Liver flukes, particularly C. sinensis, O. viverrini, O. felineus, and Metorchis spp., cause fatal liver cirrhosis and are regarded as an emerging public health issue globally, particularly in Southeast Asia. The flukes live in the hepatobiliary system and are considered to be carcinogenic agents because they are involved with the development of CCA in humans [11]. Despite tremendous efforts to control and mitigate hLF infections, the burden of food-borne trematode infections is increasing [10], and being reported in new areas. In this modern era when food security is given the first priority, a new segment of the global population is being exposed to food-borne zoonotic hLF infections [1, 2]. Since these flukes are essentially borne by snails and fishes, the most effective preventative strategy is to control or manage the spread of infectious agents through molluscan and fish intermediate hosts. We herein surveyed the MC of hLFs in common and popular lFWF. Also, we validated hLF species along with genetic analyses and the confirmation of molluscan vectors and reservoirs.

The results obtained showed that 18.7% lFWF were infected with the MC of hLFs. We very recently conducted a survey to detect the MC of FZTs in a few wild-caught small indigenous fishes (SIF), which revealed that FZTs, including hLFs, were prevalent in 66.2% of the selected SIF [2]. Although hLF infections have yet to be reported in humans living in Bangladesh, many human cases had already been reported in Southeast and Fareast countries, namely Thailand, China, Japan, Republic of Korea, Lao PDR, Cambodia, Vietnam, and Myanmar [28]. We detected the MC of C. sinensis, O. viverrini, O. felineus, and M. orientalis in 11 species of wild and cultured fishes belonging to different families, namely Cyprinidae, Heteropneustidae, Pangasiidae, Channidae, Clarridae, and Cichlidae, suggesting that they play significant roles as second intermediate hosts to complete the life-cycle of hLFs in Bangladesh. More than 100 fish species have been identified as second intermediate hosts of fish-borne trematodiosis. Among them, commonly consumed cyprinid fishes play a central role and act as the primary source of human infections in many Southeast Asian countries [29]. The large number of fish species reported to be infected with the MC of liver flukes implies the high adaptability of these flukes in nature (WHO, 1995). Along with cyprinid fishes, the MC of opisthorchiid flukes have been detected in Pangasiidae, Channidae [30] as well as in Tilapia spp. (family: Cichlidae) in Vietnam [31], suggesting that these fishes, other than cyprinids, also act as the second intermediate host of hLFs.

Since the fish species examined herein are very widely distributed in different natural and man-made water bodies in Bangladesh, they may contribute to human and animal infections. Among hLF infections, C. sinensis is the most important and significant species due to its public health impact. Existing endemic areas of clonorchiosis include China, Republic of Korea, North Vietnam, and Far East Russia [12]. C. sinensis infects not only cyprinids, but also an array of other fish families [28, 32] and 132 species of fishes (including 71 cyprinids) belonging to 11 families have been listed as second intermediate hosts with a prevalence ranging up to 95% in China [7]. In Republic of Korea, Pungtungia herzi, Zacco platypus, Carassius auratus, and Pseudogobio esocinus fishes were identified as second intermediate hosts of C. sinensis with prevalence rates of 13.5%, 12.9%, 10.8%, and 8.3%, respectively [33]. On the other hand, O. viverrini preferentially infects cyprinoid fishes. In the greater Mekong sub-region of Southeast Asia, more than 40 species of cyprinids were infected with O. viverrini with an extremely variable prevalence ranging between 2.1% and 100% [32] and 11 species of fishes have been identified as second intermediate hosts of O. viverrini in Cambodia with an average load of 74.6 MC per fish [34]. The Phu Yen province of Vietnam is regarded as a ‘hot spot’ for the parasite, where the prevalence of O. viverrini was 10–29% in the crucian carp and C. carassius [35]. In Thailand, five species of fishes, namely, Henicorhynchus siamensis, Cyclocheilichtys spp., Hampala spp., Systomus spp., and Barbonymus goniatus, were reported to be infected with O. viverrini MC [36]. Variations in infections by and the burden of MC among previous studies may be attributed to differences in the species of fishes, water reservoirs, fish biodiversity, the availability of vector snails and reservoir hosts, and snail-fish interactions as well as waste management, particularly the disposal of human waste and cropping patterns.

The present study suggests that wild and cultured lFWF are both equally hazardous for human health. In natural water bodies and aquaculture systems, the main source of infection and transmission include the contamination of water bodies with eggs from infected hosts, i.e., humans, cats, dogs, pigs, and other fish-eating mammals and birds. In Bangladesh, reservoir hosts roam freely and have easy access to natural water bodies and the vicinity of aquaculture systems. They defecate near the banks of rivers, ponds, and canals, and feces is washed into the water by rain. Additionally, ponds become contaminated through their use by animals and occasionally by human fecal waste as pond fertilizer and through run-off water from pond banks and adjoining fields; therefore, the risk of infection with the MC of hLFs is similar in fishes harvested from natural and man-made water bodies.

