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― i ―
− 編 集 委 員 会 −
編 集 委 員 長　佐藤　宏（山 口 大 学）
編 集 委 員　加藤　大智（自治医科大学）
編 集 委 員　西川　義文（帯広畜産大学）
編 集 委 員　松林　誠（大阪府立大学）
日本獣医寄生虫学会誌（ISSN 1347-961X）
第 15 巻第 1 号（平成 28 年 7 月 31 日発行）
発行者　日本獣医寄生虫学会　理事長　板垣　匡（岩手大学）
印刷者　杜陵高速印刷株式会社
岩手県盛岡市川目町 23 − 2　盛岡中央工業団地
TEL：019−651−2110 ㈹　FAX：019−654−1084
― ii ―
The Japanese Journal of Veterinary Parasitology
（Publication by the Japanese Society of Veterinary Parasitologists）
− Guide for Authors −
１. Unpublished Full papers, Notes, Review articles,
enough margin spaces（30mm）on the four sides.
and Essays, related in principle to veterinary
９. The proper noun should begin with a capital
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address. The lower half of the page should be left
of Histology, 9th ed., B. W. Saunders, Philadelphia.
blank.
４. The second page of each Full paper, Note and
2.Canpen, C. C. 1978. Tumors of the endocrine
Review article should contain an abstract（250
glands. pp.372-429. In：Tumors in Domestic
.
words or less）and Key words（5 or less）
, Univ.
Animals, 2 nd ed. （Moulton, J. E. ed.）
California Press, Berkley.
５. The description on and after the third page of
Full paper should be made in principle in the
3.Nakayama, A. 1978. Rhythm of the body tempe-
following order：INTRODUCTION, MATERIALS
rature. pp.140-145. In：Biorhythm and its Mecha-
AND METHODS, RESULTS, DISCUSSION,
nisms（Suda, M., Hayashi, O. and Nakagawa, H.
ACKNOWLEDGMENTS and REFERENCES. For
, Kodansha. Tokyo（in Japanese）
.
eds.）
Note, such captions as Abstract and Introduction
4.Pedersen, N. C., Ho, E. W., Brown, M. L. and
should not be given and References should follow
Yamamoto, J. K. 1987. Isolation of a T-lymphotr
the text.
opic virus from domestic cats with an immunodeficiency-like syndrome. Science 235：790-793.
６. The length of papers, including tables and figures,
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― iii ―
photographs should be directly marked. All the
Dr. Hiroshi Sato
Figures should be numbered through those of data
Editor-in-Chief
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Laboratory of Parasitology, Joint Faculty of
title and legends must be typewritten following
Veterinary Medicine, Yamaguchi University,
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− EDITORIAL BOARD −
Editor-in-chief：Hiroshi Sato（Yamaguchi University）
Editor：Hirotomo Kato（Jichi Medical University）
Editor：Yoshifumi Nishikawa（Obihiro University of Agriculture and Veterinary Medicine）
Editor：Makoto Matsubayashi（Osaka Prefecture University）
The Japanese Journal of Veterinary Parasitology（ISSN 1347-961X）is published semiannually
by the Japanese Society of Veterinary Parasitologists
（President Prof. Tadashi Itagaki, Iwate University）
.
― iv ―
目　次
投稿規定 …………………………………………………　i
総　説
Guide for authors ……………………………………… iii
Brief review on atypical human trypanosomiasis of
Trypanosoma lewisi
原　著
Prevalence of Eimeria infection in cattle in Japan
（Trypanosoma lewisi による非定型的人体トリパノソー
マ症）
（日本のウシにおける Eimeria 属原虫の検出状況）
Eliakunda MAFIE, Fatema Hashem RUPA,
Taihei KAWANISHI, Yuki KURAUCHI,
Aogu SETSUDA, Atsuko SAITO-ITO, and
Mikiko AOKI, and Tadashi ITAGAKI ………… 1
Hiroshi SATO……………………………………… 24
Eimeria zuernii 胞子未形成オーシストの形態学的同定
Endoparasites of Vietnamese lizards recorded in the
基準についての検討
last 50 years（1966-2015）
（A preliminary study on re-evaluation of the
characteristics for morphological identification of
Eimeria zuernii unsporulated oocysts）
谷田美和子、堀井洋一郎、野中成晃 …………… 5
（ベトナム産トカゲの内部寄生虫：過去 50 年間（19662015）の記録）
Binh Thi TRAN, Son Truong NGUYEN,
Tao Thien NGUYEN, Pham Van LUC,
Eliakunda MAFIE, Fatema Hashem RUPA,
Molecular phylogenetic relationship of Dicrocoelium
and Hiroshi SATO………………………………… 34
dendriticum from Japan and China based on nucleotide
sequences of the mitochondrial nad1
（日本および中国産 Dicrocoelium dendriticum の分子系
統解析）
解説
Veterinary Education in Nepal
（ネパール国の獣医学教育の現況）
Kei HAYASHI, Maiko OHTORI, Yuma OHARI,
Gokarna GAUTAM, and
Uday Kumar MOHANTA, WenQiang TANG,
Ishwari Prasad DHAKAL ……………………… 59
and Tadashi ITAGAKI…………………………… 12
施設紹介
短　報
大原研究所から馬原アカリ医学研究所へ　―ダニ媒介性
Molecular identification of Cryptosporidium isolates
感染症研究の断片―
from pet birds in Japan
（遺伝子解析による小鳥由来 Cryptosporidium 株の同定）
Niichiro ABE, Ikuko MAKINO,
Atsushi KOJIMA ………………………………… 19
（Brief histories of Ohara Research Laboratoy and
Mahara Institute of Medical Acarology）
藤田博己 …………………………………………… 65
Original Paper
Prevalence of Eimeria infection in cattle in Japan
Taihei KAWANISHI, Yuki KURAUCHI, Mikiko AOKI and Tadashi ITAGAKI
Laboratory of Veterinary Parasitology, Faculty of Agriculture, Iwate University
ABSTRACT
This study was designed to survey the prevalence of Eimeria spp. in cattle in Japan. Feces were collected from
382 cattle aged 9 day to 24 month in 71 farms of eight prefectures, Japan, and examined for Eimeria oocysts using
a standard sucrose flotation technique. Oocyst-positive samples were allowed to determine oocysts per gram（OPG）
using a modification of the McMaster technique. Eimeria species detected were E. alabamensis, E. auburnensis, E.
bovis, E. canadensis, E. cylindrica, E. ellipsoidalis, E. illinoiensis, E. subspherica and E. zuernii. Eimeria bovis and
E. alabamensis were detected in relatively high rates, 22.8% and 21.1%, respectively. One to six month-old cattle
showed the highest oocyst detection rates and OPG values compared to the other age cattle. Multiple Eimeria
species were detected from 68.8% of oocyst-positive feces.
Key words：Eimeria, oocysts, cattle, Japan, prevalence.
１．INTRODUCTION
２．MATERIALS AND METHODS
Bovine coccidiosis is one of the most important
Fresh fecal samples of 382 cattle（9-day-old to 24-
parasitic diseases of calves occurring worldwide and
month-old）were collected at random from 71 farms
mainly causes diarrhea. Although more than 20 Eimeria
of eight prefectures（Hokkaido, Iwate, Tochigi, Aichi,
species have been known to be causative of bovine
Hyogo, Kagawa, Ehime and Nagasaki）, Japan between
coccidiosis［6］, E. zuernii and E. bovis are highly
April and November 2009: briefly, 10 cattle of one farm
pathogenic to calves and young cattle and predominantly
in Hokkaido, 106 cattle of 34 farms in Iwate, 27 cattle
detected in severe clinical cases characterized by
of three farms in Tochigi, 31 cattle of three farms in
hemorrhagic diarrhea with sometimes fetal outcome［1］
.
Aichi, 14 cattle of one farm in Hyogo, 66 cattle of eight
However, other Eimeria species such as E. alabamensis
farms in Kagawa, 54 cattle of five farms in Ehime, and
and E. auburnnensis have been also reported to cause
74 cattle of 16 farms in Nagasaki. Fecal samples were
diarrhea［5, 13］. Economic losses due to clinical and
examined for Eimeria oocysts using a standard sucrose
sub-clinical coccidiosis in cattle and bufferlo have been
flotation technique and oocyst-positive samples were
annually estimated at 731 million US dollars［3］.
allowed to determine oocysts per gram（OPG）using a
Although bovine eimeriosis has also been severe
modification of the McMaster technique［14］. Briefly,
problems in Japan, there are only two reports on the
0.1g of each sample was mixed thoroughly with 1.7ml
prevalence in 1985［15］and 1990［9］
. The present
of sucrose solution（specific gravity 1.21）
. The mixture
study was designated to clarify recent prevalence of
was strained through a mesh to remove coarse plant
coccidiosis with identification of Eimeria species in
debris and then poured in 2 chambers with capacity
calves in Japan.
of 0.09 ml cube. Oocysts in the two chambers were
counted, summed and multiplied by the dilution factor
（100）to estimate the number of OPG. For species
identification of oocysts, 1g of positive samples was
― 1 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Prevalence of Eimeria infection in cattle in Japan
mixed with 10ml of 2.5% potassium dichromate solution
the highest detection rates and OPG values compared
in Petri dishes and kept at 26 ℃ for several days.
to the other age cattle （Figs. 1 and 2）, and the
After sporulation an aliquot of the mixtures was used
relation between oocyst detection rates and four age
for microscopic observation, and species identification
groups was confirmed in X-squared=8.3058, df=3, and
of oocysts was carried out based on their sizes and
p-value=0.0401. Although no relation of OPG values
morphological characteristics such as shape, color,
to fecal properties（normal, soft and diarrheal）was
micropyle, sporocyst size, residual, polar and stieda
observed, fecal samples in which over 1 x104 oocysts of
. Results were shown as average ± standard
body［6, 7］
E. zuernii, E. bovis, E. alabamensis, E. illinoiensis and
. The data obtained were statistically analyzed
error（SE）
E. ellipsoidalis were detected tended to be diarrheal.
using Pearson’s chi-squared test（Statistical computing
Eimeria species detected were E. alabamensis, E.
software, R ver.3.2.2）［10］, X-squared=8.3058, df=3,
auburnensis, E. bovis, E. canadensis, E. cylindrica,
p-value=0.0401, and P< 0.05 was considered significant.
E. ellipsoidalis, E. illinoiensis, E. subspherica and E.
zuernii. Detection rates of each species were high in
E. bovis（22.2%）and E. alabamensis（21.1%）, whereas
３．RESULTS
were low in E. canadensis（2.6%）and E. illinoiensis
Eimeria oocysts were detected in eight prefectures
（1.0%）
（Fig. 3）. Eimeria species detected from single
（100%）, 41 farms （54.6%） and 93 fecal samples
feces were plural（68.8%）and single（31.2%）
（Fig. 4）.
（24.3%）and the OPG value was 1403.5 ± 380.5. In
OPG values differed in Eimeria species and were high
relationships between cattle age and oocyst detection
in E. zuernii and E. subspherica（Fig. 5）.
or OPG value, 1-month to 6-month-old cattle showed
Fig. 1.Relation between detection rates of Eimeria
oocysts and cattle age（month）
. *Each bar
shows oocyst-positive feces/examined feces.
Fig. 2.Relation between Eimeria OPG and cattle age
（month）
. *OPG is shown in mean ± standard
error（SE）
.
Fig. 3. Detection rates of Eimeria species oocysts.
Fig. 4.The rates of Eimeria species detected from
single feces.
― 2 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Taihei KAWANISHI, Yuki KURAUCHI, Mikiko AOKI and Tadashi ITAGAKI
.
Fig. 5. Eimeria species detected and its OPG values. OPG is shown in mean ± standard error（SE）
for smaller sample size. However, the reason why E.
４．DISCUSSION
illinoiensis was detected and E. brasiliansis was not
This study revealed that bovine Eimeria infections
detected in this study could not be clarified. The oocysts
occur throughout Japan, although the detection rates
of E. bovis and E. zuernii, which are thought to be
（24.3%）were low compared to that（59.0%）of the
highly pathogenic species and to cause diarrhea and
previous report［9］. Difference in these rates may be
bloody feces to calves, have been detected with high
caused by the differences in sample size, localities and
OPG values from normal feces of cattle［2, 4, 8, 9］.
cattle age for sampling, as well as improved prevention
From these and the present findings, the fecal properties
measures of bovine eimeriosis.
of oocyst-positive cattle will not be connected with
Detection rates of Eimeria oocysts are the highest
Eimeria species detected and their OPG values.
in 6-month to 11-month-old calves and in 6-month to
8-month-old and thereafter, decrease with advancing
age［3, 9］. The present study also reconfirmed the
ACKNOWLEDGMENT
similar relation of cattle age to oocyst detection. On the
We sincerely thank Dr. Hiromitsu Ooba, NOSAI
other hand, no oocyst detection was observed in less
Aichi, Dr. Takehiro Hara, NOSAI Ohotsuku, Dr. Naoshi
than 1-month-old calves and low detection in the age
Yamamoto, NOSAI Hyogo, Dr. Rie Hirota, NOSAI
has been reported in Japan［9］
. These findings may
Kagawa, Dr. Taisuke Tominaga, Nagasaki, Dr. Mieko
suggest that Eimeria infection and development in the
Sugiyama, NOSAI Ehime, Dr. Youko Oku, NOSAI
host are related to feeding system of calves including
Iwate and Dr. Hideo Iso, Tochigi for providing fecal
supply of breast and artificial milk, because the highest
samples of cattle. We also thank Mr. Yuma Ohari for
oocyst detection rates were observed in 26-day to
helpful supports of statistical analyses.
30-day-old calves among 15-day to 2-month-old calves
in Argentine where weaning age of calves is 72 hours
after birth［11］. This study showed the prevalence
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low（4.5% and 5.4%, respectively）, and therefore, these
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species seem not to be detected in this study examined
on summer and winter ranges and in feedlots in
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Prevalence of Eimeria infection in cattle in Japan
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6. Levine, N. D. 1985. Apicomplexa: the coccidia
associated with infection of Eimeria zuernii in
proper. pp.130-232. In: Veterinary Protozoology.
The Iowa State University Press, Ames, Iowa.
calves. Can. J. Comp. Med. 45：34-37.
13. Svensson, C., Uggla, A. and Pehrson, B. 1994. Eimeria
7. Levine, N. D. and Ivens, V. 1970. The Coccidian
alabamensis infection as a cause of diarrhoea in
parasites（Protozoa, Sporozoa）of ruminants. Illinois
Biology Monograph No.44. University of Illinois
calves at pasture. Vet. Parasitol. 53：33-43.
14. Taira, N. 1997. ［O-ring method.］pp. 95-96.
Press, London.
［Examination Manual for Veterinary Parasitology］,
8. Neyberg, P. A., Helfer, D, H. and Knapp, S. E. 1967.
Incidence of bovine coccidian in Western Oregon.
Buneido-shuppan, Tokyo.
15. Watanabe, S. and Iwata, S. 1956. Studies on bovine
Proc. Helminthol. Soc. Wash. 34：13-14.
coccidiosis: distribution of oocysts among healthy
9. Oda, K. and Nishida, Y. 1990. Prevalence and
cattle. J. Jpn. Vet. Med. Assoc. 9：260-263.
distribution of bovine coccidian in Japan. Nippon
Juigaku Zasshi 52：71-77.
10. R Core Team. 2014. R: a language and environment
for statistical computing. R foundation for statistical
computing, Vienna, Austria, http//www. R-project.
Correspondence：Tadashi ITAGAKI, Laboratory of Veterinary
Parasitology, Faculty of Agriculture, Iwate University, Ueda
3-18-8, Morioka 020-8550, Japan.
E-mail：address: itagaki@iwate-u.ac.jp
日本のウシにおける Eimeria 属原虫の検出状況
川西　泰平、倉内　ゆき、青木　美樹子、板垣　匡
岩手大学農学部獣医寄生虫学研究室
要　約
ウシのコクシジウム症は下痢を主徴とした疾病で 20 種以上の Eimeria 属原虫が原因となる。Eimeria 種により病
原性に違いがあることが知られ、E. bovis および E. zuernii は高病原性の種として認識されている。本研究では、日
本のウシにおける Eimeria spp. 汚染状況を調査することを目的とした。8 道県の 71 農場で飼養されている 9 日齢か
ら 24 ヶ月齢のウシ 382 頭から糞便を採取し、ショ糖遠心浮遊法でオーシストの有無を検査した。オーシストが検出
された糞便は、スポロゾイト形成オーシストとして種を同定するとともにマックマスター法の変法（O リング法）に
より OPG（oocyst per gram）を計数した。その結果、9 種の Eimeria 属、すなわち E. alabamensis、E. auburnensis、E.
