Fasciola hepatica: Histological changes in the somatic and reproductive tissues of liver fluke following closantel treatment of experimentally-infected sheep

Fasciola hepatica: Histological changes in the somatic and reproductive tissues of liver fluke following closantel treatment of experimentally-infected sheep

Accepted Manuscript Title: Fasciola hepatica: histological changes in the somatic and reproductive tissues of liver fluke following closantel treatmen...

11MB Sizes 0 Downloads 39 Views

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.

1

Fasciola hepatica: histological changes in the somatic and reproductive tissues of liver fluke following closantel

2

treatment of experimentally-infected sheep.

3 4

S. Scarcella a*; R.E.B. Hanna b; G. P. Brennan c; H. Solana a; I. Fairweather c

5 a

6

Laboratorio de Biología Celular y Molecular. Centro de Investigación Veterinaria de Tandil (CIVETAN), CONICET, Facultad de Ciencias Veterinarias, UNCPBA, Tandil, Argentina

7 b

8 9

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

10 11 12

*Corresponding author:

13

[email protected], Tel: + 54 2293 439850 Int. 234, Fax: +54 2293 439850. Laboratorio de Biología Celular y Molecular.

14

Centro de Investigación Veterinaria de Tandil (CIVETAN), CONICET, Facultad de Ciencias Veterinarias, UNCPBA

15

Tandil (7000), Argentina.

16 1

17 18 19 20 21

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.

22 23 24 25

Abstract

26

Lambs infected with the Cullompton isolate of Fasciola hepatica were treated orally or subcutaneously with 10 mg/kg of closantel at

27

16 weeks post-infection. Adult flukes were recovered from the liver of individual animals at 12h, 24h, or 36h post-treatment. The

28

flukes were processed for histological analysis. In general, degenerative changes in the reproductive and somatic tissues were

29

progressive, and were most marked in flukes exposed to closantel in vivo for 36h. However, flukes from a 12h subcutaneously-

30

treated lamb showed marked deterioration of the testis, possibly because a portion of the dose has been delivered intravenously.

31

Fewer intact eggs were seen in the uterus of flukes exposed to closantel for longer times (whether administered subcutaneously or

32

orally to the host). The most conspicuous closantel-induced effect in flukes from treated hosts was progressive damage to the

33

tegumental syncytium. While the flukes from 24h-treated hosts showed relatively minor damage to limited areas of the syncytium,

34

towards the posterior end, the flukes from 36h-treated hosts (and flukes from the lamb that putatively received intravenous dosage) 2

35

had lost large areas of the surface syncytium from the posterior end and dorsal surface, although the syncytium over the anterior

36

end and the anterior ventral surface was largely spared. In areas where the syncytium had sloughed, the underlying structures such

37

as the vitelline follicles, gut profiles and testis profiles, showed marked degeneration and breakdown. Other changes included cell

38

depletion and early stage apoptosis in the testis, ovary and vitelline follicles. This study establishes a model for histological changes

39

in closantel-sensitive F. hepatica exposed to closantel in vivo.

40

Histopathological studies could be complementary to the efficacy controlled test for for closantel resistance in fluke populations.

41 42

Key words:

43

Fasciola hepatica; liver fluke; sheep; closantel in vivo; post-treatment fluke histology; fluke resistance diagnosis.

44 45 46 47

1. Introduction

48

Since its introduction in 1983, the fasciolicide triclabendazole (TCBZ) has been extensively used worldwide to control liver fluke

49

infections in sheep and cattle, mainly because it possesses a uniquely wide spectrum of activity, killing not only adult Fasciola spp.,

50

but also immature and juvenile flukes as young as 2 days post-infection (Boray et al., 1983; Fairweather and Boray,

51

1999).However, since 1995, reports of fluke resistance to the anthelmintic have appeared with increasing frequency in stock3

52

producing countries where TCBZ is used routinely (Brockwell et al., 2014; Daniel et al., 2012; Fairweather, 2005; Fairweather,

53

2009; Gordon et al., 2012; Mooney et al., 2009; Olaechea et al., 2011; Ortiz et al., 2013; Overend and Bowen, 1995). Recently, in

