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BACKGROUND Phytosanitary irradiation is used to control insect pests of quarantine concern on exported fresh horticultural products. Generic irradiation doses of 150 Gy 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 United States. 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 U.S. 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. This article is protected by copyright. All rights reserved.

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 (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.

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.

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.

Education is essential for lowering cases of Angiostrongyliasis in Hawaii. A professional development course on rat lungworm disease (RLWD) prevention was offered to Hawaii K-12 teachers during the 2018–2019 school year. Fourteen teachers, 11 from Hawaii Island and three from Maui, representing grades K, 2, 6–8, and 10−12, completed workshops and activities and received course credit. Teachers reached 652 students and educated 86 teachers and staff and approximately 900 community members including parents. A pre-course survey showed 55% of teachers agreed to a basic understanding of rat lungworm and its impact on Hawaii; this increased to 100% post-course. A sixth-grade class was the first to document the arrival of a highly competent intermediate host of the rat lungworm in a new location, an important discovery that initiated community awareness and control efforts. Six, 1-day workshops were held in 2018–2019, each on one of the main Hawaiian Islands. These were attended by 106 participants including teachers, community educators, and interested individuals from agencies, non-profits, businesses and the private sector. Of participants surveyed, 100% responded the workshop improved their overall understanding of RLWD. Efforts are being made to continue these programmes.

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).

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.

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.

The safety and efficacy of benzimidazole anthelmintics for the treatment of rat lungworm disease (neuroangiostrongyliasis) have been questioned regardless of numerous experimental animal studies and clinical reports. In this review, 40 of these experimental animal studies and 104 clinical reports are compiled with a focus on albendazole. Among the 144 articles involving an estimated 1034 patients and 2561 animals, 4.1% were inconclusive or vague regarding the use of benzimidazoles. Of the remaining 138 articles, 90.5% found benzimidazoles to be safe and effective (885 patients, 2530 animals), 4.3% as safe but ineffective (73 patients, 3 animals), and 5.0% caused adverse reactions (7 patients, 28 animals). Among those clinical reports that described a confirmed diagnosis of neuroangiostrongyliasis in which albendazole monotherapy was used, 100% reported high efficacy (743 patients, 479 animals). In those where albendazole-corticosteroid co-therapy was used, 97.87% reported it to be effective (323 patients, 130 animals).

Background Angiostrongylus cantonensis (Ac), or the rat lungworm, is a major cause of eosinophilic meningitis. Humans are infected by ingesting the 3rd stage larvae from primary hosts, snails, and slugs, or paratenic hosts. The currently used molecular test is a qPCR assay targeting the ITS1 rDNA region (ITS1) of Ac. Methods In silico design of a more sensitive qPCR assay was performed based on tandem repeats predicted to be the most abundant by the RepeatExplorer algorithm. Genomic DNA (gDNA) of Ac were used to determine the analytical sensitivity and specificity of the best primer/probe combination. This assay was then applied to clinical and environmental samples. Results The limit of detection of the best performing assay, AcanR3990, was 1 fg (the DNA equivalent of 1/100 000 dilution of a single 3rd stage larvae). Out of 127 CDC archived CSF samples from varied geographic locations, the AcanR3990 qPCR detected the presence of Ac in 49/49 ITS1 confirmed angiostrongyliasis patients, along with 15/73 samples previously negative by ITS1 qPCR despite strong clinical suspicion for angiostrongyliasis. Intermediate hosts (gastropods) and an accidental host, a symptomatic horse, were also tested with similar improvement in detection observed. AcanR3990 qPCR did not cross-react in 5 CSF from patients with proven neurocysticercosis, toxocariasis, gnathostomiasis, and baylisascariasis. AcanR3990 qPCR failed to amplify genomic DNA from the other related Angiostrongylus species tested except for Angiostrongylus mackerrasae (Am), a neurotropic species limited to Australia that would be expected to present with a clinical syndrome indistinguishable from Ac. Conclusion These results suggest AcanR3990 qPCR assay is highly sensitive and specific with potential wide applicability as a One Health detection method for Ac and Am.

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.

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.

Angiostrongylus cantonensis is the leading cause of eosinophilic meningitis worldwide, with life-threatening complications if not managed correctly. Previous in vitro studies have utilized change in motility patterns of adult female worms to assess the efficacy of anthelmintics qualitatively. However, it is the third stage larvae (L3) that are infectious to humans. With differential staining using propidium iodide penetration as the indicator of death, we can distinguish between dead and live larvae. This assay has enabled us to quantify the in vitro efficacy of nine clinically established anthelmintics on A. cantonensis L3. All drugs were tested at a 1 mm concentration. Piperazine and niclosamide were ineffective in inducing larval death; however, albendazole sulfoxide, pyrantel pamoate, diethylcarbamazine, levamisole and praziquantel were effective as compared to unexposed controls (P < 0.05). Ivermectin and moxidectin did not induce significant levels of mortality, but they considerably reduced larval motility almost immediately. This study indicates the need for further in vivo studies to determine the optimal dose and time frame for post-infection treatment with anthelmintics that demonstrated efficacy.

