Abstract
Fasciola-induced fascioliasis is a zoonotic disease with significant health and economic impacts on humans and livestock. Freshwater Lymnaea snails serve as intermediate hosts, contributing to the increasing prevalence of fascioliasis in cattle in coastal areas. The salinity tolerance of Lymnaea snails was investigated along with their distribution and Fasciola infection rates in both snails and grazing cattle in Ben Tre, Tra Vinh, and Soc Trang provinces in Mekong Delta, Vietnam, where seawater reversely enters into the paddy field during the dry season. Lymnaea snails were collected from 53 communes across the three provinces and analyzed for Fasciola larval infections. Additionally, cattle fecal samples were examined for the presence of Fasciola eggs. The salinity levels in the study areas ranged from 0.03% to 1.90%. In total, 1,152 Lymnaea snails including L. viridis and L. rubiginosa were collected. The maximum salinity levels of L. viridis and L. rubiginosa habitats were 0.64% and 0.74%, respectively. The Fasciola infection rates of L. viridis were 2.0% in low-salinity areas and 3.7% in high-salinity areas, whereas no L. rubiginosa infections were detected in either salinity area. The prevalence of cattle fascioliasis in high-salinity areas (35.7%) was similar to low-salinity areas (32.9%). This study is the first to demonstrate that L. viridis can thrive and sustain Fasciola transmission under high-salinity conditions, suggesting the salinity tolerance of these intermediate host snails as a key factor contributing to the expansion of fascioliasis in high-salinity coastal areas.
Keywords: cattle, Fasciola, high-salinity coastal area, Lymnaea snails, Mekong Delta Vietnam
INTRODUCTION
Fascioliasis is a significant yet often underreported parasitic disease affecting herbivorous livestock and humans. It is caused by liver flukes, digenean trematodes of the genus Fasciola. Freshwater snails of the genus Lymnaea serve as intermediate hosts for Fasciola species [10, 17, 21, 22]. Fascioliasis is recognized as a major zoonotic disease and affects approximately 2.4–17 million people worldwide [11, 43]. In addition to its effect on human health, this disease causes substantial economic losses in livestock farming, with annual damage exceeding $3.2 billion in developing and underdeveloped regions [14, 27].
Historically, fascioliasis was primarily associated with lowland paddy field regions, while highland and coastal areas exhibited lower prevalence rates [1, 8, 33, 35, 42, 48]. However, recent studies reported a high prevalence of fascioliasis in coastal regions. In Vietnam, fascioliasis was historically rare in coastal areas. In Ben Tre province, the prevalence ranged from 1.42% to 3.27% in 1987 [13]. In south-central coastal regions, the prevalence was reported at 4.17% in 1989, compared with a significantly higher prevalence of 44.5% in plain areas in the same period [47]. However, recent studies have revealed a notable increase in the prevalence rate in coastal areas, coinciding with the introduction of irrigation systems. In Thua Thien Hue province, a coastal region, the prevalence rate rose to 31.0% by 2016 [31]. In Ben Tre, Tra Vinh, and Soc Trang provinces, the prevalence rates reached 15.97%, 15.78%, and 14.33%, respectively, in 2016 [12]. The expansion of irrigation systems into brackish water areas was aimed at supplying freshwater for rice cultivation. However, these systems cannot fully mitigate the effects of seawater intrusion in the Mekong Delta during the dry season [6, 30]. Such conditions present significant challenges, as freshwater snails in these areas are exposed to salinity in their habitats. Consequently, the salinity tolerance of Lymnaea may play a critical role in facilitating the expansion of fascioliasis in coastal areas.
Salinity is a key abiotic factor affecting the physiology, distribution, and tolerance of aquatic organisms, including mollusks [19, 36]. Studies investigating salinity tolerance in freshwater mollusks have revealed various salinity tolerance levels. For instance, Physa acuta exhibited reduced survival and reproductive rates at 0.7% salinity and failed to survive at 0.9% salinity [38]. Similarly, Biomphalaria arabica demonstrated a salinity tolerance threshold of 0.72% [28], whereas Indoplanorbis exustus had a lethal salt concentration of 0.5% [44]. As for Lymnaea snails, L. stagnalis showed a salinity tolerance limit of 85.55 mmol/L (0.5%) [3]. However, these studies were conducted under controlled laboratory conditions, which may not fully reflect salinity tolerance in natural environments. Notably, limited information exists on the salinity tolerance of L. viridis, which is commonly reported as an intermediate host for Fasciola parasites in Asian countries, including Vietnam [9, 17, 23, 32]. To address this gap, this study aimed to investigate the salinity tolerance of Lymnaea spp. and their Fasciola parasite infectivity under varying salinity conditions, along with cattle prevalence, in the Mekong Delta region of Vietnam. These findings aim to elucidate the role of salinity tolerance in the spread of cattle fascioliasis in coastal areas.
