Fasciolosis is a parasitic disease with worldwide distribution, excluding Antarctica. In livestock, it has major economic implications with estimated worldwide economic losses amounting to USD 3,200 million, including anthelmintic treatments, control of intermediate hosts (molluscicides), research, and the implication of economic losses in dairy and meat livestock production (1, 2).

Human fasciolosis has persisted since prehistoric times (3), and currently, it has a significant global health impact in specific geographic locations. The World Health Organization (WHO) has classified fasciolosis as a neglected tropical disease (4), and it is the most geographically distributed parasitic zoonosis (5, 6). F. hepatica human infections range between 2.4 and 17 million people (7), with 91 to 180 million people at risk of infection annually (8, 9).

Currently, the control of fasciolosis in ruminants continues to be based on management measures such as pasture rotation and the use of anthelmintics (10). The continued use of anthelmintics has resulted in an increase in parasite-resistant strains for the most effective and widely used flukicides, such as triclabendazole and albendazole (11, 12). Over the past three decades, there has been a rising interest in obtaining vaccines that help prevent and control fasciolosis in ruminants (13). However, the development of vaccines against fasciolosis has been slow, partly due to the great immunomodulatory capacity of the parasite. Hence, a better understanding of the parasite-host interactions is necessary for a more rational design of new vaccine candidates (14, 15).

Fasciolosis is caused by flukes of the genus Fasciola, known as liver flukes. The two species most implicated as the etiologic agents of fasciolosis are F. hepatica, which is distributed mainly in temperate climate regions, and F. gigantica, which is located in tropical regions. Further, hybrid forms have been described in regions where the two species coexist (16, 17). Real-time PCR (qPCR) targeting ITS1 rDNA, ITS2 rDNA, and 28S rDNA have been used to differentiate the two distinct genetic signatures representing each species (18–20). The epidemiological potential of hybridization and introgression between F. hepatica and F. gigantica remains unknown; therefore, it is important to use the correct terminology consistently and not use the two terms interchangeably (21).

The life cycle of Fasciola spp. is quite complex, involving several variations. In general, it involves one or more intermediate hosts, which are the mollusks. At least 20 species of the Lymnaeidae family have been reported as intermediate hosts (22, 23). The asexual larvae undergo several multiplications (24–26) before finally infecting a definitive host in which sexual reproduction occurs.

The penetration, migration, and localization of the parasites in the bile ducts exert a traumatic action that causes a series of lesions in the liver parenchyma and in the bile ducts (27). The newly excysted juveniles (NEJs) of Fasciola spp. penetrate the intestinal mucosa and can be found in the abdominal cavity 72 h after metacercaria ingestion. NEJs migrate through the peritoneum to the liver surface and present no clinical sinology in animals (28). The destination of the majority of NEJs is the left hepatic lobe, probably due to its anatomical proximity to the duodenum and the fact that they reach less of the other hepatic lobes. Sometimes, due to massive infestations, these juveniles can have an aberrant migration to other organs, such as the diaphragm and the lung, causing pneumonia and fibrinous pleurisy (29).

Fasciolosis pathogenesis occurs in two phases—the parenchymal and biliary phases. The parenchymal phase begins when the NEJs cross the liver capsule (Glisson's capsule), continuing with the migration of the juvenile stages through the liver parenchyma. This migration causes mechanical damage through abrasion by the tegument that presents spines that help maintain the parasite's position within the liver tissues and probably by-products secreted by migrating larvae. Several pathological processes occur simultaneously within the liver parenchyma, including the migration of juvenile stages that cause necrotic and hemorrhagic lesions, which, in turn, cause inflammatory reactions activating the immune system (30). This response can be found throughout the tortuous migrating trajectory of the parasites, suggesting that the excretion and secretion of these products remain in the tissue, attracting more infiltration of inflammatory cells of an immune nature (31). The biliary phase begins when the parasites enter the bile ducts, where they exert a combined mechanical and chemical action. Through the oral sucker, adult parasites cause mechanical damage while feeding on blood and the liver parenchyma adjacent to the duct. Macerated hepatocytes have been observed inside the sucker and pharynx (27), leading to erosion of the epithelium, trauma, focal rupture of the duct, and puncture of small blood vessels. The enlargement of the bile duct can be chemically induced (32), and it has been suggested that the amino acid proline, which is essential for the synthesis of collagen by fibroblasts, is also released in large quantities by the parasite (33, 34). These two actions exerted by the adult parasite cause a severe eosinophilic and granulomatous inflammatory response, particularly when eggs reach hepatic parenchyma (35), and marked hyperplasia of the bile ducts in which the parasites lodge (36).

