2014) Echinococcus As a Model System: Biology and Epidemiology. International Journal for Parasitology, 44 (12

MURDOCH RESEARCH REPOSITORY

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Thompson, R.C.A. and Jenkins, D.J. (2014) Echinococcus as a model system: biology and epidemiology. International Journal for Parasitology, 44 (12). pp. 865-877.

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PARA 3681 No. of Pages 13, Model 5G 13 August 2014

International Journal for Parasitology xxx (2014) xxx–xxx 1 Contents lists available at ScienceDirect

International Journal for Parasitology

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2 Invited Review

5 4 Echinococcus as a model system: biology and epidemiology 6 a,⇑ b,⇑ 7 Q1 R.C.A. Thompson , D.J. Jenkins

8 a School of Veterinary and Life Sciences, Murdoch University, Murdoch, WA 6150, Australia 9 b Animal and Veterinary Sciences, Charles Sturt University, Locked Bag 588, Wagga Wagga, NSW 2678, Australia

1110 12 article info abstract 1430 15 Article history: The introduction of Echinococcus to Australia over 200 years ago and its establishment in sheep rearing 31 16 Received 11 June 2014 areas of the country inflicted a serious medical and economic burden on the country. This resulted in 32 17 Received in revised form 19 July 2014 an investment in both basic and applied research aimed at learning more about the biology and life cycle 33 18 Accepted 21 July 2014 of Echinococcus. This research served to illustrate the uniqueness of the parasite in terms of developmen- 34 19 Available online xxxx tal biology and ecology, and the value of Echinococcus as a model system in a broad range of research, 35 from fundamental biology to theoretical control systems. These studies formed the foundation for an 36 20 Keywords: international, diverse and ongoing research effort on the hydatid organisms encompassing stem cell 37 21 Echinococcus biology, gene regulation, strain variation, wildlife diseases and models of transmission dynamics. We 38 22 Development 23 Cytodifferentiation describe the development, nature and diversity of this research, and how it was initiated in Australia 39 24 Host–parasite interface but subsequently has stimulated much international and collaborative research on Echinococcus. 40 25 Taxonomy Ó 2014 Published by Elsevier Ltd. on behalf of Australian Society for Parasitology Inc. 41 26 Epidemiology 42 27 Wildlife 28 Domestic hosts 29 43 44 45 1. Introduction agricultural society following early European settlement of a con- 66 tinent with huge temperate areas perfect to exploit for raising 67 46 Echinococcus remains a major cause of zoonotic diseases of pub- livestock. 68 47 Q2 lic health and economic significance (Jenkins et al., 2005a; Budke It was the upsurge in sheep farming at the end of the 19th cen- 69 48 et al., 2006; Davidson et al., 2012; Hegglin and Deplazes, 2013). tury, with expanding exports to Europe, that contributed to the 70 49 Despite advances in control strategies, clinical management and serious and largely undocumented human health problem that 71 50 vaccine development, the parasite continues to thrive in countries existed during the late 19th and early 20th centuries (Gemmell, 72 51 throughout the world. 1990). During the first half of the 20th century, there was a high 73 52 Hydatid disease, cystic and alveolar, is a typical cyclozoonosis incidence of cystic hydatid disease in Australia, and to a lesser 74 53 that can be perpetuated in nature in wild animal cycles without extent alveolar hydatid disease, either contracted in Australia 75 54 impacting on public health but with human interference (E. granulosus) or in migrants from endemic areas overseas 76 55 (Thompson, 2013), directly or accidentally, with spillover to (E. granulosus and Echinococcus multilocularis)(Dew, 1935). There 77 56 domestic cycles can lead to severe clinical disease and death. It is was thus a need for regular surgical intervention. It was surgeons 78 57 also an important cause of economic losses to livestock industries, such as Harold Dew who had an interest in the basic biology of 79 58 particularly Echinococcus granulosus in sheep and cattle (Table 1). the parasite and who published in international journals such as 80 59 It is now well known that Echinococcus has a two-host life cycle the ‘‘British Journal of Surgery’’ and ‘‘British Medical Journal’’ that 81 60 with a sexual stage in the intestine of a carnivore definitive host led to widespread recognition of the research being undertaken 82 61 and a unique, cystic larval stage in the tissues of non-carnivorous on hydatid disease in Australia. This was enhanced by Dew’s con- 83 62 mammals and omnivores (Thompson, 1995). It is interesting that tributions in the literature (Dew, 1953) and at conferences to the 84 63 much of the research that revealed the unique features of the par- debate raging at the time on whether cystic and alveolar hydatid 85 64 asite’s way of life were undertaken in Australia. This is not a coin- disease were caused by the same or different species of Echinococ- 86 65 cidence and relates to the impact Echinococcus had on a developing cus (‘dualists’ versus ‘monists’) as well as his collaboration with 87 researchers in Europe such as Félix Dévé. 88 Dew recognised the developmental differences of the two 89 ⇑ Tel.: +61 8 9360 2466; fax: +61 8 9310 4144 (R.C.A. Thompson). Tel./fax: +61 02 6933 4179 (D.J. Jenkins). stages in the life cycle and studied the metacestode stage of the 90 E-mail addresses: [email protected] (R.C.A. Thompson), djjenkins@ cystic and alveolar forms (Fig. 1) in depth, both in human cases 91 csu.edu.au (D.J. Jenkins).

http://dx.doi.org/10.1016/j.ijpara.2014.07.005 0020-7519/Ó 2014 Published by Elsevier Ltd. on behalf of Australian Society for Parasitology Inc.

Please cite this article in press as: Thompson, R.C.A., Jenkins, D.J. Echinococcus as a model system: biology and epidemiology. Int. J. Parasitol. (2014), http:// dx.doi.org/10.1016/j.ijpara.2014.07.005 PARA 3681 No. of Pages 13, Model 5G 13 August 2014

2 R.C.A. Thompson, D.J. Jenkins / International Journal for Parasitology xxx (2014) xxx–xxx

Table 1 Current taxonomy of Echinococcus spp.

Species Strain/genotype Known intermediate hosts Known deﬁnitive hosts Infectivity to humans Echinococcus granulosus Sheep/G1 Sheep (cattle, pigs, camels, goats, macropods) Dog, fox, dingo, jackal and hyena Yes Tasmanian sheep/G2 Sheep Dog, fox Yes Buffalo/G3 Buffalo Dog Yes Echinococcus equinus Horse/G4 Horses and other equines Dog Probably not Echinococcus ortleppi Cattle/G5 Cattle Dog Yes Echinococcus canadensis Cervids/G8,G10 Cervids Wolves, dog Yes Echinococcus intermedius Camel/Pig/G6/G7 Camels, pigs, sheep Dog Yes Echinococcus felidis Lion/– Warthog (possibly zebra, wildebeest, Lion Uncertain bushpig, buffalo, various antelope, giraffe, hippopotamus) Echinococcus Some isolate Rodents, domestic and wild pig, dog, monkey Fox, dog, cat, wolf, racoon-dog, Yes multilocularis variation coyote Echinococcus shiquicus –/– Pika Tibetan fox Uncertain Echinococcus vogeli None reported Rodents Bush dog Yes Echinococcus oligarthrus None reported Rodents Wild felids Yes

Data from: Thompson et al. (1995), Thompson and McManus (2002), Jenkins et al. (2005a), Thompson (2008), and Carmena and Cardona (2014).

92 and animals (Dew, 1922, 1925, 1928, 1935). He thus comple- 93 mented much of Dévé’s research undertaken in rodents (Dévé, A 94 1919, 1946) and built on this. Dew demonstrated that an intact 95 laminated layer was a fundamental and unique component of the 96 ‘healthy’ hydatid cyst and considered this layer to be of host origin, 97 and demonstrated that elements of the cyst wall (germinal layer) 98 could regenerate (Dew, 1935). He realised that the cyst enclosed d 99 a sterile environment and was under intracystic pressure, and 100 speculated on the reasons for this being indicative of viability c 101 and a function of the germinal layer. In observations of what we 102 now know to be the metacestode of E. multilocularis, Dew b e 103 described naked prolongations of the nucleated germinal a d 104 membrane (layer) in direct contact with host tissues (Dew, 105 1935), a fundamental feature of the infiltrating metastatic meta- 106 cestode of E. multilocularis (Fig. 1) subsequently described using 107 electron microscopy 60 years later (Mehlhorn et al., 1983; and 108 see Section 2.4). Thus it was Harold Dew that led to Australia being host tissue 109 referred to as the ‘home of hydatids’ in terms of research. He paved adventitial layer E. granulosus 110 the way for other researchers and did much to establish Echinococ- laminated layer 111 cus as a model organism. ‘‘In this country of Australia we all have germinal layer 112 unrivalled opportunities to investigate this disease, both in man 113 and in animals, and it is our duty to contribute our share to future 114 advances in its study’’ (Dew, 1935). B vesicles 115 The economic impact and public health significance of cystic 116 hydatid disease in Australia worsened over the next 30 years, 117 which provided an opportunity for obtaining research funds from protrusions of germinal layer 118 organisations supporting health and livestock research. J.D. Smyth 119 obtained funds from these sources and took a physiological 120 approach in his research aimed at increasing understanding of circulation 121 the developmental biology of Echinococcus. Having forged an inter- distant 122 est in developing in vitro cultivation procedures for Schistocephalus metastatic 123 with success in establishing much of the life cycle of this cestode in foci 124 culture (Smyth, 1946, 1950), he turned his attention to Echinococ- 125 cus (see Smyth, 1990) for which the ability to study and maintain 126 the parasite in vitro would provide a great research tool for inves- E. multilocularis 127 tigating methods of control. 128 The practical and ethical difficulties of undertaking in vivo stud- 129 ies in the canid or felid definitive hosts of Echinococcus further rein- Fig. 1. Diagrams illustrating the structural differences between the metacestodes of 130 forced the need to develop in vitro systems. It was these pioneering (A) Echinococcus granulosus (a–d, stages in development of protoscoleces and brood capsule; e, daughter cyst) and (B) Echinococcus multilocularis (redrawn from 131 studies of Smyth (Smyth et al., 1966 and reviewed in Smyth and Thompson, 1995). 132 Davies, 1974a; Howell and Smyth, 1995) that demonstrated the 133 potential of Echinococcus as a model for studying principles of 134 developmental biology, differentiation, host–parasite relationships the potential of exploiting Echinococcus as a novel model system 138 135 and evolutionary biology (Smyth, 1969; Smyth et al., 1966), and for studying parasitism as distinct from a model for studies on 139 136 which have influenced research and theoretical understanding far evaluating anthelmintic or other anti-parasitic drugs (Smyth, 140 137 beyond parasitology (Thompson and Lymbery, 2013). Smyth saw 1969). He thus expanded the definition of a ‘model’ to embrace 141