MATERIALS AND METHODS
Study area
The provinces of Ben Tre, Tra Vinh, and Soc Trang are coastal regions in Mekong Delta, Vietnam (Fig. 1). The annual average temperature ranges from 25°C to 27°C, with the highest temperature occurring in April and lowest in January. The dry season lasts from December to April, whereas the rainy season occurs in the remaining months. The study area included communes in these provinces, selected based on river salinity levels [29], to compare low- and high-salinity areas. In total, 53 communes were assessed: 19 in Ben Tre, 17 in Tra Vinh, and 17 in Soc Trang.
Fig. 1.
Study area. A: Map of Vietnam showing the Ben Tre, Tra Vinh, and Soc Trang provinces shaded in gray. B: Enlarged map of these three provinces. Water reservoirs are depicted in light blue, and numbers represent the communes. The gradient color in the communes corresponds to the salinity levels, as detailed in Table 1. Communes where Lymnaea snails were found are outlined with a red border.
Salinity level measurements
Salt concentrations were measured in different water habitats, including paddy fields, canals, and pond sites, using a digital salinometer (LAQUAtwin Salt-22, HORIBA, Kyoto, Japan). Salinity level was measured at 2 or 3 points in each water body within 1 meter away from the edge. Water with a salinity level below 0.5% was classified as low-salinity, whereas water with a salinity level of ≥0.5% was classified as high-salinity.
Collection and identification of Lymnaea snails
From March to April 2024, snails were collected at sites with measured salinity levels. At each collection site, snails were collected from five 1 m2 areas to calculate density. All snails on the surface and those attached to aquatic plants were manually collected. The average snail density (snails/m2) of each site was calculated based on snail counts at the five locations where at least one snail was found. Samples were transported to the laboratory, where Lymnaea snails were identified morphologically according to the standard protocol described by Dang et al. [7] and taxonomic keys outlined by Vinarski et al. [46]. The identified snails were then counted by species. Within 7 days of collection, the snails were examined in the laboratory for the presence of Fasciola larvae.
Detection and identification of Fasciola larval forms in Lymnaea snails
Snails were crushed between two glass slides and carefully examined under a stereo microscope. When Fasciola-like larval forms (sporocysts, rediae, or cercariae) were detected, they were aspirated, placed on slides, and further examined under a light microscope at 400× magnification. The Fasciola cercariae was identified based on a trematode cercaria identification key, as outlined by Pham and Nguyen [34]. A single cercaria was preserved in 70% ethanol for molecular confirmation.
Molecular confirmation of Lymnaea snail spp. and Fasciola larvae from infected snails
Two to three snail tissue samples from each morphologically identified snail spp. were preserved in 70% ethanol for molecular confirmation. The DNA was individually extracted using Invitrogen™ DNAzol™ Reagent (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s protocol. Similarly, single ethanol-fixed Fasciola cercariae were washed with distilled water prior to DNA extraction, using the same method. The internal transcribed spacer 2 (ITS-2) region of Lymnaea snails was amplified using the primers ITS-2-NEWS (5′-TGTGTCGATGAAGAACGCAG-3′) and ITS-2-RIXO (5′-TTCTATGCTTAAATTCAGGGG-3′), as previously described by Corea et al. [5]. For Fasciola parasites, the ITS-2 region was amplified using the primers ITS2-F (5′-TGTGTCGATGAAGAGCGCAG-3′) and ITS2-R (5′-TGGTTAGTTTCTTTTCCTCCGC-3′), following the protocol outlined by Itagaki et al. [18]. PCR reactions were performed using KOD FX polymerase (Toyobo, Osaka, Japan). The PCR protocol included an initial denaturation at 94°C for 3 min, followed by 35 cycles of denaturation at 98°C for 10 sec, annealing at 55°C for 30 sec, and extension at 68°C for 45 sec, with a final extension at 68°C for 15 min. The PCR products were subsequently purified using the MagExtractor™ PCR & Gel Clean Up Kit (Toyobo). Purified fragments were sequenced by FASMAC. Co (Atsugi, Japan). Sequences from three Fasciola samples, each from the three studied provinces, were analyzed. Obtained sequences were validated using a BLAST search and aligned for phylogenetic analysis. The ITS2 regions of Lymnaea spp. and Fasciola spp. were identified by aligning with references sequences of Galba truncatula (KC248372) and F. gigantica (MF678651), respectively, and used. A neighbor-joining phylogenetic tree was constructed using MEGA v11 software [41]. The analysis was run under the Kiumura 2-parameter model with a bootstrap analysis of 1,000 replicates to evaluate reliability. Identical sequences were consolidated and represented by a single sequence in the phylogenetic tree. For sequence analysis, L. stagnalis (GenBank: FR797834) was used as the outgroup for snails, while Paragonimus westermani (GenBank: LC578473) served as the outgroup for Fasciola sp.
