Cystic echinococcosis (CE) by Echinococcosis granulosus (Eg) infection was seen in a 13 years old male lemur, found dead in a zoo in Northern Italy. Necropsy revealed several transparent cysts in the lungs and in the abdominal cavity. Freefloating cysts of varying sizes were found in the peritoneal cavity, and no protoscolex was seen microscopically. Histologically, a multifocal severe parasitic granulomatous pneumonia was observed. Confirmation of E. granulosus "sensu stricto" was reached by PCR and sequencing. In view of the absence of definitive host in the zoo, located in non-endemic region for CE, it is speculated that infection introduced through translocation of lemur from endemic region (Southern Italy zoo)

Bettini, G.

Galliani, M.

Galuppi, R.

Morandi, B.

Poglayen, G

Varcasia, A.

English

POGLAYEN, GIOVANNI

BETTINI, GIULIANO

MORANDI, BENEDETTO

GALUPPI, ROBERTA

Archivio istituzionale della ricerca - Alma Mater Studiorum Università di Bologna

121   Pakistan Veterinary Journal ISSN: 0253-8318 (PRINT), 2074-7764 (ONLINE) Accessible at: www.pvj.com.pk    Echinococcus granulosus “Sensu stricto” in a Captive Ring-Tailed Lemur (Lemur catta) in Northern Italy  G Poglayen1*, A Varcasia2, G Bettini1, B Morandi1, R Galuppi1 and M Galliani3  1Dipartimento di Scienze Mediche Veterinarie – Alma Mater Studiorum, Università di Bologna, Italy; 2Dipartimento di Medicina Veterinaria – Università di Sassari, Italy; 3Veterinary of Safari adventure “Le dune del Delta”, Ravenna, Italy *Corresponding author: giovanni.poglayen@unibo.it   ARTICLE HISTORY (15-089)    A B S T R A C T   Received: Revised: Accepted: Online available: February 21, 2015 July 15, 2015 July 29, 2015 October 07, 2016  Cystic echinococcosis (CE) by Echinococcosis granulosus (Eg) infection was seen in a 13 years old male lemur, found dead in a zoo in Northern Italy. Necropsy revealed several transparent cysts in the lungs and in the abdominal cavity. Free-floating cysts of varying sizes were found in the peritoneal cavity, and no protoscolex was seen microscopically. Histologically, a multifocal severe parasitic granulomatous pneumonia was observed. Confirmation of E. granulosus “sensu stricto” was reached by PCR and sequencing. In view of the absence of definitive host in the zoo, located in non-endemic region for CE, it is speculated that infection introduced through translocation of lemur from endemic region (Southern Italy zoo).  ©2015 PVJ. All rights reserved Key words:  Captive animals Echinococcus granulosus Lemur catta Ring-tailed lemur Zoo   To Cite This Article: Poglayen G, Varcasia A, Bettini G, Morandi B, Galuppi R and Galliani M, 2016. Echinococcus Granulosus “sensu stricto” in a Captive Ring-Tailed Lemur (Lemur catta) in Northern Italy. Pak Vet J, 36(1): 121-123.  INTRODUCTION  Echinococcus spp., are tiny flatworms belonging to the family Taeniidae and widespread all over the world. Its classification and taxonomy has been very obscuring and controversial and as far as could be ascertained, other than the ‘lion strain’10 distinct genotypes (G1-10) of E. granulosus have been reported on the basis of mitochondrial DNA sequences (Lavikainen et al., 2003). Currently, based on several morphological, biological and biomolecular criteria, 9 species are ascribed to the genus Echinococcus: E. granulosus “sensu stricto” (s.s.) (G1-G3), E. multilocularis, E. vogeli, E. oligarthrus, E. shiquicus, E. equinus (G4), E. ortleppi (G5), E. felidis and E. canadensis (G6-G10) (Nakao et al., 2007). The life cycle of E. granulosus is indirect and involves as definitive hosts domestic and wild canids, and several species of domestic and wild mammals (humans included) as intermediate hosts for the tissue-invading metacestode stage, the hydatid cyst.  