bovis、E. canadensis、E. cylindrica、E. ellipsoidalis、E. illinoiensis、E. subspherica、E. zuernii のオーシストが
確認された。Eimeria bovis および E. alabamensis の検出率はそれぞれ 22.8%、21.1% と高かった。また 1 ヶ月齢か
ら 6 ヶ月齢のウシはそれ以外の月齢ウシに比較してオーシスト検出率および OPG 共に高い値を示した。さらに、オー
シスト陽性の糞便では、その 68.8％から 2 種以上の Eimeria 種オーシストが検出された。
Key words：Eimeria 属、オーシスト、ウシ、日本、感染状況
― 4 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
原　著
Eimeria zuernii 胞子未形成オーシストの
形態学的同定基準についての検討
谷　田　美和子 1、堀　井　洋一郎 1,2、野　中　成　晃 1,2
1
宮崎大学農学部獣医学科獣医寄生虫病学研究室
2
宮崎大学産業動物防疫リサーチセンター
要　約
牛のコクシジウム症に対して、臨床現場では未成熟オーシストの大きさや形状の違いによって感染種の診断を行
うことが一般的であるが、教科書の記載に違いがあり混乱を招いている。そこで、牛に対する病原性が高い Eimeria
zuernii を対象として、2 農場の牛 2 頭の糞便から、大きさ 15.0 〜 25.0μm × 12.0 〜 20.0μm のオーシストを採取し、
個別にオーシストの形態を計測した後、ITS-1 領域の塩基配列の解読または同領域の種特異的プライマーを用いた
PCR により種同定を行い、E. zuernii 未成熟オーシストの大きさと形状を評価した。その結果、解析に供した 156 検
体のうち、53 検体のオーシストが遺伝的に E. zuernii と同定され、それらの大きさは 15.0 ～ 21.3μm × 12.5 ～ 17.5
μm であり、その形状は 53% が類円形、47% が楕円形であった。オーシストの大きさは 18 個のオーシストが示した
17.5μm × 15.0μm を中心に分布しており、16.0 ～ 20.0μm × 13.5 ～ 16.5μm に 41 個（77%）が分布していた。この
結果を既存の汎用教科書（日本語）の記載と比較したところ、どの教科書の記載にも合致しないものが認められた。
今後は、本研究のようなアプローチを用い、各種 Eimeria 未成熟オーシストの形態学的特徴を再評価する必要があ
ると考えられる。
Key words：牛、コクシジウム症、診断、Eimeria zuernii、未成熟オーシスト
行われているが、臨床現場ではオーシストを胞子形成さ
１．はじめに
せることはほとんどなく、未成熟オーシストの形態学的
牛のコクシジウム症は消化管原虫である Eimeria 属
特徴のみで同定される場合が多い。しかしながら、オー
原虫によって引き起こされる疾患であり、特に若齢牛（6
シストの大きさや形状には種内多様性が認められ、その
～ 11 カ月齢）での感染が多い［10］。コクシジウム症は
重なりから種の同定が困難となることがある。種同定の
重症の場合、水様性や出血性下痢、体重減少、また時に
参考書として用いられる汎用教科書をみても、形態の記
死亡する個体も現れることから経済的損失が大きく畜産
載に違いがみられ（表１、例：Eimeria zuernii）
、混乱
業において重要な疾患の一つである［1］。
を招く一要因となっている。これは、オーシストの大き
コクシジウム症の診断は一般的に感染牛の糞便中に排
さや形状に対する計測値や判定結果の分布パターン（度
出されたオーシストを検出することで行われる。牛にお
数分布）を示した報告がなく、そのため種の大部分が含
いて日本国内では 13 種の Eimeria 属原虫が確認されて
まれる標準形態を判断できないことが原因と考えられ
いる［5］。牛のコクシジウム感染は複数種の混合感染が
る。オーシストの形態学的特徴については 1967 年出版
一般的であり、また種によって牛に対する病原性が異な
の Levine and Ivens［9］
が詳細に報告しているが、それ
るため、コクシジウム症診断時にはオーシストの検出と
以降はオーシストの形態についての詳細な検討はなく、
合わせて種同定を行うことが重要である。
彼らの観察結果の有効性についても不明なままである。
種同定は、オーシストの形状と色、大きさ（長径と短
本 邦 で 見 つ か る 牛 の Eimeria 種 の う ち、E. zuernii
径）、ミクロパイルの有無などの形態学的特徴と好適培
（Ez）は小腸から大腸にかけて寄生し牛に対する病原性
養条件下での胞子形成時間および胞子形成後のスポロシ
が高く、重度感染で出血性下痢が観察される。しかしな
ストの形態学的特徴や内・外残体の有無などに基づいて
がら Ez のオーシストの形態は、E. alabamensis（Eal）
― 5 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Eimeria zuernii 胞子未形成オーシストの形態学的同定基準についての検討
表１．教科書および文献に記載の Eimeria zuernii のオーシストの大きさと形状
教科書・文献
オーシストの大きさ（長径×短径）
（μm）
形　状
新版 獣医臨床寄生虫学（産業動物編）［11］
16～20×15～18（平均 18×17）
類円形
最新 家畜寄生虫病学［3］
16～20×15～18
類円形
図説 獣医寄生虫学 改訂第 2 版［12］
平均 18×17
類円形
改訂 獣医寄生虫学・寄生虫病学１（総論・原虫１）［2］
12～29×10～21（平均 17～20×14～17） 類円形、類卵円形
Protozoan Parasites of Domestic Animals and of Man［7］ 15～22×13～18（平均 17.8×15.6）
類円形、丸まった楕円形
Veterinary Protozoology［8］
12～29×10～21
類円形、類卵円形、卵円形、まれに楕円形
Levine and Ivens（1967）［9］
18～23×13～19（平均 20.2×16.1）
類円形、類卵円形、卵円形、しばしば楕円形
や E. ellipsoidalis（Ee）のものと近似しており、その大
円形、短径軸と長径軸の直角の交点がいずれかの軸の中
きさと形状に重なりを持つ。本研究は、Ez のオーシス
心から外れ、かつ卵形を示すものを卵円形として判断し
トの大きさおよび形状の度数分布を観察して Ez の持つ
た。
形態学的多様性を検討し、形態学的近似種（Eal および
Ee）のものと比較することで、Ez の同定の有効性と限
2-3．DNA 抽出法
界を考察することを目的とする。なお、本研究では、我
Lalonde and Gajadhar［6］が Cyclospora cayetanensis
が国で広く使用されている教科書、新版獣医臨床寄生虫
のオーシストに用いた方法を適用した。すなわち、
学（産業動物編）および最新家畜寄生虫病学の記載値を
オーシスト 1 個を含む 1.5ml チューブに QIAamp DNA
参考基準として扱い、本研究で得られた Ez の計測結果
micro kit（QIAGEN, Hilden, Germany）の Buffer ATL
との乖離を評価するとともに、他の教科書の記載値との
を 300μl 加 え、 − 70 ℃ で 5 ～ 10 分 間 と 95 ℃ で 1 ～ 2
乖離についても考察した。
分間の凍結融解を 8 回繰り返した。次に proteinase K
を 20μl 加 え 56 ℃ で 3 時 間 加 熱 し た 後、Buffer AL を
300μl 加え 70℃で 10 分間加熱した。その後、Kit のプ
２．材料と方法
ロトコールに従い DNA 抽出を行った。なお、カラムか
2-1．オーシストの収集
らの DNA 抽出は buffer AE 30μl で行った。
2 農場（A, B 農場）で飼育されていた牛 2 頭の糞便各
5g に対してウイスコンシン変法を実施して、上清中 2
2-4．PCR
ml 中に含まれる長径 15.0 〜 25.0μm ×短径 12.0 〜 20.0
プライマーは Kawahara et al.［4］の報告にある ITS-1
μm で、類円形、楕円形または卵円形の形状（形状の定
領 域 の Eimeria genus common primer を 使 用 し た
義は 2-2 参照）と思われるオーシストを採取した。水で
（Forward : 5’-GCA AAA GTC GTA ACA CGG TTT
洗浄後、次亜塩素酸ナトリウム溶液を加えて室温で 40
CCG -3’、Reverse: 5’-CTG CAA TTC ACA ATG CGT
分間～ 1 時間静置し、蒸留水を加えて再度洗浄後、胞子
ATC GC -3’）
。
PCR 反応液は TaKaRa Ex Taq
（TAKARA
形成による DNA 抽出効率の低下を避けるため− 30℃
BIO、滋賀県草津）を用いて、10 × Ex Taq Buffer 2.0
で保存した。
μl、dNTP Mix 1.6μl、Taq 0.1μl、プライマー（10μM）
各 0.5μl、DDW 10.3μl、サンプル 5μl にて作成した。
2-2．オーシストの形態記録
サーマルサイクラーで 94℃ 30 秒加熱した後、94℃ 10
オーシスト液を氷上で融解した後、実体顕微鏡下で
秒、55℃ 30 秒、72℃ 30 秒のサイクルを 35 回繰り返し、
オーシスト 1 個を 3μl の液量と共に吸引し、スライド
72℃で 2 分間最終反応させた。
グラスに滴下して光学顕微鏡下でオーシストの長径、短
径、形状を記録した。その後オーシストを 1.5 ml チュー
2-5．電 気泳動およびアガロースゲルからの PCR 産物
の抽出
ブに回収して遺伝子解析の材料とした。なお、オーシス
トの形状については、便宜上、短径軸と長径軸が各軸の
PCR 産 物 を 電 気 泳 動 し 目 的 の バ ン ド を 確 認 し た
中心で直角に交差し、かつ短径と長径の比が 1.0 〜 1.2
後、バンドを切り出して QIAquick Gel Extraction Kit
のものを類円形、短径軸と長径軸が各軸の中心で直角に
（QIAGEN）を用いて DNA 抽出を行った。Nano Drop
交差し、かつ短径と長径の比が 1.2 より大きいものを楕
1000（Thermo Scientific, Wilmington, Delaware, USA）
― 6 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
谷　田　美和子、堀　井　洋一郎、野　中　成　晃
を用いて DNA 濃度を測定した。
価を行った。
A 農場からは 15.0 ～ 20.0μm × 12.5 ～ 17.5μm、B 農
2-6．TA クローニングおよび塩基配列の決定
場 か ら は 15.0 ～ 22.5μm × 12.5 ～ 18.8μm の 大 き さ の
ゲルより抽出した PCR 産物に対して、pGEM-T Easy
オーシストが検出された。また形状を見ると A 農場で
Vector System Ⅰ（Promega, Madison, Wisconsin, USA）
は類円形および楕円形のオーシストが、B 農場ではそれ
および E. coli DH5α Competent Cells（TAKARA BIO）
に加え卵円形のオーシストが検出された。
を用いてプロトコール通りに TA クローニングを行った。
TA クローニングによる遺伝子解析は A 農場の 66 検
コロニーを 5 個選択し、アンピシリン加 LB 培地（nacalai
体と B 農場の 55 検体の合計 121 検体について行った
tesque、京都市中京区）で培養した後、QIAprep Spin
が、A 農場 41 検体と B 農場 9 検体の合計 50 検体にお
Miniprep Kit（QIAGEN）を用いプラスミドを精製し、
い て 塩 基 配 列（ 塩 基 数 301 〜 485 base pairs） が 解 読
DNA 濃度を測定した。次に、得られた産物に対し kit
で き た。A 農 場 か ら 7 つ の ハ プ ロ タ イ プ（haplotype
付属の T7（5’-TAA TAC GAC TCA CTA TAG GG -3’）
1 〜 7： 塩 基 数 483 〜 485 base pairs）、B 農 場 か ら は
または SP6（5’-ATT TAG GTG ACA CTA TAG -3’）
5 つ の ハ プ ロ タ イ プ（haplotype 8 お よ び 9： 塩 基 数
プライマーを用いてサイクルシークエンスを行い、
301 base pairs、haplotype 10 〜 12：塩基数 450 ～ 452
®
（Applied
BigDye Terminator v3.1 Cycle Sequencing Kit
base pairs）が検出された。これらのハプロタイプを、
Biosystems, Waltham, Massachusetts, USA）および 3130
Eimeria tenella（Accession No. AF026388）をアウトグ
Genetic Analyzer（Applied Biosystems）を用いて塩基
ループとして、Kawahara et al.［4］および Kokuzawa
配列を決定した。
et al.（unpublished）が GenBank に登録した牛 Eimeria
の ITS-1 領域の塩基配列と系統樹で比較したところ、A
2-7．系統樹解析
農場のオーシスト 41 検体から得られたハプロタイプは
得 ら れ た 塩 基 配 列 と GenBank に 登 録 さ れ て い る
全て Ez のクレードに位置し、Ez と同定できた（図 1）。
牛 の Eimeria ITS-1 領 域 の 塩 基 配 列 に つ い て、CLC
haplotype 2 の塩基配列は GenBank の登録配列である
Sequence Vierwer 6、および MEGA7 を用いて NJ 法に
Accession No. AB557622 と 100% 一 致 し た。B 農 場 の
よる系統樹を作成した。
オーシスト 9 検体から得られたハプロタイプのうち、
301 base pairs のもの 2 つ（3 検体）は Eal のクレード
2-8．種特異的プライマーを用いた PCR
内に、450 ～ 452 base pairs のもの 3 つ（6 検体）は Ee
Eimeria genus common primer で PCR 反 応 陽 性 で
のクレード内に位置し、それぞれ Eal および Ee と同定
あ っ た 一 部 の サ ン プ ル に つ い て は、Kawahara et al.
できた。ただし、Ee は 2 つの異なるクレード、すなわち、
［4］の報告した Ez（Forward: 5’-AAC ATG TTT CTA
Kawahara et al.［4］
が報告した塩基配列で構成されるク
CCC ACT AC-3’; Reverse: 5’-CGA TAA GGA GGA
レードと、Kokuzawa et al. が 2013 年に GenBank に登
GGA CAA C-3’）、Eal（Forward: 5’-CAT TCA CAC
録した配列で構成されるクレードに分かれるが、今回検
ATT GTT CTT TCA G-3’; Reverse: 5’-GCT TCC AAA
出したものは後者に属した。したがって、Ee について
CTA ATG TTC TG-3’）および Ee（Forward: 5’-CAA
は今後の分類学的検証を要すると考えられる。
CGT TTT TGG TTT TGG TAT CA-3’; Reverse: 5’-ACT
これに加え、B 農場のオーシストについて種特異的プ
GCG ATG AGA GAG AGC G-3’）種特異的プライマー
ライマーを用いて Ez、Eal、Ee の種同定を行ったところ、
を用いて種同定を行った。PCR 反応液は 2-4 と同様に作
35 検体中 12 検体が Ez と判定できた。その他の検体で
成し、94℃ 30 秒加熱した後、94℃ 10 秒、55℃ 20 秒、
は PCR 増幅産物が得られず種同定を行えなかった。
72℃ 20 秒のサイクルを 35 回繰り返し、72℃で 2 分間
最後に、TA クローニングおよび種特異的プライマー
最終反応させた。
を用いた PCR により、A 農場および B 農場で種が同定
できた Ez 53 検体、Ee 6 検体および Eal 3 検体のオー
シストの形態を図 2 および表 2 に示した。Ez のオーシ
３．結　果
ストの大きさは 15.0 ～ 21.3μm × 12.5 ～ 17.5μm であっ
A 農場の牛糞便から 83 個、B 農場の牛糞便から 103
た。長径で最も度数が高かったものは 17.5μm で 23 検
個の未成熟オーシストを採取し、オーシストの形態の評
体が、短径で最も度数が高かったものは 15.0μm で 39
― 7 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Eimeria zuernii 胞子未形成オーシストの形態学的同定基準についての検討
図１．A および B 農場のオーシストから得られた塩基配列（haplotypes 1 ～ 12）と既報告の牛
Eimeria ITS-1 領域の塩基配列の比較系統樹．
検体がこれらの値を示した。オーシストの大きさは 18
個のオーシストが示した 17.5μm × 15.0μm を中心に分
４．考　察
布 し て お り、16.0 ～ 20.0μm × 13.5 ～ 16.5μm に 41 個
本研究は 2 農場で飼育されていた 2 頭の牛の糞便に含
（77%）が分布していた。また形状についても 53% が類
まれる小型オーシストを対象として行ったが、A および
円形を、47% が楕円形を示した。卵円形は検出されな
B 両農場において様々な大きさと形状のオーシストが検
かった。
出された。両農場で検出されたオーシストの形態を、新
版獣医臨床寄生虫学（産業動物編）［11］と最新家畜寄生
― 8 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
谷　田　美和子、堀　井　洋一郎、野　中　成　晃
表 2．Eimeria zuernii, E. alabamensis および E. ellipsoidalis のオーシストの大きさと形状
Eimeria 種
E. zuernii
E. alabamensis
E. ellipsoidalis
参考基準＊
改訂獣医寄生虫学・寄生虫病学１
オーシストの形態
本研究
大きさ（長径×短径 ;μm）
15.0～21.3 × 12.5～17.5（n=53）
16～20 × 15～18
12～29 × 10～21
形状
類円形、楕円形
類円形
類円形、類卵円形
大きさ（長径×短径 ;μm）
18.8～22.5 × 15.0（n=3）
17～24 × 12～16
形状
卵円形
卵円形
大きさ（長径×短径 ;μm）
21.3～22.5 × 15.0～16.3（n=6）
20～25 × 14～20
12～32 × 10～29
形状
楕円形
楕円形
長卵円形
（総論／原虫）［2］
記載無し
* 新版獣医臨床寄生虫学（産業動物編）
［11］および最新家畜寄生虫病学［3］の記載値
図２．遺 伝学的に同定できた E. zuernii オーシスト 53 検体，E. ellipsoidalis オーシスト６検体および E.
alabamensis オーシスト 3 検体の形態分布．シストの大きさを長径（横軸）および短径（縦軸）の
分布図で示し，円の色により種を区別して示した．円の大きさは同じ計測値をもつオーシストの数に
比例させて示した．また E. zuernii，E. alabamensis および E. ellipsoidalis について，参考基準にお
けるオーシストの大きさをそれぞれ橙色，紫色，水色の図形によって示した．
虫病学［3］の掲載形態（参考基準）と比較すると、両農
かになった（表 2，図 2）。また形状も類円形（53%）と
場のオーシストには Ez の参考基準（16 ～ 20μm × 15
楕円形（47%）がほぼ半数の割合で検出された。参考基
～ 18μm、類円形）に合致するもの、および Ee の参考
準の形態に合致する Ez は遺伝学的に同定されたものの
基準（20 ～ 25μm × 14 ～ 20μm、楕円形）に合致する
43%（23 検体）にすぎず、残りの 57% は参考基準から外
ものが存在した。Eal の参考基準（17 ～ 24μm × 12 ～
れたものであった。遺伝学的に Ez と同定されたオーシ
16μm、卵円形）に合致するものは B 農場でのみ検出さ
ストのうち、Eal および Ee の参考基準の大きさを持つ
れたが、両農場には形状が楕円形であるものの Eal の大
オーシストの割合は 79% および 19% であった。ただし、
きさに合致するものが存在した。また両農場で Ez、Eal
Eal のオーシストと同じ大きさを持った Ez の形状は類
または Ee のどの参考基準にも合致しないオーシストが
円形または楕円形であり、Eal の参考基準の形状である
検出された（それぞれ 11% と 10%）。
卵円形とは異なっていたため、卵円形と規定される Eal
遺伝学的に Ez と同定された 53 検体のオーシストの
のオーシストとは形状での鑑別が可能とも考えられる。
形態を評価したところ、オーシストの大きさは 15.0 ～
しかし、今回塩基配列が明らかになった Eal の 1 検体は
21.3μm × 12.5 ～ 17.5μm で、参考基準の大きさ 16 ～
楕円形を示しており、形状による厳密な鑑別は不可能で
20μm × 15 ～ 18μm よりも広い範囲であることが明ら
あることも示唆された。このオーシストの大きさは Ee
― 9 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Eimeria zuernii 胞子未形成オーシストの形態学的同定基準についての検討
の参考基準にも含まれていたため、Ee との鑑別も必要
となる。Ez と同定されたオーシストの 9％は Ez、Eal、
井壮一編），講談社，東京．
3. 今井壮一．2007．コクシジウム症．pp. 10-19. In; 最
Ee のどの種の参考基準にも含まれないものであった。
新 家畜寄生虫病学（今井壮一，板垣匡，藤崎幸蔵
一 方、 別 の 汎用 教 科 書 で あ る 改 訂 獣 医 寄 生虫学・
編），朝倉書店，東京．
寄生虫病学１（総論・原虫）［2］では、Ez の大きさは
4. Kawahara, F., Zhang, G., Mingala, C. N., Tamura,
Veterinary Protozoology［8］と同じく 12 ～ 29μm × 10
Y., Koiwa, M., Onuma, M. and Nunoya, T. 2010.
～ 21μm（平均：17 ～ 20μm × 14 ～ 17μm）と記載さ
Genetic analysis and development of species-
れており、今回参照した教科書の中では長径、短径とも
specific PCR assays based on ITS-1 region of
に最も広範囲の記載である。今回検出した Ez の大きさ
rRNA in bovine Eimeria parasites. Vet. Parasitol.
はこの教科書の記載値内に含まれる。したがって、これ
174：49-57.
らの教科書の掲載情報の方がより正確な情報であるとい
5. 小岩政照，安藤貴朗，鈴木一由，田口　清，川原史
えるかもしれないが、これらの教科書の記載にある様な
也．2009．子牛と育成牛のコクシジウム症．臨床獣
極端に小さなあるいは大きな長径や短径を持つオーシス
医 27：44-50.
トは今回検出されなかった。検査対象が限られていたこ
6. Lalonde, L. F. and Gajadhar, A. A. 2008. Highly
とに起因する可能性は否めないが、本研究と同様の検討
sensitive and specific PCR assay for reliable
を広く実施することにより、より正確な情報が得られる
detection of Cyclospora cayetanensis oocysts. Appl.
ものと思われる。
Environ. Microbiol. 74：4354-4358.
また、形状については、改訂獣医寄生虫学・寄生虫病
7. Levine, N. D. 1961. Genus Eimeria Schineider, 1875.
学１（総論・原虫）
［2］の記載は類円形と類卵円形であ
pp. 166-177. In: Protozoan Parasites of Domestic
り、今回同定した約半数の Ez オーシストが示した楕円
Animals and of Man, Burgess Pub, Minneapolis,
形は含まれていない。Veterinary Protozoology［8］では、
Minnesota, USA.
類円形および類卵円形の他に、卵円形、まれに楕円形と
8. Levine, N. D. 1985. Genus Eimeria Schineider, 1875.
の記載があり、「まれに」との注釈があるものの楕円形
pp. 142-148. In: Veterinary Protozoology, Iowa
が含まれている。
State University Press, Ames, USA.
本研究では遺伝学的同定によって主に Ez が多く検出
9. Levine, N. D. and Ivens, V. 1967. The sporulated
され、Eal および Ee の形態情報はあまり得られていな
oocysts of Eimeria illinoisensis n. sp. and of other
い。本研究で対象とした種を含めて、未成熟オーシスト
species of Eimeria of the ox. J. Prorozool. 14：351-
の形態学的同定の有効性と限界を明らかにするために
360.
は、Eimeria 各種に対して本研究のようなアプローチを
10. Oda, K. and Nishida, Y. 1990. Prevalence and
行い、Eimeria 種の形態学的特徴を再評価する必要があ
distribution of bovine coccidia in Japan. Jpn. J. Vet.
ると考えられる。
Sci. 52：71-77.
11. 新版獣医臨床寄生虫学編集委員会．1995．コクシジ
ウム．pp. 45-51. In: 新版　獣医臨床寄生虫学（産業
謝　辞
動物編）
，文永堂，東京．
本研究の実施にあたり、快く材料をご提供いただいた
12. 内田明彦，野上貞雄，黄鴻堅．2008．アイメリア．
本川和幸氏に深謝します。
メディ
pp. 35-37. In: 図説 獣医寄生虫学 改訂第 2 版，
カグローブ，弘前．
引用文献
1. Fitzgeralad, P. R. 1980. The economic impact of
coccidiosis in domestic animals. Adv. Vet. Sci.
Comp. Med. 24：121-142.
2. 石井俊雄．2007．牛のコクシジウム．pp. 63-65. In:
改訂獣医寄生虫学・寄生虫病学１（総論・原虫）
（今
連絡責任者：野中成晃、宮崎大学農学部獣医学科獣医寄生虫病
学研究室、〒 889-2192　宮崎市学園木花台西 1-1
Correspondence：Nariaki NONAKA, Laboratory of Veterinary
Parasitic Diseases, Department of Veterinary Sciences, Faculty
of Agriculture, University of Miyazaki, Gakuen-Kibanadai
Nishi 1-1, Miyazaki 889-2192, Japan.
