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The infectivity of third stage Angiostrongylus cantonensis larvae shed from drowned Achatina fulica snails and the effect of chemical agents on infectivity
602 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL M~EDICINE AND HYGIENE.
Vol. 65.
No. 5.
1971.
THE INFECTIVITY OF THIRD STAGE ANGIOSTRONGYLU$ CANTONENSI5 LARVAE SHED FROM DROWNED ACHATINA FULICA SNAILS A N D T H E E F F E C T OF C H E M I C A L A G E N T S ON INFECTIVITY JAMES R. CROOK,* SAMUEL E. F U L T O N A~CDK A M N I R D SUPANWONG
Department of Parasitology, Medical Research Laboratory, SEA TO Medical Project, Raja~ithi Road, Bangkok, Thailand Introduction Angiostrongylus cantonensis, a natural helminth parasite of rats, may cause an eosinophilic meningitis in humans when infective third stage larvae of that nematode are accidentally ingested. When pulmonate land snails, the important intermediate hosts in Thailand, drown, up to 33% of the third stage larvae are shed into the water over a period of up to 50 hours. Host snails were seen to fall into wells and congregate near household water jars in Thai residential areas. Larvae recovered from water in which naturally infected snails were drowned proved to be infective, thus suggesting a source of human infection. Chlorine water treatment has no measurable effect on infective larvae, and iodine treatment fails to provide complete protection from possible infection. Review of Literature The rat lung-worm, Angiostrongylus cantonensis (Chen, 1935; Dougherty, 1946), has been shown to cause an eosinophilic meningitis in man (NoMuRA et al., 1945; ROSEN et al., 1962). A comprehensive report on this nematode-human association described the clinical and Pacific Island epidemiological situation (ROSENet al., 1967). The intermediate hosts are various gastropods. The source of human infection with the worm in Thailand was suggested to be the operculate aquatic snarl Pila ampullacea Lin 1958 (PtrtiYAGUPTA, 1965). However, comprehensive studies in this laboratory (CROOK et al., 1968) on host snails have found only pulmonate land snails to be important natural vectors to rodents in Thailand. Investigations Observations in the North province of Chiang Mai have proved snail association with culinary water sources. Achatina fulica (Ferussac, 1818) snails were observed crawling into wells, and several were seen to fall into the water. A. fulica is a suitable intermediate host for A. cantonensis. The contamination of water by larvae released from snarls submerged in tap water was reported by CICUl~Gand ALICATA (1964). Comprehensive snail collections made around wells and household water jars showed concenThis material has been received by the Office of the Surgeon General, Department of the Army, and there is no objection to its presentation and/or publication. This review does not imply any endorsement of the opinions advanced or any recommendation of such products as may be named. *Present address: Linfield College, McMinnville, Oregon. The principles of laboratory animal care as promulgated by the National Society for Medical Research were observed.
603
JAMES R. CROOK~ S A M U E L E. F U L T O N A N D K A M N I R D S U P A N W O N G
trations of host snails in the immediate vicinity, attracted by spilled water. Around one well, 82 A. fulica were collected, and 3 proved to be infected. Routine examination of snails for infection was conducted by the homogenization of the tissues and the digestion of the homogenate in pepsin. The larvae which were released from the tissues were fed to white Rattus norvegicus, by stomach tube, and the rat brain was examined for fifth stage A. cantonensis larvae 21 days later, in order to verify larval infectivity and to allow species determination. A study was conducted to determine the possibility of water contamination by larvae released from submerged snails. A. fulica collected from Trat, Southeast Thailand, were placed in sedimentation funnels and covered with dechlorinated water. Eighteen of 30 drowned snails proved to be naturally infected. In each case of infection, larvae were found in the water of the drowned snail, and in the digestion sediment of the snail removed from the water after 60 hours (Table I). The larvae from both the water and the digestion sediment were fed to white R. norvegicus. In all cases, shed larvae recovered from the water successfully infected the rats. After 60 hours in the water, some of the larvae digested from snail tissues were infective. The uneven shedding pattern, which is apparently related to snail age, may be important in causing different levels of infection, and in influencing the length of time water can be contaminated after a snail drowns in it. TABLE I. T h i r d stage A. cantonensis larvae shed from drowned, naturally infected A. fulica.
L a r v a e collected per hour after snail was subm e r g e d in water em.
