ORIGINAL ARTICLE
Eosinophilic meningitis risk associated with raw
Ampullarium canaliculatus snails consumption
生吃福壽螺罹患嗜伊紅性腦膜炎的風險
Jiun-Jye Wang
a, Li-Yu Chung
a, Rong-Jyh Lin
a, June-Der Lee
a, Chaio-Wen Lin
b,
Chuan-Min Yen
a,*
王俊傑a,鍾麗玉a,林榮峙a,李俊德a, 林巧雯b,顏全敏a,* a
Department of Parasitology, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
b
Fourth Branch, Centers for Disease Control, Tainan, Taiwan
Received 16 September 2010; accepted 13 December 2010 Available online 21 March 2011
KEYWORDS Angiostrongylus cantonensis; Larvae infectivity; Larvae viability; Raw snail 關鍵詞 廣東住血線蟲; 蟲體感染力; 蟲體活性; 生福壽螺
Abstract In Taiwan, Angiostrongylus cantonensis infection has been reported in foreign laborers who had consumed raw Ampullarium canaliculatus snails. This study analyzed three foreign laborers who had contracted enzyme-linked immunosorbent assay-confirmed A canto-nensis infection while working in Taiwan. All three workers had consumed either roasted snails or raw snails flavored with seasoning while drinking wine. This study investigated possible risk factors for A cantonensis, including naturally occurring A cantonensis in A canaliculatus snails, viability of third-stage A cantonensis larvae in raw seasoned snails and in roasted snails, infec-tivity of larvae, and effects of alcohol while consuming snails. Positive infection rates in snails from five different irrigation canals in south Taiwan ranged from 12.3% to 29.4% and the average number of motile larvae per infected snail ranged from 36 to 65. The number of motile and coiled larvae in snail meat after 120 minutes seasoning was 93 (27.7%) and 233 (69.3%), respectively. After 20 minutes of roasting, most larvae in the snail meat were dead. The infec-tivities of motile and coiled larvae from snail meat after 60 minutes seasoning were 53.2% and 33.2%, respectively, and those from snail meat after 5 minutes roasting were 33.2% and 7.0%, respectively. Eating Taiwan A canaliculatus snails raw is extremely risky given their high infec-tion rates and infecinfec-tion intensities. Even after 120 minutes seasoning or after 20 minutes roast-ing, snail meat should be considered unsafe for human consumption. Finally, experimental rodent studies indicated that consuming alcohol while ingesting larvae does not significantly reduced infectivity.
* Corresponding author. Department of Parasitology, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Shih-Chuan 1stRoad, 80708 Kaohsiung City, Taiwan.
E-mail address:[email protected](C.-M. Yen).
a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m
j o u r n a l h o m e p a g e : h t t p : / / w w w . k j m s - o n l i n e . c o m
1607-551X/$36 Copyrightª 2011, Elsevier Taiwan LLC. All rights reserved. doi:10.1016/j.kjms.2010.12.013
摘 摘要要 生吃福壽螺肉會感染廣東住血線蟲，經訪視血清抗體陽性的外勞，瞭解他們都是喝酒時吃 烤螺肉或吃浸在辣椒醬、醋及魚醬等量混合的調味料的生螺肉，因此本研究分析田野福壽螺感染 率及帶蟲數，在浸調味料的生螺肉或螺烤後的螺肉內第三期幼蟲的活性和感染力，以及酒精對蟲 體感染的影響，來探討感染廣東住血線蟲的危險因子。結果在台灣南部5條灌溉渠道的福壽螺感 染率介於12.3%至29.4%，每個螺帶蟲數介於36至65，螺肉切成0.5公分厚浸於調味料120分鐘分別 有27.7%及69.3%第三期幼蟲呈活動及捲曲狀，螺烤20分鐘後尚有少部份蟲體成捲曲狀，螺肉浸調 味料60分鐘後活動及捲曲蟲體的感染力分別為53.2%及26.1%，而螺烤5分鐘蟲體的感染力分別為 33.2%及7.0%，因為福壽螺在台灣高感染率及高帶蟲數，因此吃浸調味料120分鐘及烤20分鐘的福 壽螺肉均不安全，而且在鼷鼠的感染實驗顯示酒精並不會降低感染風險。 Copyrightª 2011, Elsevier Taiwan LLC. All rights reserved.