Angiostrongyliasis is contracted by the ingestion of or exposure to the zoonotic parasite Angiostrongylus cantonensis, which requires two hosts to complete its life cycle. Rats are known to be the definitive hosts and mollusks are intermediate hosts. It is the intermediate host, or infected paratenic hosts that can pass the infective stage of the disease to humans and other susceptible animals. The purpose of this review is to examine the growing threat of A. cantonensis and its consequences, with particular emphasis on Hawai'i. The increase in disease cases appears to correlate with the migration of the mollusk Parmarion martensi (semi-slug) in Hawai'i. The semi-slug is a very effective host because it carries a heavy burden of the parasite. Rats in Hawai'i also carry heavy parasite burdens. Human diagnosis is difficult due to varied incubation times, multitude of symptoms which can mimic other illnesses, and lack of a low-risk effective diagnostic. Treatment varies with the severity of the symptoms. The introduction and proliferation of the invasive species A. cantonensis, along with a most efficient intermediate host P. martensi, has resulted in an increase in angiostrongyliasis in Hawai'i. In Hawai'i, consumption of local produce is encouraged and the use of rainwater harvesting is a necessity for many on Hawai'i Island, both of which elevate the risk of acquiring angiostrongyliasis. A better understanding of how to best reduce the risk of infection is needed through comprehensive research and community education.

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.

The rat lungworm (

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.

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 β 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.

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.

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.

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 contaminated water can 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. 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 can and do emerge from drowned snails and slugs, survive for extended periods of time in water, and that the potential exists that they enter the 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.

A recent increase in serious cases of rat lungworm disease impacts Hawai'i's agriculture and human health. Slugs and snails, agricultural pests, are intermediate hosts of Angiostrongylus cantonensis the rat lungworm. Infection by this parasitic nematode is the leading cause of eosinophilic meningitis globally. Infection can result from ingestion of infected produce and has caused chronic neurological problems, disability, coma, and death. There are over 200 K-12 school and youth garden, and agriculture projects throughout the Hawaiian Islands. This existing network provides an avenue for teacher and student involvement in community health education and host control programs. We collaborated with five Hawai'i Island schools connected with the Hawai'i Island School Garden Network to develop an integrated pest management plan for the control of invasive, intermediate hosts of A. cantonensis. Curricula relating to rat lungworm for grades 5-8 that support student academic achievement with a focus on science, technology, engineering, art, and math were developed. The management plan trialed the use of five different materials for shelters, which provided refuge for and easy removal of unwanted slugs and snails. Over 4,000 invasive slugs and snails were removed. Students learned how to safely dispatch pests and they collected data on species found, numbers of species removed, and shelter-type capture rates. Using the arts, students shared information at school and within their family and community. A written management plan, eleven lesson plans, and auxiliary materials are now available online. A concerted effort is needed to reduce parasite hosts if we are to reduce human cases of disease and restore public faith in local agriculture. Use of the established school garden network is an ideal avenue through which to educate the public and develop solutions for this public health problem.

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.

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 similar to 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 mu m, 10 mu m, 5 mu m, and 1 mu m filtration ratings, but not a 5 mu m 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. Max and Yetta Karasik Family Foundation National Institutes of Health National Institute of General Medical Sciences, IDeA Networks of Biomedical Research Excellence [P20GM103466] Daniel K. Inouye College of Pharmacy University of Hawai'i at Hilo Research Council State of Hawaii This work was supported by: (SJ) Donation, Dr. and Mrs. Richard Robbins representing the Max and Yetta Karasik Family Foundation, the funder did not play any role; (SJ) National Institutes of Health National Institute of General Medical Sciences, IDeA Networks of Biomedical Research Excellence, Award number: P20GM103466, the funder played no role; (SJ) Daniel K. Inouye College of Pharmacy, the funder played no role; (KH) University of Hawai'i at Hilo Research Council, the funder played no role; (SJ) State of Hawaii, the funder played no role.

