Hatching of second-stage larvae of Toxocara canis: a rapid method for processing large numbers of worms.

A method for hatching second-stage larvae of Toxocara canis is described. Whole adult worms are processed by centrifugation in the presence of hypochlorite which allows the grinding step to be eliminated. The technique is more rapid and a high yield is obtained.     

Radiolabeling and autoradiographic tracing of Toxocara canis larvae in male mice

Artificially hatched infective larvae of Toxocara canis were labeled with 75Se in Medium 199 (Gibco) containing 75Se-methionine. Male CD-1 mice were infected with radiolabeled larvae by intragastric intubation or by intraperitoneal injection. At intervals of 3-56 days mice were killed and the organs prepared for compressed organ autoradiography. Radioactivity of parasitic larvae showed an exponential decrease with time, reflecting catabolism of label with a biological half life of 26 days (effective half life of 21 days) making possible experiments lasting several months. Total body larva counts, estimated by total body autoradiography, displayed an overall downward trend, but the rate of reduction was probably not constant because no significant positive or negative trends were noted from day 14 onward in the numbers of larvae. The carcass accumulated the greatest number of larvae followed by the central nervous system, liver, and lung in that order. When the numbers of larvae were considered in relationship to the mass of tissue, there were 4 groupings: central nervous system, liver, lung, carcass, and kidney, and genito-urinary organ, pelt, and intestine. No significant difference between intragastric and intraperitoneal administration was observed in the larval distribution after the larvae had left the initial site of deposition.     

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.     

Experimental infection of mice with Toxocara canis larvae obtained from Japanese quails

The migration and distribution of Toxocara canis larvae in the tissues of Japanese quails, infected orally with 5 x 10(3) infective eggs, were studied, as well as the re-infectivity of these larvae in mice, inoculated with 50 larvae obtained from the liver of these quails. Post-infection, the highest concentrations of larvae were found to be present in the liver of quails while only a few migrated to other tissues like lungs, heart, muscle and brain. The migration and distribution of the larvae in the tissues of mice were studied by necropsy on days 6 and 12 post-infection. On both days the highest number of larvae, 11 and 10, were recovered from the carcase followed by six and seven from the leg muscles and four and eight from the brain, respectively. A few larvae were recovered from the liver, lungs and viscera. This implies that the larvae had a special affinity for the muscle and brain tissue of mice, unlike in the quails. The role of these larvae in relation to paratenism is discussed.     

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.     

Larval migration of Toxocara canis in piglets and transfer of larvae from infected porcine tissue to mice

Visceral larva migrans (VLM), caused by Toxocara canis larvae in humans, animals and birds, is now well documented throughout the world. Seven piglets were infected orally with 5 x 104 embryonated eggs and the migration and distribution of T. canis larvae in the tissues were evaluated. After artificial gastric juice digestion, larval yields at necropsy from different organs and muscles on days 1, 3, 7, 15 and 30 post-infection (DPI) revealed 3.05, 0.97, 0.21, 0.13, 0.05, 0.14% recovery from liver, lungs, heart, kidneys, skeletal muscles and brain tissues respectively, with a total of 2486 (4.97%) recovery from all tissues together. The highest number of larvae 1527 (3.05%) was recovered from the liver throughout the period (1-30 DPI), indicating a special affinity of larvae for the liver. Subsequently five mice were each infected orally with 5 g of infected pig liver and, after necropsy on 10 DPI, 20 +/- 3.62, 17 +/- 5.10, 3 +/- 1.26, 12 +/- 3.92 and 30 +/- 5.69 larvae were recovered from liver, lungs, heart, brain and muscles, respectively. Thus, primarily, the migratory potential and adaptation of T. canis larvae in porcine tissue was examined and, subsequently, their establishment in the second paratenic host, the mouse, has been successful. No influence of host sex on the migratory potential of T. canis larvae was observed. The related pathology caused by migratory larvae and its zoonotic significance through the consumption of raw or undercooked pork has been emphasized.     