Introduction
Angiostrongylus cantonensis, a nematode parasite that inhabits the lung of rats, is the major cause of eosinophilic meningitis or meningoencephalitis in humans [1,2]. The parasite is endemic to the Pacific islands, Taiwan, Thailand, and Japan[3e5]but has recently been identified in Africa, India, Australia, North America, Brazil, Germany, and China
[6e9]. Most patients are infected after eating raw or undercooked molluscan intermediate hosts and paratenic hosts that contain the infective third-stage larvae of the worm. Consuming uncooked or contaminated vegetables may also cause infection. Thus, A cantonensis is a food-borne zoonotic parasite of considerable public health importance in many countries throughout the world[10,11].
The A cantonensis is among the most important zoonotic parasites in Taiwan[12]. Hundreds of infections associated with ingestion of the Achatina fulica and Pila species of snail have been reported in indigenous populations of Taiwan and Thailand, respectively [4,13e17]. So far, few reports indicate that patients were infected with A canto-nensis by the transmission of Ampullarium canaliculatus. In recent years, three outbreaks of A cantonensis infection have occurred among foreign laborers who had consumed raw A canaliculatus (golden apple snail) [18e20]. The present study analyzed three foreign laborers with eosino-philic meningitis associated with A cantonensis infection contracted after consuming A canaliculatus while drinking wine. The workers indicated that they had prepared the snails by roasting over open flame or had eaten them raw after seasoning. This study evaluated the risk factors for eosinophilic meningitis caused by A canaliculatus by investigating the rates and intensities of A canaliculatus infection caused by A cantonensis in several areas of Taiwan. The viability of third-stage larvae was tested in snail meat prepared by the process typically used by foreign laborers. The infectivity of the larvae and the effect of consuming alcohol when ingesting larvae were studies in a mouse infection model.
Methods
PatientsThis study analyzed three male foreign laborers who had been hospitalized in a southern Taiwan medical center after contracting clinically defined eosinophilic meningitis
in November 2009. All three patients indicated that they had consumed wine with A canaliculatus snails gathered from an irrigation canal. Patient 1 and 2 had eaten sliced raw snail meat mixed with seasoning. Patient 3 had eaten meat of snails roasted for 5e10 minutes over an open fire. Spinal taps were performed on all patients, and 5 mL specimens of cerebrospinal fluid (CSF) from each patient were sent to the authors for laboratory analysis by enzyme-linked immunosorbent assay (ELISA) to detect antibodies specific to A cantonensis and Toxocara canis.
Detection of CSF antibodies
Antibodies to A cantonensis and T canis in the CSF of patients and control subjects were assayed by ELISA[21]. Briefly, CSF specimens were added to the wells of a micro-plate precoated with somatic antigens of A cantonensis or T canis and incubated at room temperature for 1 hour. After removing unbound components by washing, antihuman immunoglobulin (Gþ A þ M)-horseradish peroxidase conju-gate was added and incubated for 1 hour. After washing the wells, tetramethylbenzidine substrate solution was added and incubated for 20 minutes at room temperature. In each well, absorbance was read at 450 nm after adding 2N sulfuric acid to stop the reaction.
Field survey ofA canaliculatus infection
Live field snails were captured from five randomly selected irrigation canals in Kaohsiung County in South Taiwan. About 100 snails larger than 4 cm were randomly collected from each canal during the last week of November 2009. Third-stage A cantonensis larvae were obtained using a method described previously[22]. Briefly, after crushing the snails and removing the shells, each snail was minced individually and digested in artificial gastric juice with magnetic stirring at room temperature for 1 hour. Larvae in the sediments were counted under a dissecting microscope.
Viability of third-stage larvae in seasoned snail meat
Twenty-five infected snails larger than 4 cm were sliced into specimens 0.5 cm thick. The specimens were randomly divided into five groups. Specimens were then immersed for 5, 30, 60, or 120 minutes in the seasoning mixture typically used by foreign laborers, that is, equal volumes of chili
sauce, vinegar (4.5% acidity), and fish sauce. One unsea-soned group of specimens was used as the control group. After digestion with artificial gastric juice, the total third-stage larvae in each preparing group were examined to determine the numbers of motile, coiled, and dead larvae.
