1. Introduction
The life cycle of the common liver fluke,
Fasciola hepatica was elucidated by Leuckart (1882) [
1] and Lutz (1892, 1893) [
2,
3] and the details have recently been reviewed by Andrews (1999) [
4]. As a typical digenetic trematode, the adult fluke is hermaphroditic, exhibiting two highly branched testes located one behind the other in the posterior half of the body, and a single dendritic ovary, located on the left of the uterus, as viewed from the ventral surface. Extensively developed vitelline glands that provide the precursor shell-proteins and glycogen stores needed for egg production are located lateral and posterior to the testes. The arrangement of the gonads, accessory reproductive organs and ducts within the body of the fluke, and in particular around the ootype where the eggs are assembled, is readily visualised using carmine-stained whole mount preparations. Such preparations can provide useful information on the overall state of reproductive development in fluke populations under the influence of anthelmintics administered to the host [
5,
6,
7].
With an impressively high level of egg production, reaching 25,000 per fluke per day [
8], and with a large proportion of the body occupied by testis and vitelline tissue, it is evident that the production of spermatozoa and egg components represents the main energy consuming activity of the adult fluke [
5]. While it has been recognised that single flukes in a definitive host animal are capable of producing eggs, and hence that self-fertilization is possible [
9], it has been generally assumed that cross-fertilization is the preferred reproductive strategy when a number of individuals occur together in a single host liver. Recent studies on single-fluke infections in rats indicate that self-fertilization may occur in only about 33% of flukes without a co-inhabitant, suggesting that parthenogenesis is a possible reproductive strategy [
10]. This is supported by the fact that flukes of the triploid aspermic Cullompton isolate are highly fecund, and the eggs they produce develop and hatch normally, giving rise to viable miracidia. These are capable of infecting the molluscan intermediate host and perpetuating this clonal isolate through subsequent generations [
11].
Scanning and transmission electron microscopy have often been used to help elucidate the effects of anthelmintic drugs on specific tissues of liver flukes [
12]. However, conventional histopathological methods (including whole-mounts) have been little used until recently, despite their potential value in screening simultaneously all tissues in statistically viable samples of flukes exposed to anthelmintic drugs
in vivo or
in vitro. A primary aim of this report is to clarify aspects of the histology of the reproductive system in untreated flukes and thus provide a basis for characterising the action of anthelmintic drugs by their histological and morphological effects on the reproductive system. The latter, due to the high metabolic demands of sperm and egg production, is uniquely sensitive to adverse physiochemical and pharmacological conditions. Examination of the histological and morphological changes induced in the reproductive system of flukes after treatment of their host with anthelmintics such as triclabendazole (TCBZ) can provide useful information on the drug-resistance status of individual fluke isolates.
TCBZ, a benzimidazole anthelmintic with fasciolicidal activity, was introduced as a veterinary drug in 1983, and is active against
Fasciola hepatica,
Fasciola gigantica,
Fascioloides magna and
Paragonimus spp., but not against nematodes or cestodes [
13]. Its importance and widespread use derive mainly from the fact that it kills flukes from as early as two days post-infection through to the fully mature adult stage, thus offering effective control for both acute and chronic fasciolosis [
12,
14]. Over-reliance on this drug has, however, led to the development of resistance to TCBZ in fluke populations worldwide, the first case having been reported from Australia in 1995 [
15]. Since then resistance has been reported throughout Europe, including cases from the British Isles [
13]. Previous studies [
12,
16] have shown that TCBZ, like other anthelmintic molecules in the benzimidazole class, may act as a β-tubulin antagonist. These molecules are believed to interfere with the assembly of microtubules which have essential roles in the movement of subcellular components and metabolites within the cytoplasm, as well as spindle formation during cell division.
Studies on the efficacy of TCBZ have centered mainly on field trials involving pre- and post-treatment fluke counts, faecal egg count reduction (FECRT) and clinical chemistry. Qualitative assessment has been carried out on ultrastructural lesions induced in flukes maintained under
in vitro conditions in the presence of anthelmintic metabolites [
17,
18,
19,
20,
21] or exposed
in vivo in anthelmintic-treated sheep [
6,
22,
23,
24,
25,
26,
27]. The TCBZ resistance status of populations of
F. hepatica in field cases of fasciolosis, where treatment failure has been reported, is generally investigated by examination of faecal samples collected pre- and three weeks post-treatment with TCBZ. Tests such as FECRT and coproantigen reduction (CRT) are used [
28,
29], although these approaches are ineffective for diagnosis of the pre-bile duct stages of fasciolosis.
