Detection of Angiostrongylus cantonensis in the Blood and Peripheral Tissues of Wild Hawaiian Rats (Rattus rattus) by a Quantitative PCR (qPCR) Assay

Jarvi, Susan I.; Pitt, William C.; Farias, Margaret; Shiels, Laura; Severino, Michael; Howe, Kathleen; Jacquier, Steven; Shiels, Aaron B.; Amano, Karis K.; Luiz, Blaine; Maher, Daisy; Allison, Maureen L.; Holtquist, Zachariah; Scheibelhut, Neil

doi:10.1371/journal.pone.0123064

Cited by 25 publications

(26 citation statements)

References 23 publications

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“…The rat lungworm ( Angiostrongylus cantonensis ) is a nematode that causes human angiostrongyliasis (frequently called rat lungworm disease or RLWD). East Hawai‘i Island is the epicenter for angiostrongyliasis in the USA [ 1 ]. Symptoms of A .…”

Section: Introductionmentioning

confidence: 99%

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High prevalence of Angiostrongylus cantonensis (rat lungworm) on eastern Hawai‘i Island: A closer look at life cycle traits and patterns of infection in wild rats (Rattus spp.)

et al. 2017

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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.

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Section: Introductionmentioning

confidence: 99%

“…cantonensis , including those that were not visually positive for adult worms. In another previous study [ 1 ] 61 rats ( R . rattus ) were trapped in the Waiakea Forest Reserve in east Hawai‘i Island at 533 m elevation.…”

Section: Introductionmentioning

confidence: 99%

High prevalence of Angiostrongylus cantonensis (rat lungworm) on eastern Hawai‘i Island: A closer look at life cycle traits and patterns of infection in wild rats (Rattus spp.)

et al. 2017

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“…An under-estimation of the prevalence of A. malaysiensis has occurred because of the di culty in the morphological identi cation of adult and third-stage larva. Several polymerase chain reaction (PCR)based identi cation techniques were developed, but all were aimed at increasing the sensitivity and speci city of A. cantonensis detection in blood, the peripheral tissues of wild Hawaiian rats, and cerebrospinal uid (CSF) of patients [25][26][27]. No powerful techniques with high sensitivity and speci city have been developed for detecting and discriminating between A. cantonensis and A. malaysiensis.…”

Section: Introductionmentioning

confidence: 99%

SYBR-Green-Based Quantitative Real-Time PCR for Discriminating Between Closely Related Angiostrongylus Cantonensis and A. Malaysisensis

Jakkul¹,

Chaisiri²,

Saralamba³

et al. 2020

Preprint

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Background: Angiostrongylus cantonensis is a well-known pathogen causing human angiostrongyliasis eosinophilic meningitis. Humans, as accidental hosts, are infected by eating undercooked snails containing third-stage larvae. A. malaysiensis is closely related to A. cantonensis and has been described as a potential human pathogen. Recently, the two species have been reported to have overlapping distributions in the same endemic area, particularly in the Indochina region. Because of their similar morphological characteristics, misidentification often occurs, particularly of the third-stage larva in the snail intermediate host. Methods: We designed species-specific primers to mitochondrial cytochrome b, which was used as a genetic marker. SYBR-green quantitative real-time PCR (qPCR) was employed to quantitatively detect and identify the third-stage larvae and tissue debris in the cerebrospinal fluid (CSF) of a patient, and to quantify third-stage larvae in the snail Achatina fulica collected from the field.Results: The newly designed primers were highly specific and sensitive, even when using conventional PCR. SYBR green qPCR quantitatively detected around 10−4 ng of genomic DNA from one larva and facilitated the specific detection and identification of parasitic genetic material from the CSF of a patient with angiostrongyliasis. The method also estimated the number of larvae in A. fulica and revealed that the primary source of Angiostrongylus infection in the King Rama IX public park study area was A. malaysiensis; although, the two Angiostrongylus species each infected 10% of the snails. Conclusions: Our SYBR green qPCR method is a useful and inexpensive technique for parasite identification and has sufficient sensitivity and specificity to detect a single larva and simultaneously discriminate between A. cantonensis and A. malaysiensis. The number of larvae infecting or co-infecting the snail intermediate host can also be estimated. In future research, this qPCR method could be employed in a molecular survey of A. cantonensis and A. malaysiensis occurrence within intermediate and definitive hosts. The technique should also be applied in a study analyzing CSF specimens from patients with eosinophilic meningitis to assess the usefulness of the method for clinical diagnosis.

