Development of a recombinase polymerase amplification (RPA-EXO) and lateral flow assay (RPA-LFA) based on the ITS1 gene for the detection of Angiostrongylus cantonensis in gastropod intermediate hosts.

Angiostrongylus cantonensis is a parasitic nematode known to infect humans through the ingestion of third stage larvae which can cause inflammation and damage to the central nervous system. Currently, polymerase chain reaction (PCR) is one of the most reliable diagnostic methods for detecting A. cantonensis in humans as well as in gastropod hosts, but requires expensive and specialized equipment. Here, we compare the sensitivity and accuracy of a recombinase polymerase amplification Exo (RPA-EXO) assay, and a recombinase polymerase amplification lateral flow assay (RPA-LFA) with a traditional quantitative PCR (qPCR) assay currently available. The three assays were used to test 35 slugs from Hawai'i for the presence of A. cantonensis DNA. Consistent results among the three tests were shown in 23/35 samples (65.7%), while 7/35 (20%) were discordant in low infection level samples (<0.01 larvae per mg tissue), and 5/35 (14.3%) were equivocal. To evaluate sensitivity, a partial ITS1 gene was cloned, and serial plasmid dilutions were created ranging from 100 copies µL-1 to ~1 copy µL-1. All three assays consistently detected 50-100 copies µL-1 in triplicate and qPCR was able to detect ~13 copies µL-1 in triplicate. RPA-EXO was able to detect 25 copies µL-1 in triplicate and RPA-LFA was not able to amplify consistently below 50 copies µL-1. Thus, our RPA-EXO and RPA-LFA assays do not appear as sensitive as the current qPCR assay at low DNA concentrations; however, these tests have numerous advantages that may make them useful alternatives to qPCR.

Variation in Angiostrongylus cantonensis infection in definitive and intermediate hosts in Hawaii, a global hotspot of rat lungworm disease.

Angiostrongylus cantonensis (rat lungworm) is a tropical and subtropical parasitic nematode, with infections in humans causing angiostrongyliasis (rat lungworm disease), characterized by eosinophilic meningitis. Hawaii has been identified as a global hotspot of infection, with recent reports of high infection rates in humans, as well as rat definitive and snail intermediate hosts. This study investigated variation in A. cantonensis infection, both prevalence and intensity, in wild populations of two species of rats (Rattus exulans and R. rattus) and one species of snail (Parmarion martensi). An overall infection prevalence of 86.2% was observed in P. martensi and 63.8% in rats, with R. exulans (77.4%) greater than R. rattus (47.6%). We found infections to vary with environmental and host-related factors. Body mass was a strong predictor of infection in all three species, with different patterns seen between sexes and species of rats. Infection prevalence and intensity for R. exulans were high in May 2018 and again in February 2019, but generally lower and more variable during the intervening months. Information on sources of variability of infection in wild host populations will be a crucial component in predicting the effectiveness of future disease surveillance or targeted management strategies.

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).

In vitro comparison of treatments and commercially available solutions on mortality of Angiostrongylus cantonensis third-stage larvae.

On Hawai'i Island, an increase in human neuroangiostrongyliasis cases has been primarily associated with the accidental ingestion of Angiostrongylus cantonensis L3 in snails or slugs, or potentially, from larvae left behind in the slug's slime or feces. We evaluated more than 40 different treatments in vitro for their ability to kill A. cantonensis larvae with the goal of identifying a safe and effective fruit and vegetable wash in order to reduce the risk of exposure. Our evaluation of treatment lethality was carried out in two phases; initially using motility as an indicator of larval survival after treatment, followed by the development and application of a propidium iodide staining assay to document larval mortality. Treatments tested included common household products, consumer vegetable washes and agricultural crop washes. We found minimal larvicidal efficacy among consumer-grade fruit and vegetable washes, nor among botanical extracts such as those from ginger or garlic, nor acid solutions such as vinegar. Alkaline solutions, on the other hand, as well as oxidizers such as bleach and chlorine dioxide, did show larvicidal potential. Surfactants, a frequent ingredient in detergents that lowers surface tension, had variable results, but dodecylbenzene sulfonic acid as a 70% w/w solution in 2-propanol was very effective, both in terms of the speed and the thoroughness with which it killed A. cantonensis L3 nematodes. Thus, our results suggest promising directions for future investigation.

