Efficacy of UV-irradiated larval vaccine of Ancylostoma ceylanicum (Looss, 1911) in golden hamsters (Mesocricetus auratus)

A vaccination trial in golden hamsters with UV-irradiated infective larvae of Ancylostoma ceylanicum was attempted. One oral vaccination of hamsters with 100 infective larvae irradiated by means of UV-tube (390 nm) at different time intervals induced the development of resistance. As the time exposure of irradiation was increased, there was a corresponding decrease in the subsequent worm establishment. A high level of protection afforded by larvae irradiated for 15 min UV-exposure was recorded giving 99.0% and 95.0% worm reduction against the challenge doses of 100 and 1000 normal larvae respectively. There was no marked difference in worm establishment in hamsters vaccinated either orally or subcutaneously, followed by oral challenge. In the vaccinated hamsters, the manifestations of resistance at 15 min UV-exposure were shown by marked reduction in worm establishment and highly reduced epg in pellets with significantly higher blood haemoglobin levels compared with those given normal larvae as vaccine and challenge controls.     

Effects of age and splenectomy on heavy infection of Angiostrongylus cantonensis in rats

This study was designed to determine the effects of age and the role of spleen in rats with heavy Angiostrongylus cantonensis infection. Young rats (8 weeks) infected with 100 larvae were found to have significantly higher worm recovery rate (75.0±6.6%) than the adult (6 months) (55.7±1.5%) and the aging ones (13 months) (57.6±4.0%). Moreover, the recovery rate in adult rats with 400 larvae (33.6±10.67%) was significantly lower than those with 100 larvae (55.7±1.53%) or 200 larvae (53.3±5.4%). The splenectomized young rats with 100 larvae had a significantly higher recovery rate (84.3±2.5%) than the intact (75.0±6.6%) or sham splenectomized ones (74.4±3.8%). Although titers of antibody against A. cantonensis increased with time, those against young adults were significantly higher before week 4 whereas those against adult worms become significantly higher since week 4. Titers in the splenectomized rats were also found to be significantly lower than those in the intact ones. These finding indicate that young rats are more susceptible to A. cantonensis. Crowding effect may occur in rats with heavy infections. The effects of splenectomy on the host are independent of the intensity of infection.     

Acquired immunity in rats against Angiostrongylus cantonensis infection

Acquired immunity against Angiostrongylus cantonensis was induced by immunizing rats with somatic antigens from fifth-stage larvae and adult worms and live third-stage larvae. Rats immunized twice had significantly fewer worms than rats immunized three times. Fewer worms were recovered from rats immunized with 200 live third-stage larvae than from any other groups. Rats immunized with somatic antigens had higher enzyme-linked immunosorbent assay (ELISA) antibody levels than rats immunized with live larvae. Rats immunized with live third-stage larvae of Angiostrongylus cantonensis were more strongly protected against challenge infections (62-92%) than rats immunized with antigens extracted from fifth-stage larvae (0-30%) and adult worms (11-24%).     

The influence of Mesocestoides corti on subsequent Angiostrongylus cantonensis infections in mice.

The influence of Mesocestoides corti on subsequent Angiostrongylus cantonensis infection in mice (C57BL/6 and BALB/c) was assessed. Both strains of mice infected with M. corti demonstrated a peak blood eosinophilia at around 3 weeks post-infection (p.i.). C57BL/6 and BALB/c mice primarily infected with M. corti were given A. cantonensis infection 18 days later, but pre-existing M. corti infection did not affect the recovery of intracranial worms of A. cantonensis at day 21 p.i. BALB/c mice with mixed parasite infections showed low morbidity and mortality as compared with mice singly infected with A. cantonensis and some mice demonstrated a pulmonary migration of intracranial worms. In C57BL/6 mice, intracranial worms were killed and thus all mice survived. C57BL/6 mice with mixed parasite infections failed to resist A. cantonensis reinfection. The blastogenic responses of spleen cells against A. cantonensis antigen were lower in BALB/c than in C57BL/6 mice and mixed parasite infections also resulted in less blastogenic responses against both concanavalin A and A. cantonensis antigen than monoinfection. The recovery of M. corti biomass was significantly higher in mice with mixed parasite infections than mice with monoinfection with M. corti. These data suggest a distinct difference in response to A. cantonensis infection between C57BL/6 and BALB/c mice, and the induction of immunosuppression in both mouse strains following M. corti infection. Blood eosinophilia provoked by M. corti infection is not directly associated with the killing of worms in subsequent A. cantonensis infection.     

