- Email: [email protected]
Surveillance and management of Echinococcus multilocularis in a wildlife park
Parasitology International 65 (2016) 245–250
Contents lists available at ScienceDirect
Parasitology International journal homepage: www.elsevier.com/locate/parint
Surveillance and management of Echinococcus multilocularis in a wildlife park Gérald Umhang a,⁎, Jennifer Lahoreau b, Vanessa Hormaz a, Jean-Marc Boucher a, Amandine Guenon c, Damien Montange d, Frédéric Grenouillet d,e, Franck Boue a a
ANSES LRFSN, Wildlife Surveillance and Eco-epidemiology Unit, National Reference Laboratory for Echinococcus spp., Malzéville, France Parc Animalier de Sainte-Croix, Rhodes, France ONIRIS Ecole Nationale Vétérinaire, Agroalimentaire et de l'Alimentation Nantes-Atlantique, Nantes, France d WHO Collaborating Centre for Prevention and Treatment of Human Echinococcosis and National Reference Center for Human Alveolar Echinococcosis, CHRU University Hospital, Besançon, France e Chrono-Environnement UMR 6249 Research Team, CNRS–University of Bourgogne-Franche-Comté, Besançon, France b c
a r t i c l e
i n f o
Article history: Received 16 September 2015 Received in revised form 28 December 2015 Accepted 3 January 2016 Available online 15 January 2016 Keywords: Echinococcus multilocularis Voles Captive wildlife Lemur catta Albendazole Serology
a b s t r a c t The fox tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a severe zoonotic disease that may be fatal if untreated. A broad spectrum of mammalian species may be accidentally infected even in captivity. In April 2011, liver lesions due to E. multilocularis were observed during the necropsy of a captiveborn nutria (Myocastor coypus) in a French wildlife park, leading to initiation of a study to survey the parasite's presence in the park. A comparable environmental contamination with fox's feces infected by E. multilocularis was reported inside (17.8%) and outside (20.6%) the park. E. multilocularis worms were found in the intestines of three of the five roaming foxes shot in the park. Coprological analyses of potential definitive hosts in captivity (fox, lynx, wildcat, genet, wolf, bear and raccoon) revealed infection in one Eurasian wolf. Voles trapped inside the park also had a high prevalence of 5.3%. After diagnosis of alveolar echinococcosis in a Lemur catta during necropsy, four other cases in L. catta were detected by a combination of ultrasound and serology. These animals were treated twice daily with albendazole. The systematic massive metacestode development and numerous protoscoleces in L. catta confirmed their particular sensitivity to E. multilocularis infection. The autochthonous origin of the infection in all the captive animals infected was genetically confirmed by EmsB microsatellite analysis. Preventive measures were implemented to avoid the presence of roaming foxes, contact with potential definitive hosts and contaminated food sources for potential intermediate hosts. © 2016 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The fox tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis (AE), a severe disease that is potentially fatal if untreated. The larval stage of the parasite develops in intermediate hosts after oral ingestion of microscopic eggs released into the environment in the feces of carnivorous definitive hosts. In Western Europe, the red fox (Vulpes vulpes) is the main definitive host. It harbors E. multilocularis worms in the intestines after predation of infected intermediate hosts—mainly small rodents. Nevertheless, a broad spectrum of mammalian species—notably aquatic rodents, member of the Suidae family or primates—may be accidentally infected, in which case they are considered aberrant hosts. The parasite larva develops almost exclusively in the liver, where its growth is destructive. However, it may also affect other organs by infiltration or metastasis [1]. After developing silently for 10–15 years, human AE may be diagnosed in patients with or without symptoms. ⁎ Corresponding author. E-mail address: [email protected] (G. Umhang).
http://dx.doi.org/10.1016/j.parint.2016.01.008 1383-5769/© 2016 Elsevier Ireland Ltd. All rights reserved.
The latter include the occurrence of cholestatic jaundice, epigastric pain, asthenia, weight loss or hepatomegaly [2]. The development of infection in aberrant hosts seems to be faster in lemurs as well as in dogs and is not comparable with the development in pigs. Many cases of infection by E. multilocularis or other Echinococcus species have been reported in captive animals, and especially primates. Several species of monkey (Gorilla gorilla, Macaca sylvanus, Macaca fascicularis, Macaca nigra, Macaca fuscata, Cercopithecus diana) and lemur (Varecia rubra, Lemur catta) have been infected [3–6]. This is a very real health threat for zoos in areas endemic for E. multilocularis. For example, Basel Zoo (Switzerland) was reported to have grouped cases following the death of five cynomolgus monkeys (M. fascicularis) and a lowland gorilla (Gorilla g. gorilla). Cases were also diagnosed in three macaque species in Germany [7,8]. While such animal cases are undoubtedly of clinical interest, they are also of particular epidemiological importance as they are indicators of environmental contamination and zoonotic risk [6]. In April 2011, liver lesions were observed during the necropsy of a captive-born nutria (Myocastor coypus) in a French wildlife park, later confirmed to be due to E. multilocularis. This diagnosis was followed by the death 16 months later of a ring-tailed lemur (L. catta) massively
246
G. Umhang et al. / Parasitology International 65 (2016) 245–250
infected by the parasite [9]. Following the first nutria case, a study was initiated to survey the parasite's presence in the park. The prevalence of E. multilocularis was evaluated in wildlife around and inside the park as well as in captive animals. The origin of infection in captive intermediate hosts was genetically investigated, while preventive and curative measures were also proposed and discussed. 2. Materials and methods 2.1. Study area The wildlife park is situated in Moselle, a department (French administrative unit) in north-eastern France known to be highly endemic for E. multilocularis, with a prevalence in foxes estimated at 34% (IC95%: 25–44) [10]. The captive animals are essentially European wildlife species living in large natural enclosures. The 120-ha park is located in a rural area, and surrounded by fields and forest. 2.2. The presence of E. multilocularis in free-ranging wildlife In February 2012, experienced staff collected fox feces outside park limits within a four-kilometer radius centered on the park. Some roaming foxes are occasionally observed inside the park despite perimeter fencing. For two years, from February 2012 to January 2014, the veterinarian and animal keepers collected fox feces observed through the park. When possible, roaming foxes were shot and intestines safely collected. In April and May 2012, rodents were captured in 21 trapping sites located in different environments (forest, bank, hedge and grassland) inside the park. Each site was equipped with a 100-m line of 34 INRA live traps spaced 3 m apart on four consecutive nights. The species determination was realized visually from the rodents for Myodes glareolus and Apodemus sylvaticus and Apodemus flavicollis. Concerning other species, the skull was isolated and cleaned by hot water maceration, then determination keys for the dental morphology (especially the first lower molars teeth's for Microtus sp.) were used [11]. All suspect parasitic lesions observed during the rodents' necropsy were sampled and frozen.
lemurs suspected to have alveolar echinococcosis (lesions revealed by the ultrasound scan and positive Echinococcus serology) [2].
2.4. Laboratory analysis After one week of deep-freezing at −80 °C for decontamination, all the feces were subjected to a flotation/sieving technique (adapted from Mathis et al. [12]) and screened microscopically for the presence of taeniid eggs as previously described [13]. In the presence of taeniid eggs, DNA was extracted from the pellets (QIAGEN QIAamp DNA stool). Multiplex PCR was used to identify E. multilocularis and Taenia spp. [14]. Parasitic lesions observed in rodents were diagnosed by PCR using primers JB3-JB4.5 of the cox1 gene [15]. The amplicons obtained were sequenced by a private laboratory (Beckmann Coulter Genomics). The nucleotide sequences were aligned using the Vector NTI software program (Invitrogen) and compared with sequences available in GenBank using the BLASTn program to determine the species involved. The intestines of roaming foxes were analyzed by the Segmental Sedimentation and Counting Technique (SSCT) [16]. Five worms from each positive fox and E. multilocularis-positive samples from intermediate hosts were genotyped by EmsB microsatellite analysis [17,18]. A dendrogram was drawn up using two samples of Echinococcus granulosus sensu stricto (G1) as outgroup. The usual EmsB genetic threshold at 0.08 was used to define assemblage profiles for samples clustered under this limit. The approximately unbiased P values were calculated with multiscale bootstrap resampling (B = 1000). Echinococcus serology was performed for all lemurs using an E. granulosus ELISA (Bordier Affinity) and a commercial E. multilocularis Western blot (LDBio Diagnostic) with only one slight modification, i.e. binding using Protein A/G Alkaline phosphatase conjugate (Pierce™ Thermoscientific). Thus, strips were incubated 1 h with 0.25 μg/ml conjugate (1/5000 dilution) in optimized buffer (Tris 6 g/l, NaCl 6 g/l, NaN3 1 g/l, powdered milk 2.5 g/l, pH adjusted to 7.4 with HCl). Albendazole (ABZ) sulfoxide plasma levels were monitored in all treated animals using the HPLC method [19]. Blood samples were taken one year after ABZ treatment was initiated, 4 h after ABZ administration (peak).