Through a well-recognized pepsin digestion method, we revealed that the MC of hLFs were equally distributed in both the gills and flesh of fishes, suggesting that the entire fish is equally important for infection. However, the gills are generally not used for human consumption and are discarded during processing, but are picked up by fish-eating mammals and birds, namely, dogs, cats, foxes, and ducks, which strongly contributes to the transmission of infection to domestic and peri-domestic reservoirs. The present study proved that the FWS-fish-domestic/peri-domestic reservoir cycle of hLFs is competent enough for the survival and existence of the flukes in nature even without infecting humans. In some studies, MC were mostly recovered from the scales, fins, and tails [37]; however, we did not detect MC in the scales and fins of fishes in the present study, which may have been due to variations in the method used to collect scales. We removed and processed scales without skin or underlying tissues. However, in some territories, scales were removed with attached skin during processing.

Regarding the molecular validation of species, we used a previously standardized ITS2 multiplex PCR, which simultaneously detects C. sinensis (381 bp) and O. viverrini (375 bp); therefore, C. sinensis and O. viverrini amplicons obtained by electrophoresis were almost indistinguishable. We digested PCR products with FauI and detected both degraded and non-degraded amplicons, which confirmed the presence of both C. sinensis and O. viverrini. The targeted part of the ITS2 gene from O. viverrini contains a site that is cleaved by FauI and, thus, the enzyme produces two fragments. However, since the same segment of the ITS2 gene from C. sinensis did not have the FauI cleavage site, the amplicons retrieved from C. sinensis remained unaffected. Various types of PCR-RFLP have been developed to validate the species of hLFs and are being utilized in different countries. By employing a PCR-RFLP analysis of the ITS2 region, a cross-sectional study was initially conducted in 2009 to identify C. sinensis infection among a population of Na-Yao villagers in Thailand. The developmental stages, eggs, and adult parasites of C. sinensis were detected in the areas examined [38].

Additionally, through bioinformatic analyses, newly retrieved sequences showed few point mutations when compared with reference sequences. Reference sequences were from other countries; therefore, a few point mutations were expected. A phylogenetic analysis revealed that our sequences produced a distinct cluster only with those of O. viverrini or C. sinensis, re-confirming the validation of the species of flukes. Moreover, using self-designed, species-specific primer sets, the amplification of the COX1 gene of C. sinensis was also successful. Similarly, sequencing and subsequent bioinformatic analyses revealed that the newly retrieved sequences had the highest identity with C. sinensis or O. viverrini, providing unambiguous proof for the confirmation of the species.

A COX1-based phylogram showed that the Bangladeshi isolates produced a very closely related cluster with the isolates from Russia and China. Although a few parts of China are nearer to Bangladesh, but far from Russia. Russia is a neighboring country of China, sharing a long common border. Since Bangladesh has a strong business relationship with China and imports many goods, including fishes, the introduction of hLF-like opisthorchiid flukes to Bangladesh from China and its neighboring countries is not unlikely. The MC of O. felineus is indistinguishable from that of O. viverrini. Therefore, a set of species-specific primers was used that amplified the ITS2 gene of O. felineus. The primer did not show any cross-reactivity [24], confirming the presence of O. felineus in the study area. In addition to PCR, sequencing and bioinformatic analyses showed that the newly retrieved sequences had the highest similarity with that of O. felineus deposited previously in GenBank, which reconfirmed the presence of the fluke in Bangladesh. A phylogenetic tree generated with the new sequences also showed a distinct cluster only with that of O. felineus, providing unambiguous proof for the confirmation of species. By applying multiple molecular tools, we unambiguously demonstrated that all three important species of major hLFs, such as O. viverrini, C. sinensis, and O. felineus, were prevalent in the study area. The simultaneous occurrence of the three species of liver flukes in Bangladesh was not surprising because fishes and fish products are both imported from neighboring endemic countries, such as Vietnam, Myanmar, Thailand, China, and India. In addition, there are frequent movements of people to these endemic countries for trade, tourism, and other purposes. Furthermore, there was a large refugee settlement to Bangladesh from Myanmar recently, which is yet to be investigated, but may be another source for the transmission of these pathogens from a recognized endemic area to Bangladesh.

The presence of M. orientalis was confirmed by a self-designed primer set targeting the 28S rRNA gene. To reinforce the present results, PCR products were also sequenced and the sequences retrieved showed very high homology with those already deposited in GenBank for M. orientalis, further validating the presence of M. orientalis in Bangladesh. This fluke has a wide host range and may infect various fish-eating mammals, aquatic birds, and humans. Water bodies are co-occupied by snails, fishes, and aquatic birds; therefore, the presence of M. orientalis is not surprising but human cases have yet to be reported.

