<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.     

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.     

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.     

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.     

Toxocara canis in experimentally infected silver and arctic foxes

In two experiments, thirty-six farm foxes of two species were inoculated with various doses of infective Toxocara canis eggs or tissue larvae isolated from mice. In experiment I, six adult arctic foxes (Alopex lagopus; 11-month old) were each inoculated with 20,000 eggs and sacrificed 100, 220, or 300 days post infection (dpi), while ten silver fox cubs (Vulpes vulpes; 6–9-week old) were infected with varying doses of eggs (30–3000) and necropsied 120 dpi. In experiment II, two groups of five cubs and two groups of five adult silver foxes received both a primary inoculation and either one or two challenge inoculations: primary inoculation (day 0) with 400 embryonated eggs were administered to five cubs and five adults and another five cubs and five adults received 400 larvae. At 50 dpi, the first challenge inoculation (400 eggs) was inoculated in all animals. At 100 dpi, three animals from each group were necropsied. The remaining two animals in each group were received a second challenge inoculation of 400 tissue larvae on 100 dpi and were subsequently necropsied at 150 dpi. In both experiments, the highest numbers of larvae per gram (lpg) of tissue was found in the kidneys (100–300 dpi). In adult foxes receiving a high dose (20,000 eggs), increasing larval burdens were found in the kidneys over the course of the experiment (up to 300 dpi). The larval migration from the lungs to other tissues appeared to be dose-dependent with the highest larval burdens found in adult foxes. The faecal egg excretion, larval burden and intestinal worm burdens decreased from the first to the second challenge infection.     

Histopathologic changes and larval recovery of <Emphasis Type="Italic">Toxocara cati</Emphasis> in experimentally infected chickens

This study was made to determine the distribution pattern of Toxocara cati larvae in chickens as a paratenic host and its potential zoonotic risk by consuming infected chickens. Two groups of chickens were fed with 1,000 and 3,000 embryonated eggs of T. cati. The chickens were necropsied 3, 7, 14, and 21 days postinfection. The liver, lungs, kidneys, spleen, small intestine, and half of all the striated muscles were digested for larval recovery. Squash method was used for brain. Larvae were recovered from the liver and brain of infected chickens with 1,000 embryonated eggs. Samples of these tissues were prepared for histopathologic studies. Experimental chickens exhibited hemorrhages in the liver, lungs, and kidneys on all days postinfections (dpi). White spots on the liver surfaces that showed necrotic foci, infiltration of eosinophils, and a few lymphocytes around necrotic areas were seen on 14 and 21 dpi. Remains of larvae were present in the liver on 14 dpi. Pathologic findings showed that larvae migrated in different organs of chickens. We suggest that chickens could be paratenic hosts, and human infection with T. cati might occur after consumption of raw or undercooked meat of infected chicken with T. cati.     

Antigen-induced protection against infection withToxocara vitulorum larvae in mice

Larvae ofToxocara vitulorum hatched and migrated in the tissues of normal mice. Larvae survived in reasonable numbers, particularly in the liver and, to a lesser extent, in the lungs and kidneys, for at least 4–7 days and in muscles, albeit only in low numbers, for at least 3 weeks. Oral infection of mice on three or more occasions withT. vitulorum eggs induced protection against a challenge infection with eggs ofT. vitulorum. Prior parenteral immunization of mice with a variety ofT. vitulorum soluble antigens (extracts, excretions/secretions, or perienteric fluid and their fractions) from adult parasites and/or infective larvae induced statistically significant protection against infection. The most effective protective immunogens were three or more injections with perienteric fluid from adults (100% protection) and excretions/secretions from infective larvae ofT. vitulorum (>92% protection).     

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.     

Detection and molecular characterization of ascarid nematode infection (Toxascaris leonina and Toxocara cati) in captive Asiatic lions (Panthera leo persica)

The objective of this study was to investigate the ascarid infection in Asiatic lions using scat samples, based on microscopic analysis, PCR amplification of the ITS-2 region of ribosomal DNA and sequence analysis of the amplicons. Microscopic analysis indicated the presence of eggs of Toxascaris leonina in eleven of the sixteen scat samples analysed and in one of these eleven scats eggs of Toxocara cati were also detected. In five of the scats eggs were not detectable. The presence of T. leonina in all the infected samples was also confirmed by PCR amplification of the ITS-2 of ribosomal RNA gene and five of these also showed amplicons corresponding to T. cati, respectively. Toxocara canis infection was not observed in any of the scat samples. Nucleotide sequence analysis of the ITS-2 region indicated 97% to 99% similarity with T. leonina and T. cati, respectively. To our knowledge, this is the first molecular characterization of ascarid infection in captive Asiatic lions from a zoological garden of India. This study also indicates that Asiatic lions are more prone to infection either with T. leonina or T. cati and the parasite is not host specific.     

