Background: Hawaii is the hotspot for rat lungworm disease (angiostrongyliasis) caused by the nematode Angiostrongylus cantonensis in the USA. In humans, PCR of the CSF is typically used for diagnosis, however, collection of CSF requires hospitalization. Here, we evaluate the efficacy of two different PCR tests to detect A.cantonensis DNA in multiple tissues including blood from a rabbit presumably infected by eating contaminated lettuce. Methods: Two different PCR assays (AcanR3990, and AcanITS1) were used comparatively to test DNA extracted from slug and rabbit tissues. Assays were conducted using established protocols and were run in triplicate, with negative (dH 2 0) controls included throughout. Results: A juvenile Parmarian martensi (semi-slug) found in local lettuce tested positive for the presence of Angiostrongylus cantonensis DNA. A family and their two domestic rabbits ( Oryctolagus cuniculus ) consumed this lettuce twice within the five days preceding testing. One rabbit exhibited symptoms consistent with eosinophilic meningitis 3-6 days after being fed the lettuce. Appropriate veterinary treatment was ineffective and the rabbit was subsequently euthanized. This study comparatively applies two different PCR assays to detect A. cantonensis DNA in the peripheral blood, cerebrospinal fluid, brain, heart, and lung tissue of this rabbit, and provides data implicating parasite transmission via contaminated home-grown lettuce. Six of the nine brain DNA samples, as well as the CSF sample, tested positive in replicate or triplicate for A. cantonensis DNA with both PCR assays. The AcanR3990 assay also detected A. cantonensis DNA from the lung, heart septum, all nine samples from the brain, and blood products (plasma, EDTA-treated whole blood, and buffy coat/red blood cells) in replicate or triplicate.

AbstractAngiostrongylus 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.

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 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 functional mechanism of anti-merozoite antibodies.Antibodies to merozoite surface proteins can mediate several eff ector mechanisms, including complement fi xation due to cytophillic antibodies that result in merozoite lysis of C3b opsonization; inhibition of merozoite invasion into the RBC; phagocytosis of IgG-opsonized merozoites; production of reactive oxygen species (ROS) or Nitric oxide (NO) in response to opsonized parasites

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 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.

Cold storage is used to preserve fruit quality after harvest during transportation in marketing channels. Low temperature can be a stressor for insects that reduces survivorship, and cold storage may contribute to the efficacy of postharvest quarantine treatments such as irradiation against quarantine insect pests. The combined effect of irradiation and cold storage was examined in a radiation-tolerant fruit fly, Bactrocera cucurbitae Coquillet (melon fly), and a radiation-intolerant fruit fly, Ceratitis capitata (Wiedemann) (Mediterranean fruit fly) (Diptera: Tephritidae). Third instars on diet or in papaya were treated with a sublethal radiation dose of 30 Gy and stored at 4 or 11 degrees C for 3-13 d and held for adult emergence. For both fruit fly species, survival of third instars to the adult stage generally decreased with increasing cold storage duration at 4 or 11 degrees C in diet or papaya. Survivorship differences were highly significant for the effects of substrate (diet > papaya), temperature (11 > 4 degrees C), and irradiation (0 > 30 Gy). Few Mediterranean fruit flies survived in any cold storage treatment after receiving a radiation dose of 30 Gy. No melon fly larvae survived to the adult stage after irradiation and 11 d cold storage at 4 or 11 degrees C in papayas. Cold storage enhances the efficacy and widens the margin of security in postharvest irradiation treatments. Potentially irradiation and cold storage can be used in combination to reduce the irradiation exposure requirements of quarantine treatments.

Irradiation is a quarantine treatment option for stored products pests. Dose response tests were conducted to identify a post-harvest radiation treatment that would control rice weevil, Sitophilus oryzae (L.) (Coleoptera: Curculionidae) in rice. Rice infested with adult or immature weevils was treated at radiation doses of 30, 60, 90, or 120 Gy, or left untreated as a control. Live and dead beetles were counted weekly for 15-24 weeks. Treatment of adult weevils at a radiation dose of 120 Gy resulted in no live adults after two weeks, indicating that this radiation dose caused adult mortality and sterility, whereas a total of 1261 adult beetles emerged during 24 weeks in the untreated controls. Treatment of immature life stages (a mixture of eggs, larvae and pupae) with a radiation dose of 90 or 120 Gy resulted in no adults emerging after five or two weeks, respectively, indicating that these doses prevented reproduction, whereas a total of 4275 adults emerged throughout 15 weeks in the untreated controls. Weight loss of rice infested with immature or adult weevils was significantly reduced by irradiation treatment at 60 Gy and 120 Gy. In a large-scale confirmatory test, a radiation dose of 120 Gy applied to 38,025 adult weevils in rice resulted in no reproduction. Irradiation at 120 Gy will provide quarantine security for rice weevil, and prevent post-irradiation weight loss caused by insect feeding in the commodity. Irradiation may be particularly helpful in controlling phosphine-resistant populations, and could help manage resistance by preventing the spread of resistant weevils in exported grains. Published by Elsevier Ltd.