The involvement of antigen-presenting cells and suppressor cells in the ultraviolet radiation-induced inhibition of secondary cytotoxic T cell sensitization

When mice were treated with ultraviolet (UV) radiation before immunization with the skin sensitizers, trinitro-chlorobenzene or dinitrofluorobenzene, they showed greatly depressed levels of priming for a secondary in vitro cytotoxic response against haptenated cells. Because the irradiation and skin painting were done on separate sites, these results suggest that the UV radiation had a systemic effect. The priming response of irradiated mice to allogeneic stimulation was normal, indicating some antigen selectivity to the inhibitory effect of UV radiation. A defect in antigen-presenting cells, previously demonstrated in UV-irradiated mice, was found to be largely responsible for the depressed priming response observed in these animals. In addition, the UV-irradiated, immunized mice possessed suppressor cells that were capable of blocking priming for cytotoxic responses against haptenated cells in normal mice.     

Experimental infection routes of Angiostrongylus cantonensis in mice.

Stomach intubation is the most common method used in the experimental infection of animals with Angiostrongylus cantonensis. In order to compare the effectiveness of other possible transmission methods, groups of BALB/c mice were given infective third-stage larvae of A. cantonensis by different routes including intraperitoneal or subcutaneous injections, and penetration of anal mucosa, vaginal mucosa, conjunctival mucosa, lacerated skin, unabraded skin, foot pad and tail skin, while stomach intubation was used as control. Recovery of fifth-stage larvae was higher in mice inoculated with third-stage larvae subcutaneously. Successful infections were established through all experimental transmission routes except tail skin penetration. This study suggests that oral infection may not be the only route for the transmission of human angiostrongyliasis, and subcutaneous infection may be a better method for experimental infection.     

Curcumin alleviates eosinophilic meningitis through reduction of eosinophil count following albendazole treatment against Angiostrongylus cantonensis in mice

Angiostrongylus cantonensis (A. cantonensis) is the most common cause of parasitic eosinophilic meningitis worldwide. By using an animal model of BALB/c mice infected with A. cantonensis, previous studies indicated that the anthelmintic drug, albendazole, could kill A. cantonensis larvae and prevent further infection. However, the dead larvae will induce severe immune responses targeting at brain tissues. To alleviate the detrimental effects caused by the dead larvae, we administered curcumin, a traditional anti-inflammatory agent, as a complementary treatment in addition to albendazole therapy, to determine whether curcumin could be beneficial for treatment. The results showed that although curcumin treatment alone did not reduce worm number, combined treatment by albendazole and curcumin helped to reduce eosinophil count in the cerebrospinal fluid, better than using albendazole alone. This alleviating effect did not affect albendazole treatment alone, since histological analysis showed similar worm eradication with or without addition of curcumin. Nevertheless, curcumin treatment alone and combined albendazole-curcumin treatment did not inhibit MMP-9 expression in the brain tissue. In conclusion, curcumin, when used as a complementary treatment to albendazole, could help to alleviate eosinophilic meningitis through suppression of eosinophil count in the cerebrospinal fluid.     

Prevalence and intensity of Angiostrongylus cantonensis in freshwater snails in relation to some ecological and biological factors

The purpose of the study was to record different intermediate hosts of A. cantonensis and to determine the infection prevalence and intensity of this parasite in freshwater snails in relation to some ecological and biological factors. The study was conducted at Al-Salam irrigation Canal and Al-Abtal village (north Sinai) for one year, from March 2004 to February 2005. Thirteen species of freshwater snails of nine families were examined for A. cantonensis infection. Six species were found infected with A. cantonensis larvae. These species were L. carinatus, C. bulimoides, C. cyclostomoides, B. alexandrina, L. natalensis and M. tuberculta. The infection prevalence of A. cantonensis in the examined snails ranged from 0.63 to 2.24%. L. carinatus snail had the highest prevalence, mean abundance and mean intensity of A. cantonensis infection. Positive correlations were found between both prevalence and mean abundance of A. cantonensis and host size in L. carinatus and M. tuberculata. Negative correlations were detected between salinity and prevalence, mean abundance and mean intensity of larvae of A. cantonensis. The results demonstrated seasonal and spatial variation in the prevalence, mean abundance and mean intensity of infection among examined snails. In this study, A. cantonensis larvae were found in a wide range of freshwater snails and M. tuberculata snail was recorded as a new intermediate host for the first time. In conclusion, further investigations in other areas and controlled laboratory experiments of infection approaches are required to evaluate the possible threat of this parasite on humans.     