Anthelmintic efficacy of tinidazole against the progression of Toxocara canis larvae to the brain in mice

The anthelmintic effect of tinidazole (100 mg/kg per day for 3 successive days) was tested in male Swiss CF-1 mice infected with second-stage Toxocara canis larvae at challenge doses of 250, 500, 1000, and 1500 embryonated eggs per mouse. The drug was given orally on days 3–5 postinfection (p.i.) to one-half of the animals, and all mice were killed on day 40 p.i. The number of larvae recovered from each mouse's brain and skeletal muscle was then scored in both groups. Tinidazole yielded a highly significant reduction in the total recovery of larvae from the test animals' brains at the second and third inoculum levels but no statistically significant reduction at the highest larval dose as compared with the values obtained in the untreated control animals. Received: 8 March 1999 / Accepted: 22 March 1999     

A survey of helminths in stray cats from Copenhagen with ecological aspects

At autopsy of 230 adult stray cats, 120 from backyards and 110 from gardens, the intestinal tract was scrutinized for helminths. The prevalence ofToxocara cati, Taenia taeniaeformis andDipylidium caninum was found to be 79%, 11% and 14%, respectively. Comparisons were made with the results of previous Danish investigations. The prevalence ofToxocara cati was found to be independent of time of collection and the sex and habitat of the cats and identical in cats with or withoutTaenia. This indicates that paratenic hosts do not play an important epizootiological role in the transmission ofT. cati. The intensity ofToxocara per cat followed a negative binomial pattern. The high prevalence ofT. cati combined with most cats having a low wormload shows that the cat population generally possesses a high degree of resistance against superimposed infections. The intensity of maleToxocara increases with the size of the worm population. This we consider to be an expression of increasing susceptibility of the cats. The prevalence ofT. taeniaeformis was significantly higher in garden cats, due to their greater opportunity for catching mice.D. caninum, however, was significantly more frequent in backyard cats, probably owing to better living condition for the flea larvae in backyards. For bothT. taeniaeformis andD. caninum a higher frequency was found in female cats, which is thought to be associated with their care for the kittens.     

Faecal water content and egg survival of goat gastro-intestinal strongyles under dry tropical conditions in Guadeloupe

Faeces from naturally infected goats were deposited on a natural grassland during the dry season in Guadeloupe (French West Indies) at different times throughout the day. The grass was either 7 or 20–30 cm tall. After a period of between several hours and 7 days, the number of viable strongyle eggs and the faecal water content were measured. Faecal temperature was recorded continuously. Faecal temperature was >40°–45°C at midday and dehydration was rapid between 8 a.m. and 2 p.m. Egg mortality was greater on short than on tall grass and higher in morning than in evening deposits. Minimal faecal water content during the first 36 h explained the 74%, 55% and 38% mortality rate for eggs ofOesophagostomum columbianum (OC),Haemonchus contortus (HC) andTrichostrongylus colubriformis (TC), respectively. In all, 5%–22% of the eggs of the latter species remained viable in a state of anhydrobiosis after 7 days on the ground. A delay of only 2 days between goat departure and irrigation would be sufficient to ensure that >95% ofO. columbianum andH. contortus eggs and 70% ofT. colubriformis eggs are destroyed.     

Zur serologischen Differenzierung vonAscaris suum- undToxocara canis-Infektionen beim Schwein

Zusammenfassung Je 8 Miniaturschweine wurden mit 1500 embryonierten Eiern vonAscaris suum oderToxocara canis bzw. mit je 1000 Eiern der beiden Spulwurmarten infiziert. Seren der Tiere wurden vor der Infektion sowie 3 und 6 Wochen post infectionem mit dem ELISA in Mikrotiterplatten gegen Antigene verschiedener Entwicklungsstadien untersucht. Bei Verwendung von Extrakten adulterAscaris undToxocara als Antigene waren sowohl bei den infizierten als auch bei den nichtinfizierten Kontrollen deutliche Reaktionen nachweisbar. Antigene aus embryonierten Eiern bzw. Larven beider Askaridenarten erzeugten nur bei den infizierten Tieren deutliche positive Reaktionen einschließlich bei den heterolog infizierten Schweinen, jedoch nicht bei den nicht infizierten Kontrolltieren.Nach Absättigung der Seren mit den jeweils heterologen Antigenen konnten mit homologen Antigenextrakten deutliche Antikörperreaktionen nachgewiesen werden. Auf diese Weise gelang es, Infektionen mit den beiden Nematodenarten serologisch zu differenzieren.