Viability of third-stage larvae in roasted snail
Infected A canaliculatus snails were placed on a typical netted aluminum roasting plate with a handle and roasted over a fire fueled with dry tree branches. Five snails of each group were roasted for 5, 10, or 20 minutes. All snails were turned over every 1e2 minutes during roasting and were treated with artificial gastric juice after roasting. All third-stage larvae in each group of snail meat were analyzed to determine the numbers of motile, coiled, and dead larvae remaining after the digestion process.
Infectivity of each larvae group
Experiments were performed using male 6-week-old BALB/C mice that had been bred and maintained at the laboratory animal center at this institution. Each animal was infected with 30 third-stage larvae from snail meat subjected to one of the preparations via a stomach tube after slight ether anesthesia. In additional experimental groups, the mice were orally given 30 third-stage larvae from snail meat sub-jected to one of the treatments followed by 1.5 mL/kg of a local alcoholic drink (40% alcohol) via a stomach tube. The mice were sacrificed on Day 21 after infection, and
young-adult worms were recovered from the brains. Larval infec-tivity was measured by the rate at which young-adult worms were recovered.
Statistics
Significant group differences in infection rates of A canaliculatus and viability of third-stage larvae were identified by chi-square test. Significant group differences in infection intensities of A canaliculatus and third-stage larvae infectivity were tested by analysis of variance. Significant group differences in third-stage larvae infec-tivity in mice with or without alcoholic drink were tested by Student t test. A p value less than 0.05 was considered significant.
Results
Examination of CSF antibodies and worms
Antibodies in the CSF of patients and control subjects were examined by ELISA using antigens of A cantonensis and T canis, respectively. The levels of CSF antibodies were expressed as ELISA values. Table 1 shows that, in three patients, the ELISA values increased for those observed in the A cantonensis positive control subject but did not significantly exceed for those observed in the T canis negative control subjects. Two and one young adult A cantonensis worms were found in CSF of Patient 1 and 2, respectively.
Table 1 Results for CSF specimens examined for young adult Angiostrongylus cantonensis by dissecting microscope and examined for antibodies to A cantonensis and Toxocara canis by ELISA
Patients Specific antibodies to Number of A cantonensis young-adult
worm A cantonensis T canis 1 1.07 0.19 2 2 1.09 0.20 1 3 0.91 0.16 0 (þ) control 1.13 0.98 2 () control 0.18 0.15 0
ELISAZ enzyme-linked immunosorbent assay.
Table 2 Rates and intensities of infection with Angiostrongylus cantonensis in Ampullarium canaliculatus collected from five defined irrigation canals in south Taiwan
Defined irrigation Number of examined canals
Positive, n(%) Number of larvae per infected snail, mean SD A 109 32 (29.4) 60 8 B 112 20 (17.9) 49 5 C 115 30 (26.1) 65 9 D 98 12 (12.3) 36 5 E 101 15 (14.9) 53 6
Chi-square test of infected snails among five canals,c2Z 14.7, p < 0.01.
Number of larvae per infected snail, one way analysis of variance, FZ 41.2, p < 0.01. SDZ standard deviation.
Natural infection ofA canaliculatus
As shown inTable 2, the positive rate of third-stage A can-tonensis larvae in A canaliculatus collected from irrigation canals AeE were 12.3%e29.4%; the average number of motile larvae per infected snail ranged from 36 to 65. The foreign laborers with eosinophilic meningitis in this study had gathered A canaliculatus from Canal C.
Viability of third-stage larvae in seasoned snail meat
Table 3compares the number of larvae and their activity in snail meat after different durations of seasoning. The number of motile larvae significantly decreased from 323 in unseasoned snail meat to 93 in snail meat seasoned for 120 minutes (p< 0.001). However, the longer the snail meat was immersed in seasoning, the more coiled larvae were found.
Viability of third-stage larvae in roasted snail meat
As shown inTable 4, motile larvae in the snails significantly decreased from 323 to 0 after 20 minutes roasting (p< 0.0001). The number of coiled larvae in the snails rapidly peaked (5 minutes after roasting) and then gradu-ally decreased. However, the number of dead larvae in the snail meat was significantly and positively associated with snail roasting time (p< 0.0001). Roasting for 10 and 20 minutes resulted in 74.5% (228/306) and 87.5% (246/281) larvae death, respectively.