Validation of a death assay for Angiostrongylus cantonensis larvae (L3) using propidium iodide in a rat model (Rattus norvegicus).

Angiostrongylus cantonensis is a pathogenic nematode and the cause of neuroangiostrongyliasis, an eosinophilic meningitis more commonly known as rat lungworm disease. Transmission is thought to be primarily due to ingestion of infective third stage larvae (L3) in gastropods, on produce, or in contaminated water. The gold standard to determine the effects of physical and chemical treatments on the infectivity of A. cantonensis L3 larvae is to infect rodents with treated L3 larvae and monitor for infection, but animal studies are laborious and expensive and also raise ethical concerns. This study demonstrates propidium iodide (PI) to be a reliable marker of parasite death and loss of infective potential without adversely affecting the development and future reproduction of live A. cantonensis larvae. PI staining allows evaluation of the efficacy of test substances in vitro, an improvement upon the use of lack of motility as an indicator of death. Some potential applications of this assay include determining the effectiveness of various anthelmintics, vegetable washes, electromagnetic radiation and other treatments intended to kill larvae in the prevention and treatment of neuroangiostrongyliasis.

Characterization of class II ß chain major histocompatibility complex genes in a family of Hawaiian honeycreepers: 'amakihi (Hemignathus virens).

Hawaiian honeycreepers (Drepanidinae) have evolved in the absence of mosquitoes for over five million years. Through human activity, mosquitoes were introduced to the Hawaiian archipelago less than 200 years ago. Mosquito-vectored diseases such as avian malaria caused by Plasmodium relictum and Avipoxviruses have greatly impacted these vulnerable species. Susceptibility to these diseases is variable among and within species. Due to their function in adaptive immunity, the role of major histocompatibility complex genes (Mhc) in disease susceptibility is under investigation. In this study, we evaluate gene organization and levels of diversity of Mhc class II ß chain genes (exon 2) in a captive-reared family of Hawaii 'amakihi (Hemignathus virens). A total of 233 sequences (173 bp) were obtained by PCR+1 amplification and cloning, and 5720 sequences were generated by Roche 454 pyrosequencing. We report a total of 17 alleles originating from a minimum of 14 distinct loci. We detected three linkage groups that appear to represent three distinct haplotypes. Phylogenetic analysis revealed one variable cluster resembling classical Mhc sequences (DAB) and one highly conserved, low variability cluster resembling non-classical Mhc sequences (DBB). High net evolutionary divergence values between DAB and DBB resemble that seen between chicken BLB system and YLB system genes. High amino acid identity among non-classical alleles from 12 species of passerines (DBB) and four species of Galliformes (YLB) was found, suggesting that these non-classical passerine sequences may be related to the Galliforme YLB sequences.

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.

Analysis of the trap gene provides evidence for the role of elevation and vector abundance in the genetic diversity of Plasmodium relictum in Hawaii.

BACKGROUND: The avian disease system in Hawaii offers an ideal opportunity to investigate host-pathogen interactions in a natural setting. Previous studies have recognized only a single mitochondrial lineage of avian malaria (Plasmodium relictum) in the Hawaiian Islands, but cloning and sequencing of nuclear genes suggest a higher degree of genetic diversity. METHODS: In order to evaluate genetic diversity of P. relictum at the population level and further understand host-parasite interactions, a modified single-base extension (SBE) method was used to explore spatial and temporal distribution patterns of single nucleotide polymorphisms (SNPs) in the thrombospondin-related anonymous protein (trap) gene of P. relictum infections from 121 hatch-year amakihi (Hemignathus virens) on the east side of Hawaii Island. RESULTS: Rare alleles and mixed infections were documented at three of eight SNP loci; this is the first documentation of genetically diverse infections of P. relictum at the population level in Hawaii. Logistic regression revealed that the likelihood of infection with a rare allele increased at low-elevation, but decreased as mosquito capture rates increased. The inverse relationship between vector capture rates and probability of infection with a rare allele is unexpected given current theories of epidemiology developed in human malarias. CONCLUSIONS: The results of this study suggest that pathogen diversity in Hawaii may be driven by a complex interaction of factors including transmission rates, host immune pressures, and parasite-parasite competition.