E-mail：nnonaka@cc.miyazaki-u.ac.jp
― 10 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
谷　田　美和子、堀　井　洋一郎、野　中　成　晃
A preliminary study on re-evaluation of the
characteristics for morphological identification of
Eimeria zuernii unsporulated oocysts
Miwako TANIDA 1, Yoichiro HORII 1,2, Nariaki NONAKA 1,2
1
Laboratory of Veterinary Parasitic Diseases, Department of Veterinary Sciences,
Faculty of Agriculture, and 2 Center for Animal Disease Control, University of Miyazaki.
ABSTRACT
In clinical situation, determination of infected species in bovine coccidiosis relys on the size and shape of
unsporulated oocysts. However, different description of the morphology among reference textbooks incurs
confusion. In this study, we collected Eimeria oocysts with size of 5.0 to 25.0μm × 12.0 〜 20.0μm from two cattle
raised at two farms, recorded their size and shape, and identified their species molecularly by sequencing matching
or species-specific PCR assay. In result, 53 oocysts were identified as E. zuernii. The size of oocysts were within
range of 15.0 ～ 21.3μm × 12.5 ～ 17.5μm, and the shape of 53% were spherical but the rest were ellipsoidal. Most
frequent size observed was 17.5μm × 15.0μm that was recognized in 18 oocysts, and 41 oocysts（77%）showed
their size within 16.0 ～ 20.0μm × 13.5 ～ 16.5μm. When the results were checked using the Japanese wide-use
textbooks, the morphology of some oocysts were recognized not being matched to the description in any textbooks.
Considering those preliminary results, further studies using similar approach like this study should be necessary
conducted to re-evaluate the morphological characteristics of unsporulated oocysts of Eimeria species.
Key words：cattle, coccidiosis, diagnosis, Eimeria zuernii, oocyst.
― 11 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Original Paper
Molecular phylogenetic relationship of Dicrocoelium
dendriticum from Japan and China based on nucleotide
sequences of the mitochondrial nad1
Kei HAYASHI 1,2, Maiko OHTORI 1, Yuma OHARI 1,2, Uday Kumar MOHANTA 1,2,
WenQiang TANG 3 and Tadashi ITAGAKI 1,2
1
2
Laboratory of Veterinary Parasitology, Faculty of Agriculture, Iwate University
Department of Pathogenic Veterinary Science, United Graduate School of Veterinary Science, Gifu University
3
Institute of Animal Science, Tibet Academy of Agricultural and Animal Husbandry Sciences
ABSTRACT
The aim of this study was to analyze the phylogenetic relationship between Dicrocoelium dendriticum
populations in Japan and China based on molecular markers. Fourteen lancet flukes collected from Japan and China
were identified as D. dendriticum based on their testes orientation and nucleotide sequences of ribosomal ITS2.
The flukes were then analyzed phylogenetically by the nucleotide sequences of a mitochondrial nad1 gene. In the
resultant phylogenetic tree, the nad1 haplotypes from Japan and China were divided into two separated clades,
and the nucleotide variations between the clades were extremely high, hypothesizing that the D. dendriticum
populations from Japan and China have conspicuously differed in genetic characters. This study provides
the nucleotide sequences of nad1 gene of D. dendriticum in Japan for the first time, and may provide useful
information for elucidating phylogenetic relationship of the species between Japan and China.
Key words：China, Dicrocoelium dendriticum, ITS2, Japan, nad1.
and D. chinensis have been distinguished on the basis
１．INTRODUCTION
of the testes orientation［10, 19］. However, it is quite
Lancet flukes of the genus Dicrocoelium include
difficult to identify the morphologically ambiguous flukes
three species, D. dendriticum, D. chinensis and D. hospes
with diagonal orientation of the testes. Therefore,
which are well-known causative agents of dicrocoeliasis
molecular methods based on the nucleotide sequence
in domestic and wild ruminants. The disease causes
of the ribosomal internal transcribed spacer 2（ITS2）
occasional economic loss in the livestock industry
have recently been developed for precise identification
by reducing productivity due to liver disorder［11］.
of Dicrocoelium spp.［9, 10］
. In addition, the nucleotide
Dicrocoelium dendriticum is distributed in Europe,
sequence of mitochondrial NADH dehydrogenase
Asia, North and South America, Australia and northern
subunit 1 （nad1） has been used for intraspecific
Africa［12, 13］, whereas D. chinensis is distributed in
phylogenetic analysis in many helminth species, due
Eastern Asia and Europe［11, 14］
, and D. hospes in
to its rapid evolutionary and mutation rates［2, 5］.
Africa［13］. Dicrocoelium dendriticum infection has
However, information about the molecular markers
been reported mainly in wild ruminants, sika deer
of D. dendriticum was limited to date. In this study,
（Cervus nippon）and Japanese serow（Capricornis
we identified lancet flukes from Japan and China as
crispus）in Japan［8, 9, 18, 19］
, whereas the species
D. dendriticum based on the morphological features
occurs in domestic sheep and goat in China［17, 22］.
and nucleotide sequence of ITS2, and analyzed the
Dicrocoelium dendriticum（synonym for D. lanceatum）
intraspecific variation and phylogenetic relationship
― 12 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Kei HAYASHI, Maiko OHTORI, Yuma OHARI, Uday Kumar MOHANTA, WenQiang TANG, Tadashi ITAGAKI
between the populations from Japan and China on the
including partial 5.8S and 28S, and partial nad1
basis of nad1 sequences.
fragment were amplified by PCR in a final volume of
50μL, containing 1μL of template DNA, 0.2 pM of
primers, 1.25 U of Tks Gflex DNA Polymerase（TaKaRa
２．MATERIALS AND METHODS
Bio Inc., Otsu, Japan）and the manufacturer-supplied
2-1．Lancet fluke collections and morphological
reaction buffer. The following primer sets were used:
identification
ITS2-F and ITS2-R for ITS2［3］and Dicro-nad1-F（5’-
Eight lancet flukes were collected from the bile ducts
GGAGTGTGGTGTTTTGGTTT-3’）and Dicro-nad1-R
of three Japanese serows in Iwate, Japan from October,
（5’-AACAACGAACTAACCCAAGC-3’） for nad1,
2003 to March, 2005, and 6 flukes were recovered from
which were designed based on complete mitochondrial
a yellow cattle（Bos taurus）in Dingxi and a yak（Bos
sequences of D. dendriticum（GenBank accession no.
grunniens）in Yushu, China from August to September,
KF318787）and D. chinensis（KF318786）
［6］
. The thermal
2013（Table 1）. The flukes were fixed in 70% ethanol
cycling conditions for the amplification consisted of an
and transported to the laboratory for further studies.
initial denaturation step at 94 ℃ for 1 min; followed by
The anterior part of each fluke was removed for
30 cycles at 98 ℃ for 10 sec, 55 ℃ for 15 sec, and 68
DNA extraction［7］. Then, the testes orientation of
℃ for 30 sec. The amplicons were directly sequenced
the flukes from China were observed after staining
in both directions on an ABI 3500 Genetic Analyzer
with hematoxylin–carmine solution and morpho-
（Applied Biosystems, Foster City, CA, USA）with a
metric studies were conducted［4, 20, 21］. The testes
BigDye Terminator v3.1 Cycle Sequence Kit（Applied
orientation of the flukes from Japan has been analyzed
Biosystems）using the same primers as those for PCR.
previously［9］.
The resultant sequences were initially assembled
using ATGC ver. 6.0.3（Genetyx Co., Tokyo, Japan）,
2-2．DNA analyses
and the haplotypes were distinguished by GENETYX
Total DNA was extracted using High Pure PCR
ver. 10（Genetyx Co.）. Multiple sequence alignment of
Template Preparation Kit（Roche, Mannheim,
the ITS2 sequences（239 bp）along with those of D.
Germany）according to the manufacturer’s instructions
chinensis（AB367790）and D. hospes（EF102026）was
and stored at －20 ℃ until use. The ITS2 region
performed using GENETYX ver. 10.
Table 1. The molecular characters of D. dendriticum analyzed in this study.
Country
Locality
Host code
Japan
Iwate
Japanese serow #1
China
Dingxi
Yushu
Fluke code
Nuclear ITS2 genotype
Mitochondrial nad1 haplotype
Code
Code
Accession no.
Accession no.
fluke #1
ITS2-DD-1
AB367789
ND-DD-J1
LC159517
fluke #2
ITS2-DD-1
AB367789
ND-DD-J2
LC159518
fluke #3
ITS2-DD-1
AB367789
ND-DD-J3
LC159519
fluke #4
ITS2-DD-1
AB367789
ND-DD-J3
LC159519
fluke #5
ITS2-DD-1
AB367789
ND-DD-J3
LC159519
fluke #6
ITS2-DD-2
LC159512
ND-DD-J2
LC159518
Japanese serow #2
fluke #7
ITS2-DD-3
LC159513
ND-DD-J2
LC159518
Japanese serow #3
fluke #8
ITS2-DD-1
AB367789
ND-DD-J4
LC159520
Yellow cattle #4
Yak #5
fluke #9
ITS2-DD-4
LC159514
ND-DD-C1
LC159521
fluke #10
ITS2-DD-5
LC159515
ND-DD-C2
LC159522
fluke #11
ITS2-DD-5
LC159515
ND-DD-C2
LC159522
fluke #12
ITS2-DD-4
LC159514
ND-DD-C4
LC159524
fluke #13
ITS2-DD-5
LC159515
ND-DD-C3
LC159523
fluke #14
ITS2-DD-6
LC159516
ND-DD-C5
LC159525
― 13 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Molecular phylogenetic relationship of Dicrocoelium dendriticum from Japan and China based on nucleotide sequences of the mitochondrial nad1
2-3．Molecular phylogenetic analyses
similarity was 99.2–99.6% within the genotypes from
Phylogenic tree of the nad1 sequences（659 bp）
Japan and China, while that was 98.3–99.6% between
was constructed with the Neighbor-joining method
genotypes from Japan and China. On the other hand,
in MEGA version 6.06［16］using the Tamura and
the genotypes showed a relatively low level of similarity
Nei model with gamma distribution［15］, which was
to other Dicrocoelium species; 95.4–95.8% to D. chinensis
selected with the maximum likelihood test based
and 86.6-87.9% to D. hospes, suggesting that all the
on A.I.C criteria. Node support was assessed with
14 flukes analyzed in this study were identified as D.
1,000 bootstrap replicates. The nad1 sequences of D.
dendriticum based on the molecular marker（Table 1
chinensis（LC164821）and Eurytrema pancreaticum
and Fig. 1）.
（KP241855）were used as reference sequences in the
The partial nucleotide sequences of nad1（659 bp）
phylogeny construction. The median-joining network
yielded 75 substitution sites, and 9 haplotypes were
inferred from the nad1 haplotypes was generated with
distinguished: ND-DD-J1 to ND-DD-J4 （LC159517–
Network 4.6.1.2 software［1］.
LC159520）from Japan and ND-DD-C1 to ND-DD-C5
（LC159521–LC159524）from China（Table 1）
. In the
phylogenetic tree, the nad1 haplotypes from Japan
３．RESULTS
and China were clearly divided into two separated
All the 14 flukes were morphologically identified as
clades with 89% support value（Fig. 2）
. The median-
D. dendriticum on the basis of the testes orientation
joining network showed that ND-DD-J2 and ND-DD-J3
［4, 21］. The nucleotide sequences of ITS2（239 bp）
might be main haplotypes in Japan. The haplotypes
yielded 5 substitution sites, and 6 genotypes were
detected from Japan have only one to three nucleotide
distinguished: ITS2-DD-1 to ITS2-DD-3（AB367789,
substitutions among them. On the other hand, the
LC159512 and LC159513）from Japan and ITS2-DD-4
haplotypes detected from China have 2–35 substitutions
to ITS2-DD-6（LC159514–LC159516）from China. ITS2-
among them: 2–35 substitutions within haplotypes
DD-6 was identical to a genotype（DQ379986）which
from Dingxi, and 27–35 substitutions between those
has been detected from Germany and Italy［10］. The
from Dingxi and Yushu. The nucleotide substitutions
Fig. 1.A multiple sequence alignment of the ITS2 genotypes（239 bp）detected in Dicrocoelium dendriticum
from Japan and China with Dicrocoelium chinensis（AB367790）and Dicrocoelium hospes（EF102026）.
― 14 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Kei HAYASHI, Maiko OHTORI, Yuma OHARI, Uday Kumar MOHANTA, WenQiang TANG, Tadashi ITAGAKI
between the haplotypes from Japan and China were
These results hypothesized that the D. dendriticum
48–70.
populations in Japan and China have been genetically
differentiated. In addition, the nad1 haplotypes detected
from China showed extremely higher variation than
４．DISCUSSION
the haplotypes from Japan, hypothesizing that the
In this study, the lancet flukes were identified as
D. dendriticum population in China is more ancient
D. dendriticum based on the morphology of testes
population than that in Japan（Fig. 3）. However, the
orientation［4, 21］and the nucleotide sequences of
numbers of localities, hosts and flukes analyzed in this
ITS2［9, 10］
. The genetic variation in ITS2 seemed
study were not enough to confirm those hypotheses.
to be intraspecific in comparison to the interspecific
Again, no fluke from sika deer was analyzed in this
level with D. chinensis and D. hospes. In addition, the
study, although the ruminant also can be a definitive
genotypes from Japan and China were identical or very
host of D. dendriticum along with Japanese serow in
similar to D. dendriticum from Germany and Italy
Japan［19］
. Therefore, further studies using additional
［10］. Therefore, the nucleotide sequence of ITS2 is
lancet flukes are required to elucidate the phylogenetic
considered to be a suitable marker for discriminating D.
relationship of D. dendriticum populations between
dendriticum from other Dicrocoelium species（Fig. 1）.
Japan and China, and to unveil their genetic history.
In the nad1 phylogenetic tree, D. dendriticum
This study provided the nucleotide sequences of nad1
haplotypes from Japan and China were clearly divided
gene of D. dendriticum in Japan for the first time, and
into two separated clades（Fig. 2）
, and the variation
the molecular features may provide useful information
between the haplotypes was extremely high（Fig. 3）.
for further phylogenetic studies on the species.
Fig. 2.Phylogenetic tree of Dicrocoelium dendriticum with Dicrocoelium chinensis（LC164821）and Eurytrema
pancreaticum（KP241855）based on the partial sequence（659 bp）of the nad1 region. The tree was
constructed with the Neighbor-joining method and the Tamura and Nei model with gamma distribution.
The node support was calculated with 1,000 bootstrap replicates.
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Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Molecular phylogenetic relationship of Dicrocoelium dendriticum from Japan and China based on nucleotide sequences of the mitochondrial nad1
Fig. 3.A median-joining network based on the mitochondrial nad1 haplotypes of Dicrocoelium dendriticum.
Each haplotype from Iwate, Dingxi and Yushu are shown in white, stripe and gray circle, respectively.
The haplotype codes are shown adjacent to the circles, and number on each circle and node indicates
the number of flukes and the number of substitutions, respectively. No label means only one substitution.
Small, dark circles on the node represent median vectors.
5. Lavikainen, A., Haukisalmi, V., Lehtinen, M. J.,
ACKNOWLEDGMENT
Henttonen, H., Oksanen, A. and Meri, S. 2008. A
This study was supported in part by the Grants-in-Aid
phylogeny of members of the family Taeniidae
for Science Research（B）and（C）
（grant nos. 23405044
based on the mitochondrial cox1 and nad1 gene
and 24580420）from the Ministry of Education, Culture,
data. Parasitology 135：1457-1467.
Sports, Science and Technology of Japan.
6. Liu, G. H., Yan, H. B., Otranto, D., Wang, X. Y., Zhao,
G. H., Jia, W. Z. and Zhu, X. Q. 2014. Dicrocoelium
chinensis and Dicrocoelium dendriticum（Trematoda:
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phylogenies. Mol. Biol. Evol. 16：37-48.
Mol. Phylogenet. Evol. 79：325-331.
7. Mohanta, U. K., Ichikawa-Seki, M., Hayashi, K.
2. Hayashi, K., Ichikawa-Seki, M., Mohanta, U. K.,
and Itagaki, T. 2015. Morphological and molecular
Singh, T. S., Shoriki, T., Sugiyama, H. and Itagaki, T.
characterization of Eurytrema cladorchis parasitizing
2015. Molecular phylogenetic analysis of Fasciola
cattle（Bos indicus）in Bangladesh. Parasitol. Res.
flukes from the eastern India. Parasitol. Int. 64：
114：2099-2105.
334-338.
8. Nakamura, T., Nakahari, J., Machida, N., Kiryu, K.
3. Itagaki, T. and Tsutsumi, K. 1998. Triploid form of
and Machida, M. 1984. Dicroceliasis in the wild
Fasciola in Japan: genetic relationships between
Japanese serow, Capricornis crispus. Jpn. J. Vet.
Fasciola hepatica and Fasciola gigantica determined
Sci. 46：405-408.
by ITS-2 sequence of nuclear rDNA. Int. J. Parasitol.
9. Ohtori, M., Aoki, M. and Itagaki, T. 2014. Distinct
28：777-781.
distribution of Dicrocoelium dendriticum and D.
4. Lapage, G. 1962. Mönnig’s veterinary helminthology
chinensis in Iwate prefecture, Japan, and a new
and entomology, 5th ed., Bailliere Tindall and Cox,
final host record for D. chinensis. J. Vet. Med. Sci.
London.
76：1415-1417.
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Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Kei HAYASHI, Maiko OHTORI, Yuma OHARI, Uday Kumar MOHANTA, WenQiang TANG, Tadashi ITAGAKI
10. Otranto, D., Rehbein, S., Weigl, S., Cantacessi, C.,
2725-2729.
Parisi, A., Lia, R. P. and Olson, P. D. 2007. Morphological
17. Tang, C. and Tang, L. 1985. Investigation on the
and molecular differentiation between Dicrocoelium
biology of a few Dicrocoelium species from sheep
dendriticum（Rudolphi, 1819）and Dicrocoelium
on Qinghai plateau. Acta Zool. Sin. 31：254-262.（in
chinensis（Sudarikov and Ryjikov, 1951）Tang and
Chinese）
Tang, 1978（Platyhelminthes: Digenea）. Acta Trop.
18. Yagisawa, M. 1978. Studies on zoonotic helminthes
104：91-98.
from mammals in northern Honshu, Japan. Hirosaki
11. Otranto, D. and Traversa, D. 2002. A review of
Med. J. 30：239-284.
dicrocoeliosis of ruminants including recent advances
19. Yamaguti, S. 1933. Studies on the helminth fauna
in the diagnosis and treatment. Vet. Parasitol.
of Japan. Part1. Trematodes of birds, reptiles and
107：317-335.
mammals. Jpn. J. Zool. 5：1-134.
12. Otranto, D. and Traversa, D. 2003. Dicrocoeliosis
20. Yamaguti, S. 1958. Digenea of mammals. pp. 800-
of ruminants: a little known fluke disease. Trends
979. In: Systema helminthum, Vol. 1. The digenetic
Parasitol. 19：12-15.
trematodes of vertebrates. Interscience Publishers
13. Soulsby, E. J. L. 1968. Helminths, arthropods and
protozoa of domesticated animals, 6th ed. Bailliere,
Inc., New York.
21. Yamaguti, S. 1971. Synopsis of digenetic trematodes
London.
of vertebrates, Keigaku publishing Ltd, Tokyo.
14. Taira, K., Shirasaka, S., Taira, N., Ando, Y. and
22. Zhao, G. H., Bian, Q. Q., Ren, W. X., Jia, Y. Q.,
Adachi, Y. 2006. Morphometry on lancet flukes
Cheng, W. Y., Fang, Y. Q., Song, J. K. and Lin,
found in Japanese sika deer（Cervus nippon centralis）
Q. 2013. Genetic variability among Dicrocoelium
captured in Iwate prefecture. J. Vet. Med. Sci.
dendriticum isolates from different regions in
68：375-377.
Shaanxi province, China revealed by sequences
15. Tamura, K and Nei, M. 1993. Estimation of the
of three mitochondrial genes. Mitochondrial DNA
number of nucleotide substitutions in the control
24：683-688.
region of mitochondrial DNA in humans and
chimpanzees. Mol. Biol. Evol. 10：512-526.
16. Tamura, K., Stecher, G., Peterson, D., Filipski, A. and
Kumar, S. 2013. MEGA6: molecular evolutionary
genetics analysis version 6.0. Mol. Biol. Evol. 30：
Correspondence：Tadashi ITAGAKI, Laboratory of Veterinary
Parasitology, Faculty of Agriculture, Iwate University, Ueda
3-18-8, Morioka 020-8550, Japan.
