The detection of Schistosoma mekongi infections in a natural population of Neotricula aperta at Khong Island, Laos, and the control of Mekong schistosomiasis

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J. Moll. Stud. (2001), 67, 400–405 © The Malacological Society of London 2001
Mekong schistosomiasis is transmitted by the caenogastro-
pod snail Neotricula aperta (Gastropoda: Pomatiopsidae)
(Temcharoen 1971). The disease has been known in the lower
Mekong Basin since 1957
1,2,3,4
and has presented a serious
public health problem throughout its history. The causative
agent of Mekong schistosomiasis is the blood fluke Schisto-
soma mekongi (Trematoda: Digenea) Voge, Bruckner and
Bruce 1978. All known endemic areas lie along the Mekong
river and include Champassac Province, of southern Laos,
and Cambodia north of Kratié. Southern Laos and Cam-
bodia, together with parts of Thailand and Vietnam, com-
prise the lower Mekong Basin (Fig. 1). In 1990 over 150,000
persons were estimated to inhabit areas where the transmis-
sion of Mekong schistosomiasis was endemic and at least
80,000 of these were thought to be infected.
5,6
After nearly a
decade of mass chemotherapy and education the number of
persons known to be infected has fallen to around 7,000;
however, even such prolonged control efforts have been
unable to break transmission in any of the provinces affected.
The failure of control may be attributed to the lack of atten-
tion paid to limiting populations of N. aperta acting as inter-
mediate host for S. mekongi.
Three strains (,  and ) were originally described for
N. aperta in Northeast Thailand and southern Laos
7
and all
are to varying degrees () capable of transmitting
schistosomiasis, although the -strain alone is epidemiolog-
ically significant.
8
N. aperta so far appears restricted to the
lower Mekong river, which here refers to the Mekong and its
tributaries in the lower Mekong Basin. Ecological studies
have been hampered by problems of snail culture in the
laboratory, and the severe annual flood (spate) in the lower
Mekong which interrupts direct observation of the snails.
The stream flow of the Mekong river and its tributaries shows
marked seasonal variations. Generally the river rises in June
following the onset of the Southwest monsoon, with peak
flow in September, and low water conditions are restored
in February. In the Mekong and Mul rivers, N. aperta is
apparently semelparous, the eggs take 4–5 weeks to hatch
and longevity is unlikely to exceed 15 months.
9
The snails are
also relatively small, with mean (maximum) shell length
ranging from 2.0 mm (-strain) to 3.5 mm (-strain).
Prior to 1962 the only well documented focus of Mekong
schistosomiasis was along the Mekong river at Khong Island,
Khong District, in Champassac Province, Laos, some 25 km
from the border with Cambodia (Fig. 2). The 1969 epidemio-
logical survey at Khong Island, conducted by a Smithsonian
Institute-Mahidol University team
10,11
revealed the shallow
water near the market at Ban Xieng-Wang, Khong Town
(14°630N; 105°5145E) to be a site of intense transmission
of schistosomiasis. The overall prevalence was 14.4% in
humans, with the peak of 63.3% in the 7–15 year-old cohort,
and 11% in dogs. More recently the prevalence in the 7–14
year cohort was estimated to be 30.2%
12
with an overall
prevalence of around 10%.
13
The disease causes severe mor-
bidity in Laos and hospitals in Khong District reported 30
deaths in 1990 due to ascites, jaundice or gastrointestinal
bleeding.
5
Schistosomiasis control in Khong District began
in 1989 under the Ministry of Health, Laos, with funding and
technical assistance from the World Health Organisation
(WHO). The baseline prevalence in children was 50.7% based
on Kato-Katz faecal smear examination. Administration
of Praziquantel (single oral dose of 40 mg/kg body weight)
began immediately after the baseline survey in October 1989,
when coverage reached 88% of the target (child) population,
and continued until November 1995. The final round of mass
treatment was performed in May 1998 and covered all
endemic districts. Follow up surveys in June 1999 were
encouraging in that the prevalence in school children had
fallen to 0.8%. Despite this the prevalence in one village, Hat-
Xai-Khoun on the east bank of the Mekong river, remained
high (26.8% in children and 12.6% in pigs
14
). In view of the
severity and persistence of the disease, the present study
aimed to quantify the prevalence of S. mekongi in N. aperta
around Khong Island.
