Angiostrongylus cantonensis is the causative agent for neuroangiostrongyliasis (rat lungworm disease). While any slug or snail can serve as an intermediate host, the semi-slug Parmarion martensi is a highly efficient intermediate host in Hawai`i [1, 2, 3]. There have been several studies involving experimental infections with A. cantonensis in rabbits. These primarily involve the brain or central nervous system (CNS), as rabbits are considered non-permissive hosts and the larvae are not expected to leave the CNS [4]. Wang et al [5] conducted a long-term MRI study of the brains of rabbits infected with 200 A. cantonensis third-stage larvae (L3). Changes in the brain were monitored from day 0 to day 207 post-infection (PI). Lesions were first observed on day (D) 22 PI, and severe abnormalities were noted on D35 PI. They reported pathological changes in the brain tissues through D207 PI. Wang et al [6] conducted experimental infections on rabbits to evaluate the effects of albendazole treatment on pathological changes in the brain. Histopathological changes were observed in untreated rabbits infected with 400 A. cantonensis larvae between 21-28 days PI; however, data were not collected prior to D21 PI. Alicata [7] evaluated the effects of larval dosage in a study that included four groups of rabbits: groups 1-3 (n=4) received 100, 200, and 500 larvae respectively, and group 4 (n=2) received 1000 larvae. Animals in group 4 developed paralysis of the hind quarters within 24 hours and died D2 PI, while none of the other groups showed these early, severe neurological symptoms. Jindrak and Magnusson [8] also evaluated the effects of larval dose on symptom onset and severity in rabbits. One rabbit received a dose of 500 larvae and at D3 PI showed mild weakness in its left hind leg. Two other rabbits received a dose of 1000 larvae each and at D4 PI showed a slight heaviness of both hind legs. At a dose of 1500 larvae, another rabbit showed weakness in its hind legs at D3 PI, and by D6 PI showed paralysis in its right forelimb. Two other rabbits received 2000 larvae; one was paralyzed by D3 PI and the other was comatose and paralyzed by D5 PI. These studies suggest a positive correlation between larval dose and neurological symptoms in rabbits.
In humans, early diagnosis of neuroangiostrongyliasis is difficult due to the range of symptoms and variation in incubation rates [9]. In the USA, although symptomatology and exposure history are considered, confirmed diagnosis is primarily via PCR using an ITS1-based assay (AcanITS1) for the detection of A. cantonensis DNA in the cerebrospinal fluid (CSF) of humans [10, 11, 12]. Unfortunately for the patient, it takes time for the parasite to reach the CNS and thus become detectable by PCR [13], time during which neurological damage has likely already occurred. Using an AcanITS1 assay developed for qPCR [2], A. cantonensis DNA was detected in the peripheral blood of rats at various time points including very early PI [14]. Recent development of a novel qPCR assay (AcanR3990) based on tandem repeats in genomic DNA has been shown to be ~100-1,000 times more sensitive than the AcanITS1 assay [15]. In Sears et al. [15], the AcanR3990 assay detected A. cantonensis DNA in CSF, blood, and brain tissues from a horse exhibiting clinical symptoms of infection, whereas the AcanITS1 assay detected A. cantonensis DNA only in the brain. Here, we apply both the AcanITS1 and the AcanR3990 assays for the detection of A. cantonensis DNA in CSF, various blood products, and multiple organs from a domestic rabbit unintentionally infected through apparent consumption of contaminated local lettuce in a household in Hawai`i.
In the laboratory under sterile conditions, the shell and mantle of the P. martensi (semi-slug) were excised and discarded using sterile forceps and scalpel blade. The remainder of the slug (0.2128g) was placed in a 2.0 mL screw top vial with 500 uL DNA lysis buffer (0.1 M Tris HCl, 0.1 M EDTA, 2% SDS) containing 0.2 g 0.5 zirconia-silica beads (BioSpec Products, Bartlesville, OK, USA) and six 3.0 mm zirconia beads (OPS Diagnostics, Bridgewater, NJ, USA). An Omni Bead Rupter 24 Elite (Omni International, Kennesaw GA, USA) was used for 45 seconds at 8.0m/s to homogenize the tissue. A subsample of 50 µL of the resulting homogenate was used for DNA extraction using the DNeasy Blood & Tissue Kit (Qiagen) with the Tissue Protocol with one modification: 100 µL of elution buffer was used instead of the recommended 200 µL to increase the concentration of eluted DNA. The extraction was quantified by BioSpec-nano (Shimadzu Biotech) at 102.5 µg/µL DNA.
The P. martensi DNA sample was subjected to real-time PCR using a Custom TaqMan Gene Expression Assay (Life Technologies, Grand Island, NY) on a StepOne Plus RealTime PCR system (Life Technologies). The cycling conditions, primers (AcanITS1F1 and AcanITS1R1) and probe (AcanITS1P1) were based on the ITS1 gene region as described [11]. PCR reactions were carried out as described [2] with the modification of 50 cycles instead of 40, in 20 µL total volume, and included 0.25 µM probe, 0.9 µM forward and reverse primers and 1X TaqMan Environmental Master Mix 2.0 (Life Technologies). Another aliquot of the same DNA sample was used as template in quantitative PCR using the AcanR3990 assay as described with minor modification [15]. Briefly, qPCR was performed in 10 µL reactions containing 1X TaqMan Fast Advanced Master Mix (ThermoFisher Scientific, Waltham MA), and 5 µL template DNA. Thermocycling was performed on a StepOne system as above with cycling conditions of 95°C for 20 seconds, 40°C for 1 second, and 60°C for 20 seconds for 40 cycles. Negative controls using dH2O instead of DNA template were run simultaneously for all reactions.