2.3. Surveillance and follow-up of E. multilocularis in captive wildlife 3. Results Fecal samples were regularly taken from all the captive carnivores in the park: one red fox (V. vulpes), three wildcats (Felis s. silvestris), three lynx (Lynx lynx), four genets (Genetta genetta), one pack of seven Alaskan tundra wolves (Canis lupus tundrarum), two packs of seven Eurasian wolves (Canis lupus lupus) each, four brown bears (Ursus arctos) and four raccoons (Procyon lotor). Feces were collected monthly from December 2011 to April 2012, and repeated only for the three packs of wolves from November 2012 to April 2013. An individual sampling was not possible for most of the species due to the large enclosures and the presence of up to seven animals inside. The collect was then realized trying to obtain the best coverage of the population representation in each enclosure every month. Following observation of the first case of E. multilocularis infection in a ring-tailed lemur [9], the other 11 L. catta, four V. rubra and three Eulemur rubriventer underwent abdominal palpation to detect any enlargement of the cranial abdomen. From December 2012 to January 2013, all 11 L. catta had an ultrasound examination by a veterinarian. All the other lemurs were similarly examined in July 2013. A prospective serological survey was implemented from November 2012 to February 2014 for all the lemurs. A retrospective analysis of sera sampled previously, in May 2011 upon the admittance to the park and in February 2012, was also performed to date the occurrence of seroconversion. Blood was collected from the femoral vein, centrifuged and stored at − 20 °C until sent to the laboratory. In accordance with the results, albendazole (ABZ) (Valbazen Dix — Zoetis France) was given orally at a dosage of 10 mg/kg/day, in 2 divided doses, on a piece of banana to
3.1. Prevalence in foxes around and inside the park Of the 97 fox feces collected around the park, the presence of taeniid eggs was observed in 37 samples (38.1%), of which 20 (20.6%, IC95%: 13.1–30.0) were confirmed to contain E. multilocularis. Other taeniid infections were attributed to Taenia polyacantha and Taenia crassiceps. Inside the park, the presence of E. multilocularis was diagnosed in eight (17.8%, IC95%: 8.0–32.1) of the 45 fox feces collected. Two were co-infected with T. polyacantha and in another, taeniid eggs were identified as Hydatigera taeniaeformis. Five foxes were shot in the park during the study. E. multilocularis worms were observed in three of them, with a worm burden evaluated at 45, 110 and 1310.
3.2. Infection in potential definitive hosts in captivity During the first year of surveillance of potentially-infected captive carnivores, 194 feces from red fox (n = 3), wildcats (n = 15), lynx (n = 18), genets (n = 8), Alaskan tundra wolves (n = 35), Eurasian wolves (n = 75), brown bears (n = 20) and raccoons (n = 20) were analyzed. Only one fecal sample from an Eurasian wolf collected in March 2012 contained numerous taeniid eggs. E. multilocularis infection was diagnosed using molecular analysis. Despite the next year's surveillance of all three wolf packs, no other taeniid infection was observed among the 125 feces investigated.
G. Umhang et al. / Parasitology International 65 (2016) 245–250
3.3. Rodents trapped inside the park A total of 227 rodents were trapped on 21 trapping sites. Voles were mainly represented, with 135 M. glareolus and 16 Microtus agrestis. The other species concerned were 32 A. sylvaticus, 29 A. flavicollis and 15 Mus domesticus. Twenty-one voles exhibited parasitic lesions which were investigated by PCR. Seven M. glareolus and one M. agrestis were infected by E. multilocularis leading to a prevalence in voles of 5.3% (IC95%: 2.3– 10.2) and 3.5% (IC95%: 1.5–6.8) taking all rodent species into consideration. Concerning infected rodents, very small lesions were observed except for three M. glareolus. The presence of protoscolex was microscopically confirmed for the two of them (Fig. 1). The other 13 infections were due to Taenia (Versteria) mustelae. Five of the eight rodents infected by E. multilocularis were trapped in the forest area neighboring the nutria and lemurs enclosures. Furthermore, a rat (Rattus norvegicus) found dead in an annexed barn in 2013 also had multiple liver cysts confirmed as E. multilocularis by PCR. 3.4. Investigations of alveolar echinococcosis in lemurs In August 2012, a massive infection by E. multilocularis was observed during the necropsy of a ring-tailed lemur (index case) which drowned in the water surrounding its island enclosure [9]. This first case and an overview of other AE cases in captive primates in the literature led us to investigate all the other lemurs for the presence of the parasite. Ultrasound examination and serological investigations were used to identify other infected lemurs. Serological methods using a non-specific protein A/G enzyme-conjugated binding were chosen due to a lack of available specific labeled anti-lemur antiglobulin. The index AE case and four other live lemurs had positive serology results, with highly positive
Fig. 1. Myodes glareolus trapped inside the park showing fertile alveolar lesions of E. multilocularis in the liver (in the red circle). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
247
ELISA and positive Western blot, including at least three positive bands (7 kDa and 26–28 kDa bands in each case, associated with either 16–18 kDa or 17 kDa bands) (Fig. 2). The ultrasound examination of these four lemurs showed pseudo-tumor and pseudo-cystic lesions typical of AE. Retrospective analysis of sera sampled when these five animals were admitted to the park showed that no animal was seropositive at this date, and that seroconversion occurred during their stay in the park. Another L. catta was positive for an E. granulosus ELISA but only weakly positive with a Western blot (only 26–28 kDa bands). No lesions were detected in these animals using ultrasound. An ABZ treatment was given to the four infected animals and to the fifth serologically positive L. catta. Since the latter passed from positive to negative in both immunological methods after one year of follow-up, the ABZ treatment was stopped for this particular lemur. The serology of infected animals did not show any significant change after one year of treatment. The monitoring of ABZ treatment proved that its absorption in treated lemurs was optimal, as ABZ sulfoxide levels ranged from 3.8 to 13.7 μmol/l. Lemurs presenting an albendazole sulfoxide peak (T4) equal to 13.7 μmol/l showed trough plasma concentrations (T0) before ABZ intake of 1.3 μmol/l. No adverse effects of ABZ were observed (neither hepatotoxicity, nor alopecia). Despite ABZ treatment, a second L. catta diagnosed with AE died in September 2014 after showing a loss of form and marked apathy for one day. Postmortem examination revealed a creamy flocculent abdominal fluid due to the rupture of a liver abscess. More than 75% of the liver
Fig. 2. Western blot of lemur sera using protein A/G binding. Strip 10: positive control serum (human sera from AE patient). Strips 12 and 15: sera from Lemur catta with AE (Echinococcus specific 7 and 26–28 kDa bands, weak E. multilocularis 16 and 18 kDa bands). Strip 18: serum from Lemur catta without AE (negative serology).
248
G. Umhang et al. / Parasitology International 65 (2016) 245–250
was replaced by masses formed by a partially calcified outer wall with a centro-parasitic heterogeneous necrosis (i.e. parasite degeneration). Several abdominal lymph nodes were enlarged and purulent. A histopathological examination confirmed peritonitis and severe chronic granulomatous hepatitis with numerous degenerated protoscoleces. A severe lymphoid hyperplasia was also observed on the lymph nodes and spleen. Four months later, it was decided to euthanize one of the three remaining infected L. catta due to a massive enlargement of the abdomen causing increasingly frequent dyspnea and difficulties in moving (Fig. 3). Necropsy revealed a severe hepatomegaly (liver weight represented more than 40% of the total body weight) with extensive pseudo-tumoral proliferations. White multifocal infiltrations were present in the spleen, on the visceral surface of the diaphragm, on the renal capsule and on the mesentery. Enlargement of lymph nodes with a necrotic center and thick cystic wall was seen in the thorax and abdominal cavity. A diagnostic confirmation of AE was obtained by PCR for both animals and the presence of protoscoleces confirmed by necropsy.