Cattle fecal sample collection
Fecal samples were collected from domestic yellow cattle raised in the study areas, where Lymnaea snails were present, between March and April 2024. The cattle were randomly selected, regardless of age or sex. Cattle fecal samples were collected immediately after defecation to ensure freshness and individuality. To avoid the double-counting of individuals on different sampling days, samples were collected only once per commune. A minimum of 10 fecal samples were collected from each commune, except for Tan Thanh (3) and My Hung (12) in Ben Tre province where only 9 and 7 samples were obtained, respectively, due to the limited number of cattle. For each sampled cattle, the owner provided age estimates, and the sex of each animal was recorded, although these data were not separately analyzed. Fresh fecal samples, collected within 5–10 min post-defecation, were immediately placed in individual plastic bags without preservatives. The samples were stored at 4–8°C and processed for coprological examination within 1 week of collection.
Coprological examination
The sedimentation technique was used to detect Fasciola eggs in fecal samples. Briefly, 3 g of feces were suspended in 800 mL of tap water and filtered through a 250-µm pore sieve to remove large debris. The suspension was allowed to settle for 15 min, after which the supernatant was carefully decanted. The sediment was resuspended in 800 mL of tap water, and the sedimentation process was repeated three times. Finally, the remaining 30 mL of the fecal suspension was thoroughly mixed, and 10 mL aliquots were transferred into Petri dishes for microscopic examination at 10× magnification. A sample was considered positive if a Fasciola egg with the correct morphology, as described by Hussein et al. [15], Andrews et al. [2], and Valero et al. [45], was detected. The examination process continued until a Fasciola egg was found or the entire 30 mL of the suspension had been analyzed.
Statistical analysis
Statistical analysis was performed using R Statistical Software (version 4.3.1; R Core Team 2023). The χ2 test was applied where appropriate, and a P value of <0.05 was considered significant.
RESULTS
Salinity level in study areas
The salinity levels were assessed in different habitats (Table 1). In low-salinity areas including 17 communes, the salinity levels ranged from 0.03% to 0.15%. By contrast, high-salinity areas with 36 communes exhibited greater variability, with salinity levels ranging from 0.54% to 1.9%. Salinity levels were measured in each water body where Lymnaea snails were detected, with an average of two to three water bodies recorded per habitat. Paddy fields in the area exhibited a maximum salinity level of 0.80%. Canal salinity ranged from 0.05% to 0.87%. Meanwhile, salinity levels ranging from 1.00% to 1.90% were observed only in shrimp cultivation ponds. In this study, Lymnaea snails were found in a canal in My Hoa, with a maximum salinity level of 0.74% (30).
Table 1. Salinity levels in the study areas along with the presence or absence of snails.