Adult Neotricula aperta were collected from within 2 m of
shore along the Mekong river at 3 villages around Khong
Island. The collections were restricted to areas of human
water contact, such as laundry areas and boat moorings.
Three such collection sites were chosen on the basis of earlier
reports of endemic schistosomiasis;
5,10
these sites and the
numbers of snails collected are given in Table 1. The Mekong
river is 1.05 km wide at Ban Hat-Xai-Khoun (Fig. 2). The
river was deep in May 2000, as a result of the early onset of
the rainy season, and collection was difficult. Small brushes
and forceps were used to remove snails from stones found in
the river. The snails were taken to Bangkok within 3 days
of collection and maintained in the dark until screening.
Beginning one week after collection, the snails were placed
individually into the wells of microtitre plates containing
de-chlorinated water. The plates were then exposed to white
light for 4 h. Each snail was subsequently examined for the
release of furcocercous cercariae (also lacking eye-spots),
which was judged as a sign of a patent infection with S.
mekongi. Exposure of white mice to these cercariae yielded
adult Schistosoma after 6 weeks. Examination of the recov-
ered worms indicated S. mekongi, as did the appearance of
RESEARCH NOTES
The detection of Schistosoma mekongi infections in a natural population of Neotricula
aperta at Khong Island, Laos, and the control of Mekong schistosomiasis
S.W. Attwood,
1,2
E.S. Upatham
2,3
and V.R. Southgate
1
1
Department of Zoology, The Natural History Museum, Cromwell Road, London SW7 5BD. UK.
2
Department of Biology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
3
Department of Medical Science, Faculty of Science, Burapha University, Bangsaen, Chonburi 20131, Thailand.

RESEARCH NOTES
401
Figure 1. The lower Mekong Basin showing the Mekong and Mul rivers. The location of Khong Island, , is also shown. Scale and inter-
national boundaries are approximate.

RESEARCH NOTES
402
Figure 2. The Mekong river around Khong Island, Champassac Province, southern Laos. Scale is approximate.

RESEARCH NOTES
403
the eggs found in liver preparations from the mice.
15,16,17
Screening took place between 08:00 and 13:00 h; this was
based on observations of the peak period of cercarial emer-
gence. Screening was terminated 15 weeks after the field
collection. The screening period was considered sufficient in
view of earlier findings which indicated that all patent infec-
tions in N. aperta were apparent after 8 weeks.
8,18
Snails shed-
ding cercariae were denoted as positive and were isolated in a
separate maintenance culture. Negative snails were returned
to the original culture until the next screening.
Table 2 presents all data relating to infection and mortality
for N. aperta sampled at Ban Hat-Xai-Khoun. All the posi-
tive snails were detected upon first screening, one week after
collection. Shedding of S. mekongi cercariae generally com-
mences between 6 and 12 weeks post-exposure.
8,19
Con-
sequently, the present data suggest that exposure to miracidia
in the Mekong river occurred between mid-February and
early April in the year of collection. The Ban Xieng-Wang
and Ban Na samples were screened according to the schedule
in Table 2 but no positive snails were found in these samples.
In all 3 cases a mortality of 30 ± 5% was observed over the
15 week period; these data are provided in order to afford
comparison with other studies, as increasing mortality results
in a greater chance of the death of an infected snail before
cercariae are shed. The positive snails shed an average of 23
cercariae per day as estimated on the fourth week of patency.
The prevalence of 0.22% estimated in the present study is
comparable with the level of 0.3% first observed at Khong
Island in 1973 by Kitikoon.
20
It is interesting to note that
infection was detected at Ban Hat-Xai-Khoun only, the site
reported by the Laotian Ministry of Health as having a
significant post-intervention prevalence in the human popu-
lation. The data thus support the conclusion that up to 9
years of mass chemotherapy have failed to break the trans-
mission of human schistosomiasis in Khong District. A per-
sistent reservoir of infection is apparent at the level of
both the definitive host (probably including pigs and other
domestic animals) and larval populations in the intermediate
host.
