Detection of Rat Lungworm in Intermediate, Definitive, and Paratenic Hosts Obtained from Environmental Sources

Abstract

Angiostrongylus cantonensis is the most common parasite causing human eosinophilic meningitis worldwide. The geographical distribution of this disease has changed dramatically in the last few decades. Various methods have been used to detect A. cantonensis in host animals around the world. A survey of mollusks collected on the island of Hawai‘i in 2005 using PCR showed an infection rate of 24–78% depending on the mollusk species. In this study, samples from intermediate, definitive, and paratenic hosts were analyzed to further determine the presence of A. cantonensis in the United States. All samples were from Hawai‘i, except for the apple snails (Pomacea maculata) that were collected in New Orleans, Louisiana. Angiostrongylus cantonensis was detected in the majority of species examined, including the apple snails from New Orleans and flatworms (planarians) from Hawai‘i. Among the mollusks examined, the semi-slug Parmarion martensi had the highest parasite load, with an average larval burden of 445 larvae in 25 mg of tissue, as estimated by real-time PCR. In contrast, slime excreted from these highly infected mollusks contained no or very little A. cantonensis DNA. Analysis of definitive hosts (Rattus spp.) showed discrepancies between morphological and PCR-based identification; 54% of the rats were positive based on morphology, while 100% of tissue samples from these animals were positive by real-time PCR. This indicates that necropsies of rodents could underestimate the infection rates in definitive hosts of A. cantonensis.

Introduction

From its original range in southeastern China, A. cantonensis spread throughout many tropical and sub-tropical regions of the world during the 20th century.¹ It now occurs in Southeast Asia, many Pacific Islands, Australia, India, Sri Lanka, the state of Louisiana in the United States, the Canary Islands, and some countries in the Caribbean, South and Central America, and Africa.^2–9 This rapid geographical spread has coincided with globalization and has probably been facilitated by the unintentional transport of infected host animals in cargo ships and planes.¹ Once introduced into a new area, the parasite may easily establish itself in the local fauna since a large number of mollusk species can act as intermediate hosts and rats are ubiquitous. Thus, it is plausible that A. cantonensis will continue to spread to new regions, especially coastal cities, increasing the risk for cerebral angiostrongyliasis in humans. Isolated cases of A. cantonensis eosinophilic meningitis have been reported in captive animals in Mississippi and Florida but since there have been no surveys of rodents and mollusks in those areas it is unclear to what extent the nematode has spread into these states.^10,11 Ascertaining the geographic presence of the parasite is an important public health need to prevent additional cases of the disease and to inform diagnosis of patients with eosinophilic meningitis. This report gives an overview of common detection methods and their use to detect A. cantonensis in biological samples obtained from environmental sources. It also presents data from surveys of intermediate (mollusks), definitive (rats) and paratenic (flatworm) hosts, mainly collected in Hawai‘i.

Morphology-based Detection Methods

The ultimate evidence for occurrence is to find adult worms infecting the local rodent population. Traditionally, this is done by removal of adult worms from the pulmonary arteries of rodents during a careful necropsy and identification of species-specific morphological features.¹² Alternatively, formalin-fixed tissues from the heart, lungs, and brain can be used to prepare hematoxylin and eosin stained microscopy slides.³ For situations in which trapping and killing rodents is not desirable, A. cantonensis infection of rodents can be inferred by detecting first-stage larvae (L1) in animal feces.¹³ Isolation of L1 from fecal material can be accomplished by soaking the feces in water and collecting the migrating larvae using a Baermann apparatus.¹² Detection of A. cantonensis in intermediate and paratenic hosts can be performed by digesting their tissues with a HCl-pepsin solution and identifying the released third-stage larvae (L3).¹⁴ However, this procedure requires access to live animals so it must be performed shortly after sample collection.

Morphological identification of A. cantonensis in host animals has two general disadvantages. First, it is time- and labor-consuming and requires highly trained parasitologists with skills to recognize the parasite's diagnostic features. Second, identification to the species level can only be done on adult male worms with intact posterior ends. Larval stages may display features allowing identification to the family or genus level, depending on stage and isolation technique. If detailed identification is required from larval stages, they can be passed through appropriate laboratory animals to develop into adult worms. Thus, L1 isolated from rat droppings can be used to infect mollusks so that they reach the L3 stage,^15–17 which can be used to infect either mice or rats, to produce immature adults developing in the mouse brain^15,18 or fully mature adult worms in the rat pulmonary arteries.^{15,17,19–21}

A rapid method to detect A. cantonensis in large snails such as Pomacea canaliculata and Achatina fulica is to perform a visual inspection of the snail lung tissue.^22,23 The lungs are removed from the snail body, spread open and examined under a dissecting microscope for the characteristic nodules containing the L3 larvae.