| Ben Tre |
Tra Vinh |
Soc Trang |
||||||
|---|---|---|---|---|---|---|---|---|
| Communes | Salinitya
(%) |
Lymnaea snail | Communes | Salinity (%) |
Lymnaea snail | Communes | Salinity (%) |
Lymnaea snail |
| Phong My (1)b | 0.03 | (+)c | Nhi Long (20) | 0.06 | (−)c | Long Hung (37) | 0.05 | (+) |
| Binh Thanh (2) | 0.06 | (−) | Tan An (21) | 0.07 | (+) | My Tu (38) | 0.05 | (+) |
| Giong Trom (3) | 0.06 | (−) | Dai Phuc (22) | 0.08 | (−) | My Huong (39) | 0.06 | (+) |
| Tan Thanh (4) | 0.08 | (+) | Binh Phu (23) | 0.09 | (−) | My Thuan (40) | 0.07 | (−) |
| Binh Hoa (5) | 0.08 | (−) | Huyen Hoi (24) | 0.15 | (+) | Thuan Hung (41) | 0.07 | (−) |
| Hung Nhuong (6) | 0.10d | (+) | Thanh Hoa Son (25) | 0.58 | (+) | An Ninh (42) | 0.08 | (−) |
| Thanh Phu Town (7) | 0.54 | (+) | Don Xuan (26) | 0.60 | (+) | Tham Don (43) | 0.54 | (+) |
| Vinh Hoa (8) | 0.55 | (+) | Ngu Lac (27) | 0.61 | (+) | Dai Tam (44) | 0.56 | (+) |
| Phu Ngai (9) | 0.61 | (+) | Don Chau (28) | 0.62 | (+) | My Xuyen Town (45) | 0.57 | (+) |
| Vinh An (10) | 0.64 | (+) | My Long Bac (29) | 0.64 | (+) | Vien An (46) | 0.62 | (+) |
| Hoa Loi (11) | 0.65 | (+) | My Hoa (30) | 0.74d | (+) | Lich Hoi Thuong Town (47) | 0.63 | (+) |
| My Hung (12) | 0.73 | (+) | Hiep My Tay (31) | 0.81e | (−) | Lieu Tu (48) | 0.69d | (+) |
| An Hoa Tay (13) | 0.73 | (−) | My Long Nam (32) | 1.00f | (−) | Hoa Tu (49) | 0.74 | (−) |
| An Thuan (14) | 1.48f | (−) | Hiep My Dong (33) | 1.30f | (−) | Ngoc Dong (50) | 0.80 | (−) |
| Bao Thuan (15) | 1.52f | (−) | Long Toan (34) | 1.80f | (−) | Ngoc To (51) | 0.87e | (−) |
| Bao Thanh (16) | 1.60f | (−) | Long Vinh (35) | 1.80f | (−) | Trung Binh (52) | 1.56f | (−) |
| Tan Thuy (17) | 1.60f | (−) | Long Huu (36) | 1.90f | (−) | Lich Hoi Thuong (53) | 1.70f | (−) |
| An Thanh (18) | 1.62f | (−) | ||||||
| Binh Thanh (19) | 1.80f | (−) | ||||||
a Salinity levels of paddy fields were presented unless no water remained in paddy field or snails found only in canals. b Number in the parentheses after commune names correspond to the number in Fig. 1 which indicates the place of the communes. c (+) means snails were found, (−) means no snails were found. d Salinity level of canal from where snails were collected was presented. e Salinity levels of canals were presented due to no water in paddy fields. f Salinity levels of pond were presented due to no paddy fields and canals.
Lymnaea snail distribution in salinity measured study area
Lymnaea snails were found in 27 of 53 surveyed communes (Table 1). This included nine communes in low-salinity areas, with salinity levels ranging from 0.03% to 0.15%, and 18 communes in high-salinity areas, with salinity levels ranging from 0.54% to 0.74% (Table 1). Subsequent analyses focused on communes where Lymnaea snails were detected. A total of 1,152 snails were collected and morphologically identified as L. viridis and L. rubiginosa. Various aquatic habitats, including paddy fields, canals, and ponds, were surveyed for the presence of these snails, with no snails detected in pond habitats. L. viridis snails were exclusively found in paddy fields in 20 communes, whereas L. rubiginosa snails were detected in both paddy fields and canals in eight communes. The distribution of Lymnaea snails is summarized in Table 2. Paddy fields were the primary habitat for Lymnaea snails in both low- and high-salinity areas. L. viridis snails were found in paddy fields, with 198 snails detected in low-salinity regions and 648 snails in high-salinity regions. The density of L. viridis varied substantially, ranging from a minimum of 3.0 snails/m2 in Lich Hoi Thuong Town (47) to a maximum of 27.8 snails/m2 in Phu Ngai (9). L. rubiginosa was also present in paddy fields, with 65 snails detected in low-salinity areas and 128 snails in high-salinity areas. The highest density of L. rubiginosa in paddy fields was observed in My Hung (12) at 14.8 snails/m2. In canal habitats, only L. rubiginosa was detected in both low- and high-salinity areas. In low-salinity canals, 56 L. rubiginosa snails were recorded in Hung Nhuong (6). In high-salinity regions, 57 snails were found in My Hoa (30) and Lieu Tu (48). The highest density of L. rubiginosa in canal habitats was recorded in Hung Nhuong (6) at 11.2 snails/m2. L. viridis was found at the highest salinity level of 0.64% in My Long Bac (29) paddy field, whereas L. rubiginosa was observed in My Hoa (30) canal, with a maximum salinity of 0.74%.