One of the two rabbits exposed to the contaminated lettuce, a 4.5 year-old Lionhead neutered male, began exhibiting neurological symptoms (listlessness, progressing to hind limb ataxia and forelimb ataxia) consistent with eosinophilic meningitis three to six days after eating the contaminated lettuce. Exact date of exposure is not known, as the rabbit was fed the lettuce on two different days. Treatment was initiated 5-8 days after presumed exposure with an antibiotic (Baytril Flavor Tabs 22.7 mg/12 hrs), a steroid (PrednisOLONE Suspension 0.4mL/12 hours for 3D, then 0.4mL/24 hours for 3D), and the antiparasitic drug fenbendazole (Panacur 50 mg/ml/24 hours for 5D). The rabbit did not respond to treatment and was euthanized 8-11 days after presumed exposure (3D after initiation of treatment). A necropsy was performed and multiple tissue samples collected. Samples were transported to the lab on ice and stored at -80°C. DNA extraction was performed on subsections of the samples using a DNeasy Blood & Tissue Kit per Tissue Protocol for brain, heart, lung, and pulmonary artery tissue and per Blood Protocol for CSF, EDTA-treated whole blood, plasma, and buffy coat/RBC samples. Amplification of A. cantonensis DNA from tissues was carried out as described above using both the AcanITS1 and AcanR3990 assays. Samples were tested in separate PCR runs, using the same conditions in each assay. Negative controls using dH2O instead of DNA template were run simultaneously for all reactions.
The rabbit in this study developed symptoms between 3-6 days after presumed exposure, and was obtunded and euthanized 5 days after developing symptoms (8-11 days after presumed exposure). The PCR results of the P. martensi and O. cuniculus tissues tested are provided in Table 1. The negative controls for all reactions remained negative. The P. martensi sample was tested with the AcanITS1 PCR assay and found to contain significant amounts of A. cantonensis DNA with a relatively low CT value of 19.36. The tissues collected from the O. cuniculus necropsy were tested with both the AcanR3990 and the AcanITS1 PCR assays. 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. Additionally, among the six samples that amplified in replicate or triplicate with both sets of primers, the average CT value was significantly lower among the samples tested using AcanR3990 (x̅ CT = 23.69) as compared with the CT values of the same samples tested with AcanITS1 (x̅ CT = 31.69) (P = 0.024). The mean CT of the 11 samples that amplified with AcanR3990 but not AcanITS1 was relatively high (x̅ CT = 37.06), suggesting lower A. cantonensis DNA concentrations in those samples. The CT values of the samples that did not replicate were also relatively high (39.05-40.79). PCR results with a minimum of two replicates and a standard deviation (SD) of < 0.5 are graphically depicted in Figure 1.
Table 1
Results of PCR testing of the Parmarion martensi and multiple tissues from Oryctolagus cuniculus using the AcanR3990 and AcanITS1 PCR assays (0.25 threshold). The number of positive replicates (# Pos Reps), mean CT values and standard deviations (SD) are as indicated. R = right, L = left.
| PCR assay AcanR3990 | | PCR assay AcanITS1 |
Sample | PCR reaction | # Pos Reps | Mean CT | CT SD | | PCR reaction | # Pos Reps | Mean CT | CT SD |
P. martensi | | | | | | 279 | 3 | 19.36 | 0.062 |
O. cuniculus | | | | | | | | | |
R cranial lung | 12 | 2 | 38.75 | 5.78* | | 306 | 0 | | - |
L caudal lung | 12 | 2 | 39.33 | 1.205* | | 306 | 0 | | - |
R cranial lung | 12 | 2 | 39.64 | 0.333 | | 306 | 0 | | - |
Pulmonary artery | 12 | 1 | 39.05* | - | | 306 | 0 | | - |
R heart | 12 | 1 | 39.22* | - | | 306 | 0 | | - |
L heart | 12 | 0 | | - | | 306 | 0 | | - |
heart septum | 12 | 2 | 39.30 | 0.218 | | 306 | 0 | | - |
Brain 1 | 12 | 3 | 17.55 | 0.09 | | 306 | 3 | 24.99 | 0.018 |
Brain 2 | 12 | 2 | 35.04 | 0.387 | | 306 | 0 | | - |
Brain 3 | 12 | 3 | 26.45 | 0.113 | | 306 | 3 | 34.50 | 0.139 |
Brain 4 | 12 | 3 | 19.09 | 0.117 | | 306 | 3 | 26.61 | 0.023 |
Brain 5 | 12 | 3 | 32.58 | 0.336 | | 306 | 1 | 39.06* | - |
Brain 6 | 12 | 2 | 34.24 | 0.054 | | 306 | 0 | | - |
Brain 7 | 12 | 3 | 32.61 | 0.217 | | 306 | 0 | | - |
Brain 8 | 12 | 3 | 27.28 | 0.335 | | 306 | 3 | 36.03 | 0.458 |
Brain 9 | 12 | 3 | 22.20 | 0.073 | | 306 | 3 | 29.64 | 0.112 |
CSF | 12 | 3 | 29.59 | 0.182 | | 307 | 2 | 38.40 | 0.453 |
plasma | 4 | 2 | 36.77 | 0.344 | | 280 | 1 | 40.79* | - |
EDTA blood | 13 | 2 | 36.04 | 0.089 | | 280 | 0 | | - |
Buffy coat and RBCs | 13 | 2 | 43.35 | 4.133* | | 280 | 0 | | - |
*Data with SD > 0.5 or non-replicated results. | | | | | | |