Cystic echinococcosis (CE), represents a public health problem and plays an important socio-economic role in many areas of the world and in particular in the Mediterranean Region (MR) where it is currently considered among the five most frequently diagnosed zoonosis, along with brucellosis, rabies, leishmaniasis and food-borne zoonotic infection (Sadjjadi, 2006). Cystic echinococcosis is endemic in different regions of Italy. Prevalence in livestock is high in Sicily and Sardinia, medium-high in the central and southern regions, and negligible in the remaining areas (Varcasia et al. 2011). The reports of CE in wild intermediate hosts are very rare. Our report is referred to the metacestodosis in a captive Ring-tailed lemur (Lemur catta). This non-human, arboreal primate is endemic to islands of Madagascar. It is an omnivorous, terrestrial and diurnal animal. The Ring-tailed lemur is highly social, living in groups of up to 30 individuals and it is an endangered species according to the IUCN Red List of Threatened SpeciesTM (Andriaholinirina et al., 2014).  Case history and clinical examination: A 13 years old male lemur, host of a colony in a zoo of the province of Ravenna (Northern Italy: 44°19’35.5’’N; 12°16’29.9’’E) was found dead. The colony originated from animals previously imported from another zoo in Southern Italy and was composed of 12 animals. The diet was based on vegetables and fruits industrially produced in the area and tap water. During the necropsy of the dead animal, innumerable transparent cysts were observed in the lungs and in the abdominal cavity. The cysts were of different size from 4 cm to few mm, many were free floating in the peritoneal cavity (Fig.1). The microscopic exam of cystic fluid revealed  non-fertile   cysts  (without   any  protoscolices).  CASE REPORT Pak Vet J, 2016, 36(1): 121-123.  122  Fig. 1: Cysts of different size found during necropsy.    Fig. 2: Crumpled fragments of the parasitic membrane surrounded by severe granulomatous reaction, with multinucleated giant cells, palisading epitheliod cells, macrophages and lymphocytes. Lung, HE, bar=100µm.  Histologically, the lung cysts were outlined by a 100-200µm thick lamellar hyaline layer, and were internally lined by a thin germinal layer, but did not contain any protoscolices. Externally, the cysts were surrounded by an infiltrate of mixed inflammatory cells, including multinucleated giant cells, palisading epitheliod cells, macrophages and lymphocytes. Multifocally, there were crumpled fragments of the outer lamellar membrane enclosed by a particularly severe inflammatory infiltrate. The lung tissue showed also scattered peribronchial lymphocytic nodules (Fig. 2). Molecular identification was carried out by PCR by amplifying fragments within 2 mitochondrial gene cytochrome C oxidase 1 (cox1) and NADH dehydro-genase 1 (ND1) using DNA extracted from the cysts.  PCR products were purified using the High Pure PCR product purification kit  (Roche Diagnostics, Mannheim, Germany) and commercially sequenced by MWG-Biotech (Ebersberg, Germany) using the PCR primers. Nucleotide sequences were compared to those available in GenBank® through the use of the basic local alignment search tool (BLAST) (http://www.ncbi.nlm.nih. gov/BLAST/).  Molecular analyses of cox 1 and ND1 mitochondrial genes respectively shown an homology of 100% with Echinococcus granulosus s.s. sequences deposited in GenBank (accession numbers: GQ502212.1;  GQ502217.1) revealing an involvement of  E. granulosus s.s. as cause of CE in lemur examined in the present survey. The Cox1 sequence was deposited in GenBank archive, accession number KT282119. DISCUSSION  This, to our knowledge, is the first report of E. granulosus “sensu stricto” in a Ring-tailed lemur. Previously, case of hydatidosis in a captive Lemur catta has been reported from Zoological Center of Tel-Aviv (Shahar et al., 1995), however, organism was not genotyped. Contrary to our finding, cysts observed in this case were, fertile, that may be ascribed to involvement of different strain. Other reports in the literature of echinococcosis in Lemur catta have been referred caused only by E. multilocularis (Palotay and Uno, 1975; Kondo et al., 1996; Deplazes and Eckert, 2001, Umhang et al., 2013). The last three reports were from captive animals in zoo located in endemic zone of Japan, Switzerland and France, respectively, and as infectious route, even if not clear, was suspected eggs-contaminated food or free-roaming foxes. The two cases described by Palotay and Uno (1975) were from a zoo in Oregon (USA) and was supposed that this primates were infected in the wild.  