Table 2. Habitats of collected Lymnaea snails in the study areas along with their species and densities (snails/m2).
| Province | Salinitya | Commune | Habitats |
|||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Paddy fields |
Canal |
|||||||||
| L. viridis | L. rubiginosa | L. viridis | L. rubiginosa | |||||||
| Ben Tre | Low | Phong My (1)b, 0.03c | 23 | (4.6) | 0 | 0 | 0 | |||
| Tan Thanh (4), 0.08 | 37 | (7.4) | 0 | 0 | 0 | |||||
| Hung Nhuong (6), 0.10 | 0 | 0 | 0 | 56 | (11.2) | |||||
| High | Thanh Phu Town (7), 0.54 | 77 | (15.4) | 21 | (4.2) | 0 | 0 | |||
| Vinh Hoa (8), 0.55 | 16 | (3.2) | 0 | 0 | 0 | |||||
| Phu Ngai (9), 0.61 | 139 | (27.8) | 0 | 0 | 0 | |||||
| Vinh An (10), 0.64 | 33 | (6.6) | 0 | 0 | 0 | |||||
| Hoa Loi (11), 0.65 | 0 | 33 | (6.6) | 0 | 0 | |||||
| My Hung (12), 0.73 | 0 | 74 | (14.8) | 0 | 0 | |||||
| Tra Vinh | Low | Tan An (21), 0.07 | 32 | (6.4) | 0 | 0 | 0 | |||
| Binh Phu (23), 0.09 | 26 | (5.2) | 0 | 0 | 0 | |||||
| Huyen Hoi (24), 0.15 | 0 | 19 | (3.8) | 0 | 0 | |||||
| High | Thanh Hoa Son (25), 0.58 | 44 | (8.8) | 0 | 0 | 0 | ||||
| Don Xuan (26), 0.60 | 44 | (8.8) | 0 | 0 | 0 | |||||
| Ngu Lac (27), 0.61 | 117 | (23.4) | 0 | 0 | 0 | |||||
| Don Chau (28), 0.62 | 38 | (7.6) | 0 | 0 | 0 | |||||
| My Long Bac (29), 0.64 | 6 | (1.2) | 0 | 0 | 0 | |||||
| My Hoa (30), 0.74 | 0 | 0 | 0 | 23 | (4.6) | |||||
| Soc Trang | Low | Long Hung (37), 0.05 | 0 | 46 | (9.2) | 0 | 0 | |||
| My Tu (38), 0.05 | 26 | (5.2) | 0 | 0 | 0 | |||||
| My Huong (39), 0.06 | 54 | (10.8) | 0 | 0 | 0 | |||||
| High | Tham Don (43), 0.54 | 45 | (9.0) | 0 | 0 | 0 | ||||
| Dai Tam (44), 0.56 | 23 | (4.6) | 0 | 0 | 0 | |||||
| My Xuyen Town (45), 0.57 | 30 | (6.0) | 0 | 0 | 0 | |||||
| Vien An (46), 0.62 | 21 | (4.2) | 0 | 0 | 0 | |||||
| Lich Hoi Thuong Town (47), 0.63 | 15 | (3.0) | 0 | 0 | 0 | |||||
| Lieu Tu (48), 0.69 | 0 | 0 | 0 | 34 | (6.8) | |||||
| Subtotal | Low | 198 | 65 | 0 | 56 | |||||
| High | 648 | 128 | 0 | 57 | ||||||
| Total | 846 | 193 | 0 | 113 | ||||||
a Salinity level below 0.5% was classified as low-salinity, whereas water with a salinity level of ≥0.5% was classified as high-salinity. b Number in the parentheses after commune names correspond to the number in Fig. 1 which indicates the place of the communes. c Salinity level where snails were found.
Infectivity of Fasciola sp. in Lymnaea snails in Mekong Delta, Vietnam
Overall, 939 Lymnaea snails were examined for Fasciola larval infection. Only Fasciola cercariae were found. Fasciola infection was only detected in L. viridis snails, whereas no Fasciola infection was observed in L. rubiginosa snails (Table 3). Only Echinostoma sp. cercariae and metacercariae were detected other than Fasciola larvae (data not shown). Moreover, Fasciola infections were found in L. viridis snails from low- and high-salinity areas. A total of 663 L. viridis snails—198 from low-salinity areas and 465 from high-salinity areas—were examined. Among these, four snails from low-salinity areas and 17 snails from high-salinity areas were infected. The highest Fasciola infectivity (6.7%) rate was documented in Tham Don (43), at a salinity level of 0.54% (Table 3). The average Fasciola infection rates in L. viridis snails were 2.0% in low-salinity areas and 3.7% in high-salinity areas. Statistical analysis showed no significant difference in Fasciola infection rates between low- and high-salinity areas (χ2=0.737, P>0.05).