Reversion to virulence and efficacy of an attenuated Canarypox vaccine in Hawai'i 'Amakihi (Hemignathus virens).

Vaccines may be effective tools for protecting small populations of highly susceptible endangered, captive-reared, or translocated Hawaiian honeycreepers from introduced Avipoxvirus, but their efficacy has not been evaluated. An attenuated Canarypox vaccine that is genetically similar to one of two passerine Avipoxvirus isolates from Hawai'i and distinct from Fowlpox was tested to evaluate whether Hawai'i 'Amakihi (Hemignathus virens) can be protected from wild isolates of Avipoxvirus from the Hawaiian Islands. Thirty-one (31) Hawai'i 'Amakihi were collected from high-elevation habitats on Mauna Kea Volcano, where pox transmission is rare, and randomly divided into two groups. One group was vaccinated with Poximune C, whereas the other group received a sham vaccination with sterile water. Four of 15 (27%) vaccinated birds developed life-threatening disseminated lesions or lesions of unusually long duration, whereas one bird never developed a vaccine-associated lesion or "take." After vaccine lesions healed, vaccinated birds were randomly divided into three groups of five and challenged with either a wild isolate of Fowlpox (FP) from Hawai'i, a Hawai'i 'Amakihi isolate of a Canarypox-like virus (PV1), or a Hawai'i 'Amakihi isolate of a related, but distinct, passerine Avipoxvirus (PV2). Similarly, three random groups of five unvaccinated 'Amakihi were challenged with the same virus isolates. Vaccinated and unvaccinated 'Amakihi challenged with FP had transient infections with no clinical signs of infection. Mortality in vaccinated 'Amakihi challenged with PV1 and PV2 ranged from 0% (0/5) for PV1 to 60% (3/5) for PV2. Mortality in unvaccinated 'Amakihi ranged from 40% (2/5) for PV1 to 100% (5/5) for PV2. Although the vaccine provided some protection against PV1, both potential for vaccine reversion and low efficacy against PV2 preclude its use in captive or wild honeycreepers.

Larvicidal Efficacy of Ozone and Ultrasound on Angiostrongylus cantonensis (Rat Lungworm) Third-Stage Larvae.

The parasitic nematode Angiostrongylus cantonensis (rat lungworm) is the leading cause of human eosinophilic meningitis worldwide. Most human infections occur through the accidental consumption of A. cantonensis hidden within produce as infectious third-stage larvae (L3), yet little research has been published addressing possible methods to mitigate this means of transmission. Here, we describe our tests of ozone gas-an oxidizing agent-and ultrasound, both used for disinfection of food and municipal water supplies and in industrial cleaning. We found that exposure to ozone, produced using two different commercially available ozone generators over varying durations of time and concentrations, was capable of achieving 100% larval mortality. In addition, we evaluated the impact of different sound frequencies on A. cantonensis L3 survival using two different commercially available ultrasonic cleaners, and found that 60 s of 40 kHz produced 46% mortality within 2 h. The combined use of ultrasound and ozone gas simultaneously resulted in a minimum of 89% normalized mean percent mortality within 2 h of treatment. Our study suggests that both ozone and ultrasound show high larvicidal efficacy, both independently and together, and thus show promise as methods for reducing the risk of rat lungworm infection via accidental consumption.

Occurrence of Rat Lungworm (Angiostrongylus cantonensis) in Invasive Coqui Frogs (Eleutherodactylus coqui) and Other Hosts in Hawaii, USA.

