Effect of egg dosage and host genotype on liver trapping in murine larval toxocariasis

In this study we examined the effect of various initial sensitizing doses of infective Toxocara canis eggs and the effect of murine host genotype on the level of trapping of larvae in the liver after larval challenge. In the initial experiments, C57BL/6J mice were infected with a sensitization dose of 5, 25, 75, 125, or 250 infective T. canis eggs on day 0 postinfection (PI). On day 28 PI all mice were challenged with 500 infective eggs. On days 7, 14, and 21 postchallenge (PC) larval numbers within individual livers were determined. Trapping of larvae was observed in mice receiving a sensitization dose of 25 or more eggs. At 7 and 14 days PC the level of trapping increased with sensitization egg dose up to a dose of 125 eggs. At 21 days PC the level of trapping reached a plateau at a sensitization dose of 75 eggs. The peak level of larval trapping was observed on day 7 and day 14 PC following sensitization doses of 125 and 250 eggs, respectively. In the subsequent experiments, mice of various strains and H-2 haplotypes were inoculated with an initial sensitization dose of 125 eggs and a challenge dose of 500 eggs on day 0 and day 28 PI, respectively. Larval trapping within the liver was determined on day 14 PC. C57BL/6J mice trapped significantly more larvae than DBA/2J mice (P less than 0.01); all other strains trapped larvae at a lower, but statistically similar, level to the C57BL6/J mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of inducible nitric oxide synthase in murine visceral larva migrans: effects on lung and liver damage

The roles of nitric oxide production and oxidative process were studied in mice infected with Toxocara canis and treated with aminoguanidine which is a specific inhibitor of inducible nitric oxide synthase (iNOS). Relations of nitric oxide synthase inhibition and tissue pathology were assessed by biochemical, histological and immunohistochemical methods. In experiments, Balb/c albino mice were inoculated with T. canis eggs either with or without aminoguanidine treatment or alone, at 24th, 48th hours and on 7th days. LPx and SOD values in liver tissue and plasma were measured. Liver and lung tissues were evaluated for the pathological lesions. The expression of eNOS and iNOS in both tissues were studied with immunohistochemistry in the same intervals. We observed significant differences between T. canis infected and aminoguanidine treated animals. Larval toxocarosis led to oxidative stress elevation in plasma. Microscopic examination of the liver histological sections revealed pathological lesions in the hepatic parenchyma in infected mice. In the mice received T. canis eggs plus aminoguanidine, the sinusoidal areas were enlarged. Histological lesions were more severe at 48 hours after infection. Numbers of eNOS and iNOS expressing epithelial cells were increased in the T. canis infected mice. The activities of eNOS and iNOS were also observed in the body of the larvae which have migrated to lung and liver. As a result, we have demonstrated that in vivo production of eNO and iNO during T. canis infection cause direct host damages and it is strongly related to the oxidative stress. We propose that larval NO can also be effective in larval migration, but it needs further investigation on distribution of NO in larvae.     

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.     

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.     

Visceral larva migrans: migratory pattern of Toxocara canis in pigs.

The migratory pattern of Toxocara canis was investigated following infection of pigs with 60000 infective eggs. Groups of six pigs were slaughtered at 7, 14 and 28 days after infection (p.i.), and the number of larvae in selected organs and muscles was determined by digestion. A group of uninfected pigs was used as negative controls for blood parameters and weight gain. Toxocara canis migrated well in the pig, although the relative numbers of larvae recovered decreased significantly during the experiment. On day 7 p.i., high numbers of larvae were recovered from the lymph nodes around the small intestine and to some extent also from the lymph nodes around the large intestine, and from the lungs and the liver. On day 14, the majority of larvae were recovered from the lungs and the lymph nodes around the small intestine, and by day 28 p.i. most larvae were found in the lungs. Larvae were recovered from the brain on days 14 and 21, with a maximum on day 14 p.i. No larvae were found in the eyes. Severe pathological changes were observed in the liver and lungs, especially on day 14 p.i.; also, development of granulomas was observed in the kidneys. Finally, a strong specific antibody response towards T. canis L2/L3 ES products was observed from day 14 p.i. until termination of the experiment, and the maximum eosinophil response was observed 14 days p.i. The pig is a useful non-primate model for human visceral larva migrans, since T. canis migrate well and induce a strong immunological response in the pig. However, the importance of the pig as a paratenic host is probably minor, because of the relatively early death of most of the 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.     

