Accepted Manuscript Title: Fasciola hepatica: histological changes in the somatic and reproductive tissues of liver fluke following closantel treatment of experimentally-infected sheep Author: S. Scarcella R.E.B. Hanna G.P. Brennan H. Solana I. Fairweather PII: DOI: Reference:
S0304-4017(15)30066-2 http://dx.doi.org/doi:10.1016/j.vetpar.2015.10.029 VETPAR 7819
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
30-3-2015 28-10-2015 30-10-2015
Please cite this article as: Scarcella, S., Hanna, R.E.B., Brennan, G.P., Solana, H., Fairweather, I., Fasciola hepatica: histological changes in the somatic and reproductive tissues of liver fluke following closantel treatment of experimentallyinfected sheep.Veterinary Parasitology http://dx.doi.org/10.1016/j.vetpar.2015.10.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Fasciola hepatica: histological changes in the somatic and reproductive tissues of liver fluke following closantel
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treatment of experimentally-infected sheep.
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S. Scarcella a*; R.E.B. Hanna b; G. P. Brennan c; H. Solana a; I. Fairweather c
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Laboratorio de Biología Celular y Molecular. Centro de Investigación Veterinaria de Tandil (CIVETAN), CONICET, Facultad de Ciencias Veterinarias, UNCPBA, Tandil, Argentina
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c
Veterinary Sciences Division, Agri-Food and Biosciences Institute (AFBI), Stormont, Belfast BT4 3SD, United Kingdom
Parasite Therapeutics Research Group, School of Biological Sciences, Medical Biology Centre, The Queen’s University of Belfast, Belfast BT9 7BL, United Kingdom
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*Corresponding author:
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[email protected], Tel: + 54 2293 439850 Int. 234, Fax: +54 2293 439850. Laboratorio de Biología Celular y Molecular.
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Centro de Investigación Veterinaria de Tandil (CIVETAN), CONICET, Facultad de Ciencias Veterinarias, UNCPBA
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Tandil (7000), Argentina.
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Highlights
Histological changes were examined in closantel-treated Fasciola hepatica from lambs. Thetegumental syncytium deteriorated progressively with treatment time. The testis, ovary and vitelline follicles showed progressive cell depletion. Histological changes were related to inhibition of intermediary metabolism.
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Abstract
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Lambs infected with the Cullompton isolate of Fasciola hepatica were treated orally or subcutaneously with 10 mg/kg of closantel at
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16 weeks post-infection. Adult flukes were recovered from the liver of individual animals at 12h, 24h, or 36h post-treatment. The
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flukes were processed for histological analysis. In general, degenerative changes in the reproductive and somatic tissues were
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progressive, and were most marked in flukes exposed to closantel in vivo for 36h. However, flukes from a 12h subcutaneously-
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treated lamb showed marked deterioration of the testis, possibly because a portion of the dose has been delivered intravenously.
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Fewer intact eggs were seen in the uterus of flukes exposed to closantel for longer times (whether administered subcutaneously or
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orally to the host). The most conspicuous closantel-induced effect in flukes from treated hosts was progressive damage to the
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tegumental syncytium. While the flukes from 24h-treated hosts showed relatively minor damage to limited areas of the syncytium,
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towards the posterior end, the flukes from 36h-treated hosts (and flukes from the lamb that putatively received intravenous dosage) 2
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had lost large areas of the surface syncytium from the posterior end and dorsal surface, although the syncytium over the anterior
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end and the anterior ventral surface was largely spared. In areas where the syncytium had sloughed, the underlying structures such
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as the vitelline follicles, gut profiles and testis profiles, showed marked degeneration and breakdown. Other changes included cell
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depletion and early stage apoptosis in the testis, ovary and vitelline follicles. This study establishes a model for histological changes
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in closantel-sensitive F. hepatica exposed to closantel in vivo.
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Histopathological studies could be complementary to the efficacy controlled test for for closantel resistance in fluke populations.
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Key words:
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Fasciola hepatica; liver fluke; sheep; closantel in vivo; post-treatment fluke histology; fluke resistance diagnosis.
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1. Introduction
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Since its introduction in 1983, the fasciolicide triclabendazole (TCBZ) has been extensively used worldwide to control liver fluke
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infections in sheep and cattle, mainly because it possesses a uniquely wide spectrum of activity, killing not only adult Fasciola spp.,
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but also immature and juvenile flukes as young as 2 days post-infection (Boray et al., 1983; Fairweather and Boray,
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1999).However, since 1995, reports of fluke resistance to the anthelmintic have appeared with increasing frequency in stock3
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producing countries where TCBZ is used routinely (Brockwell et al., 2014; Daniel et al., 2012; Fairweather, 2005; Fairweather,
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2009; Gordon et al., 2012; Mooney et al., 2009; Olaechea et al., 2011; Ortiz et al., 2013; Overend and Bowen, 1995). Recently, in
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Northern Ireland (NI), 13 sheep farms were surveyed for TCBZ resistance. It was shown that, in every flock where FECs indicated
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the presence of significant chronic Fasciola hepatica infection, substantial resistance to TCBZ was also present, but closantel and
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nitroxynil effectively eliminated the adult fluke burden (Hanna et al., 2015). It is likely, therefore, that the emphasis in
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chemotherapeutic control of fasciolosis in highly endemic areas will switch from routine use of TCBZ for treatment both of acute
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and chronic infections, to strategic use of drugs such as closantel, which have a narrower spectrum of activity, limited to late
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immature and adult F. hepatica, in an attempt to minimise pasture contamination with fluke eggs, and reduce the risk of acute
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infection in the next season’s lamb flock (Hanna et al., 2015).