3.5. EmsB microsatellite genotyping Successful EmsB amplifications were obtained for all of the 15 collected E. multilocularis worms from foxes and for metacestode samples from all captive and free-ranging intermediate hosts except one M. glareolus. Of the six different EmsB profiles found, three concern captive intermediate hosts (Fig. 4). Samples from captive animals (the three lemurs and the nutria) systematically clustered with wildlife sampled inside the park (voles and/or foxes). The nutria shared the same EmsB profile as six worms from two foxes. The index lemur case (Lemur_1) has the same EmsB profile as two voles trapped several hundred meters from the lemur island and the nutria's enclosure. The same EmsB profile was identified in samples from the last two lemurs to die (Lemur_2 and
Fig. 4. Dendrogram constructed with EmsB data from captive aberrant hosts in the park (nutria and L. catta) and free-ranging wildlife samples (worms from foxes and voles). The approximately unbiased P values (red numbers on nodes, in percent) were calculated with multiscale bootstrap resampling (B = 1000). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Lemur_3) and also clustered with two voles trapped at the opposite end of the park and eight worms from the three sampled foxes. 4. Discussion
Fig. 3. Massive Echinococcus multilocularis infection in a captive Lemur catta. Metacestode development had deformed the abdomen, causing dyspnea leading to euthanasia. Liver weight represented more than 40% of the total body weight.
A general expansion of the distribution of E. multilocularis across Europe has been observed over the last 15 years [20–23]. During the same period, there is evidence of an increase in the parasite's prevalence in many endemic areas in Germany, France or Poland, for example [10,24,25], resulting in a more contaminated environment. Associated with an increased awareness of the parasite, many infections of aberrant hosts have been reported even among captive animals. In this study, five L. catta and one nutria from a wildlife park in a highly endemic area for E. multilocularis were identified as aberrant hosts, while one Eurasian wolf was also described as a definitive host. The presence of E. multilocularis in fox feces around and inside the park was evaluated at an expected prevalence value considering the infection observed at French departmental level It shows evidence of
G. Umhang et al. / Parasitology International 65 (2016) 245–250
the parasite's presence from surrounding foxes as the observation of infected foxes shot inside the park. Nevertheless the density of foxes and feces remained much lower inside the park, resulting in a much lower level of environmental contamination. While foxes can be infected by predation of rodents outside the park, the very high prevalence of infected rodents inside the park highlights the possibility of roaming foxes to become also infected inside the park. All these elements demonstrate the existence of a sylvatic E. multilocularis lifecycle inside the park. The wild rodent population constitutes a good sentinel of the presence of environmental contamination with E. multilocularis eggs inside the park. Arvicola sherman and Microtus arvalis are the two E. multilocularis intermediate hosts in Europe. These two species were already described in the Moselle department but their presences were not reported here. Concerning M. arvalis, this absence inside the park may be explained by grassland area only constituting the cervid enclosure leading to an intensive trampling. The size and type of the INRA traps excluded the possibility to capture A. sherman, nevertheless no evidence of their presence was ever observed arguing for the absence or presence at very low level of this species inside the park. In the absence of the two latter rodent species, the infection was observed in two species not considered as key species for the transmission of E. multilocularis. The presence of protoscolex was assessed in two M. glareolus suggesting a role of intermediate host in the wild lifecycle of E. multilocularis inside the park, while the potential role of M. agrestis would require more investigations. An important preventive measure would be to try to limit access to the park by roaming foxes. The park is delimited by a 4.5 km wire fence, easy to cross by foxes. Because of the length of the fence and the forest habitat, the installation of a new fence totally preventing the entry of foxes is technically and financially impossible. Preventing the entry of rodents is even more difficult. The most effective tool for significantly lowering the infection pressure due to parasite eggs would be to establish an anthelmintic baiting strategy. This was envisaged, but it is necessary to implement long-term baiting campaigns and to tailor the baiting strategy (principally the frequency of treatment) to the local situation in order to obtain good results [26,27]. Initially this option of control was not retained, but may be an interesting way to reduce the pressure of infection inside the park in the future. Thus, fox shooting needs to be continued both to eliminate invasive foxes and to maintain the “landscape of fear” in order to avoid or at less reduce fox's intrusion [28]. Food sources that attract foxes also need to be controlled. Considering anthropogenic food sources, visitors are only allowed to eat in dedicated areas where garbage is eliminated every day in order to prevent access to foxes during the night. The Eurasian wolf is known to be a possible definitive host for E. multilocularis, and infections have been sporadically reported in Europe [29,30]. This case is the first report in captivity for this species. The infection is due to the predation and ingestion of wild rodents inside the enclosure, located near the forest area where infected rodents were reported. Since the enclosures of other carnivores are also accessible to wild rodents, the potential infection of all these captive animals must be envisaged even if it appears infrequent. Care must be taken to avoid the potential infection of captive definitive hosts. This problem needs to be addressed by implementing preventive measures. It is impossible to limit access to the enclosures by rodents. A monthly individual deworming of captive carnivores wasn't decided due to difficulties in individual administration (for some species and animals, it would require capture or remote injection) and especially as infection appears to be very infrequent. Safety precautions need to be taken when handling the animals during veterinary care or when removing feces from the enclosures during cleaning operations. After the infection reported in a nutria, all the other E. multilocularis infections of captive intermediate hosts were observed in ring-tailed lemurs. Curiously, no infection was observed in the other two species of lemur (E. rubriventer and V. rubra), which arrived in the park in the
249
same week and which share the same environment. While L. catta appears to be particularly susceptible to infection and development of AE, with massive infection and numerous protoscoleces, notably in comparison to humans or gorillas [3], no relevant information is available concerning the distinction between lemur species. Infection of a V. rubra by Echinococcus equinus was reported in the UK but almost all reported cases of infection by E. multilocularis or even other taeniid infections (e.g. Taenia martis or T. crassiceps) concern the ring-tailed lemur, which is also the main lemur species in European zoos [3,4,6, 31–34]. Another possible hypothesis may be that this species spends more time on the ground, even in the wild, thus increasing the risk of contamination. L. catta is the most common lemur species in the park and they were more frequently on the island, which is the only place where they are in contact with the ground. Unlike the nutria, born in the park, all the lemurs were imported from different parks, mainly in France but also from Germany, Poland and Belgium. The parks where the infected lemurs came from are situated in areas considered to be free or with a very low endemic level of E. multilocularis. Moreover, all infected L. catta were seronegative for Echinococcus upon their arrival in the park, but were observed with an Echinococcus seroconversion one year later. Since the three EmsB profiles identified in the lemurs and the nutria were also those found in the wild animals captured inside the park, contamination is considered to be acquired inside the park. In September 2014, a second L. catta died after 22 months of the twice-daily albendazole treatment. More recently, in January 2015, a third lemur was euthanized after showing breathing and locomotion difficulties due to a significantly enlarged abdomen. Except for an enlargement of the cranial abdomen, the other two lemurs did not show any signs of disease. It is difficult to evaluate the real impact of the treatment notably due to the logistical difficulties in performing regular imaging. The proof of optimal ABZ absorption was obtained by high ABZ levels compared to the recommended therapeutic range in humans (1.5 to 1–3 μmol/L 4 h after morning drug intake) (Brunetti, 2010). The infected lemurs did not appear to be disturbed by the treatment with an ABZ liquid suspension, enable such plasma levels to be reached. To prevent the infection of intermediate hosts, feces of roaming foxes in the park may be eliminated