The control of N. aperta is likely to prove effective in the
control of human schistosomiasis when combined with
chemotherapy of villagers in endemic areas. However, con-
trol of the intermediate host has been attempted on only one
occasion, by a WHO team on Khong Island at Ban Xieng-
Wang. This small scale trial took place in May 1991 during
the low water period of the Mekong river; the time of maxi-
mum snail population density.
21
Beginning a little over 20 m
from the shore, twenty 1 kg sacs containing Niclosamide
were suspended across the Mekong river near the district hos-
pital (Fig. 2); this achieved an approximate concentration of
0.5 ppm downstream after 2 h. On the day following treat-
ment the N. aperta population density 10 m downstream had
fallen to only 2 m
2
, around 1% of the initial density. The
population density 150 m downstream fell to zero from an
initial 1.9 m
2
. Less encouragingly, five days after treatment
the density 10 m downstream rose to 2,400% of its original
value and then fell to its pre-treatment value over the next
four days. The density 150 m downstream rose to only 280%
of initial levels after 5 days, however, there were increases to
over 1,200% of pre-treatment values at this station by day 10.
These less than satisfactory results were attributed to an
insufficient level of Niclosamide and a greater influence of the
river’s current than first thought.
21
The overall effects of the
molluscicide trial appear to have been a marked increase in
snail population density in areas of initial paucity, with little
effect on originally high density foci. It is likely that local
extirpation of snails left vacant habitats which were particu-
larly vulnerable to recolonisation from outside the treatment
area; this may be due to an epilithic algal bloom, or growth of
some other food item, in the vacant habitats.
22
Habitats in
Table 1. Locations and dates of Neotricula aperta collec-
tions in Khong District, Champassac Province, southern
Laos. The number of snails collected is also shown.
Village name Date No. of snails
Ban Xieng-Wang 27/04/00 2,100
Ban Na 28/04/00 1,000
Ban Hat-Xai-Khoun 01/05/00 1,800
Table 2. Results of screening field collected Neotricula aperta for cercariae of
Schistosoma mekongi. The snails were collected in the Mekong river at Ban
Hat-Xai-Khoun, Khong District, southern Laos. The week number is counted from
the date of snail collection. The number of snails collected is given at the head
of column 2. A positive snail is one exhibiting signs of patency, i.e. shedding
cercariae. The infection rate (%) equals (total no. of positive snails/ no. of snails
initially screened)  100.
Week no. No. of snails surviving No. of new snails Infection rate (%)
(1,800) becoming positive
1 1,766 4 0.22
5 1,607 0 0.22
8 1,508 0 0.22
15 1,298 0 0.22

RESEARCH NOTES
404
which the snail population density was initially high are
probably never fully cleared of snails and therefore did not
achieve such a ‘bloom’. As the 1991 trial was small scale and
of only 10 days duration, it is not possible to determine the
long-term effects of such snail control methods and therefore
more extensive trials are required.
The potential of N. aperta for dispersal by drifting with the
current is a major problem facing any control programme.
Colonisation rates exceeding 90 snails/m
2
/h have been report-
ed for the -strain in the Mekong river in May.
23
In addition,
the range of N. aperta, which until recently had been thought
restricted to the Mekong and Mul rivers,
9,24
is now known
to include several Mekong tributaries in Laos.
25
N. aperta
appears able to survive the spate and lay eggs once low water
conditions are restored. Copulation may occur before the
spate in some individuals but after the spate in the majority of
snails. In years when the spate is most severe local N. aperta
populations may be destroyed leaving vacant habitats, which
are later recolonised by snails from elsewhere in the river and
probably also from the tributaries draining the mountains of
central Laos.
26
That recolonisation from such tributaries has
occurred is further supported by the genetic distances, as high
as 1.01 (Nei’s 1978 minimum standard distance
27
), between
Mekong river populations separated by only 200 km.