SUMMARYNext-generation 454 sequencing techniques were used to re-examine diversity of mitochondrial cytochrome b lineages of avian malaria (Plasmodium relictum) in Hawaii. We document a minimum of 23 variant lineages of the parasite based on single nucleotide transitional changes, in addition to the previously reported single lineage (GRW4). A new, publicly available portal (Integroomer) was developed for initial parsing of 454 datasets. Mean variant prevalence and frequency was higher in low elevation Hawaii Amakihi (Hemignathus virens) with Avipoxvirus-like lesions (P = 0·001), suggesting that the variants may be biologically distinct. By contrast, variant prevalence and frequency did not differ significantly among mid-elevation Apapane (Himatione sanguinea) with or without lesions (P = 0·691). The low frequency and the lack of detection of variants independent of GRW4 suggest that multiple independent introductions of P. relictum to Hawaii are unlikely. Multiple variants may have been introduced in heteroplasmy with GRW4 or exist within the tandem repeat structure of the mitochondrial genome. The discovery of multiple mitochondrial lineages of P. relictum in Hawaii provides a measure of genetic diversity within a geographically isolated population of this parasite and suggests the origins and evolution of parasite diversity may be more complicated than previously recognized.

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.

Introduction of the West Nile virus (WNV) to Hawai’i will undoubtedly devastate many populations of critically endangered avian species indigenous to Hawai’i. The protective efficacy of a protein-based WNV subunit vaccine formulated with adjuvant was evaluated in domestic geese as a surrogate species for the endangered Nēnē, the state bird of Hawai’i. Prevention of viremia following viral infection of vaccinated birds was used as the clinical endpoint of protection. ELISA and plaque reduction neutralization tests demonstrate that significant levels of vaccine antigen-specific antibody were produced in groups of birds vaccinated with 5 or 10 μg of the WN-80E antigen formulated with ISA720 adjuvant. Moreover, after challenge with WNV, no viremia was detected in vaccinated birds, whereas viremia was detected up to 4 days after and virus was detected by oral swab for 6 days after infection among control groups. Safe and effective vaccination of managed or captive endangered bird populations will protect species with critically low numbers that could not survive the added mortality of introduced disease.

We cultured avian pox (Avipoxvirus spp.) from lesions collected on Hawai‘i, Maui, Moloka‘i, and ‘Oahu in the Hawaiian Islands from 15 native or non-native birds representing three avian orders. Phylogenetic analysis of a 538 bp fragment of the gene encoding the virus 4b core polypeptide revealed two distinct variant clusters, with sequences from chickens (fowlpox) forming a third distinct basal cluster. Pox isolates from one of these two clusters appear closely related to canarypox and other passerine pox viruses, while the second appears more specific to Hawai‘i. There was no evidence that birds were infected simultaneously with multiple pox virus variants based on evaluation of multiples clones from four individuals. No obvious temporal or geographic associations were observed and strict host specificity was not apparent among the 4b-defined field isolates. We amplified a 116 bp 4b core protein gene fragment from an ‘Elepaio (Chasiempis sandwichensis) collected in 1900 on Hawai‘i Island that clustered closely with the second of the two variants, suggesting that this variant has been in Hawai‘i for at least 100 years. The high variation detected between the three 4b clusters provides evidence for multiple, likely independent introductions, and does not support the hypothesis of infection of native species through introduction of infected fowl. Preliminary experimental infections in native Hawai‘i ‘Amakihi (Hemignathus virens) suggest that the 4b-defined variants may be biologically distinct, with one variant appearing more virulent. These pox viruses may interact with avian malaria (Plasmodium relictum), another introduced pathogen in Hawaiian forest bird populations, through modulation of host immune responses.

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.

This study documents the presence ofPlasmodium spp. in landbirds ofcentral Polynesia. Blood samples collectedfrom eight native and introduced species fromthe island of Tutuila, American Samoa wereevaluated for the presence of Plasmodiumspp. by nested rDNA PCR, serology and/ormicroscopy. A total of 111/188 birds (59%)screened by nested PCR were positive. Detection of Plasmodium spp. was verifiedby nucleotide sequence comparisons of partial18S ribosomal RNA and TRAP(thrombospondin-related anonymous protein)genes using phylogenetic analyses. All samplesscreened by immunoblot to detect antibodiesthat cross-react with Hawaiian isolates of Plasmodium relictum (153) were negative. Lack of cross-reactivity is probably due toantigenic differences between the Hawaiian andSamoan Plasmodium isolates. Similarly,all samples examined by microscopy (214) werenegative. The fact that malaria is present,but not detectable by blood smear evaluation isconsistent with low peripheral parasitemiacharacteristic of chronic infections. Highprevalence of apparently chronic infections,the relative stability of the native land birdcommunities, and the presence of mosquitovectors which are considered endemic andcapable of transmitting avian Plasmodia,suggest that these parasites are indigenous toSamoa and have a long coevolutionary historywith their hosts.