when observed by the zoo keepers, but this is fastidious and needs to comply with strict safety conditions. Since lemurs appear to be particularly susceptible to E. multilocularis infection, several specific prevention measures were implemented. It can be assumed that the lemurs were contaminated when the lemurs were introduced to their island enclosure, which was artificially constructed with earth from the park and accessible to roaming foxes before the lemurs' arrival. The absence of any new cases in lemurs supports this hypothesis of previously contaminated ground, but some control measures have been implemented nonetheless. The branches of trees (mostly willows) are no longer directly laid on or dragged along the ground when crossing the park to be brought to the lemurs in order to avoid potential infection from taeniid eggs on the ground. Access to the lemurs' island enclosure has also been surrounded in winter by a wire fence in order to prevent foxes accessing the island when the surrounding water is frozen. Furthermore, the serological and clinical follow-up of lemurs will enable any new cases to be detected early on. The diagnosis of E. multilocularis in the first two animals has required informing all the animal care staff about the lifecycle, species concerned, zoonotic risk and associated preventive measures. The staff was proposed a medical follow-up through an ultrasound examination of the liver every two years. Faced with the increase in alveolar echinococcosis cases in captive animals and their potentially high severity, it would be helpful to undertake diagnostic tests by imaging and/or serological means, particularly for very sensitive captive animals such as lemurs living in endemic areas [4]. In the same way, an effective non-lethal diagnosis based on an ultrasound scan in combination with a laparoscopy feasible under field conditions was recently developed for beavers, in order to prevent
250
G. Umhang et al. / Parasitology International 65 (2016) 245–250
the introduction of E. multilocularis during translocations to nonendemic areas [35]. Parks and zoos in highly endemic areas need to address the presence of E. multilocularis, particularly by implementing relevant control measures to prevent the presence of foxes and food potentially infected sources for potential intermediate hosts. Acknowledgments The authors wish to thank the staff of the wildlife park for being able to complete this study, along with Marie Moinet, Jean-Michel Demerson and Christophe Caillot from ANSES LRFSN for their participation in collecting fox feces, David Leroy for the rodent trapping, Florence Grenouillet from Besançon University Hospital for the immunological analyses, Dr. Alexandra Nicolier from Vet Diagnostics for histological analysis and finally Dr. Oméro Sessini and Dr. Vanessa Alerte for the ultrasound examinations. References [1] J. Eckert, M.A. Gemmell, F.X. Meslin, Z.S. Pawlowski, WHO/OIE Manual on Echinococcosis in Humans and Animals: A Public Health Problem of Global Concern, O.I.E. — O.M.S, Paris, 2001. [2] E. Brunetti, P. Kern, D.A. Vuitton, Expert consensus for the diagnosis and treatment of cystic and alveolar echinococcosis in humans, Acta Trop. 114 (2010) 1–16. [3] H. Kondo, Y. Wada, G. Bando, M. Kosuge, K. Yagi, Y. Oku, Alveolar hydatidosis in a gorilla and a ring-tailed lemur in Japan, J. Vet. Med. Sci. 58 (1996) 447–449. [4] B. Boufana, M.F. Stidworthy, S. Bell, J. Chantrey, N. Masters, S. Unwin, et al., Echinococcus and Taenia spp. from captive mammals in the United Kingdom, Vet. Parasitol. 190 (2012) 95–103. [5] K. Yamanos, H. Kouguchi, K. Uraguchi, T. Mukai, C. Shibata, H. Yamamoto, et al., First detection of Echinococcus multilocularis infection in two species of nonhuman primates raised in a zoo: a fatal case in Cercopithecus diana and a strongly suspected case of spontaneous recovery in Macaca nigra, Parasitol. Int. 63 (2014) 621–626. [6] P. Deplazes, J. Eckert, Veterinary aspects of alveolar echinococcosis—a zoonosis of public health significance, Vet. Parasitol. 98 (2001) 65–87. [7] P. Rehmann, A. Gröne, B. Gottstein, H. Sager, N. Müller, J. Völlm, et al., Alveolar echinococcosis in the Zoological Garden Basle, Schweiz. Arch. Tierheilk. 147 (2005) 498–502. [8] D. Tappe, K. Brehm, M. Frosch, A. Blankenburg, A. Schrod, F.J. Kaup, et al., Echinococcus multilocularis infection of several old world monkey species in a breeding enclosure, Am. J.Trop. Med. Hyg. 77 (2007) 504–506. [9] G. Umhang, J. Lahoreau, A. Nicolier, F. Boue, Echinococcus multilocularis infection of a ring-tailed lemur (Lemur catta) and a nutria (Myocastor coypus) in a French zoo, Parasitol. Int. 62 (2013) 561–563. [10] B. Combes, S. Comte, V. Raton, F. Raoul, F. Boue, G. Umhang, et al., Westward spread of echinococcus multilocularis in foxes, France, 2005–2010, Emerg. Infect. dis. 18 (2012) 2059–2062. [11] J.P. Quere, H. Le Louarn, Les rongeurs de France, Faunistique et Biologie, 3è ed.Editions Quae, Versailles, 2011. [12] A. Mathis, P. Deplazes, J. Eckert, An improved test system for PCR-based specific detection of Echinococcus multilocularis eggs, J. Helminthol. 70 (1996) 219–222. [13] G. Umhang, V. Raton, S. Comte, V. Hormaz, J.-M. Boucher, B. Combes, et al., Echinococcus multilocularis in dogs from two French endemic areas: no evidence of infection but hazardous deworming practices, Vet. Parasitol. 188 (2012) 301–305.
[14] D. Trachsel, P. Deplazes, A. Mathis, Identification of taeniid eggs in the faeces from carnivores based on multiplex PCR using targets in mitochondrial DNA, Parasitology 134 (2007) 911–920. [15] J. Bowles, D. Blair, D.P. McManus, Genetic variants within the genus Echinococcus identified by mitochondrial DNA sequencing, Mol. Biochem. Parasitol. 54 (1992) 165–173. [16] G. Umhang, N. Woronoff-Rhen, B. Combes, F. Boue, Segmental sedimentation and counting technique (SSCT): an adaptable method for qualitative diagnosis of Echinococcus multilocularis in fox intestines, Exp. Parasitol. 128 (2011) 57–60. [17] J. Knapp, J.M. Bart, M.L. Glowatzki, A. Ito, S. Gerard, S. Maillard, et al., Assessment of use of microsatellite polymorphism analysis for improving spatial distribution tracking of Echinococcus multilocularis, J. Clin. Microbiol. 45 (2007) 2943–2950. [18] G. Umhang, J. Knapp, V. Hormaz, F. Raoul, F. Boue, Using the genetics of Echinococcus multilocularis to trace the history of expansion from an endemic area, Infect. Genet. Evol. 22 (2014) 142–149. [19] T. Zeugin, T. Zysset, J. Cotting, Therapeutic monitoring of albendazole: a highperformance liquid chromatography method for determination of its active metabolite albendazole sulfoxide, Ther. Drug Monit. 12 (1990) 187–190. [20] J. Eckert, F.J. Conraths, K. Tackmann, Echinococcosis: an emerging or re-emerging zoonosis? Int. J. Parasitol. 30 (2000) 1283–1294. [21] T. Romig, Echinococcus multilocularis in Europe—state of the art, Vet. Res. Commun. 33 (Suppl. 1) (2009) 31–34. [22] T. Romig, A. Dinkel, U. Mackenstedt, The present situation of echinococcosis in Europe, Parasitol. Int. 55 (Suppl) (2006) S187–S191. [23] B. Gottstein, M. Stojkovic, D.A. Vuitton, L. Millon, A. Marcinkute, P. Deplazes, Threat of Alveolar Echinococcosis to Public Health — A Challenge for Europe, Trends Parasitol, 2015 in press. [24] A. König, T. Romig, D. Thoma, K. Kellermann, Drastic increase in the prevalence of Echinococcus multilocularis in foxes (Vulpes vulpes) in southern Bavaria, Germany, Eur. J. Wildl. Res. 51 (2005) 277–282. [25] J. Karamon, M. Kochanowski, J. Sroka, T. Cencek, M. Rózycki, E. Chmurzyńska, et al., The prevalence of Echinococcus multilocularis in red foxes in Poland — current results (2009–2013), Parasitol. Res. 113 (2014) 317–322. [26] S. Comte, V. Raton, F. Raoul, D. Hegglin, P. Giraudoux, P. Deplazes, et al., Fox baiting against Echinococcus multilocularis: contrasted achievements among two medium size cities, Prev. Vet. Med. 111 (2013) 147–155. [27] D. Hegglin, P. Deplazes, Control of Echinococcus multilocularis: strategies, feasibility and cost—benefit analyses, Int. J. Parasitol. 43 (2013) 327–337. [28] J.W. Laundré, L. Hernández, W.J. Ripple, The landscape of fear: ecological implications of being afraid, Open Ecol. J. 3 (2010) 1–7. [29] K. Martínek, L. Kolářová, E. Hapl, I. Literák, M. Uhrin, Echinococcus multilocularis in European wolves (Canis lupus), Parasitol. Res. 87 (2001) 838–839. [30] G. Bagrade, M. Kirjuina, K. Vismanis, J. Ozoli, Helminth parasites of the wolf Canis lupus from Latvia, J. Helminthol. 83 (2009) 63–68. [31] J.L. Palotay, H. Uno, Hydatid disease in four nonhuman primates, J. Am. Vet. Med. Assoc. 167 (1975) 615–618. [32] M. Luzon, C. de la Fuente-Lopez, E. Martinez-Nevado, J. Fernandez-Moran, F. PonceGordo, Taenia crassiceps cysticercosis in a ring-tailed lemur (Lemur catta), J. Zoo Wildl. Med. 41 (2010) 327–330. [33] B.C. Shock, J.W. Brooks, E. Pokorny, T. Mattive, M.J. Yabsley, Clinical challenge, J. Zoo Wildl. Med. 41 (2010) 374–376. [34] C. De Liberato, F. Berrilli, R. Meoli, K.G. Friedrich, P. Di Cerbo, C. Cocumelli, et al., Fatal infection with Taenia martis metacestodes in a ring-tailed lemur (Lemur catta) living in an Italian zoological garden, Parasitol. Int. 63 (2014) 695–697. [35] R. Campbell-Palmer, J. Del Pozo, B. Gottstein, S. Girling, J. Cracknell, G. Schwab, et al., Echinococcus multilocularis detection in live Eurasian beavers (Castor fiber) using a combination of laparoscopy and abdominal ultrasound under field conditions, PLoS ONE 10 (2015) e0130842.