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142 studies on all the biological activities that supported the parasite support observations made from in vivo and population genetic 204 143 life style of Echinococcus, as well as the potential of this model sys- studies of the importance of the host intestinal ecosystem in 205 144 tem for broader studies of a more fundamental nature in biology, providing the correct physical and physiochemical conditions 206 145 for example stem cells (Smyth, 1969, 1987). As such the promotion necessary to support fertilisation (Smyth, 1982; Smyth and 207 146 of such a eukaryotic, metazoan model was at the time somewhat of Davies, 1974a; Lymbery and Thompson, 1988, 2012; Lymbery 208 147 a ‘first’. et al., 1989; Howell and Smyth, 1995). 209 148 Smyth worked closely with his Australasian colleague Michael The success with cultivating Echinococcus and the elucidation of 210 149 Gemmell, who also had an interest in Echinococcus but who was conditions that would support maturation stimulated studies on 211 150 keen to understand the epidemiology of hydatid disease, its eco- other taeniids including Taenia pisiformis and Taenia crassiceps 212 151 nomic impacts and impediments to control (Gemmell, 1990). As (Esch and Smyth, 1976; Osuna-Carrillo and Mascaró-Lazcano, 213 152 such, he was the first to apply mathematical models to Echinococ- 1982), as well as Mesocestoides corti (Barrett et al., 1982; 214 153 cus and the concept of the basic reproductive rate (R0) in order to Thompson et al., 1982). These largely supported the observations 215 154 define the conditions under which transmission of Echinococcus made with Echinococcus but also served to illustrate the unique- 216 155 and other taeniids occur, and the regulatory role of immunity ness of the Echinococcus model system. 217 156 (Gemmell, 1990; Gemmell and Roberts, 1995). During the 25 years Smyth and his colleagues spent studying 218 157 In this review, we describe the research, particularly that and optimising strobilate development and maturation of Echino- 219 158 undertaken in Australia, which has demonstrated the value of Echi- coccus in vitro, many valuable observations were made that have 220 159 nococcus as a model system influencing advances in biology and served to establish Echinococcus as a model system not only in 221 160 epidemiology of parasites and parasitic infections. terms of developmental biology, but also for cytodifferentiation 222 and the host–parasite interface. 223

161 2. Developmental biology 2.2. Developmental plasticity 224

162 2.1. Growth and maturation Studies on the in vitro cultivation of Echinococcus from the 225 larval protoscolex to the adult worm demonstrated the inherent 226 163 Echinococcus has two developmental stages in its life cycle; the plasticity of the parasite – a phenomenon eloquently described 227 164 adult tapeworm, which is the sexual stage, and the cystic or infil- as ‘heterogeneous morphogenesis’ (Smyth et al., 1967; Smyth, 228 165 trative larval metacestode that reproduces asexually (Fig. 1). The 1987), unique in most metazoan organisms apart from coelenter- 229 166 metacestode of E. granulosus, which was easily accessible from ates such as Hydra, and making Echinococcus an unusual model 230 167 infected livestock in the abattoir or from human surgical cases, for differentiation studies. Depending upon the culture conditions, 231 168 was the logical starting point for experimental manipulations. Ini- protoscoleces can either develop in a cystic or adult direction. Cys- 232 169 tially, these were undertaken in rodents where the asexual prolif- tic development itself may take a variety of paths, again influenced 233 170 erative nature of the parasite was first observed, a phenomenon by environmental conditions. These alternative developmental 234 171 that was subsequently exploited with the development of rodent pathways have been described in detail by Howell and Smyth 235 172 models of secondary hydatidosis (Dévé, 1919). These provided a (1995). Protoscoleces can also develop into an adult tapeworm 236 173 means to maintain the parasite virtually indefinitely in vivo by under different conditions to those supporting cystic development, 237 174 serial passage (e.g., Thompson, 1976; Whitfield and Evans, 1983; most importantly the need for a diphasic medium incorporating a 238 175 Kamiya et al., 1985). solid supporting substrate thought to provide nutrients and/or a 239 176 Although a number of workers had reported vesicular/cystic contact stimulus (see Section 2.1). However, observations on the 240 177 development and proliferation of protoscoleces in vitro, a major development of adult E. granulosus and E. multilocularis demon- 241 178 goal was to provide appropriate conditions to stimulate and sup- strated that in addition to ‘normal’ worms, cultures often con- 242 179 port development in an adult (strobilate) direction. It was not until tained worms that had matured but not segmented (monozoic) 243 180 Smyth and his colleagues refined procedures with the aim to and worms with more than one scolex and other malformations 244 181 reproduce the conditions that protoscoleces would be exposed to (reviewed in Howell and Smyth, 1995). Interestingly, adult worms 245 182 in the gut of the definitive host that strobilate development was that have developed from protoscoleces can ‘de-differentiate’ in a 246 183 obtained (Smyth et al., 1966). Smyth took a physiological approach cystic direction under adverse conditions (see Sections 2.3 and 247 184 to this research, with the cyst as a starting point. Its sterile environ- 2.4; Smyth, 1969; Thompson and Lymbery, 1990; Thompson 248 185 ment provided the perfect ‘entry point’ if accessed aseptically to et al., 1990). These observations raised two important and funda- 249 186 carefully remove essentially ‘dormant’ invaginated protoscoleces mental questions: how is this complex and plastic development 250 187 and then expose them to appropriate physiochemical conditions controlled and what cells have the potential to develop in such a 251 188 (Smyth and Davies, 1974a). Of particular significance was the pro- myriad of different routes? 252 189 vision, in addition to the liquid medium, of a solid proteinaceous 190 base in the culture flask (diphasic medium) which was found to 2.3. Developmental control 253 191 be important in stimulating proglottisation and segmentation in 192 E. granulosus with maturation to the pre-fertilisation stage Smyth developed a hypothetical yet logical model to explain 254 193 (reviewed in Smyth and Davies, 1974a; Howell and Smyth, how genes regulate differentiation and developmental shifts in 255 194 1995). Similar results were obtained with E. multilocularis but Echinococcus (Smyth, 1969). This was based on the Jacob–Monod 256 195 proglottisation without segmentation will take place without a model of genetic expression and showed the complex interactions 257 196 solid proteinaceous base (Smyth and Davies, 1975; Smyth, 1979; and regulatory switches that may be involved in controlling devel- 258 197 Thompson et al., 1990). This and other developmental abnormali- opment and differentiation in Echinococcus. Since then a number of 259 198 ties observed in E. multilocularis were considered to reflect a differ- studies have isolated and characterised regulatory genes from Echi- 260 199 ence in gene expression between the two species (Howell and nococcus (reviewed in Thompson, 1995 and Parkinson et al., 2012) 261 200 Smyth, 1995 and see Section 2.3). Although the conditions devel- but how they fit together in a control network has yet to be deter- 262 201 oped supported reproducible strobilate development, fertilisation mined. Recently, Parkinson et al. (2012) identified long non-coding 263 202 and egg production eluded Smyth and other workers. However, (nc)RNAs that may be involved in the regulation of gene expression 264 203 they served to emphasise the complexity of the process and to in response to environmental cues in the host. They also identified 265