E-mail：address: itagaki@iwate-u.ac.jp
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Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Molecular phylogenetic relationship of Dicrocoelium dendriticum from Japan and China based on nucleotide sequences of the mitochondrial nad1
日本および中国産 Dicrocoelium dendriticum の分子系統解析
林　慶 1,2、鳳　麻衣子 1、尾針　由真 1,2、モハンタ・ウドイ・クマル 1,2、唐　文强 3、板垣　匡 1,2
1
2
岩手大学　農学部　獣医寄生虫学研究室、
岐阜大学大学院　連合獣医学研究科、3 チベット農牧科学院
要　約
日本および中国間における槍形吸虫 Dicrocoelium dendriticum の分子遺伝学的関係について解析を行った。解析
には日本のニホンカモシカ 3 頭および中国のヤク 1 頭、黄牛 1 頭から得られた槍形吸虫 14 隻を用いた。まず、精巣
の位置およびリボソーム DNA の ITS2 領域（239 bp）の塩基配列に基づき、全虫体の種同定を行った。その結果、
解析に用いた 14 隻はいずれも D. dendriticum と同定された。次に、ミトコンドリア DNA の nad1 領域（659 bp）
を用いた系統樹およびネットワークを作製した。その結果、日本および中国の D. dendriticum は系統樹において
異なるクレードを形成した。また、各クレード間の塩基置換率は非常に大きく、日本および中国の間において D.
dendriticum は遺伝的に分化している可能性があると考えられた。本研究は日本の D. dendriticum における nad1 領
域の塩基配列を初めて報告した。
Key words：槍形吸虫、Dicrocoelium dendriticum、中国、日本、ITS2、nad1
― 18 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Note
Molecular identification of Cryptosporidium isolates
from pet birds in Japan
Niichiro ABE 1, Ikuko MAKINO 2, Atsushi KOJIMA 3
1
Department of Microbiology, Osaka City Institute of Public Health and Environmental Sciences
2
3
Fujisawa Avian Clinic, Fujisawa, Kanagawa
Little Animal and Bird Clinic Little Bird, Setagaya-ku, Tokyo
ABSTRACT
Cryptosporidium spp. are important pathogens for humans and animals. Cases of infection by C. parvum, C.
hominis, C. meleagridis, C. andersoni, and C. muris with zoonotic potential have also been reported in domestic and
wild birds. Recent studies have revealed the presence of at least 13 host-adapted genotypes in birds. It is generally
difficult to discern their oocysts accurately by morphology among Cryptosporidium species and genotypes. In
Japan, 2 species（C. baileyi and C. meleagridis）and 2 genotypes（avian genotypes Ⅲ and Ⅴ）have been identified
molecularly in pet（cockatiels and peach-faced lovebirds）and domestic（chickens）birds, but the presence of
other species and/or genotypes in birds remains unclear. In this study, we attempted to identify 7 isolates from
3 cockatiels, 1 budgerigar, 1 masked lovebird, 1 Pacific parrotlet, and 1 Java sparrow, which were raised by
individual owners, using sequence analysis of Cryptosporidium actin locus. Analysis identified avian genotype V in
cockatiels and a budgerigar, avian genotype Ⅲ in a masked lovebird, C. galli in a Pacific parrotlet, and C. baileyi
in a Java sparrow. This report is the first of a study identifying the presence of avian genotype Ⅴ and C. baileyi
in budgerigar and Java sparrow in Japan, respectively. This study also demonstrated Pacific parrotlet（Forpus
coelestis）as a new host record of C. galli.
Key words：Cryptosporidium, avian genotype Ⅴ, avian genotype Ⅲ, C. baileyi, C. galli.
Seven species of Cryptosporidium, an important
renal or cloacal illness［7, 13, 17］.It is generally difficult
pathogen in humans and animals, have been reported
to discriminate accurately among Cryptosporidium
in domestic birds: C. meleagridis, C. baileyi, C. galli,
species and genotypes using light microscopy because
C. parvum, C. hominis, C. muris, and C. andersoni
of the morphological similarity of their oocysts. The
［17］.The five species aside from C. baileyi and C. galli
host specificity of Cryptosporidium genotypes found
have also been recognized as zoonotic Cryptosporidium
in birds remains unclear. Moreover, it is important
［21］. In addition to these valid species, the following
to identify the avian isolates accurately, particularly
13 genotypes are known: avian genotypes Ⅰ-Ⅵ
pet birds that are in close contact with humans in
（identified in many avian species）, goose genotypes
everyday life and which could be a source of human
（Branta canadensis）Ⅰ-Ⅴ, black duck（Anas rubripes）
infection. Cryptosporidium parasite was first isolated
genotype, and Eurasian woodcock（Scolopax rusticola）
from chickens in domestic birds［10］.Later, this isolate
genotype［6, 17］. Among these species and genotypes,
was identified as C. baileyi using multilocus sequence
C. meleagridis, C. baileyi, C. galli, avian genotype
analysis［11］. Cryptosporidium parasites were also
Ⅲ , and possibly avian genotype V are recognized as
, but
found histopathologically in Japanese quail［14］
important pathogens associated with mortality, weight
they have not been identified. Recently, several isolates
loss, diarrhea, respiratory illness, chronic vomiting, and
from pet birds in Japan were identified molecularly.
― 19 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Molecular identification of Cryptosporidium isolates from pet birds in Japan
Infection of C. baileyi, C. meleagridis, avian genotype
using a QIAamp DNA Stool Mini Kit and DNeasy
Ⅲ or Ⅴ in cockatiels and peach-faced lovebirds has
Blood and Tissue Kit（Qiagen GmbH, Hilden, Germany）
,
been reported［1-3, 13］
. This study identified seven
respectively, according to the manufacturer’s instructions.
isolates from five species of pet birds using sequence
The Cryptosporidium actin gene fragment（approximately
analysis of Cryptosporidium actin locus.
1100 bp） was amplified following the nested PCR
Fecal samples from seven pet birds（three cockatiels,
protocol［23］. PCR amplification was performed in a
one budgerigar, one masked lovebird, one Pacific
volume of 50μl containing 1× PCR buffer, 2 mM MgCl2,
parrotlet, and one Java sparrow）were collected during
250μM of each dNTP, 0.5μM of each primer, 1.25
2012–2014 at Little Animal and Bird Clinic Little Bird
units of TaKaRa Ex Taq Hot Start Version（Takara
and Fujisawa Avian Clinic（Table 1）. They were found
Shuzo Co. Ltd., Otsu, Shiga, Japan）, and 5μl of DNA
to be positive for Cryptosporidium infection by light
sample. Reactions were performed using a GeneAmp
microscopy using sucrose centrifugal flotation. All
PCR System 9700 thermocycler（Applied Biosystems,
birds had been kept at separate households. The two
CA, U.S.A.）
. The PCR products were purified using the
cockatiels（original hosts of isolate codes 0306-01 and
QIAquick Gel Extraction or QIAquick PCR Purification
120420-1）died during treatment. One cockatiel（0306-
Kit（QIAGEN GmbH, Hilden, Mettmann, Germany）,
01）was provided for autopsy. Small pieces of each
and were sequenced in both directions on an automated
internal organ（crop, proventriculus, small intestine,
sequencer（ABI 3130; Applied Biosystems, Foster City,
cloaca, trachea, kidney, liver）collected using disposable
. Sequence chromatograms
Carlsbad, California, U.S.A.）
tweezers were put into individual 1.5-ml tubes
from each strand were inspected using the SEQUENCHER
containing 70% ethanol. These samples were provided
Version 4.1（Gene Codes Corp., Ann Arbor, MI, U.S.A.）.
for identification of the location of Cryptosporidium
Nucleotide similarity searching of the obtained partial
found in a fecal sample of this cockatiel by PCR sequence
gene sequences was performed using the FASTA
analysis, as reported previously ［3］. DNAs were
program（EMBL; http://www.ebi.ac.uk/ Tools/fasta33/
extracted and purified from fecal and tissue samples
nucleotide.html）
.
Table 1. Cryptosporidium isolates from pet birds identified in the present study
Isolate code
0306-01
0016-01
120420-1
3503-58
733-3
0018-03
0224-01
a
Host detailsa
Nymphicus hollandicus; cockatiel; 2 months;
emaciation, diarrhea and death
Nymphicus hollandicus; cockatiel; 3 months;
dispepsia
Nymphicus hollandicus; cockatiel; 2 months;
emaciation, diarrhea and death
Melopsittacus undulatus; budgerigar; 2 months;
diarrhea and vomiting
Agapornis personata; masked lovebird; 6 years;
no symptom
Forpus coelestis; Pacific parrotlet; >2 years;
diabetes and proventriculus distension
Padda oryzivora; Java sparrow; 2 months;
diarrehea
Identification at actin locus
avian genotype Ⅴ
avian genotype Ⅴ
avian genotype Ⅴ
avian genotype Ⅴ
avian genotype Ⅲ
100% identity（978/978 bp）to those from avian
genotype Ⅴ（AB471660, AB471661, JQ320301）
100% identity（978/978 bp）to those from avian
genotype Ⅴ（AB471660, AB471661, JQ320301）
100% identity（978/978 bp）to those from avian
genotype Ⅴ（AB471660, AB471661, JQ320301）
100% identity（978/978 bp）to those from avian
genotype Ⅴ（AB471660, AB471661, JQ320301）
100% identity（1000/1000 bp）to those from avian
genotype Ⅲ（AB471655-AB471659）
100%（987/987 bp, 921/921 bp）or 99.9%（961/962
C. galli
bp, 927/928 bp）identity to those from C. galli
（AY163901, EU53265-EU543267, EU543265）
C. baileyi
Descibed in order of scientific name, common name, age of the bird, and clinical symptoms.
― 20 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Most highest nucleotide sequence identity
100% identity（990/990 bp）to those from C. baileyi
（AF382346, EU741840-EU741852）
Niichiro ABE, Ikuko MAKINO, Atsushi KOJIMA
Partial actin gene nucleotide sequences（978–1000
resulted from an infection of avian genotype V because
bp） were obtained from all isolates examined in
we were unable to examine other pathogens such as
this study. Sequences from four isolates from three
Candida spp., Escherichia coli, Salmonella spp., and
cockatiels and one budgerigar were identical among
various viruses that are recognized as concurrent
those isolates and were also identical to those of
pathogens of intestinal cryptosporidiosis ［5, 12］.
Cryptosporidium avian genotype V（Table 1）.The PCR
The site of infection in avian hosts of this genotype
was positive in six samples（crop, proventriculus, small
has been found by scanning electron microscopical
intestine, cloaca, trachea, kidney）except for the liver.
or histopathological examination to be the ileum,
The sequences from those amplicons were mutually
cecum, ureter, and cloaca［7, 9］. In the present study,
identical and also identical to that of avian genotype
Cryptosporidium DNA was detected in the trachea,
. The sequence of the isolate from a
V（AB471660）
crop, proventriculus, small intestine, cloaca, and kidney
masked lovebird was identical to that of avian genotype
collected from a dead cockatiel（0306-01）. Recently, we
Ⅲ. The sequences of the two isolates from a Pacific
also identified the trachea and cloaca as the possible
parrotlet and a Java sparrow were also identical to
location of this genotype in avian hosts by PCR for
those of C. galli and C. baileyi, respectively（Table 1）.
actin and 18S ribosomal DNA loci using the tissue
Results demonstrated that intragenotype or intraspecies
DNA samples from a severely emaciated dead cockatiel
variations are low or absent at the actin locus in
［3］. Considering both the present and the previous［3］
Cryptosporidium［23］. Therefore, we identified the
PCR data, we strongly presume the possible location
present isolates as Cryptosporidium avian genotypes Ⅲ,
of this genotype as the trachea, which is true also for
V, C. galli, and C. baileyi.
C. baileyi. Further histological examination of trachea
Cryptosporidium avian genotype V, recently proposed
in birds infected with avian genotype V must be
, was
as a new Cryptosporidium species（C. avium）
［9］
conducted to examine this possibility.
first found in fecal samples from two cockatiels caged
In Japan, C. baileyi has been identified only in
at the same pet shop in Japan［2］. Subsequently, it has
domestic chickens and pet cockatiels ［1, 11］. In
been identified in cockatiels and a budgerigar in China
addition, the avian genotype V has been identified only
［19, 24］and in a blue-fronted parrot in Brazil［16］, in
in pet cockatiels［2, 3］. Therefore this report is the
a pet Major Mitchell’s cockatoo in U.S.A.［7］. Recently,
first of a study identifying the presence of C. baileyi
we also identified this genotype in a cockatiel［3］. In
and avian genotype V in Java sparrow and budgerigar,
the present study, the infection with this genotype was
respectively in Japan. Reports of the literature show
confirmed in the three cockatiels. Although we were
that C. galli has been identified in at least 32 avian
unable to perform a follow-up survey of the origin, avian
, but it has not been
species［4, 6, 8, 15, 16, 18-20, 22］
genotype V might be widely distributed throughout
found in the Pacific parrotlet （Forpus coelestis）.
the cockatiel population of Japan. The pathogenicity
Therefore, the present report also identified a new host
of this genotype in avian hosts has remained unclear
of C. galli.
because of a lack of clinical reports associated with
About 5,000 psittaciform birds have been introduced
this genotype infection［2, 9, 16, 19, 24］. All birds
annually into Japan during the most recent five years
infected with this genotype examined in the present
（2011–2015）. These birds are mainly imported from
study showed mainly digestive symptoms（Table 1）. A
Belgium, the United States of America, the Philippines,
recent report has also described a fatal case with renal
and Singapore（This information is available at the
and cloacal cryptosporidiosis caused by this genotype
following URLS, but all descriptions at these sites are in
in a Major Mitchell’s cockatoo（Cacatua leadbeteri）
［7］,
Japanese: http://www.mhlw.go.jp/stf/ seisakunitsuite/
suggesting possible pathogenicity of avian genotype
bunya/0000069864.html）. In addition, a few birds are
V. However, it was not possible to ascertain whether
bred in households or breeder farms in Japan. Because
the digestive symptoms found in the present study
no survey of Cryptosporidium infection in birds has
― 21 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Molecular identification of Cryptosporidium isolates from pet birds in Japan
been performed in exporting countries and/or in Japan,
Mitchell’s cockatoo（Lophochroa leadbeateri）. J.
the origins of Cryptosporidium- parasite-infected pet
Zoo. Wildl. Med. 46：934-937.
birds in Japan remain unclear. Global epizootiological
8. da Silva, D. C., Homem, C. G., Nakamura, A. A.,
studies of Cryptosporidium infection in pet birds must
Teixeira, W. F. P., Perri, S. H. V. and Meireles, M.
be undertaken to control Cryptosporidium infection
V. 2010. Physical, epidemiological, and molecular
among pet bird populations, and to clarify the infection
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sources and routes of transmission.
Passeriformes. Parasitol. Res. 107：271-277.
9. Holubová, N., Sak, B., Horčičková, M., Hlásková, L.,
Kvĕtoňová, D., Menchaca, S., McEvoy, J. and Kváč
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16. Nakamura, A. A., Homem, C. G., da Silva, A. M.
7. Curtiss, J. B., Leone, A. M., Wellehan, J. F. X.,
J. and Meireles, M. V. 2014. Diagnosis of gastric
Emerson, J. A., Howerth, E. W. and Farina, L.
cryptosporidiosis in birds using a duplex real-time
L. 2015. Renal and cloacal cryptosporidiosis
PCR assay. Vet. Parasitol. 205：7-13.
（Cryptosporidium avian genotype V）in a major
17. Nakamura, A. A. and Meireles, M. V. 2015.
― 22 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Niichiro ABE, Ikuko MAKINO, Atsushi KOJIMA
Cryptosporidium infections in birds - a review.
Guimarães, M. B., Souza, S. O., Allegretti, L.,
Braz. J. Vet. Parasitol. 24：253-267.
Sinhorini, J. A., Duarte, V. V. and Soares, R. M.
18. Ng, J., Pavlasek, I. and Ryan, U. 2006. Identification
2011. Genotyping of Cryptosporidium spp. from
of novel Cryptosporidium genotypes from avian
free-living wild birds from Brazil. Vet. Parasitol.
hosts. Appl. Environ. Microbiol. 72：7548-7553.
175：27-32.
19. Qi, M., Wang, R., Ning, C., Li, X., Zhang, L., Jian,
23. Sulaiman, I. M., Lal, A. A. and Xiao, L. 2002.
F., Sun, Y. and Xiao, L. 2011. Cryptosporidium spp.
Molecular phylogeny and evolutionary relationships
in pet birds: genetic diversity and potential public
of Cryptosporidium parasites at the actin locus. J.
health significance. Exp. Parasitol. 128：336-340.
Parasitol. 88：388-394.
20. Ryan, U. M., Xiao, L., Read, C., Sulaiman, I. M.,
24. Zhang, X. X., Zhang, N. Z., Zhao, G. H., Zhao, Q.
Monis, P., Lal, A. A., Fayer, R. and Pavlasek, I.
and Zhu, X. Q. 2015. Prevalence and genotyping of
2003. A redescription of Cryptosporidium galli
Cryptosporidium infection in pet parrots in North
Pavlasek, 1999（Apicomplexa: Cryptosporidiidae）
China. BioMed. Res. Int. 2015：1-6.
from birds. J. Parasitol. 89：809-813.
21. Ryan, U., Fayer, R. and Xiao, L. 2014. Cryptosporidium
species in humans and animals: current understanding
and research needs. Parasitology 141：1667-1685.
22. Sevá, A. P., Funada, M. R., Richtzenhain, L.,
Correspondence：Niichiro ABE, Osaka City Institute of Public
Health and Environmental Sciences, Tennoji–ku, Osaka 5430026, Japan.
E-mail：niichiro@gmail.com
遺伝子解析による小鳥由来 Cryptosporidium 株の同定
阿部　仁一郎 1、牧野　幾子 2、小嶋　篤史 3
1
2
大阪市立環境科学研究所微生物保健グループ、
ふじさわアビアンクリニック、3 鳥と小動物の病院リトル・バード
要　約
クリプトスポリジウムはヒトと動物に寄生する原虫で、鳥類では人獣共通寄生性 5 種と少なくとも 13 の遺伝子型
の報告がある。日本では、C. baileyi（オカメインコ、鶏）、C. meleagridis（オカメインコ）、avian genotype Ⅲ（コ
ザクラインコ）
、Ⅴ（オカメインコ）が確認されていたが他種寄生状況は不明であった。今回、本原虫の actin 遺伝子
領域における塩基配列の相同性検索により鳥類由来 7 株の同定を試みたところ、オカメインコ由来 3 株とセキセイイ
ンコ由来 1 株を avian genotype Ⅴ、キエリクロボタンインコ由来 1 株を avian genotype Ⅲ、
マメルリハ由来 1 株を C.
galli、文鳥由来 1 株を C. baileyi と同定した。国内のセキセイインコと文鳥に avian genotype V と C. baileyi が各々
寄生していることを初めて確認し、マメルリハを C. galli の新宿主として報告した。
Key words：Cryptosporidium、avian genotype Ⅴ、avian genotype Ⅲ、C. baileyi、C. galli
― 23 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Review
Brief review on atypical human trypanosomiasis of
Trypanosoma lewisi
Eliakunda MAFIE 1, Fatema Hashem RUPA 1, Aogu SETSUDA 1,
Atsuko SAITO-ITO 2, Hiroshi SATO 1
1
Laboratory of Parasitology, United Graduate School of Veterinary Science, Yamaguchi University
2
Section of Microbiology, School of Pharmacy, Hyogo University of Health Sciences
ABSTRACT
Trypanosomes（Kinetoplastea: Trypanosomatida: Trypanosomatidae）, particularly salivarian trypanosome
species as well as stercorarian Trypanosoma cruzi, are important parasites of humans and other animals that cause
often fatal diseases. Stercorarian T. lewisi is known as a rat-specific species of the subgenus Herpetosoma. Rodent
trypanosomes cause latent infections, and T. lewisi infection beyond the genus border, e.g. infection in mice, is
considered to be virtually impossible. Nevertheless, nine human cases of T. lewisi infection have been reported
in recent decades, with an increased incidence（five cases）in the last two decades. In the present review, we
summarize the records of atypical human trypanosomiasis ascribed to T. lewisi infection and provide information
on the background of disease incidences and possible PCR-based diagnostic approaches.