The rat lungworm (Angiostrongylus cantonensis) has emerged as an important human and animal health concern in Hawaii, US. Although the life cycle of the parasite requires both rat and gastropod hosts, other animals acting as paratenic hosts, such as frogs and centipedes, have been identified as sources of infection. We investigated the occurrence of rat lungworm infections in potential paratenic hosts in Hawaii to provide information on how they might be involved in transmission of angiostrongyliasis. We confirmed the presence of rat lungworm in 87% (21/24) of introduced Puerto Rican coqui frogs (Eleutherodactylus coqui) in Hilo, Hawaii, by real-time PCR. Additionally, four Cuban greenhouse frogs (Eleutherodactylus planirostris), two cane toads (Rhinella marina), and three centipedes (Scolopendra subspinipes) were found to be infected. In the frogs and toads, multiple tissue types were positive, including stomach and intestine, muscle, liver, heart, and brain, indicating larval migration. We identified rat lungworm infections in frogs, toads, and centipedes in Hawaii and highlighted the lack of knowledge of the role paratenic hosts may be playing in the transmission and life cycle maintenance of rat lungworm in Hawaii.

Estimating Human Exposure to Rat Lungworm (Angiostrongylus cantonensis) on Hawai'i Island: A Pilot Study.

Angiostrongylus cantonensis is a zoonotic, parasitic nematode causing angiostrongyliasis or rat lungworm disease. Clinical diagnosis in humans is currently confirmed by detection of parasite DNA in cerebrospinal fluid. This study estimated human exposure to A. cantonensis in volunteer participants solicitated via public venues on east Hawai'i Island using blood-based tests. Antibodies were screened in sera by crude antigen ELISA, followed by a 31-kDa dot-blot test developed and validated in Thailand. Human participants (n = 435) donated blood samples and completed a questionnaire to self-report relevant symptomology or clinical diagnosis. Among symptoms reported by participants diagnosed by licensed clinicians, headaches, high eosinophil counts, stiff neck, fatigue, and joint pain were most severe during the initial 3 months of infection. ELISA results revealed 22% of the serum samples as positive, 46% as equivocal, and 32% as negative. A subset of 186 samples was tested by dot blot, with 30% testing positive and 70% testing negative. A significantly higher mean ELISA value was found among recently (2014-2015) clinically diagnosed participants as than among those with a diagnosis before 2010 (P = 0.027). All dot-blot positives were also ELISA positive and were significantly associated with higher ELISA values compared with dot-blot negatives (P = 0.0001). These results suggest that an ELISA using crude antigen isolated from adult A. cantonensis from Hawai'i may be an effective initial screening method for estimating exposure to A. cantonensis in Hawai'i and likewise suggest that dot-blot tests using the 31-kDa antigen exhibit efficacy as a diagnostic for exposure.

A nucleotide-constrained single base extension method for improved detection of minority alleles in Plasmodium.

In genetically mixed Plasmodium infections, minority alleles may have a significant role in drug resistance and other responses to selective pressures. Many available methods to detect single nucleotide polymorphisms representing minority alleles either are not sensitive enough to detect these rare alleles or are limited in the number of loci to be screened in a single reaction. In order to achieve highly sensitive, multiplex SNP genotyping, we have developed a nucleotide-constrained method based on the traditional single base extension approach. Here, we report results when using standard and nucleotide-constrained reactions to determine alleles at nine SNP loci in the trap gene of Plasmodium relictum. This innovative method offers great improvements in detection limits while maintaining the accuracy and multiplex capabilities of single base extension SNP genotyping.

Angiostrongyliasis (Rat Lungworm Disease): Viewpoints from Hawai'i Island.

Hawai'i, particularly east Hawai'i Island, is the epicenter for angiostrongyliasis in the United States. Case numbers have been increasing and appear to parallel the introduction and spread of the semislug (Parmarion martensi) to east Hawai'i. The infective larvae in rainwater catchment as a source for household and agricultural water may also play a role. The spread of Angiostrongylus cantonensis as well as the potential introduction of the semislug P. martensi should be a concern to the mainland United States. The State of Hawai'i should recognize the seriousness of this growing problem and thus collaborate to fund studies to address the growing challenges surrounding angiostrongyliasis.