54

Northern Ireland (NI), 13 sheep farms were surveyed for TCBZ resistance. It was shown that, in every flock where FECs indicated

55

the presence of significant chronic Fasciola hepatica infection, substantial resistance to TCBZ was also present, but closantel and

56

nitroxynil effectively eliminated the adult fluke burden (Hanna et al., 2015). It is likely, therefore, that the emphasis in

57

chemotherapeutic control of fasciolosis in highly endemic areas will switch from routine use of TCBZ for treatment both of acute

58

and chronic infections, to strategic use of drugs such as closantel, which have a narrower spectrum of activity, limited to late

59

immature and adult F. hepatica, in an attempt to minimise pasture contamination with fluke eggs, and reduce the risk of acute

60

infection in the next season’s lamb flock (Hanna et al., 2015).

61

Closantel is a salicylanilide anthelmintic that binds extensively to plasma albumin (Michiels et al., 1987). As a result, its activity is

62

mainly directed against blood-feeding internal parasites such as F. hepatica, Haemonchus contortus, Oestrus ovis and

63

Oesophagostomum larvae. Its activity is associated with disruption of energy metabolism, specifically uncoupling of oxidative

64

phosphorylation, a mechanism different from the anti-microtubule action of TCBZ (Fairweather and Boray, 1999). Unlike TCBZ,

65

closantel is much less effective on young immature flukes than on adults and late immature flukes that have reached the bile ducts

66

(Boray, 1997; Fairweather and Boray, 1999). However, in experiments with both sheep and rats, (Maes et al., 1988) showed that,

67

as well as removing a high percentage of adult flukes, and to a lesser extent immature flukes, the size of any flukes that survived

68

treatment was stunted. This finding was supported by the work of Hanna et al. (Hanna et al., 2006), who showed that in vivo 4

69

closantel treatment of late immature, 5-week-old F. hepatica infections in cattle resulted in reduction of the fluke burden by more

70

than 50%, with the surviving parasites showing stunting, reduced histological development of the reproductive organs, and

71

attenuation of egg output.

72

To date, there is limited evidence of development of fluke resistance to closantel, although in endemic areas of New South Wales,

73

where closantel and rafoxanide have long been used to control H. contortus as well as F. hepatica, resistant strains of the

74

nematode have been reported (Rolfe et al., 1990; Van Wyk and Malan, 1988), and instances of suspected salicylanilide resistance

75

in F. Hepatica have been recorded in England and Wales (Fairweather and Boray, 1999). With increasing use of closantel in areas

76

where TCBZ resistance is established (McMahon, 2015), it is expected that fluke resistance to the former will also emerge.

77

Diagnosis of anthelmintic resistance in local fluke populations is complicated by a number of factors, including the long pre-patent

78

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),

79

and the retention of eggs in the gall bladder of the host for some time after the flukes themselves have been removed by successful

80

drug treatment, which can yield false-positive FECs (Chowaniec and Darski, 1970; Flanagan et al., 2011a; Flanagan et al., 2011b;

81

Mitchell et al., 1998). It is considered advisable, therefore, that diagnosis of flukicide resistance in field situations should be based

82

on the results of several diverse methods, rather than relying solely on faecal egg count reduction tests (FECRT). Confirmatory

83

tests might include the coproantigen reduction test (CRT) by ELISA (Flanagan et al., 2011a; Flanagan et al., 2011b; Gordon et al.,

84

2012; Gordon et al., 2012b; Mezo et al., 2004;Valero et al., 2009)

5

85

post-treatment fluke histology (Hanna et al., 2010), or egg hatch assay (EHA) (Alvarez et al., 2009; Canevari et al., 2014;

86

Fairweather, 2011b; Fairweather et al., 2012).

87

The purpose of the present investigation is to describe the histological changes that occur in closantel-sensitive F. hepatica,

88

exposed to the anthelmintic in recently-treated hosts. Few morphological studies have been carried out on closantel action.