Molecular DNA-based Detection Methods

Molecular detection using the polymerase chain reaction (PCR) has been used to circumvent the problems associated with morphological identification of Angiostrongylus worms. Genomic DNA suitable for PCR detection can be extracted from various types of material, including intact worms in all developmental stages, tissues from intermediate, definitive and paratenic hosts, and rat droppings. The first published PCR methods for A. cantonensis detection used broadly reactive PCR primers to amplify a genetic region from nematode worms, followed by either restriction length fragment polymorphism analysis or DNA sequencing to provide a species-specific identification. These methods targeted the small subunit ribosomal gene (SSU rRNA),^24,25 the internal transcribed spacer 2 (ITS2),²⁶ or the mitochondrial cytochrome oxidase I gene (CO1).²⁶ As DNA sequencing data became available from more Angiostrongylus species it has been possible to design species-specific PCR assays. Thus, an A. cantonensis-specific real-time PCR assay amplifying the ITS1 region detects only this species in infected mollusks and slime trails.²⁷ Another real-time PCR assay based on species-specific regions of ITS2 was developed to detect A. cantonensis L1 in rat feces.²⁸ More recently, the loop-mediated isothermal DNA amplification (LAMP) technique has been used to detect A. cantonensis in invasive snail species in China.^29,30 The main advantage compared to PCR is that the LAMP technique does not require special equipment; amplification of the target DNA can be performed in a water bath or heat block and the end-point analysis can be achieved by visual inspection of turbidity directly in the reaction tube. This makes LAMP very suitable for field studies.

Quantitative Assessment of Larval Burden in Intermediate and Paratenic Hosts

The ITS1 real-time PCR assay²⁷ was used to assess the larval burden in intermediate and paratenic hosts. In order to create a standard curve to use in the quantification, L3 larvae were obtained from naturally infected Parmarion martensi semi-slugs from the island of Hawai‘i by pepsin digestion. The larvae were counted under a dissecting microscope and transferred to new tubes, so that the final numbers of larvae were 1, 5, 10, 100, 500, and 1000 per tube. Figure 1 shows a standard curve obtained by plotting the Ct values from the samples containing known number of larvae against the larvae counts. To test the ability of the standard curve to correctly estimate larval burden, foot tissue from two infected slugs was analyzed both morphologically (counting the larvae released after pepsin treatment) and by quantitative real-time PCR (using the standard curve developed). One slug contained 237 larvae based on the microscopic evaluation and 476 larvae estimated from the real-time PCR results, while the corresponding values for the other slug were 14 and 11 larvae. The difference in counts between the two methods could be due to uneven distribution of larvae in the tissue and the fact that real-time PCR detected DNA from different stages, including dead worms, while only live L3 larvae were included in the morphological method.

Figure 1.

Standard curve for the quantitative estimation of larval burdens. The threshold cycle (Ct) values obtained from samples containing known numbers of larvae were plotted against the larval counts. The regression line has a slope of −3.302, corresponding to a PCR amplification efficiency of 101%, and a coefficient of determination R2 of 0.987.

The standard curve was then used to estimate the larval burden in mollusks and flatworms from Hawai‘i and New Orleans. Most of these samples were screened for A. cantonensis in previous studies and have thus been described in detail elsewhere.^25,27,31 Locations of collection sites in Hawai‘i are shown in Figure 2; details of those in New Orleans are given by Teem, et al.³⁴ In this study, 79% of P. martensi semi-slugs were infected with A. cantonensis. It seems that P. martensi is a common host of A. cantonensis; recent surveys found a 78% infection rate in Hawai‘i and 20% in Japan.^23,31 Furthermore, quantification by real-time PCR indicated that P. martensi had much higher parasite loads than the other species examined, with an average burden of 445 larvae in 25 mg of tissue, compared to 1 to 205 larvae for other species (Table 1). In 17% (13 of 77) of the positive P. martensi samples, the real-time PCR produced a Ct value that was outside the range covered in the standard curve, indicating that this species frequently contained more than 1,000 larvae in 25 mg of tissue. This high parasite load means that even a relatively small piece might contain enough infective larvae to cause severe infection if accidentally ingested. Although high percentages of some other molluscan species were positive for A. cantonensis, none had estimated parasite loads as high as P. martensi (Table 1).