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Closantel is a salicylanilide anthelmintic that binds extensively to plasma albumin (Michiels et al., 1987). As a result, its activity is
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mainly directed against blood-feeding internal parasites such as F. hepatica, Haemonchus contortus, Oestrus ovis and
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Oesophagostomum larvae. Its activity is associated with disruption of energy metabolism, specifically uncoupling of oxidative
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phosphorylation, a mechanism different from the anti-microtubule action of TCBZ (Fairweather and Boray, 1999). Unlike TCBZ,
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closantel is much less effective on young immature flukes than on adults and late immature flukes that have reached the bile ducts
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(Boray, 1997; Fairweather and Boray, 1999). However, in experiments with both sheep and rats, (Maes et al., 1988) showed that,
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as well as removing a high percentage of adult flukes, and to a lesser extent immature flukes, the size of any flukes that survived
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treatment was stunted. This finding was supported by the work of Hanna et al. (Hanna et al., 2006), who showed that in vivo 4
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closantel treatment of late immature, 5-week-old F. hepatica infections in cattle resulted in reduction of the fluke burden by more
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than 50%, with the surviving parasites showing stunting, reduced histological development of the reproductive organs, and
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attenuation of egg output.
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To date, there is limited evidence of development of fluke resistance to closantel, although in endemic areas of New South Wales,
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where closantel and rafoxanide have long been used to control H. contortus as well as F. hepatica, resistant strains of the
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nematode have been reported (Rolfe et al., 1990; Van Wyk and Malan, 1988), and instances of suspected salicylanilide resistance
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in F. Hepatica have been recorded in England and Wales (Fairweather and Boray, 1999). With increasing use of closantel in areas
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where TCBZ resistance is established (McMahon, 2015), it is expected that fluke resistance to the former will also emerge.
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Diagnosis of anthelmintic resistance in local fluke populations is complicated by a number of factors, including the long pre-patent
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period (at least 6 weeks in sheep and 8 weeks in cattle), which can give rise to false-negative results in faecal egg counts (FEC),
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and the retention of eggs in the gall bladder of the host for some time after the flukes themselves have been removed by successful
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drug treatment, which can yield false-positive FECs (Chowaniec and Darski, 1970; Flanagan et al., 2011a; Flanagan et al., 2011b;
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Mitchell et al., 1998). It is considered advisable, therefore, that diagnosis of flukicide resistance in field situations should be based
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on the results of several diverse methods, rather than relying solely on faecal egg count reduction tests (FECRT). Confirmatory
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tests might include the coproantigen reduction test (CRT) by ELISA (Flanagan et al., 2011a; Flanagan et al., 2011b; Gordon et al.,
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2012; Gordon et al., 2012b; Mezo et al., 2004;Valero et al., 2009)
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post-treatment fluke histology (Hanna et al., 2010), or egg hatch assay (EHA) (Alvarez et al., 2009; Canevari et al., 2014;
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Fairweather, 2011b; Fairweather et al., 2012).
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The purpose of the present investigation is to describe the histological changes that occur in closantel-sensitive F. hepatica,
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exposed to the anthelmintic in recently-treated hosts. Few morphological studies have been carried out on closantel action.
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Ultrastructural changes to the tegument and gut were described by Verheyen et al. (1980), Skuce (1987) and Skuce and
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Fairweather (1990) and histological changes to the reproductive organs by Maes et al. (1988).These studies on adult fluke
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established the time-course of successful drug action in vivo and helped to explain how the morphological changes led to the
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elimination of the flukes from the host. In the present investigation, fluke material was collected at an early time-point (12 h post-
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treatment [pt]), as well as at 24 h and 36 h pt. The flukes were examined by histology, to determine when the changes to the
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reproductive system become evident. This information will help provide a model for subsequent investigations of closantel
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resistance using pt fluke histology.
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2. Materials and methods
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2.1. Experimental protocol
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Animal procedures and management protocols were approved by the Ethics Committee according to the Animal Welfare Policy
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(Act 087/02) of the Faculty of Veterinary Medicine, Universidad Nacional del Centro de la Provincia de Buenos Aires (UNCPBA),
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Tandil, Argentina (http://www.vet.unicen.edu.ar ), and to internationally accepted Animal Welfare Guidelines (A.V.M.A, 2001).
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Fourteen parasite-free Corriedale weaned lambs were each inoculated orally with 200 metacercariae of F. hepatica contained in a 6
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gelatin capsule. The isolate used for this experiment was the Cullompton isolate, which is TCBZ-susceptible: for details of its
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provenance please see (Fairweather, 2011a). The presence of liver fluke in the lambs was confirmed 15 weeks after infection by
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the finding of eggs in the faeces, and liver damage was estimated indirectly by measurement of serum Glutamate Dehydrogenase
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and Gamma Glutamyl Transferase activities, as described previously (Solana et al., 2001). Sixteen weeks after oral inoculation, the
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animals were assigned to three experimental groups, based on their clinical condition and body weight, and were treated orally or
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by subcutaneous injection with closantel, as detailed in Table 1.
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Treated animals in groups 2 and 3 were stunned and exsanguinated at 12 h, 24 h and 36 h pt, following the W.A.A.V.P. guidelines for
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evaluating antiparasitic treatments in ruminants (Wood et al., 1995). Flukes were recovered from the common bile duct of each lamb
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and rinsed thoroughly with warm (37 oC), sterile saline solution (0.9% w/v, sodium chloride) to remove bile and/or adhering
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materials. Likewise, flukes were collected from the untreated control animals in group 1.Total number of flukes recovered from each
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animal is disclosed in Table 2. The flukes obtained from each animal were examined grossly and two representative specimens
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were selected for detailed histological examination from each of the experimental treatments.
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2.2. Preparation of flukes for histology
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Flukes were fixed in 10% (v/v) neutral-buffered formalin for 24 h. Histological processing and embedding in wax was carried out by
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conventional techniques. Sections 3 µm in thickness were cut from each block face and stained with haematoxylin and eosin using
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standard histological protocols. Sections were examined and the tissues photographed using a Leica DM LBZ microscope fitted
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with a Nikon Coolpix 5000 camera system. Amore detailed protocol is given by (Hanna et al., 2008; Hanna et al., 2010; Hanna et
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al., 2015).
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3. Results
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The report on the histology was based on a combined assessment of the two specimens examined from each treatment.