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266 a number of genes reflecting specificities of particular stages, different gene expression patterns (Koziol et al., 2014), thus con- 330 267 including those whose expression is up-regulated by pepsin-acid firming Smyth’s predictions. 331 268 activation. It is to be hoped that Smyth’s models will help to pro- 269 vide functionality to the genomic data that is now available for 2.5. Host–parasite interface 332 270 both E. granulosus and E. multilocularis (Tsai et al., 2013; Zheng 271 et al., 2013). However, differences in development between E. 2.5.1. Definitive host 333 272 granulosus and E. multilocularis highlighted by studies in vitro are Observations that contact of the scolex with a proteinaceous 334 273 considered to reflect differences in the control of gene expression base stimulated strobilate development in E. granulosus led to 335 274 (Howell and Smyth, 1995). studies on the nature of the interface both in vitro and in vivo, 336 and the discovery of the rostellar gland which comprises a modi- 337 275 2.4. Cytodifferentiation fied group of tegumental cells situated in the apical rostellum 338 (Smyth, 1964; Smyth et al., 1969; Thompson et al., 1979; 339 276 Slais (1973) demonstrated that the post-oncospheral develop- Thompson and Eckert, 1983; Fig. 2). The rostellar gland releases 340 277 ment of Echinococcus was initiated by the growth and division of secretory material by a holocrine process into the interface 341 278 primary germinal cells, and Swiderski (1983) described five pairs between parasite and host (Thompson et al., 1979). The origin 342 279 of these cells in the posterior pole of the oncosphere. Observations and site of synthesis of the secretion has still to be determined, 343 280 on the development of Echinococcus in vitro demonstrated that the although large amounts occur in both the perinuclear and distal 344 281 fundamental processes of germinal and somatic differentiation, cytoplasm of the tegument as well as in the tegumental nuclei 345 282 comprising cystic development, proglottisation, maturation, (Thompson et al., 1979; Herbaut et al., 1988). The secretion is pro- 346 283 growth and segmentation (strobilisation), can take place indepen- teinaceous, containing cystine and lipid. It is not known if there are 347 284 dently. This not only illustrated the complexity of cytodifferentia- one or more proteins secreted but Siles-Lucas et al. (2000) demon- 348 285 tion but also the possible existence of several primitive cell lines. strated that the secretion contains a regulatory protein (14-3-3) 349 286 However, to date all the evidence suggests that only one primitive that is released into the host–parasite interface. 350 287 morphological cell type exists as a pool of uncommitted, undiffer- The rostellar gland of Echinococcus is seemingly unique to Echi- 351 288 entiated multipotent germinal, or stem, cells in both the adult and nococcus. Although rostellar secretions have been described in lar- 352 289 metacestode (Smyth, 1969; Gustafsson, 1976; Thompson et al., val T. crassiceps (Krasnoshchekov and Pluzhnnikov, 1981), no gland 353 290 1990; Thompson, 1995; Koziol et al., 2014). The undifferentiated has been described. Rostellar glands have been described in other 354 291 germinal cells are a component of the syncytial germinal layer of adult cestodes, particularly proteocephalids, but their function is 355 292 the metacestode and neck region of the adult cestode. Ultrastruc- also unclear and structurally they are different to the rostellar 356 293 tural studies reveal unremarkable rounded cells of variable size gland in Echinococcus (McCullough and Fairweather, 1989; 357 294 Q3 of approximately ? lm diameter (Gustafsson, 1976; Mehlhorn Zd’árská and Nebesárová, 2003). There is clearly a need for more 358 295 et al., 1983; Albani et al., 2010). Cell proliferation derives from studies on the interface of attached adult cyclophyllidean cestodes 359 296 the continuous replicative activity of these dividing stem cells (Pospekhova and Bondarenko, 2014). 360 297 located in the germinal layer or neck region of the adult tapeworm This intimate association of the rostellar gland and its secre- 361 298 (Gustafsson, 1976; Galindo et al., 2003). They have considerable tions suggests a role(s) that enhances the host–parasite relation- 362 299 proliferative potential (Eckert et al., 1983; Mehlhorn et al., 1983; ship in favour of the parasite, which may be regulatory, 363 300 Galindo et al., 2003; Martínez et al., 2005), and are the only prolif- nutritional and/or protective. The relationship between rostellar 364 301 erating cells in Echinococcus (Koziol et al., 2014). This is particularly gland activity and localised humoral and cellular reactions 365 302 well illustrated by the capacity of the parasite for indefinite perpet- (Deplazes et al., 1993) is not known but such localised reactions 366 303 uation in the larval stage by passage of protoscoleces or germinal demonstrate stimulation of host immune effector mechanisms. 367 304 layer material in rodents (secondary hydatidosis; Howell and The secretory molecules would seem to be obvious candidates 368 305 Smyth, 1995). In alveolar hydatid disease caused by the metaces- for exploitation in vaccine studies since a focus on prophylaxis of 369 306 tode of E. multilocularis, the proliferating larval parasite has an the definitive host may be more attractive than the intermediate 370 307 infiltrative capacity to establish distant foci of infection due to host, particularly for the control of E. multilocularis, from both com- 371 308 the distribution via blood or lymph of detached germinal cells mercial and practical perspectives. As pointed out by Heath (1995) 372 309 (Ali-Khan et al., 1983; Eckert et al., 1983; Mehlhorn et al., 1983; the scolex is in intimate contact with the systemic circulation even 373 310 Fig. 1). in the Peyers patches and would appear to maintain its privileged 374 311 Problems with host cell contamination dogged early attempts integrity by suppression of cytotoxic and effector cell activity in 375 312 to establish germinal cell lines of E. granulosus and E. multilocularis the region of the scolex. 376 313 (reviewed in Howell and Smyth, 1995). In addition, their isolation The few studies on definitive host vaccines do not seem to have 377 314 from the germinal layer, and their in vitro propagation, could have targeted rostellar gland secretions and have produced results 378 315 been hampered by the fact that the germinal layer is a syncytium. which are controversial and open to contrasting interpretation 379 316 However, the establishment and long-term perpetuation of Echino- (Zhang et al., 2006; Petavy et al., 2008; Torgerson, 2009; 380 317 coccus germinal cells has now been achieved for both species Kouguchi et al., 2013). The studies by Petavy et al. (2008) demon- 381 318 (Yamashita et al., 1997; Spiliotis and Brehm, 2009; Spiliotis et al., strated a strong inflammatory response in the intestine of vacci- 382 319 2008; Albani et al., 2010). The germinal cells behave similarly to nated compared with infected controls but did not show if this 383 320 classical stem cells with the formation of cell aggregates and clus- was localised to where worms were situated. More recently, 384 321 ters with cavity formation, and there is cytological evidence of Kouguchi et al. (2013) used a surface glycoprotein from E. multiloc- 385 322 transformation (Spiliotis et al., 2008; Albani et al., 2013). ularis as a vaccine in dogs which induced significant protection 386 323 Galindo et al. (2003) found evidence of the regionalisation of when administered via a mucosal route and demonstrated anti- 387 324 DNA and protein synthesis in developing stages of Echinococcus, bodies raised by their vaccine on the surface of the suckers, rostella 388 325 although no morphological evidence has been found of different and hooks. These results emphasise the importance of characteris- 389 326 primitive or germinal cell lines to explain the concept of heteroge- ing the secretions produced by the rostellar gland of Echinococcus 390 327 neous morphogenesis (Smyth, 1969). However, such heterogeneity which will contribute to the further development of vaccines 391 328 has now been confirmed at the molecular level. Germinal cells are against the adult parasite. Unfortunately in vivo studies require 392 329 in fact heterogeneous, with the existence of subpopulations with research on the definitive host, usually dogs, which is ethically 393

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Fig. 2. Diagram illustrating adult Echinococcus in situ in the intestine, with suckers on the scolex grasping the epithelium at the base of the villi. Rostellum is deeply inserted into a crypt of Lieberkühn (A, B) and extensibility of the apical rostellar region is shown in B. Rostellar gland with secretory material is anterior to the rostellar pad (r).

394 challenging and hazardous if patent infections are maintained. to date (Lin et al., 2012). There is also increasing evidence of the 430 395 Thus reports of successful patent infections in experimentally immune-regulatory role of the laminated layer, probably in associ- 431 396 infected immunosuppressed rodents (Kamiya and Sato, 1990a,b) ation with the immuno-modulatory activities of the glycoproteins 432 397 were regarded as an important breakthrough (Howell and Smyth, known as Antigen 5 and Antigen B (Diaz et al., 2011b). 433 398 1995; Thompson, 1995). Unfortunately, these results have not 399 been further exploited and should perhaps form the basis for 400 future research. 2.6. Taxonomy and speciation 434

One of the most important observations made as a result of 435 401 2.5.2. Intermediate host studies on the in vitro cultivation of Echinococcus was the failure 436 402 A unique interface is also found in the intermediate host where of protoscoleces collected from hydatid cysts in horses to develop 437 403 the laminated layer presents a physiochemical barrier with appar- in the same way as those of sheep origin. Protoscoleces from horses 438 404 ent multifunctionality and a structure whose biosynthesis has evaginated and increased in length but failed to undergo proglotti- 439 405 become a model system for carbohydrate chemistry (Diaz et al., sation or segmentation, even though they were grown in exactly 440 406 2011a,b; Parkinson et al., 2012). The laminated layer is a specia- the same diphasic medium (Smyth and Davies, 1974b). This fairly 441 407 lised extracellular matrix unique to Echinococcus (Fig. 1), whose simple observation resulted in radical shifts in our understanding 442 408 synthesis is a major metabolic activity of the much thinner germi- of the epidemiology of hydatid disease and transmission of the 443 409 nal layer (Diaz et al., 2011a; Lin et al., 2012; Parkinson et al., 2012). aetiological agents as well as their taxonomy and phylogenetic 444 410 The origin of the laminated layer was controversial for some time, relationships. The results demonstrated that there were funda- 445 411 i.e. whether it is entirely of parasite origin or if there is a host con- mental physiological differences between E. granulosus of sheep 446 412 tribution. Holcman and Heath (1997) demonstrated that it is and horse origin and the coining of the term ‘physiological strain 447 413 entirely of parasite (germinal layer) origin by studying the early differences’ (Smyth and Davies, 1974b; Smyth, 1982). This had a 448 414 stages of cyst development from oncospheres in vitro. broad influence beyond Echinococcus, and in particular the impor- 449 415 Considerable metabolic activity in the germinal layer is tance of combining phenotypic and genetic differences in the char- 450 416 required to synthesise and maintain the interfacial barrier of the acterisation and description of parasites at the intraspecific level 451 417 laminated layer (Parkinson et al., 2012). The role of the laminated (Thompson and Lymbery, 1990, 1996; Lymbery and Thompson, 452 418 layer would appear to be one of protection since cyst survival is 2012). 453 419 dependent upon its integrity (Gottstein et al., 2002). Whether this In terms of Echinococcus, the observation of physiological differ- 454 420 is purely physical or if there is selective permeability is not known. ences between the two parasites of sheep and horse origin comple- 455 421 Monteiro et al. (2010) identified several molecules in hydatid cyst mented earlier epidemiological and taxonomic studies on 456 422 fluid that could play a role in host evasion. Smyth commented on Echinococcus of horse origin (Williams and Sweatman, 1963). These 457 423 the significance of the presence of a human blood group P-like sub- demonstrated morphological differences between the two forms 458 424 stance in the laminated layer of Echinococcus and its significance as which were considered to be taxonomically significant, and to 459 425 a model system in better understanding the immunological basis reflect differences in host specificity and the life cycles which 460 426 of the host-parasite relationship. This P1 blood-antigen motif has maintained the two parasites. This was subsequently shown to 461 427 since attracted much attention and has been further characterised be correct with the sympatric occurrence of distinct sheep- and 462 428 as a protein-carbohydrate, trisaccharide/mucin complex contain- horse-dog cycles in several European countries (Thompson and 463 429 ing galactosamine, yet no biological function has been described Smyth, 1975; Thompson, 2001; Gonzalez et al., 2002). In addition, 464