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.     

Initial stage of development and migratory behavior of Toxocara canis larvae in BALB/c mouse experimental model

In the present study, the initial developmental stage of Toxocara canis eggs and larvae, and number of recovered larvae from BALB/c mouse-infected organs are described. In vitro culture of T. canis detects the frequencies of interphasic, mitotic and embryonated eggs only within a 7-day period. Analysis by egg counting was carried out for 32 days. The results showed that at 7 days after cultivation, the frequency of larvae was 50.4% and that this frequency reached 52.8% in 32 days. In the experimental infection of BALB/c mice with T. canis, the number of recovered larvae statistically increased in the brain and liver, with doses of approximately 200 and 1000 eggs. After 7 days of infection, a larger number of larvae were obtained in the lung and liver, although a maximum amount was found in the brain after a 15- or 30-day post-infection period.     

The influence of inoculum size and time post-infection on the number and position of Toxocara canis larvae recovered from the brains of outbred CD1 mice

Outbred CD1 mice were administered doses of 1000 and 3000 Toxocara canis eggs and postmortem took place on days 7, 42 and 120 post-infection. Mice were killed by cervical dislocation and brains were sagitally bisected and fixed in 10% neutral buffered formalin prior to histological preparation and examination. The number of T. canis larvae were counted per brain and per section and the number of larvae cited for the first time per section were also recorded. These observations were compared by dose administered and by day of postmortem. The total number of larvae per brain and per section was higher for the 3000 dose compared to the 1000 dose. A different pattern emerged for the number of larvae observed in the brain over the three postmortem days depending upon the dose received. For the 1000 dose larval numbers increase from day 7 to day 120 whereas for the 3000 dose the opposite trend occurs. Larvae were assigned to one of five regions in the brain - the telencephalon, diencephalon, cerebellum, medulla, pons and brain stem and the olfactory bulb. Larvae did not show a random distribution in the brain. The majority of larvae were recorded from the telencephalon and the cerebellum. The percentage of sections with larvae in them is higher for the 3000 dose compared to the 1000 dose for all regions of the brain. For the majority of regions, the percentage of sections with larvae in them increases between day 7 and 42 and then decreases by day 120 and this is most pronounced for the cerebellum. For the telencephalon and diencephalon only, more larvae were detected on the right hand side of the brain compared to the left hand side. Statistical analysis revealed that dose and brain region are significant factors which influence the number of larvae observed in histological sections of the brain but day post-infection is not.     

The morphogenesis of Ascaris suum to the infective third-stage larvae within the egg.

Studies of the morphology of Ascaris suum larvae developing in the egg during embryonation in vitro at room temperature showed that 2 molts take place within the egg. The first larval stage (L1) appeared in the egg after 17-22 days of cultivation, the first molt to the second larval stage (L2) took place from day 22 to day 27, and the second molt to the third larval stage (L3) started on day 27 and continued during the 60-day observation period. Infectivity of the eggs was studied by oral egg inoculation in mice and showed that the L3 are the infective stage for mice. Molting to the L3 stage occurs gradually over a period of 2-6 wk, and it is recommended to have an additional maturation period so the infectivity of an egg batch may reach maximum level.     