Contents lists available at ScienceDirect
Parasitology International journal homepage: www.elsevier.com/locate/parint
Surveillance and management of Echinococcus multilocularis in a wildlife park Gérald Umhang a,⁎, Jennifer Lahoreau b, Vanessa Hormaz a, Jean-Marc Boucher a, Amandine Guenon c, Damien Montange d, Frédéric Grenouillet d,e, Franck Boue a a
ANSES LRFSN, Wildlife Surveillance and Eco-epidemiology Unit, National Reference Laboratory for Echinococcus spp., Malzéville, France Parc Animalier de Sainte-Croix, Rhodes, France ONIRIS Ecole Nationale Vétérinaire, Agroalimentaire et de l'Alimentation Nantes-Atlantique, Nantes, France d WHO Collaborating Centre for Prevention and Treatment of Human Echinococcosis and National Reference Center for Human Alveolar Echinococcosis, CHRU University Hospital, Besançon, France e Chrono-Environnement UMR 6249 Research Team, CNRS–University of Bourgogne-Franche-Comté, Besançon, France b c
a r t i c l e
i n f o
Article history: Received 16 September 2015 Received in revised form 28 December 2015 Accepted 3 January 2016 Available online 15 January 2016 Keywords: Echinococcus multilocularis Voles Captive wildlife Lemur catta Albendazole Serology
a b s t r a c t The fox tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a severe zoonotic disease that may be fatal if untreated. A broad spectrum of mammalian species may be accidentally infected even in captivity. In April 2011, liver lesions due to E. multilocularis were observed during the necropsy of a captiveborn nutria (Myocastor coypus) in a French wildlife park, leading to initiation of a study to survey the parasite's presence in the park. A comparable environmental contamination with fox's feces infected by E. multilocularis was reported inside (17.8%) and outside (20.6%) the park. E. multilocularis worms were found in the intestines of three of the five roaming foxes shot in the park. Coprological analyses of potential definitive hosts in captivity (fox, lynx, wildcat, genet, wolf, bear and raccoon) revealed infection in one Eurasian wolf. Voles trapped inside the park also had a high prevalence of 5.3%. After diagnosis of alveolar echinococcosis in a Lemur catta during necropsy, four other cases in L. catta were detected by a combination of ultrasound and serology. These animals were treated twice daily with albendazole. The systematic massive metacestode development and numerous protoscoleces in L. catta confirmed their particular sensitivity to E. multilocularis infection. The autochthonous origin of the infection in all the captive animals infected was genetically confirmed by EmsB microsatellite analysis. Preventive measures were implemented to avoid the presence of roaming foxes, contact with potential definitive hosts and contaminated food sources for potential intermediate hosts. © 2016 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The fox tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis (AE), a severe disease that is potentially fatal if untreated. The larval stage of the parasite develops in intermediate hosts after oral ingestion of microscopic eggs released into the environment in the feces of carnivorous definitive hosts. In Western Europe, the red fox (Vulpes vulpes) is the main definitive host. It harbors E. multilocularis worms in the intestines after predation of infected intermediate hosts—mainly small rodents. Nevertheless, a broad spectrum of mammalian species—notably aquatic rodents, member of the Suidae family or primates—may be accidentally infected, in which case they are considered aberrant hosts. The parasite larva develops almost exclusively in the liver, where its growth is destructive. However, it may also affect other organs by infiltration or metastasis [1]. After developing silently for 10–15 years, human AE may be diagnosed in patients with or without symptoms. ⁎ Corresponding author. E-mail address: [email protected] (G. Umhang).
http://dx.doi.org/10.1016/j.parint.2016.01.008 1383-5769/© 2016 Elsevier Ireland Ltd. All rights reserved.
The latter include the occurrence of cholestatic jaundice, epigastric pain, asthenia, weight loss or hepatomegaly [2]. The development of infection in aberrant hosts seems to be faster in lemurs as well as in dogs and is not comparable with the development in pigs. Many cases of infection by E. multilocularis or other Echinococcus species have been reported in captive animals, and especially primates. Several species of monkey (Gorilla gorilla, Macaca sylvanus, Macaca fascicularis, Macaca nigra, Macaca fuscata, Cercopithecus diana) and lemur (Varecia rubra, Lemur catta) have been infected [3–6]. This is a very real health threat for zoos in areas endemic for E. multilocularis. For example, Basel Zoo (Switzerland) was reported to have grouped cases following the death of five cynomolgus monkeys (M. fascicularis) and a lowland gorilla (Gorilla g. gorilla). Cases were also diagnosed in three macaque species in Germany [7,8]. While such animal cases are undoubtedly of clinical interest, they are also of particular epidemiological importance as they are indicators of environmental contamination and zoonotic risk [6]. In April 2011, liver lesions were observed during the necropsy of a captive-born nutria (Myocastor coypus) in a French wildlife park, later confirmed to be due to E. multilocularis. This diagnosis was followed by the death 16 months later of a ring-tailed lemur (L. catta) massively
246
G. Umhang et al. / Parasitology International 65 (2016) 245–250
infected by the parasite [9]. Following the first nutria case, a study was initiated to survey the parasite's presence in the park. The prevalence of E. multilocularis was evaluated in wildlife around and inside the park as well as in captive animals. The origin of infection in captive intermediate hosts was genetically investigated, while preventive and curative measures were also proposed and discussed. 2. Materials and methods 2.1. Study area The wildlife park is situated in Moselle, a department (French administrative unit) in north-eastern France known to be highly endemic for E. multilocularis, with a prevalence in foxes estimated at 34% (IC95%: 25–44) [10]. The captive animals are essentially European wildlife species living in large natural enclosures. The 120-ha park is located in a rural area, and surrounded by fields and forest. 2.2. The presence of E. multilocularis in free-ranging wildlife In February 2012, experienced staff collected fox feces outside park limits within a four-kilometer radius centered on the park. Some roaming foxes are occasionally observed inside the park despite perimeter fencing. For two years, from February 2012 to January 2014, the veterinarian and animal keepers collected fox feces observed through the park. When possible, roaming foxes were shot and intestines safely collected. In April and May 2012, rodents were captured in 21 trapping sites located in different environments (forest, bank, hedge and grassland) inside the park. Each site was equipped with a 100-m line of 34 INRA live traps spaced 3 m apart on four consecutive nights. The species determination was realized visually from the rodents for Myodes glareolus and Apodemus sylvaticus and Apodemus flavicollis. Concerning other species, the skull was isolated and cleaned by hot water maceration, then determination keys for the dental morphology (especially the first lower molars teeth's for Microtus sp.) were used [11]. All suspect parasitic lesions observed during the rodents' necropsy were sampled and frozen.
lemurs suspected to have alveolar echinococcosis (lesions revealed by the ultrasound scan and positive Echinococcus serology) [2].
2.4. Laboratory analysis After one week of deep-freezing at −80 °C for decontamination, all the feces were subjected to a flotation/sieving technique (adapted from Mathis et al. [12]) and screened microscopically for the presence of taeniid eggs as previously described [13]. In the presence of taeniid eggs, DNA was extracted from the pellets (QIAGEN QIAamp DNA stool). Multiplex PCR was used to identify E. multilocularis and Taenia spp. [14]. Parasitic lesions observed in rodents were diagnosed by PCR using primers JB3-JB4.5 of the cox1 gene [15]. The amplicons obtained were sequenced by a private laboratory (Beckmann Coulter Genomics). The nucleotide sequences were aligned using the Vector NTI software program (Invitrogen) and compared with sequences available in GenBank using the BLASTn program to determine the species involved. The intestines of roaming foxes were analyzed by the Segmental Sedimentation and Counting Technique (SSCT) [16]. Five worms from each positive fox and E. multilocularis-positive samples from intermediate hosts were genotyped by EmsB microsatellite analysis [17,18]. A dendrogram was drawn up using two samples of Echinococcus granulosus sensu stricto (G1) as outgroup. The usual EmsB genetic threshold at 0.08 was used to define assemblage profiles for samples clustered under this limit. The approximately unbiased P values were calculated with multiscale bootstrap resampling (B = 1000). Echinococcus serology was performed for all lemurs using an E. granulosus ELISA (Bordier Affinity) and a commercial E. multilocularis Western blot (LDBio Diagnostic) with only one slight modification, i.e. binding using Protein A/G Alkaline phosphatase conjugate (Pierce™ Thermoscientific). Thus, strips were incubated 1 h with 0.25 μg/ml conjugate (1/5000 dilution) in optimized buffer (Tris 6 g/l, NaCl 6 g/l, NaN3 1 g/l, powdered milk 2.5 g/l, pH adjusted to 7.4 with HCl). Albendazole (ABZ) sulfoxide plasma levels were monitored in all treated animals using the HPLC method [19]. Blood samples were taken one year after ABZ treatment was initiated, 4 h after ABZ administration (peak).