The life cycle of the nematode Angiostrongylus cantonensis involves rats as the definitive host and slugs and snails as intermediate hosts. Humans can become infected upon ingestion of intermediate or paratenic (passive carrier) hosts containing stage L3 A. cantonensis larvae. Here, we report a quantitative PCR (qPCR) assay that provides a reliable, relative measure of parasite load in intermediate hosts. Quantification of the levels of infection of intermediate hosts is critical for determining A. cantonensis intensity on the Island of Hawaii. The identification of high intensity infection ‘hotspots’ will allow for more effective targeted rat and slug control measures. qPCR appears more efficient and sensitive than microscopy and provides a new tool for quantification of larvae from intermediate hosts, and potentially from other sources as well.

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.

Sequence information from 28 CHD1 gene fragments reveals that a primary source of variability in CHD1-W genes is a variable intron microsatellite; a single-codon deletion was found in the 3′ exon in one species. Sequence variation of CHD1-Z genes was detected in males that altered polymerase chain reaction (PCR) fragment length. Three sets of CHD1-based primers were evaluated for sex determination in 12 endemic and 8 alien Hawaiian species, including one of the last po’o-uli. Combined, these primers provide a reliable means of sex determination in most species (including the po’o-uli), and have produced a valuable reference database for future expanded population-level studies.

Introduced vector-borne diseases, particularly avian malaria (Plasmodium relictum) and avian pox virus (Avipoxvirus spp.), continue to play significant roles in the decline and extinction of native forest birds in the Hawaiian Islands. Hawaiian honeycreepers are particularly susceptible to avian malaria and have survived into this century largely because of persistence of high elevation refugia on Kaua‘i, Maui, and Hawai‘i Islands, where transmission is limited by cool temperatures. The long term stability of these refugia is increasingly threatened by warming trends associated with global climate change. Since cost effective and practical methods of vector control in many of these remote, rugged areas are lacking, adaptation through processes of natural selection may be the best long-term hope for recovery of many of these species. We document emergence of tolerance rather than resistance to avian malaria in a recent, rapidly expanding low elevation population of Hawai‘i ‘Amakihi (Hemignathus virens) on the island of Hawai‘i. Experimentally infected low elevation birds had lower mortality, lower reticulocyte counts during recovery from acute infection, lower weight loss, and no declines in food consumption relative to experimentally infected high elevation Hawai‘i ‘Amakihi in spite of similar intensities of infection. Emergence of this population provides an exceptional opportunity for determining physiological mechanisms and genetic markers associated with malaria tolerance that can be used to evaluate whether other, more threatened species have the capacity to adapt to this disease.

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 Hawai‘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 Hawai‘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 Hawai‘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.

Although West Nile Virus (WNV) has not been reported in Hawai'i, eventual introduction appears unavoidable with potential adverse effects on avian species. Nēnē (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 Nēnē and does not produce adverse biologic effects. Six captive, nonbreeding Nēnē were immunized with two 10-μg doses (4 wk apart) of the WN-80E recombinant protein adjuvanted with Montanide ISA720. Two Nēnē 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 Nēnē. 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.

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.