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3.1. Control flukes from untreated lambs
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The histological features of the reproductive organs in untreated Cullompton isolate flukes were described previously by Hanna et
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al. (2010) and by Hanna (2015). In the untreated flukes examined in this study, there was no evidence of post-mortem degenerative
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changes, and therefore they were considered to be valid controls for comparison with flukes from treated animals. The tegumental
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syncytium was unbroken, unvacuolated, and was attached at all points to the underlying basal lamina and musculature. The
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tegumental perikarya were unvacuolated and appeared normal (Fig. 1a-c). The gut profiles contained abundant yellow-brown
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granular content (haematin), together with intact host erythrocytes (RBCs) and some leukocytes (WBCs) (Fig. 1a and b). The
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parenchymal tissue was eosinophilic. The uterus in each fluke was well-filled with normal-appearing shelled eggs, but no
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spermatozoa were present in the lumen, as was to be expected for flukes of the Cullompton isolate, which is aspermic and triploid
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(Fletcher et al., 2004; Hanna et al., 2008) (Fig. 1a). The testis profiles were densely packed with spermatogonia, and primary
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spermatocytes were also present, many showing condensed or fragmented chromatin, cytoplasmic eosinophilia or abnormal
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cytokinesis (features suggestive of apoptosis) (Fig. 1d). No later stages in spermatogenesis were represented. There were a few
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peripheral vacuoles in the testis profiles. Sections of ovary, where seen, displayed oogonia and oocytes in the normal distribution 8
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pattern, with little inter-cellular space (Fig. 1e). The Mehlis’ gland cells (S1- and S2- types), displayed rather uniform unvacuolated,
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granular, basophilic or pale eosinophilic cytoplasm, and the tubules connecting these cells to the ootype were also unvacuolated
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(Fig. 1f). The vitelline follicles appeared to be normal, with all stages in vitelline cell development well represented (stem cells; early
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and late intermediate cells and mature vitelline cells) (Fig. 1c).
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3.2. Twelve hours post-treatment (subcutaneous) flukes
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There was vacuolation beneath the tegumental syncytium, giving a somewhat striated appearance, and the tegumentalperikarya
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were rather shrunken and individualised (Fig. 1g). The tegumental syncytium was intact and attached to the basement membrane
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only over the anterior and mid-ventral parts of the sections (Fig. 2a-c). Behind the level of the uterus and common vitelline duct, the
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dorsal tegumental syncytium showed progressive fragmentation and detachment/sloughing from the basal lamina, leaving the latter
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and the underlying musculature largely exposed (Fig. 2a, e-g). The ventral syncytium remained attached to the basal lamina over
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the anterior 2/3 of the ventral surface of each section (Fig. 2a, d, f, g) but, in the posterior 1/3 of each section, the syncytium was
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fragmented or missing over both the dorsal and the ventral surface (Fig. 2a, h-j). Gut profiles were empty apart from a little
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haematin, rather contracted, and contained no blood cells (Fig. 2b, d-f,), while some profiles towards the posterior of the sections
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contained fibrillar basophilic material and cellular debris, suggesting breakdown of the gastrodermis (Fig. 2g-i). The uterus
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contained normal, or fewer than normal, numbers of well-shelled eggs (Fig. 2b and c). The testis profiles contained numerous
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rather shrunken and individualised spermatogonia and spermatocytes (including apoptotic cells), with much space between cells,
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and overall depletion of cell numbers (Fig. 2d-g).Profiles of ovary contained normal-looking oogonia and oocytes, but there was 9
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slightly more space between cells than in the untreated control sections. Mehlis’ gland cells, where seen, appeared normal (Fig.
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2c). The vitelline follicles were rather shrunken and most cells were either mature or late intermediate, containing numerous shell
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protein granules. Stem cells and early intermediate cells were less evident (Fig. 2g-j) than in control sections.
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3.3. Twelve hours post-treatment (oral) flukes
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In each fluke, the tegumental syncytium was intact and unbroken round the entire section profile, well attached to the basal lamina.
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Beneath the basal lamina there was vacuolation, giving a rather striated appearance to this zone. Gut profiles were empty and
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rather contracted, with only a little haematin in a few crypts of the gastrodermis. Normal eggs were present in the uterine lumen, but
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free vitelline cells were also seen. The testis profiles were generally well packed with spermatogonia and spermatocytes, but some
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profiles appeared rather depleted of cells, and in these tubules the cells were rounded, individualised, and well separated. The
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parenchyma, ovary and Mehlis’ gland were substantially normal. In the vitelline follicles mature vitelline cells tended to
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predominate, with rather fewer stem cells and early intermediate cells than in control material.
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3.4. Twenty-four hours post-treatment (subcutaneous) flukes
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The tegumental syncytium was intact and unbroken throughout the sections, with little vacuolation below the basal lamina. Gut
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profiles were contracted, with only a small amount of haematin in the gastrodermal crypts of some profiles. The parenchyma
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appeared normal. The uterus contained numerous shelled, normal-appearing eggs, but the most proximal coils also contained
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some free vitelline cells. The testis profiles were generally well populated with densely packed spermatogonia and spermatocytes,
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but peripheral vacuolation was more marked than in the control flukes. In profiles of ovary, the intercellular space was increased, 10
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separating individual oocytes and oogonia. Mehlis’ gland cells were vacuolated. Vitelline follicles were similar to those in the
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control flukes, but mature cells and intermediate cells were rather more abundant, and stem cells rather less abundant than in the
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untreated flukes.
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3.5. Twenty-four hours post-treatment (oral) flukes
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The tegumental syncytium in the flukes was largely intact, unbroken, and attached to the basal lamina all around the profiles of the
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sections, but there were occasional areas of breakdown and sloughing of the syncytium near the posterior end and, in some other
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areas, the basal infoldings of the syncytium were swollen, giving a striated pattern of vacuolation. The gut profiles were largely
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empty, rather contracted, and contained only a small amount of haematin in the gastrodermal crypts. In the uterus, some shelled
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eggs were present, together with shell debris and free vitelline cells. The testis profiles were rather depleted of cells, with rounding
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and separation of the spermatogonia and spermatocytes, appearance of empty space between cells, and breakdown of cells, with
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fragmentary debris present. The ovary showed increased intercellular space and some separation of oogonia and oocytes. The
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Mehlis’ gland cells were vacuolated. The vitelline follicles featured more mature and late intermediate cells, and relatively fewer
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stem cells (Fig. 3a) than seen in the untreated controls.