Infectivity of third-stage larvae and effects of alcohol on mouse infection
Infectivity of third-stage larvae was expressed as the ratio of young adult worms recovered from the brain of the mouse to the number of third-stage larvae consumed by the mouse.Table 5shows that the infectivity of motile larvae from untreated snail meat was significantly higher than that of larvae obtained from snail meat seasoned for 60 minutes or from snail roasted for 5 minutes (p< 0.001). In snail meat treated with seasoning or roasting, coiled larvae can still cause infection, but dead larvae cannot. In mice that ingested larvae orally, the infectivity of third-stage larvae from each snail meat preparation was unaffected by concurrent alcohol consumption.
Discussion
Since Nomura and Lin reported the first human case of eosinophilic meningitis caused by A cantonensis in 1945[23], many epidemiological surveys of this parasitic disease have been reported. Terrestrial and aquatic mollusks, including several species of snails and slugs, are known to be natural intermediate hosts of A cantonensis in Taiwan[5,24e26]. Other potential paratenic or transport hosts include planarian, crabs, shrimp, fish, frogs, and toads, although their roles in human infection have not been elucidated [27]. Obviously, food-borne A cantonensis infection is still a major public health concern in this country.
A cantonensis infection is endemic to south Taiwan, where the giant African snail is the major intermediate host
[4,14]. The incidence of human cases is highest during the Table 3 Viability of Angiostrongylus cantonensis third-stage larvae in meat of Ampullarium canaliculatus after four different durations of seasoning
Third-stage larvae activity Numbers of larvae in snail meat seasoning for different durations
Untreated 5 min 30 min 60 min 120 min
Motile 323 319 307 165 93
Coiled 6 5 47 140 233
Dead 1 1 3 7 10
Total 330 325 357 312 336
Chi-square test of motile and coiled larvae from snail meat after four different seasoning treatments, c2Z 45.8, p < 0.001 and c2Z 50.5, p < 0.001, respectively.
Table 4 Viability of Angiostrongylus cantonensis third-stage larvae in meat of Ampullarium canaliculatus after three different durations of roasting
Third-stage larvae activity Numbers of larvae in snail meat for varying durations of roasting
Untreated 5 min 10 min 20 min
Motile 323 97 9 0
Coiled 6 177 69 35
Dead 1 73 228 246
Total 330 347 306 281
Chi-square test of motile and coiled larvae from snail meat after three different durations of roasting,c2Z 111.4, p < 0.0001 and
MayeSeptember rainy season [13], when giant African snails are abundant and active in the field[28]. The rate and intensity of A cantonensis infection is reportedly associated with the size of the snail [28]. People gather giant African snails for cooking. The viscera and mucus of snails, which reportedly contain 95% of A cantonensis larvae
[25]are often thrown on the ground during preparation and may be inadvertently ingested by children playing nearby. Thus, about 66% of reported cases are children younger than 9 years who live in rural and mountainous areas of south Taiwan[13]. In adults, infection usually results either from eating partially cooked snails or from eating foods or using dishes that have been contaminated during the cooking process. Snails are rarely eaten raw in Taiwan
[4,13].
In contrast to the indigenous patients infected with A cantonensis in Taiwan, the three outbreaks in foreign laborers working in southern Taiwan involved adults who had ingested raw golden apple snails rather than giant African snails. In northeastern areas of Thailand, the high incidence of human infection with A cantonensis affects the life, health, and productivity of the indigenous people [10]. Koi-hoi, a traditional uncooked snail dish, is eaten with relish by the indigenous population in these areas. Koi-hoi is prepared from Pila snails, which are reportedly a major cause of cerebral angiostrongyliasis. A survey indicated that 0.9% of 423 Pila polita snails in northeast Thailand were infected with A can-tonensis [29]. Because A canaliculatus and Pila sp have a similar in size and appearance, we hypothesize that foreign laborers in Taiwan may consume A canaliculatus snails because of mistaking them for P polita snails.