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465 epidemiological evidence has not only demonstrated distinct dif- and livestock, mainly sheep, during European settlement 527 466 ferences in intermediate host specificity but also that, unlike the (Gemmell, 1990). Domestically, the parasite spread rapidly 528 467 sheep strain (E. granulosus), the horse strain (Echinococcus equinus) through sheep and dog populations (Gemmell, 1990), due mainly 529 468 (Table 1) does not appear to be infective to humans (Thompson to a complete lack of understanding of the association between 530 469 and Lymbery, 1988, 1991). taeniid tapeworms in dogs and cysts in sheep. It was not until 531 470 The concept of host-adapted strains of E. granulosus led to stud- 1851, (63 years after the first European settlers and their animals 532 471 ies on other forms of the parasite in other species of intermediate arrived in Australia) that infection experiments were undertaken, 533 472 hosts such as cattle, pigs, camels and cervids. These studies not demonstrating the relationship between cysts and tapeworms. 534 473 only confirmed the existence of a number of host-adapted life Küchenmeister, working in Germany, fed metacestodes of T. pisi- 535 474 cycles in different parts of the world but also provided additional formis to foxes and obtained tapeworms (Küchenmeister, 1851) 536 475 data on developmental differences between strains which may and in 1853 fed tapeworm eggs to sheep and obtained metaces- 537 476 impact on control (reviewed in Thompson and Lymbery, 1988; todes. In the next few years von Siebold and Küchenmeister, work- 538 477 Thompson et al., 1995; Thompson, 2008). ing independently, fed protoscoleces of Echinococcus to dogs and 539 478 Subsequent molecular characterisation of host-adapted strains obtained tapeworms (von Siebold, 1853; Küchenmeister, 1855). 540 479 of Echinococcus, coupled with molecular epidemiological studies An additional important infection experiment, also undertaken in 541 480 in endemic areas, has confirmed their genetic and morphological Germany, was that of Naunyn in 1863 who fed protoscoleces from 542 481 distinctness and revealed phylogenetic relationships which sup- a human cyst to dogs and obtained adult E. granulosus, reporting 543 482 port a robust, meaningful taxonomy based on a previously sug- his results the same year. This experiment demonstrated for the 544 483 gested nomenclature (Table 1; Bowles et al., 1994; Thompson first time the association between hydatid cysts of sheep and 545 484 et al., 1995, 2006; Cruz-Reyes et al., 2007; Harandi et al., 2002; humans and the role of dogs in the life cycle of human hydatidosis. 546 485 Thompson and McManus, 2002; Lavikainen et al., 2003; Jenkins However it was not until 1876 that Leuckart demonstrated eggs of 547 486 et al., 2005a; Romig et al., 2006; Moks et al., 2008; Thompson, Echinococcus when fed to intermediate hosts (pigs) led to the 548 487 2008; Huttner et al., 2009; Saarma et al., 2009; Nakao et al., development of hydatid cysts (Leuckart, 1876). A detailed review 549 488 2013). Interestingly, the nomenclature used for these ‘species’ con- of the elucidation of the Taenia and Echinococcus life cycles can 550 489 forms to that proposed by observational parasitologists in the be found in Grove (1990). Word of these discoveries took time to 551 490 1920s–1960s, before molecular tools were available to confirm reach Australia but it was not long before experimental studies 552 491 their morphological descriptions and epidemiological observations were undertaken in Australia that confirmed Naunyn’s data 553 492 (Thompson et al., 1995; Thompson and McManus, 2002; (Thomas, 1884). 554 493 Thompson, 2008). Hydatid disease soon became a major public health problem in 555 the growing population of new Australians (Gemmell, 1990) caus- 556 ing serious illness and leading to the deaths of many colonists. 557 494 3. Epidemiology of E. granulosus in Australia 3.2. Wildlife hosts of E. granulosus in Australia 558 495 3.1. Introduction Data generated in studies on wildlife in Australia have world- 559 496 Much of the basic knowledge regarding the epidemiology and wide relevance, an example being the work of Barnes (Barnes 560 497 control of E. granulosus in Australia was generated by Michael et al., 2007a,b, 2008, 2011) investigating the health impacts and 561 498 Gemmell. His contribution to the field has been immense; the growth rate of hydatid cysts in macropodids. Barnes showed that 562 499 breadth of his studies were wide, covering the impact of climate hydatid cysts have a major impact on respiration capacity in mac- 563 500 on egg longevity, immune responses against the parasite in defin- ropods, indicating infected animals to be more susceptible to pre- 564 501 itive and intermediate hosts, prevalence in domestic dogs and live- dation by wild dogs. This work complements and validates the 565 502 stock, studies on infection in wildlife and mathematical modelling studies of Mech (1966) and Jolly and Messier (2004) in the USA 566 503 of transmission as an aid to more effective control (Gemmell and with respect to the impact of hydatid disease on moose (Alces 567 504 Lawson, 1986). Gemmell’s work has provided a solid platform for alces), rendering infected animals more susceptible to predation 568 505 many of the current studies reviewed below. However, probably by wolves (Canis lupus). Another good example is the study on 569 506 his greatest legacy was his contribution to the development of the encroachment of E. granulosus-infected foxes (Vulpes vulpes) 570 507 strategies for the control of E. granulosus transmission. Neverthe- and wild dogs into urban areas in Australia and associated public 571 508 less, in the Australian situation, although control of E. granulosus, health implications (Jenkins and Craig, 1992; Jenkins et al., 572 509 domestically, has largely been achieved (Jenkins et al., 2014), the 2008). These studies complement and support work undertaken 573 510 presence of an extensive wildlife reservoir on mainland Australia in Europe with the encroachment into urban centres of foxes 574 511 ensures the compete eradication of E. granulosus is unlikely to ever infected with E. multilocularis (Deplazes et al., 2004) and studies 575 512 become a reality. Further, unlike the situation with E. multilocularis in Alberta, Canada on E. multilocularis in urban coyotes (Canis 576 513 and Echinococcus canadensis which are maintained in natural wild- latrans)(Catalano et al., 2012). 577 514 life cycles, the wildlife cycle in Australia is an artificial one estab- Australian native wildlife species evolved in isolation from E. 578 515 lished as a result of anthropogenic activities and spillover from granulosus; consequently, they were highly susceptible to infection 579 516 domestic cycles (Thompson, 2013). with this new parasite and wildlife species were soon acting as a 580 517 The work of Gemmell had broad ramifications internationally in major sylvatic reservoir for the perpetuation of E. granulosus in 581 518 terms of the epidemiological principles of hydatid control. Many Australia (Durie and Riek, 1952), a situation that prevails today 582 519 subsequent hydatid control programmes world-wide looked at (Jenkins and Morris, 2003). 583 520 Gemmell’s critical analysis of the epidemiology in Australia and Initially, transmission of E. granulosus to macropodids occurred 584 521 New Zealand and based their programmes on his data. These through accidental consumption of eggs spread in the environment 585 522 served as a foundation for the development of control programmes by the dogs of colonists. Infection in dingoes eventuated through 586 523 in many countries, and the epidemiological principles he devel- predation of sheep on the recently established sheep farms. How- 587 524 oped similarly have been used as a model (Eckert et al., 2001). ever, it would not have been long before the dingoes themselves 588 525 Echinococcus granulosus is the only member of the genus to began to spread eggs throughout their territories, exposing an ever 589 526 occur in Australia, having been introduced with domestic dogs increasing population of macropodids to infection with hydatid 590