Disseminated toxoplasmosis in magpie geese (Anseranas semipalmata) with large numbers of tissue cysts in livers

Fatal disseminated Toxoplasma gondii infection was diagnosed in 2 captive magpie geese (Anseranas semipalmata) from a zoo in Texas. Both geese died suddenly, without apparent clinical signs. Lesions associated with T. gondii tachyzoites were seen in lungs, pancreas, liver, adrenals, bursa of Fabricius, spleen, brain, and kidneys. Toxoplasmic pneumonia and hepatitis were considered to be the primary cause of death. An unusual feature was the presence of numerous tissue cysts in hepatocytes of both geese. The diagnosis was confirmed immunohistochemically. Antibodies to T. gondii were found in 2 of 11 other geese from the zoo examined using the modified agglutination test. This is the first report of T. gondii infection in magpie geese (Anseranas semipalmata).     

A cell-free culture system for development of large numbers of Brugia pahangi larvae.

Techniques used in the in vitro culture of massive numbers of Brugia pahangi third-stage larvae (L3) are described. Procedures for larval preparation and four culture conditions, with or without animal cell co-cultures, were studied, resulting in the adoption of a relatively simple cell-free culture system for routine harvesting of larval moulting excretory/secretory product.     

Toxocara canis: proteolytic enzymes secreted by the infective larvae in vitro

Second-stage larvae of the dog nematode Toxocara canis are infective to man and cause the syndromes of visceral larva migrans and ocular toxocariasis. Larvae cultured in vitro secrete proteases which degrade components of a model of extracellular matrix and basement membranes. These enzymes have been characterized using a variety of techniques. Multiple enzyme activities were demonstrated by substrate gel electrophoresis, associated with proteins of molecular weights of 120 and 32 kDa. The enzyme activity was inhibited both in substrate gels and in a radiogelatin microplate assay by phenylmethylsulfonyl fluoride. Optimal activity occurred at pH 9, with minor activities apparent at pH 5 and 7; the relationship between these proteolytic activities is currently under investigation.     

Simian immunodeficiency virus (SIV)-specific CTL are present in large numbers in livers of SIV-infected rhesus monkeys

The immunopathogenesis of AIDS-associated hepatitis was explored in the SIV/rhesus monkey model. The livers of SIV-infected monkeys showed a mild hepatitis, with a predominantly CD8+ T lymphocyte infiltration in the periportal fields and sinusoids. These liver-associated CD8+ T cells were comprised of a high percentage of SIV-specific CTL as defined by MHC class I/Gag peptide tetramer binding and Gag peptide epitope-specific lytic activity. There was insufficient viral replication in these livers to account for attracting this large number of functional virus-specific CTL to the liver. There was also no evidence that the predominant population of CTL were functionally end-stage cells trapped in the liver and destined to undergo apoptotic cell death in that organ. Interestingly, we noted that liver tetramer-binding cells showed an increased expression of CD62L, an adhesion molecule usually only rarely expressed on tetramer-binding cells. This observation suggests that the expression of specific adhesion molecules by CTL might facilitate the capture of these cells in the liver. These results demonstrate that functional SIV-specific CD8+ T cells are present in large numbers in the liver of chronically SIV-infected monkeys. Thus, the liver may be a trap for virus-specific cytotoxic T cells.     

Observations on Toxocara pteropodis infections in mice

Infection in mice with Toxocara pteropodis was investigated. In mice fed infective eggs, third-stage larvae hatched out and penetrated the mucosa, predominantly that of the lower intestine. They travelled via the portal vein to the liver, where they remained at least 14 months. They grew in length from 430 +/- 15 micron, at three days post infection (p.i.), to 600 +/- 50 micron, at six to nine weeks p.i., after which time growth ceased. Blood eosinophilia appeared at 28 days p.i., and eosinophil levels continued to rise gradually beyond this time. In female mice the larvae did not migrate from the liver in response to pregnancy or lactation. When infective eggs were inoculated subcutaneously or intra-peritoneally, larvae hatched out and ultimately appeared in the liver in larger numbers than seen with oral infections.     

Biosynthetic labelling of the excretory and secretory antigens of Toxocara canis larvae

Toxocara canis larvae were cultured in vitro in medium containing [35S-]methionine for six days. The medium and the larval tissues were analysed for biosynthetically labelled polypeptides by sodium dodecyl sulphate polyacrylamide gel electrophoresis and autoradiography. Immunoprecipitates with positive and negative human antiserum were similarly analysed, using Staphylococcus aureus to absorb immunocomplexes. The larvae secrete biosynthetically labelled polypeptides into the medium, with three major polypeptides of molecular weights between 99 and 110 X 10(3) the major constituents. Both of these react strongly with human IgG in human positive sera. Many polypeptides become labelled in the larval tissue, but only one polypeptide with similar molecular weight to the ES antigens, strongly reacted with human IgG.     