Development of brain injury in mice by Angiostrongylus cantonensis infection is associated with the induction of transcription factor NF-kappaB, nuclear protooncogenes, and protein tyrosine phosphorylation

Eosinophilic meningitis or meningoencephalitis caused by Angiostrongylus cantonensis is endemic to the Pacific area of Asia, especially Taiwan, Thailand, and Japan. Although eosinophilia is an important clinical manifestation of A. cantonensis infection, the role of eosinophils in the progress of the infection remains to be elucidated. In this experiment, we showed that A. cantonensis-caused eosinoplia and inflammation might lead to the induction of NF-kappaB and protooncogene expression via activation of the tyrosine phosphorylation signal pathway. After mice were infected daily with 30 third-stage larvae of A. cantonensis by oral adminstration for 6 weeks, no significant differences PKC-alpha, MEK-1, ERK-2, JNK, and p38 protein expression were found between the control and infected mice. However, the protein tyrosine phosphorylation levels, NF-kappaB, and iNOS protein products were significantly increased by 3.5-, 3.3-, and 6.3-fold, respectively, after 3 weeks of A. cantonensis infection. The same pattern was found for c-Myc, c-Jun, and c-Fos proteins, which were elevated by 3.2-, 2.3-, and 3.4-fold, respectively, compared to control animals after 3 weeks. The expression potency of these proteins started increasing in week 1, reaching maximal induction in week 3, and then declining in week 5 after A. cantonensis infection. Another consistent result was noted in the pathological observations, including eosinophilia, leukocyte infiltration, granulomatous reactions, and time responses in brain tissues of infected mice. These data suggest that the development of brain injury by eosinophlia of A. cantonensis infection is associated with NF-kappaB and/or nuclear protooncogenes expression, which is activated by the tyrosine phosphorylation pathway.     

Studies on anthelmintic effects of flubendazole and mebendazole on the rat lungworm Angiostrongylus cantonensis in mice and rats

Flubendazole and mebendazole given orally at 10 mg/kg/day 5-7 days post-infection (total 30 mg/kg) were found to eliminate 93-100% of Angiostrongylus cantonensis larvae in mice and rats. No significant difference was observed between the effects of the 2 drugs. The effectiveness of the drugs decreased with the increase in days post-infection on which medication was administered. It was found possible to treat A. cantonensis adults in rats by administering flubendazole or mebendazole at 10 mg/kg/day for 10 consecutive days. The drugs exhibited better anthelmintic efficacy in a divided dosing regimen than in a single dosing regimen.     

Development of Angiostrongylus cantonensis in the lung of Achatina fulica (author's transl)

Twenty-four Achatina fulica from Pai-sha of the Pescardores Islands, where no Angiostrogylus cantonensis has been found in A. fulica(4), were inoculated with the first stage larvae of A cantonensis. One snail was killed every day after 24 days following inoculation. The lung of each snail was separated and examined for larvae under microscope. The same lung was then digested with pepsin. The larvae recovered after pepsin digestion were examined for more detailed morphology. This study confirmed that the first stage larvae of A. cantonensis passed through the respiratory pore to the lung of A. fulica and developed into infectious form (3rd stage larvae) in the lung. The second and third stage larvae were found in the lung on the 7th and 17th day respectively after inoculation. Worm capsules were found in the lung on the 12th day after inoculation and the size of capsule was in proportion to the duration of the infection.     

Observations on the transmission of Angiostrongylus cantonensis from snail to rodent

A survey of Angiostrongylus cantonensis was carried out to investigate the mode of transmission from mollusc to rat in a fixed study area of Yoron Island from 1979 to 1982. Rattus rattus was found to be infected with a small number of worms in spite of heavy infection with third-stage larvae in Achatina fulica and an abundance of this snail in the area. Natural infection and/or susceptibility with A. cantonensis were confirmed in three small snail species. Bradybaena circulus, Fruticicola despecta and Luchuena reticulata. Young A. fulica was found to be infected with fewer third-stage larvae than mature A. fulica. It was concluded that molluscs which were infected with a small number of third-stage larvae of A. cantonensis play an important role in maintaining the life cycle of A. cantonensis. The percentage of rat stomachs containing mollusc tissue was relatively low, and the incidence and infection was low in rats. Infection with A. cantonensis did not occur very often in R. rattus in nature.     