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Serological differentiation ofAscaris suum andToxocara canis infections in pigs

Groups of eight miniature pigs were infected either with 1,500 embryonated eggs each ofAscaris suum orToxocara canis or with 1,000 eggs each of both nematodes. Sera were sampled before the infection as well as three and six weeks postinfection and then investigated in the ELISA on microtitre plates against antigens of various developmental stages. When extracts of adultAscaris andToxocara worms were used as antigens, distinct reactions were registered both in the infected and in the uninfected control groups. Antigens isolated from either embryonated eggs or larvae of both worms produced distinctly positive reactions with sera of the infected animals, including those pigs with heterologous infections but not with sera from uninfected controls. After saturation with heterologous antigens, the sera showed distinct antibody reactions against homologous antigen extracts and in this way infections with the two nematode species could be serologically differentiated.

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Herrn Prof. Dr. G. Piekarski zum 70 Geburtstag giwidmet     

Effects of heterologous helminth infections on passive transfer of immunity using a mouse monoclonal IgE antibody againstSchistosoma japonicum

Passive transfer of immunity using a mouse monoclonal IgE antibody againstSchistosoma japonicum was found to be enhanced by heterologous helminth infections. BALB/c mice were infected withToxocara canis orNippostrongylus brasiliensis so as to induce eosinophilia prior to a challenge infection withS. japonicum. Recovery of adult schisotomes decreased in a group of mice that had been infected withT. canis and challenged with cercariae at the cutaneous site of sensitization with the IgE antibody as compared with that in mice that had been similarly treated with normal serum in the absence ofT. canis infection. Histological examinations revealed a close association of polymorphonuclear cells, including eosinophils, with damaged schistosomula in the skin ofT. canis-infected mice that had received the IgE antibody. An enhancement in worm reduction was also observed in mice harboring either of both nematodes when the monoclonal antibody had been injected intraperitoneally during the phase of migration of schistosomula from the skin to the lungs. In vitro studies on macrophage-mediated damage to schistosomula suggested that the enhancement in worm reduction was at least partly due to the activation of macrophages induced by the heterologous infections.     

Growth of a Japanese isolate ofTaenia crassiceps in intermediate and definitive hosts

Higher infection rates were observed in gerbils and voles than in ICR mice after oral inoculation with eggs of a Japanese isolate ofTaenia crassiceps. Asexual reproduction ofT. crassiceps cysticerci was observed in all gerbils and voles infected i.p. with the cysticerci. However, ICR mice and Wistar rats were not suitable for the asexual proliferation ofT. crassiceps. The hooks of cysticerci from mice were smaller than those from gerbils. In experimentally infected puppies, parasite development was noted as follows: strobilation and initial differentiation of the genital primordia on day 7 postinoculation (p.i.), appearance of the testes on day 9, observation of the ovaries on day 10, and development of the lateral branches of the uterus on day 15. The prepatent period was 27–31 days. After day 15 p.i., most of the worms were recovered from the middle third of the small intestine. The number of proglottids shed per day by each strobila was about 1. The number of eggs contained in a gravid segment was about 13000.     

A preliminary investigation on the infectivity of<Emphasis Type="Italic"> Trichinella</Emphasis> larvae in traditional preparations of walrus meat

This study evaluated the infectivity of Trichinella nativa in freshly frozen walrus meat and traditionally aged walrus meat (igunaq) associated with two human outbreaks of trichinellosis in the Canadian Arctic. Trichinella larvae recovered from walrus meat stored at –20°C for up to 20 months remained infective for guinea pigs inoculated with 135 or 716 larval doses. However, none of the 4–5 and 10-month-old igunaq preparations contained infective T. nativa larvae as measured by bioassays using mice and guinea pigs at inoculation doses ranging from 6 to 500 larvae. This indicates that the degradation process that occurred in the field can be sufficient to either kill Trichinella larvae or render them non-infective for mice and guinea pigs. Further research is needed to evaluate the food safety risk of traditional walrus igunaq aged under different field conditions and storage times.     

