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.

In vivo efficacy of pyrantel pamoate as a post-exposure prophylactic for rat lungworm (Angiostrongylus cantonensis).

Rat lungworm (Angiostrongylus cantonensis) is a neurotropic nematode, and the leading cause of eosinophilic meningitis worldwide. The parasite is usually contracted through ingestion of infected gastropods, often hidden in raw or partially cooked produce. Pharmaceutical grade pyrantel pamoate was evaluated as a post-exposure prophylactic against A. cantonensis. Pyrantel pamoate is readily available over-the-counter in most pharmacies in the USA and possesses anthelmintic activity exclusive to the gastrointestinal tract (GIT). Administering pyrantel pamoate immediately after exposure should theoretically paralyze the larvae in the GIT, causing the larvae to be expelled via peristalsis without entering the systemic circulation. In this study, pyrantel pamoate (11 mg/kg) was orally administered to experimentally infected rats at 0, 2-, 4-, 6-, or 8-h post-infection. The rats were euthanized six weeks post-infection, and worm burden was evaluated from the heart-lung complex. This is the first in vivo study to evaluate its efficacy against A. cantonensis. This study demonstrates that pyrantel pamoate can significantly reduce worm burden by 53-72% (P = 0.004), and thus likely reduce the severity of infection that is known to be associated with worm burden. This paralyzing effect of pyrantel pamoate on the parasite may also be beneficial for delaying the establishment of infection until a more suitable anthelmintic such as albendazole is made available to the patient.

Oral Rabies Vaccination of Small Indian Mongooses (Urva auropunctata) with ONRAB via Ultralite Baits.

The Ontario Rabies Vaccine (ONRAB) is a human adenovirus rabies glycoprotein recombinant oral vaccine immunogenic for small Indian mongooses when delivered by direct instillation into the oral cavity. We offered Ultralite baits containing ~1.8 mL 109.5 TCID50 ONRAB oral rabies vaccine to 18 mongooses, while 6 mongooses were offered identical baits in placebo form. We collected sera from individual mongooses at days 0, 14 and 30 post vaccination (pv) and quantified rabies virus neutralizing antibodies (RVNA) using the rapid fluorescent focus inhibition test, with titers greater than or equal to 0.1 IU/mL considered positive. All study subjects were RVNA negative prior to bait offering. Bait consumption was variable: all 6 sham and 13 of 18 (72%) treatment animals consumed/punctured the baits offered. By day 30 pv, RVNA were detected among 11 of 13 (84.6%) of treatment mongooses that consumed/punctured baits, whereas sham-vaccinated mongooses remained RVNA negative throughout the study. We conclude ONRAB is immunogenic for mongooses by Ultralite bait delivery, although the bait design may need further optimization.

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.

Immunogenicity of Ontario Rabies Vaccine for Small Indian Mongooses (Herpestes auropunctatus).

Oral rabies vaccination is the principal strategy used to control rabies in wildlife. No oral rabies vaccine is licensed for small Indian mongooses (Herpestes auropunctatus). The Ontario Rabies Vaccine Bait (ONRAB) is a human adenovirus type-5 rabies glycoprotein recombinant vaccine licensed for rabies control in striped skunks (Mephitis mephitis) in Canada and is under experimental evaluation in the US. We evaluated varying doses of ONRAB vaccine by direct instillation into the oral cavity with three groups of 10 mongooses: Group 1 received 109.5 TCID50, group 2 received 108.8 TCID50, and group 3 received 108.5 TCID50 of vaccine. Six control mongooses were sham-vaccinated with culture media. We collected a serum sample prior to vaccination and on days 14 and 30 postvaccination (PV). We quantified the level of rabies virus neutralizing antibodies (RVNA) from mongoose sera and compared titers among vaccinated groups and time points PV, where values greater than or equal to 0.1 IU/mL were considered positive. On day 14 PV, 87% (26 of 30, 95% confidence interval 70-95%) of vaccinates had seroconverted, whereas all vaccinates demonstrated RVNA by day 30 PV. There was a marginal effect of vaccine dose on group means of log-transformed RVNA titers at day 14 PV (F=2.5, P=0.099), but not day 30 PV. Sham-vaccinated animals were seronegative during all time points.

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.

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.

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose (Herpestes Auropunctatus) Sera for Use as an Oral Rabies Vaccination Biological Marker.

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases reported annually. The control of RABV circulation in wildlife reservoirs is typically accomplished by a strategy of oral rabies vaccination (ORV). Currently no wildlife ORV program exists in Puerto Rico. Research into oral rabies vaccines and various bait types for mongooses has been conducted with promising results. Monitoring the success of ORV relies on estimating bait uptake by target species, which typically involves evaluating a change in RABV neutralizing antibodies (RVNA) post vaccination. This strategy may be difficult to interpret in areas with an active wildlife ORV program or in areas where RABV is enzootic and background levels of RVNA are present in reservoir species. In such situations, a biomarker incorporated with the vaccine or the bait matrix may be useful. We offered 16 captive mongooses placebo ORV baits containing ethyl-iophenoxic acid (et-IPA) in concentrations of 0.4% and 1% inside the bait and 0.14% in the external bait matrix. We also offered 12 captive mongooses ORV baits containing methyl-iophenoxic acid (me-IPA) in concentrations of 0.035%, 0.07% and 0.14% in the external bait matrix. We collected a serum sample prior to bait offering and then weekly for up to eight weeks post offering. We extracted Iophenoxic acids from sera into acetonitrile and quantified using liquid chromatography/mass spectrometry. We analyzed sera for et-IPA or me-IPA by liquid chromatography-mass spectrometry. We found adequate marking ability for at least eight and four weeks for et- and me-IPA, respectively. Both IPA derivatives could be suitable for field evaluation of ORV bait uptake in mongooses. Due to the longevity of the marker in mongoose sera, care must be taken to not confound results by using the same IPA derivative during consecutive evaluations.

Predator water balance alters intraguild predation in a streamside food web.

Previous work suggests that animal water balance can influence trophic interactions, with predators increasing their consumption of water-laden prey to meet water demands. But it is unclear how the need for water interacts with the need for energy to drive trophic interactions under shifting conditions. Using manipulative field experiments, we show that water balance influences the effects of top predators on prey with contrasting ratios of water and energy, altering the frequency of intraguild predation. Water-stressed top predators (large spiders) negatively affect water-laden basal prey (crickets), especially male prey with higher water content, whereas alleviation of water limitation causes top predators to switch to negatively affecting energy-rich midlevel predators (small spiders). Thus, the relative water and energy content of multiple prey, combined with the water demand of the top predator, influences trophic interactions in ways that can alter the strength of intraguild predation. These findings underscore the need for integration of multiresource approaches for understanding implications of global change for food webs.