Figure 2.

Location of collection sites for intermediate and paratenic hosts on the islands of O‘ahu (top) and Hawai‘i (bottom). Open circles: collection sites for mollusks only in 2005–2011. Closed circles: collection sites for flatworms and mollusks in 2009 and 2011.

Table 1.

Quantitative real-time PCR estimations of larval burden in environmental samples.

Sample	Origin	Number of positives (%)	Estimated larval burden in 25 mg tissuea
			Average	Median	Maximum
Parmarion martensi (n = 97)	Hawai‘i Island, Hawai‘i	77 (79)	445	110	>1,000
Veronicella cubensis (n = 71)	Hawai‘i Island, Hawai‘i	27 (38)	35	<1	468
Pallifera sp. (n = 5)	Hawai‘i Island, Hawai‘i	2 (40)	<1	<1	<1
Laevicaulis alte (n = 5)	Hawai‘i Island, Hawai‘i	4 (80)	205	2	819
Achatina fulica (n = 8)	Hawai‘i Island, Hawai‘i	7 (88)	5	1	18
Achatina fulica (n = 9)	O‘ahu, Hawai‘i	5 (56)	7	3	25
Other mollusksb (n = 14)	Hawai‘i Island, Hawai‘i	6 (43)	<1	<1	<1
slime from infected P. martensi (n = 13)	Hawai‘i Island, Hawai‘i	1 (8)	-	-	<1
Flatworms (planarians) (n=12)	Hawai‘i Island, Hawai‘i	8 (67)	4	<1	30
Pomacea maculata (n=31)	New Orleans, Louisiana	5 (16)	19	2	71

^aValues lower or higher than the range included in the standard curve are presented as <1 or >1,000, respectively.

^bIncluded three Euglandina rosea, three Ovachlamys fulgens, one Bradybaena similaris, and seven unidentified mollusks; two of the O. fulgens and four of the unidentified mollusks were positive for A. cantonensis by PCR.

It has been debated whether the slime trail from infected mollusks could transfer infective L3 larvae to humans if ingested. The real-time PCR assay gave a positive result on the slime from one of 13 naturally infected P. martensi; however, quantification using the standard curve indicated that this slime contained DNA corresponding to much less than one intact worm. This is in agreement with previous findings that slime from infected mollusks seemed to contain no or few larvae under normal circumstances, even when the mollusks themselves had a high parasite burden.^16,32 Nevertheless, more studies are needed to clarify the role of mollusk slime in human disease transmission, especially in regards to different environmental conditions.

The finding of Pomacea maculata (formerly known as Pomacea insularum)³³ from New Orleans infected with A. cantonensis concurs with previous reports about the potential for angiostrongyliasis transmission in this area.^5,16 These preliminary data prompted a more detailed assessment of the distribution of A. cantonensis in P. maculata populations in other states in the Southeastern United States, results of which are reported by Teem, et al.³⁴

Flatworms (planarians) from Hawai‘i were also included in this study. Eight out of twelve flatworms tested positive for A. cantonensis, with larval burdens estimated by real-time PCR of up to 30 larvae in 25 mg of tissue. Although the flatworms were not identified to species, at least one of them was almost certainly Platydemus manokwari, a species that feeds on slugs and snails and that has been widely disseminated across the Pacific in ecologically ill-considered attempts to control populations of the giant African snail.³⁵ Predatory flatworms that ingest infected mollusks are known to be paratenic (carrier) hosts of A. cantonensis.²³ In Okinawa, P. manokwari was suspected to be an important source of infection for humans because they hide in leafy vegetables and can contaminate fresh salads with larvae of A. cantonensis if these flatworms are sliced open during food preparation.