28
Such
genetic distances could not have arisen by random drift alone
over the time scales involved. Further, N. aperta has not been
found in the more turbid Mekong river waters and eroded
substrata stretching for 100 km upstream of Khong Island;
this again suggests that colonisation of the Mekong in Khong
District is via tributaries originating in Laos. In view of this,
snail control in areas such as Khong Island should include
potential source populations in upstream Mekong tribu-
taries. A Mekong river habitat left vacant in March or April
following mollusciciding is particularly vulnerable to rapid
recolonisation by snails from source populations, and the
result could be population densities exceeding pre-control
levels (we suggest through growth of the algal epilithon). The
problem is particularly serious in that snails from likely
source populations in central Laos have greater compati-
bility with S. mekongi than some Mekong river strains.
25
In
addition, there is the possibility that source populations also
harbour S. mekongi and could introduce the parasite to pre-
viously unaffected areas of the Mekong river.
The present data indicated a period between mid-February
and early April as the transmission season for S. mekongi at
Khong Island and this corresponds to the early low water
period in the Mekong river. Low water conditions, when
snails are most abundant and human water contact most
frequent, are thought to facilitate transmission.
21
In view of
this, and the fact that N. aperta life-cycles in some Laotian
tributaries are ahead of those at Khong, any programme of
mollusciciding is likely to achieve success if begun in the
tributaries in early February and extended to the Mekong
river itself by late February or March. In keeping with other
members of the Schistosoma japonicum group, the cercarial
output of S. mekongi in N. aperta is relatively low.
20
Fecundity
also appears relatively low in S. mekongi and an estimated
95 eggs per day (per worm) were released into the stools or
rectal mucosa, compared with 250 eggs for S. japonicum.
29
In
contrast, snail infection rates in the laboratory are over 20%
for N. aperta,
8
compared with only 5% in some S. japonicum-
Oncomelania hupensis associations.
30
It is therefore possible
that the high population densities of N. aperta and miracidial
success compensate for the lower productivity in S. mekongi.
Consequently, the control of N. aperta should not be ruled
out in the planning of future anti-schistosomiasis pro-
grammes in Laos.
It has been suggested that the construction of dams, such
as that at Pak-Mul on the Mul river close to its juncture with
the Mekong in Northeast Thailand, could (or even has) led to
the establishment of S. mekongi in Thailand.
31
However,
there is as yet there is no reliable evidence for S. mekongi in
Thailand. Populations of the -strain of N. aperta above
Pak-Mul have increased only slightly since operation of the
dam began in 1994, and it is not yet clear if this is to be a
sustained trend.
32
Available ecological data
7,9
suggest that N.
aperta will not thrive in the deeper, slower flowing and more
turbid waters found just upstream of the dam. To date N.
aperta has not been found downstream near the Pak-Mul
dam, although thorough surveys are still required. The ques-
tion of the impact of such dams is an important one, and
requires further investigation as several mainstream projects
are now planned along the lower Mekong river. The detec-
tion of S. mekongi in natural populations of N. aperta is of
further significance in the context of future parasitological
surveys. The extensive field work of Yasuraoka et al.
33,34
failed to detect S. mekongi in N. aperta populations sampled
across Khong District, despite ongoing transmission. These
authors used a snail crushing technique to detect infection,
rather than the observation of shed cercariae; this suggests
that the crushing technique is inadequate or is difficult to use
effectively.
In conclusion, the present study has demonstrated natural
S. mekongi infection in N. aperta for the first time since the
early 1970s. The findings support those of the 1999 WHO
team in southern Laos, which indicated the control of human
schistosomiasis in all surveyed areas of Khong District,
excepting Ban Hat-Xai-Khoun. The transmission period at
Khong Island during 2000 was estimated to lie between mid-
February and early April and this is relevant to the timing of
schistosomiasis control, particularly during such years of
severe flooding. A reservoir of infection has been demon-
strated after 9 years of mass chemotherapy in human popula-
tions. It is proposed that future intervention involve both
chemotherapy and snail control. Future control measures
should also be properly co-ordinated with respect to factors
of snail distribution and ecology, in particular source popula-
tions in Mekong river tributaries should be monitored.
We thank the staff of the Centre for Applied Malacology &
Entomology, Mahidol University, Bangkok, for providing
facilities for snail maintenance, and the people of Khong
Island, Champassac Province for their enthusiasm and hos-
pitality. This work was supported by Wellcome Trust Project
Grant number 058932.

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