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.     

Migration behaviour and pathogenesis of five ascarid nematode species in the Mongolian gerbil Meriones unguiculatus

To understand the characteristic features of the Mongolian gerbil, Meriones unguiculatus, as an animal model of ascarid infections, the migration behaviour and pathogenesis of larvae were investigated in experimentally infected gerbils. Embryonated eggs from each of Toxocara canis, Baylisascaris procyonis, B. transfuga, Ascaris suum, and A. lumbricoides were orally inoculated into gerbils and larvae were recovered from various organs at designated periods. In T. canis-infected gerbils, larvae were present in the liver 3 days after infection and in the skeletal muscle and brain via the heart and lungs at a similar rate. In B. procyonis- and B. transfuga-infected gerbils, larvae were present in the lungs within 24 h after infection, with some having reached the brain by that time. After 24 h, larvae of B. procyonis tended to accumulate in the brain, while those of B. transfuga accumulated in skeletal muscles. In A. suum- and A. lumbricoides-infected gerbils, larvae remained in the liver on day 5 post-infection and elicited pulmonary haemorrhagic lesions, which disappeared 7 days after initial infection. Thereafter, no larvae of any type were recovered. Ocular manifestations were frequently observed in T. canis- and B. procyonis infected gerbils, but were rare in B. transfuga-infected gerbils. In the cases of A. suum and A. lumbricoides, migration to the central nervous system and eyes was extremely rare, and larvae had disappeared by 2 weeks post-infection. Fatal neurological disturbances were observed in B. procyonis-infected gerbils, whereas irreversible non-fatal neurological symptoms were observed in the case of B. transfuga.     

Toxocara canis: impact of preweaning nutritional deprivation on the pathogenesis of pneumonia in the mouse

The present study was conducted to better understand the immure response to Toxocara canis pneumonia in mice with preweaning nutritional deprivation. Breast-fed Swiss mice, undernourished due to large litter size (up to 15 pups) and paired controls with only 5-8 pups were used. At 21 days old, both groups were infected with T. canis larvae. Liver retinol, retinyl palmitate, and inflammatory infiltrate in lungs were compared in both groups. Significantly lower levels of retinol and retinyl palmitate in liver tissue confirmed the hypovitaminosis A (P < 0.0001 for both comparisons) in the nutritionally deprived animals. Histological analysis showed similar eosinophilic infiltration in both groups at day 3 but was significantly more severe in undernourished mice at day 20 post-infection (P = 0.01). The present findings indicate that preweaning undernourishment is associated with a more severe inflammation in response to T. canis pneumonia. It suggests that vitamin A deficiency that persists after nutritional rehabilitation, may contribute to the severity of T. canis infection. The authors suggest that nutritional status should be carefully investigated in patients with more severe clinical findings.     

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.     

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 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.     

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.     

Toxocara canis: search for a potential drug amongst beta-carboline alkaloids--in vitro and mouse studies

The goal of this study was to search for new treatments for Toxocara canis using both in vitro and in vivo experiments. We specifically looked for a treatment for T. canis larva migrans, and examined beta-carboline alkaloids (17 compounds) with various structural modifications, both in in vitro and in vivo experiments. In the in vitro experiments, screening for nematocidal activity on the T. canis second stage larvae, cytotoxic activity, and immune activity in the host were undertaken. Compound 17 was selected, as it exhibited nematocidal activity for T. canis larvae and did not have any cytotoxic or immunosuppressive activity in the host. The effectiveness of compound 17 was then examined using T. canis larvae infected mice in in vivo experiments. To evaluate the anthelmintic effect, the relative mobility value for the larvae was examined in addition to the number of larvae in the brain, skeletal muscle, and liver. Compound 17 was also examined in both free and liposome-entrapped (LE) forms. Polyethylene glycol (PEG)-LE compound 17 showed an anthelmintic effect in which the number of larvae in the brain was decreased compared free albendazole. PEG-LE compound 17 also effectively suppressed the mobility of the larva in brain and skeletal muscle. The experimental procedure employed assisted in the discovery of this potential candidate and is a promising approach for finding alternative therapeutic regimens for T. canis larva migrans.     

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.     

Kinetics of liver trapping of infective larvae in murine toxocariasis

Mice sensitized by prior infection with Toxocara canis eggs trap many larvae of a challenge infection within the liver. In this study the distribution of challenge larvae in sensitized mice was examined to determine the earliest onset of liver trapping and to establish if the previously described phenomenon truly represented larval trapping. In all experiments, C57BL/6J mice were infected with a sensitization dose of 125 infective T. canis eggs on day 0 postinfection (PI) and challenged with 500 infective eggs on day 28 PI. In the initial experiments, larval numbers were determined within the intestinal contents, intestinal wall, mesenteric tissues, liver, lungs, skeletal muscle, and brain of each mouse on days 0.5, 1, 2, 3, 5, and 6 postchallenge (PC). Migration patterns were similar among the test and control groups except the peak of larval numbers in the liver, seen at 1 day PC in control mice, was delayed until 3 days PC in the test group. Larval trapping occurred within the liver of test mice at least by day 5 PC. In subsequent experiments, larval numbers were determined within the liver, skeletal muscle, brain of each mouse, and within the eyes of each mouse group at 4, 8, 12, and 16 wk PC. Larval numbers within the liver of test mice were similar both at 5 days PC and 16 wk PC, implying that larvae were trapped in this organ rather than delayed in their migration to other body sites. Liver trapping did not protect the eyes or brain of sensitized mice from larval migration, nor did it result in larval killing.     

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.     

Uptake and degradation of mast-cell granules by mouse peritoneal macrophages.