1-5 hr. L ~
D 2
4.5
11
__s
5-2
100
-
-
-
-
5"4 5.6
100
6"2
I00
6"5
59
-
-
-
-
6-ghr. L
D
10-24 hr. L
338 27
--
3
14
--
30
13
--
10
14
--
17
179
--
118
D
L
D
~
46
--
-
--
-
5
134
7' 2
68
--
2
7' 5
34
4
7" 5
13
4
7"6 346
--
8" 6
89
8"6
115
86
31 18
4
66
--
370
100
32
326
100
29
67
100
23
356
8
111
57
170
350
100
272
53
100
33
466
914
100
27
100
49
450
300
100
89
50
22
125
380
100
26
1
2
1
3
--
* S u b m e r g e d 60 hours, **Digested after 60 hours i n water. XCollected living. 2CoUected dead. ~Dashes equal zero in this a nd all following tables.
34
34
4
34
12
242
86
100
Ra t died
14
4
14
5
261
82
i00
I0
100
31
100
65
1415
161
100
71
I32
43
480
210
100
78
100
70
511 2
109
31
37
14
114
132 --
100
516
159
474
20
283
24
117
--
Recovered
27
--
160
Fed
35
37
--
Live D e a d
100
--
33
R a t feeding confirmation
49
12
128
Sediment larvae
123
201
--
Recovered
100
--
1
Fed
132
8
2
--
53
Digested**
R a t feeding confirmation
395
--
8"6 22
Live D e a d
59
351 99
D
131 8
7"9 8-2
--
-
272
7"0
L
2
-
6"8
9" 2
25-29hr.
Drowned*
S h e d larvae 30-53 hr . total
403
132
181
4
181
103
34
1
30
3
1128
231
100
34
23
4
260
80
100
34
23
750
604
ANGIOSTRONGYLUS
LARVAE
A further consideration was investigated concerning the effect of water purification chemicals on shed infective larvae. Laboratory experiments showed no appreciable effect of free residual chlorine at 1.0 ppm. (0-tolidine dihydrochloride colorometric deterruination) on larvae either shed or digested from snail tissues. The most frequently used water purification chemical among field workers in this area, including military, is tetraglycine hydroperiodide. The instructions for use note that the most turbid water is to be treated with two tablets per quart, which release a total of 16.0 rag. of iodine. One quart of water containing from 100 to 300 larvae shed from naturally infected A. fulica was treated with 2 tablets. At the end of 30 minutes the treated water was placed in sedimentation funnels, the descending larvae were collected and some of the living larvae were fed to rats. Other larvae shed from the same drowned snail were left in tap water during the iodine treatment of snail-mates and were fed to rats at the same time as the treated, thus serving as infectivity controls. In each of 7 experiments numerous living larvae were recovered from iodine treated water. Four of the rat feedings developed into brain infections; however, most of the larvae were rendered uninfective by the chemical (Table II). Control larvae were infective within normal percentages. TABLE II. A. cantonensis third stage larvae treated with 16.0 rag. iodine per quart of water. Exposed to iodine
Larvae fed to rats
5th stage larvae recovered from rat
Iodine treated
Iodine treated
No. of
larvae hal qt.
No. recovered post treatment
1Control
L 2
D 3
300
63
72
63
100
100
34
27
34
100
200
69
58
69
100
1
1.4
200
111
38
111
111
3
2.7
200
61
55
61
61"
1
1"6
86
69
86*
86
44
48
44
44
200 150
[
i
Control
%
No.
%
No.
6
9.5
65
65
33
33
31
31
57
51 "3
31
36
i i
7
15 "9
r
1The first 3 experimental control feedings were 100 larvae even; the last 4 were equal to the number of larvae recovered after treatment. 2Collected living. 8Collected dead. *Rat died before the 21 day examination time. Conclusions
The belief that human cases of A. cantonensis eosinophilic meningitis can be caused by a very few worms (RosE~ et al., 1967) lends hnportance to the possibility that human infection can occur from water contaminated with larvae shed from drowned pulmonate land snails. It is here demonstrated that A. cantonensis infections can be contracted from contaminated water. This observation is particularly applicable in regions where private
JAMES R. CROOK, SAMUEL E. FULTON AND KAMNIRD SUPANWONG
605
and public culinary water sources are often subject to snail contamination. This study demonstrates that these larvae survive in chlorine-treated water. It should also be remembered that routine water treatment with iodine does not completely attenuate A. cantonensis larvae, and that infections may be contracted from this source. REFERENCES CHENG, T. C., ALIGATA, J. E. (1964). J. Parasit., 50, 39. CROOK, J., FULTON, S. & SUPANWONG,K. (1968). Ann. trop. Med. Parasit., 82, 27. NOMURA, S. & LIN, P. H. (1945). Taiwan No Ikai, 3, 589. PUNYAGUPTA,S. (1965). Am. ~. trop. Med. Hyg., 14, 370. ROSEN, L., CHAPPELL, R., WALLACE, G. L. & WEINSTEIN, P. P. (1962). J. Am. med. Ass., 1799 620. ~ LoISON, G., LAIGRET,J. & WALLACE,G. D. (1967). Am. ~. Epidemiology, 85, 17.
