to bats by the oral route.     

<Emphasis Type="Italic">Toxocara canis</Emphasis> larvae reinfecting BALB/c mice exhibit accelerated speed of migration to the host CNS

Using a small animal imaging system, migratory activity of Toxocara canis larvae stained by carboxyfluorescein succinimidyl ester (CFSE) was observed post primary infection (PPI) and post reinfection (PR) of BALB/c mice. Each infection was performed with 1,000 larvae per mouse. Primary infections were performed with labeled larvae, while for challenge infections the reinfecting larvae were stained by CFSE. The worm burden in mouse organs was determined during a period from 6 h to 21 days and 4 months PPI and PR. In comparison with primary infections that led to the first larvae appearance in the brain after 60 h, greatly accelerated migration of the parasites administered 3 weeks PPI to the CNS and eyes of challenged mice was noted—in both organs the larvae appeared 6 h PR. In all challenged mice, reinfecting larvae prevailed in the resident parasite population. Preliminary experiments with Toxocara cati larvae also revealed early brain involvement in primarily infected mice. Staining of T. canis larvae by CFSE had no effect on the development of a humoral antibody response against T. canis excretory–secretory antigens. In ELISA, elevated levels of specific IgG and IgG1 were noted on day 14 PPI and the levels of antibodies increased till the end of experiment. Reinfection induced an increase in the levels of both antibodies. In terms of optical density, IgG1 antibodies gave higher values in all sera examined. In ELISA for IgG antibodies, an increase in the avidity index of around 50% was detected 1 month PPI; higher-avidity antibodies were also detected in sera of reinfected animals.     

Insulin Binding Proteins in Normal Serum. The in vitro Association of Homologous125I Labelled Insulin and Normal Serum Proteins

By incubation in vitro of normal human serum with ¹²⁶I labelled insulin, association of radioactivity with serum protein fractions was demonstrated. Previous findings of an association with two macroglobulins were confirmed and an association with a third fraction observed. The macro-globulins were identified as alpha-2-M and beta-2-M globulin by starch gel electrophoresis, immunoelectrophoresis, chromatography on Sephadex G 200, immunoprecipitation with specific antisera and precipitation with ammoniumsulphate and trichloroacetic acid. The third serum protein fraction was characterized by starch gel electrophoresis as an alpha-one globulin migrating immediately behind albumin. By gel filtration it was eluted with albumin and it was soluble in 80 % ammonium sulphate, but completely precipitated by tricholoracetic acid and by Rivanol. The proportion of the radioactivity incorporated into the three fractions was independent of the amount of insulin present but proportional to the duration of the incubation. The results indicate that the incorporation of radioactivity into the alpha-2-M and alpha-one globulin continues throughout the incubation period with a constant rate of 1 and 0.5 per cent per 24 hrs respectively, except [or a rapid initial association of approximately 4 per cent to the alpha-one globulin. In contrast, the incorporation into the beta-2-M globulin reaches a constant level within the first week. About 10 per cent of the radioactivity in the macroglobulin fraction and 32 per cent of that in the alpha-one globulin fraction could be precipitated by immunoprecipitation for insulin.     

Immunomodulative effect of muramyldipeptide in mice with larval toxocarosis

The phagocytic ability of polymorphonuclear leukocytes, the metabolic activity of peritoneal macrophages, the proliferative response of T- and B-cells, and the production of specific anti-Toxocara antibodies were studied in paratenic hosts with experimental larval toxocarosis after treatment with the immunomodulator muramyldipeptide for 119 days. Peroral infection of mice with 2,500 Toxocara canis eggs partially reduced the numbers of cells and inhibited the activity of all cellular immune-response parameters studied. During most of the experiment the greatest suppression was recorded for the peritoneal-macrophage metabolic activity. Parenteral administration of muramyldipeptide to mice at two doses prior to and after infection restored and significantly stimulated the parasite-inhibited phagocytic ability of blood polymorphonuclear leukocytes, metabolic activity of macrophages, and T-lymphocyte proliferative response. It also elevated the production of specific circulating anti-Toxocara antibodies. The immunomodulator significantly reduced the count of migrating T. canis larvae in the host organism (30.6%) and decreased by half the percentage of larvae in the brain. Received: 31 May 1999 / Accepted: 28 July 1999     