High prevalence of Angiostrongylus cantonensis (rat lungworm) on eastern Hawai'i Island: A closer look at life cycle traits and patterns of infection in wild rats (Rattus spp.).

The nematode Angiostrongylus cantonensis is a zoonotic pathogen and the etiological agent of human angiostrongyliasis or rat lungworm disease. Hawai'i, particularly east Hawai'i Island, is the epicenter for angiostrongyliasis in the USA. Rats (Rattus spp.) are the definitive hosts while gastropods are intermediate hosts. The main objective of this study was to collect adult A. cantonensis from wild rats to isolate protein for the development of a blood-based diagnostic, in the process we evaluated the prevalence of infection in wild rats. A total of 545 wild rats were sampled from multiple sites in the South Hilo District of east Hawai'i Island. Adult male and female A. cantonensis (3,148) were collected from the hearts and lungs of humanely euthanized Rattus rattus, and R. exulans. Photomicrography and documentation of multiple stages of this parasitic nematode in situ were recorded. A total of 45.5% (197/433) of rats inspected had lung lobe(s) (mostly upper right) which appeared granular indicating this lobe may serve as a filter for worm passage to the rest of the lung. Across Rattus spp., 72.7% (396/545) were infected with adult worms, but 93.9% (512/545) of the rats were positive for A. cantonensis infection based on presence of live adult worms, encysted adult worms, L3 larvae and/or by PCR analysis of brain tissue. In R. rattus we observed an inverse correlation with increased body mass and infection level of adult worms, and a direct correlation between body mass and encysted adult worms in the lung tissue, indicating that larger (older) rats may have developed a means of clearing infections or regulating the worm burden upon reinfection. The exceptionally high prevalence of A. cantonensis infection in Rattus spp. in east Hawai'i Island is cause for concern and indicates the potential for human infection with this emerging zoonosis is greater than previously thought.

Detection of Angiostrongylus cantonensis in the Blood and Peripheral Tissues of Wild Hawaiian Rats (Rattus rattus) by a Quantitative PCR (qPCR) Assay.

The nematode Angiostrongylus cantonensis is a rat lungworm, a zoonotic pathogen that causes human eosinophilic meningitis and ocular angiostrongyliasis characteristic of rat lungworm (RLW) disease. Definitive diagnosis is made by finding and identifying A. cantonensis larvae in the cerebral spinal fluid or by using a custom immunological or molecular test. This study was conducted to determine if genomic DNA from A. cantonensis is detectable by qPCR in the blood or tissues of experimentally infected rats. F1 offspring from wild rats were subjected to experimental infection with RLW larvae isolated from slugs, then blood or tissue samples were collected over multiple time points. Blood samples were collected from 21 rats throughout the course of two trials (15 rats in Trial I, and 6 rats in Trial II). In addition to a control group, each trial had two treatment groups: the rats in the low dose (LD) group were infected by approximately 10 larvae and the rats in the high dose (HD) group were infected with approximately 50 larvae. In Trial I, parasite DNA was detected in cardiac bleed samples from five of five LD rats and five of five HD rats at six weeks post-infection (PI), and three of five LD rats and five of five HD rats from tail tissue. In Trial II, parasite DNA was detected in peripheral blood samples from one of two HD rats at 53 minutes PI, one of two LD rats at 1.5 hours PI, one of two HD rats at 18 hours PI, one of two LD rats at five weeks PI and two of two at six weeks PI, and two of two HD rats at weeks five and six PI. These data demonstrate that parasite DNA can be detected in peripheral blood at various time points throughout RLW infection in rats.

PCR diagnostics underestimate the prevalence of avian malaria (Plasmodium relictum) in experimentally-infected passerines.