Table 3. Fasciola infectivity of Lymnaea snails in Ben Tre, Tra Vinh, Soc Trang provinces.
| Province | Salinitya | Commune |
L. viridis
|
L. rubiginosa |
||||
|---|---|---|---|---|---|---|---|---|
| No. Examined |
No. Infected |
Infectivity (%) |
No. Examined |
No. Infected |
Infectivity (%) |
|||
| Ben Tre | Low | Phong My (1)b | 23 | 0 | 0 | - | - | - |
| Tan Thanh (4) | 37 | 1 | 2.7 | - | - | - | ||
| Hung Nhuong (6) | - | - | - | 50 | 0 | 0 | ||
| High | Thanh Phu Town (7) | 50 | 3 | 6.6 | 21 | 0 | 0 | |
| Vinh Hoa (8) | 16 | 1 | 6.3 | - | - | - | ||
| Phu Ngai (9) | 50 | 0 | 0 | - | - | - | ||
| Vinh An (10) | 33 | 2 | 6.1 | - | - | - | ||
| Hoa Loi (11) | - | - | - | 33 | 0 | 0 | ||
| My Hung (12) | - | - | - | 50 | 0 | 0 | ||
| Tra Vinh | Low | Tan An (21) | 32 | 1 | 3.1 | - | - | - |
| Binh Phu (23) | 26 | 0 | 0 | - | - | - | ||
| Huyen Hoi (24) | - | - | - | 19 | 0 | 0 | ||
| High | Thanh Hoa Son (25) | 44 | 2 | 4.5 | - | - | - | |
| Don Xuan (26) | 44 | 1 | 2.3 | - | - | - | ||
| Ngu Lac (27) | 50 | 2 | 4.4 | - | - | - | ||
| Don Chau (28) | 38 | 1 | 2.6 | - | - | - | ||
| My Long Bac (29) | 6 | 0 | 0 | - | - | - | ||
| My Hoa (30) | - | - | - | 23 | 0 | 0 | ||
| Soc Trang | Low | Long Hung (37) | - | - | - | 46 | 0 | 0 |
| My Tu (38) | 26 | 0 | 0 | - | - | - | ||
| My Huong (39) | 54 | 2 | 3.7 | - | - | - | ||
| High | Tham Don (43) | 45 | 3 | 6.7 | - | - | - | |
| Dai Tam (44) | 23 | 1 | 4.3 | - | - | - | ||
| My Xuyen Town (45) | 30 | 1 | 3.3 | - | - | - | ||
| Vien An (46) | 21 | 0 | 0 | - | - | - | ||
| Lich Hoi Thuong Town (47) | 15 | 0 | 0 | - | - | - | ||
| Lieu Tu (48) | - | - | - | 34 | 0 | 0 | ||
| Subtotal | Low | 198 | 4 | 2 | 115 | 0 | 0 | |
| High | 465 | 17 | 3.7 | 161 | 0 | 0 | ||
| Total | 663 | 21 | 3.2 | 276 | 0 | 0 | ||
a Salinity level below 0.5% was classified as low-salinity, whereas water with a salinity level of ≥0.5% was classified as high-salinity. b Number in the parentheses after commune names correspond to the number in Fig. 1 which indicates the place of the communes.
Molecular confirmation of Lymnaea snail sp. and Fasciola cercaria from infected snails
The ITS-2 sequences of L. viridis and L. rubiginosa supported the morphological identification and clustered with L. viridis and L. rubiginosa, respectively (Fig. 2). In the phylogenetic tree made by neighbor-joining method displayed in Fig. 2, two L. viridis sequences from this study (LC856635) were identical and clustered with Austropeplea viridis (KF042387, synonym of L. viridis and Orientogalba viridis) isolates from Vietnam. Similarly, three ITS-2 sequences of L. rubiginosa in this study (LC856763) were identical and grouped with Radix rubiginosa (synonym of L. rubiginosa), most closely aligning with an isolate from the Kien Giang Province in Mekong Delta, Vietnam (LC659106). The species of the isolated snails in this study was also confirmed by the phylogenic analysis using maximum likelihood method (Supplementary Fig. 1).
Fig. 2.
Morphological features and phylogenetic trees based on the internal transcribed spacer 2 sequences of Lymnaea viridis (A) and L. rubiginosa (B) in Mekong Delta. L. stagnalis sequence (FR797834) was used as an outgroup for analysis. Evolutionary history was inferred using the neighbor-joining method. The optimal tree is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is presented next to the branches.
