Demographics of the semi-slug Parmarion martensi, an intermediate host for Angiostrongylus cantonensis in Hawai'i, during laboratory rearing.

The semi-slug, Parmarion martensi, is an intermediate host of the zoonotic nematode, Angiostrongylus cantonensis, the aetiological agent of neuroangiostrongyliasis or rat lungworm disease in humans. Rearing methods were developed for P. martensi to facilitate studies on nematode transmission and control. Parmarion martensi exhibited high survivorship when reared on a diet of dog food and fresh fruits and vegetables in temperature-controlled cabinets at 21.4°C, 98% relative humidity and 12:12 L:D cycle. Rearing containers were lined with moist paper towels for substrate and plastic pots were provided for hiding/resting and egg-laying. Under these conditions, time to first reproduction was 165.3 ± 12.3 days, fecundity was approximately 34.5 ± 7.8 eggs per adult, and hatch rate was 52.7 ± 3.2%. Survivorship post egg hatch was 86.2 ± 2.9% at 30 days (neonates had a mortality rate of about 14%) and 99% thereafter for up to a year. The demographics of laboratory-reared and wild-caught P. martensi were similar except for the weight of reproductive adults, which was significantly higher in laboratory-reared adults (4.0 ± 0.2 g) than in field-collected adults (1.5 ± 0.1 g).

Efficacy of X-Ray Phytosanitary Irradiation on the Infectivity and Reproduction of Angiostrongylus cantonensis in Experimentally Infected Rats.

Angiostrongylus cantonensis is a globally distributed nematode and the leading cause of eosinophilic meningitis in humans. As a global hotspot for this disease, Hawaii's agricultural exports may be contributing to the spread of A. cantonensis. Phytosanitary irradiation doses of 150 or 400 Gy provide quarantine security against multiple insect pests. We evaluated the in vitro and in vivo effects of phytosanitary irradiation on infectious, third-stage, A. cantonensis larvae. In vitro experiments directly exposed larvae to irradiation doses ranging from 200 to 1,000 Gy. Results showed low mortality and no dose response across all treatments 27 days post-irradiation. In vivo studies isolated larvae from wild-caught Parmarion martensi after exposure to x-ray irradiation at doses of 0, 150, and 400 Gy and infected them into laboratory rats. Fourteen rats were assigned to each treatment and infected with 50 larvae from their assigned irradiation dose. Results at 3 and 6 weeks post-infection demonstrated a significant negative dose response in regard to the number of larvae that migrated to the brain and adults found in the pulmonary artery. No irradiated larvae that grew into adults were able to produce eggs. These findings indicate that x-ray irradiation does not result in the direct mortality of A. cantonensis larvae; however, it does affect the infectivity and reproduction of A. cantonensis within its definitive host, the rat. Phytosanitary irradiation at doses ≥150 Gy appears to be an effective means of preventing the establishment of viable populations of A. cantonensis, thus reducing the potential for global spread due to agricultural exports from Hawaii.

Immunodiagnostic Detection of Angiostrongylus cantonensis Exposure on Hawaii Island Using Isogeographic 31-kDa Antigen.

Angiostrongylus cantonensis is the leading cause of neuroangiostrongyliasis worldwide, and east Hawaii Island is a hotspot for the disease in the United States. A combination of glycoproteins with molecular weight of 31 kDa has been used as antigen to evaluate antibody response in human serum samples in Thailand with high specificity and sensitivity. In a previous pilot study, the Thailand-isolated 31-kDa proteins showed efficacy in dot-blot tests using serum samples from 435 human volunteers on Hawaii Island. However, we hypothesized that native antigen isolated from Hawaii A. cantonensis may exhibit higher specificity than the Thailand-isolated 31-kDa antigen due to potential minor variation in epitopes between isolates. In this study, 31-kDa glycoproteins were isolated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis from adult A. cantonensis nematodes collected from rats captured on east Hawaii Island. The resultant proteins were purified by electroelution, pooled, bioanalyzed, and quantified. A subset of 148 samples from human participants of the original cohort of 435 was consented for this study, including 12 of the original 15 clinically diagnosed participants. Results of ELISA using the Hawaii-isolated 31-kDa antigen were compared with results of the same serum samples previously tested with both crude Hawaii antigen ELISA and Thailand 31-kDa antigen dot blot. This study shows a seroprevalence in the general population of East Hawaii Island of 25.0%, similar to previous findings of 23.8% seroprevalence in this cohort using crude antigen from Hawaii A. cantonensis and 26.5% using Thailand 31-kDa antigen.