The effect of these two phases causes a series of lesions in the liver parenchyma, which is widely correlated with the infective dose; a high dose causes more severe lesions that are more acute and even fatal. However, different studies carried out in sheep (35) and goats (37) have also shown that small repetitive doses (trickle infections) caused more severe hepatic damage than a single dose using the same total number of metacercariae. These findings suggest that the mechanical and enzymatic activities of the parasite may be the initial cause of liver damage. Therefore, the immune response or healing, as well as simultaneous infection at different stages and the immune response to the first infection, play an important role in the pathogenesis of fasciolosis (31).

The inflammatory reaction in fasciolosis is one of the points to be treated primarily to understand the immune response and its evasion. Since metacercariae are excysted in the gut lumen, NEJs are exposed to the host immune response to kill the parasite. However, Fasciola spp. has developed a variety of strategies to evade the host response in the different compartments where they stay during the early and late stages of infection, which allows the parasite to live for years within the host. Some of these strategies may be considered passive, as the protection conferred by the tegument, which consists of a syncytial layer covering the entire body of the parasite, formed by a plasma membrane that serves as a support for the outer glycocalyx and a basement membrane that is connected through channels. These structures allow the passage of the components needed for the replacement of the tegument. The rapid replacement of the glycocalyx that covers the tegument—which takes place every 2 to 3 h—may also be an obstacle for products released by inflammatory cells to reach the parasite tegument (84), which is composed of at least 369 proteins. Additionally, the presence of abundant N-glycosylated proteins and glycolipids has made it difficult to characterize its physiological and immune regulatory functions (85).

The majority of strategies used by the parasite to evade the host response may be considered active since they imply the release of a large amount of parasite molecules into the parasite vicinity. These molecules can be released free or within extracellular vesicles (EVs) that are covered by a membrane, and they can be internalized by the host cells, causing their modulation (84, 85). EVs are produced by all developmental stages of F. hepatica, and they are considered efficient transporters of parasite molecules to different host compartments, preventing the action of antibodies due to the membrane surrounding the parasite molecules contained in EVs (86). In EVs from F. hepatica, up to 618 proteins have been identified, which gives us an idea of how important EVs are for the parasite to interact with the host (87).

Fasciola spp. not only use proteins to modulate the host immune response, but EVs also contain microRNAs (miRNAs), molecules with modulating gene expression capacity. miRNAs are abundant in both metacercariae, juvenile and adult F. hepatica worms and may play a main role in regulating the developmental and metabolic processes of the parasite, as well as in host-parasite interactions (88–90). The miRNA content in the EVs is different when they are produced by adult or juvenile parasites, leading to different influences in the host cells. These data support the hypothesis that miRNAs are the mediators of the previously demonstrated immune modulatory function of the EVs. However, current data do not allow a fundamental understanding of their regulatory mechanisms in different processes of host-parasite interaction (88–91).

Another mechanism used by liver fluke to survive, migrate, obtain nutrients, and evade the immune response of the host, is the release of excretory secretory products (ESP) (92). FhESP from adult F. hepatica contains up to 160 different proteins, including proteases such as cathepsins B and L (FhCB and FhCL), leucine aminopeptidase and carboxypeptidase, fatty acid-binding protein (FABP), and the F. hepatica saposin-like protein (FhSAP), all of them necessary for its metabolism (93) (Table 1). FhESP also contains numerous antioxidant enzymes to protect the parasite from reactive oxygen species released by eosinophils and macrophages, such as superoxide dismutase (SOD), glutathione-S-transferase (GST), thioredoxin peroxidase (TPx), and peroxiredoxin (Px) (Table 1). These enzymes not only participate in inactivating reactive oxygen species but also in several important metabolic processes important for parasite survival, such as the excyst of the metacercariae, tissue migration, feeding, and immune evasion (92, 105, 106). Some strategies that Fasciola spp. use to evade the host response are discussed below.