89

Ultrastructural changes to the tegument and gut were described by Verheyen et al. (1980), Skuce (1987) and Skuce and

90

Fairweather (1990) and histological changes to the reproductive organs by Maes et al. (1988).These studies on adult fluke

91

established the time-course of successful drug action in vivo and helped to explain how the morphological changes led to the

92

elimination of the flukes from the host. In the present investigation, fluke material was collected at an early time-point (12 h post-

93

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

94

reproductive system become evident. This information will help provide a model for subsequent investigations of closantel

95

resistance using pt fluke histology.

96

2. Materials and methods

97

2.1. Experimental protocol

98

Animal procedures and management protocols were approved by the Ethics Committee according to the Animal Welfare Policy

99

(Act 087/02) of the Faculty of Veterinary Medicine, Universidad Nacional del Centro de la Provincia de Buenos Aires (UNCPBA),

100

Tandil, Argentina (http://www.vet.unicen.edu.ar ), and to internationally accepted Animal Welfare Guidelines (A.V.M.A, 2001).

101

Fourteen parasite-free Corriedale weaned lambs were each inoculated orally with 200 metacercariae of F. hepatica contained in a 6

102

gelatin capsule. The isolate used for this experiment was the Cullompton isolate, which is TCBZ-susceptible: for details of its

103

provenance please see (Fairweather, 2011a). The presence of liver fluke in the lambs was confirmed 15 weeks after infection by

104

the finding of eggs in the faeces, and liver damage was estimated indirectly by measurement of serum Glutamate Dehydrogenase

105

and Gamma Glutamyl Transferase activities, as described previously (Solana et al., 2001). Sixteen weeks after oral inoculation, the

106

animals were assigned to three experimental groups, based on their clinical condition and body weight, and were treated orally or

107

by subcutaneous injection with closantel, as detailed in Table 1.

108

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

109

evaluating antiparasitic treatments in ruminants (Wood et al., 1995). Flukes were recovered from the common bile duct of each lamb

110

and rinsed thoroughly with warm (37 oC), sterile saline solution (0.9% w/v, sodium chloride) to remove bile and/or adhering

111

materials. Likewise, flukes were collected from the untreated control animals in group 1.Total number of flukes recovered from each

112

animal is disclosed in Table 2. The flukes obtained from each animal were examined grossly and two representative specimens

113

were selected for detailed histological examination from each of the experimental treatments.

114

2.2. Preparation of flukes for histology

115

Flukes were fixed in 10% (v/v) neutral-buffered formalin for 24 h. Histological processing and embedding in wax was carried out by

116

conventional techniques. Sections 3 µm in thickness were cut from each block face and stained with haematoxylin and eosin using

117

standard histological protocols. Sections were examined and the tissues photographed using a Leica DM LBZ microscope fitted

7

118

with a Nikon Coolpix 5000 camera system. Amore detailed protocol is given by (Hanna et al., 2008; Hanna et al., 2010; Hanna et

119

al., 2015).

120

3. Results

121

The report on the histology was based on a combined assessment of the two specimens examined from each treatment.

122

3.1. Control flukes from untreated lambs

123

The histological features of the reproductive organs in untreated Cullompton isolate flukes were described previously by Hanna et

124

al. (2010) and by Hanna (2015). In the untreated flukes examined in this study, there was no evidence of post-mortem degenerative

125

changes, and therefore they were considered to be valid controls for comparison with flukes from treated animals. The tegumental

126

syncytium was unbroken, unvacuolated, and was attached at all points to the underlying basal lamina and musculature. The

127

tegumental perikarya were unvacuolated and appeared normal (Fig. 1a-c). The gut profiles contained abundant yellow-brown

128

granular content (haematin), together with intact host erythrocytes (RBCs) and some leukocytes (WBCs) (Fig. 1a and b). The

129

parenchymal tissue was eosinophilic. The uterus in each fluke was well-filled with normal-appearing shelled eggs, but no

130

spermatozoa were present in the lumen, as was to be expected for flukes of the Cullompton isolate, which is aspermic and triploid

131

(Fletcher et al., 2004; Hanna et al., 2008) (Fig. 1a). The testis profiles were densely packed with spermatogonia, and primary

132

spermatocytes were also present, many showing condensed or fragmented chromatin, cytoplasmic eosinophilia or abnormal

133

cytokinesis (features suggestive of apoptosis) (Fig. 1d). No later stages in spermatogenesis were represented. There were a few