2.3. Surveillance and follow-up of E. multilocularis in captive wildlife 3. Results Fecal samples were regularly taken from all the captive carnivores in the park: one red fox (V. vulpes), three wildcats (Felis s. silvestris), three lynx (Lynx lynx), four genets (Genetta genetta), one pack of seven Alaskan tundra wolves (Canis lupus tundrarum), two packs of seven Eurasian wolves (Canis lupus lupus) each, four brown bears (Ursus arctos) and four raccoons (Procyon lotor). Feces were collected monthly from December 2011 to April 2012, and repeated only for the three packs of wolves from November 2012 to April 2013. An individual sampling was not possible for most of the species due to the large enclosures and the presence of up to seven animals inside. The collect was then realized trying to obtain the best coverage of the population representation in each enclosure every month. Following observation of the first case of E. multilocularis infection in a ring-tailed lemur [9], the other 11 L. catta, four V. rubra and three Eulemur rubriventer underwent abdominal palpation to detect any enlargement of the cranial abdomen. From December 2012 to January 2013, all 11 L. catta had an ultrasound examination by a veterinarian. All the other lemurs were similarly examined in July 2013. A prospective serological survey was implemented from November 2012 to February 2014 for all the lemurs. A retrospective analysis of sera sampled previously, in May 2011 upon the admittance to the park and in February 2012, was also performed to date the occurrence of seroconversion. Blood was collected from the femoral vein, centrifuged and stored at − 20 °C until sent to the laboratory. In accordance with the results, albendazole (ABZ) (Valbazen Dix — Zoetis France) was given orally at a dosage of 10 mg/kg/day, in 2 divided doses, on a piece of banana to
3.1. Prevalence in foxes around and inside the park Of the 97 fox feces collected around the park, the presence of taeniid eggs was observed in 37 samples (38.1%), of which 20 (20.6%, IC95%: 13.1–30.0) were confirmed to contain E. multilocularis. Other taeniid infections were attributed to Taenia polyacantha and Taenia crassiceps. Inside the park, the presence of E. multilocularis was diagnosed in eight (17.8%, IC95%: 8.0–32.1) of the 45 fox feces collected. Two were co-infected with T. polyacantha and in another, taeniid eggs were identified as Hydatigera taeniaeformis. Five foxes were shot in the park during the study. E. multilocularis worms were observed in three of them, with a worm burden evaluated at 45, 110 and 1310.
3.2. Infection in potential definitive hosts in captivity During the first year of surveillance of potentially-infected captive carnivores, 194 feces from red fox (n = 3), wildcats (n = 15), lynx (n = 18), genets (n = 8), Alaskan tundra wolves (n = 35), Eurasian wolves (n = 75), brown bears (n = 20) and raccoons (n = 20) were analyzed. Only one fecal sample from an Eurasian wolf collected in March 2012 contained numerous taeniid eggs. E. multilocularis infection was diagnosed using molecular analysis. Despite the next year's surveillance of all three wolf packs, no other taeniid infection was observed among the 125 feces investigated.
G. Umhang et al. / Parasitology International 65 (2016) 245–250
3.3. Rodents trapped inside the park A total of 227 rodents were trapped on 21 trapping sites. Voles were mainly represented, with 135 M. glareolus and 16 Microtus agrestis. The other species concerned were 32 A. sylvaticus, 29 A. flavicollis and 15 Mus domesticus. Twenty-one voles exhibited parasitic lesions which were investigated by PCR. Seven M. glareolus and one M. agrestis were infected by E. multilocularis leading to a prevalence in voles of 5.3% (IC95%: 2.3– 10.2) and 3.5% (IC95%: 1.5–6.8) taking all rodent species into consideration. Concerning infected rodents, very small lesions were observed except for three M. glareolus. The presence of protoscolex was microscopically confirmed for the two of them (Fig. 1). The other 13 infections were due to Taenia (Versteria) mustelae. Five of the eight rodents infected by E. multilocularis were trapped in the forest area neighboring the nutria and lemurs enclosures. Furthermore, a rat (Rattus norvegicus) found dead in an annexed barn in 2013 also had multiple liver cysts confirmed as E. multilocularis by PCR. 3.4. Investigations of alveolar echinococcosis in lemurs In August 2012, a massive infection by E. multilocularis was observed during the necropsy of a ring-tailed lemur (index case) which drowned in the water surrounding its island enclosure [9]. This first case and an overview of other AE cases in captive primates in the literature led us to investigate all the other lemurs for the presence of the parasite. Ultrasound examination and serological investigations were used to identify other infected lemurs. Serological methods using a non-specific protein A/G enzyme-conjugated binding were chosen due to a lack of available specific labeled anti-lemur antiglobulin. The index AE case and four other live lemurs had positive serology results, with highly positive
Fig. 1. Myodes glareolus trapped inside the park showing fertile alveolar lesions of E. multilocularis in the liver (in the red circle). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
247
ELISA and positive Western blot, including at least three positive bands (7 kDa and 26–28 kDa bands in each case, associated with either 16–18 kDa or 17 kDa bands) (Fig. 2). The ultrasound examination of these four lemurs showed pseudo-tumor and pseudo-cystic lesions typical of AE. Retrospective analysis of sera sampled when these five animals were admitted to the park showed that no animal was seropositive at this date, and that seroconversion occurred during their stay in the park. Another L. catta was positive for an E. granulosus ELISA but only weakly positive with a Western blot (only 26–28 kDa bands). No lesions were detected in these animals using ultrasound. An ABZ treatment was given to the four infected animals and to the fifth serologically positive L. catta. Since the latter passed from positive to negative in both immunological methods after one year of follow-up, the ABZ treatment was stopped for this particular lemur. The serology of infected animals did not show any significant change after one year of treatment. The monitoring of ABZ treatment proved that its absorption in treated lemurs was optimal, as ABZ sulfoxide levels ranged from 3.8 to 13.7 μmol/l. Lemurs presenting an albendazole sulfoxide peak (T4) equal to 13.7 μmol/l showed trough plasma concentrations (T0) before ABZ intake of 1.3 μmol/l. No adverse effects of ABZ were observed (neither hepatotoxicity, nor alopecia). Despite ABZ treatment, a second L. catta diagnosed with AE died in September 2014 after showing a loss of form and marked apathy for one day. Postmortem examination revealed a creamy flocculent abdominal fluid due to the rupture of a liver abscess. More than 75% of the liver
Fig. 2. Western blot of lemur sera using protein A/G binding. Strip 10: positive control serum (human sera from AE patient). Strips 12 and 15: sera from Lemur catta with AE (Echinococcus specific 7 and 26–28 kDa bands, weak E. multilocularis 16 and 18 kDa bands). Strip 18: serum from Lemur catta without AE (negative serology).
248
G. Umhang et al. / Parasitology International 65 (2016) 245–250
was replaced by masses formed by a partially calcified outer wall with a centro-parasitic heterogeneous necrosis (i.e. parasite degeneration). Several abdominal lymph nodes were enlarged and purulent. A histopathological examination confirmed peritonitis and severe chronic granulomatous hepatitis with numerous degenerated protoscoleces. A severe lymphoid hyperplasia was also observed on the lymph nodes and spleen. Four months later, it was decided to euthanize one of the three remaining infected L. catta due to a massive enlargement of the abdomen causing increasingly frequent dyspnea and difficulties in moving (Fig. 3). Necropsy revealed a severe hepatomegaly (liver weight represented more than 40% of the total body weight) with extensive pseudo-tumoral proliferations. White multifocal infiltrations were present in the spleen, on the visceral surface of the diaphragm, on the renal capsule and on the mesentery. Enlargement of lymph nodes with a necrotic center and thick cystic wall was seen in the thorax and abdominal cavity. A diagnostic confirmation of AE was obtained by PCR for both animals and the presence of protoscoleces confirmed by necropsy.