Mouse strain difference in visceral larva migrans of Toxocara canis

The mode of larval migration (visceral larva migrans) in Toxocara canis infection was compared for BALB/c, C57BL/6, C3H/He, DBA/2, NC and BALB/c nude mice following oral infection with 400 eggs. The mean recovery of larvae from the liver on day 2 post infection (PI) was not different in terms of the strain, age or sex of the mice. The number of larvae recovered from the liver decreased in all strains on days 6, 12 and 21 PI, but the mean for BALB/c and (NC X BALB/c) F1 mice was significantly higher than that for C567BL/6, NC and BALB/c nude mice, unless the total number of larvae in the carcasses on day 21 PI was the same among those strains including athymic nude mice. The mean recovery of larvae from the liver on day 6 PI increased with age in both NC and BALB/c mice, although no sex difference was observed. From these results, it is emphasized that the age and strain of animals should be properly selected for animal experimentation with T. canis infection.     

Influence of mouse strain, infective dose and larval burden in the brain on activity in Toxocara-infected mice

Outbred LACA mice and inbred NIH mice were administered low (100 ova), medium (1000 ova), high (3000 ova) and trickle (4x250 ova) doses of Toxocara canis ova and the effect of infection on activity was examined with respect to: (i) the dose of ova administered and (ii) the number of larvae recovered from the brain. Larval recovery from the brain was significantly reduced in NIH mice compared to LACA mice for the 1000, 3000 and trickle doses. Mice from each strain were divided into larval intensity groupings based upon the number of larvae recovered from their brain. Activity for each mouse was measured pre- and post-infection by observing its behaviour in the home cage. Activity was assessed by monitoring six different independent categories of murine behaviour - ambulation, grooming, rearing, digging, climbing and immobility. Within each behavioural category, the duration of time spent at each behaviour per mouse within one thousandth of a second, the number of short bouts performed and the number of long bouts of behaviour performed were recorded over a 20 min period. Activity of LACA and NIH mice differed prior to infection. LACA mice spent more time immobile compared to NIH mice, which ambulated and climbed more. Variations in activity were also observed between groups of mice prior to infection. The effect of infection differed by strain, by dose and by larval intensity. Post-infection LACA mice became more immobile and ambulated less. NIH mice showed reduced immobility, but while ambulation decreased digging and climbing increased post-infection. Short bouts of activity remained unchanged among LACA mice post-infection but showed an increase for some behaviours in NIH mice.     

Larval recovery of Toxocara canis in organs and tissues of experimentally infected Rattus norvegicus

The aim of this note was to record for the first time the recovery of Toxocara canis larvae from tissues and organs of Rattus norvegicus (Berkenhout, 1769), Wistar strain, until the 60th day after experimental infection. Rats were orally infected with embryonated T. canis eggs, killed on days 3, 5, 8, 10, 15, 30, and 60 after inoculation and larvae were recovered from liver, lungs, kidneys, brain, and carcass after acid digestion, showing a pattern of migration similar of that previously observed in mice.     

Eosinophilia, granuloma formation, migratory behaviour of second stage larvae in murine Toxocara canis infection. Effect of the inoculum size

Experimental infection of mice with Toxocara canis provides one of the best models for immunological and pathological studies of the visceral larva migrans syndrome. Blood eosinophilia, the migratory behaviour of second stage larvae and granuloma formation were studied in Swiss mice infected with Toxocara canis. Eosinophilia, spleen, liver and lung indexes were followed during a primary infection with different inoculum sizes (500 and 1500 eggs) while the migratory behaviour of larvae was studied in a primary infection with 1500 eggs over a period of 4 months. In mice infected with three challenges of 1500 eggs in order to elicit a strong inflammatory reaction in the tissues, a histopathological study was carried out. The results showed that eosinophilia, spleen and lung indexes (but not the liver index) were influenced by the parasite inoculum size. The migratory behaviour study showed that larval recovery was maximal three days post-infection, from the liver and lungs; the peak recovery from the skeletal muscles and brain being on days 15 and 30 post-infection, respectively. The histopathological study revealed the formation of granulomas in all the tissues examined (liver, lungs, kidneys, spleen, lymph nodes, myocardium etc.) but not in nervous tissue or in the retina of the eye. Granulomas in the lungs were larger than those found in the liver. The implications of these results are discussed considering host-parasite inter-relations.     