Key words：Trypanosoma lewisi, atypical human trypanosomiasis, Herpetosoma, rodent trypanosome, rDNA.
rodents（ca. 50 nominal species）［7］
. Members of
１．INTRODUCTION
Herpetosoma exhibit an almost identical morphology
Hemoflagellates of the genus Trypanosoma
of bloodstream trypomastigote forms. However, they
（Kinetoplastea: Trypanosomatida: Trypanosomatidae）
usually have a strict host specificity, so no infection
have a wide range of hosts（fish, amphibians, reptiles,
occurs beyond the families, subfamilies, or genera of
birds, and mammals）and are transmitted by blood-
host rodents［7］. By use of special rodent hosts such
feeding invertebrates. Members of the genus are
as Mongolian jirds（Meriones unguiculatus）treated
conveniently divided into two major groups, i.e. Salivaria
with immunosuppressants, the partial host barrier
and Stercoraria, based on their mode of transmission
to Herpetosoma infection can be overcome. This was
［7］. Salivarian trypanosomes are transmitted to the
demonstrated by the successful experimental infection
recipient via the saliva of vectors, whereas stercorarian
of T. grosi and T. lewisi, which take Apodemus
trypanosomes are transmitted via vector feces or
spp. or Rattus spp. as natural hosts, respectively, in
vectors themselves. The former group of trypanosomes
immunosuppressed Mongolian jirds［23-25］
.
（e.g. Trypanosoma brucei, T. vivax, and T. congolense）
,
transmitted by tsetse flies, is of medical and/or
veterinary importance. The diseases caused by the
latter group（stercorarians）
, with the exception of T.
２．HUMAN CASES OF T. lewisi
INFECTION
cruzi, are latent.
Human cases of T. lewisi infection have been reported
Trypanosoma lewisi with a cosmopolitan distribution
in different parts of the world. The first noted infection
is the type species of the subgenus Herpetosoma,
was in 1933 in a four-month-old infant living in Malaysia
which includes a variety of trypanosome species of
（Table 1）
［10, 31, 33］. More than 40 years later, two
― 24 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Eliakunda MAFIE, Fatema Hashem RUPA, Aogu SETSUDA, Atsuko SAITO-ITO, Hiroshi SATO
Table 1. Human cases of Trypanosoma lewisi or T. lewisi-like trypanosome infection ＊
Case No. Country
Age and sex of patient
Trypanosome
species
Year of notice
Method of species
identification
Symptom
Prognosis
Reference
1
Malaysia
4-month-old infant
T. lewisi
1933
Morphology
Fever
Self cure
［10］
2
India（Madhya Pradesh）
One of a married couple
T. lewisi
1974
Morphology
Fever
Self cure
［30］
3
India（Madhya Pradesh）
One of a married couple
T. lewisi
1974
Morphology
Fever
Self cure
［30］
4
Gambia
a baby
T. lewisi-like
2003
Genetic（ITS1）
Fever
Cure
［8］
5
Thailand
45-day-old infant
T. lewisi-like
2003
Genetic
Fever
Cure
［22］
6
India（Mumbai）
1.5-month-old girl
T. lewisi
2006
Morphology
Fever
Self cure
［13, 28］
7
India（Pune）
57-year-old man
T. lewisi
2007
Genetic
Fever
Death
［5］
8
India（Bagpat）
37-day-old infant
T. lewisi
2010
Genetic
Fever
Cure
［37］
9
India（Madhya Pradesh）
40-year-old man ＊＊
T. lewisi
2014
Morphology
Fever
Cure
［38］
＊
Modified after Truc et al.［33］.
＊＊
Immunocompromised person with HIV infection and intermittent febrile episodes for 12 months due to chronic malaria.
other cases of T. lewisi infection were reported – a
that the actual prevalence is probably underestimated
married couple living in a village in Madhya Pradesh,
based on the following study. From a seroepidemiological
India［30］. In these three cases, the trypanosomes in
survey conducted in a village where a patient with T.
blood films were identified morphologically, and all
evansi infection was identified, 22.7% of the residents
the cases were spontaneously cured. After another
（410/1,806）were found to be antibody-positive by a
extensive time period, six more human cases of T.
card agglutination test for T. evansi（CATT/T. evansi;
lewisi infection were reported（Gambia, one case in
Institute of Tropical Medicine, Antwerp, Belgium）with
2003; Thailand, one case in 2003; and India, four cases
a 1:4 dilution of whole blood. Positive residents were
in 2006, 2007, 2010, and 2014）
［5, 8, 13, 22, 28, 37, 38］.
subsequently subjected to serosurvey using serum
Major symptoms of these T. lewisi-infected patients
samples and a card agglutination test for trypanosomiasis
included fever, coughing, anorexia, and depression［12,
. The prevalence
（CATT; Institute of Tropical Medicine）
22, 28, 37］. In the most recent cases, anti-trypanosome
of confirmed sero-positive residents was reduced to 4.5%
drugs were effectively used with a good prognosis［33,
（81/1,806）and no trypanosomes were microscopically
38］.
detected in them［29］, suggesting substantial exposure
In addition to these nine human cases of T. lewisi
occasions of human residents to T. evansi in the
infection, atypical human trypanosomiasis has been
surveyed area. Considering the recent geographical
caused by T. brucei brucei（four cases in 1930, 1947,
expansion of T. evansi in North Africa, Middle East,
1987, and 2003）, T. congolense（one case in 1998）,
southern Eurasia, South Asia, and South America［4,
and T. evansi（five cases in 1977, 1999, 2004, 2005,
15］,it is highly possible that human exposure occasions
and 2010）
［11, 33］. The five human cases of T. evansi
to the trypanosomes might be increased in the near
infection were reported in India（three cases）, Sri
future, as is the case with domestic and wild animals at
Lanka（one case）, and Egypt（one case）, and other
present.
human cases of salivarian animal trypanosomes have
In India, epidemiological surveys of T. lewisi in
been reported in the Sub-Saharan region［33］. Patients
rats, conducted in areas surrounding patients’ houses,
infected with salivarian animal trypanosomes had a
showed prevalences of around 20%［5, 37］. These
fluctuating trypanosome parasitemia associated with
values are similar to those from other field studies
febrile episodes for a long time period, e.g. five months
in other areas in different countries such as Brazil,
［12］.
Ecuador, Cambodia, Lao PDR, Thailand, and China［9,
Due to an increasing number of reports of patients
14, 20, 21, 32］. Maia de Silva et al.［17］reported T.
with atypical trypanosomiasis in the last two decades,
lewisi infection in Brazilian monkeys in captivity at a
T. evansi and T. lewisi are now regarded as emerging
rate of 3/160（two Callithrix jacchus and one Alouatta
protozoan pathogens in humans. Truc et al.［33］proposed
fusca）.
― 25 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Brief review on atypical human trypanosomiasis of Trypanosoma lewisi
brucei could not survive in 0.0001% NHS or 0.1 μg/ml
３．FACTOR（S）RESPONSIBLE
FOR HOST SPECIFICITY OF
TRYPANOSOMES
rhapoL-I. Furthermore, their in vivo experiments using
T. lewisi- and T. brucei brucei- infected rats and NHS
injections supported their in vitro results, revealing
Trypanosoma lewisi and other rodent trypanosomes,
that T. lewisi might be fully resistant to NHS［16］.
often referred to as T. lewisi-like due to their similar
Apart from this finding, it is thought that the inability
morphology, are transmitted by fleas whereby rats
of T. lewisi to infect a range of mammals is associated
are infected by either licking flea feces on their fur
with the trypanolytic activity of both the complement
or ingesting infected fleas［1, 7］. Human infection
. Consequently, immature
and granulocytes ［1, 6］
requires an infestation of fleas or flea feces, with most
or depressed immune systems allow the host to be
human cases being associated with patients living in
infected with rat trypanosomes as an opportunistic
non-hygienic houses with potential exposure to rat
parasite. Therefore, considering that five out of the
fleas and/or their feces. Once infected, human innate
nine aforementioned human cases of T. lewisi infection
immunity against atypical trypanosomes must work to
were reported in unweaned babies between 1.5 and
eliminate the hemoflagellates.
4 months of age（Table 1）, it appears that atypical
Typical human trypanosomiasis is caused by T. brucei
human trypanosomiasis requires not only exposure
gambiense which causes chronic sleeping sickness in
chances to infected fleas but also a defective innate
West Africa, T. brucei rhodesiense which causes acute
immunity in hosts.
sleeping sickness in East Africa, and T. cruzi which
causes Chagas disease in South America. These human
trypanosomes are capable of infecting humans because
they are resistant to normal human serum（NHS）,
４．MOLECULAR DIAGNOSIS OF
ATYPICAL TRYPANOSOMIASIS
i.e. trypanolysis induced by human serum protein
The levels of parasitemia in atypical trypanosomiasis
apolipoprotein L-1（apoL-I）, while animal trypanosomes
are usually too low to provide sufficient numbers of
are susceptible to this protein and cannot survive
bloodstream trypomastigotes for a specific diagnosis
in human blood ［19, 34］. A lack of apoL-I was
based on morphology. In addition, as mentioned above,
demonstrated in patients with clinical manifestation of
rodent trypanosomes of the subgenus Herpetosoma
atypical trypanosomiasis due to T. evansi［35］.
show an almost identical morphology, making specific
The apoL-I protein contains an ionic pore-forming
identification difficult. Hence, molecular diagnosis of
domain and an adjacent pH-sensitive membrane-
atypical trypanosomiasis is essentially necessary for all
addressing domain［19］. After endocytosis, the protein
cases［33］
. Cases of atypical trypanosomiasis caused
triggers the formation of anion-selective pores in the
by T. lewisi or T. lewisi-like trypanosomes in human
lysosomal membrane, inducing osmotic swelling of the
patients and non-human primates have been diagnosed
lysosomes and subsequent trypanolysis［19, 34, 36］
.
by molecular technologies using PCR amplification and/
Human trypanosomes such as T. brucei rhodesiense
or nucleotide sequencing of the ribosomal RNA gene
inhibit apoL-I activity by expressing a truncated form
（rDNA）
［17, 22］.
of the variant surface glycoprotein, termed serum
As illustrated in Figures 3 and 5 of Sato et al.［26］,
resistance-associated protein, which is a lysosomal
almost the full length of the 18S rDNA has been
.
protein［34］
sequenced and deposited for only a portion of the rodent
Lun et al.［16］recently reported that T. lewisi is
trypanosomes. Out of approximately 50 nominal species
resistant to NHS containing trypanolytic apoL-I. This
of the subgenus Herpetosoma［7］, at present just eight
perception is based on their findings that T. lewisi
of them are contained in the database provided by the
could resist up to 90% NHS or 30μg/ml recombinant
National Center for Biotechnology Information（NCBI;
human apoL-I（rhapoL-I）in vitro. In contrast, T. brucei
http://www.ncbi.nlm. nih.gov/taxonomy）
. However, a few
― 26 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Eliakunda MAFIE, Fatema Hashem RUPA, Aogu SETSUDA, Atsuko SAITO-ITO, Hiroshi SATO
Fig. 1. Maximum likelihood（ML）phylogenetic tree based on the 18S rDNA sequences.
The nucleotide sequences of rodent trypanosomes（the subgenus Herpetosoma）were retrieved from the DDBJ/ EMBL/
GenBank databases, and ML analysis was performed as described in one of our previous studies［27］. Regions judged to be
poorly aligned and characters with a gap in any sequences were excluded from subsequent analyses; 2,109 characters, of
which 49 were variable, remained for subsequent analysis. The probability of inferred branch was assessed by the approximate
likelihood-ratio test as previously described［27］. Two trypanosome species of Sciuridae（two isolates of T. otospermophili and
one isolate of T. kuseli）were used as an outgroup for the construction of the ML phylogenetic tree.
more sequences of other nominal rodent trypanosomes
（such as T. nabiasi and T. evotomys）and multiple
even more than the interspecific variations among the
aforementioned Herpetosoma species.
anominal species have been deposited. Previous studies
The ITS1 region of T. lewisi and T. lewisi-like
on the genetic characterization of the 18S rDNA
trypanosomes is characterized by multiple repeats of
and/or the glycosomal glyceraldehyde-3-phosphate
single, double, or longer nucleotide units such as‘TA’
,
dehydrogenase（gGAPDH）gene were conducted to
‘TG’
, and‘AC’. The numbers of repeats of such few
clarify the interspecific relationships of mammalian
nucleotide units often vary even in a single parasite
trypanosomes. To improve the resolution of species
individual or an isolate of some helminths［18, 27］;
identification, the internal transcribed spacer 1（ITS1）
however, little is known in the case of trypanosomes.
region of rDNA has recently been used to discriminate
Tang et al.［32］reported that 16 T. lewisi isolates
T. lewisi and its isolates from atypical trypanosomiasis
from Guangzhou（Southern China）had the same ITS1
in humans and non-human primates［2, 3, 17, 22］.
sequence, which was slightly different from other
As shown in Fig. 1 and Table 2, as far as is currently
isolates from Jilin（Northern China）and Thailand.
known, multiple nominal species such as T. lewisi, T.
Table 3 summarizes the nucleotide differences in
musculi, and T. blanchardi have an almost identical
the ITS1 region among T. lewisi and T. lewisi-like
18S rDNA nucleotide sequence with few nucleotide
trypanosomes（only those closest to T. lewisi, i.e. T.
substitutions. Furthermore, Brazilian T. lewisi
blanchardi and T. rabinowitschae）. Although these
isolates consistently display a nucleotide substitution
sequences are only representative at present, it would
in comparison to ones from other places（USA, UK,
appear that ITS1 nucleotide sequences can successfully
and Asia）
（Table 2）. Contrastingly, T. grosi isolates
discriminate T. lewisi from closely related T. lewisi-like
from Apodemus spp. in the Far East show substantial
species such as T. blanchardi and T. rabinowitschae.
intraspecific nucleotide variations, comparable to or
When eliminating nucleotide unit repeats, 11 nucleotide
― 27 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
TryBiIDN203
TryRoIDN204
TryCC 34
TryCC 35
TryCC 44
TryCC 1148
Af
TryCC 43
TryCC 124
T. lewisi
T. lewisi
T. lewisi
T. lewisi
T. lewisi
T. lewisi
T. lewisi
T. lewisi
T. lewisi
― 28 ―
SESUJI
AKHA
HANTO
T. grosi
T. grosi
T. grosi
Russia
Japan
Russia
China
France
China
France
UK
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Indonesia
Indonesia
Indonesia
AB175623
AB175624
AB175622
FJ694763
AY491765
AB242274
AY491764
AJ223568
GU252214
GU252212
GU252209
GU252215
GU252213
GU252211
GU252210
AB242273
AB242273
AB242273
Sequence
2219 bp
2219 bp
2219 bp
2159 bp
2155 bp
2147 bp
2155 bp
2187 bp
2168 bp
2168 bp
2168 bp
2168 bp
2168 bp
2168 bp
2168 bp
2147 bp
2147 bp
2147 bp
2147 bp
2155 bp
2187 bp
length
・
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40
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135
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186
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T
272
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274
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277
C
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・
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T
289
・
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C
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T
345
A
A
A
A
・
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T
501
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559
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T
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620
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T
706
C
C
C
C
・
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T
764
C
C
C
C
・
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T
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・
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G
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・
・
・
・
・
・
・
・
・
・
・
・
・
・
A
・
・
・
A
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
T
・
・
・
C
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
T
C
C
C
C
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
T
814 1023 1085 1323 1411 1477 1503 1540 1769 1965 2016 2127 2132
18S rDNA＊
Twenty-one 18S rDNA nucleotide sequences of T. lewisi and T. lewisi-like trypanosomes shown above were aligned by Clustal W multiple alignment program, with subsequent manual adjustment. By this processing, nucleotide positions were settled, and
Apodemus peninsulae
Apodemus speciosus
AJ009156
AJ223566
Accession no.
Philippines AB242273
UK
USA
Locality
which separate T. lewisi in Brazil from T. lewisi in other places are encased in a rectangle.
nucleotide position is expressed relative to the 5'-terminus of T. grosi SESUJI（DDBJ/EMBL/GenBank accession no. AB175622）. Dots denote an identical nucleotide to that of T. lewisi（AJ223566）, and gaps are indicated by '—'. Nucleotide substitutions
＊
Apodemus agrarius
Cha1
T. grosi
Apodemus agrarius
Cricetus cricetus
Niviventer confucianus
T. rabinowitschae
Eliomys quercinus
TryNcCHN503
Trypanosoma sp.
Mus musculus
Alouatta fusca
Rattus norvegicus
Rattus rattus
Rattus rattus
Rattus rattus
Rattus omanicus
Bandicota indica
Rattus argentiventer
Rattus rattus
T. blanchardi
LUM 343
Callithrix jacchus
TryRaIDN202
T. lewisi
T. musculi
Aotus sp.
TryRrPHL201
T. lewisi
Rattus norvegicus
Rattus sp.
ATCC 30085
Molteno B3
T. lewisi
Host
T. lewisi
Isolate
Species
Table 2. Nucleotide variations in the18S rDNA of T. lewisi and closely related T. lewisi-like trypanosomes
Brief review on atypical human trypanosomiasis of Trypanosoma lewisi
lewisi
lewisi
lewisi
lewisi
lewisi
lewisi
lewisi
lewisi
lewisi
T.
T.
T.
T.
T.
T.
T.
T.
T.
Af
TryCC 124
TryCC 34
TryCC 43
Rn02
rattus
norvegicus
norvegicus
rattus
Eliomys quercinus
Cricetus cricetus
Rattus norvegicus
Rattus norvegicus
Alouatta fusca
Callithrix jacchus
Rattus rattus
Aotus sp.
Rattus tanezumi
Rattus norvegicus
Homo sapiens
Rattus
Rattus
Rattus
Rattus
Host
France
France
China
China
Brazil
Brazil
Brazil
Brazil
Thailand
China
Thailand
Brazil
Brazil
China
Brazil
Locality
GU252223
GU252224
FJ011094
EU861192
GU252216
GU252221
GU252217
GU252219
HQ437158
FJ011095
DQ345394
GU252220
GU252222
EU599639
GU252218
Accession no.
A
A
・
・
・
・
・
・
・
・
・
16
T
・
・
・
・
・
G
G
G
G
G
G
G
G
G
24
A
・
・
・
（TA）×5
（TA）×5
（TA）×5
（TA）×5
（TA）×5
（TA）×4
（TA）×4
（TA）×4
・
T （TA）×1
T （TA）×1
T
T
T
T
T
T
T
T
T
33
35–44
C （TA）×3
・
・
・
・
・
・
C
C
・
・
・
・
・
・
・
・
・
—CA
—CC
・
・
・
—CA
—CA
CAC
—CA
・
・
69
T
・
・
・
47–49
— —A
・
・
・
・
・
・
・
・
・
・
・
・
・
・
83–84
——
・
TG
TG
・
・
A
A
A
A
A
A
A
A
A
89
G
・
・
・
G
G
G
G
G
G
G
G
G
G
G
93
T
・
・
・
ITS1 ＊
G
G
・
・
・
・
・
・
・
・
・
94
A
・
・
・
G
G
・
・
・
・
・
・
・
・
・
101
A
・
・
・
・
・
・
・
・
・
・
・
・
・
G
104
A
・
・
・
・
—
・
・
・
・
・
・
・
・
・
121
T
・
・
・
・
・
・
・
・
・
・
・
G
・
・
124
A
・
・
・
・
・
・
・
・
・
・
・
・
・
・
143
C
・
T
・
・
・
・
・
・
・
・
・
・
C
・
147
—
・
・
・
C
C
・
・
・
・
・
・
・
・
・
149
T
・
・
・
・
・
・
・
・
・
・
・
・
・
—
162
C
・
・
・
G
G
・
・
・
・
・
・
・
・
・
167
A
・
・
・
lewisi
lewisi
lewisi
lewisi
lewisi
lewisi
lewisi
lewisi
lewisi
T.
T.
T.
T.
T.
T.
T.
T.
T.
T. blanchardi
T. rabinowitschae
＊
See above.
GU252220
GU252222
EU599639
GU252218
lewisi
lewisi
lewisi
lewisi
T.
T.
T.
T.
GU252223
GU252224
FJ011094
EU861192
GU252216
GU252221
GU252217
GU252219
HQ437158
FJ011095
DQ345394
Accession no.