3.5. EmsB microsatellite genotyping Successful EmsB amplifications were obtained for all of the 15 collected E. multilocularis worms from foxes and for metacestode samples from all captive and free-ranging intermediate hosts except one M. glareolus. Of the six different EmsB profiles found, three concern captive intermediate hosts (Fig. 4). Samples from captive animals (the three lemurs and the nutria) systematically clustered with wildlife sampled inside the park (voles and/or foxes). The nutria shared the same EmsB profile as six worms from two foxes. The index lemur case (Lemur_1) has the same EmsB profile as two voles trapped several hundred meters from the lemur island and the nutria's enclosure. The same EmsB profile was identified in samples from the last two lemurs to die (Lemur_2 and
Fig. 4. Dendrogram constructed with EmsB data from captive aberrant hosts in the park (nutria and L. catta) and free-ranging wildlife samples (worms from foxes and voles). The approximately unbiased P values (red numbers on nodes, in percent) were calculated with multiscale bootstrap resampling (B = 1000). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Lemur_3) and also clustered with two voles trapped at the opposite end of the park and eight worms from the three sampled foxes. 4. Discussion
Fig. 3. Massive Echinococcus multilocularis infection in a captive Lemur catta. Metacestode development had deformed the abdomen, causing dyspnea leading to euthanasia. Liver weight represented more than 40% of the total body weight.
A general expansion of the distribution of E. multilocularis across Europe has been observed over the last 15 years [20–23]. During the same period, there is evidence of an increase in the parasite's prevalence in many endemic areas in Germany, France or Poland, for example [10,24,25], resulting in a more contaminated environment. Associated with an increased awareness of the parasite, many infections of aberrant hosts have been reported even among captive animals. In this study, five L. catta and one nutria from a wildlife park in a highly endemic area for E. multilocularis were identified as aberrant hosts, while one Eurasian wolf was also described as a definitive host. The presence of E. multilocularis in fox feces around and inside the park was evaluated at an expected prevalence value considering the infection observed at French departmental level It shows evidence of
G. Umhang et al. / Parasitology International 65 (2016) 245–250
the parasite's presence from surrounding foxes as the observation of infected foxes shot inside the park. Nevertheless the density of foxes and feces remained much lower inside the park, resulting in a much lower level of environmental contamination. While foxes can be infected by predation of rodents outside the park, the very high prevalence of infected rodents inside the park highlights the possibility of roaming foxes to become also infected inside the park. All these elements demonstrate the existence of a sylvatic E. multilocularis lifecycle inside the park. The wild rodent population constitutes a good sentinel of the presence of environmental contamination with E. multilocularis eggs inside the park. Arvicola sherman and Microtus arvalis are the two E. multilocularis intermediate hosts in Europe. These two species were already described in the Moselle department but their presences were not reported here. Concerning M. arvalis, this absence inside the park may be explained by grassland area only constituting the cervid enclosure leading to an intensive trampling. The size and type of the INRA traps excluded the possibility to capture A. sherman, nevertheless no evidence of their presence was ever observed arguing for the absence or presence at very low level of this species inside the park. In the absence of the two latter rodent species, the infection was observed in two species not considered as key species for the transmission of E. multilocularis. The presence of protoscolex was assessed in two M. glareolus suggesting a role of intermediate host in the wild lifecycle of E. multilocularis inside the park, while the potential role of M. agrestis would require more investigations. An important preventive measure would be to try to limit access to the park by roaming foxes. The park is delimited by a 4.5 km wire fence, easy to cross by foxes. Because of the length of the fence and the forest habitat, the installation of a new fence totally preventing the entry of foxes is technically and financially impossible. Preventing the entry of rodents is even more difficult. The most effective tool for significantly lowering the infection pressure due to parasite eggs would be to establish an anthelmintic baiting strategy. This was envisaged, but it is necessary to implement long-term baiting campaigns and to tailor the baiting strategy (principally the frequency of treatment) to the local situation in order to obtain good results [26,27]. Initially this option of control was not retained, but may be an interesting way to reduce the pressure of infection inside the park in the future. Thus, fox shooting needs to be continued both to eliminate invasive foxes and to maintain the “landscape of fear” in order to avoid or at less reduce fox's intrusion [28]. Food sources that attract foxes also need to be controlled. Considering anthropogenic food sources, visitors are only allowed to eat in dedicated areas where garbage is eliminated every day in order to prevent access to foxes during the night. The Eurasian wolf is known to be a possible definitive host for E. multilocularis, and infections have been sporadically reported in Europe [29,30]. This case is the first report in captivity for this species. The infection is due to the predation and ingestion of wild rodents inside the enclosure, located near the forest area where infected rodents were reported. Since the enclosures of other carnivores are also accessible to wild rodents, the potential infection of all these captive animals must be envisaged even if it appears infrequent. Care must be taken to avoid the potential infection of captive definitive hosts. This problem needs to be addressed by implementing preventive measures. It is impossible to limit access to the enclosures by rodents. A monthly individual deworming of captive carnivores wasn't decided due to difficulties in individual administration (for some species and animals, it would require capture or remote injection) and especially as infection appears to be very infrequent. Safety precautions need to be taken when handling the animals during veterinary care or when removing feces from the enclosures during cleaning operations. After the infection reported in a nutria, all the other E. multilocularis infections of captive intermediate hosts were observed in ring-tailed lemurs. Curiously, no infection was observed in the other two species of lemur (E. rubriventer and V. rubra), which arrived in the park in the
249
same week and which share the same environment. While L. catta appears to be particularly susceptible to infection and development of AE, with massive infection and numerous protoscoleces, notably in comparison to humans or gorillas [3], no relevant information is available concerning the distinction between lemur species. Infection of a V. rubra by Echinococcus equinus was reported in the UK but almost all reported cases of infection by E. multilocularis or even other taeniid infections (e.g. Taenia martis or T. crassiceps) concern the ring-tailed lemur, which is also the main lemur species in European zoos [3,4,6, 31–34]. Another possible hypothesis may be that this species spends more time on the ground, even in the wild, thus increasing the risk of contamination. L. catta is the most common lemur species in the park and they were more frequently on the island, which is the only place where they are in contact with the ground. Unlike the nutria, born in the park, all the lemurs were imported from different parks, mainly in France but also from Germany, Poland and Belgium. The parks where the infected lemurs came from are situated in areas considered to be free or with a very low endemic level of E. multilocularis. Moreover, all infected L. catta were seronegative for Echinococcus upon their arrival in the park, but were observed with an Echinococcus seroconversion one year later. Since the three EmsB profiles identified in the lemurs and the nutria were also those found in the wild animals captured inside the park, contamination is considered to be acquired inside the park. In September 2014, a second L. catta died after 22 months of the twice-daily albendazole treatment. More recently, in January 2015, a third lemur was euthanized after showing breathing and locomotion difficulties due to a significantly enlarged abdomen. Except for an enlargement of the cranial abdomen, the other two lemurs did not show any signs of disease. It is difficult to evaluate the real impact of the treatment notably due to the logistical difficulties in performing regular imaging. The proof of optimal ABZ absorption was obtained by high ABZ levels compared to the recommended therapeutic range in humans (1.5 to 1–3 μmol/L 4 h after morning drug intake) (Brunetti, 2010). The infected lemurs did not appear to be disturbed by the treatment with an ABZ liquid suspension, enable such plasma levels to be reached. To prevent the infection of intermediate hosts, feces of roaming foxes in the park may be eliminated