Over the past two decades, there have been considerable advances in identifying potential vaccine molecules for the control of fasciolosis in livestock. However, despite some promising results with some vaccine candidates in ruminants, a consistent efficacy required for commercialization has not yet been reached (13). A major obstacle to developing vaccines for fasciolosis is the immune suppression/modulation induced by Fasciola spp. that prevents the induction of a protective immune response (Figure 1), evidenced by the lack of immunity observed in naturally and experimentally infected sheep (31, 70, 129). In cattle, natural or experimental infections have been shown to induce certain protection against reinfection, which is maintained long-term (up to 26 weeks post-infection). It has been attributed to the severe fibrosis induced by the primary infection that makes the hepatic migration difficult during the secondary infection (130) or by an increase of intestinal eosinophil and mucosal mast cells (47). Some studies have also reported evidence that protection against F. hepatica is inducible in rats, sheep, or cattle by passive transfer of immune sera and cells (131). However, other studies have reported no resistance to reinfection measured by fluke burdens (75). Moreover, no differences in fluke burdens, fecal egg counts, humoral response (specific IgG1 and IgG2), and cell-mediated immune response (IFN-γ production) were reported in calves challenged with F. hepatica after single or trickle infection (48, 57, 75) suggesting that reinfections do not induce protection. Experimental studies reported no protection against reinfection in sheep (35, 81, 132) and goats (37), although the host response was different; thus, primo-infected sheep showed a mixed Th1/Th2/Th17 response while reinfected ones presented a more Th2 polarized response (81) and a lower humoral response (132).

It has been reported that in protective vaccines in sheep (133) and goats (134), a mixed Th1/Th2 response was found with higher levels of IFN-γ and lower levels of IL-4 in vaccinated groups than in the infected control group (133). In sheep immunized with a non-protective vaccine, the host immune response showed a predominantly Th2 profile during chronic stages of the infection, similar to that found in non-vaccinated and infected animals (80). The challenge is to identify the specific antigens that are the targets of this protective immunity and incorporate these in vaccine formulations that induce a mixed Th1/Th2 response to enhance vaccine efficacy (135). It has been estimated that a vaccine with an efficacy of 50–60% in fluke reduction would likely be beneficial in numerous countries to significantly reduce economic losses, and it also would have a positive impact on epidemiology by reducing eggs in pasture (13).

Several strategies have been used to design vaccine candidates for fasciolosis in livestock. The first vaccine trials used native proteins isolated using conventional biochemical methods from the excreted/secreted (ES) proteins of adult parasites (136, 137). Despite good protection being found in sheep and cattle in these trials using native FhCL1 and FhGST, the use of native proteins in a commercial vaccine for fasciolosis in livestock is not feasible, which is why the majority of subsequent vaccine trials have been carried out using recombinant proteins of different stages of the parasite (13). Some vaccine trials using recombinant proteins reported high protection of up to 89% in fluke reduction (Tables 2, 3); however, this high protection has not been reproducible in different labs and conditions. A combination of recombinant vaccines (cocktail vaccines) has also been used recently with variable efficacy (Table 3). The majority of vaccine trials have used the subcutaneous or intramuscular administration route. However, a few trials have used mucosal vaccine delivery with promising results. For instance, Norbury et al. (154) administered a cocktail vaccine containing FhCL5 and FhCB2 by an intranasal method in sheep, obtaining a 40.5% fluke reduction and a 92% egg viability reduction, while the same vaccine administered intramuscularly did not induce protection. The oral route has also been used to administer freeze-dried transgenic lettuce expressing the cysteine proteinase of F. hepatica (CPFhW) in sheep and cattle, inducing significant protection in cattle (56.2%) and 35.5% fluke reduction (not significant) in sheep (142).

Most vaccine trials in ruminants have used proteases, antioxidant enzymes, or fatty acid-binding proteins as antigens (Tables 2, 3). However, these proteins are quite abundant in Fasciola spp, and blocking one or several of them by a vaccine probably does not cause serious problems to the worm since it has other proteins with similar functions. This might be a reason for the limited efficacy obtained in the numerous vaccine trials conducted with these antigens in ruminants.

The slow progress to date in developing a protective vaccine to be used in the control of fasciolosis in livestock suggests that new approaches should be investigated, such as the use of new antigens, evaluation of immunity induced by recombinant proteins, use of different adjuvants, formulations, and delivery systems. Despite important advances in the knowledge of host-parasite interactions in fasciolosis, a more rational vaccine candidate design requires a deeper knowledge of the mechanisms and molecules involved in host-parasite cross-talk in relevant target host species (sheep, cattle, goats, buffalo). The progress of the -omics technologies and the immunoinformatic/immunoproteomic approaches should provide useful data in the next few years. An example is the new proteomic technologies applied to NEJs after crossing the gut (158) or during the early stages of hepatic migration, which may be useful to select new vaccine candidates directed against NEJs, a stage of the parasite that it is more exposed to the host immune system than adult ones located within the bile ducts.

LF-V: Writing—original draft. MR-C: Writing—original draft. GH-T: Writing—review & editing. ÁM-M: Writing—review & editing. FM-M: Writing—review & editing. RZ: Writing—review & editing. LB: Writing—review & editing. PR-M: Writing—review & editing. VM-H: Conceptualization, Writing—original draft, Writing—review & editing. JP: Conceptualization, Writing—original draft, Writing—review & editing.