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3.6. Thirty-six hours post-treatment (subcutaneous) flukes
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The tegumental syncytium was intact and attached to the basal lamina only in the anterior 1/3 of the sections. In the posterior 2/3 of
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the sections, the tegumental syncytium was missing in places, fragmenting and sloughing from the basal lamina. In those areas
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where the syncytium was missing, there was disintegration of the vitellaria, gut profiles, testis and parenchyma, with loss of 11
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basophilic nuclear staining, and reduced eosinophilic staining of cytoplasm. Profiles of gut in the anterior portions of the flukes were
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empty, rather contracted, and contained only small amounts of haematin associated with the gastrodermis. In the posterior parts of
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the sections the gut profiles were disorganised, the gastrodermis was broken down and fragments occupied the lumen. There was
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marked loss of cellular detail. In the uterine lumen, few shelled eggs were present, but shell debris and free vitelline cells were
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evident (Fig. 3b). Testis profiles at the anterior end of the flukes showed depletion of cells, with marked peripheral vacuolation and
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some rounding and separation of spermatogonia and spermatocytes. These changes were more marked towards the posterior end
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of the flukes (Fig. 3c). The vitelline follicles towards the anterior of the flukes showed more mature vitelline cells and fewer stem
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cells than normal, and the stem cells tended to be rounded and individualised. Towards the posterior of the flukes the vitelline
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follicles showed disorganisation, disintegration of cells and loss of cellular detail, with shell protein granules appearing in irregular
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clumps and masses (Fig. 3d). Sections of ovary showed some depletion and rounding of oocytes, with increased inter-cellular
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space (Fig. 3e). Mehlis’ gland cells were vacuolated (Fig. 3f).
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3.7. Thirty-six hours post-treatment (oral) flukes
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The tegument was intact and attached to the basal lamina only over the anterior end of the sections, while it was missing, sloughing
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or fragmented over large areas of the posterior end of the sections. Where the tegumental syncytium was missing, the underlying
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vitelline follicles, testis profiles, gut profiles and parenchyma were disintegrating, and lacked basophilic nuclear staining and cellular
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detail. Where the tegument remained, the underlying structures retained more cellular detail and nuclear staining. In the uterus, no
12
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eggs were present, only free vitelline cells. Testis, ovary, vitelline follicles and gut profiles in areas of the sections that retained the
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tegumental syncytium were similar to those described for 36 h pt (subcutaneous) flukes (Section 3.6).
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The histological findings are summarised in Table 3.
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4. Discussion
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In general, there was a progressive increase in the severity of histological changes seen in the somatic and reproductive tissues of
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liver flukes with increasing time of in vivo exposure to closantel (12- 36 h), whether administered to the host by the oral or the
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subcutaneous route. A marked exception was the appearance of severe deterioration in the tegument and testis of flukes from a
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lamb treated by the subcutaneous route 12 h before slaughter (see Table 3). In this case, it is possible that a proportion of the dose
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was inadvertently delivered directly into a subcutaneous blood vessel, meaning that flukes in the bile ducts, feeding on blood,
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acquired a significant quantity of the active anthelmintic drug into the gut very soon after the host was dosed, in contrast to the
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situation following oral or subcutaneous administration. In the blood, closantel binds strongly to plasma albumin, achieving a peak
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level of approximately 55 µg/ml within 24 h after a 10 mg/kg oral dose, or 24–48 h after subcutaneous treatment
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Considering the findings summarised in Table 3, and discounting the anomalous 12h results, oral dosing of the host appears to
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have had a more severe and immediate effect on the flukes than subcutaneous dosing. This is evident in the 24h findings for
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tegument and testis. The ovary was the least affected of all tissues after both oral and subcutaneous dosing. However, examination
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of the uterus indicated that egg production was more affected by oral dosing than by subcutaneous dosing, with abnormalities
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evident from 12h onwards. The impact of closantel on egg production may have had more to do with effects on the vitellaria and 13
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Mehlis’ gland than on the ovary. The early appearance of changes in the tegument and gut, from 12h pt onwards, may indicate that
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primary drug effects on these tissues triggered more widespread effects on the internal organs, with the testis and vitellaria most
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severely affected because of their relatively high energy demand for cell differentiation and turn-over.
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Closantel, one of the salicylanilide group of anthelmintics, is believed to act mainly as an uncoupler of oxidative phosphorylation in
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flukes, although the initial effect may be on glycolysis (Fairweather and Boray, 1999; Kane et al., 1980; Rohrer et al., 1986; Van
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den Bossche et al., 1979). Closantel causes rapid spastic paralysis of F. hepatica, which may reflect changes in calcium ion levels
225
within muscle cells, rather than disruption of energy metabolism (Fairweather, 1997; Fairweather and Boray, 1999; Skuce and
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Fairweather, 1990). This paralytic effect of closantel on flukes may be the more significant action, because it may cause
227
detachment from the food source (blood vessels in the bile duct wall) and probably inhibits the pumping action of the pharyngeal
228
muscles, both effects leading to starvation, metabolic stress and enforced mobilisation of glycogen reserves in the parenchyma
229
(Fairweather and Boray, 1999).
230
Considering that closantel binds strongly to plasma albumin (Michiels et al., 1987; Mohammed-Ali and Bogan, 1987), it is likely that
231
the main route of entry of the drug into flukes is through the gut, following blood feeding. Paralysis of gut activity may cause pooling
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of the drug in the gut caeca, with subsequent distribution to other tissues. Cessation of normal feeding activity probably occurred
233
soon after treatment, as host blood cells were absent from the gut caeca 12 h onwards. In the later stages of treatment, sloughing
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of the gastrodermal lamellae and breakdown of the gastrodermis, particularly in the posterior region, was noted (Verheyen et al.,
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1980) related the posterior disruption of flukes from closantel-treated sheep to accumulation of the drug in the posterior gut caeca, 14
236
and found that morphological changes to the gut and tegument were evident from 4-8h pt, while changes to the reproductive
237
tissues became evident only after 24h treatment. Thus, while closantel is believed to have an over-arching impact on energy
238
metabolism in treated flukes, its action is compounded by paralysis, starvation, osmoregulatory effects and tegumental loss,
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meaning that the sequence of toxicological effects is difficult to interpret.