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591 disease. Transmission of E. granulosus to wildlife from domestic Reports of E. granulosus-infected wild dog encroachment into 653 592 animals in the more remote areas of Australia was likely to have outer suburban areas have begun to appear; Townsville (Brown 654 593 been enhanced through transhumance grazing (King, 1959). Trans- and Copeman, 2003), Maroochy Shire (Jenkins et al., 2008), the 655 594 humance grazing was undertaken for over 100 years in the states north-western suburbs of Brisbane in the Pine River Shire, Queens- 656 595 of New South Wales and Victoria, with the last ‘‘Snow Lease’’ in land and the outer suburbs of Katoomba in New South Wales (D.J. 657 596 New South Wales being revoked in 1972. In this process hundreds Jenkins, unpublished data). In a number of urban areas, wild dogs 658 597 of thousands of sheep (and tens of thousands of cattle), accompa- have become numerous and a public nuisance raiding garbage 659 598 nied by drovers and their dogs, were moved to the higher altitudes bins, killing domestic pets and menacing residents. They have 660 599 of the Great Dividing Range each summer to graze the alpine pas- become such a problem in a number of places that local authorities 661 600 tures. This was a highly organised process where families leased have employed professional wild dog controllers to remove them 662 601 defined areas of pasture for their animals to graze for 3 - 4 months using lethal methods. Examination of the intestines of some of 663 602 each year. Therefore, annually these pastures would have become these euthanised wild dogs from several locations has revealed 664 603 contaminated with eggs from infected sheep dogs acting as a high prevalences and heavy worm burdens of E. granulosus 665 604 source of infection of both sheep and macropodids. Dingoes would (Brown and Copeman, 2003; Jenkins et al., 2008). The establish- 666 605 have been infected through predating on sheep and opportunist ment of peri-urban transmission cycles are unlikely, but regular 667 606 scavenging of dead sheep and offal discarded by the drovers when encroachment of E. granulosus-infected wildlife definitive hosts 668 607 they killed a sheep to eat whilst staying on the lease. into outer suburbs of urban centres has been demonstrated 669 (Brown and Copeman, 2003; Jenkins et al., 2008), which is difficult 670 to control and likely to be an ongoing issue for local councils to 671 608 3.2.1. Dingoes manage. 672 609 The Australian wildlife top-order predator coincidentally was a Hitherto, it was supposed urban human populations in Australia 673 610 placental canid, the dingo (Canis lupus dingo), introduced from were insulated from exposure to E. granulosus, and clearly, with the 674 611 south-eastern Asia by visiting seafarers (Corbett, 1995). Dingoes encroachment of E. granulosus-infected wild dogs (and foxes, see 675 612 were medium sized wild dogs (11–17 kg) capable of killing sheep. Section 3.7.1) into urban areas this is no longer the case. 676 613 Settlers moving to Australia also brought their domestic dogs 614 and it soon became evident that dingoes were able to breed with 3.4. Australian native carnivores as definitive hosts for E. granulosus 677 615 domestic dogs and produce fertile hybrid offspring. The result 616 has been that currently in most areas of remaining suitable habitat Infection with adult E. granulosus has never been reported from 678 617 in south-eastern and eastern Australia the resident top order pred- Australian native marsupial carnivores. In 2005, Jenkins et al. 679 618 ator populations no longer consist of pure-bred dingoes but mainly undertook a small study investigating the faeces from wild-caught 680 619 dingo/domestic dog hybrids with occasional pure-bred dingoes spotted-tailed quolls (Dasyurus maculatus) for coproantigens of E. 681 620 (Claridge et al., in press). Animals in these populations are referred granulosus and reviewed the literature regarding experimental 682 621 to as wild dogs. Pure-bred dingoes are mostly restricted to remote infection of dasyurids with E. granulosus. None of the quoll faeces 683 622 areas, especially in northern parts of Western Australia. Neverthe- tested were found to be positive for coproantigens of E. granulosus 684 623 less, there is no indication that hybrid dingoes are any more or less (Jenkins et al., 2005b) and in none of the attempted experimental 685 624 susceptible to infection with E. granulosus than pure-bred animals infections of several species of dasyurid have E. granulosus been 686 625 since no difference in the range of worm burdens has been noted recovered. From these data, Jenkins et al. (2005b) concluded that 687 626 between hybrid and pure-bred dingoes (Jenkins and Morris, dasyurids are refractory to infection with E. granulosus. 688 627 2003; Jenkins et al., 2008). The hunting and pack behaviours of 628 dingo hybrids compared with dingoes also appear to be similar 3.5. Wildlife intermediate hosts 689 629 (Claridge et al., in press). The one important physiological differ- 630 ence between dogs and dingoes is breeding capacity; female Macropodid marsupials are highly susceptible to infection with 690 631 domestic dogs come into oestrus twice each year whilst for din- the intermediate stage of E. granulosus (Jenkins and Morris, 2003; 691 632 goes it is only once. Data collected on the breeding behaviour of Banks, 2006), often carrying multiple large metacestodes (hydatid Q4 692 633 dingo hybrids does not indicate they are having more litters per cysts). This is especially the case in a number of wallaby species, 693 634 year than pure-bred dingoes, but there have been reports that litter namely swamp wallabies (Wallabia bicolor) in south-eastern Aus- 694 635 sizes in hybrids are larger than those of pure-bred dingoes and the tralia (Jenkins and Morris, 2003). Swamp wallabies are commonly 695 636 size of wild dogs has increased approximately 20% during the last infected with E. granulosus (Jenkins and Morris, 2003) and are also 696 637 40 years (Claridge et al., in press). a favourite dietary item for wild dogs (Claridge et al., in press), 697 638 Anecdotally, in many areas wild dog population numbers making them pivotal in the transmission of E. granulosus in wildlife 698 639 appear to be increasing, a likely combination of increased litter size in south-eastern Australia. Other wallaby species appear to be of 699 640 and increased food resources due to environment modification. similar importance in other parts of Australia (Banks et al., 2006a). 700 641 Provision of more pasture and watering points for livestock has Curiously, on the island state of Tasmania macropodid marsupi- 701 642 led to major increases in macropodid populations in many areas, als never become infected with E. granulosus (Howkins, 1966), 702 643 providing increased food supply for wild dogs. With increased wild despite there having been high levels of transmission in domestic 703 644 dog populations it is reasonable to assume that the biomass and animals. This may have been associated with the absence of din- 704 645 rate of transmission of E. granulosus in wildlife is also increasing. goes and the presence of a dasyurid top order predator, the thyla- 705 cine (Thylacine cynocephalus), refractory to infection with E. 706 646 3.3. Echinococcus granulosus-infected wild dog encroachment into granulosus (see Section 3.4). Nevertheless, human hydatidosis 707 647 urban areas became such a major public health problem in Tasmania that a 708 program of intense control was introduced that continued for 709 648 With increasing wild dog numbers and the spread of urbanisa- 30 years (Beard et al., 2001). 710 649 tion into areas of hitherto wild dog habitat has displaced young Other species of native wildlife susceptible to infection with E. 711 650 and old animals seeking new uncontested habitat. These animals granulosus are wombats (Wombatus ursinus). However, hydatid 712 651 are encroaching into outer urban areas and bringing E. granulosus infection in wombats has only been reported once (Grainger and 713 652 with them (Brown and Copeman, 2003; Jenkins et al., 2008). Jenkins, 1996). Wombats are consumed periodically by wild dogs 714