Species
Table 3.（continued）
・
・
・
・
・
・
・
・
・
・
—
169
C
・
・
・
・
・
・
・
・
・
・
・
・
C
・
170
A
・
・
・
・
・
・
・
・
・
・
・
・
T
・
189
G
・
・
・
・
・
・
・
・
・
・
・
・
・
G
191
A
・
・
・
C
C
・
・
・
・
・
・
・
・
・
193
T
・
・
・
A
A
・
・
・
・
・
・
・
・
・
212
G
・
・
・
・
・
・
・
・
・
・
・
C
・
・
218
T
・
・
・
・
・
A
・
・
・
・
・
・
G
・
219
—
・
・
・
・
・
・
・
・
・
・
・
・
T
・
223
—
・
・
・
C
C
・
・
・
・
・
・
・
・
・
230
A
・
・
・
G
G
・
・
・
・
・
・
・
・
・
（TA）×3
（TA）
×3
（TA）×4 +T
（TA）×5
（TA）×6
（TA）×6
（TA）×6
（TA）×7
（TA）×6
（TA）×4 +T
（TA）×6
236
241–254
A
（TA）×2
・
・
・ （TA）×5 +T
・
（TA）×7
———
———
・
・
・
・
・
・
・
・
・
—————
—————
・
・
・
・
・
・
・
・
・
ITS1 ＊
255–257
260–264
CAT
ATATA
・
・
———
・
・
・
G
G
・
・
・
・
・
・
・
・
・
352
A
・
・
・
・
・
G
G
G
G
G
G
G
G
G
・
・
（AT）×0
（AT）×0
（AT）×0
（AT）×0
（AT）×0
（AT）×0
（AT）×0
（AT）×0
（AT）×0
364
370–373
A （AT）×2
・
・
G （AT）×0
G （AT）×0
GT
GT
・
・
・
・
・
・
・
・
・
（AT）×4
（AT）×4
（AT）×0
（AT）×0
（AT）×0
（AT）×0
（AT）×0
（AT）×0
（AT）×0
（AT）×0
（AT）×0
374–375
376–383
——
（AT）×1
・
・
・
（AT）×0
・
（AT）×0
・
・
・
・
・
・
・
・
・
G
・
387
A
・
・
・
・
・
・
・
・
・
・
・
・
・
T
412
A
・
・
・
・
・
・
・
・
・
・
・
・
・
T
414
G
・
・
・
・
・
・
・
・
・
・
・
・
—
・
428
T
・
・
・
・
・
・
・
・
・
・
・
・
—
・
437
G
・
・
・
＊
Fifteen ITS1 nucleotide sequences of either T. lewisi, T. blanchardi or T. rabinowitschae shown above were aligned by Clustal W multiple alignment program, with subsequent manual adjustment. By this processing,
444 nucleotide positions were settled, and nucleotide position is expressed relative to the 5'-terminus of T. lewisi（DDBJ/EMBL/GenBank accession no. GU252220）. Dots denote an identical nucleotide to that of T. lewisi
（GU252220）,and gaps are indicated by '—'. T. blanchardi
T. rabinowitschae
TryCC 44
TryCC 1148
lewisi
lewisi
lewisi
lewisi
T.
T.
T.
T.
TryCC 35
Isolate
Species
Table 3. Nucleotide variations in the ITS1 region of T. lewisi and closely related T. lewisi-like trypanosomes（T. blanchardi and T. rabinowitschae）
Eliakunda MAFIE, Fatema Hashem RUPA, Aogu SETSUDA, Atsuko SAITO-ITO, Hiroshi SATO
― 29 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Brief review on atypical human trypanosomiasis of Trypanosoma lewisi
positions（16, 69, 94, 101, 149, 167, 193, 212, 230, 236, and
in number in the last two decades. Considering the
352 in Table 3）are relatively significant to discriminate
geographical expansion in distribution and increased
T. lewisi from these two closely related species, and five
prevalence of T. evansi in the Middle East, India,
nucleotide positions（24, 33, 89, 93, and 364 in Table 3）
Asia, and South America, and the wide spectrum of
are important to discriminate major genotypes of T.
vectors transmitting the trypanosomes（including the
lewisi. The almost identical ITS1 nucleotide sequences
genera Tabanus, Stomoxys, Haematopota, Chrysops,
of T. blanchardi and T. rabinowitschae（see Table 3）
and Lyperosia［4, 15］
）, the chance of human exposure
are in contrast to the more divergent 18S rDNA nucleotide
to infective T. evansi is likely to be increased at
sequences of these two species（see Table 2）. Thus,
the present time and in the future regardless of
the collection of more ITS1 nucleotide sequences of
the hygienic status of human living. In contrast, as
Herpetosoma trypanosomes is required to clarify the
vectors of T. lewisi are rat fleas and transmission of
significance of this region in inter- and intraspecific
T. lewisi occurs by human ingestion of flea feces or
discrimination. Fig. 2 illustrates the relationships among
fleas themselves, the hygienic status of human living
T. lewisi isolates collected to date based on their ITS1
is closely related to the occurrence of atypical human
nucleotide sequences.
trypanosomiasis regardless of geographical location
Finally, we would like to emphasize that phylogenetic
because the host （rats） and parasite （T. lewisi）
t r e e s us ing th e IT S re g i o n o f c l o s e l y relat ed
are cosmopolitan in distribution. Little is known in
Herpetosoma trypanosome species may only be valid
relation to how T. lewisi is able to infect humans.
if they are constructed extremely carefully. Usually it
However, it is apparent that some humans and non-
is not appropriate to use ITS-based phylogenetic trees
human primates, particularly lactating babies, are
to determine the relationships among trypanosomes in
more susceptible to the infection and/or the disease
general or even in limited categories. We also need to
. Although
can manifest itself more easily［17, 33］
surmount the difficulties of sequencing the ITS1 region
microscopic discrimination of T. lewisi infection
with its multiple repeats of certain nucleotide units.
in atypical hosts is extremely difficult, molecular
approaches using the rDNA sequence are emerging.
However, due to a low social interest in non-pathogenic
５．CONCLUSIVE REMARKS
trypanosomes, the current number of basic studies of
Records of atypical human trypanosomiasis caused
rodent trypanosomes of the subgenus Herpetosoma
by T. evansi and T. lewisi appear to have increased
［7］, particularly their genetic characterization, is not
Fig. 2. Maximum likelihood（ML）phylogenetic tree of T. lewisi isolates based on the ITS1 region of rDNA sequences.
The nucleotide sequences of T. lewisi and its closely related T. lewisi-like species (T. blanchardi and T. rabinowitschae)
were retrieved from the DDBJ/EMBL/GenBank databases, and ML analysis was performed as stated in the legend for Fig. 1.
Regions judged to be poorly aligned and characters with a gap in any sequences were excluded from subsequent analyses;
392 characters, of which 27 were variable, remained for subsequent analysis. Two trypanosome species (T. blanchardi and T.
rabinowitschae) were used as an outgroup for the construction of the ML phylogenetic tree.
― 30 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Eliakunda MAFIE, Fatema Hashem RUPA, Aogu SETSUDA, Atsuko SAITO-ITO, Hiroshi SATO
sufficient, and only a limited number of nucleotide
5. Doke, P. P. and Kar, A. 2011. A fatal case of
sequences is available for a limited number of rodent
Trypanosoma lewesi in Maharashtra, India. Ann.
trypanosome species. Presently, specific discrimination
Trop. Med. Public Health 4：91-95.
of the subgenus Herpetosoma largely relies on the host
6. Ferrante, A. 1985. Trypanolytic activity, agglutinins,
category since the morphology of trypomastigotes in
and opsonins in sera from animals refractory to
the bloodstream is almost identical（hence the term
Trypanosoma lewisi. Infect. Immun. 49：378-382.
“T. lewisi-like”）and little is known about the biology
7. Hoare, C. A. 1972. The trypanosomes of mammals:
of each species. As shown in Table 2, the intraspecific
A zoological monograph. Blackwell Scientific
nucleotide variations of T. grosi exceed the interspecific
Publications, Oxford, U.K., p.749.
nucleotide variations between, for example, T. blanchardi
8. Howie, S., Guy, M., Fleming, L., Bailey, W., Noyes.
and T. lewisi or T. musculi. In order to provide the
H., Faye, J. A., Pepin, J., Greenwood, B., Whittle,
basis for an accurate specific discrimination in atypical
H., Molyneux, D. and Corrah, T. 2006. A Gambian
human trypanosomiasis, greater effort in both the
infant with fever and an unexpected blood film.
field and laboratory needs to be directed towards the
PLoS Med. 3：e355 ［DOI：10.1371/ journal.
collection of more rodent trypanosome isolates for
pmed.0030355］.
genetic and biological characterization.
9. Jittapalapong, S., Inpankaew, T., Sarataphan, N.,
Herbreteau, V., Hugot, J. P., Morand, S. and Stich,
R. W. 2008. Molecular detection of divergent
ACKNOWLEDGEMENT
trypanosomes among rodents of Thailand. Infect.
This study was supported in part by a Grant-in-Aid
for International Collaboration Research in Asia 2016
Genet. Evol. 8：445-449.
10. Johnson, P. D. 1933. A case of infection by
from the Heiwa Nakajima Foundation.
Trypanosoma lewisi in a child. Trans. R. Soc.
Trop. Med. Hyg. 26：467-468.
11. Joshi, P. P. 2013. Human trypanosomiasis in India:
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56：1118-1121.
Med. Hyg. 68：143-144.
23. Sato, H., Ishita, K., Matsuo, K., Inaba, T., Kamiya, H.
31. Sousa, M. A. 2014. On opportunist infections by
― 32 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Eliakunda MAFIE, Fatema Hashem RUPA, Aogu SETSUDA, Atsuko SAITO-ITO, Hiroshi SATO
Trypanosoma lewisi in humans and its differential
Human Trypanosoma evansi infection linked to a
diagnosis from T. cruzi and T. rangeli. Parasitol.
lack of apolipoprotein L-1. N. Engl. J. Med. 355：
Res. 113：4471-4475.
2752-2756.
32. Tang, H., Lan Y., Wen Y., Zhang, X., Desquesnes, M.,
36. Vanwalleghem, G., Fontaine, F., Lecordier, L.,
Yang, T., Hide, G. and Lun, Z. 2012. Detection of
Tebabi, P., Klewe, K., Nolan, D. P., Yamaryo-
Trypanosoma lewisi from wild rats in Southern China
Botte, Y., Botte, C., Kremer, A., Burkard, G. S.,
and its genetic diversity based on the ITS1 and
Rassow, J., Roditi, I., Perez-Morga, D. and Pays,
ITS2 sequences. Inf. Genet. Evol. 12：1046-1051.
E. 2015. Coupling of lysosomal and mitochondrial
33. Truc, P., Büscher, P., Cuny, G., Gonzatti, M. I.,
membrane permeabilization in trypanolysis by
Jannin, J., Joshi, P., Juyal, P., Lun, Z.-R., Mattioli,
APOL1. Nat. Commun. 6：8078［DOI：10.1038/
R., Pays, E., Simarro, P. P., Teixeira, M. M. G.,
ncomms9078］.
Touatier, L., Vincendeau, P. and Desquesnes,
37. Verma, A., Manchanda, S., Kumar, N., Sharma,
M. 2013. Atypical human infections by animal
A., Goel, M., Banerjee, P. S., Garg, R., Singh, B. P.,
trypanosomes. PLoS Negl. Trop. Dis. 7：e2256
Balharbi, F., Lejon, V., Deborggraeve, S., Singh
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［DOI：10.1371/journal.pntd. 0002256］
Rana, U. V. and Pullyel, J. 2011. Trypanosoma
34. Vanhamme, L., Paturiaux-Hanocq, F., Poelvoorde, P.,
lewisi or T. lewisi-like infection in a 37-day-old
Nolan, D. P., Lins, L., Van Den Abbeele, J., Pays, A.,
Indian infant. Am. J. Trop. Med. Hyg. 85：221-224.
Tebabi, P., Van Xong, H., Jacquet, A., Moguilevsky,
38. Warpe, B. M. and More, S. V. 2014. A rare Indian case
N., Dieu, M., Kane, J. P., De Baetseller, P., Brasseur,
of human trypanosomiasis caused by Trypanosoma
R. and Pays, E. 2003. Apolipoprotein L-I is the
lewisi-like parasites. J. Biosci. Technol. 5：564-567.
trypanosome lytic factor of human serum. Nature
422：83-87.
35. Vanhollebeke, B., Truc, P., Poelvoorde, P., Pays, A.,
Joshi, P. P., Katti, R., Jannin J. G. and Pays, E. 2006.
Correspondence：Hiroshi SATO, Laboratory of Parasitology, Joint Faculty of Veterinary Medicine, 1677-1 Yoshida,
Yamaguchi 753-8515, Japan.
E-mail：sato7dp4@yamaguchi-u.ac.jp
Trypanosoma lewisi による非定型的人体トリパノソーマ症
マフィエ・エリアクンダ 1、ルパ・ファテムハシム 1、説田　景 1、斎藤　あつ子 2、佐藤　宏 1
1
山口大学 大学院 連合獣医学研究科、2 兵庫医療大学 薬学部 微生物学分野
要　約
トリパノソーマ（Kinetoplastea: Trypanosomatida: Trypanosomatidae）
、殊にサリバリアに分類される種やクルー
ズトリパノソーマは人および動物の健康を脅かし、しばしば致命的な感染を引き起こすことが知られている。ステル
コリアンに区分される Trypanosoma lewisi はラット寄生の Herpetosoma 亜属トリパノソーマである。この亜属に分
類される齧歯類寄生種の感染は潜伏的で、また、齧歯類間でもその属を超えた感染はほぼ不可能と考えられている。
ところが、T. lewisi の人体感染症例は過去に 9 件報告され、そのうち 5 症例はこの 20 年間に発生していることから、
新興感染症として考える向きもある。本稿では T. lewisi による非定型的人体トリパノソーマ症についてその発生状
況と背景、および PCR を活用した分子生物学的感染診断の現況について概要を記した。
Key words：ラットトリパノソーマ（Trypanosoma lewisi）
、非定型的人体トリパノソーマ症、Herpetosoma 亜属、
齧歯類トリパノソーマ、rDNA.
― 33 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Review
Endoparasites of Vietnamese lizards recorded
in the last 50 years（1966−2015）
Binh Thi TRAN 1, Son Truong NGUYEN 2, Tao Thien NGUYEN 3, Pham Van LUC 3,
Eliakunda MAFIE 4, Fatema Hashem RUPA 4, Hiroshi SATO 4
1
Department of Parasitology, and 2 Department of Veterinary Zoology, Institute of Ecology and
Biological Resources（IEBR）
, and 3 Vietnam National Museum of Nature,
Vietnam Academy of Science and Technology（VAST）
4
Laboratory of Parasitology, United Graduate School of Veterinary Science, Yamaguchi University
ABSTRACT
At present, there is a limited knowledge of amphibian and reptile parasites in Vietnam. To date, 45 species of
endoparasite in Vietnamese lizards have been recorded. These species consist of 11 cestode, 12 trematode, 18
nematode, one acanthocephalan and three pentastomid species from 10 host species. As Vietnam is one of the
global hot spots for herpetofauna diversity（a recent report documented 385 reptiles and 181 amphibians in the
, it appears that only a fraction of the parasites of lizards in this richly biodiverse territory has been
country）
recorded. To facilitate the accurate taxonomical identification of parasites and clarify the taxonomic relationship
of parasites from Vietnamese lizards with those from Oriental lizards or lizards of other geographical regions,
parasites should be characterized both morphologically and phylogenetically.
Key words：Vietnam, lizard, parasite, helminth, pentastomid, record.
or lizard fauna, little is known about the parasites of
１．INTRODUCTION
lizards endemic in Vietnam. To date, parasitological
Vietnam is one of the global hot spots for reptilian
studies have been conducted on only 10 host species,
and amphibian diversity. Nguyen and Ho［90］recorded
with 45 endoparasite species being found, of which
258 reptiles and 82 amphibians as the herpetofauna
80.0%（36 species）were parasites of the spiny-tailed
of Vietnam in 1996. More recent active surveys
house gecko Hemidactylus frenatus（Schlegel, 1836）,
on the herpetofauna in the country by Vietnamese
clouded monitor Varanus nebulosus（Gray, 1831）and
herpetologists and collaborative overseas scientists
. Due
water monitor Varanus salvator（Laurenti, 1768）
have disclosed more and more species. Nguyen et al.
to multiple records being published in Vietnamese or
［92］recorded 368 reptiles and 177 amphibians in 2009,
in domestic conference books in Vietnam, overseas
while Ziegler and Nguyen［133］reported 385 reptiles
researchers outside of Vietnam may experience
and 181 amphibians in 2010. The lizard group, such as
difficulties accessing them. To address this, the present
the agamids, geckos and skinks, currently comprises
study lists the parasite records from Vietnamese
a total of more than 120 species, of which at least
lizards for the benefit of researchers interested in this
57 species were described during the period 1996 to
topic.
2010［81, 90-92, 133］. The major reptile families in
Vietnam are Gekkonidae（42 species）and Scincidae（46
species）.
In contrast to the active research on herpetofauna
２. VIETNAMESE LIZARDS EXAMINED
FOR THEIR ENDOPARASITES
We surveyed conference records and scientific
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Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Binh Thi TRAN, Son Truong NGUYEN, Tao Thien NGUYEN, Pham Van LUC, Eliakunda MAFIE, Fatema Hashem RUPA, Hiroshi SATO
Fig. 1.Map of Vietnam illustrating 61 provinces. The provinces coloured grey denote localities where parasites
have been recorded in lizards.
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Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
― 36 ―
＊
Blank denotes no record.
Pseudocalotes brevipes (Werner, 1904)
Vietnamese false bloodsucker
Leiolepis reevesii Gray, 1831
Eastern butterfly lizard
Calotes versicolor (Daudin, 1802)
Garden fence lizard
Calotes emma Gray, 1845
Emma Gray’s forest lizard
Acanthosaura lepidogaster Cuvier, 1829
Scale-bellied tree lizard
Agamidae
Eutropis longicaudata Hallowell, 1856 Long-tailed mabuya
Scincidae
Hemidactylus frenatus Schlegel, 1836
Spiny-tailed house gecko
Gekko badenii Szezerbak et Nekrasova, 1994
Golden gecko
Gekkonidae
Varanus salvator Laurenti, 1768
Water monitor
Varanus nebulosus Gray, 1831
Clouded monitor
Varanidae
Lizard species
7
20
No data
6
32
38
149
65
20
23
Number of
lizards
examined
Oochoristica chinensis; Oochoristica
tuberculata; Oochoristica sp. 1;
Oochoristica sp. 2
Acanthotaenia beddardi; Acanthotaenia
nilotica; Acanthotaenia sp.; Kapsulotaenia
sandgroundi; Duthiersia expansa;
Scyphocephalus bisulcatus
Acanthotaenia shipleyi; Acanthotaenia
beddardi; Acanthotaenia nilotica;
Acanthotaenia sp.; Duthiersia expansa
Cestoda
Paradistomum orientalis
Plagiorchis molini; Parabascus lepidotus;
Postorchigenes ovatus
Encyclometra colubrimumorum;
Singhiatrema vietnamensis;
Euparadistomum varani; Diplodiscus
mehrai; Mesocoelium brevicaecum;
Meristocotyle provitellaria; Haplorchis
pumilio
Strongyluris calotis
Thelandros vietnamensis
Abbreviata deschiensi
Strongyluris calotis
Cosmocercoides tonkinensis
Oswaldocruzia sp. 1; Meteterakis mabuyae
Oswaldocruzia sp. 1; Skrjabinodon
azerbajdzanicus
Spauligodon vietnamensis;
Pharyngodon duci
Oswaldocruzia sp. 2; Raillietascaris
varani; Tanque tiara
Strongyloides mirzai; Kalicephalus sp.;
Oswaldcruzia sp. 2; 'Herpetostrongylus
varani' sensu Nguyen 2002;
Raillietascaris varani; Meteterakis
varani; Hastospiculum varani; Piratuba
varanicola
Nematoda
Recorded parasites ＊
Encyclometra colubrimumorum;
Singhiatrema vietnamensis; Mesocoelium
brevicaecum; Haplorchis pumilio;
Artyfechinostomum surfrartyfex
Trematoda
Table 1. Endoparasites of lizards in Vietnam recorded during the period of 1966 − 2015.
Pseudoacanthocephalus
nguyenthileae
Acanthocephala
Raillietiella affinis
Raillietiella frenatus
Raillietiella orientalis
Arthropoda
Endoparasites of Vietnamese lizards recorded in the last 50 years（1966 − 2015）
Binh Thi TRAN, Son Truong NGUYEN, Tao Thien NGUYEN, Pham Van LUC, Eliakunda MAFIE, Fatema Hashem RUPA, Hiroshi SATO
Fig. 2.Shadow pictures of lizards that have been recorded as hosts for endoparasites in
Vietnam. Lizards of different snout-vent length（SVL）are shown without reflecting
their sizes. Approximate SVLs are as follows: V. nebulosus, 120 cm; V. salvator, 80
cm; A. lepidogaster, L. reevesii and E. longicaudata, 14 cm; and the remaining five
species, 8 cm.
publications in domestic and international journals.