Studies on the development and role of some cell-mediated immune responses in experimental toxocarosis in mice

The studies were undertaken to investigate the development of some cell-mediated immune responses in experimental toxocarosis in mice and to assess the influence of these responses on the course of infection. Mice were infected orally with 350 eggs of Toxocara canis and reinfected with the same dose of parasites after 8 weeks. Groups of infected animals were killed each week of the experiments to obtain spleens, livers and brains for further studies. Lymphocytes from removed spleens were analysed by flow-cytometry for CD4 and CD8 expression and cultured in vitro to measure their responses to Concanavalin A and excretory-secretory (ES) antigen of T. canis in a lymphocyte transformation test. Pieces of livers were used to prepare paraffin sections to be stained later with haematoxylin and eosin, whereas whole brains of the infected animals were examined for the presence of parasite larvae. The results of the studies showed depression of T-cell responses to ConA in early stages of infection and significant increase in the blastogenic responses to the ES antigen from week 4 following infection. The depression of T-cell responses was accompanied by lowered CD4+/CD8+ ratio resulting from increased percentages of CD8+ T cells. Histopathological examination of liver sections revealed trapping of larvae in T. canis reinfected mice. The intensity of infection as measured by larval recoveries from the brains of mice increased gradually up to the 8th week of infection, but did not show significant changes after reinfection, testifying to the development of long-lasting protective immunity during primary infection.     

Evaluation of the immunosuppressive effect of cyclophosphamide and dexamethasone in mice with visceral toxocariasis

Visceral toxocariasis is a serious public health problem with a cosmopolitan distribution. Children are susceptible due to their immature immune system and high risks of infection. Nevertheless, the few completed studies about immunosuppression have had controversial results. To evaluate the effect of two immunosuppressive drugs on the larval burden of Toxocara canis, four groups of ten Swiss strain mice each were inoculated on day 0 with 1,200 embryonated T. canis eggs. Fifteen days before the experimental infection, group 1 (control) was treated via intraperitoneal injection (IP) with sterile distilled water and groups 2 and 3 were treated with dexamethasone (DEX) at 1 and 5 mg/kg/day, respectively. Additionally, group 4 was treated IP with cyclophosphamide (CY) at 50 mg/kg at two times per week for 2 weeks. Sixty days following infection, the mice were euthanised to recover the larvae by means of the tissue digestion technique. The levels of antibodies detected by indirect ELISA were not associated with the larval burden. Administration of CY (50 mg/kg) and DEX (5 mg/kg) resulted in an increase of the larval burden of 162.1% and 50.8%, respectively, in relation to the control group. These two treatments, especially CY (50 mg/kg), promoted immunosuppression and the establishment of a significant larval burden, supporting its further utilisation in studies related to immunosuppression in visceral toxocariasis.     

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     

Vertical transmission of<Emphasis Type="Italic"> Toxocara cati</Emphasis> Schrank 1788 (Anisakidae) in the cat

In eight cats and their offspring the mode of transmission of Toxocara cati following natural and experimental infection was investigated in three experiments. In experiments 1 and 2 the kittens of four cats with a chronic natural infection and of four cats with an acute experimental infection, respectively, were examined. In experiment 3 two queens of experiment 2 were mated again to examine whether in the adult cat dormant larvae exist in the tissue, that can be reactivated during pregnancy or lactation to infect the offspring. Additionally, the muscle tissue and organs of two adult cats, one with chronic one with acute infection, were examined for hypobiotic larvae. Pre-natal infections with T. cati did not occur in experiments 1 or 2. In none of the kittens that were examined directly after birth were larvae found. In the offspring of experiment 1 one single larva of T. cati was found 28 days post-partum. Whereas in the kittens of experiment 2 up to 333 larvae were found in one animal. Lactogenic transmission of larvae occurs after acute infection of the queen during late pregnancy but not during chronic natural infection. There is no evidence for the existence of arrested somatic larvae in the adult cat as an important host-finding strategy in the life cycle of T. cati. Following milk-borne infections, the majority of larvae seem to undergo direct development in the intestine without tracheal migration. Only a small number of larvae was found in other organs.