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715 but appear to only be an occasional host for E. granulosus, therefore E. granulosus. More recently, E. granulosus have been recovered 777 716 they cannot be regarded as an important component of the E. gran- from a number of naturally infected foxes in various studies 778 717 ulosus life cycle in Australia. (Thompson et al., 1985; Obendorf et al., 1989; Jenkins and Craig, 779 1992; Reichel et al., 1994; Jenkins and Morris, 2003). Almost 780 718 3.6. Factors contributing to the transmission success of E. granulosus in exclusively, worm burdens have been small, usually less than 50 781 719 Australian wildlife tapeworms/fox. However, in one study (Reichel et al., 1994) several 782 thousand E. granulosus in each of two foxes from Victoria were 783 720 Hydatid disease of macropodids is almost exclusively confined reported. These data are so at odds with all other existing fox E. 784 721 to the lungs, causing major respiratory impairment (Jenkins and granulosus worm burden data that they should be regarded 785 722 Morris, 2003; Barnes et al., 2007a,b, 2008, 2011) that may lead to cautiously. However, if corroborated then the role of foxes in the 786 723 the death of the host (Johnson et al., 1998; Barnes et al., 2008). transmission of E. granulosus may have to be re-evaluated. The cur- 787 724 Why hydatid infection in macropods should be mainly confined rent consensus is that foxes act as definitive hosts for E. granulosus 788 725 to the lungs is unclear, however, this is also seen with hydatid but are of minor importance in the transmission of the parasite in 789 726 infection in cervids (Schantz et al., 1995). Hydatid-infected macro- rural areas of Australia. However, foxes infected with E. granulosus 790 727 podids are rendered highly susceptible to predation by dingoes, a are of potential public health importance when infected animals 791 728 similar situation to that with the interaction of wolves and hyda- encroach into urban areas (Jenkins and Craig, 1992). 792 729 tid-infected moose (A. alces) in the USA (Mech, 1966; Jolly and 730 Messier, 2004). In addition, cysts in macropodids develop quickly, 3.7.2. Feral cats 793 731 becoming fertile (containing protoscoleces) in 8–9 months (Barnes Infection of Australian feral cats with adult E. granulosus has 794 732 et al., 2007a), compared with sheep where the earliest time to fer- never been reported. Jenkins and Morris (2003) examined the 795 733 tility is approximately 2 years (Slias, 1980). The increased suscep- intestines of 23 feral cats collected in an area of high E. granulosus 796 734 tibility of hydatid-infected macropodids to predation by wild dogs transmission in the local wildlife and none was found infected. 797 735 means that wild dogs are catching and consuming a disproportion- Jenkins also fed protoscoleces recovered from a wallaby to two cats 798 736 ately large number of infected animals which is likely to be a major and two fox cubs, and small numbers of E. granulosus were 799 737 contribution to the high worm burdens seen in many wild dogs. recovered from both foxes but nothing was found in the cats 800 738 Within all wild dog populations surveyed, a proportion of the sam- (D.J. Jenkins, unpublished data). It is generally considered that feral 801 739 ple has been wild dogs with worm burdens in excess of 100,000 E. cats in Australia are not definitive hosts for E. granulosus. 802 740 granulosus (Jenkins and Morris, 1991, 2003; Jenkins et al., 2008). 741 These animals are the ‘‘super spreaders’’ of the population 3.7.3. Feral pigs, goats, deer, horses and rabbits 803 742 (Gemmell and Lawson, 1986), ensuring large numbers of eggs Hydatid disease occurs in Australian feral pigs (Jenkins and 804 743 being released into the environment. The home range of wild dogs Morris, 2003; Lidetu and Hutchinson, 2007). The study of Jenkins 805 744 varies depending on the sex of the animal and availability of food and Morris (2003) was conducted in south-eastern New South 806 745 and water; in eastern Australia home ranges average approxi- Wales; 204 pigs were examined, 22.5% contained hydatid cysts 807 746 mately 100 km2 (Claridge et al., in press). Therefore large areas and depending on the survey area between 15 and 22% of the cysts 808 747 can become contaminated with eggs from a single animal, but were fertile. In contrast, the study of Lidetu and Hutchinson (2007) 809 748 since packs of wild dogs consisting of several animals usually was conducted in tropical northern Queensland; 74 pigs were 810 749 occupy these spaces (Claridge et al., in press), contamination of examined and 31.1% contained hydatid cysts, but the rate of fertil- 811 750 the environment with faeces can become heavy in areas used com- ity was almost three times that of the pigs collected in New South 812 751 monly by a resident pack. Wales. The reason for this difference in cyst fertility is unclear. 813 752 The longevity of eggs of E. granulosus in the environment is There is no doubt feral pigs could contribute to transmission of E. 814 753 thought to be several months to perhaps approximately 1 year, granulosus in wildlife but the degree to which they contribute is 815 754 so long as the environment is not too hot and dry (Eckert and debatable. Adult feral pigs (the animals most likely to harbour fer- 816 755 Deplazes, 2004). However, in a study in Argentina (Thevenet tile cysts) are big and strong and difficult for wild dogs to catch and 817 756 et al., 2005), E. granulosus egg-laden dog faeces were left in the kill; consequently, wild dogs mainly predate on piglets, animals 818 757 environment for 41 months. After this time eggs were separated least likely to contain fertile cysts. Inevitably, adult pigs eventually 819 758 from the faeces and fed to each of four sheep. Hydatid cysts devel- die and their carcasses become available for scavenging by wild 820 759 oped in each of the sheep, suggesting that the longevity of eggs of dogs and foxes. However, particularly in summer, scavengers 821 760 E. granulosus in Australia under optimal conditions may be longer would need to find the carcass soon after death before fertile cysts 822 761 than 12 months. Studies reported in Gemmell (1958) revealed became non-infective due to putrefaction. Therefore, the role of 823 762 the most important environmental conditions required for trans- pigs in the transmission of E. granulosus in Australian is likely to 824 763 mission for E. granulosus in Australia to be at least 25 mm of rain be minor. 825 764 for 6 months of the year with temperatures up to 30 °C. Conse- Hydatid infection in goats has never been reported except for an 826 765 quently, E. granulosus has become more prevalent in eastern Aus- anecdotal report from Western Australia (R.C.A. Thompson, unpub- 827 766 tralia, in areas associated with the Great Dividing Range, and in lished data). Periodically, one of the current authors (D.J. Jenkins) 828 767 an elevated area of Western Australia (Thompson et al., 1988; has examined small numbers of feral goats, collected from various 829 768 Jenkins and Macpherson, 2003)(Fig. 3). areas of south-eastern New South Wales, and has never found any 830 infected with hydatid cysts (D.J. Jenkins, unpublished data). The 831 769 3.7. Echinococcus granulosus in introduced wildlife species absence of hydatid infection in Australian feral goats is curious 832 because in many countries goats are important intermediate hosts 833 770 3.7.1. Foxes (Schantz et al., 1993). 834 771 The first creditable report of E. granulosus infection in foxes in Hydatid cysts have never been reported from feral horses or 835 772 Australia was by Gemmell (1959a) where a single terminal deer in Australia. Therefore, until data to the contrary are available, 836 773 segment was recovered from the intestine of one of 41 animals feral goats, deer and horses appear not to be part of the lifecycle of 837 774 collected in New South Wales. Nevertheless, based on the available E. granulosus in Australia. 838 775 experimental infection and survey data, Gemmell (1959a) felt that Rabbits naturally infected with hydatid disease have rarely 839 776 Australian foxes were not acting as a definitive host for been reported. The two reports, (cited in Gemmell, 1959b) are 840

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Fig. 3. Map depicting areas of high, low, rare and no transmission of Echinococcus granulosus in Australia (red, high transmission; orange, low transmission; beige, rare transmission; grey, no transmission). (Modiﬁed version of the map published by Jenkins and Macpherson (2003)).

841 thought to be a mis-identification of metacestodes of Taenia serial- samples and 7.8% of Tasmanian samples). Some coproantigen-posi- 871 842 is. Wild Australian rabbits have been shown to be susceptible to tive faecal samples were also positive in an E. granulosus coproPCR 872 843 experimental infection with hydatid disease (Jenkins and (Jenkins et al., 2014). 873 844 Thompson, 1995). The cysts established in the lungs and appeared 845 to be developing normally. However, the rabbits were given a large 846 dose of eggs, far higher than anything they would normally be 3.8.2. Sheep 874 847 exposed to in the wild, which may have been the reason they Hydatid disease prevalence data are not collected in Australian 875 848 became infected. abattoirs, but in Tasmania occurrence of infection in livestock is 876 monitored and traced back to the property of origin. Hydatid dis- 877 849 3.8. Current prevalence of E. granulosus in Australian domestic dogs, ease still occurs periodically in Australian sheep, however the 878 850 sheep and cattle on mainland Australia and the island state of prevalence has declined steadily during the last 30 years (D.J. Jen- 879 851 Tasmania kins, personal observations), no doubt a reflection of the decline of 880 E. granulosus infection in rural domestic dogs (Jenkins et al., 2014). 881 852 3.8.1. Dogs Sheep populations now most at risk of contracting hydatid disease 882 853 The most recently published study on intestinal helminths in are those grazed on pasture abutting national and state parks and 883 854 Australian mainland dogs is that of Palmer et al. (2008) where forests containing populations of wild dogs (Grainger and Jenkins, 884 855 1400 faecal samples collected in vet clinics were sent to the 1996). These wild dogs periodically enter pastures to predate on 885 856 authors for testing. These samples were from urban dog owners the sheep but whilst there also defecate, contaminating the pasture 886 857 with a close interest in the health of their pets; unsurprisingly, with eggs of E. granulosus. Nothing recent has been published 887 858 no taeniid cestodes were identified in any of the samples collected. regarding the prevalence of hydatid disease in mainland sheep. 888 859 The most recent study of intestinal helminths in rural dogs (Jenkins The most up-to-date data have been collected by the National 889 860 et al., 2014) screened faeces from 1425 rural dogs living in eastern Sheep Health Monitoring Program (Animal Health Australia, 890 861 Australia (1119 mainland; 306 Tasmania) with faecal flotation, 2011, http://www.animalhealthaustralia.com.au/programs/dis- 891 862 molecular methods and the E. granulosus coproantigen test ease-surveillance/the-national-sheep-health-monitoring-project/). 892 863 (Jenkins et al., 2000). Surprisingly, taeniid eggs in faeces were also Routine monitoring of slaughtered sheep for hydatid infection is 893 864 uncommon, being recovered from the faeces of only 11 dogs. This still undertaken in Tasmania. Since the declaration of provisional 894 865 is likely to be because more than 98% of owners reported feeding eradication in 1996, there have been two reports of infection in 895 866 dry commercial dog food exclusively or as a major component of sheep; one in 2011 in sheep sent to a mainland abattoir for 896 867 their dog’s diet and approximately 50% of owners either de- slaughter (Jenkins et al., in press) the other, more recently in Q5 897 868 wormed their dog(s) 2 monthly or 4 monthly with a de-wormer 2013 (D.J. Jenkins, unpublished data) in a single animal, resident 898 869 containing praziquantel. Nevertheless, an additional 45 dogs were on a property in the midlands area of Tasmania. Currently, the ori- 899 870 positive in the E. granulosus coproantigen test (1.9% of mainland gins of this animal are still being investigated. 900