According to these sources, parasitological surveys
on Vietnamese lizards were often conducted at
３．PARASITES RECORDED IN
VIETNAM- ESE LIZARDS
several places in the Red River Delta, where the
As mentioned above, 45 species parasitic to lizards
Red River flows from Yunnan in southwest China
were recorded in Vietnam. These species comprised
through northern Vietnam to the Gulf of Tonkin, and
11 cestodes, 12 trematodes, 18 nematodes, one
mountainous provinces of central and southern regions
acanthocephalan and three pentastomids［2, 6, 22,
of Vietnam as well as unknown places in the southern
68, 82-88, 99, 100, 117-120, 122, 125］. Among them, six
region（Fig. 1）. We found 45 parasite species recorded
species were described as new to science at the time
from 10 host lizard species, and more than 360 host
of publication: Abbreviata deschiensi Le et Nguyen,
individuals were examined by Vietnamese researchers
1966; Pharyngodon duci Tran et al., 2007; Spauligodon
as well as their overseas collaborators（Table 1）.
vietnamensis Tran et al., 2007; Thelandros vietnamensis
These host lizards were classified into four families
Bui et al., 2009; Cosmocercoides tonkinensis Tran et al.,
（Table 1 and Fig. 2）: Varanidae（Varanus nebulosus
2015; and Pseudoacanthocephalus nguyenthileae Amin
and Varanus salvator）; Gekkonidae（Gekko badenii
et al., 2008. In the following subsections, localities, when
and Hemidactylus frenatus）; Scincidae （Eutropis
defined, are shown by the name of the province and
longicaudata）; and Agamidae（Acanthosaura lepidogaster,
the number plotted on the map（Fig. 1）in parentheses.
Calotes emma, Calotes versicolor, Pseudocalotes brevipes
The incidence（prevalence）and intensity are shown
and Leiolepis reevesii）
.
for parasite species where these data were provided,
although it was found that this information was
generally missing from the Vietnamese reports.
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Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Endoparasites of Vietnamese lizards recorded in the last 50 years（1966 − 2015）
3-1．Cestoda
Incidence and intensity: 2.7% （4/149） with 1–3
Eleven recorded species were classified into three
worms/host
families: Anoplocephalidae Cholodkowsky, 1902
Comment: This cestode is the type species of
（Oochoristica Lühe, 1898 – four spp.）; Proteocephalidae
the genus and is distributed widely in Eurasia and
La Rue, 1911（Acanthotaenia von Linstow, 1903 – four
northern Africa（Palaearctic region）by parasitism of a
spp. and Kapsulotaenia Freze, 1965 – one sp.）; and
variety of lizards（at least 31 species of 23 genera）as
Diphyllobothriidae Lühe, 1910（Duthiersia Perrier, 1873
well as snakes（nine species of nine genera）
［40, 131］.
– one sp. and Scyphocephalus Riggenbach, 1898 – one
This species recorded from common house geckos in
sp.）.
Vietnam had 28–30 testes per segment, different from
the aforementioned O. chinensis with 17–22 testes per
3-1-1．Oochoristica chinensis Jensen, Schmidt et
segment.
Kuntz, 1983
Host and location: Hemidactylus frenatus, small
3-1-3．Oochoristica sp. 1
intestine［85, 118］
Host and location: Hemidactylus frenatus, small
Locality: Yen Bai Province（5）; additionally, Hanoi
intestine［85, 118］
Province（14）
［unpublished］
Locality: Yen Bai Province（5）
Incidence and intensity: 4.7% （7/149） with 1–5
Incidence and intensity: 1.3% （2/149） with 1–3
worms/host
worms/host
Comment: Anoplocephalid cestodes of the genus
Comment: This cestode was small in size（7–20 mm
Oochoristica, ca. 80 species at present, are cosmopolitan
in length and 1.3 mm in width）and had 20–24 testes
in distribution and predominantly parasitize lizards, but
per segment and a small cirrus sac extending 16–20%
also snakes, turtles and marsupials［31］. The present
of the width of the mature segment. The cirrus sacs of
species was recorded from Japalura swinhonis（the
the two former species, O. chinensis and O. tuberculata,
type host）and Eutropis longicaudata（syn. Mabuya
extended 25–30% and 18–20% of the segment width,
longicaudata）in Taiwan［52, 93］. The morphological
respectively. Although morphological characters were
features of the isolate from common house geckos
recorded in detail, the exact taxonomic situation of this
（H. frenatus）in Vietnam corresponded well with the
species was uncertain［118］.
.
original description［52］
Since assumptions of strict host specificity and
3-1-4．Oochoristica sp. 2
geographical isolation had apparently been used as
Host and location: Hemidactylus frenatus, small
criteria in determining species of this genus, Criscione
intestine［85, 118］
and Font［31］conducted an experimental infection
Locality: Yen Bai Province（5）
of Oochoristica javaensis of lizard hosts distributed in
Incidence and intensity: 2.0% （3/149） with no
a non-endemic region of the cestode and concluded
intensity information
that members of the Oochoristica may lack strict host
Comment: This cestode was small in size（7–9 mm
specificity.
in length and 0.3 mm in width）and had 12–14 testes
per segment and a cirrus sac extending 50–58% of the
3-1-2．Oochoristica tuberculata（Rudolphi, 1819）
width of the mature segment. Although morphological
Lühe, 1898
characters were recorded in detail, the exact taxonomic
Syn. Skrjabinochora sobolevi Spasskii, 1948
situation of this species was uncertain［118］.
Host and location: Hemidactylus frenatus, small
In addition, Nguyen et al.［85］recorded another
intestine［85, 118］
Oochoristica sp. from Eutropis longicaudata at the
Locality: Yen Bai Province（5）; additionally, Hanoi
same localities in northern Vietnam. This species was
Province（14）
［unpublished］
different from the four aforementioned Oochoristica
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Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Binh Thi TRAN, Son Truong NGUYEN, Tao Thien NGUYEN, Pham Van LUC, Eliakunda MAFIE, Fatema Hashem RUPA, Hiroshi SATO
spp. from H. frenatus. No detailed information is
aforementioned species, A. shipleyi, had 40–65 testes
available.
per segment and a uterus with 29–36 lateral branches.
3-1-5．Acanthotaenia shipleyi von Linstow, 1903
3-1-7．Acanthotaenia nilotica Beddard, 1913
Host and location: Varanus nebulosus, small intestine
Host and location: Varanus nebulosus and Varanus
［117］
salvator, small intestine［117］
Locality: Southern Vietnam（not specified）
Locality: Vietnam（not specified）
Incidence and intensity: 17.4% （4/23） with no
Incidence and intensity: 27.9%（12/43）with no intensity
intensity information
information
Comment: Members of the genus Acanthotaenia
Comment: This species was originally described in
have a scolex with an apical muscular organ（piercing
Varanus niloticus from Africa, having 75–92 testes per
organ）
. The scolex and anterior part of the strobila
segment and a uterus with 17–25 lateral branches on
are covered with a dense network of spines［103］.
each side.
The uterus has numerous, irregular diverticula.
Acanthotaenia spp. are parasitic to varanid reptiles
3-1-8．Acanthotaenia sp.
in Africa, Australia and the Indo-Pacific region［103,
Host and location: Varanus nebulosus and Varanus
128］, and A. shipleyi collected from Varanus salvator
salvator, small intestine［117］
in Sri Lanka is the type species of the genus. Since
Locality: Vietnam（not specified）
the original description of the species was made using
Incidence and intensity: 14.0%（6/43）with no intensity
immature worms, Yamaguti［127］redescribed it using
information
mature cestodes collected from Varanus salvator on
Comment: This species was characterized to have
Sulawesi Island, formerly known as Celebes, Indonesia.
150–195 testes per segment and a uterus with 25–52
Recently, de Chambrier et al. ［37］ conducted
lateral branches on each side, distinct from any known
phylogenetic analyses of proteocephalid cestodes
Acanthotaenia spp.
（110 taxa of 54 genera classified in all 13 currently
recognized subfamilies, including A. shipleyi）using
3-1-9．Kapsulotaenia sandgroundi（Carter, 1943）
Freze, 1965
almost complete 28S ribosomal RNA gene（rDNA）
nucleotide sequences. Their findings led them to
Syn. Proteocephalus sandgroundi Carter, 1943
propose a need to revise the systematics of the family
Host and location: Varanus salvator, small intestine
based on phylogenetic achievements or newly defined
［117］
morphological characters suitable for the division of
Locality: Northern Vietnam（not specified）
subgroups.
Incidence and intensity: 15.0%（3/20）with no intensity
information
3-1-6．Acanthotaenia beddardi（Woodland, 1925）
Comment: The genera Kapsulotaenia and Acanthotaenia
Schmidt et Kuntz, 1974
are closely related, although formation of membranous
Host and location: Varanus nebulosus and Varanus
egg capsules only occurs in the former genus［103］.
salvator, small intestine［117］
The type species of the genus is the present species,
Locality: Vietnam（not specified）
which was fully redescribed by de Chambrier［36］on
Incidence and intensity: 18.6% （8/43） with no
the basis of the type specimen from Varanus komodoensis
intensity information
on Komodo Island, Indonesia, and museum materials
Comment: This species was originally described
from varanid lizards on the Lesser Sunda Islands,
in Varanus bengalensis in India. It was defined as
Indonesia, and Australia.
having 60–77 testes per segment and a uterus with
15–20 lateral branches on each side, whereas the
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Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Endoparasites of Vietnamese lizards recorded in the last 50 years（1966 − 2015）
3-1-10．Duthiersia expansa Perrier, 1873
［125］
Syns. Duthiersia crassa Woodland, 1938; Duthiersia
Locality: Nghe An Province（27）
venusta Woodland, 1938
Incidence and intensity: No information
Host and location: Varanus nebulosus and Varanus
Comment: The genus Scyphocephalus is characterized
salvator, small intestine［117］
by a special scolex with rudimentary bothria and an
Locality: Vietnam（not specified）
invaginated anterior end forming a sucking organ［21］.
Incidence and intensity: 18.6%（8/43）with no intensity
This cestode is the type species of the genus, described
information
based on worms from Varanus salvator in Java,
Comment: Members of the genus Duthiersia are
Indonesia［104］
. As detailed above, S. secundus from
small worms from varanid lizards, not exceeding 200
Varanus salvator on Leyte Island, Philippines［124］
mm in length, and having a broad and fan-like scolex
was synonymized by Vlnová［125］.‘Scyphocephalus
with bothrial margins frilled or crenulated ［21］.
jadhavi’ described from the same host species in
Woodland［126］examined numerous specimens from
Andhra Pradesh, India［60］appears to be a species of
varanids in Africa and Asia, and divided them into D.
a distinct genus.
fimbriata（Diesing, 1854）and D. expansa Perrier, 1873,
respectively, with the suppression of D. elegans Perrier,
3-2．Trematoda
1873, D. robusta Woodland, 1938, and D. latissima
Twelve recorded species were classified into
Woodland, 1938, as junior synonyms of D. fimbriata,
10 families: Plagiorchidae Lühe, 1901 （Plagiorchis
and similarly D. crassa Woodland, 1938, and D. venusta
; Encyclometridae Mehra,
Lühe, 1899 – one sp.）
Woodland, 1938, as junior synonyms of D. expansa.
1931 （Encyclometra Baylis et Cannon, 1924 – one
Woodland［126］indicated that the critical differences
sp.）; Philophthalmidae Looss, 1899 （Singhiatrema
between these two species, or African and Asian
Simha, 1954 – one sp.）; Dicrocoeliidae Odhner, 1911
forms, were few except for the shapes of scoleces and
（Euparadistomum Tubangui, 1931 – one sp. and
the absence or presence of posterior pore openings
; Phaneropsolidae
Paradistomum Kossak, 1910 – one sp.）
of the bothrial grooves. Current taxonomy follows his
Mehra, 1935（Parabascus Looss, 1907 – one sp. and
division. Along with the aforementioned D. expansa
; Diplodiscidae
Postorchigenes Tubangui, 1928 – one sp.）
specimens, Tran［117］reported the collection of‘D.
Cohn, 1904 （Diplodiscus Diesing, 1836 – one sp.）;
fimbriata’from Vietnamese varanids at an incidence
Mesocoeliidae Dollfus, 1929（Mesocoelium Odhner,
of 16.3%（7/43）. Since the basis for the separation
1910 – one sp.）; Meristocotylidae Fischthal et Kuntz,
between these two species was not shown in the work
1964（Meristocotyle Fischthal et Kuntz, 1964 – one
［117］,the present study omits this record.
sp.）; Heterophyidae Leiper, 1909（Haplorchis Looss,
The Asian form, i.e. D. expansa, is the type species
of the genus. It is commonly found in a variety of
1899 – one sp.）; and Echinostomatidae Dietz, 1909
（Testisacculus Bhalerao, 1927 – one sp.）
.
Asian Varanus spp.（V. bengalensis, V. flavescens, V.
komodoensis, V. marmoratus, V. nebulosus, V. nuchalis,
3-2-1．Plagiorchis molini Lent et Freitas, 1946
V. salvator and V. salvadorii） or Iguana sp. and
Host and location: Hemidactylus frenatus, intestine
Cyclura stejnegeri（Iguanidae）in Indonesia, Thailand,
［100］
Philippines, China, Malaysia, India, Sri Lanka, Pakistan
Locality: Quang Tri Province（30）
and Afghanistan［128］
.
Incidence and intensity: 3.0%（1/33）with 3 worms/
host
3-1-11．Scyphocephalus bisulcatus Riggenbach, 1898
Comment: This plagiorchid species is often found in
Syns. Scyphocephalus secundus Tubangui, 1968;
lacertid lizards such as sand lizards（Lacerta agilis）and
Scyphocephalus longus Sawada et Kugi, 1973
common wall lizards（Podarcis muralis）distributed
Host and location: Varanus salvator, small intestine
widely in Europe［70, 71］. Okulewicz et al.［95］found
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Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Binh Thi TRAN, Son Truong NGUYEN, Tao Thien NGUYEN, Pham Van LUC, Eliakunda MAFIE, Fatema Hashem RUPA, Hiroshi SATO
P. molini in a Chinese water dragon（Physignathus
Members of the genus have pyriform bodies with a
cocincinus）imported into Poland, suggesting that this
head-collar with a row of spines, interrupted dorsally
species is also distributed in China and Southeast Asia.
［61］. A well-developed ventral sucker is located
Plagiorchis elegans（Rudolphi, 1802）Braun, 1902, has
centrally and the intestinal caeca end at the posterior
also been reported from lacertid lizards［131］.
margin of the ventral sucker.
3-2-2．Encyclometra colubrimurorum（Rudolphi, 1819）
3-2-4．Euparadistomum varani Tubangui, 1931
Dollfus, 1929
Host and location: Varanus salvator, gall bladder［99］
Host and location: Varanus nebulosus and Varanus
Locality: Hanoi Province（14）
salvator, oesophagus and intestine［99］
Incidence and intensity: No information
Locality: Hanoi Province（14）and southern Vietnam
Comment: This discoid dicrocoeliid species with
（not specified）
almost full occupation of the body by uterine coils and
Incidence and intensity: No information
two testes situated symmetrically at anterolateral
Comment: Members of the genus Encyclometra are
positions to the acetabulum was originally described in
trematodes that dwell in the oesophagus, stomach
Varanus salvator from the Philippines, then other places
and intestine of snakes in Eurasia［116］. Gupta and
in Southeast Asia, Pacific Islands and Madagascar［28,
Mehrotra［49］differentiated three valid species: E.
44］. In Vietnam, this species was found in the Asian
colubrimurorum（testes tandem or obliquely tandem,
water monitor（V. salvator）in 2003［99］, and more
with equal intestinal caeca）; E. bungara Srivastava
recently in the Asian house shrew（Suncus murinus）
et Ghosh, 1968（testes almost symmetrically placed）;
［80］.
and E. asymmetrica Wallace, 1936（testes tandem or
obliquely tandem, with very unequal intestinal caeca）.
3-2-5．Paradistomum orientalis（Narain et Das, 1929）
Bhalerao, 1936
Encyclometra japonica Yoshida et Ozaki, 1929, and
E. vitellata Gupta, 1954, were synonymized with E.
Syns. Dicrocoelium orientale Narain et Das, 1929;
colubrimurorum and E. asymmetrica, respectively［49］.
Paradistomoides orientale（Narain et Das, 1929）Yamaguti,
Metacercariae of E. colubrimurorum are often found
1958
in amphibians［7］. In Vietnam, this encyclometrid
Host and location: Eutropis longicaudata and Hemida-
species was found in snakes（Xenopeltis unicolor and
ctylus frenatus, gall bladder［83, 85］
Xenochrophis piscator）, as reported in various snakes
Locality: Hanoi Province（14）and Yen Bai Province（5）
（Ptyas mucosus, Natrix natrix and Natrix piscator）from
.
other Eurasian areas［51］
Incidence and intensity: No information
Comment: This oval dicrocoeliid species is a common
trematode in the gall bladder of various lizards including
3-2-3．Singhiatrema vietnamensis Curran et al., 2001
Calotes versicolor and Hemidactylus flaviviridis in India.
Host and location: Varanus nebulosus and Varanus
Examining C. versicolor in India, Arora and colleagues
salvator, oesophagus and intestine［99］
［8, 9］found a high prevalence（67/74）of the species
Locality: Hanoi Province（14）and southern Vietnam
（not specified）
in the gall bladder, and from their studies on the
intraspecific variations of the specimens concluded
Incidence and intensity: No information
several described species to be junior synonyms of P.
Comment: This species was originally described from
orientalis. Studying the monthly population dynamics of
the small intestine of Chinese water snakes（Enhydris
P. orientalis in C. versicolor in India, Madhavi et al.［76］
chinensis）and rice paddy snakes（Enhydris plumbea）
reported that the frequency distribution of the fluke in
in Vietnam［32］. It also parasitizes other snakes such
the host followed the overdispersion pattern and that
as the Taiwan cobra（Naja atra）, Chinese ratsnake
crowding effects serve as a major regulatory force
（Ptyas korros）and banded krait（Bungarus fasciatus）.
for maintaining the equilibrium of parasite densities
― 41 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Endoparasites of Vietnamese lizards recorded in the last 50 years（1966 − 2015）
throughout the year. Killick and Beverley-Burton
nigrovittata, Quasipaa verrucospinosa and Paramesotriton
［64］made a taxonomical evaluation of 21 nominal
deloustali［86, 110］. The family Diplodiscidae is a small
Paradistomum spp. described from Southeast Asian
group of paramphistomoids found predominantly in
lizards.
amphibians, but also in reptiles and fish, and characterized
by a single testis and a ventroterminal sucker［53］.
3-2-6．Parabascus lepidotus Looss, 1907
Host and location: Hemidactylus frenatus, intestine
［83, 85］
3-2-9．Mesocoelium brevicaecum Ochi, 1930
Syn. Mesocoelium pearsei sensu Pham et Nguyen, 2003
Locality: Hanoi Province（14）and Yen Bai Province（5）
Host and location: Varanus nebulosus and Varanus
Incidence and intensity: No information
salvator, small intestine［82, 99］
Comment: This trematode is the type species of the
Locality: Hanoi Province（14）and southern Vietnam
genus, originally described from Pipistrellus kuhlii（syn.
（not specified）
Vesperugo kuhlii; Chiroptera: Vespertilionidae）in Cairo,
Incidence and intensity: No information
Egypt［74］. This Kuhl’s pipistrelle bat is distributed
Comment: Pham and Nguyen［99］collected a small-
widely around Mediterranean regions （southern
sized oval mesocoeliid species from the small intestine
Europe and northern Africa）to India, and there are
of varanid lizards in Vietnam and identified it as
records of this trematode in the regions with the host
Mesocoelium pearsei Goto et Ozaki, 1930. Subsequently,
distribution.
however, Nguyen and Ha［82］reidentified it as M.
brevicaecum. This species was originally described
3-2-7．Postorchigenes ovatus Tubangui, 1928
from the small intestine of various amphibians in Japan,
Syn. Parabascus ovatus （Tubangui, 1928） sensu
such as Bufo japonicus（syn. Bufo vulgaris japonicus）,
Nguyen et Pham, 2005
,
Pelophylax nigromaculatus（syn. Rana nigromaculata）
Host and location: Hemidactylus frenatus, intestine
Glandirana rugosa （syn. Rana rugosa）, Lithobates
［83, 85, 96］
, Elaphe quadrivirgata
catesbeianus（syn. Rana catesbeiana）
Locality: Yen Bai Province（5）
and Plestiodon latiscutatus（syn. Eumeces latiscutatus）
Incidence and intensity: No information
. Similarly, the trematode was found in Duttaphrynus
［94］
Comment: This oval trematode is the type species
melanostictus and Hylarana guentheri（syn. Rana guentheri）
of the genus, originally described from Hemidactylus
.
from Vietnam and D. melanostictus from Taiwan［39］
frenatus in the Philippines［74, 129］.This species is one
As too many species in the genus Mesocoelium have
of the very common trematodes of geckos in Southeast
been described, Dronen et al.［39］recently undertook
Asian countries such as Indonesia, Thailand, Vietnam
their evaluation based on intensive morphological
and Philippines［14, 64, 78］.
criteria and consequently proposed synonymies of
multiple species.
