Advance Publication

The Journal of Veterinary Medical Science

Accepted Date: 7 Dec 2012 J-STAGE Advance Published Date: 21 Dec 2012 1 Category: Pathology

2 Note

3

4 First case of Echinococcus multilocularis infection in a zoo-housed

5 flying squirrel (Pteromys volans orii)

6

7 Eri Oikawa1), Ryoji Shimura2), Maki Nishimura3), Hidefumi Furuoka1)

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9 1)Division of Pathobiological Science, Department of Basic

10 Veterinary Medicine and 3)National Research Center for Protozoan

11 Diseases, Obihiro University of Agriculture and Veterinary Medicine,

12 Obihiro 080-8555; 2)Kushiro City Zoo, Kushiro 085-0201, Japan

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14

15 Correspondence to: Hidefumi Furuoka

16 Division of Pathobiological Science, Department of Basic Veterinary

17 Medicine, Obihiro University of Agriculture and Veterinary Medicine,

18 Obihiro 080-8555

19 E-mail: [email protected],

20 Phone: 0155-49-5360,

21 Fax: 0155-49-5359

22

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24

25

26 Running head: ECHINOCOCCOSIS IN A FLYING SQUIRREL 27 ABSTRACT. A thirty-three month-old male flying squirrel kept in a

28 zoo developed progressive dyspnea and died. Macroscopically, the

29 liver and lung were enlarged with numerous nodular vesicles.

30 Histologically, these organs were replaced by numerous collapsed

31 vesicles demarcated by fibrous tissues. The cysts lined by a

32 cellular, germinal layer contained numerous brood capsules with

33 abundant production of well-developed protoscolices. Protoscolices

34 were about 80-100 μ m in diameter, and had hooks being visible as

35 refractive structures. This zoo locates in the east of Hokkaido

36 where is an endemic area of Echinococcus multilocularis infection.

37 From epidemiology and pathological findings, this animal was

38 diagnosed as E.multilocularis infection. This report describes the

39 pathology of the first case of E. multilocularis infection in a flying

40 squirrel.

41

42 Key words; Echinococcus multilocularis, flying squirrel, Hokkaido,

43 new host record, pathology

2 44 Echinococcosis, also known as hydatid disease, is one of the most

45 important zoonotic diseases, causing human morbidity and mortality

46 worldwide [3]. Generally, six species of Echinococcus are

47 recognized, and four of them are of public health concern:

48 Echinococcus granulosus, E. multilocularis, E. vogeli, and E.

49 oligarthrus [6, 9-11]. E.granulosus is characterized by the

50 presence of unilocular cysts. In contrast, E.multilocularis which

51 causes alveolar echinococcosis, and E.vogeli, and E.oligarthrus are

52 associated with the multicystic lesion. While E.vogeli and

53 E.oligarthrus are restricted to South and Central America [5], the

54 geographical distribution of E.granulosus is worldwide, and that of

55 E. multilocularis is wide in the northern hemisphere such as central

56 Europe, Russia, central Asia, China, Alaska and Canada [9-11]. The

57 life cycle of E. granulosus involves dogs and other canids as

58 definitive hosts, and domestic and wild ungulates as intermediate

59 hosts. Meanwhile E. multilocularis exploits predator-prey

60 relationships between canids and rodents [2]. In Hokkaido, an

61 endemic area of E. multilocularis in Japan, the Ezo red fox (Vulpes

62 vulpes chrenchki) and red-backed vole (Myodes rufocanus bedfordiae)

63 mainly maintain its life cycle [7]. In foreign countries, the common

64 vole (Microtus arvalis), the water vole (Arvicola terrestris) and

65 the muskrat (Ondatra zibethicus) are important intermediate host for

66 this species [2]. In addition to voles, grounds squirrels (Citellus

67 daurious, C. erythrogenys, C. ungugalatus and C. alashanicus), red

68 squirrel (Sciurus vulgaris), bobak marmot (Marmota bobak) and

69 Prevost’s squirrel belonging to the suborder Sciuromorpha have been

3 70 reported rarely as the intermediated host [2, 12, 13]. This paper

71 describes the first case of Ezo flying squirrel (Pteromys volans orii)

72 affected with larval E. multilocularis.

73 A thirty-three month-old male Ezo flying squirrel, which was born

74 and kept in the lair at a logged timber soon after birth to one

75 month-old, and then housed in a metal cage enclosure with an outdoor

76 area in Kushiro City Zoo. The animal developed progressive dyspnea

77 and died on three days after the onset of dyspnea, then dissected

78 by a zoo veterinarian. Main organs including liver, spleen, kidney,

79 heart, lung, colon and testes were fixed in 15% formalin and submitted

80 to our laboratory. Samples were embedded in paraffin wax, and

81 sections (4 μ m) were stained with hematoxylin and eosin (HE) or

82 periodic acid-Schiff (PAS). At necropsy, the thoracic cavity was

83 filled with enlarged lung containing vesicular cysts. One third of

84 the liver was replaced by a mass sized head of the thumb consisting

85 of conglomerate cysts (Fig.1). These cysts showed multilocular

86 vesiculation containing white to yellow gruel-like fluid.