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901 3.8.3. Cattle in Australia. However, the research that has been conducted in 962 902 ‘‘High levels’’ of hydatid disease in slaughtered cattle have been Australia has contributed much to the international research effort 963 903 reported, anecdotally, from abattoirs in south-eastern Queensland, on Echinococcus and hydatid disease, in particular the value of the 964 904 north-eastern New South Wales and eastern Victoria. No recent organism as a model in studies on developmental biology and 965 905 data have been published regarding hydatid disease prevalence in epidemiology, not just concerned with Echinococcus but more 966 906 Australian cattle, but according to abattoir management prevalence broad-ranging in terms of basic biology and theoretical models of 967 907 is high enough to be causing substantial financial losses to the transmission dynamics. We hope that Australia will continue to 968 908 abattoirs through condemnation of offal. An estimated loss to the contribute to research on Echinococcus and that this will have an 969 909 northern Queensland beef industry of $6 million per year due to impact internationally and sustain the collaborations that have 970 910 hydatid-infected offal was reported for 2004, with prevalence levels developed with research groups and centres world-wide. 971 911 ranging between 20–51% in cattle from coastal areas and 3% in 912 those from inland areas (Banks et al., 2006b). The source of infection 5. Uncited references 972 913 in these cattle is likely from wildlife since cattle are commonly 914 grazed in areas inhabited by wild dogs because predation impacts (Gemmell et al., 1986; Harris and Revfeim, 1980; Lavikainen 973 915 are less severe than if sheep were grazed in the same areas. et al., 2006). Q6 974 916 As with sheep, hydatid disease is monitored in slaughtered cat- 917 tle in Tasmania. Since declaration of provisional eradication in Acknowledgements 975 918 1996, over 200 hydatid-infected cattle have been identified. Most 919 of these animals proved to be imports from the mainland, mainly We are very grateful to Mark Preston (Graphic Designer, 976 920 Gippsland in Victoria, an area where hydatid infection in cattle is Murdoch University, Australia) for producing the graphical images 977 921 common. However, 31 were animals less than 3 years old and and to Craig Poynter (Spacial Analysis Unit, Charles Sturt 978 922 had never left Tasmania (Jenkins et al., 2014). DNA was extracted University, Australia) for preparing the map. 979 923 and sequenced from cysts removed from four of the infected cattle; 924 their infections proved to be the G1 sheep strain of E. granulosus References 980 925 (Jenkins et al., unpublished data). The G1 sheep strain not only 926 infects sheep but can also infect cattle, pigs, goats and macropodid Albani, C.M., Elissondo, M.C., Cumino, A.C., Chisari, A., Denegri, G.M., 2010. Primary 981 927 marsupials. Less than 5% of G1 hydatid cysts in Australian cattle cell culture of Echinococcus granulosus developed from the cystic germinal layer: 982 928 are fertile (Kumaratilake and Thompson, 1982), therefore cysts biological and functional characterization. Int. J. Parasitol 40, 1269–1275. 983 984 929 from infected cattle, if consumed by dogs, dingoes or their hybrids, Albani, C.M., Cumino, A.C., Elissondo, M.C., Denegri, G.M., 2013. Development of a cell line from Echinococcus granulosus germinal layer. Acta Trop. 128, 124–129. 985 930 commonly do not contribute to transmission of E. granulosus. Ali-Khan, Z., Siboo, R., Gomersall, M., Faucher, M., 1983. Cystolytic events and the 986 931 The 24 Tasmanian domestic dogs that tested positive in the E. possible role of germinal cells in metastasis in chronic alveolar hydatidosis. 987 988 932 granulosus coproantigen ELISA and the hydatid-infection data from Ann. Trop. Med. Parasitol. 77, 497–512. Banks, D.J., Copeman, D.D., Skerratt, L.F., 2006a. Echinococcus granulosus in northern 989 933 sheep and cattle found since declaration of provisional freedom in Queensland. 2. Ecological determinants of infection in beef cattle. Aust. Vet. J. 990 934 1996 indicate that hydatid disease has not been completely eradi- 84, 308–311. 991 935 cated from the island as was previously thought but continues to Banks, D.J., Copeman, D.D., Skerratt, L.F., Molina, E.C., 2006b. Echinococcus granulosus 992 in northern Queensland. 2. Prevalence in cattle. Aust. Vet. J. 84, 303–307. 993 936 be transmitted at low levels (Jenkins et al., 2014). Barnes, T.S., Hinds, L.A., Jenkins, D.J., Coleman, G.T., 2007a. Precious development of 994 hydatid cysts in a macropod host. Int. J. Parasitol. 37, 1379–1389. 995 Barnes, T.S., Morton, J.M., Coleman, G.T., 2007b. Clustering of hydatid infection in 996 937 3.9. Human prevalence macropods. Int. J. Parasitol. 37, 943–952. 997 Barnes, T.S., Goldizen, A.W., Morton, J.M., Coleman, G.T., 2008. Cystic Echinococcus in 998 999 938 The prevalence of human hydatidosis on mainland Australia is a wild population of the brush-tailed rock-wallaby (Petrogale penicillata)a threatened macropodid. Parasitology 135, 715–723. 1000 939 unknown, because in most jurisdictions human hydatid disease Barnes, T.S., Hinds, L.D., Jenkins, D.J., Bielefeldt-Ohmann, H., Lightowlers, M.W., 1001 940 has ceased to be notifiable. The last published data on human Coleman, G.T., 2011. Comparative pathology of pulmonary hydatid cysts in 1002 1003 941 hydatid disease on mainland Australia was that of Jenkins and macropods and sheep. J. Comp. Pathol. 144, 113–122. Barrett, N.J., Smyth, J.D., Ong, S.J., 1982. Spontaneous sexual differentiation of 1004 942 Power (1996) but these data were only collected in New South Mesocestoides corti tetrathyridia in vitro. Int. J. Parasitol. 12, 315–322. 1005 943 Wales and the Australian Capital Territory and are now out of date. Beard, T., Bramble, A.J., Middleton, M.J., 2001. Eradication in Our Time: a Log Book of 1006 944 Nevertheless, new cases continue to be identified annually on the Hydatid Control Program 1962–1996. Department of Primary Industry, 1007 Water and Environment, Hobart. 1008 945 mainland Australia. A proportion of these new cases are in recently Bowles, J., Blair, D., McManus, D.P., 1994. Molecular genetic characterisation of the 1009 946 arrived immigrants from a range of countries who were infected in cervid strain (‘‘northern form’’) of Echinococcus granulosus. Parasitology 109, 1010 947 their country of origin. In the study of Jenkins and Power (1996), 215–221. 1011 Brown, B., Copeman, D.B., 2003. Zoonotic importance of parasites in wild dogs 1012 948 60% of cases diagnosed in residents living in major urban centres caught in the vicinity of Townsville. Aust. Vet. J. 81, 700–702. 1013 949 were recent immigrants. There has been a recent publication on Budke, C.M., Deplazes, P., Torgerson, P.R., 2006. Global socioeconomic impact of 1014 950 human hydatid disease in Tasmania (O’Hern and Cooley, 2013). cystic echinococcosis. Emerg. Infect. Dis. 12, 296–303. 1015 Carmena, D., Cardona, G.A., 2014. Echinococcosis in wild carnivorous species: 1016 951 The authors described human hydatid disease in Tasmania since epidemiology, genotypic diversity, and implications for veterinary public 1017 952 the declaration of provisional freedom in 1996. Transmission of health. Vet. Parasitol. 202, 69–94. 1018 953 hydatid disease to humans in Tasmania has now ceased, although Catalano, S., Lejeune, M., Liccioli, S., Verocai, G.G., Gesy, K.M., Jenkins, E.J., Kutz, S.J., 1019 1020 954 one or two new cases are diagnosed annually. However, all of these Fuentealba, C., Duignan, P.J., Mossolo, A., 2012. Echinococcus multilocularis in urban coyotes, Alberta, Canada. Emerg. Infec. Dis. 18, 1625–1628. 1021 955 new cases are in elderly people who were thought to have been Claridge, A.W., Spencer, R.J., Wilton, A.N., Jenkins, D.J., Dall, D., Lapidge, S.J., in press. 1022 956 infected pre-1996. When is a dingo not a dingo? Hybridisation with domestic dogs. In: Glen, A.S., 1023 Dickman, C.R. (Eds), Carnivores of Australia. CSIRO Publishing, Collingwood, 1024 Australia. Q7 1025 1026 957 4. Conclusions Corbett, L., 1995. The Dingo in Australia and Asia. University of New South Wales Press, Sydney, Australia. 1027 Cruz-Reyes, A., Constantine, C.C., Boxell, A.C., Hobbs, R.P., Thompson, R.C.A., 2007. 1028 958 Australia has a long history of research on hydatid disease and Echinococcus granulosus from Mexican pigs is the same strain as that in Polish 1029 1030 959 the causative agent Echinococcus. Much of this was borne from pigs. J. Helminthol. 81, 287–292. Davidson, R.K., Romig, T., Jenkins, E., Tryland, M., Robertson, L.J., 2012. The impact of 1031 960 necessity, given the impact the parasite had on public health and globalisation on the distribution of Echinococcus multilocularis. Trends Parasitol. 1032 th th 961 the agricultural economy in the late 19 and early 20 centuries 28, 239–247. 1033

