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.

Determination of residue levels of rodenticide in rodent livers offered novel diphacinone baits by liquid chromatography-tandem mass spectrometry.

A specific and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the determination of the anticoagulant rodenticide diphacinone (DPN) in mouse and rat liver. Tissue samples were extracted with a mixture of water and acetonitrile containing ammonium hydroxide. The extracted sample was cleaned up with a combination of liquid-liquid partitioning and dispersive solid phase extraction. Chromatographic separation was achieved using a Waters X-Bridge BEH C-18 LC column (50 mm, 2.1 mm ID, 2.5 µm particle size) with detection on a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode. The monitored transition for DPN was m/z 339.0 → 167.0 for quantitation and 339.0 → 172.0 and 339.0 → 116.0 for confirmation. The linear range was 0.5 to 375 ng/mL. The average precision of DPN, represented by the relative standard deviation of the observed concentrations, was 7.2% (range = 0.97% - 20.4%) and the average accuracy, represented by the relative error, was 5.8% (range = 1.06% - 14.7%). The recovery of DPN fortified at 3 different levels averaged 106% in rat liver and 101% in mouse liver. The established method was successfully used to determine DPN residue levels in Polynesian rats (Rattus exulans) and mice (Mus musculus) fed two different formulated baits containing DPN. The observed residue levels were consistent with values observed in other rodent studies. However, the amount of bait consumed was lower for the novel baits evaluated in this study.

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.

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.

Efficacy of rodenticide baits for the control of three invasive rodent species in Hawaii.

We tested the efficacy and palatability of nine commercial rodenticide bait formulations on Polynesian rats (Rattus exulans), roof rats (R. rattus), and house mice (Mus musculus). Efficacy varied by rodenticide tested and rodent species. Generally, rodenticides were more effective against mice than for either of the rat species, and mice tended to consume more rodenticide bait than the laboratory chow alternative food. Efficacy was generally highest for the second-generation anticoagulants tested; however, this varied across products and one-first-generation rodenticide had similar effectiveness. Bait acceptance (palatability) also varied both by rodenticide and by rodent species. Acceptance was the lowest for the acute rodenticides. Bait acceptance appeared to substantially affect the efficacy of rodenticides; materials that were not well accepted produced lower mortality rates. Rodenticide products currently registered for use in Hawaii performed less effectively in this study than other available products not yet registered. Although markets for rodent control products for use on islands are limited, there are advantages to having additional products registered for island use in agriculture, conservation, and public health.

A comparison of plotless density estimators using Monte Carlo simulation on totally enumerated field data sets.

BACKGROUND: Plotless density estimators are those that are based on distance measures rather than counts per unit area (quadrats or plots) to estimate the density of some usually stationary event, e.g. burrow openings, damage to plant stems, etc. These estimators typically use distance measures between events and from random points to events to derive an estimate of density. The error and bias of these estimators for the various spatial patterns found in nature have been examined using simulated populations only. In this study we investigated eight plotless density estimators to determine which were robust across a wide range of data sets from fully mapped field sites. They covered a wide range of situations including animal damage to rice and corn, nest locations, active rodent burrows and distribution of plants. Monte Carlo simulations were applied to sample the data sets, and in all cases the error of the estimate (measured as relative root mean square error) was reduced with increasing sample size. The method of calculation and ease of use in the field were also used to judge the usefulness of the estimator. Estimators were evaluated in their original published forms, although the variable area transect (VAT) and ordered distance methods have been the subjects of optimization studies. RESULTS: An estimator that was a compound of three basic distance estimators was found to be robust across all spatial patterns for sample sizes of 25 or greater. The same field methodology can be used either with the basic distance formula or the formula used with the Kendall-Moran estimator in which case a reduction in error may be gained for sample sizes less than 25, however, there is no improvement for larger sample sizes. The variable area transect (VAT) method performed moderately well, is easy to use in the field, and its calculations easy to undertake. CONCLUSION: Plotless density estimators can provide an estimate of density in situations where it would not be practical to layout a plot or quadrat and can in many cases reduce the workload in the field.

Determination of diphacinone residues in Hawaiian invertebrates.

A reversed-phase ion-pair liquid chromatographic analysis combined with a solid-phase extraction clean-up method is used to assess the quantity of diphacinone residue found in invertebrates. Three invertebrate species are exposed to commercially available diphacinone-fortified bait used for rat control. The invertebrate samples are collected, frozen, and shipped to the laboratory. The samples are homogenized after cryogenic freezing. A portion of the homogenized samples are extracted with acidified chloroform-acetone, followed by cleanup with a silica solid-phase extraction column. Diphacinone is detected by UV absorption at 325 nm after separation by the chromatographic system. The method limit of detection (MLOD) for snail and slug samples averaged 0.055 and 0.066 mg/kg, respectively. Diphacinone residues in snail tissue ranges from 0.83 to 2.5 mg/kg for Oxychilus spp. The mean recoveries from snails at 0.20 and 2.0 are 97 +/- 21% and 84 +/- 6%. Diphacinone residues in slug tissue ranges from 1.3 to 4.0 mg/kg for Deroceras laeve and < MLOD to 1.8 mg/kg for Limax maximus, respectively. The mean recoveries from slugs at 0.20 and 2.0 mg/kg are 91% +/- 15% and 86% +/- 5%.

Probabilistic risk assessment for snails, slugs, and endangered honeycreepers in diphacinone rodenticide baited areas on Hawaii, USA.

Three probabilistic models were developed for characterizing the risk of mortality and subacute coagulopathy to Poouli, an endangered nontarget avian species, in broadcast diphacinone-baited areas on Hawaii, USA. For single-day exposure, the risk of Poouli mortality approaches 0. For 5-d exposure, the mean probability of mortality increased to 3% for adult and 8% for juvenile Poouli populations. For Poouli that consume snails containing diphacinone residues for 14 d, the model predicted increased levels of coagulopathy for 0.42 and 11% of adult and juvenile Poouli populations, respectively. Worst-case deterministic risk characterizations predicted acceptable levels of risk for nonthreatened or endangered species such as northern bobwhite quail and mallards. Also, no acute toxicity was noted for snails and slugs that feed on diphacinone baits.