The effects of irradiation on egg, larval, and pupal development, and adult reproduction in light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae),were examined. Eggs, neonates, third instars, fifth instars, and early stage pupae were irradiated at target doses of 60, 90, 120, or 150 Gy or left untreated as controls in replicated factorial experiments and survival to the adult stage was recorded. Tolerance to radiation generally increased with increasing age and developmental stage. A radiation dose of 120 Gy applied to eggs and neonates prevented adult emergence. A dose of 150 Gy prevented adult emergence in larvae at all stages. In large-scale validation tests, a radiation dose of 150 Gy applied to fifth instars in diet, apples or peppers resulted in no survival to the adult stage in 37,947 treated individuals. Pupae were more radio tolerant than larvae, and late stage pupae were more tolerant than early stage pupae. Radiation treatment of late pupae at 350 and 400 Gy resulted in three and one fertile eggs in 4,962 and 4,205 total eggs laid by 148 and 289 mating pairs, respectively. For most commodities, the fifth instar is the most radio tolerant life stage likely to occur with the commodity; a minimum radiation dose of 150 Gy will prevent adult emergence from this stage and meets the zero tolerance requirement for market access. For traded commodities such as table grapes that may contain E. postvittana pupae, a radiation dose > 400 Gy may be necessary to completely sterilize emerging adults. After review of the literature, a generic radiation treatment of 250 Gy is proposed for tortricid eggs and larvae in regulated commodities.

Trail pheromone disruption of invasive ants is a novel tactic that builds on the development of pheromone-based pest management in other insects. Argentine ant trail pheromone, ( Z )-9-hexadecenal, was formulated as a micro-encapsulated sprayable particle and applied against Argentine ant populations in 400 m 2 field plots in Hawai‘i Volcanoes National Park. A widely dispersed point source strategy for trail pheromone disruption was used. Traffic rates of ants in bioassays of treated filter paper, protected from rainfall and sunlight, indicated the presence of behaviorally significant quantities of pheromone being released from the formulation for up to 59 days. The proportion of plots, under trade wind conditions (2–3 m s −1 ), with visible trails was reduced for up to 14 days following treatment, and the number of foraging ants at randomly placed tuna-bait cards was similarly reduced. The success of these trail pheromone disruption trials in a natural ecosystem highlights the potential of this method for control of invasive ant species in this and other environments.

BACKGROUND:Dual epidemics of the malaria parasite Plasmodium and HIV-1 in sub-Saharan Africa and Asia present a significant risk for co-infection in these overlapping endemic regions. Recent studies of HIV/Plasmodium falciparum co-infection have reported significant interactions of these pathogens, including more rapid CD4+ T cell loss, increased viral load, increased immunosuppression, and increased episodes of clinical malaria. Here, we describe a novel rhesus macaque model for co-infection that supports and expands upon findings in human co-infection studies and can be used to identify interactions between these two pathogens.METHODOLOGY/PRINCIPAL FINDINGS:Five rhesus macaques were infected with P. cynomolgi and, following three parasite relapses, with SIV. Compared to macaques infected with SIV alone, co-infected animals had, as a group, decreased survival time and more rapid declines in markers for SIV progression, including peripheral CD4+ T cells and CD4+/CD8+ T cell ratios. The naïve CD4+ T cell pool of the co-infected animals was depleted more rapidly than animals infected with SIV alone. The co-infected animals also failed to generate proliferative responses to parasitemia by CD4+ and CD8+ T cells as well as B cells while also having a less robust anti-parasite and altered anti-SIV antibody response.CONCLUSIONS/SIGNIFICANCE:These data suggest that infection with both SIV and Plasmodium enhances SIV-induced disease progression and impairs the anti-Plasmodium immune response. These data support findings in HIV/Plasmodium co-infection studies. This animal model can be used to further define impacts of lentivirus and Plasmodium co-infection and guide public health and therapeutic interventions.

Species within the cockroach genus Parcoblatta are sexually dimorphic for wing length; females have reduced wings and are flightless, while males have long wings that are used in flight. We predicted that Parcoblatta females would release a volatile sex pheromone to attract the more mobile males. Nymphs of the broad wood cockroach, P. lata, and the Caudell's wood cockroach, P. caudelli, were collected in forested areas in North Carolina, USA, and reared in the laboratory for observations of sexual behavior and for pheromone analysis. After several days of sexual maturation, virgin females of both species exhibited distinct calling behaviors. In females of P. lata, calling commenced 6 days after adult emergence. Under a light–dark photoperiod regime, calling behavior in both species was restricted to the scotophase. Calling consisted of a repeated pattern of raising and lowering the abdomen with occasional exposure of the genital vestibulum. To test whether calling behavior is associated with the release of pheromone, volatiles from calling and noncalling females were collected on Super-Q and tested by electroantennogram (EAG) and behavioral assays. Volatile collections from calling females elicited higher male-specific EAG responses than collections from noncalling females of the same physiological stage. In an olfactometer choice test (Y-tube), males preferred volatiles from calling females over those from noncalling females. To determine the anatomical source of the pheromone, solvent extracts of various body parts were analyzed by EAG. The first through seventh tergites were the only body parts that elicited male-specific EAG responses in both species. In P. lata, the activity of the extract increased from 1- to 7-day-old females, but was lower in mated than in virgin females of the same age. The putative pheromone gland appears to consist of numerous class-3 secretory units, each composed of a secretory cell connected to a cuticular pore via a tubular duct. We conclude that female P. lata and P. caudelli produce sex-specific volatile pheromones that are emitted during calling behavior.