Infection with the roundworm Toxocara canis leads to exacerbation of experimental allergic airway inflammation

Background Epidemiological studies performed in developing as well as in western countries suggest that infection with Toxocara canis contributes to the development of atopic diseases. Objectives To investigate the association between infection with this helminth and allergy, we examined the effect of T. canis infection on experimental allergic airway inflammation. Methods BALB/c mice were infected by oral administration with 500 embryonated T. canis eggs followed by ovalbumin (OVA) sensitization and challenge to induce allergic airway inflammation. Results Infection with T. canis in combination with OVA treatment leads to exacerbation of pulmonary inflammation, eosinophilia, airway hyperresponsiveness, OVA specific and total IgE. Relative quantification of cytokine expression in the lungs of these mice showed increased expression of IL‐4 compared with mice that were only T. canis infected or OVA treated. Increased expression of IL‐5 and IL‐10 was measured in the lungs of T. canis‐infected or OVA‐treated mice compared with controls; however, combining infection and OVA treatment did not significantly change the expression of these cytokines. Conclusion A previous infection with T. canis leads to exacerbation of experimental allergic airway inflammation. These results have important consequences for findings on the helminths–allergy association. Several factors, including parasite species, infection of definitive vs. accidental host, parasite load and timing of infection, may influence whether an infection with helminths protects one from or enhances allergic manifestations.     

Evaluation of the initial and chronic phases of toxocariasis after consumption of liver treated by freezing or cooling

Human toxocariasis is a neglected parasitic zoonosis of worldwide distribution. The consumption of raw or undercooked meat and offal from paratenic hosts of the Toxocara canis nematode can cause infection in humans, but there have been a lack of studies examining specific prophylactic measures to combat this mode of transmission. The aim of this study was to evaluate the establishment of infection by T. canis larvae at the initial and chronic phases of visceral toxocariasis after the consumption of mouse liver subjected to cold treatment. This study was divided into two stages using groups (G) of five donor mice inoculated with 2,000 eggs of T. canis. Two days post-inoculation, the livers of donor mice in G1 and G2 were kept at ?20 °C and between 0 and 4 °C, respectively, for 10 days. In the first stage of the study, the livers of mice from G1, G2, and G3 (control) were subjected to a tissue digestion technique and found to be positive for infection. In the second stage, which evaluated infection in mice that had consumed livers from donor mice, receiver mice of G4 and G7 were fed with livers of donor mice from G1 (freezing), receiver mice of G5 and G8 were fed with livers of donor mice from G2 (cooling), and receiver mice of G6 and G9 with livers from G3 (control). Then, the tissue digestion technique was performed for recovering larvae from organs and carcasses of mice, at 2 days (G4, G5, and G6) and 60 days after liver consumption (G7, G8, and G9). It was observed that freezing inhibited the viability of 100 % of the larvae, while cooling promoted 87.7 and 95.7 % reductions in the intensity of infection at 2 and 60 days after liver consumption, respectively. Under the studied conditions, cold treatment shows great potential to help control this parasitosis, both in the initial and chronic phases of toxocariasis.     

Susceptibility of rats of infection withToxocara pteropodis

Infective eggs ofToxocara pteropodis were administered to Wistar rats via oral and parenteral routes. Third-stage larvae were recovered from the livers of suckling young 8 days after oral infection, and from livers and lungs after intraperitoneal or subcutaneous inoculation of eggs. These larvae were short-lived as none were found in suckling mice killed 2 weeks post-infection. Larvae were not recovered from tissues of rats aged 22 days or more when inoculated orally, indicating that refractoriness to infection develops rapidly with growth. Small numbers of larvae were recovered from the lungs of older rats 4 days after subcutaneous but not after oral inoculation. Adult male Buffalo and Fisher rats were also totally resistant to oral infection. Hence, rats differ from mice in their susceptibility toT. pteropodis.