Phytosanitary irradiation using X-rays prevents reproduction in the semi-slug Parmarion martensi (Stylommatophora: Ariophantidae), a host of the human pathogenic nematode Angiostrongylus cantonensis (Rhabditida: Angiostrongylidae).

BACKGROUND: Phytosanitary irradiation is used to control insect pests of quarantine concern on exported fresh horticultural products. Generic irradiation doses of 150 and 400 Gy are approved for tephritid fruit flies and all other insects, respectively. Other invertebrates such as gastropods (snails and slugs) may be classified as quarantine pests and require a disinfestation treatment. Parmarion martensi Simroth (Stylommatophora: Ariophantidae) is a semi-slug quarantine pest sometimes found on fresh sweet potatoes and other fruits and vegetables exported from Hawai'i to the continental USA. Also, P. martensi is a host of the parasitic nematode Angiostrongylus cantonensis (Rhabditida: Angiostrongylidae), the causative agent of neuroangiostrongyliasis or rat lungworm disease in humans. We conducted a study to determine if phytosanitary irradiation could control P. martensi and thereby reduce the risk of transmitting A. cantonensis in the USA. RESULTS: Two-, 12-, and 21-week-old P. martensi were treated with X-ray radiation at a dose of 150 or 400 Gy or left untreated as controls then held in the laboratory for up to 250 days. Survivorship and reproduction were recorded every 2-3 days and individual weights were measured biweekly. Irradiation at 150 and 400 Gy reduced growth and increased the mortality rate compared to untreated controls and prevented reproduction. CONCLUSION: Phytosanitary irradiation treatment at doses ≥150 Gy will prevent the establishment of viable populations of P. martensi. The literature on radiation tolerance in gastropods suggests that the internationally approved generic dose for tephritid fruit flies of 150 Gy may be effective against many slug and snail pest species.

Water transmission potential of Angiostrongylus cantonensis: Larval viability and effectiveness of rainwater catchment sediment filters.

Neuroangiostrongyliasis, caused by Angiostrongylus cantonensis, has been reported in Hawai'i since the 1950's. An increase in cases is being reported primarily from East Hawai'i Island, correlated with the introduction of the semi-slug Parmarion martensi. Households in areas lacking infrastructure for water must use rainwater catchment as their primary domestic water supply, for which there is no federal, state, or county regulation. Despite evidence that slugs and snails can contaminate water and cause infection, regulatory bodies have not addressed this potential transmission route. This study evaluates: 1) the emergence of live, infective-stage A. cantonensis larvae from drowned, non-native, pestiforous gastropods; 2) larvae location in an undisturbed water column; 3) longevity of free-living larvae in water; and 4) effectiveness of rainwater catchment filters in blocking infective-stage larvae. Larvae were shed from minced and whole gastropods drowned in either municipal water or rainwater with ~94% of larvae recovered from the bottom of the water column 72-96 hours post drowning. Infective-stage larvae were active for 21 days in municipal water. Histological sectioning of P. martensi showed proximity of nematode larvae to the body wall of the gastropod, consistent with the potential for shedding of larvae in slime. Gastropod tissue squashes showed effectivity as a quick screening method. Live, infective-stage larvae were able to traverse rainwater catchment polypropylene sediment filters of 20 µm, 10 µm, 5 µm, and 1 µm filtration ratings, but not a 5 µm carbon block filter. These results demonstrate that live, infective-stage A. cantonensis larvae emerge from drowned snails and slugs, survive for extended periods of time in water, and may be able to enter a catchment user's household water supply. This study illustrates the need to better investigate and understand the potential role of contaminated water as a transmission route for neuroangiostrongyliasis.