Several polymerase chain reaction (PCR)-based methods have recently been developed for diagnosing malarial infections in both birds and reptiles, but a critical evaluation of their sensitivity in experimentally-infected hosts has not been done. This study compares the sensitivity of several PCR-based methods for diagnosing avian malaria (Plasmodium relictum) in captive Hawaiian honeycreepers using microscopy and a recently developed immunoblotting technique. Sequential blood samples were collected over periods of up to 4.4 yr after experimental infection and rechallenge to determine both the duration and detectability of chronic infections. Two new nested PCR approaches for detecting circulating parasites based on P. relictum 18S rRNA genes and the thrombospondin-related anonymous protein (TRAP) gene are described. The blood smear and the PCR tests were less sensitive than serological methods for detecting chronic malarial infections. Individually, none of the diagnostic methods was 100% accurate in detecting subpatent infections, although serological methods were significantly more sensitive (97%) than either nested PCR (61-84%) or microscopy (27%). Circulating parasites in chronically infected birds either disappear completely from circulation or to drop to intensities below detectability by nested PCR. Thus, the use of PCR as a sole means of detection of circulating parasites may significantly underestimate true prevalence.

Control measures for slug and snail hosts of Angiostrongylus cantonensis, with special reference to the semi-slug Parmarion martensi.

Slugs and snails (class Gastropoda) are the obligate intermediate hosts of the rat lungworm, Angiostrongylus cantonensis. This nematode is the causative agent of human angiostrongyliasis and the most common cause of human eosinophilic meningoencephalitis. Humans can become infected by accidental consumption of slugs or snails and possibly flatworms (or a portion of one of these animals) in fresh produce, but the slime from these animals can contain nematodes and may also constitute a disease risk. Gastropod carriers in Hawa'i include, among other species, giant African snails, veronicellid slugs, and the semi-slug Parmarion martensi. This latter species was first discovered on the island of Hawa'i in 2004 and is now common in the area where the majority of the state's documented cases of human angiostrongyliasis occurred between 2005 and 2011. This species is considered a high risk carrier of A. cantonensis because of its climbing behavior, abundance around human dwellings, and high worm burdens. One individual collected from east Hawa'i Island contained >6,800 infective third stage A. cantonensis larvae. Common and efficient control methods for slugs and snails include sanitation (eg, removal of objects that serve as hiding places) and the use of poison food baits, such as those containing metaldehyde and iron. An iron-containing bait that is relatively safe to non-target organisms was effective in controlling semi-slugs in cage experiments, although it killed more slowly than a metaldehyde-containing bait and the majority of slugs affected did not die until 1-2 weeks following ingestion.

Vaccination of captive nene (Branta sandvicensis) against West Nile virus using a protein-based vaccine (WN-80E).

Although West Nile Virus (WNV) has not been reported in Hawai'i, eventual introduction appears unavoidable with potential adverse effects on avian species. Nene (Branta sandvicensis) are endemic endangered Hawaiian geese that are susceptible to WNV. We demonstrate that a vaccine developed against WNV for humans (WN-80E) is also highly immunogenic in Nene and does not produce adverse biologic effects. Six captive, nonbreeding Nene were immunized with two 10-µg doses (4 wk apart) of the WN-80E recombinant protein adjuvanted with Montanide ISA720. Two Nene were similarly injected with "mock" preparation as controls. Blood samples were collected before the first dose, then 2 wk and 6 mo after the second dose. WNV-specific antibody titers were determined by an endpoint enzyme-linked immunosorbent assay. An unpaired t-test demonstrated significantly higher geometric mean titers for immunized vs. control groups 2 wk after dose 2 (4,129 and 100, respectively, P=0.010) and 6 mo after dose 2 (246 and 63, respectively, P=0.002). Daily observations revealed no swelling at the site of injection and no serious adverse biological effects from the immunization. The vaccine containing the WN-80E and Montanide ISA720 adjuvant appears to be safe and immunogenic in Nene. This protein-based WNV vaccine may be safer for use in Hawai'i than killed virus and live chimeric or recombinant canarypox-vectored vaccines because it cannot cause disease.