134

peripheral vacuoles in the testis profiles. Sections of ovary, where seen, displayed oogonia and oocytes in the normal distribution 8

135

pattern, with little inter-cellular space (Fig. 1e). The Mehlis’ gland cells (S1- and S2- types), displayed rather uniform unvacuolated,

136

granular, basophilic or pale eosinophilic cytoplasm, and the tubules connecting these cells to the ootype were also unvacuolated

137

(Fig. 1f). The vitelline follicles appeared to be normal, with all stages in vitelline cell development well represented (stem cells; early

138

and late intermediate cells and mature vitelline cells) (Fig. 1c).

139

3.2. Twelve hours post-treatment (subcutaneous) flukes

140

There was vacuolation beneath the tegumental syncytium, giving a somewhat striated appearance, and the tegumentalperikarya

141

were rather shrunken and individualised (Fig. 1g). The tegumental syncytium was intact and attached to the basement membrane

142

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

143

dorsal tegumental syncytium showed progressive fragmentation and detachment/sloughing from the basal lamina, leaving the latter

144

and the underlying musculature largely exposed (Fig. 2a, e-g). The ventral syncytium remained attached to the basal lamina over

145

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

146

fragmented or missing over both the dorsal and the ventral surface (Fig. 2a, h-j). Gut profiles were empty apart from a little

147

haematin, rather contracted, and contained no blood cells (Fig. 2b, d-f,), while some profiles towards the posterior of the sections

148

contained fibrillar basophilic material and cellular debris, suggesting breakdown of the gastrodermis (Fig. 2g-i). The uterus

149

contained normal, or fewer than normal, numbers of well-shelled eggs (Fig. 2b and c). The testis profiles contained numerous

150

rather shrunken and individualised spermatogonia and spermatocytes (including apoptotic cells), with much space between cells,

151

and overall depletion of cell numbers (Fig. 2d-g).Profiles of ovary contained normal-looking oogonia and oocytes, but there was 9

152

slightly more space between cells than in the untreated control sections. Mehlis’ gland cells, where seen, appeared normal (Fig.

153

2c). The vitelline follicles were rather shrunken and most cells were either mature or late intermediate, containing numerous shell

154

protein granules. Stem cells and early intermediate cells were less evident (Fig. 2g-j) than in control sections.

155

3.3. Twelve hours post-treatment (oral) flukes

156

In each fluke, the tegumental syncytium was intact and unbroken round the entire section profile, well attached to the basal lamina.

157

Beneath the basal lamina there was vacuolation, giving a rather striated appearance to this zone. Gut profiles were empty and

158

rather contracted, with only a little haematin in a few crypts of the gastrodermis. Normal eggs were present in the uterine lumen, but

159

free vitelline cells were also seen. The testis profiles were generally well packed with spermatogonia and spermatocytes, but some

160

profiles appeared rather depleted of cells, and in these tubules the cells were rounded, individualised, and well separated. The

161

parenchyma, ovary and Mehlis’ gland were substantially normal. In the vitelline follicles mature vitelline cells tended to

162

predominate, with rather fewer stem cells and early intermediate cells than in control material.

163

3.4. Twenty-four hours post-treatment (subcutaneous) flukes

164

The tegumental syncytium was intact and unbroken throughout the sections, with little vacuolation below the basal lamina. Gut

165

profiles were contracted, with only a small amount of haematin in the gastrodermal crypts of some profiles. The parenchyma

166

appeared normal. The uterus contained numerous shelled, normal-appearing eggs, but the most proximal coils also contained

167

some free vitelline cells. The testis profiles were generally well populated with densely packed spermatogonia and spermatocytes,

168

but peripheral vacuolation was more marked than in the control flukes. In profiles of ovary, the intercellular space was increased, 10

169

separating individual oocytes and oogonia. Mehlis’ gland cells were vacuolated. Vitelline follicles were similar to those in the

170

control flukes, but mature cells and intermediate cells were rather more abundant, and stem cells rather less abundant than in the

171

untreated flukes.