The present study recorded PC from snails belonging to Bithynia spp., but not from other snail species, suggesting that only bithynid snails acted as the first intermediate host of opisthorchiid flukes in Bangladesh. To the best of our knowledge, this is the first study to investigate the molluscan intermediate hosts of opisthorchiid flukes in Bangladesh. Trematodes of the families Heterophyidae, Opisthorchiidae, and Cryptogonimidae produce PC [39]. By employing molecular tools, we confirmed the PC of opisthorchiid flukes from bithynid snails. Several operculate snails, such as Bithynia spp., Thaira spp., and Viviparus spp., have been reported to act as the first intermediate hosts of opisthorchiid flukes [22]. A previous study reported that the prevalence of cercarial infection in bithyniid snails in Thailand was 8.4%, and found that Bithynia siamensis harbored seven different types of cercariae, including PC [40]. In Lao PDR, PC (0.9%) were detected in Bithynia siamensis snails [41]. In India, virgulate and xiphidiocercous cercariae were detected in Bithynia pulchella snails, suggesting that Bithynia snails act as the intermediate host of various trematodes, including hLFs. In the present study, Bithynia spp. were mainly found to be attached to submerged hard objects, such as stones, boats, logs, and the leaves of aquatic plants. However, bithynid snails are frequently distributed in ponds, streams, canals, and paddy fields [42]. Their location may be deemed a suitable setting for cercariae because these habitats are co-occupied by both FWS and fishes. The co-existence of the first (snails) and second (fishes) intermediate hosts facilitates the survival of hLF in a particular area. After aggressing cercariae from FWS, cercariae easily enter fishes, in which they encyst into MC.

In the present study, the eggs of opisthorchiid flukes were detected in the feces of street dogs and cats, suggesting that these animals acted as reservoirs of opisthorchiid flukes in the areas examined. Canines and felines mainly act as the reservoirs of Opisthorchis spp. and C. sinensis. In a recent study, opisthorchiid eggs were detected in the feces of dogs and cats in other parts of Bangladesh [43]. Approximately 50 years ago, opisthorchiid flukes, such as O. tenuicollis and O. felineus, were recovered from wild cats and street dogs in Bangladesh [16]. The roles of dogs, cats, and foxes in the transmission cycle of hLFs have been evaluated in different countries. Intensive epidemiological surveillance conducted in Thailand, China, and Republic of Korea detected the ova of hLFs in fecal samples collected from dogs and cats [44,45,46]. In the study areas, dogs and cats roam freely and have easy access to natural water bodies as well as man-made fish farms. These reservoir hosts also eat raw fishes and raw fish leftovers. Their feces easily contaminate natural and man-made water bodies and, thus, plays a significant role in the transmission of hLFs and their existence in Bangladesh. In addition to canid and felid reservoirs, aquatic birds, particularly ducks, act as reservoir of M. orientalis. In the present study, we also isolated and identified M. orientalis from the livers of ducks. Ducks are the second most common and popular poultry in Bangladesh. There are approximately 66.0 million [47] ducks in Bangladesh, most of which are reared in scavenging and semi-scavenging systems. Ducks are more common in low lying marshy areas, where they have free access to paddy fields, rivers, canals, and ponds. They consume the fishes available in natural and man-made water bodies, which increases their risk of getting infection by the MC of Metorchis spp. Through this multifaceted study, we assume that the “FWS-FWF-duck” cycle plays a vital role in the existence of M. orientalis in Bangladesh.

In the present study, we only isolate adult M. orientalis from ducks but we could not isolate other adult flukes, including O. viverrini, C. sinesis, and O. felineus, because adult flukes can only be detected in a postmortem examination of reservoir hosts, such as street dogs, cats, and wild carnivores (e.g., foxes and jackals). At present in Bangladesh, the killing of street dogs, cats, and wild animals is strictly prohibited. Therefore, we were unable to isolate adult flukes from these reservoirs. Alternatively, we collected feces from street dogs and cats, detected eggs of the flukes by coprological examinations, and confirmed the species of liver flukes by copro-PCR. Another limitation of the present study is that we could not collect samples, particularly fish samples, from the entire country due to funding constrain. However, the socio-economic status, knowledge, attitude and practice of the people, geo-climatic condition, aquaculture and management prevailed in the selected study areas are almost similar throughout the country. In addition, Mymensingh and Kishoreganj are the main hub of fresh water fishes of the country and from these two areas caught fishes are mainly distributed to other parts of Bangladesh.