Vol. 65.
No. 5.
1971.
THE INFECTIVITY OF THIRD STAGE ANGIOSTRONGYLU$ CANTONENSI5 LARVAE SHED FROM DROWNED ACHATINA FULICA SNAILS A N D T H E E F F E C T OF C H E M I C A L A G E N T S ON INFECTIVITY JAMES R. CROOK,* SAMUEL E. F U L T O N A~CDK A M N I R D SUPANWONG
Department of Parasitology, Medical Research Laboratory, SEA TO Medical Project, Raja~ithi Road, Bangkok, Thailand Introduction Angiostrongylus cantonensis, a natural helminth parasite of rats, may cause an eosinophilic meningitis in humans when infective third stage larvae of that nematode are accidentally ingested. When pulmonate land snails, the important intermediate hosts in Thailand, drown, up to 33% of the third stage larvae are shed into the water over a period of up to 50 hours. Host snails were seen to fall into wells and congregate near household water jars in Thai residential areas. Larvae recovered from water in which naturally infected snails were drowned proved to be infective, thus suggesting a source of human infection. Chlorine water treatment has no measurable effect on infective larvae, and iodine treatment fails to provide complete protection from possible infection. Review of Literature The rat lung-worm, Angiostrongylus cantonensis (Chen, 1935; Dougherty, 1946), has been shown to cause an eosinophilic meningitis in man (NoMuRA et al., 1945; ROSEN et al., 1962). A comprehensive report on this nematode-human association described the clinical and Pacific Island epidemiological situation (ROSENet al., 1967). The intermediate hosts are various gastropods. The source of human infection with the worm in Thailand was suggested to be the operculate aquatic snarl Pila ampullacea Lin 1958 (PtrtiYAGUPTA, 1965). However, comprehensive studies in this laboratory (CROOK et al., 1968) on host snails have found only pulmonate land snails to be important natural vectors to rodents in Thailand. Investigations Observations in the North province of Chiang Mai have proved snail association with culinary water sources. Achatina fulica (Ferussac, 1818) snails were observed crawling into wells, and several were seen to fall into the water. A. fulica is a suitable intermediate host for A. cantonensis. The contamination of water by larvae released from snarls submerged in tap water was reported by CICUl~Gand ALICATA (1964). Comprehensive snail collections made around wells and household water jars showed concenThis material has been received by the Office of the Surgeon General, Department of the Army, and there is no objection to its presentation and/or publication. This review does not imply any endorsement of the opinions advanced or any recommendation of such products as may be named. *Present address: Linfield College, McMinnville, Oregon. The principles of laboratory animal care as promulgated by the National Society for Medical Research were observed.
603
JAMES R. CROOK~ S A M U E L E. F U L T O N A N D K A M N I R D S U P A N W O N G
trations of host snails in the immediate vicinity, attracted by spilled water. Around one well, 82 A. fulica were collected, and 3 proved to be infected. Routine examination of snails for infection was conducted by the homogenization of the tissues and the digestion of the homogenate in pepsin. The larvae which were released from the tissues were fed to white Rattus norvegicus, by stomach tube, and the rat brain was examined for fifth stage A. cantonensis larvae 21 days later, in order to verify larval infectivity and to allow species determination. A study was conducted to determine the possibility of water contamination by larvae released from submerged snails. A. fulica collected from Trat, Southeast Thailand, were placed in sedimentation funnels and covered with dechlorinated water. Eighteen of 30 drowned snails proved to be naturally infected. In each case of infection, larvae were found in the water of the drowned snail, and in the digestion sediment of the snail removed from the water after 60 hours (Table I). The larvae from both the water and the digestion sediment were fed to white R. norvegicus. In all cases, shed larvae recovered from the water successfully infected the rats. After 60 hours in the water, some of the larvae digested from snail tissues were infective. The uneven shedding pattern, which is apparently related to snail age, may be important in causing different levels of infection, and in influencing the length of time water can be contaminated after a snail drowns in it. TABLE I. T h i r d stage A. cantonensis larvae shed from drowned, naturally infected A. fulica.
L a r v a e collected per hour after snail was subm e r g e d in water em.
1-5 hr. L ~
D 2
4.5
11
__s
5-2
100
-
-
-
-
5"4 5.6
100
6"2
I00
6"5
59
-
-
-
-
6-ghr. L
D
10-24 hr. L
338 27
--
3
14
--
30
13
--
10
14
--
17
179
--
118
D
L
D
~
46
--
-
--
-
5
134
7' 2
68
--
2
7' 5
34
4
7" 5
13
4
7"6 346
--
8" 6
89
8"6
115
86
31 18
4
66
--
370
100
32
326
100
29
67
100
23
356
8
111
57
170
350
100
272
53
100
33
466
914
100
27
100
49
450
300
100
89
50
22
125
380
100
26
1
2
1
3
--
* S u b m e r g e d 60 hours, **Digested after 60 hours i n water. XCollected living. 2CoUected dead. ~Dashes equal zero in this a nd all following tables.