Seasonal intensity of Angiostrongylus cantonensis in the intermediate host, Laevicaulis alte.

The slug, Laevicaulis alte, is a vector of Angiostrongylus cantonensis in the Greater Bombay area. The seasonal prevalence of A. cantonensis was investigated to determine the maximum intensity of the parasite in a field population occupying an area of approximately 400 m2. The maximum intensity of larvae was observed in the rainy months, June-November. Investigations showed that individual L. alte could tolerate an infecting dose of 150 first-stage A. cantonensis larvae before mortalities occurred.     

Antigen-presenting activity of draining lymph node cells from mice painted with a contact allergen during ultraviolet carcinogenesis.

The induction of skin cancers in mice by chronic UV irradiation is accompanied by a decrease in the numbers of Ia+ and Thy-1+ dendritic cells in the epidermis early in the course of UV irradiation. Subsequently, the number of Ia+ cells, but not Thy-1+ cells, increases until the time of tumor development. To assess the functional significance of these changes in cutaneous immune cells, and to help define the role these cells may play in immune surveillance against skin cancers, we tested the afferent immunologic capability of the skin during the development of UV-B radiation-induced skin cancers. Afferent immune function was measured by testing the Ag-presenting capacity of draining lymph node (DLN) cells from mice sensitized epicutaneously with dinitrofluorobenzene. A reduced contact hypersensitivity response was induced in mice immunized with DLN cells from UV-irradiated mice that had been sensitized with hapten on UV-irradiated skin. This decreased reactivity was present during the entire latent period of tumor development. However, in tumor-bearing mice, the DLN cells from UV-irradiated, sensitized animals exhibited normal Ag-presenting activity. DLN cells from UV-irradiated mice sensitized on ventral, unirradiated skin exhibited normal Ag-presenting activity. The lowest amount of Ag-presenting activity in the draining lymph nodes of UV-irradiated mice correlated temporally with the lowest number of Ia+, adenosine triphosphatase+ dendritic epidermal cells in the UV-irradiated skin. At least during the early part of the tumor latent period, an increase in the number of these cells was paralleled by an increase in the Ag-presenting activity of the DLN cells. In contrast, the number of Thy-1+ dendritic epidermal cells in UV-irradiated skin did not correlate with the Ag-presenting activity. Thus, the decrease in the number of identifiable epidermal Langerhans cells early in the course of chronic UV irradiation correlated with a decrease in Ag-presenting activity after sensitization through the UV-irradiated skin. These studies demonstrate that the afferent arm of the cutaneous immune response is impaired in the site of tumor development throughout the latent period of UV carcinogenesis.     

Surface antigens of Angiostrongylus cantonensis developing in permissive and non-permissive hosts

The immunofluorescent antibody test and immunocytochemical method were employed to study the surface antigens of Angiostrongylus cantonensis obtained from infected rats, mice and guinea pigs. Positive results with intense fluorescence and brownish peroxidase staining were observed on the cuticular surface of A. cantonensis recovered from rats 22 days (late cerebral phase) and 34 days (lung phase) post-infection when tested with antisera against host (normal rat serum) antigens as well as crude extracts of A. cantonensis. However, host antigens were not observed on the surface of the nematode recovered from the brain of mice and guinea pigs 15 days post-infection.     

Efficacy of albendazole combined with a marine fungal extract (m2-9) against Angiostrongylus cantonensis-induced meningitis in mice

The pathogenesis of angiostrongyliasis, resulting from Angiostrongylus cantonensis invasion of the human central nervous system, remains elusive. Anthelmintics are usually used to kill worms, although dead worms in the brain may cause severe inflammation which will lead to central nervous system damage. Therefore, combination therapy with anthelmintics and anti-inflammatory drugs in the treatment of human angiostrongyliasis needs further study. To evaluate the efficacy of albendazole combined with a marine fungal extract (m2-9) in A. cantonensis infection, BALB/c mice infected by the third-stage larvae of A. cantonensis were divided into three groups: mice treated with albendazole or m2-9 alone or in combination from day 5 post-inoculation (PI). Several efficacy parameters were recorded, including weight change, worm recovery, neurological function, behavioural analysis, eosinophil and leucocyte counts. The results showed that combination therapy increased body weight, reduced worm burden, improved learning ability, memory and action, decreased neurological dysfunction and leucocyte response in these mice. The combination of albendazole and m2-9 treatment significantly decreased leucocyte response and increased the frequency of rearing, compared to infected mice treated with either drug alone. Therefore, m2-9 is a natural product with potentially significant therapeutic value for angiostrongyliasis and is worthy of further study.     