No other paratenic hosts were included in this study. Many species of amphibians, fish, and crustaceans can act as paratenic hosts for infective L3 and pose a threat to human health if ingested raw or undercooked. In Thailand several cases of angiostrongyliasis were attributed to the consumption of yellow tree monitor lizards, with A. cantonensis larvae in 21 out of 22 lizards collected from the wild in five provinces.³⁶ Through careful dissection of various organs it was determined that the liver was the main organ where L3s were found in these lizards. Species of amphibians have been surveyed in New Caledonia and Japan and found to be paratenic hosts for A. cantonensis.^23,37 A recent national survey in China looked for L3 in various paratenic hosts from markets and restaurants, including frogs, shrimps, crabs, toads, and fish but all samples were negative for A. cantonensis.³⁸

Detection in Rats in Hawai‘i

Rats were trapped at six sites on the island of Hawai‘i (Figure 3). Three species of rats were trapped: 26 Rattus rattus, 10 R. exulans, and one R. norvegicus. The arteries of their lungs and hearts were examined for the presence of adult A. cantonensis worms, and if present they were removed and counted. The remaining lung tissues were saved for real-time PCR detection. The majority, 20 out of 37 (54%), of the rats were positive for adult A. cantonensis worms: 14 of the R. rattus, 5 of the R. exulans, and the single R. norvegicus. The infection rate in rats in other countries varies between 3 and 100%.⁸ Between 1 and 30 adult worms were detected in each positive rat in Hawai‘i. It is not uncommon for rats to have many worms in their bodies; previous studies in other countries have found up to 100 worms in individual rats.²⁸

Figure 3.

Locations of rat traps on Hawai‘i Island (closed circles). Rats were trapped in 2009 and 2011.

The lungs from all examined rats were positive for A. cantonensis based on real-time PCR, including the rats that were negative for adult worms according to the morphological examination. In general, tissue from the rats in which adult worms had been detected resulted in a stronger real-time PCR signal compared to tissue from rats without adult worms. PCR does not distinguish between the different life stages of the parasite and will produce a positive result for any material containing A. cantonensis DNA, including eggs, fragments of worms, and residual cells shed from adult worms. The lung tissues contained enough genetic material to produce a real-time PCR signal, even after all visible worms had been removed from the arteries. The lungs from rats in which no adult worms were detected could have contained eggs and/or L1 larvae left by adult worms that had since died or escaped detection during the necropsy. Alternatively, these rats could have been recently infected so that L3 larvae were present in their blood stream and were passing through the pulmonary arteries on their way to the brain. In any case, all the rats examined in this study displayed evidence of active or recent infection with A. cantonensis.

Future Developments

Molecular detection by PCR or LAMP offers the possibility of screening samples from environmental sources more efficiently for the presence of A. cantonensis. However, environmental samples are often challenging for DNA amplification, since they may contain substances that co-purify with the DNA during the extraction process and inhibit downstream applications.^39,40 Also, environmental samples must be collected in a very systematic way to avoid cross-contamination and therefore false-positive results. Since high-quality DNA is essential for successful molecular detection, it is crucial to carefully validate the DNA extraction method to avoid false negative results. In this study, PCR inhibition was common in DNA extracted from mollusks and required dilution or further purification of some samples before accurate PCR amplification could occur. PCR inhibitors were detected by including a PCR reaction in a separate vessel spiked with DNA extracted from adult A. cantonensis worms. Another way of testing for PCR inhibition is to co-amplify an internal control in every sample. This internal control can be a genetic target in the sample matrix or an artificial template that is added to the sample prior to extraction.⁴¹

Another challenge for environmental surveys is that they often involve large sample sizes. One way to facilitate testing of large numbers of samples could be to pool samples and analyze them in batches.^42,43 This has the potential to substantially reduce the number of procedures required. However, pooling samples can also compromise the performance of the detection method, especially reducing the sensitivity, so the pooling strategy has to be carefully evaluated.⁴⁴

In Hawai‘i and other regions where it is not customary to eat raw snail meat, the route of infection in humans often remains unknown. The most commonly proposed route of infection is accidental ingestion of infected neonate mollusks or mollusk pieces hidden in food items such as leafy vegetables.^8,23,45,46 The role of slime trails and mollusk feces in the transmission of infective larvae to humans is still uncertain. It has also been speculated that humans can become infected by consuming water contaminated with larvae released from decaying mollusks.^47,48 These hypotheses could be addressed by exposing laboratory rats or an animal model to contaminated foods. Molecular detection methods are of limited use for direct examination of potential contamination in foods and water, since they do not distinguish between infective and non-infective larvae.

Finally, to facilitate collaborations and comparisons of studies it would be beneficial to standardize the methodology to detect A. cantonensis in samples from environmental sources. Such standardized methods should incorporate quality controls and be validated in several laboratories around the world. A recent multi-center validation study for the standardization of PCR for Chagas disease could serve as a model for such a project.⁴⁹

Conflict of Interest

None of the authors identifies any conflict of interest.