Toxocara canis: interaction of human blood eosinophils with the infective larvae

This study was carried out to investigate the nature of the immunological responses which took place in a child who had recently recovered from toxocariasis. She had developed a marked eosinophilia and had high titers of toxocara antibodies. Experiments were performed to examine whether Toxocara canis infective larvae could be killed in the presence of her serum and human eosinophils. Eosinophils with human complement, or this patient's serum, adhered to the surface of the larvae within 10 min. By 40 min, using both light and electron microscopy, it was shown that the cells had flattened against the cuticle and degranulated. However, by 3 hr, eosinophils had begun to detach, and the larvae remained alive for at least 1 week afterward. Further addition of serum or of eosinophils, which were shown to be able to immobilize T. spiralis infective larvae, failed to kill the T. canis larvae. It was concluded that, in this patient, the development of an inflammatory response to a T. canis infection was not associated with the appearance of antibodies capable of inducing eosinophil dependent toxicity to the larvae in vitro. Eosinophil dependent killing mechanisms may be less important than other components of the immune response, in immunity to this parasite in humans.     

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.     

Effects of irradiation on the biology of the infective larvae of Toxocara canis in the mouse

Mice were infected with either 2,000 normal or irradiated embryonated eggs of Toxocara canis and the number of larvae in their livers, lungs, brains, and carcasses investigated at 5, 20, and 33 days of infection. Mortality of mice infected with normal eggs was 33% between day 4 and 8 postinfection but there was no mortality among mice infected with irradiated eggs. Irradiation with 60, 90, or 150 kr of X-rays inhibited the migration of larvae from the livers and lungs and their accumulation in brain and carcass in proportion to the irradiation dose. By day 33 of infection, the ratio of larvae in liver and lungs to larvae in brain and carcass was 0.16 in normal mice, 0.42 in 60-kr mice, 0.98 in 90-kr mice, and 23.3 in 150-kr mice. Irradiated larvae, particularly those migrating through the peritoneal cavity, died faster than normal larvae until day 20. Irradiation favored survival after day 20. By days 20 and 33 postinfection the total parasite load was 29% and 8%, respectively, of the administered dose in control mice, 18% and 12% in 60-kr mice, 8% and 4% in 90-kr mice, and 0.9% and 0.3% in 150-kr mice. Irradiation of infective T. canis larvae, then, reduces their pathogenicity, inhibits their migration from liver and lungs, kills some of the parasites during the first 3 weeks of infection, but favors their late survival in the host.     

In vivo inhibition of inducible nitric oxide and evaluation of the brain tissue damage induced by Toxocara canis larvae in experimentally infected mice

Nitric oxide (NO) is known to be produced by macrophages, endothelial cells and neurons and synthesized by an enzyme called nitric oxide synthase (NOS). Various effector mechanisms and infections can affect the NO production. Excessive amount of NO will lead to biochemical reactions, which cause toxic effects. In this study the role of NO has been evaluated in larval toxocarosis, which is a systemic parasite infection caused by T. canis larvae. Infection was established in the Balb/c mice with or without inducible NOS (iNOS) inhibition and the effects of infection and NOS inhibition were observed according to the results of SOD and LPx measurements in brain tissue and NADPH-diaphorase (NADP-d) histochemistry. Results of NADPH-d histochemistry indicate that iNOS inhibition has protective effect on the brains of infected mice and that larval T. canis infection could be related to oxidative stress, and NO production and iNOS inhibition can protect the tissue from damage in this infection.     

Estimation of the numbers and infectivity of Nippostrongylus larvae

Experimental investigations of the subcutaneous infection of third-stage larvae of Nippostrongylus brasiliensis reveal discrepancies between the actual and intended inoculum, and also variability between replicate inocula. Emphasis is thus placed on the importance of obtaining accurate estimates of the level of confidence associated with inoculum size. Under specified laboratory conditions, the retention of infectivity of N. brasiliensis larvae was found to be age-dependent, with a maximum of between 120 and 156 days. The motility of the larvae was not found to be indicative of their ability to complete migration and to become established in the small intestine of the host following subcutaneous inoculation.