As shown in Fig. 3, the ITS-2 sequence of Fasciola cercaria isolated from infected snails (LC856764) clustered with F. gigantica. The sequence was most closely related to an isolate from Ninh Thuan, Vietnam (MT429181). The Fasciola species in this study was also confirmed by the phylogenic analysis using maximum likelihood method (Supplementary Fig. 2).
Fig. 3.
Phylogenetic relationship of Fasciola parasites based on the internal transcribed spacer 2 sequences. Evolutionary history was inferred using the neighbor-joining method. The optimal tree is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are presented next to the branches. This analysis involved 15 Fasciola spp. sequences with one Paragonimus westermani sequence (LC578473) as the outgroup.
Prevalence of cattle fascioliasis
In total, 471 cattle fecal samples were collected to assess the prevalence of fascioliasis in low- and high-salinity areas (Table 4). Fasciola eggs were detected in cattle from 25 out of 27 communes sampled. The highest prevalence rate was observed in the high-salinity area of Thanh Phu Town (7), where 62.5% of cattle tested positive for Fasciola at a salinity level of 0.54%, with both L. viridis and L. rubiginosa present. Don Chau (28) closely followed this at a prevalence of 60.0% and salinity level of 0.62% (Table 4). The average prevalence of Fasciola spp. was slightly higher in high-salinity areas (35.7%) compared with that in low-salinity areas (32.9%), although the differences were not significant (χ2=0.239, P>0.05).
Table 4. Prevalence of cattle fascioliasis where Lymnaea snails were found.
| Province | Salinitya | Commune | No. Examined | No. Positive | Prevalence% | Snail b |
|---|---|---|---|---|---|---|
| Ben Tre | Low | Phong My (1)c | 12 | 6 | 50 | V |
| Tan Thanh (4) | 9 | 0 | 0 | V | ||
| Hung Nhuong (6) | 14 | 0 | 0 | R | ||
| High | Thanh Phu Town (7) | 24 | 15 | 62.5 | V, R | |
| Vinh Hoa (8) | 15 | 3 | 20 | V | ||
| Phu Ngai (9) | 24 | 12 | 50 | V | ||
| Vinh An (10) | 18 | 6 | 33.3 | V | ||
| Hoa Loi (11) | 30 | 6 | 20 | R | ||
| My Hung (12) | 7 | 2 | 28.6 | R | ||
| Tra Vinh | Low | Tan An (21) | 18 | 9 | 50 | V |
| Binh Phu (23) | 15 | 6 | 40 | V | ||
| Huyen Hoi (24) | 18 | 9 | 50 | R | ||
| High | Thanh Hoa Son (25) | 12 | 3 | 25 | V | |
| Don Xuan (26) | 15 | 6 | 40 | V | ||
| Ngu Lac (27) | 21 | 9 | 42.9 | V | ||
| Don Chau (28) | 15 | 9 | 60 | V | ||
| My Long Bac (29) | 15 | 3 | 20 | V | ||
| My Hoa (30) | 18 | 6 | 33.3 | R | ||
| Soc Trang | Low | Long Hung (37) | 21 | 6 | 28.6 | R |
| My Tu (38) | 21 | 6 | 28.6 | V | ||
| My Huong (39) | 18 | 6 | 33.3 | V | ||
| High | Tham Don (43) | 12 | 6 | 50 | V | |
| Dai Tam (44) | 18 | 6 | 33.3 | V | ||
| My Xuyen Town (45) | 21 | 3 | 14.3 | V | ||
| Vien An (46) | 24 | 12 | 50 | V | ||
| Lich Hoi Thuong Town (47) | 15 | 3 | 20 | V | ||
| Lieu Tu (48) | 21 | 6 | 28.6 | R | ||
| Subtotal | Low | 146 | 48 | 32.9 | ||
| High | 325 | 116 | 35.7 | |||
| Total | 471 | 164 | 34.8 | |||
a Salinity level below 0.5% was classified as low-salinity, whereas water with a salinity level of ≥0.5% was classified as high-salinity. b Snails species collected in each commune, V and R indicated L. viridis and L. rubiginosa, respectively. c Number in the parentheses after commune names correspond to the number in Fig. 1 which indicates the place of the communes.
DISCUSSION
The salinity levels in the water bodies of Ben Tre, Tra Vinh, and Soc Trang provinces ranged from 0.03% to 1.90% (Table 1). The highest salinity was observed in Long Huu (36), at 1.90%, where seawater was introduced for shrimp cultivation. This value is comparable to the maximum salinity level of 1.82% observed in the fields during the same period (1–10 April 2024) across three provinces, as documented in the saline intrusion forecast report in southern Vietnam [29].