87 Histologically, the liver and lung were replaced by numerous

88 collapsed vesicles demarcated by fibrous tissues (Fig. 2). These

89 fibrous tissues in both organs were not so broad, and thinned

90 especially at the periphery of the vesicles projecting to the

91 parenchyma. In the liver, fibrosis was associated with bile-duct

92 proliferation. The outermost of the vesicles was a homogeneous

93 layer in HE-stained sections, and stained strongly with PAS. This

94 structure showed often fragmentation and convolution. The inner

95 cavity of the vesicles lined by a cellular, germinal layer was

4 96 composed of plural brood capsules containing numerous protoscolices.

97 Protoscolices were about 80-100 μ m in diameter, and had hooks being

98 visible as refractive structures. Numerous calcareous corpuscles

99 were observed within the germinal layer as well as protoscolices (Fig.

100 3). A dense accumulation of germinal cells, showing budding form,

101 were sometimes observed (Fig. 4). Many multinucleated giant cells

102 being reactive to the vesicles were observed, associated with

103 numerous lymphocytes and foamy macrophages (Fig. 5). No

104 pathological lesions were observed in other organs.

105 In this study, alveolar echinococcosis was diagnosed in a flying

106 squirrel at postmortem examination. In Japan, Hokkaido, the

107 northern island, is the only region with documented endemic

108 occurrence of E. multilocularis [2]. Red foxes (Vulpues vulpes),

109 domestic dogs and raccoon-dogs (Nyctereutes procyonoides) have been

110 identified as definitive host in Hokkaido [7]. Macroscopical

111 finding showed marked enlargement of the liver and lung characterized

112 by polycystic or multiloculated-cystic structures, and were

113 consistent with alveolar echinococcosis. Histologically,

114 metacestode origin of these cysts showing multivesicular,

115 infiltrating structures consisted of a bladder with numerous

116 protoscoleces. In addition to the epidemiology, these morphological

117 criteria confirm the diagnosis of E. multilocularis infection.

118 E. multilocularis lesion is characterized by a granulomatous host

119 reaction that includes infiltration of cells including lymphocytes,

120 epithelioid cells and multinucleated giant cells and fibrosis [4,

121 13]. These periparasitic reactions surrounding the metacestode

5 122 vesicles were not markedly different in the small mammal intermediate

123 hosts including the experimental murine model and our flying squirrel

124 case [2, 8]. The metacestode evades immune responses and

125 proliferates progressively, and is capable to produce large numbers

126 of protoscoleces. The periparasitic granuloma and fibrosis protect

127 the host against the parasite’s growth. In experimental murine model

128 and human cases [4, 13], the correlation between resistance and

129 development of fibrosis suggests that fibrogenesis is an important

130 component of the immunologically mediated effector mechanisms that

131 limit larval growth. Since thin fibrosis in the periphery of the

132 metacestode expanding growth does not seem to inhibit the

133 infiltrative growth in this case, the flying squirrel may have high

134 susceptibility to infection. Although lung involvement is rare in

135 E. multilocularis infections in human [1], pulmonary lesions with

136 metacestode tissue are sometimes observed in the intermediate animals

137 [2]. In this animal, the pulmonary lesions caused by hematogenous

138 metastasizing spread from primary hepatic lesions induced clinically

139 breathing difficulty and cyanosis, and finally death.

140 Infection of intermediate hosts occurs by the ingestion of eggs.

141 The zoo maintaining a flying squirrel in this study is surrounded

142 by a fox-tight perimeter fence, and direct contact with foxes can

143 be excluded. Generally, metacestodes of E. multilocularis develop

144 rapidly in intermediate host animals, and death of the intermediate

145 host can occur, usually around five months after infection [2]. From

146 these findings, the infection route in this animal may be ingestion

147 of feeds or some branches from wild trees or shrubs contaminated by

6 148 E.multilocularis.

149 Based on the histological findings, flying squirrel may be one of

150 the most suitable intermediate hosts for E. multilocularis as

151 red-backed voles, and could contribute to the transmission of this

152 disease by chance.

153

7 154 REFERENCES

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205

10 206 Figure legends

207 Fig. 1. The liver and lung have a mass sized head of the thump

208 associated with numerous nodular vesicles.

209 Fig. 2. The greater part of the liver is replaced by numerous

210 collapsed vesicles demarcated by fibrous tissues. HE stain.

211 Fig. 3. Liver. A vesicles lined by the homogenous laminated layer

212 and the germinal layer contains numerous protoscolices and calcareous

213 corpuscles. HE stain.

214 Fig. 4. Liver. Budding of the cells from the germinal layer. The

215 cells of the germinal layer focally proliferate and form internal,

216 small, protruding bud. HE stain.

217 Fig. 5. Lung. Numerous multinucleated ginat cells are reactive to

218 the vesicles. Interstitial tissues are infiltrated by numerous

219 lymphocytes and plasma cells, associated with fibrosis. HE stain.

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