Histological examination of flukes following
in vivo exposure to anthelmintics such as TCBZ offers the possibility of conveniently screening large numbers of flukes, or entire fluke populations from individual experimental or field infections. It can yield quantitative data relating to each and every parasite tissue. Such information can supplement and augment conventional coprological testing and aid or direct electron microscopy and stereology, which can be time-consuming, expensive and restricted in terms of numbers of fluke examined and range of tissues sampled in individual flukes. A histological approach was recently used to complement FECRT and CRT in a comparative study of the relative efficacies of TCBZ, nitroxynil and closantel in the control of fasciolosis on sheep farms in Northern Ireland [
30].
4. Discussion
A number of investigations have been carried out on the morphological changes induced in
F. hepatica by exposure to TCBZ and its active metabolites. These studies, reviewed by Fairweather and Boray (1999) [
12] and Fairweather (2009) [
16], have mainly involved the use of Scanning and Transmission Electron Microscopy on flukes exposed
in vitro to physiological concentrations of the molecules in question. They have yielded much useful information relating to the possible mechanisms of drug action on the parasite. Most of the changes described are consistent with the known anti-tubulin effects of benzimidazoles, but, in addition, inhibition of protein synthesis has also been evoked as a possible mode of action [
44].
The histological features of the testis in TCBZ-R flukes and in TCBZ-S flukes from untreated hosts described here were found to be broadly consistent with the ultrastructural findings of Stitt and Fairweather (1990) [
37]. In the testis tubules of TCBZ-S flukes from sheep treated with TCBZ there was a marked and progressive increase in the proportion of cells that displayed degenerative features such as multiple nuclei, pyknotic or karyorrhectic nuclei, eosinophilic cytoplasm and rounded profiles. These changes are consistent with apoptosis, the fundamental phenomenon of “internally-programmed cell death” that occurs in all metazoans, and serves to eliminate cells that are irreparably damaged, particularly if that damage affects the DNA [
45]. Many of the cells showing morphological changes associated with apoptosis in the testis of TCBZ-S flukes exposed to metabolites of the drug
in vivo, also gave a strong positive labelling with the TUNEL method. This confirms the occurrence of endonuclease-induced DNA strand breaks, a marker of apoptosis, in these abnormal cells, and supports the concept that TCBZ activity may target spindle formation in dividing cells, triggering the cascade of events that leads to apoptosis. Recently, it has been shown that sustentacular tissue is located in the peripheral zone of the testis tubules in
F. hepatica [
38]. A primary function of this tissue appears to be the scavenging of effete cells and cytoplasmic debris, as well as recycling of useful molecules. This is carried out by a process of heterophagic digestion using lysosomal enzymes generated in the cytoplasm of the sustentacular tissue. Presumably the sustentacular tissue may have a role in scavenging cells damaged by TCBZ action.
Progressively fewer cells were present in the testis tubules of TCBZ-S flukes from treated sheep than in those of flukes from untreated sheep, with a corresponding increase in “empty” space. This probably followed from inhibition of mitotic division in the primary, secondary and tertiary spermatogonia by the metabolites of TCBZ. The activity of the benzimidazole group of anthelmintics, to which TCBZ belongs, is believed to lie in their selective ability to bind β-tubulin, thus inhibiting microtubule-mediated processes such as spindle formation during mitosis [
46]. Fairweather (2005, 2009) [
13,
16] has reviewed the data linking the
in vitro effects of TCBZ metabolites on
F. hepatica with those of established microtubule inhibitors such as tubulozole-C. As observed in the present study, with inhibition of mitosis, it is likely that the testis tubules become progressively depopulated as the degenerating cellular contents exit through the vasa deferentia and genital pore. As expected, the histological features of the testis in TCBZ-R flukes were unaffected by anthelmintic treatment of the host, consistent with the findings of McConville
et al. (2009a) [
6].