Margaret E. M. Farias et al..., Background Avipoxvirus sp. is a significant threat to endemic bird populations on several groups of islands worldwide, including Hawaìi, the Galapagos Islands, and the Canary Islands. Accurate identification and genotyping of Avipoxvirus is critical to the study of this disease and how it interacts with other pathogens, but currently available methods rely on invasive sampling of pox-like lesions and may be especially harmful in smaller birds. Methodology/Principal Findings Here, we present a nested TaqMan Real-Time PCR for the detection of the Avipoxvirus 4b core protein gene in archived blood samples from Hawaiian birds. The method was successful in amplifying Avipoxvirus DNA from packed blood cells of one of seven Hawaiian honeycreepers with confirmed Avipoxvirus infections and 13 of 28 Hawaìi `amakihi (Hemignathus virens) with suspected Avipoxvirus infections based on the presence of pox-like lesions. Mixed genotype infections have not previously been documented in Hawaìi but were observed in two individuals in this study. Conclusions/Significance We anticipate that this method will be applicable to other closely related strains of Avipoxvirus and will become an important and useful tool in global studies of the epidemiology of Avipoxvirus., Funding for this study was provided by: U.S. Geological Survey, Pacific Island Ecosystems Research Center (biology.usgs.gov/pierc/); U.S. Geological Survey Wildlife (biology.usgs.gov/wter/) and Invasive Species (biology.usgs.gov/invasive/) Programs; National Science Foundation (DEB0083944, www.nsf.gov); NIH/NCRR IDeA Networks of Biomedical Research Excellence (INBRE), P20RR016467 (http://www.ncrr.nih.gov/research_infrast​ructure/institutional_development_award/​idea_networks_of_biomedical_research_exc​ellence/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The native Hawaiian honeycreepers represent a classic example of adaptive radiation and speciation, but currently face one the highest extinction rates in the world. Although multiple factors have likely influenced the fate of Hawaiian birds, the relatively recent introduction of avian malaria is thought to be a major factor limiting honeycreeper distribution and abundance. We have initiated genetic analyses of class II beta chain Mhc genes in four species of honeycreepers using methods that eliminate the possibility of sequencing mosaic variants formed by cloning heteroduplexed polymerase chain reaction products. Phylogenetic analyses group the honeycreeper Mhc sequences into two distinct clusters. Variation within one cluster is high, with dNdS and levels of diversity similar to other studies of Mhc (B system) genes in birds. The second cluster is nearly invariant and includes sequences from honeycreepers (Fringillidae), a sparrow (Emberizidae) and a blackbird (Emberizidae). This highly conserved cluster appears reminiscent of the independently segregating Rfp-Y system of genes defined in chickens. The notion that balancing selection operates at the Mhc in the honeycreepers is supported by transpecies polymorphism and strikingly high dN/dS ratios at codons putatively involved in peptide interaction. Mitochondrial DNA control region sequences were invariant in the i'iwi, but were highly variable in the 'amakihi. By contrast, levels of variability of class II beta chain Mhc sequence codons that are hypothesized to be directly involved in peptide interactions appear comparable between i'iwi and 'amakihi. In the i'iwi, natural selection may have maintained variation within the Mhc, even in the face of what appears to a genetic bottleneck.

Background The relatively recent introduction of a highly efficient mosquito vector and an avian pathogen (Plasmodium relictum) to an isolated island ecosystem with naïve, highly susceptible avian hosts provides a unique opportunity to investigate evolution of virulence in a natural system. Mixed infections can significantly contribute to the uncertainty in host-pathogen dynamics with direct impacts on virulence. Toward further understanding of how host-parasite and parasite-parasite relationships may impact virulence, this study characterizes within-host diversity of malaria parasite populations based on genetic analysis of the trap (thrombospondin-related anonymous protein) gene in isolates originating from Hawaii, Maui and Kauai Islands. Methods A total of 397 clones were produced by nested PCR amplification and cloning of a 1664 bp fragment of the trap gene from two malarial isolates, K1 (Kauai) and KV115 (Hawaii) that have been used for experimental studies, and from additional isolates from wild birds on Kauai, Maui and Hawaii Islands. Diversity of clones was evaluated initially by RFLP-based screening, followed by complete sequencing of 33 selected clones. Results RFLP analysis of trap revealed a minimum of 28 distinct RFLP haplotypes among the 397 clones from 18 birds. Multiple trap haplotypes were detected in every bird evaluated, with an average of 5.9 haplotypes per bird. Overall diversity did not differ between the experimental isolates, however, a greater number of unique haplotypes were detected in K1 than in KV115. We detected high levels of clonal diversity with clear delineation between isolates K1 and KV115 in a haplotype network. The patterns of within-host haplotype clustering are consistent with the possibility of a clonal genetic structure and rapid within-host mutation after infection. Conclusion Avian malaria (P. relictum) and Avipoxvirus are the significant infectious diseases currently affecting the native Hawaiian avifauna. This study shows that clonal diversity of Hawaiian isolates of P. relictum is much higher than previously recognized. Mixed infections can significantly contribute to the uncertainty in host-pathogen dynamics with direct implications for host demographics, disease management strategies, and evolution of virulence. The results of this study indicate a widespread presence of multiple-genotype malaria infections with high clonal diversity in native birds of Hawaii, which when coupled with concurrent infection with Avipoxvirus, may significantly influence evolution of virulence. Reviewers This article was reviewed by Joseph Schall (nominated by Laura Landweber), Daniel Jeffares (nominated by Anthony Poole) and Susan Perkins (nominated by Eugene Koonin).

The application of molecular techniques to conservation genetics issues can provide important guidance criteria for management of endangered species. The results from this study establish that PCR-based approaches for sex determination developed in other bird species (Griffiths and Tiwari 1995; Griffiths et al. 1996, 1998; Ellegren 1996) can be applied with a high degree of confidence to at least four species of Hawaiian honeycreepers. This provides a rapid, reliable method with which population managers can optimize sex ratios within populations of endangered species that are subject to artificial manipulation through captive breeding programmes or geographic translocation.