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1034 Deplazes, P., Thompson, R.C.A., Constantine, C.C., Penhale, W.J., 1993. Primary Holcman, B., Heath, D.D., 1997. The early stages of Echinococcus granulosus 1120 1035 infection of dogs with Echinococcus granulosus: systemic and local (Peyer’s development. Acta Trop. 64, 5–17. 1121 1036 patches) immune responses. Vet. Immunol. Immunopathol. 40, 171–184. Howell, M.J., Smyth, J.D., 1995. Cultivation. In: Thompson, R.C.A., Lymbery, A.J. 1122 1037 Deplazes, P., Hegglin, D., Gloor, S., Romig, T., 2004. Wilderness in the city: the (Eds.), Echinococcus and Hydatid Disease. CAB International, Wallingford, Oxon, 1123 1038 urbanisation of Echinococcus multilocularis. Trends Parasitol. 20, 77–84. UK, pp. 200–232. 1124 1039 Dévé, F., 1919. Secondary echinococcosis. Lancet ii, 835–838. Howkins, A.B., 1966. The role of macropodidae in Tasmania as intermediate hosts in 1125 1040 Dévé, F., 1946. L’Echinococcose Secondaire. Masson et Cie, Paris. hydatid disease. Aust. Vet J. 42, 240–241. 1126 1041 Dew, H.R., 1922. Observations on the mode of development of brood capsules and Huttner, M., Siefert, L., Mackenstedt, U., Romig, T., 2009. A survey of Echinococcus 1127 1042 scolices in the encysted stage of Taenia Echinococcus. Med. J. Aust. 2, 381–384. species in wild carnivores and livestock in East Africa. Int. J. Parasitol. 39, 1269– 1128 1043 Dew, H.R., 1925. The histogenesis of the hydatid parasite (Taenia echinococcus)in 1276. 1129 1044 the pig. Med. J. Aust. I, 101–110. Jenkins, D.J., Craig, N.A., 1992. Role of foxes (Vulpes vulpes) in the epidemiology 1130 1045 Dew, H.R., 1928. Hydatid Disease, Its Pathology, Diagnosis and Treatment. of Echinococcus granulosus in urban environments. Med. J Aust. 157, 754– 1131 1046 Australian Medical Publishing Company, Sydney. 756. 1132 1047 Dew, H.R., 1935. Advances in our knowledge of hydatid disease during the Jenkins, D.J., Macpherson, C.N.L., 2003. Transmission ecology of Echinococcus 1133 1048 twentieth century. Br. Med. J. 2, 620–622. granulosus in wildlife in Australia and Africa. Parasitology 127, S63–S72. 1134 1049 Dew, H.R., 1953. Hydatid disease in Australia. Arch. Int. Hidatidosis 13, 1319–1332. Jenkins, D.J., Morris, B., 1991. Unusually heavy infections of Echinococcus granulosus 1135 1050 Diaz, A., Casaravilla, C., Irigoin, F., Lin, G., Previato, J.O., Ferreira, F., 2011a. in wild dogs in south-eastern Australia. Aust. Vet. J. 68, 36–37. 1136 1051 Understanding the laminated layer of larval Echinococcus I: structure. Trends Jenkins, D.J., Morris, B., 2003. Echinococcus granulosus in wildlife in and around the 1137 1052 Parasitol. 27, 204–213. Kosciuszko National Park, southeastern New South Wales. Aust. Vet. J. 86, 81– 1138 1053 Diaz, A., Casaravilla, C., Allen, J.E., Sim, R.B., Ferreira, F., 2011b. Understanding the 85. 1139 1054 laminated layer of larval Echinococcus II: immunology. Trends Parasitol. 27, Jenkins, D.J., Power, K., 1996. Human hydatidosis in New South Wales and the 1140 1055 263–272. Australian Capital Territory, 1987–1992. Med. J. Aust. 164, 18–21. 1141 1056 Durie, P.H., Riek, R.F., 1952. The role of the dingo and wallaby in the infestation of Jenkins, D.J., Thompson, R.C.A., 1995. Hydatid cysts in an experimentally infected 1142 1057 cattle with hydatids (Echinococcus granulosus Batch 1786) (Rudolphi 1805) in rabbit. Vet. Rec. 137, 148–149. 1143 1058 Queensland. Aust. Vet. J. 28, 249–254. Jenkins, D.J., Fraser, A., Bradshaw, H., Craig, P.S., 2000. Detection of Echinococcus 1144 1059 Eckert, J., Deplazes, P., 2004. Biological, epidemiological and clinical aspects of granulosus coproantigens in Australian canids with natural or experimental 1145 1060 Echinococcus, a zoonosis of increasing concern. Clin. Microbiol. Rev. 17, 107– infection. J. Parasitol. 86, 140–145. 1146 1061 135. Jenkins, D.J., Murray, A.J., Claridge, A.W., Story, G.L., Bradshaw, H., Craig, P.S., 2005a. 1147 1062 Eckert, J., Thompson, R.C.A., Mehlhorn, H., 1983. Proliferation and metastases The contribution of spotted-tailed quolls in the transmission of Echinococcus 1148 1063 formation of larval Echinococcus multilocularis. I. Animal model, macroscopical granulosus in the Byadbo Wilderness Area of the Kosciuszko National Park, 1149 1064 and histological findings. Z. Parasitol. 69, 737–748. Australia. Wild. Res. 32, 37–41. 1150 1065 Eckert, J., Meslin, F.-X., Pawlowski, Z.S. (Eds.), 2001. WHO/OIE Manual on Jenkins, D.J., Romig, T., Thompson, R.C.A., 2005b. Emergence/re-emergence of 1151 1066 Echinococcosis in Humans and Animals: a Public Health Problem of Global Echinococcus spp. – a global update. Int. J. Parasitol. 35, 1205–1219. 1152 1067 Concern. World Organisation for Animal Health and World Health Organisation, Jenkins, D.J., Allen, L., Goullet, M., 2008. Encroachment of Echinococcus granulosus 1153 1068 Geneva. into urban areas of eastern Queensland, Australia. Aust. Vet. J. 86, 294–300. 1154 1069 Esch, G.W., Smyth, J.D., 1976. Studies on the in vitro culture of Taenia crassiceps. Int. Jenkins, D.J., Lievaart, J.J., Boufana, B., Lett, W., Bradshaw, H., Armua-Fernandes, M.T., 1155 1070 J. Parasitol. 6, 143–149. 2014. Echinococcus granulosus and other intestinal helminths in rural dogs from 1156 1071 Galindo, M., Paredes, R., Marchant, C., Miño, V., Galanti, N., 2003. Regionalization of eastern Australia, the current situation. Aust. Vet. J. 92, 292–298. 1157 1072 DNA and protein synthesis in developing stages of the parasitic platyhelminth Johnson, P.M., Speare, R., Beveridge, I., 1998. Mortality in wild and captive rock 1158 1073 Echinococcus granulosus. J. Cell. Biochem. 90, 294–303. wallabies and nailtail wallabies due to hydatid disease caused by Echinococcus 1159 1074 Gemmell, M.A., 1958. Hydatid disease in Australia III. Observations on the incidence granulosus. Aust. Mammal. 20, 419–423. 1160 1075 and geographical distribution of hydatid disease in sheep in New South Wales. Jolly, D.O., Messier, F., 2004. The distribution of echinococcosis in moose: evidence 1161 1076 Aust. Vet. J. 34, 269–280. for parasite-induced vulnerability to predation to wolves? Cecolodia 140, 586– 1162 1077 Gemmell, M.A., 1959a. Hydatid disease in Australia. VI. Observations on the 590. 1163 1078 carnivore of New South Wales as definitive hosts for Echinococcus granulosus Kamiya, M., Sato, H., 1990a. Survival, strobilation and sexual maturation of 1164 1079 (Batch 1786), (Rudolphi 1801) and their role of the spread of hydatidosis in Echinococcus multilocularis in the small intestine of golden hamsters. 1165 1080 domestic animals. Aust. Vet. J. 35, 450–455. Parasitology 100, 125–130. 1166 1081 Gemmell, M.A., 1959b. Hydatid disease in Australia VII. An appraisal of the present Kamiya, M., Sato, H., 1990b. Complete life cycle of the canid tapeworm, Echinococcus 1167 1082 position and some problems in control. Aust. Vet. J. 35, 505–514. multilocularis, in laboratory rodents. FASEB J. 4, 3334–3339. 1168 1083 Gemmell, M.A., 1990. Australasian contributions to an understanding of the Kamiya, M., Ooi, H.-K., Oku, Y., Yagi, K., Ohbayashi, M., 1985. Growth and 1169 1084 epidemiology and control of hydatid disease caused by Echinococcus development of Echinococcus multilocularis in experimentally infected cats. 1170 1085 granulosus-past, present and future. Int. J. Parasitol. 20, 431–456. Jpn. J. Vet. Res. 33, 135–140. 1171 1086 Gemmell, M.A., Lawson, R.J., 1986. Epidemiology and control of hydatid disease. In: King, H.W.H., 1959. Transhumance grazing in the snow belt of New South Wales. 1172 1087 Thompson, R.C.A. (Ed.), The Biology of Echinococcus and Hydatid Disease. George Aust. Geogr. 7, 129–140. 1173 1088 Allen and Unwin, London, UK, pp. 179–216. Kouguchi, H., Matsumoto, J., Nakao, R., Yamano, K., Oku, Y., Yagi, K., 2013. 1174 1089 Gemmell, M.A., Roberts, M.G., 1995. Modelling Echinococcus life cycles. In: Characterization of a surface glycoprotein from Echinococcus multilocularis and 1175 1090 Thompson, R.C.A., Lymbery, A.J. (Eds.), Echinococcus and Hydatid Disease. CAB its mucosal vaccine potential in dogs. PLoS ONE 8, e69821. 1176 1091 International, Wallingford, UK, pp. 333–354. Koziol, U., Rauschendorfer, T., Rodríguez, L.Z., Krohne, G., Brehm, K., 2014. The 1177 1092 Gemmell, M.A., Lawson, J.R., Roberts, M.G., 1986. Population dynamics in unique stem cell system of the immortal larva of the human parasite 1178 1093 echinococcosis and cysticercosis: biological parameters of Echinococcus Echinococcus multilocularis. EvoDevo 5, 10. 1179 1094 granulosus in dogs and sheep. Parasitology 92, 599–620. Krasnoshchekov, G.P., Pluzhnnikov, L.T., 1981. 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