The successful formation of a normal shelled egg in the ootype of
F. hepatica represents the culmination of a complex sequence of cytokinetic and cytological events involving cells originating from the ovary and the vitelline follicles. There is also input of proteinaceous secretory material, including lipoproteins, from the Mehlis’ gland cells that envelop the ootype [
12,
47,
48]. The co-ordination of cell movements and release of secretory products in the ootype region is achieved by neural and neurosecretory influences. While the neuroanatomy of the region has been mapped in elegant studies by Fairweather
et al. (1987) [
49] and Magee
et al. (1989) [
50], the exact mechanism awaits clarification. Clearly, drug-induced defects in the function and output of the ovary, vitelline system and Mehlis’ gland, as well as the neural/peptidergic mechanisms controlling the activity of the ootype [
12,
48], are likely to be reflected in the appearance and integrity of the eggs emerging from the ootype and entering the proximal coils of the uterus. The histological features of the uterus and its contents therefore provide an early and sensitive indication of the emergence of malfunctions throughout the female reproductive tract. In each and every TCBZ-S fluke exposed to TCBZ metabolites
in vivo for 48 h or 72 h, no normal eggs were observed in the uterus. Instead, there were free vitelline cells (lacking peripheral shell globule clusters), free ova and irregular masses of coagulated shell protein material in the uterine lumen. In the ootype, the mechanism involved in laying down a coherent uniform layer of shell protein material to form a shell around each group of de-granulated vitelline cells, with their single associated ovum, had clearly failed. The underlying reason for this failure lay perhaps in abnormalities in the shell protein precursors, abnormalities in the Mehlis’ gland secretions or in disorganisation of the mechanical activity of the ootype. The onset of abnormal ovigenesis in these individuals occurs within 24h of administration of TCBZ to the host [
10]. In flukes exposed for 72 h in TCBZ-treated hosts and in degenerating flukes from animals treated 96 h before slaughter with TCBZ, the uterine coils were largely empty. This reflected, not only loss of the content via the genital pore, but also cessation of the movement of vitelline cells and ova into the ootype. TCBZ-R flukes retain a full complement of maturing eggs in the uterus, reflecting uninterrupted ovigenesis and integrity of all components of the female reproductive tract, throughout the treatment period.
Within each vitelline follicle of
F. hepatica, mitotic activity in the stem cells at the periphery provides a continuous supply of cells destined for shell protein production and glycogen storage. At the same time it ensures continuity of the stem cell line. Maturing vitelline cells differentiate progressively towards the core of the follicle, undergoing a phase of protein synthesis, during which shell globule clusters accumulate in the cytoplasm. Finally they reorganise as intracellular glycogen storage sites) [
42,
51]. As in the testis tubules, disruption of mitosis by the action of TCBZ metabolites
in vivo results in apoptosis of stem cells, yielding discrete labelling of individual cells at the periphery of the follicles in TUNEL preparations. The break in supply of immature vitelline cells leads to progressive disappearance of early and intermediate stages in the follicles, as pre-formed cells mature. Production of shell protein globules and deposition of glycogen apparently continues in the presence of TCBZ metabolites
in vivo. However, increasing numbers of mature vitelline cells within the follicles show evidence of disruption, with release and coalescence of the shell protein globules. The material so released assumes an unusually dark and condensed appearance. This possibly suggests that the enzyme-mediated chemical processes involved with quinone-tanning of the egg-shell [
12,
48] have been initiated prematurely. The role of TCBZ metabolites in the apparent disintegration of mature vitelline cells is unclear, although failure in onward propulsion through the vitelline ducts may be a factor.
In vitro and
in vivo treatment of
F. hepatica with TCBZ metabolites has been shown to disrupt muscle fibres [
20,
21,
24,
25]. Therefore, lack of muscle tone in the duct walls, coupled with failure of cell pressure following inhibition of mitosis at the periphery of the follicles, may delay the exit of mature vitelline cells and result in partial disintegration. Consequently, precocious tanning of the shell protein material proceeds. As expected, TCBZ-R flukes taken from TCBZ- treated hosts showed no histological abnormalities in the vitelline tissues.