172

3.5. Twenty-four hours post-treatment (oral) flukes

173

The tegumental syncytium in the flukes was largely intact, unbroken, and attached to the basal lamina all around the profiles of the

174

sections, but there were occasional areas of breakdown and sloughing of the syncytium near the posterior end and, in some other

175

areas, the basal infoldings of the syncytium were swollen, giving a striated pattern of vacuolation. The gut profiles were largely

176

empty, rather contracted, and contained only a small amount of haematin in the gastrodermal crypts. In the uterus, some shelled

177

eggs were present, together with shell debris and free vitelline cells. The testis profiles were rather depleted of cells, with rounding

178

and separation of the spermatogonia and spermatocytes, appearance of empty space between cells, and breakdown of cells, with

179

fragmentary debris present. The ovary showed increased intercellular space and some separation of oogonia and oocytes. The

180

Mehlis’ gland cells were vacuolated. The vitelline follicles featured more mature and late intermediate cells, and relatively fewer

181

stem cells (Fig. 3a) than seen in the untreated controls.

182

3.6. Thirty-six hours post-treatment (subcutaneous) flukes

183

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

184

the sections, the tegumental syncytium was missing in places, fragmenting and sloughing from the basal lamina. In those areas

185

where the syncytium was missing, there was disintegration of the vitellaria, gut profiles, testis and parenchyma, with loss of 11

186

basophilic nuclear staining, and reduced eosinophilic staining of cytoplasm. Profiles of gut in the anterior portions of the flukes were

187

empty, rather contracted, and contained only small amounts of haematin associated with the gastrodermis. In the posterior parts of

188

the sections the gut profiles were disorganised, the gastrodermis was broken down and fragments occupied the lumen. There was

189

marked loss of cellular detail. In the uterine lumen, few shelled eggs were present, but shell debris and free vitelline cells were

190

evident (Fig. 3b). Testis profiles at the anterior end of the flukes showed depletion of cells, with marked peripheral vacuolation and

191

some rounding and separation of spermatogonia and spermatocytes. These changes were more marked towards the posterior end

192

of the flukes (Fig. 3c). The vitelline follicles towards the anterior of the flukes showed more mature vitelline cells and fewer stem

193

cells than normal, and the stem cells tended to be rounded and individualised. Towards the posterior of the flukes the vitelline

194

follicles showed disorganisation, disintegration of cells and loss of cellular detail, with shell protein granules appearing in irregular

195

clumps and masses (Fig. 3d). Sections of ovary showed some depletion and rounding of oocytes, with increased inter-cellular

196

space (Fig. 3e). Mehlis’ gland cells were vacuolated (Fig. 3f).

197

3.7. Thirty-six hours post-treatment (oral) flukes

198

The tegument was intact and attached to the basal lamina only over the anterior end of the sections, while it was missing, sloughing

199

or fragmented over large areas of the posterior end of the sections. Where the tegumental syncytium was missing, the underlying

200

vitelline follicles, testis profiles, gut profiles and parenchyma were disintegrating, and lacked basophilic nuclear staining and cellular

201

detail. Where the tegument remained, the underlying structures retained more cellular detail and nuclear staining. In the uterus, no

12

202

eggs were present, only free vitelline cells. Testis, ovary, vitelline follicles and gut profiles in areas of the sections that retained the

203

tegumental syncytium were similar to those described for 36 h pt (subcutaneous) flukes (Section 3.6).

204

The histological findings are summarised in Table 3.

205

4. Discussion

206

In general, there was a progressive increase in the severity of histological changes seen in the somatic and reproductive tissues of

207

liver flukes with increasing time of in vivo exposure to closantel (12- 36 h), whether administered to the host by the oral or the

208

subcutaneous route. A marked exception was the appearance of severe deterioration in the tegument and testis of flukes from a

209

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

210

was inadvertently delivered directly into a subcutaneous blood vessel, meaning that flukes in the bile ducts, feeding on blood,

211

acquired a significant quantity of the active anthelmintic drug into the gut very soon after the host was dosed, in contrast to the

212

situation following oral or subcutaneous administration. In the blood, closantel binds strongly to plasma albumin, achieving a peak

213

level of approximately 55 µg/ml within 24 h after a 10 mg/kg oral dose, or 24–48 h after subcutaneous treatment