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The most striking change in treated fluke was the widespread loss of the tegument, particularly in the posterior region of the
241
fluke, in all the 36 h-treated flukes and in the 12 h subcutaneously-treated group. This left the basal lamina and underlying
242
musculature exposed at the surface. The sloughing was most likely the result of swelling of the basal infolds in the syncytium and
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consequent disruption of the energy-dependent osmoregulatory role of the tegument (Fairweather and Boray, 1999). That the
244
posterior region of the fluke was most severely affected may be due to the accumulation of drug in the gut, as suggested by
245
Verheyen et al. (1980). Regional variation in drug effects have been noted in studies on other flukicides and preservation of the
246
anterior end (which contains “essential” organs such as the main nerve ganglia, suckers and pharynx) observed ,for references,
247
see (Toner et al., 2010).
248
In the uterus of closantel-treated flukes, normal shelled eggs were present, albeit in decreasing numbers, until 36 h pt. This is
249
consistent with the results obtained of an egg viability test efficacy. The results showed that 89.5% of eggs embryonated at 12 h pt,
250
86.75% at 24 h pt and 67.5% at 36 h pt (Solana et al., 2014). However, greater numbers of free vitelline cells and irregular masses
251
of shell debris accumulated in the uterine lumen with increasing time of treatment. This suggests that the normal process of egg
252
assembly in the ootype may have decelerated and possibly stopped completely by 36 h pt, possibly due to restriction of the supply 15
253
of energy intermediates imposed by the metabolic effects of the anthelmintic. Neuromuscular inhibition of the uterine walls may
254
have led to retention of eggs within the uterine lumen, partially disguising earlier blockage of egg assembly. With TCBZ-sensitive F.
255
hepatica isolates, in vivo treatment with TCBZ resulted in rapid cessation of egg assembly in the ootype, and the uterine coils of 24
256
h-treated flukes contained free vitelline cells and non-cellular shell debris, but no shelled eggs (Hanna et al., 2010; Hanna et al.,
257
2012).
258
Whatever the underlying drug mechanism, disruption of egg production in closantel-treated flukes will be the result of gross
259
changes in the different tissues involved, namely, the ovary, Mehlis’ gland and vitellaria. The ovary was the least affected of all the
260
reproductive organs, but there was a progressive depletion in the numbers of oogonia and primary oocytes. In the 36 h-treated
261
individuals, many oocytes appeared rather rounded and individualised, with condensed cytoplasm, possibly suggestive of
262
apoptosis, as described in the ovary of TCBZ-sensitive flukes treated in vivo with TCBZ (Hanna et al., 2013). It is likely that cell
263
depletion in the ovary reflected failure of the rate of mitosis in the oogonia to replace exiting oocytes, due to restriction in the supply
264
of high energy intermediates of metabolism. The Mehlis’ gland cells are not replaced by stem cell division but, with progressively
265
longer exposure to closantel, they displayed vacuolation in the cell cytoplasm and in the tubules that convey their secretions to the
266
ootype. Mehlis’ gland secretions are known to be involved in the release of shell protein material from the vitelline cells in the
267
ootype and the laying down of the eggshell (Colhoun et al., 1998; Fairweather et al., 1999), so disruption of the cells would greatly
268
impair egg formation. There is a sophisticated neuromuscular mechanism that co-ordinates the complex and rapid events
269
associated with egg production, as alluded to above, and its inhibition would only (serve to) compound the situation. 16
270
Adult F. hepatica each produce approximately 25,000 eggs per day (Happich and Boray, 1969), and approximately 30 vitelline cells
271
are incorporated in each egg to provide the shell-protein precursor molecules and the glycogen stores necessary for embryogenesis
272
(Fairweather et al., 1999). Therefore, cell multiplication and differentiation in the vitelline follicles, which occupy a larger proportion of
273
the body than any other tissue, probably consume the largest proportion of the energy generated by intermediary metabolism (Hanna
274
et al., 2006), and would be expected to be particularly sensitive to closantel-induced energy restriction. In 5-week-old flukes, closantel
275
appeared to block both cell division and differentiation, resulting in marked stunting of the vitelline follicles (Hanna et al., 2006).
276
However, in the adult flukes examined here, the primary effect of in vivo closantel treatment appeared to be blockage of division in
277
the stem cells, resulting in a decline in their number, while pre-formed intermediate-type vitelline cells continued their development for
278
some time, ultimately packing the follicles with swollen mature cells. In 36 h-treated flukes, mature vitelline cells had often
279
disintegrated within the follicles, possibly because of increasing cell pressure and blockage of exit from the follicles due to the energy
280
deficit or to neuromuscular inhibition of the lining of the vitelline ducts. As with the gut and testis, histological change and
281
disintegration in the vitelline follicles was most marked towards the posterior end of the treated flukes, especially where the
282
tegumental syncytium was disrupted or absent.
283
The primary effect of in vivo closantel treatment on the testis of Cullompton-isolate F. hepatica was depletion of the cellular content.
284
The cells affected were mainly spermatogonia, with fewer primary and secondary spermatogonia present at the periphery of the
285
tubules. The tertiary spermatogonia became progressively dispersed, individualised and often rounded, displaying unusually dense
286
nuclei, in closantel-treated worms. Such changes may be indicative of apoptosis (Cohen et al., 1992; Hanna et al., 2013; Hanna et 17
287
al., 2012), but the characteristic morphological changes seen in these closantel-treated flukes were less conspicuous than those
288
seen in the testis of Cullompton flukes exposed to in vivo TCBZ treatment (Hanna et al., 2010; Hanna et al., 2013). It is likely that
289
depletion of cell populations in the testis of closantel-treated flukes resulted from a failure of the spermatogonia to undergo mitosis,
290
due to the restriction in energy metabolism. Pre-formed tertiary spermatogonia may be similarly blocked from further development,
291
precipitating the reactions of the caspase cascade, and triggering apoptosis (Kumar et al., 2005). The effects of closantel in causing
292
stunting and inhibition of gametogenesis and vitellogenesis in late immature (5-week-old) F. hepatica, were likewise attributed to
293
blockage of intermediary metabolism in the developing flukes (Hanna et al., 2006). In flukes of the triploid Cullompton isolate used
294
in this study, meiosis cannot be completed, and cells representing developmental stages later than primary spermatocytes were
295
rarely seen. Even in untreated flukes, the primary spermatocytes appeared abnormal, exhibiting karyorrhexis, failure of cytokinesis,
296
and apoptosis, consistent with previous descriptions (Hanna et al., 2010).