A canaliculatus was introduced in Taiwan from Argentina in 1979 as a food source. Since then, the species has propagated in paddy field and drainage ditches throughout the island. In 1985, Yen et al. [26] reported a successful experimental infection of the snail with A cantonensis. Field surveys in the present study showed that 12e29% of A canaliculatus were infected with third-stage A cantonensis larvae, which is similar to the results reported 20 years earlier[22]. The number of larvae per infected snail ranged from 36 to 65. Clearly, the rate and intensity of A cantonensis infection are significantly higher in the A canaliculatus snail in Taiwan than that in the P polita snail in Thailand. Thus, consuming raw A canaliculatus snail meat in Taiwan is extremely risky.
Although many inhabitants in rural areas of Taiwan collect A canaliculatus for food, the snail is rarely consumed raw. In Thailand, the traditional koi-hoi dish is prepared from chopped raw snail meat flavored with seasoning and vegetables. Third-stage A cantonensis larvae in chopped snail meat are easily exposed to the seasoning used in koi-hoi. After snail meat is seasoned for 10 minutes or longer, the number of motile third-stage larvae in coiled state increases[30]. However, a mouse infection model in a recent study revealed infectivity in coiled third-stage larvae in snail meat even after 1 hour seasoning [31]. Foreign laborers in Taiwan may have difficulty finding the ingredients they traditionally use to flavor raw snail meat and may therefore modify the ingredients. They usually slice the snail meat to a thickness of about 0.5 cm before seasoning with the modified ingredients. They typically consume the snail meat immediately after seasoning with the modified ingredients, before the seasoning can permeate. Within 5 minutes after exposure, almost all A cantonensis larvae observed in snail meat in this study were motile, and infectivity was 68.7%. Longer exposure to seasoning decreased the numbers of motile larvae in the snail meat and increased the numbers of coiled larvae. However, either motile or coiled larvae can still cause infection in mice, and even coiled larvae in snail meat seasoned for 60 minutes had 26.1% infectivity. Clearly, the seasoning ingredients used by foreign laborers in Taiwan only partially inhibit the viability of third-stage A cantonensis larvae.
A literature review reveals no studies of the viability of third-stage A cantonensis larvae in A canaliculatus snails roasted over open flame. Foreign laborers in Taiwan often gather after work for dining, drinking, and chatting. On such occasions, they typically use a simple roasting net to roast snails over an open fire started from tree branches. This study showed that, in A canaliculatus snails larger than 4 cm, snails roasted over an open flame for 20 minutes are safer for consumption compared with snails eaten raw. According to interviews with the three hospitalized patients analyzed in this study, most foreign laborers believe that raw snail meat is safe if eaten with alcoholic drinks. However, analysis of mice infected with third-stage A cantonensis larvae in this study indicated that consuming alcohol did not confer significant protection from infection. Table 5 Infectivity of Angiostrongylus cantonensis third-stage larvae from Ampullarium canaliculatus snails after seasoning or roasting in BALB/c mice and effect of alcoholic drink given to mice immediately after infection with larvae
Snail meat Alcoholic drink Infectivity of third-stage larvae with different activity
Motile Coiled Dead
Untreated 68.7 7.1 ND ND
Control þ 65.6 5.7 ND ND
Treated with seasoning for 60 min
53.2 6.1 26.1 4.6 ND
þ 52.5 5.0 25.3 3.5 ND
Treated with roasting for 5 min 33.2 4.6 7.0 2.0 0
þ 31.5 4.3 6.2 1.9 0
Infectivity of motile larvae from each group of snails, one-way analysis of variance, FZ 26.2, p < 0.001. Infectivity of larvae from each group of snails in mice with or without alcoholic drink, Student t test, p> 0.05. NDZ insufficient larvae to infect mice.
In conclusion, the high rate of A cantonensis infection in A canaliculatus, the consumption of the snail, and the method of preparation are major risk factors for A cantonensis infec-tion in foreign laborers in Taiwan. Some authors recommend that foreign laborers be legally prohibited from consuming raw snail meat to reduce the risk of A cantonensis infection. However, changing dietary habits that have existed for generations in foreign laborers would be difficult. To reduce infection risk, the authors therefore suggest an informational program to advise foreign laborers in safely preparing snail meat for human consumption (i.e. seasoning for longer than 2 hours and roasting for longer than 20 minutes).
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