The survey revealed that L. viridis and L. rubiginosa snails could survive salinity levels of 0.64% and 0.74%, respectively (Table 2). This study is the first to identify freshwater snail habitats under such salinity conditions. These thresholds are similar to those reported in previous studies on other freshwater snails, including P. acuta (0.7%) [38], and B. arabica (0.72%) [28], but were higher compared with those for L. stagnalis (0.5%) [3] and I. exustus (0.5%) [44] under controlled experimental conditions. The variations in salinity tolerance among species may be attributed to differences in snail size. Suresh et al. reported that larger Indoplanorbis and Lymnaea snails have significantly lower sodium concentrations in their body fluids compared with smaller snails [39]. The higher salinity tolerance of L. viridis and L. rubiginosa snails compared with L. stagnalis snails may be due to their generally smaller size [24, 25, 37]. In this study, the absence of snails at salinity levels higher than 0.74% suggests that this salt concentration might be lethal for L. viridis and L. rubiginosa snails.
The absence of Lymnaea snails in nine low-salinity communes (Table 1) may be attributed to agricultural practices. During the survey period, which was scheduled during the dry season and post-harvest phase of rice cultivation, many paddy fields were drying out. Additionally, farmers had applied molluscicides in paddy fields [16] to control apple snails (Pomacea sp.), which are highly destructive to rice crops.
In this study, paddy fields were the predominant habitat for Lymnaea snails in both low- and high-salinity areas (Table 2). L. viridis snails were found in shallow areas, whereas L. rubiginosa snails were predominantly detected in deeper water areas, often floating on the water surface. By contrast, canals served as a habitat exclusively for L. rubiginosa snails, which were found in both low- and high-salinity areas. This finding highlights the L. rubiginosa snails’ ability to occupy various aquatic habitats compared with that of L. viridis snails. The presence of L. rubiginosa snails in canals may indicate their higher salinity tolerance, as these canals are linked to high-salinity rivers owing to seawater intrusion and exhibit relatively higher salinity levels compared with the surrounding paddy fields [26, 31].
The detection of Fasciola larvae in infected L. viridis from both low-salinity (2.0%) and high-salinity areas (3.7%) demonstrates the ability of this snail species to act as an intermediate host under these salinity conditions (Table 3). All L. viridis snails were found in the paddy fields (Table 2), which serve as post-harvest grazing areas. Considering that L. viridis snails in paddy fields have a higher likelihood of encountering Fasciola-infected cattle, they may play a significant role in driving the Fasciola life cycle, particularly in high-salinity areas. In contrast, Fasciola infection was not observed in L. rubiginosa snails in this study. L. rubiginosa snails have been reported as vectors of Fasciola infection in Thailand [4, 20, 26, 40]. Kaset et al. reported that in Thailand, Fasciola-infected L. rubiginosa was exclusively found in paddy fields and not in canals [20]. One possible explanation for the absence of infections in L. rubiginosa from canals (Table 2) may be due to the fewer opportunities for exposure to Fasciola miracidia compared with those found in paddy fields. The flow in canals might also interfere the miracidia infection to snails. Charoenchai et al. documented an infection rate of 3.5% (60/1,735) for L. rubiginosa with Fasciola larvae in Thailand [4]. In Vietnam, Dung et al. reported the absence of Fasciola infection in L. rubiginosa snails after examining 1,000 snails [10]. In this study, 285 L. rubiginosa snails were collected. L. rubiginosa snails may not be a suitable vector in Vietnam. However, owing to its ability to thrive in saline habitats, the potential of Fasciola infection in L. rubiginosa snails should still be considered.
The present study demonstrated that Lymnaea snails inhabit high-salinity areas and may contribute to the spread of fascioliasis in these high-salinity coastal regions. However, if their salinity tolerance supported their survival in high-salinity areas, the question arises as to why the Fasciola prevalence was low (1.42–3.27%) in 1987 [13], before the introduction of irrigation. The recent elevation in cattle fascioliasis prevalence associated with the increased abundance of Lymnaea snails in high-salinity coastal areas may be linked to irrigation practices. Irrigation could have diluted the paddy field salinity from lethal levels (>0.8%) to sublethal levels. Future research should focus on the long-term monitoring of the interactions between the Fasciola parasite, its intermediate host, and cattle in high-salinity areas to gain insights on the risk of fascioliasis in high-salinity coastal regions.
CONFLICT OF INTEREST
The authors declare there is no conflict of interest in this study.
Supplementary
Acknowledgments
The Japan Science and Technology Agency partially supported this research under the Fostering Advanced Human Resources to Lead Green Transformation project at the University of Tokyo (grant number: JPMJSP2108).
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