214

Considering the findings summarised in Table 3, and discounting the anomalous 12h results, oral dosing of the host appears to

215

have had a more severe and immediate effect on the flukes than subcutaneous dosing. This is evident in the 24h findings for

216

tegument and testis. The ovary was the least affected of all tissues after both oral and subcutaneous dosing. However, examination

217

of the uterus indicated that egg production was more affected by oral dosing than by subcutaneous dosing, with abnormalities

218

evident from 12h onwards. The impact of closantel on egg production may have had more to do with effects on the vitellaria and 13

219

Mehlis’ gland than on the ovary. The early appearance of changes in the tegument and gut, from 12h pt onwards, may indicate that

220

primary drug effects on these tissues triggered more widespread effects on the internal organs, with the testis and vitellaria most

221

severely affected because of their relatively high energy demand for cell differentiation and turn-over.

222

Closantel, one of the salicylanilide group of anthelmintics, is believed to act mainly as an uncoupler of oxidative phosphorylation in

223

flukes, although the initial effect may be on glycolysis (Fairweather and Boray, 1999; Kane et al., 1980; Rohrer et al., 1986; Van

224

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

226

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

232

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

234

of the gastrodermal lamellae and breakdown of the gastrodermis, particularly in the posterior region, was noted (Verheyen et al.,

235

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,

239

meaning that the sequence of toxicological effects is difficult to interpret.

240

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

243

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

References

319

A.V.M.A, 2001. 2000 Report of the A.V.M.A Panel on Euthanasia. J. Am. Ve.t Med. Assoc 218, 669-696.

19

320 321 322 323 324 325 326

Alvarez, L., Moreno, G., Moreno, L., Ceballos, L., Shaw, L., Fairweather, I., Lanusse, C., 2009. Comparative assessment of albendazole and triclabendazole ovicidal activity on Fasciola hepatica eggs. Vet. Parasitol 164, 211-216. Boray, J.C., 1997. Chemotherapy of infections with Fasciolidae, In: AGVET, M. (Ed.) Immunology, Pathobiology and Control of Fasciolosis.Rahway,N J, pp. 83-97. Boray, J.C., Crowfoot, P.D., Strong, M.B., Allison, J.R., Schellenbaum, M., Von Orelli, M., Sarasin, G., 1983. Treatment of immature and mature Fasciola hepatica infections in sheep with triclabendazole. Vet. Rec 113, 315-317. Brockwell, Y.M., Elliott, T.P., Anderson, G.R., Stanton, R., Spithill, T.W., Sangster, N.C., 2014. Confirmation of Fasciola hepatica

327

resistant to triclabendazole in naturally infected Australian beef and dairy cattle. International Journal for Parasitology: Drugs

328

and Drug Resistance 4, 48-54.

329

Canevari, J., Ceballos, L., Sanabria, R., Romero, J., Olaechea, F., Ortiz, P., Cabrera, M., Gayo, V., Fairweather, I., Lanusse, C.,

330

2014. Testing albendazole resistance in Fasciola hepatica: validation of an egg hatch test with isolates from South America

331

and the United Kingdom. J. Helminthol 88, 286-292.

332 333 334 335

Cohen, G.M., Sun, X.M., Snowden, R.T., Dinsdale, D., Skilleter, D.N., 1992. Key morphological features of apoptosis may occur in the absence of internucleosomal DNA fragmentation. Biochem..J 286, 331-334. Colhoun, L., Fairweather, I., Brennan, G., 1998. Observations on the mechanism of eggshell formation in the liver fluke, Fasciola hepatica. Parasitology 116, 555-567.

20

336 337 338 339 340 341

Chowaniec, W., Darski, J., 1970. Investigations on excretion time of liver fluke eggs after killing the parasite. Bull. Vet. Inst. Pulway 14, 108-110. Daniel, R., Van Dijk, J., Jenkins, T., Akca, A., Mearns, R., Williams, D., 2012. Composite faecal egg count reduction test to detect resistance to triclabendazole in Fasciola hepatica. Vet Rec 171, 153. Fairweather, I., 1997. The quest for an understanding of fasciolicidal action: Holy Grail or poisoned chalice?, In: AGVET, M. (Ed.) Immunology, Pathobiology and Control of Fasciolosis.Rahway, New Jersey, pp. 99-130.