Larvicidal effect of flubendazole on Angiostrongylus cantonensis in mice with various worm burdens

Flubendazole given orally at 50 mg/kg to mice inoculated with 50, 250 or 500 larvae of Angiostrongylus cantonensis showed almost complete larvicidal effect, irrespective of the worm burden.     

Further studies on the tumor-specific suppressor cells induced by ultraviolet radiation.

Recent studies have suggested that the immunologic unresponsiveness of UV-irradiated mice against UV-induced fibrosarcomas might be due to the presence of suppressor lymphoid cells. In these experiments, we present additional evidence that suppressor lymphoid cells are present in lymph nodes and spleens of UV-irradiated mice and demonstrate that these cells are enriched after incubation on nylon wool columns, that they are inactive after treatment with anti-theta serum and complement, and that they are effective for at least 7 weeks after transfer to lethally x-irradiated mice. Splenectomy of UV-treated mice before tumor challenge did not restore their anti-tumor reactivity. The UV-induced suppressor cells appear to be specific for syngeneic UV-induced tumors, since they did not suppress the rejection of an allogeneic UV-induced tumor or two chemically induced syngeneic tumors.     

In vitro study of the effects of Angiostrongylus cantonensis larvae extracts on apoptosis and dysfunction in the blood-brain barrier (BBB)

It has been hypothesized that blood-brain barrier (BBB) dysfunction in Angiostrongylus cantonensis infection might be due to the apoptosis of the hosts' BBB cells. Here, we evaluated this hypothesis through several methods, all based on an in vitro mouse BBB model consisting of primary culture brain microvascular endothelial cells (BMECs) and brain astrocytic cells (BACs). In the present study, a four-hour percolation and HRP permeability experiment showed that A. cantonensis larvae extracts can increase the permeability of the BBB. Apoptosis among BMECs and BACs after exposure to larvae extracts was monitored by TUNEL and annexin-V-FITC/PI double staining. A. cantonensis larvae extracts were found to induce apoptosis in both BMECs and BACs. For this reason, we concluded that the induction of apoptosis might participate in the BBB dysfunction observed during angiostrongyliasis. Improved fundamental understanding of how A. cantonensis induces apoptosis may lead to new approaches to the treatment or prevention of this parasitic disease.     

Cross-reactions with Ascaris suum antigens of sera from mice infected with A. suum, Toxocara canis, and Angiostrongylus cantonensis

Ascaris suum larval excretory-secretory (AsES) antigen and larval (AsLA) as well as adult somatic antigen (AsAA) which were thought to be possibly helpful in the diagnosis of visceral larva migrans (VLM) due to A. suum infection were investigated in the present study. Serum taken from mice orally inoculated with approximately 250 embryonated eggs of A. suum or Toxocara canis, or 40 third-stage larvae of Angiostrongylus cantonensis were assessed by enzyme-linked immunosorbent assay (ELISA) using the AsES antigen, AsLA or AsAA at 1, 2, 3, 4, 6 and 8 weeks post infection (WPI). The titer of serum IgG from mice infected with A. suum increased from 1 WPI and a peak at 4 WPI was observed when it reached approximately three times the level of uninfected control mice. Thereafter, it decreased gradually but remained high as found from 6 to 8 WPI. No cross-reactions of heterologous serum IgG against AsES antigen was observed, whereas heterologous serum IgM exhibited significant cross-reactions to AsES antigen. Cross-reactivities to AsLA and AsAA by heterologous serum IgG as well as IgM antibodies were also observed in the trial. Altogether, the AsES antigen apparently seemed to be superior to the other two somatic antigens when used in the diagnosis of A. suum-induced VLM with serum IgG as tested by ELISA. Moreover, it was the first report to test the possibly antigenic cross-reactivity between A. suum and A. cantonensis.