In contrast to the situation in the testis, vitelline follicles and ovary of
F. hepatica, no mitotic or meiotic activity occurs in the Mehlis’ gland complex. Therefore, it is not surprising that the histological features associated with anti-tubulin action in TCB- treated hosts, namely apoptosis and progressive decline in cell population, are not seen in the Mehlis’ gland. However, the apparent shrinkage of the S1 and S2 cells and loss of secretory material from the cytoplasm and connecting tubules is consistent with decline or cessation of the normal protein or lipoprotein secretory activity. It is also possible that the disruption of synthetic activity in the Mehlis’ gland complex is due to failure of energy metabolism. Fairweather and Boray (1999) [
12] have reviewed evidence that TCBZ metabolites are capable of inhibiting protein synthesis in
F. hepatica, and that benzimidazoles may be implicated in uncoupling oxidative phosphorylation. However, bearing in mind the well-established anti-tubulin activity of anthelmintics in this class, general disruption of metabolism in the Mehlis’ gland cells, and indeed ultrastructural disorganisation of organelles such as the GER, mitochondria and Golgi complexes leading to cytoplasmic vacuolation, may well be secondary to failure of microtubule-mediated intracellular processes.
The viscous alkaline lipoprotein secretions from the Mehlis’ gland cells are believed to play a key role in the uniform spreading and coalescence of the shell precursor proteins during egg-shell formation in the ootype. This is by virtue of the interface they form with the less viscous acidic fluid surrounding the vitelline cells [
12,
48]. Alteration in the physicochemical properties of Mehlis’ gland secretion might possibly reflect preferential accumulation of the hydrophobic molecules of the anthelmintic metabolites in the lipid component. Clearly, even minor alterations in the physicochemical constitution of the Mehlis’ gland secretions would be liable to have a major impact on proper formation of the egg-shell. Indeed, the disruption of this process, with the appearance of loose vitelline cells and aberrant coagulated masses of shell protein, was a prominent histological feature in the uterus of TCBZ-S flukes from the earliest stages of exposure to TCBZ
in vivo.
The main histological changes seen in the ovaries of TCBZ-S flukes from hosts treated with TCBZ before slaughter, included progressive decline in cell population (with resulting constriction of the ovarian tubules and appearance of vacuoles). Furthermore, there was development of apoptotic changes in the remaining cell population. The TUNEL labelling pattern in the ovarian tubules of TCBZ-treated sensitive flukes probably reflects apoptosis in oogonia attempting to initiate mitosis, and oocytes in the initial stages of the first meiotic division. Primary oocytes move out of the ovary at the end of prophase of the first meiotic division, completing their development in the proximal coils of the uterus [
41]. Failure of mitosis in the oogonia, resultant decline in cell output and initiation of apoptosis are consistent with defective spindle formation, initiated by TCBZ action. No histological abnormalities developed in the ovaries of the TCBZ-R flukes collected from treated sheep, as was the case with all the other reproductive structures.
While the contribution of electron microscopical techniques to the analysis of drug-induced lesions in
F. hepatica is well established, conventional histopathological methods have not often been employed. This is despite their potential value in screening simultaneously all tissues in representative samples of flukes exposed to anthelmintic drugs
in vivo or
in vitro. The present investigation seeks to provide a description and overview of lesions that develop in the reproductive organs of TCBZ-S liver flukes in host animals treated with TCBZ. Given the ease and rapidity with which large numbers of liver flukes can be processed for histology, it is possible to apply a semi-quantitative scoring system. This enables the overall severity of drug induced abnormality to be calculated for each treatment regimen [
31]. A more rigorous quantitative approach might be achieved by the use of an “image analysis” programme. This would enable statistical comparison of results, and might prove particularly interesting in cases where TCBZ resistance was partial. It might also have a role in comparative assessment of the activities of putative anthelmintic molecules in field trials. The utility of histological analysis in aiding the choice of appropriate anthelmintic for the control of fasciolosis in field situations has been demonstrated. A recent study compared the relative efficacies of TCBZ, nitroxynil and closantel in reducing the fluke burden in sheep on farms throughout Northern Ireland [
30]. This study also exemplified the use of histology as one of several complementary methods (FECRT; CRT; fluke histology; comparative anthelmintic efficacy testing) for the confirmation of a diagnosis of fluke drug resistance, as recommended by Fairweather (2011) [
52].