297
In summary, progressive histological changes were seen in adult Cullompton-isolate F. hepatica exposed to closantel in lambs that
298
had been treated by oral or subcutaneous administration of the drug 12 – 36 h before slaughter. These changes could, in general,
299
be attributed to anthelmintic-induced blockage of intermediary metabolism and neuromuscular paralysis, which have previously
300
been implicated as the main mechanisms of fasciolicide action for closantel. In the fluke tegument, inhibition of osmoregulation,
301
induced by the energy deficit, led to swelling, vacuolation and sloughing of the surfacelayer, particularly in the posterior and dorsal
302
regions, thus contributing to disintegration of the underlying tissues. In the gut, rapid neuromuscular paralysis ensured cessation of
303
feeding activity, contributing to energy starvation. In those tissues such as testis, ovary and vitelline follicles, where maintenance of 18
304
output requires active cell division, defects in closantel-treated flukes were aligned with failure in the energy-demanding processes
305
of mitosis and differentiation.
306
The importance of this study is that it establishes a model for closantel-induced histological changes in F. hepatica from treated sheep,
307
and can inform the development of supplementary histology-based diagnostic methods for the identification of closantel resistance in
308
fluke, as has been described for the identification of TCBZ resistance in field isolates (Hanna et al., 2015). While closantel resistance
309
in F. hepatica is not widespread or a concernat present, it has been described in endemic areas of New South Wales, and cases have
310
been reported in Great Britainin the past (Fairweather and Boray, 1999; Rolfe et al., 1990; Van Wyk and Malan, 1988). With increasing
311
use of the drug to replace TCBZ, closantel resistance may become (more of) an issue in the future.
312
Conflict of interest statement
313
No actual or potential conflict of interest was identified that could inappropriately influence, or be perceived to influence the
314
outcome of this work.
315
Acknowledgements
316
Thanks are due to the staff of the Histopathology Section and to Mr C. Mason, Photographer, VSD, AFBI, Stormont for expert
317
assistance. No external funding was obtained for this work.
318
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319
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Hanna, R.E.B., Edgar, H.W.J., McConnell, S., Toner, E., McConville, M., Brennan, G.P., Devine, C., Flanagan, A., Halferty, L.,
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G.P., 2015. Fasciola hepatica: A comparative survey of adult fluke resistance to triclabendazole, nitroxynil and closantel on
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selected upland and lowland sheep farms in Northern Ireland using faecal egg counting, coproantigen ELISA testing and
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Hanna, R.E.B., Scarcella, S., Solana, H., Mc Connell, S., Fairweather, I., 2012. Early onset of changes to the reproductive system of Fasciola hepatica following in vivo treatment with triclabendazole. Vet. Parasitol 184, 341-347. 23
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Mitchell, G.B., Maris, L., Bonniwell, M.A., 1998. Triclabendazole-resistant liver fluke in Scottish sheep. Vet. Rec 143, 399.
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Mohammed-Ali, N., Bogan, J., 1987. The pharmacodynamics of the flukicidal salicylanilides, rafoxanide, closantel and
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Olaechea, F., Lovera, V., Larroza, M., Raffo, F., Cabrera, R., 2011. Resistance of Fasciola hepatica against triclabendazole in cattle in Patagonia (Argentina). Vet. Parasitol 178, 364-366. Ortiz, P., Scarcella, S., Cerna, C., Rosales, C., Cabrera, M., Guzmán, M., Lamenza, P., Solana, H., 2013. Resistance of Fasciola
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Overend, D.J., Bowen, F.L., 1995. Resistance of Fasciola hepatica to triclabendazole. Aust. Vet. J 22, 275-276.
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Rohrer, S.P., Saz, H.J., Nowak, T., 1986. 31 P-NMR studies of the metabolisms of the parasitic helminths Ascaris suum and
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Fasciola hepatica. Arch. Biochem. Biophys 248, 200-209. Rolfe, P.F., Boray, J.C., Fitzgibbon, C., Parsons, G., Kemsley, P., Sangster, N., 1990. Closantel resistance in Haemonchus contortus from sheep. Aust. Vet. J 67, 29-31. Skuce, P.J., Fairweather, I., 1990. The effect of the hydrogen ionophore closantel upon the pharmacology and ultrastructure of the adult liver fluke Fasciola hepatica. Parasitol. res 76, 241-250. Solana, H.D., Rodriguez, J.A., Lanusse, C.E., 2001. Comparative metabolism of albendazole and albendazole sulphoxide by different helminth parasites. Parasitol. Res 87, 275-280. Solana, M.V., Scarcella, S., E, M.-M., Solana, H., 2014. In vivo assessment of Closantel ovicidal activity in Fasciola hepatica eggs. In: 13 th international Congress of Parasitology, Mexico, p. 1140.
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Toner, E., Brennan, G., Hanna, R.E.B., Edgar, H.W.J., Fairweather, I., 2010. Tegumental surface changes in adult Fasciola hepatica in response to treatmentin vivo with triclabendazole in the sheep host. Vet. Parasitol 172, 238-248. Van den Bossche, H., Verhoeven, H., Vanparijs, O., Lauwers, H., Thienpont, D., 1979. Closantel, a new antiparasitic hydrogen ionophore Arch. Int. Physiol. Biochim 87, 851. Van Wyk, J.A., Malan, F.S., 1988. Resistance of field strains of Haemonchus contortus to ivermectin, closantel, rafoxanide and the benzimidazoles in South Africa. Vet. Rec 123, 226-228. Verheyen, A., Vanparijs, O., Lauwers, H., Thienpont, D., Van den Bossche, H., 1980. The influence of closantel administration to
425
sheep on the ultrastructure of the adult liver fluke Fasciola hepatica In: The host-invader interplay.(Proc. 3rd Internat. Symp.