342

Fairweather, I., 2005. Triclabendazole:new skills to unravel an old (ish) enigma. J. Helminthol., 227-234.

343

Fairweather, I., 2009. Triclabendazole progress report, 2005–2009: an advancement of learning? . J. Helminthol., 139–150.

344

Fairweather, I., 2011a. Liver fluke isolates: a question of provenance. Vet Parasitol. 176, 1-8.

345

Fairweather, I., 2011b. Reducing the future threat from (liver) fluke: realistic prospect or quixotic fantasy? Vet. Parasitol 180, 133-

346 347 348 349

143. Fairweather, I., Boray, J.C., 1999. Mechanisms of fasciolicide action and drug resistance in Fasciola hepatica, In: Dalton, J.P. (Ed.) Fasciolosis. CABI Publishing, Wallingford, Ox, pp. 225-276. Fairweather, I., McShane, D.D., Shaw, L., Ellison, S.E., O’Hagan, N.T., York, E.A., Trudgett, A., Brennan, G.P., 2012. Development

350

of an egg hatch assay for the diagnosis of triclabendazole resistance in Fasciola hepatica: Proof of concept. Vet. Parasitol

351

183, 249-259.

21

352 353 354

Fairweather, I., Threadgold, L., Hanna, R., 1999. Development of Fasciola hepatica in the mammalian host, In: Fasciolosis. CAB International, Wallingford, pp. 47-111. Flanagan, A., Edgar, H.W.J., Gordon, A., Hanna, R.E.B., Brennan, G.P., Fairweather, I., 2011a. Comparison of two assays, a

355

faecal egg count reduction test (FECRT) and a coproantigen reduction test (CRT), for the diagnosis of resistance to

356

triclabendazole in Fasciola hepatica in sheep. Vet. Parasitol 176, 170-176.

357

Flanagan, A.M., Edgar, H.W.J., Forster, F., Gordon, A., Hanna, R.E.B., McCoy, M., Brennan, G.P., Fairweather, I., 2011b.

358

Standardisation of a coproantigen reduction test (CRT) protocol for the diagnosis of resistance to triclabendazole in Fasciola

359

hepatica. Vet. Parasitol 176, 34-42.

360 361 362 363 364 365 366 367

Fletcher, H., Hoey, E., Orr, N., Trudgett, A., Fairweather, I., Robinson, M., 2004. The occurrence and significance of triploidy in the liver fluke, Fasciola hepatica. Parasitol. 128, 69-72. Gordon, D., Zadoks, R., Skuce, P., Sargison, N., 2012. Confirmation of triclabendazole resistance in liver fluke in the UK. Vet Rec 171, 159-160. Gordon, D.K., Zadoks, R.N., Stevenson, H., Sargison, N.D., Skuce, P.J., 2012b. On

farm evaluation of the coproantigen ELISA

and coproantigen reduction test in Scottish sheep naturally infected with Fasciola hepatica. Vet. Parasitol.187, 436-444. Hanna, R.E.B., Cromie, L., Taylor, S.M., Couper, A., 2006. The effect of a parenteral ivermectin/closantel injection on the growth and reproductive development of early immature Fasciola hepatica in cattle. Vet. Parasitol 142, 78-90.

22

368

Hanna, R.E.B., Edgar, H., Moffett, D., McConnell, S., Fairweather, I., Brennan, G.P., Trudgett, A., Hoey, E.M., Cromie, L., Taylor,

369

S.M., 2008. Fasciola hepatica: Histology of the testis in egg-producing adults of several laboratory-maintained isolates of

370

flukes grown to maturity in cattle and sheep and in flukes from naturally infected hosts. Vet. Parasitol 157, 222-234.