Trapping of large numbers of larvae in the livers of Toxocara canis-reinfected mice

Congenitally athymic nude mice (BALB/c-nu/nu) and BALB/c-nu/+ were infected with 500 embryonated Toxocara canis eggs. Six weeks later they were reinfected with the same number of eggs. The liver and other organs were examined for numbers of 2nd-stage larvae at 2, 4, and 6 weeks after reinfection. Far more larvae were trapped in the liver after reinfection than after the primary infection but fewer were found in the livers of BALB/c-nu/nu than in BALB/c-nu/+ mice.     

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

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

Alternative migration routes of Ascaris suum in the pig

Experiments were conducted to investigate possible alternative routes of extraintestinal migration of Ascaris suum larvae in the pig. Pigs were infected with A. suum via injection of newly hatched larvae into cecal veins (i.v.), into cecal lymph nodes (LN), or intraperitoneally (i.p.), and control animals were inoculated orally with infective eggs (p.o.). Two pigs per inoculation route were necropsied on days 1, 4, and 13 postinoculation. The numbers of liver lesions and the percentage of larvae recovered was considerably greater in pigs inoculated i.v. or p.o. on each necropsy day. However, irrespective of inoculation route, at least a proportion of larvae passed through the livers and were able to complete migration to the small intestine by day 13. The results indicate that larval penetration of the intestinal wall is not necessary for liver-lung migration and that passage through the liver may be favorable for migrating A. suum larvae, although a delayed arrival in the small intestine cannot be ruled out for larvae following alternative routes.     

Effect of immune reconstitution on resistance to Brugia pahangi in congenitally athymic nude mice

The dichotomy of resistance to Brugia pahangi (Nematoda: Filarioidea) between nonsusceptible, euthymic C3H/HeN mice, heterozygotic for the "nu" gene (+/nu), and susceptible, congenitally-athymic "nude" (nu/nu) C3H/HeN mice, suggests that resistance is thymus-dependent. To test this hypothesis, the effect of syngeneic neonatal thymus grafts and neonatal thymus cell suspensions on recovery of worms at day 40 PI, and responses to Concanavalin A (Con A) were examined in reconstituted nudes. Nude recipients of a thymus graft 7 or 14 wk before subcutaneous inoculation with 50 infective larvae (L3) yielded no worms and responded strongly to Con A. Serum from these mice reacted in two lines of identity with serum from similarly-infected heterozygotes by double radial immunodiffusion against an adult worm saline extract. Nude recipients of a thymus 2 days or 3 wk before inoculation harbored an average of three or two worms, respectively. Intravenous injection of nude recipients with 10(7) or 10(8) neonatal thymus cells seven weeks before inoculation was less effective in conferring resistance to B. pahangi and responsiveness to Con A. Complete resistance to B. pahangi could be adoptively transferred to nude mice by 10(8) spleen cells obtained from infection-primed heterozygotes and injected intravenously on the day of larval inoculation. The same numbers of worms were significantly reduced. less effective when injected 3 wk before inoculation, although numbers of worms were significantly reduced. Passive transfer of primed heterozygote serum, containing high titers of antibodies to adult worm and larval antigens, failed to protect nude recipients against a larval inoculum in the absence of cellular reconstitution.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Observations on the post-mortem migration of nematode larvae and its role in tissue digestion techniques

The importance of larval viability in relation to the success of recovery techniques and to the interpretation of histological and migration studies was tested using mice infected with Toxocara pteropodis, T. canis and T. cati. Little difference was found in total numbers of viable larvae recovered from tissues incubated for 18 hours in 0.85% saline, 2% trypsin or 1% pepsin, but the rates of larval egress did vary. The recovery of dead larvae by digestion techniques was much lower. Larvae migrated in tissues undergoing fixation, which influenced histological findings. Larvae of T. canis did not undergo significant peritoneal migration in acutely-infected mice, but did penetrate their intestinal walls in large numbers within 4 hours of death. The implications of these findings in experimental studies are discussed.