when observed by the zoo keepers, but this is fastidious and needs to comply with strict safety conditions. Since lemurs appear to be particularly susceptible to E. multilocularis infection, several specific prevention measures were implemented. It can be assumed that the lemurs were contaminated when the lemurs were introduced to their island enclosure, which was artificially constructed with earth from the park and accessible to roaming foxes before the lemurs' arrival. The absence of any new cases in lemurs supports this hypothesis of previously contaminated ground, but some control measures have been implemented nonetheless. The branches of trees (mostly willows) are no longer directly laid on or dragged along the ground when crossing the park to be brought to the lemurs in order to avoid potential infection from taeniid eggs on the ground. Access to the lemurs' island enclosure has also been surrounded in winter by a wire fence in order to prevent foxes accessing the island when the surrounding water is frozen. Furthermore, the serological and clinical follow-up of lemurs will enable any new cases to be detected early on. The diagnosis of E. multilocularis in the first two animals has required informing all the animal care staff about the lifecycle, species concerned, zoonotic risk and associated preventive measures. The staff was proposed a medical follow-up through an ultrasound examination of the liver every two years. Faced with the increase in alveolar echinococcosis cases in captive animals and their potentially high severity, it would be helpful to undertake diagnostic tests by imaging and/or serological means, particularly for very sensitive captive animals such as lemurs living in endemic areas [4]. In the same way, an effective non-lethal diagnosis based on an ultrasound scan in combination with a laparoscopy feasible under field conditions was recently developed for beavers, in order to prevent
250
G. Umhang et al. / Parasitology International 65 (2016) 245–250
the introduction of E. multilocularis during translocations to nonendemic areas [35]. Parks and zoos in highly endemic areas need to address the presence of E. multilocularis, particularly by implementing relevant control measures to prevent the presence of foxes and food potentially infected sources for potential intermediate hosts. Acknowledgments The authors wish to thank the staff of the wildlife park for being able to complete this study, along with Marie Moinet, Jean-Michel Demerson and Christophe Caillot from ANSES LRFSN for their participation in collecting fox feces, David Leroy for the rodent trapping, Florence Grenouillet from Besançon University Hospital for the immunological analyses, Dr. Alexandra Nicolier from Vet Diagnostics for histological analysis and finally Dr. Oméro Sessini and Dr. Vanessa Alerte for the ultrasound examinations. References [1] J. Eckert, M.A. Gemmell, F.X. Meslin, Z.S. Pawlowski, WHO/OIE Manual on Echinococcosis in Humans and Animals: A Public Health Problem of Global Concern, O.I.E. — O.M.S, Paris, 2001. [2] E. Brunetti, P. Kern, D.A. Vuitton, Expert consensus for the diagnosis and treatment of cystic and alveolar echinococcosis in humans, Acta Trop. 114 (2010) 1–16. [3] H. Kondo, Y. Wada, G. Bando, M. Kosuge, K. Yagi, Y. Oku, Alveolar hydatidosis in a gorilla and a ring-tailed lemur in Japan, J. Vet. Med. Sci. 58 (1996) 447–449. [4] B. Boufana, M.F. Stidworthy, S. Bell, J. Chantrey, N. Masters, S. Unwin, et al., Echinococcus and Taenia spp. from captive mammals in the United Kingdom, Vet. Parasitol. 190 (2012) 95–103. [5] K. Yamanos, H. Kouguchi, K. Uraguchi, T. Mukai, C. Shibata, H. Yamamoto, et al., First detection of Echinococcus multilocularis infection in two species of nonhuman primates raised in a zoo: a fatal case in Cercopithecus diana and a strongly suspected case of spontaneous recovery in Macaca nigra, Parasitol. Int. 63 (2014) 621–626. [6] P. Deplazes, J. Eckert, Veterinary aspects of alveolar echinococcosis—a zoonosis of public health significance, Vet. Parasitol. 98 (2001) 65–87. [7] P. Rehmann, A. Gröne, B. Gottstein, H. Sager, N. Müller, J. Völlm, et al., Alveolar echinococcosis in the Zoological Garden Basle, Schweiz. Arch. Tierheilk. 147 (2005) 498–502. [8] D. Tappe, K. Brehm, M. Frosch, A. Blankenburg, A. Schrod, F.J. Kaup, et al., Echinococcus multilocularis infection of several old world monkey species in a breeding enclosure, Am. J.Trop. Med. Hyg. 77 (2007) 504–506. [9] G. Umhang, J. Lahoreau, A. Nicolier, F. Boue, Echinococcus multilocularis infection of a ring-tailed lemur (Lemur catta) and a nutria (Myocastor coypus) in a French zoo, Parasitol. Int. 62 (2013) 561–563. [10] B. Combes, S. Comte, V. Raton, F. Raoul, F. Boue, G. Umhang, et al., Westward spread of echinococcus multilocularis in foxes, France, 2005–2010, Emerg. Infect. dis. 18 (2012) 2059–2062. [11] J.P. Quere, H. Le Louarn, Les rongeurs de France, Faunistique et Biologie, 3è ed.Editions Quae, Versailles, 2011. [12] A. Mathis, P. Deplazes, J. Eckert, An improved test system for PCR-based specific detection of Echinococcus multilocularis eggs, J. Helminthol. 70 (1996) 219–222. [13] G. Umhang, V. Raton, S. Comte, V. Hormaz, J.-M. Boucher, B. Combes, et al., Echinococcus multilocularis in dogs from two French endemic areas: no evidence of infection but hazardous deworming practices, Vet. Parasitol. 188 (2012) 301–305.
[14] D. Trachsel, P. Deplazes, A. Mathis, Identification of taeniid eggs in the faeces from carnivores based on multiplex PCR using targets in mitochondrial DNA, Parasitology 134 (2007) 911–920. [15] J. Bowles, D. Blair, D.P. McManus, Genetic variants within the genus Echinococcus identified by mitochondrial DNA sequencing, Mol. Biochem. Parasitol. 54 (1992) 165–173. [16] G. Umhang, N. Woronoff-Rhen, B. Combes, F. Boue, Segmental sedimentation and counting technique (SSCT): an adaptable method for qualitative diagnosis of Echinococcus multilocularis in fox intestines, Exp. Parasitol. 128 (2011) 57–60. [17] J. Knapp, J.M. Bart, M.L. Glowatzki, A. Ito, S. Gerard, S. Maillard, et al., Assessment of use of microsatellite polymorphism analysis for improving spatial distribution tracking of Echinococcus multilocularis, J. Clin. Microbiol. 45 (2007) 2943–2950. [18] G. Umhang, J. Knapp, V. Hormaz, F. Raoul, F. Boue, Using the genetics of Echinococcus multilocularis to trace the history of expansion from an endemic area, Infect. Genet. Evol. 22 (2014) 142–149. [19] T. Zeugin, T. Zysset, J. Cotting, Therapeutic monitoring of albendazole: a highperformance liquid chromatography method for determination of its active metabolite albendazole sulfoxide, Ther. Drug Monit. 12 (1990) 187–190. [20] J. Eckert, F.J. Conraths, K. Tackmann, Echinococcosis: an emerging or re-emerging zoonosis? Int. J. Parasitol. 30 (2000) 1283–1294. [21] T. Romig, Echinococcus multilocularis in Europe—state of the art, Vet. Res. Commun. 33 (Suppl. 1) (2009) 31–34. [22] T. Romig, A. Dinkel, U. Mackenstedt, The present situation of echinococcosis in Europe, Parasitol. Int. 55 (Suppl) (2006) S187–S191. [23] B. Gottstein, M. Stojkovic, D.A. Vuitton, L. Millon, A. Marcinkute, P. Deplazes, Threat of Alveolar Echinococcosis to Public Health — A Challenge for Europe, Trends Parasitol, 2015 in press. [24] A. König, T. Romig, D. Thoma, K. Kellermann, Drastic increase in the prevalence of Echinococcus multilocularis in foxes (Vulpes vulpes) in southern Bavaria, Germany, Eur. J. Wildl. Res. 51 (2005) 277–282. [25] J. Karamon, M. Kochanowski, J. Sroka, T. Cencek, M. Rózycki, E. Chmurzyńska, et al., The prevalence of Echinococcus multilocularis in red foxes in Poland — current results (2009–2013), Parasitol. Res. 113 (2014) 317–322. [26] S. Comte, V. Raton, F. Raoul, D. Hegglin, P. Giraudoux, P. Deplazes, et al., Fox baiting against Echinococcus multilocularis: contrasted achievements among two medium size cities, Prev. Vet. Med. 111 (2013) 147–155. [27] D. Hegglin, P. Deplazes, Control of Echinococcus multilocularis: strategies, feasibility and cost—benefit analyses, Int. J. Parasitol. 43 (2013) 327–337. [28] J.W. Laundré, L. Hernández, W.J. Ripple, The landscape of fear: ecological implications of being afraid, Open Ecol. J. 3 (2010) 1–7. [29] K. Martínek, L. Kolářová, E. Hapl, I. Literák, M. Uhrin, Echinococcus multilocularis in European wolves (Canis lupus), Parasitol. Res. 87 (2001) 838–839. [30] G. Bagrade, M. Kirjuina, K. Vismanis, J. Ozoli, Helminth parasites of the wolf Canis lupus from Latvia, J. Helminthol. 83 (2009) 63–68. [31] J.L. Palotay, H. Uno, Hydatid disease in four nonhuman primates, J. Am. Vet. Med. Assoc. 167 (1975) 615–618. [32] M. Luzon, C. de la Fuente-Lopez, E. Martinez-Nevado, J. Fernandez-Moran, F. PonceGordo, Taenia crassiceps cysticercosis in a ring-tailed lemur (Lemur catta), J. Zoo Wildl. Med. 41 (2010) 327–330. [33] B.C. Shock, J.W. Brooks, E. Pokorny, T. Mattive, M.J. Yabsley, Clinical challenge, J. Zoo Wildl. Med. 41 (2010) 374–376. [34] C. De Liberato, F. Berrilli, R. Meoli, K.G. Friedrich, P. Di Cerbo, C. Cocumelli, et al., Fatal infection with Taenia martis metacestodes in a ring-tailed lemur (Lemur catta) living in an Italian zoological garden, Parasitol. Int. 63 (2014) 695–697. [35] R. Campbell-Palmer, J. Del Pozo, B. Gottstein, S. Girling, J. Cracknell, G. Schwab, et al., Echinococcus multilocularis detection in live Eurasian beavers (Castor fiber) using a combination of laparoscopy and abdominal ultrasound under field conditions, PLoS ONE 10 (2015) e0130842.