371

Hanna, R.E.B., Edgar, H.W.J., McConnell, S., Toner, E., McConville, M., Brennan, G.P., Devine, C., Flanagan, A., Halferty, L.,

372

Meaney, M., Shaw, L., Moffett, D., McCoy, M., Fairweather, I., 2010. Fasciola hepatica: Histological changes in the

373

reproductive structures of triclabendazole (TCBZ)-sensitive and TCBZ-resistant flukes after treatment in vivo with TCBZ and

374

the related benzimidazole derivative, Compound Alpha. Vet. Parasitol 168, 240-254.

375

Hanna, R.E.B., Forster, F.I., Brennan, G.P., Fairweather, I., 2013. Fasciola hepatica: histological demonstration of apoptosis in the

376

reproductive organs of flukes of triclabendazole-sensitive and triclabendazole-resistant isolates, and in field-derived flukes

377

from triclabendazole-treated hosts, using in situ hybridisation to visualise endonuclease-generated DNA strand breaks. Vet.

378

Parasitol 191, 240-251.

379

Hanna, R.E.B., McMahon, C., Ellison, S., Edgar, H.W., Kajugu, P.E., Gordon, A., Irwin, D., Barley, J.P., Malone, F.E., Brennan,

380

G.P., 2015. Fasciola hepatica: A comparative survey of adult fluke resistance to triclabendazole, nitroxynil and closantel on

381

selected upland and lowland sheep farms in Northern Ireland using faecal egg counting, coproantigen ELISA testing and

382

fluke histology. Vet. Parasitol 207, 34-43.

383 384

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

385 386 387 388 389 390 391 392 393 394 395 396

Happich, F.A., Boray, J.C., 1969. Quantitative diagnosis of chronic fasciolosis. The estimation of daily total egg production of Fasciola hepatica. Aust. Vet. J 45, 329-331. Kane, H.J., Behm, C.A., Bryant, C., 1980. Metabolic studies on the new fasciolicidal drug, closantel. Molec. Biochem. Parasitol 1, 347-355. Kumar, V., Abbas, A.K., Fausto, N., 2005. Cellular responses to stress and toxic insults: adaption, injury and death, In: Saunders, t.e.E. (Ed.) Robbins and Cotran Pathologic Basis of Disease.Philadelphia, PA, USA, pp. 26-32. Maes, L., Lauwers, H., Deckers, W., Vanparijs, O., 1988. Flukicidal action of closantel against immature and mature Fasciola hepatica in experimentally infected rats and sheep. Res. vet. sci 44, 229-232. McMahon, C., 2015. PhD Thesis: Anthelmintic resistance in parasites of sheep in Northern Ireland and the strategic control of parasitic diseases. Queen’s University of Belfast, Belfast. Michiels, M., Meuldermans, W., Heykants, J., 1987. The metabolism and fate of closantel (Flukiver) in sheep and cattle. Drug Metab. Rev 18, 235-251.

397

Mitchell, G.B., Maris, L., Bonniwell, M.A., 1998. Triclabendazole-resistant liver fluke in Scottish sheep. Vet. Rec 143, 399.

398

Mohammed-Ali, N., Bogan, J., 1987. The pharmacodynamics of the flukicidal salicylanilides, rafoxanide, closantel and

399 400 401

oxyclosanide. Journal of veterinary pharmacology and therapeutics 10, 127-133. Mooney, L., Good, B., Hanrahan, J.P., Mulcahy, G., de Waal, T., 2009. The comparative efficacy of four anthelmintics against a natural acquired Fasciola hepatica infection in hill sheep flock in the west of Ireland. Vet. Parasitol, 201-205. 24

402 403 404

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

405

hepatica against Triclabendazole in cattle in Cajamarca (Peru): A clinical trial and an in vivo efficacy test in sheep. Vet.

406

Parasitol.

407

Overend, D.J., Bowen, F.L., 1995. Resistance of Fasciola hepatica to triclabendazole. Aust. Vet. J 22, 275-276.

408

Rohrer, S.P., Saz, H.J., Nowak, T., 1986. 31 P-NMR studies of the metabolisms of the parasitic helminths Ascaris suum and

409 410 411 412 413 414 415 416 417

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.

25

418 419 420 421 422 423 424

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

429

edition of guidelines for evaluating the efficacy of anthelmintics in ruminants (bovine, ovine, caprine). Vet. Parasitol 58, 181-

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.

38

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