Moreover, E. granulosus (G1) and E. equinus (formally G4), have been documented in other genus of lemur, Cercopithecus ascanius and Varecia rubra, respectively (Boufana et al., 2012).  In the latter animal, the presence of free-floating hydatids was also described similarly in our case. These primates were from a zoo of UK, where the E. granulosus (G1) and E. equinus are known to be endemic however the source of infection was uncertain. In the present report, no suitable definitive hosts were present in the zoo and the lemurs lived in a status of permanent isolation form other animals. The food-water-borne infection (i.e. with contaminated vegetables/water) was excluded as the lemur were feed with vegetables and fruits produced industrially in the same area and tap water. The zoo is located in an area where CE has very low prevalence, so the most probable way of infection could be ascribed to the fact that infected lemur belonged to a colony that was previously hosted in another zoo in southern Italy, where CE is hyperendemic. All these consideration allow to reassure staff and visitors of the zoo in respect of this important zoonosis agent but E. granulosus s.s. must be taken into account in exotic captive animals, for the possibility to administer to their contaminated food/water from endemic areas.   Author’s contribution: GP, BM and RG collected sample, analyzed the data, conceived and designed the paper, AV performed molecular identification; GB performed histological analysis; MG performed necropsy. All authors interpreted the data and approved the final version   Acknowledgement: Financial support was provided by research contract DSMVet Rep n. 35/2013 with Zoosafari “Le Dune del Delta“.  REFERENCES  Andriaholinirina N, Baden A, Blanco M, Chikhi L, Cooke A et al., 2014. Lemur catta. The IUCN Red List of Threatened Species. Version 2015.2. <www.iucnredlist.org>. Downloaded on 10 July 2015. Boufana B, Stidworthy MF, Bell S, Chantrey J, Masters N et al., 2012. Echinococcus and Taenia spp. from captive mammals in the United Kingdom. Vet Parasitol, 190: 95-103. Pak Vet J, 2016, 36(1): 121-123.  123Deplazes P and Eckert J, 2001. Veterinary aspects of alveolar echinococcosis- a zoonosis of public health significance. Vet Parasitol, 98: 65-87. Kondo H, Wada Y, Bando G, Kosuge M, Yagi K et al., 1996. Alveolar hydatidosis in a Gorilla and a Ring-Tailed Lemur in Japan. J Vet Med Sci, 58: 447-449. Lavikainen A, Lehtinen MJ, Meri T, Hirvela-Koski V and Meri S, 2003. Molecular genetic characterization of the Fennoscandian cervid strain, a new genotypic group (G10) of Echinococcus granulosus. Parasitology, 127: 207-215. Nakao M, McManus DP, Schantz PM, Craig PS and Ito A, 2007. A molecular phylogeny of the genus Echinococcus inferred from complete mitochondrial genomes. Parasitology, 134: 713-722. Palotay JL and Uno H, 1975. Hydatid disease in four nonhuman primates.  J Am Vet Med Assoc, 167: 615-618. Sadjjadi SM, 2006. Present situation of echinococcosis in the Middle East and Arabic North Africa. Parasitol Int, 55: 197-202. Shahar R, Horowitz H and Aizenberg I, 1995. Disseminated hydatidosis in a Ring Tailed lemur (Lemur catta): a case report. J Zool Wildl Med, 26: 119-122. Umhang G, Lahoreau J, Nicolier A and Boué F, 2013. Echinococcus multilocularis infection of a ring-tailed lemur (Lemur catta) and a nutria (Myocastor coypus). Parasitol Int, 62: 561-563. Varcasia A, Tanda B, Giobbe M, Solinas C, Pipia AP et al., 2011. Cystic echinococcosis in Sardinia: farmers' knowledge and dog infection in sheep farms. Vet Parasitol, 181: 335-340.  

Echinococcus granulosus "sensu stricto" in a captive ring-tailed lemur (Lemur catta) in Northern Italy

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Echinococcus granulosus "sensu stricto" in a captive ring-tailed lemur (Lemur catta) in Northern Italy

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