426
on the biochemistry of parasites and host-parasite relationships, Beerse, Belgium, 30 June to 3 July 1980.), pp. 705-708.
427
Wood, I.B., Amaral, N.K., Bairden, K., Duncan, J.L., Kassai, T., Malone, J.B., Pankavich, J.A., Reinecke, R.K., Slocombe, O.,
428
Taylor, S.M., Vercruysse, J., 1995. World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) Second
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430
213.
431 432
Legends for Tables:
433
Table 1.
434
Summary of treatments administered to groups of experimental animals. 26
435
Table 2.
436
Total number of flukes recovered from each animal.
437
Table 3.
438
Summary of the histological changes occurring in the tissues of Cullompton isolate Fasciola hepatica collected from sheep treated
439
with closantel orally or by subcutaneous injection 12 h, 24 h or 36 h prior to slaughter, compared to flukes collected from untreated
440
sheep. The severity of change is indicated by box shading, thus:
441
Unshaded = normal histology
442
Pale shading = mild to moderate abnormality
443
Dark shading = moderate to severe abnormality
444 445 446
Legends for Figures
447
Fig. 1.(a - f)
448
Fasciola hepatica: H&E-stained sections of Cullompton isolate flukes from an untreated sheep; (g)H&E-stained section of a
449
Cullompton isolate fluke from a sheep treated 12h previously by subcutaneous injection with closantel. (a) The surface syncytium of
450
the tegument (Ts) at the anterior end is deeply scalloped and bears numerous spines (arrowed), while the tegumentalperikarya (Tc)
451
lie in a discontinuous layer beneath the somatic musculature (M). The uterus (U) is packed with well-shelled eggs (E), and a section 27
452
of gut caecum (G) contains partially digested host blood, together with haematin. Profiles of testis (Te) are densely packed with
453
spermatogonia, and profiles of the excretory system appear as unstained spaces (Ex) in the eosinophilic parenchyma (P). (b) The
454
tegumental syncytium (Ts) is continuous round the posterior end of the fluke, but is relatively smooth, and bears few spines. Some
455
gut profiles (G) contain host blood, while some are empty. Vitelline follicles (V) are abundant in the eosinophilic parenchyma (P).
456
(c)Each vitelline follicle contains stem cells peripherally (Vs), early and late intermediate cells (Vi), characterised by the presence of
457
shell protein globules, and mature vitelline cells (Vm), each of which is swollen with unstained glycogen. The tegumental syncytium
458
(Ts) is firmly attached to the basal lamina (BL) and is not vacuolated. Tegumentalperikarya (Tc) lie beneath the muscle layers. (d)
459
Profiles of testis (Te) are densely packed with spermatogonia (Sg), and primary spermatocytes (Sc), some of which exhibit
460
karyorrhexis or apoptosis (arrowed) in flukes of this isolate. Occasional peripheral vacuoles (Sv) are evident. P = parenchyma. (e)
461
Ovarian tubules (O) are demarked by a thick muscular wall (M) and contain oogonia (Og) with dense nuclei located peripherally,
462
while the core is densely packed with oocytes (Oc), each containing one or two conspicuous nucleoli (arrowed). (f) Mehlis’ gland
463
cells (Mg) have a granular unvacuolated cytoplasm, with elongated tubules leading towards the ootype. A portion of the vitelline
464
reservoir (Vr) is visible. (g) Although the tegumental syncytium (Ts) is firmly attached to the basal lamina (BL), there is marked
465
vacuolation (arrowed) between the syncytium and the muscle layers (M). Tc = tegumentalperikarya; Tsp = tegumental spine.
466
Fig. 2.
467
Fasciola hepatica: H&E-stained mid-sagittal section of a Cullompton isolate fluke from a sheep treated 12h previously by
468
subcutaneous injection with closantel. On the low magnification image (2a), the location of the high magnification images (2b – j) 28
469
are indicated by boxes. In (a), the oral sucker (OS) and the acetabulum (ventral sucker, VS) are located anterior to the uterus (U)
470
and ovarian tubules (O). Profiles of testis (Te) are seen in the mid region of the body, while vitelline follicles (V) extend from the mid
471
region to the posterior. Profiles of gut (G) are evident at all levels of the section. The tegumental syncytium (arrows) is intact and
472
attached around the anterior end of the section, but is fragmented and missing from the mid-dorsal region, progressively towards
473
the posterior. The syncytium is missing from both the dorsal and ventral surfaces in the posterior third of the section. (b) Dorsal
474
surface. The uterus (U) contains shelled eggs (E) and the tegumental syncytium (Ts), which bears spines (Tsp), is firmly attached
475
to the basal lamina, although some basal vacuolation is evident (arrowed). G = gut caecum. (c)Dorsal surface.Some shell debris
476
and free vitelline cells (arrowed), as well as shelled eggs, are apparent in the uterus (U). A section of oviduct (Ov) is partially
477
surrounded by Mehlis’ gland cells (Mg). (d) Ventral surface. The tegumental syncytium (Ts) is intact and well attached to the basal
478
lamina. The testis profiles (Te) show depleted numbers of spermatogonia, with a corresponding increase in intercellular space. G =
479
gut caecum; Tc = tegumentalperikarya. (e)Dorsal surface.The tegumental syncytium (Ts) is fragmenting and with the spines (Tsp)
480
is detaching from the basal lamina (BL), which is left exposed at the surface. The parenchyma (P) is highly vacuolated. G = gut
481
caecum; Te = testis. (f, g) The tegumental syncytium (Ts) remains largely attached on the ventral surface but is fragmenting and
482
detaching from the dorsal surface. G = gut caeca; Te = testis; V = vitelline follicles. ( h-j) The tegumental syncytium is missing from
483
both the dorsal and the ventral surface, leaving the basal lamina exposed (BL). The gut caeca (G) contain fibrillar material or
484
sloughed gastrodermal cells. The vitelline follicles (V) are shrunken and breaking down. The parenchyma (P) is highly vacuolated.
485 29
486
Fig. 3.