35S-labelled mast-cell granules isolated from mouse mastocytomas were added to mouse macrophages in vitro. The granules were avidly phagocytosed, and subsequently the radioactivity was released to the medium as inorganic [35S]sulphate. After pulse-labelling, a total of about 80% of the cell-associated radioactivity was thus released in the course of 24 h, indicating an extensive breakdown of the sulphated polysaccharides, mainly heparin, present in the granules. The uptake of the mast-cell granules caused pronounced, but reversible, spreading of the macrophages.     

Pathological and haematological responses of cats experimentally infected with Toxocara canis larvae

Responses of eight adult cats to one or two infections with larvae of Toxocara canis were studied up to 39 days post infection (DPI). Clinically, all cats remained normal throughout the study. The major necropsy finding was multifocal, white to grey nodules mainly within the liver, lungs and kidneys; live larvae were found in liver nodules. Histologically, the nodules were eosinophilic granulomas. Granulomas containing a larval section were observed mainly within the liver. All infected cats had variably severe, eosinophilic arteritis and bronchiolitis and medial hypertrophy and hyperplasia of the pulmonary arteries. No inflammatory eye lesions were detected. Circulating eosinophil levels increased in all infected cats; peak values of 15,790 and 10,050 eosinophils microliters-1 were observed at 25 or 32 DPI in cats receiving a single or double infection, respectively. Bone marrow of all infected cats exhibited marked eosinophilic hyperplasia which did not correlate with the level of circulating eosinophilia. Thus, infection of cats by the larvae of T. canis causes disseminated eosinophilic and granulomatous disease with marked pulmonary artery and airway lesions.     

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.     

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.     

The expression analysis of inflammatory and antimicrobial genes in the goldfish (Carassius auratus L.) infected with Trypanosoma carassii

We report results of a comprehensive analysis of inflammatory gene expression during the course of infection of Trypanosoma carassii in the goldfish. We observed significant increases in mRNA levels of genes encoding pro-inflammatory cytokines IFN-γ, TNFα1 and TNFα2; IL-1β-1 and IL-1β-2; IL-12-p35 and IL-12-p40; CCL1; CXCL8, anti-inflammatory cytokines IL-10 and TGFβ and iNOS A and iNOS B, using quantitative PCR. Expression levels and profiles of these cytokines and iNOS isoforms varied in the different tissues (kidney, spleen, liver) of goldfish during the course of T.?carassii infection. The expression of majority of genes that encode pro- and anti-inflammatory cytokines were up-regulated during the acute phase of infection (days 7-21 post-infection). The mRNA levels of these cytokines returned to normal levels or were down-regulated during the elimination phase of infection (days 28-56), with exception of IL-10 in the spleen and liver of infected fish. A parallel up-regulation of IFN-γ and IL-10 mRNA levels were observed in all tissues of infected fish during the acute phase of the infection. The expression of iNOS genes (iNOS A and B) was significantly delayed (day 14?pi) in the kidney, liver and spleen of infected fish. These results provide insights into the interaction between T.?carassii and goldfish, and suggest that Th1/Th2-like responses may be important for controlling T.?carassii infection in the goldfish.     

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.     

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.     

Peroxide sensitivity of endothelin responses in coronary artery smooth muscle: ETA vs. ETB pathways

The aim of this study was to investigate the effect of pyridoxine (Vitamine B6) deficiency on the immunological response of BALB/c mice infected with the parasite T. spiralis. Specific anti-parasite IgM and IgG immunoglobulins were detected by ELISA method in the serum of treated animals at different periods for 60 days post infection.Vitamin B6-deficiency was induced in two separate groups of mice by either (1) maintaining the mice on a Vitamin B6-deficient synthetic pellet diet for 40 days before infection, or (2) by daily intraperitoneal injection of 8 ×105 M/100 g of 4-Deoxypyridoxine (4-DPD), a potent antagonist of Vitamin B6 for 20 days prior to infection. These two groups of mice were then injected with 100 larvae (L1-T. spiralis) per os.Parasite burdens in the mice were observed by light microscopy. Cysts were present in the diaphragms of the mice after 60 days post-infection. Parasite specific IgG, as well as IgG. levels were determined in the sera of infected mice fed a normal diet. These levels were found to be lower in the 4-DPD-treated mice compared to the untreated mice. The inhibition started from the 10th day and continued to the 60th day, and in the 4-DPD treated group the inhibition initiated after 24 h to 60 days. IgM level also was depressed by 4-DPD, starting from 24 h after injection of the compound. In mice fed Vitamin B6-deficient diets the levels of IgG were lower than in mice fed normal diets.These results show that BALB/c mice infected with T. spiralis and fed either a Vitamin B6-deficient diet or a diet which included the Vitamin B6-antagonist, 4-DPD, both influence the course of IgG, IgGI and IgM production.     

Experimental infection of the cockroach Periplaneta americana with Toxocara canis and the establishment of patent infections in pups

The possible role of the cockroach Periplaneta americana in the transmission of Toxocara canis eggs and larvae via faeces and tissue migration was studied. Cockroaches fed with 3 x 105 and 5 x 105 embryonated eggs were found to harbour viable eggs and larvae from days 1 to 5 post-infection (DPI). At necropsy on 5 DPI, eggs and larvae were also recovered from the rectal contents but not from the tissues of cockroaches. In addition patent infections were established in pups fed on infected faeces of cockroaches, with eggs first appearing in the faeces of pups at 38 DPI. Adult worms of T. canis were also recovered at necropsy. Therefore the importance of cockroaches as good mechanical disseminators of ascarid eggs, especially T. canis, is discussed.