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“…While exact numbers are not known, it was estimated that 30,000-60,000 people relied on catchment water when the Guidelines on Rainwater Catchment Systems for Hawai‘i manual was written in 2010 [21]. In the Puna District, where most recent cases have originated [16, 22, 23], there are large subdivisions which were developed in the late 1950’s to mid 1970’s with little or no infrastructure for water [24]. Today, the majority of households in this district rely on rainwater catchment for their household water supply and there is no state or federal agency that oversees the use or management of catchment systems [25].…”

Section: Introductionmentioning

confidence: 99%

Water transmission potential of Angiostrongylus cantonensis: larval viability, and effectiveness of rainwater catchment sediment filters

Howe

Kaluna

Fisher

et al. 2018

Preprint

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16 Neuroangiostrongyliasis, caused by Angiostrongylus cantonensis, has been reported in Hawaiʻi 17 since the 1950's. An increase in cases is being reported primarily from East Hawaiʻi Island, 18 correlated with the introduction of the semi-slug Parmarion martensi. Households in areas 19 lacking infrastructure for water must use rainwater catchment as their primary domestic water 20 supply, for which there is no federal, state, or county regulation. Despite evidence that 21 contaminated water can cause infection, regulatory bodies have not addressed this potential 22 transmission route. This study evaluates: 1) the emergence of live, infective-stage A. cantonensis 23 larvae from drowned, non-native, pestiforous gastropods; 2) larvae location in an undisturbed 24 water column; 3) longevity of free-living larvae in water; and 4) effectiveness of rainwater 25 catchment filters in blocking infective-stage larvae. Larvae were shed from minced and whole 26 gastropods drowned in either municipal water or rainwater with >94% of larvae recovered from 27 the bottom of the water column. Infective-stage larvae were active for 21 days in municipal 28 water. Histological sectioning of P. martensi showed proximity of nematode larvae to the body 29 wall of the gastropod, consistent with the potential for shedding of larvae in slime. Gastropod 2 30 tissue squashes showed effectivity as a quick screening method. Live, infective-stage larvae were 31 able to traverse rainwater catchment polypropylene sediment filters of 20 µm, 10 µm, 5 µm, and 32 1 µm filtration ratings, but not a 5 µm carbon block filter. These results demonstrate that live, 33 infective-stage A. cantonensis larvae can and do emerge from drowned snails and slugs, survive 34 for extended periods of time in water, and that the potential exists that they enter the household 35 water supply. This study illustrates the need to better investigate and understand the potential 36 role of contaminated water as a transmission route for neuroangiostrongyliasis. 37 38 Introduction 39 The nematode Angiostrongylus cantonensis is established throughout the main Hawaiian Islands 40 with the possible exception of Lānaʻi [1, 2, 3]. The complex lifecycle of this parasite has been 41 well-described in the literature [4, 5, 6, 7]. In Hawaiʻi, Rattus rattus and Rattus exulans are 42 important definitive hosts, and many gastropod species are effective intermediate hosts including 43 Achatina fulica, Euglandia rosea, Laevicaulis alte, Limax maximus, Parmarion martensi and 44 Veronicella cubensis [1, 2, 8]. The third stage larva (L3) is harbored in the intermediate host, and45 it is this larval stage that is infective to rats and accidental hosts, including humans, as the L3 46 larvae can safely pass through the acidic environment of the mammalian gut. There are also 47 paratenic hosts that can carry the infective stage larvae; these include shrimp, prawns, crabs, 48 frogs, water monitor lizards, centipedes, and some planarians [7. 9, 10, 11, 12]. Of planarians, 49 the predacious Platydemou...

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Detection of Angiostrongylus cantonensis in the Blood and Peripheral Tissues of Wild Hawaiian Rats (Rattus rattus) by a Quantitative PCR (qPCR) Assay

Cited by 25 publications

References 23 publications

High prevalence of Angiostrongylus cantonensis (rat lungworm) on eastern Hawai‘i Island: A closer look at life cycle traits and patterns of infection in wild rats (Rattus spp.)

High prevalence of Angiostrongylus cantonensis (rat lungworm) on eastern Hawai‘i Island: A closer look at life cycle traits and patterns of infection in wild rats (Rattus spp.)

SYBR-Green-Based Quantitative Real-Time PCR for Discriminating Between Closely Related Angiostrongylus Cantonensis and A. Malaysisensis

Water transmission potential of Angiostrongylus cantonensis: larval viability, and effectiveness of rainwater catchment sediment filters

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