487
Fasciola hepatica: H&E-stained sections of Cullompton isolate flukes from sheep treated 24h previously by oral administration of
488
closantel (a) or 36h previously with closantel by subcutaneous injection (b-f). In (a), the vitelline follicles contain mainly swollen
489
mature vitelline cells (Vm) with unstained cytoplasm, while stem cells (Vs) and immature vitelline cells are less evident than normal.
490
Tc = tegumentalperikarya. (b) Profiles of uterus (U) in the anterior end of the body contain a few shelled eggs (E), together with
491
shell debris and free vitelline cells (arrowed). The gut caeca (G) are empty, and the tegumental syncytium (Ts) is attached to the
492
basal lamina. P = parenchyma. (c) In the testis (Te), there is a marked increase in intercellular space, especially peripherally, with
493
rounding, separation and depletion of spermatogonia (Sg). Spermatocytes (Sc), with marginalised nuclei are evident. (d) The
494
vitelline follicles (V), which contain mainly mature vitelline cells (Vm), are disintegrating, with release of protein globules,
495
cytoplasmic and nuclear debris. The gut caecum (G) contains haematin in the gastrodermal crypts. (e) In the ovary (O), some of the
496
peripheral oogonia (Og) are rounded and individualised, while oocytes (Oc) are rounded and depleted, with a corresponding
497
increase in intercellular space. P = parenchyma. (f) The Mehlis’ gland cells (Mg) and their elongated connections (arrowed) to the
498
ootype are vacuolated. Od = terminal oviduct.
499
30
500 501
Fig. 1
502 503 31
504 505
Fig. 2
506 507 32
508 509
Fig. 3
510 511 33
512 513 514
Table 1
515
Experimental animals groups.
516
Group
Animals
Dose
Days post-infection before treatment
1
2
no treatment
... ... ... ... ... ..
2
6
10 mg/kg b.w. Oral
112
3
6
10 mg/kg b.w.
112
subcutaneously 517 518 519 520 521 34
522
Table 2
523
Total number of flukes recovered from each animal.
524
Group
Time post treatment (PT)
Flukes recovered
Untreated
-
46
Oral
12 H
20
Oral
24 H
16
Oral
36 H
6
Subcutaneous
12 H
7
Subcutaneous
24 H
22
Subcutaneous
36 H
8
525 526 527
35
528 529 530 531
Table 3.
532
Summary of the histological changes occurring in the tissues of Cullompton isolate Fasciola hepatica collected from sheep treated with
533
closantel orally or by subcutaneous injection 12 h, 24 h or 36 h prior to slaughter, compared to flukes collected from untreated sheep. The
534
severity of change is indicated by box shading, thus:
535
Unshaded = normal histology
536
Pale shading = mild to moderate abnormality
537
Dark shading = moderate to severe abnormality
538 539 540 541 542 36
Fluke tissue
0 hour
12 hour
24 hour
36 hour
untreated
post-treatment
post-treatment
post-treatment
control
543 544 545 37
Subcut. Tegument
Oral
Subcut.
Oral Syncytium mainly intact and attached to BL, but some sloughing towards posterior.
Syncytium intact and attached to basal lamina (BL).
Syncytium intact and attached to BL only anteriorly.
Syncytium intact and attached to BL throughout.
Syncytium intact and attached to BL throughout.
Not vacuolated.
Fragmented/detached posteriorly.
Some vacuolation below syncytium.
Little vacuolation below syncytium.
Some vacuolation.
Small amount of haematin only.
Small amount of haematin only.
Small amount of haematin only.
Subcut.
Oral
Intact and attached to BL anteriorly.
Intact and attached to BL anteriorly.
Fragmented or sloughed from posterior.
Fragmented or sloughed from posterior.
Empty anteriorly.
Empty anteriorly.
Breakdown of gastrodermis posteriorly.
Breakdown of gastrodermis posteriorly.
Basal vacuolation. Gut
Contains haematin, RBCs and WBCs.
Small amount of haematin only. Some gastrodermis breakdown posteriorly.
Uterus
Full of well shelled eggs.
Few to normal numbers of eggs.
Few to normal numbers of eggs +/- vitelline cells.
Numerous normal eggs +/- vitelline cells.
Some eggs present +/shell debris +/- vitelline cells.
Few eggs +/- shell debris +/- vitelline cells
Only free vitelline cells present.
Depletion of cell numbers.
Most profiles normal.
Densely packed with Sg and Sc.
Cell depletion.
Cell depletion.
Cell depletion.
Shrinkage of cells.
A few profiles with cell depletion and increased intercellular space.
Rounding and separation of Sg and Sc.
Peripheral vacuolation.
Peripheral vacuolation.
Rounding and separation of Sg and Sc.
Rounding and separation of Sg and Sc.
Changes most evident posteriorly.
Changes most evident posteriorly.
Increased intercellular space.
Increased intercellular space.
No spermatozoa. No vitelline cells. Testis
Densely packed with spermatogonia (Sg) and spermatocytes (Sc, many apoptotic). Little peripheral vacuolation.
Ovary
Densely packed with oogonia and oocytes.
Increase in intercellular space.
Marked peripheral vacuolation.
Fragmentation of cells.
Normal appearance.
Generally normal.
Separation of cells.
Separation of cells.
Slight increase in intercellular space.
Small increase in intercellular space.
Increase in intercellular space.
Increased intercellular space.
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Mehlis’ gland Vitellaria
Gland cells and tubules not vacuolated.
Normal appearance.
Normal appearance.
Gland cells vacuolated.
Gland cells vacuolated.
Gland cells vacuolated.
Gland cells vacuolated.
All developmental stages well represented, viz. stem cells, early and late intermediate cells, mature cells .
Increased proportion of mature and late intermediate cells.
Increased proportion of mature and late intermediate cells.
Increased proportion of mature and late intermediate cells.
Increased proportion of mature and late intermediate cells.
Increased proportion of mature cells.
Increased proportion of mature cells.
Stem cells numbers decreased, often shrunken.
Decreased stem cell numbers.
Proportion of stem cells decreased.
Proportion of stem cells decreased.
Proportion of stem cells decreased.
Proportion of stem cells decreased.
Disintegration of follicles posteriorly.
Disintegration of follicles posteriorly.
546 547 548
39