34
34
4
34
12
242
86
100
Ra t died
14
4
14
5
261
82
i00
I0
100
31
100
65
1415
161
100
71
I32
43
480
210
100
78
100
70
511 2
109
31
37
14
114
132 --
100
516
159
474
20
283
24
117
--
Recovered
27
--
160
Fed
35
37
--
Live D e a d
100
--
33
R a t feeding confirmation
49
12
128
Sediment larvae
123
201
--
Recovered
100
--
1
Fed
132
8
2
--
53
Digested**
R a t feeding confirmation
395
--
8"6 22
Live D e a d
59
351 99
D
131 8
7"9 8-2
--
-
272
7"0
L
2
-
6"8
9" 2
25-29hr.
Drowned*
S h e d larvae 30-53 hr . total
403
132
181
4
181
103
34
1
30
3
1128
231
100
34
23
4
260
80
100
34
23
750
604
ANGIOSTRONGYLUS
LARVAE
A further consideration was investigated concerning the effect of water purification chemicals on shed infective larvae. Laboratory experiments showed no appreciable effect of free residual chlorine at 1.0 ppm. (0-tolidine dihydrochloride colorometric deterruination) on larvae either shed or digested from snail tissues. The most frequently used water purification chemical among field workers in this area, including military, is tetraglycine hydroperiodide. The instructions for use note that the most turbid water is to be treated with two tablets per quart, which release a total of 16.0 rag. of iodine. One quart of water containing from 100 to 300 larvae shed from naturally infected A. fulica was treated with 2 tablets. At the end of 30 minutes the treated water was placed in sedimentation funnels, the descending larvae were collected and some of the living larvae were fed to rats. Other larvae shed from the same drowned snail were left in tap water during the iodine treatment of snail-mates and were fed to rats at the same time as the treated, thus serving as infectivity controls. In each of 7 experiments numerous living larvae were recovered from iodine treated water. Four of the rat feedings developed into brain infections; however, most of the larvae were rendered uninfective by the chemical (Table II). Control larvae were infective within normal percentages. TABLE II. A. cantonensis third stage larvae treated with 16.0 rag. iodine per quart of water. Exposed to iodine
Larvae fed to rats
5th stage larvae recovered from rat
Iodine treated
Iodine treated
No. of
larvae hal qt.
No. recovered post treatment
1Control
L 2
D 3
300
63
72
63
100
100
34
27
34
100
200
69
58
69
100
1
1.4
200
111
38
111
111
3
2.7
200
61
55
61
61"
1
1"6
86
69
86*
86
44
48
44
44
200 150
[
i
Control
%
No.
%
No.
6
9.5
65
65
33
33
31
31
57
51 "3
31
36
i i
7
15 "9
r
1The first 3 experimental control feedings were 100 larvae even; the last 4 were equal to the number of larvae recovered after treatment. 2Collected living. 8Collected dead. *Rat died before the 21 day examination time. Conclusions
The belief that human cases of A. cantonensis eosinophilic meningitis can be caused by a very few worms (RosE~ et al., 1967) lends hnportance to the possibility that human infection can occur from water contaminated with larvae shed from drowned pulmonate land snails. It is here demonstrated that A. cantonensis infections can be contracted from contaminated water. This observation is particularly applicable in regions where private
JAMES R. CROOK, SAMUEL E. FULTON AND KAMNIRD SUPANWONG
605
and public culinary water sources are often subject to snail contamination. This study demonstrates that these larvae survive in chlorine-treated water. It should also be remembered that routine water treatment with iodine does not completely attenuate A. cantonensis larvae, and that infections may be contracted from this source. REFERENCES CHENG, T. C., ALIGATA, J. E. (1964). J. Parasit., 50, 39. CROOK, J., FULTON, S. & SUPANWONG,K. (1968). Ann. trop. Med. Parasit., 82, 27. NOMURA, S. & LIN, P. H. (1945). Taiwan No Ikai, 3, 589. PUNYAGUPTA,S. (1965). Am. ~. trop. Med. Hyg., 14, 370. ROSEN, L., CHAPPELL, R., WALLACE, G. L. & WEINSTEIN, P. P. (1962). J. Am. med. Ass., 1799 620. ~ LoISON, G., LAIGRET,J. & WALLACE,G. D. (1967). Am. ~. Epidemiology, 85, 17.