3-2-8．Diplodiscus mehrai Pande, 1937
Host and location: Varanus salvator, intestine［99］
3-2-10．Meristocotyle provitellaria Liu et al., 2002
Locality: Hanoi Province（14）
Syn. Meristocotyle sp. sensu Pham et Nguyen, 2003
Incidence and intensity: No information
Host and location: Varanus salvator, small intestine［99］
Comment: This species was originally described from
Locality: Hanoi Province（14）
Euphlyctis cyanophlyctis（syn. Rana cyanophlyctis）
Incidence and intensity: No information
in India［129］, followed by records in various frogs
Comment: Fischthal and Kuntz［44］erected a new
in India［50］. Similarly in Vietnam, this trematode
genus for their new species Meristocotyle varani from
was found in various amphibians including Fejervarya
Varanus salvator in the Philippines. Trematodes of this
limnocharis（syn. Rana limnocharis）, Duttaphrynus
genus are characterized by the unusual bipartite nature
melanostictus （syn. Bufo melanostictus）, Hylarana
of the ventral sucker; in other words, a horizontally
― 42 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Binh Thi TRAN, Son Truong NGUYEN, Tao Thien NGUYEN, Pham Van LUC, Eliakunda MAFIE, Fatema Hashem RUPA, Hiroshi SATO
divided ventral sucker with two lumina. Later, Liu et
the Indian spiny-tailed lizard Saara hardwickii（syn.
al.［72］described the second species, M. provitellaria,
Uromastyx hardwickii）in India［18, 129］
. Simha and
from the same host species in China. The morphological
Deshpande［112］collected a dozen specimens from the
characteristics of Meristocotyle sp. from V. salvator in
intestine of a single Varanus bengalensis and described
Vietnam provided by Pham and Nguyen［99］coincided
a new species, namely Artyfechinostomum varanum.
well with those of M. provitellaria detailed by Liu et
Bhardwaj［19］collected and examined two specimens
al.［72］
, whereas they were less coincident with those
from Varanus bengalensis in Jabalpur, India, and erected
of M. varani reported by Fischthal and Kuntz［44］.
a new genus and species, Pseudoartyfechinostomum
These two studies from the Philippines and China
larueiformis. Premvati and Pande［101］synonymized all
documented that worms were collected from the lungs
described species under the genera Artyfechinostomum,
in addition to the intestine.
Neoartyfechinostomum and Pseudoartyfechinostomum,
and retained only A. malayanum （Leiper, 1911）
3-2-11．Haplorchis pumilio Looss, 1896
Mendheim, 1943. Kostadinova［65］essentially followed
Host and location: Varanus nebulosus and Varanus
their revision, but elected A. sufrartyfex Lane, 1915
salvator, small intestine［99］
（syn. A. malayanum）as the type species of the genus.
Locality: Hanoi Province（14）and southern Vietnam
（not specified）
Based on these interpretations, we decided to replace
the name of the parasites‘Testisacculus indicus’
Incidence and intensity: No information
collected by Pham and Nguyen［99］with A. sufrartyfex
Comment: In Southeast Asia including Vietnam,
in this article.
human infections with fishborne zoonotic intestinal
trematodes have been increasingly reported in the
3-3．Nematoda
last two decades, with one of the major causes being
Eighteen recorded species were classified into 12
Haplorchis pumilio［123］. This species is common not
families: Cosmocercidae Travassos, 1925（Cosmocercoides
only in humans but also in mammals, birds and varanid
Wilkie, 1930 – one sp.）; Strongyloididae Chitwood et
lizards through the consumption of raw freshwater
McIntosh, 1934（Strongyloides Grassi, 1879 – one sp.）;
fish［67, 114］. The original description was made using
Diaphanocephalidae Travassos, 1920（Kalicephalus Molin,
specimens from white pelicans（Pelecanus onocrotalus）
1861 – one sp.）; Molineidae（Skrjabin et Schulz, 1937,
in Egypt［129］.
subfam.）Durette-Desset et Chabaud, 1977（Oswaldocruzia
Travassos, 1917 – two spp.）; Herpetostrongylidae
3-2-12．Artyfechinostomum sufrartyfex Lane, 1915
（Skrjabin et Schulz, 1937, subfam.）Durette-Desset et
Syns. Testisacculus indicus Bhalerao, 1931; Artyfechino-
Chabaud, 1981（Herpetostrongylus Baylis, 1931 – one
stomum varanum Simha et Deshpande, 1964; Testisacculus
; Ascaridida, Ascaridoidea（Raillietascaris Sprent,
sp.）
indicus sensu Pham et Nguyen, 2003
1985 – one sp.）;Gnathostomatidae Railliet, 1895（Tanqua
Host and location: Varanus nebulosus, small intestine
von Linstow, 1897– one sp.）; Heterakidae Railliet et
［99］
Henry, 1914（Meteterakis Karve, 1930 – two spp. and
Locality: Southern Vietnam（not specified）
Strongyluris Müller, 1894 – one sp.）; Diplotriaenidae
Incidence and intensity: No information
（Skrjabin, 1916, subfam.）Anderson, 1958（Hastospiculum
Comment: Echinostomatid trematodes of the genus
Skrjabin, 1923 – one sp.）
; Onchocercidae Leiper,
Artyfechinostomum Lane, 1915（syn. Testisacculus
;
1911（Piratuboides Bain et Sulahian, 1974 – one sp.）
Bhalerao, 1927）are characterized by a spinose tegument
Pharyngodonidae Travassos, 1919 （Spauligodon
with very large scale-like spines comparable in size to
Skrjabin, Schikhobalova et Lagodovskaja, 1960 –
collar-spines, dorsal collar-spines in double rows, and
two spp.; Pharyngodon Diesing, 1861– one sp.; and
deeply branched testes［65］.The species was originally
Thelandros Wedl, 1862 – one sp.）; and Physalopteridae
described using specimens from the duodenum of
Railliet, 1893（Abbreviata Travassos, 1919 – one sp.）
.
― 43 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Endoparasites of Vietnamese lizards recorded in the last 50 years（1966 − 2015）
3-3-1．Cosmocercoides tonkinensis Tran et al., 2015
the Neotropical region. Zhang et al.［132］reported
Host and location: Acanthosaura lepidogaster, large
two Kalicephalus spp., i.e. K. guangdongensis and K.
intestine［120］
schadi fotedari, from the intestine of Varanus salvator
Locality: Cao Bang（8）, Son La（4）
, Bac Giang（17）,
from Guangdong Wildlife Rescue Centre, Guangdong
Thanh Hoa（26）Provinces
Province, South China, although these two species
Incidence and intensity: 31.3%（10/32）with 1–29
resembled each other except for spicule length and
（geomean 4.1）worms/host
other morphometric values, and length and position
Comment: Currently, ca. 20 species of the genus with
of the externodorsal ray. As the measurements and
caudal rosette papillae, not combined with plectanes,
drawings provided by Nguyen［87］are not sufficient
have been described mainly in amphibians worldwide.
to determine the species, in this article we describe
The morphology of this species was well characterized
the species from clouded monitors in Vietnam as
using both light and scanning electron microscopies.
Kalicephalus sp. Re-examination of the specimens in
The phylogenetic relationships with related species
the future should yield a more precise identification. In
with available genetic data（Cosmocercoides pulcher
addition, Kalicephalus（Kalicephalus）costatus indicus
and Cosmocercoides dukae）were also reported［120］.
Ortlepp, 1923, from Varanus bengalensis in India, K.
This is the third species from lizard hosts after C.
（K.）megacephalus Schad, 1962, from Varanus indicus
variabilis in North America and C. sauria in Brazil［10,
in India and V. salvator in the Philippines, and K.（K.）
23, 47］.
schadi Ogden, 1966, from V. bengalensis in India or Sri
.
Lanka（London Zoo）have been recorded［12］
3-3-2．Strongyloides mirzai Singh, 1954
Furthermore, a variety of Kalicephalus spp. has also
Host and location: Varanus nebulosus, intestine［87］
been recorded from snakes in Southeast and South
Locality: Vietnam（not specified）
Asia［66, 73, 77, 102, 108, 130］. Le and Pham［69］, and
Incidence and intensity: No information
Oshmarin and Demshin［96］recorded the following
Comment: This species was originally described
Kalicephalus spp. from snakes in Vietnam: K. alatospiculus
using specimens from Indian sand boas（Eryx johnii）
from Chinese ratsnakes Ptyas korros（syn. Zamenis
and Oriental ratsnakes（Ptyas mucosa）in India［113］.
korros）, K. viperae chungkingensis from Indian cobras
Likewise in Vietnam, the species was found in a
Naja naja, K. indicus from Chinese ratsnakes and radiated
variety of snakes such as Chinese cobra（Naja atra）,
ratsnakes Coelognathus radiata（syn. Elaphe radiata）,
many-banded krait（Bungarus multicinctus）, banded
K. bungari （syn. K. najae after ［12, 108］） from
krait（Bungarus fasciatus）and Chinese ratsnake（Ptyas
Indian cobras, K. natricis from Sri Lankan keelbacks
korros）
［86］.
Xenochrophis asperrimus（syn. Natrix piscator）and
Kalicephalus sp. from banded kraits Bungarus fasciatus.
3-3-3．Kalicephalus sp.
Kalicephalus natricis Yamaguti, 1935, originally from
Syn. Kalicephalus macrovulvus sensu Nguyen, 2002,
Rhabdophis tigrinus（syn. Natrix tigrina）and Elaphe
nec Caballero, 1954
quadrivirgata in Japan, is considered to be a‘species
Host and location: Varanus nebulosus, oesophagus［87］
inquirenda’,since multiple species, probably K. costatus
Locality: Vietnam（not specified）
indicus, K. brachycephalus, K. sinensis or K. viperae
Incidence and intensity: No information
chungkingensis, were described under the name‘K.
Comment: Nguyen ［87］recorded Kalicephalus
［12, 108］.
natricis’
nematodes from the oesophagus of clouded monitors
（Varanus nebulosus）in Vietnam as K. macrovulvus
3-3-4．Oswaldocruzia sp. 1
Caballero, 1954, originally isolated from Agkistrodon
Syn. Oswaldocruzia agamae sensu Nguyen et Bui, 2007
bilineatus in Guatemala. Schad［108］reclassified the
Host and location: Hemidactylus frenatus and Eutropis
species as K. inermis macrovulvus distributed in
longicaudata, intestine［85, 88］
― 44 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Binh Thi TRAN, Son Truong NGUYEN, Tao Thien NGUYEN, Pham Van LUC, Eliakunda MAFIE, Fatema Hashem RUPA, Hiroshi SATO
Locality: Hanoi Province（14）and Yen Bai Province（5）
Incidence and intensity: No information
Incidence and intensity: No information
Comment: Although the specific name‘Herpeto-
Comment: Nominal species of the genus Oswaldocruzia
strongylus varani’assigned by Nguyen［87］to the
are currently more than 80 in number［17, 48, 106］.
herpetostrongylid nematodes from clouded monitors
Ben Slimane et al.［17］attempted to subdivide the
in Vietnam is retained in this article, an exact specific
genus into five groups based on the disposition of the
identification is required in the future. At this time, we
caudal bursal rays, the morphology of the synlophe
are unable to ascertain the exact taxonomy of the present
and the anatomy of the spicules: 1）Oriento-Ethiopian
specimens due to Nguyen’s measurements and drawings
species, 10 spp.; 2）Neo-Ethiopian species, 11 spp.; 3）
［87］being insufficient for species determination. For
Holarctic species, 24 spp.; 4）Continental Neotropical
reasons outlined in the next paragraph, it is particularly
species, 21 spp.; and 5）Caribbean Neotropical species,
important to evaluate its possible classification in the
8 spp. From Vietnam, O. mitunagai Durette-Desset,
genus Vaucherus Durette-Desset, 1980.
Nasher et Ben Slimane, 1992, and O. hoepplii Hsü, 1935,
Herpetostrongylus varani Baylis, 1931, was originally
both from D. melanostictus and Ranidae, were noted in
recorded from Gould’s monitors（Varanus gouldii）in
the identification key of this comprehensive taxonomic
Townsville, North Queensland, Australia［16］. At the
study by Ben Slimane et al.［17］
. Oswaldocruzia hoepplii
same time, another species, Herpetostrongylus pythonis
sensu Moravec et Sey, 1985, nec Hsü, 1935, was considered
Baylis, 1931, was described from Morelia spilota（syn.
to be a junior synonym of O. mitunagai（the renamed
, and later recorded
Python spilotes）in Australia［16］
species of O. hoepplii sensu Yamaguti et Mitunaga,
in Varanus salvator from Palawan, Philippines［109］.
1943, nec Hsü, 1935）. Following the systematics of Ben
Durette-Desset［41］erected a new genus, Vaucherus,
Slimane et al.［17］, O. agamae Sandground, 1930, is a
for three herpetostrongylid nematode species from
parasite of agamid lizards distributed in Liberia. As the
Indian and Asian varanid hosts: V. vaucheri Durette-
measurements and drawings provided by Nguyen and
Desset, 1980, from Varanus rudicollis in Kuala Lumpur,
Bui［88］are not sufficient to determine the species,
Malaysia; V. leiperi（Sharief, 1957）Durette-Desset,
in this article we describe the species from geckos in
1980, from Varanus indicus in Hyderabad, India; and V.
Vietnam as Oswaldocruzia sp. Re-examination of the
indicus（Deshmukh, 1969）Durette-Desset, 1980, from
specimens in the future should yield a more precise
Varanus indicus in Aurangabad, India. Durette-Desset
identification.
et al.［42］differentiated the genus Vaucherus from
the genus Herpetostrongylus based on differences in
3-3-5．Oswaldocruzia sp. 2
bursal ray arrangements; 2-2-1 type with hypertrophied
Host and location: Varanus salvator and Varanus
ray 2, and 1-3-1 type with rays 2 and 3 more strongly
nebulosus, intestine［87］
developed than 5 and 6, respectively.
Locality: Vietnam（not specified）
3-3-7．Raillietascaris varani（Baylis et Daubney, 1922）
Incidence and intensity: No information
Sprent, 1985
Comment: This species was differentiated from
the former species based on multiple morphological
Syns. Amplicaecum varani Baylis et Daubney, 1922;
features, e.g. different development of caudal bursa and
Amplicaecum monitor Khera, 1954; Amplicaecum
rays, different morphology of spicules and different egg
iguaneae Wahid, 1961
dimensions（0.07–0.10 mm by 0.05–0.06 mm vs. 0.06–0.07
Host and location: Varanus salvator and Varanus
.
mm by 0.03–0.04 mm, respectively）
nebulosus, stomach and intestine［87］
Locality: Vietnam（not specified）
3-3-6．‘Herpetostrongylus varani’sensu Nguyen, 2002
Incidence and intensity: No information
Host and location: Varanus nebulosus, intestine［87］
Comment: This species was originally described
Locality: Vietnam（not specified）
as Amplicaecum varani using specimens from the
― 45 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Endoparasites of Vietnamese lizards recorded in the last 50 years（1966 − 2015）
intestine of Varanus salvator in Calcutta, India, and
3-3-10．Meteterakis mabuyae Chakravarty, 1944
several different species from Indian varanids were
Host and location: Eutropis longicaudata, intestine
recorded in the decades that followed［130］
. Sprent
［115］erected a new genus, Raillietascaris, and unified
［85, 88］
Locality: Hanoi Province（14）and Yen Bai Province（5）
such species from varanids as junior synonyms of R.
Incidence and intensity: No information
varani（Baylis et Daubney, 1922）.This species was also
Comment: This was the fourth species recorded
recorded from Varanus rudicollis in Borneo, Indonesia,
in Vietnam after M. striatura, M. japonica and M.
and imported Chinese water dragons in Poland［35, 95,
varani. In addition, at least two Meteterakis spp. were
.
107］
detected in the large intestine of scale-bellied tree
lizards, Acanthosaura lepidogaster, in Vietnam. Specific
3-3-8．Tanqua tiara（von Linstow, 1879）Blanchard,
1904
identification of these species is currently in progress
.
（unpublished）
Host and location: Varanus salvator, stomach［87］
Locality: Vietnam（not specified）
3-3-11．Strongyluris calotis Baylis et Daubney, 1923
Incidence and intensity: No information
Syns. Ascaridia japalurae Yamaguti et Mitunaga,
Comment: This species was originally described
1935; Strongyluris brevicaudata sensu Hsü et Hoeppli,
using specimens from the stomach of varanid lizards in
1931, nec Müller, 1894
South Africa. It was subsequently isolated from Varanus
Host and location: Pseudocalotes brevipes and Calotes
salvator in Sumatra, Varanus gouldii in Australia or
emma, large intestine［122］
New Guinea, Varanus bengalensis in Sri Lanka and
Locality: Phu Tho Province （12） and Bac Kan
Varanus niloticus in the White Nile ［15, 29, 130］.
Province（7）
Gibbons and Keymer［45］redescribed the species in
Incidence and intensity: 57.1% （4/7） with 1–6
detail, along with a list of previous records of T. tiara
worms/host（P. brevipes）
; 16.7%（1/6）with no intensity
from varanids in Africa through to Asia. Phylogenetic
information（C. emma）
analyses of geographical isolates distributed widely
Comment: This species is distributed widely in agamid
would be very interesting.
lizards from the Oriental region. Tran et al.［122］
confirmed for the first time the distribution of the
3-3-9．Meteterakis varani（Maplestone, 1931）Skrjabin
et al., 1961
species in Vietnam. Furthermore, their morphological
studies of two isolates from P. brevipes and C. emma
Host and location: Varanus nebulosus, intestine［87］
in Vietnam demonstrated differences in numbers and
Locality: Vietnam（not specified）
arrangements of caudal papillae. However, in combination
Incidence and intensity: No information
with genetic characterization, their conspecificity was
Comment: The genus Meteterakis Karve, 1930,
. There are at least four nominal
shown［121, 122］
currently contains 25 nominal species from Oriental
Strongyluris spp. in agamid lizards in the Oriental
amphibians and reptiles. In Vietnam, Meteterakis
region: S. chamaeleonis Baylis et Daubney, 1922; S.
striatura（Oshmarin et Demshin, 1972）from yellow pond
bengalensis Chakravorty, 1936; S. karawirensis Karve,
turtle Mauremys mutica（syn. Clemmys mutica）and
1938; and S. japalurae Jiang et Lin, 1980. As there
Meteterakis japonica（Wilkie, 1930）from a yellowcheek
are few critical morphological differences among
（Elopichthys bambusa）have been recorded［79, 96］,
these described species, they need to be genetically
although the latter record was considered to be an
characterized to confirm the validity of their taxonomy.
accidental infection［79］. Meteterakis varani was the
third Meteterakis species recorded in Vietnam.
3-3-12．Hastospiculum varani Skrjabin, 1923
Host and location: Varanus nebulosus, body cavity［87］
Locality: Vietnam（not specified）
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Binh Thi TRAN, Son Truong NGUYEN, Tao Thien NGUYEN, Pham Van LUC, Eliakunda MAFIE, Fatema Hashem RUPA, Hiroshi SATO
Incidence and intensity: No information
information（Gia Lai Province）
Comment: There are five Hastospiculum spp. recorded
Comment: Currently, 51 species have been described
from varanid lizards［20, 130］
: H. varani Skrjabin,
worldwide in the genus Spauligodon Skrjabin,
1923, from Varanus griseus in Turkistan and Varanus
Schikhobalova et Lagodovskaja, 1960［27］. Spauligodon
indicus in India; H. bipinnatum von Linstow, 1899,
vietnamensis recorded in golden geckos from Vietnam
from Varanus griseus（syn. Psammosaurus griseus）in
was the 44th species assigned to the genus［119］.
northeastern Africa; H. macrophallos Parona, 1889, or
Golden geckos （Gekko badenii） have a limited
its junior synonym H. spinigerum Chandler, 1929, from
distribution in the high mountains of central and
Varanus spp. in Myanmar, India and Russia; H. gouldi
southern Vietnam, i.e. Tay Ninh, Kon Tum and Gia Lai
Yorke et Maplestone, 1926, from Varanus gouldii in
Provinces［34, 92］. As two scientific names for golden
Australia; and H. spiralis Bolette, 1998, from Varanus
geckos, G. badenii and G. ulikovskii, were published
indicus in Indonesia. As the record in Vietnam was
independently in different scientific journals on 15 May
,
based on a single male specimen from the host［87］
and 15 June, 1994, respectively, the former scientific
more specimens need to be examined for an accurate
name has priority.
identification of the species.
3-3-15．Skrjabinodon azerbajdzanicus（Sharpio, 1974）
3-3-13．Piratuboides varanicola（Mackerras, 1962）
Bain et Sulahian, 1974
Bursey et Goldberg, 1999
Syn. Spauligodon azerbajdzanicus Sharpio, 1974
Syn. Piratuba varanicola Mackerras, 1962
Host and location: Hemidactylus frenatus, intestine
Host and location: Varanus nebulosus, lungs［87］
［85, 88］
Locality: Vietnam（not specified）
Locality: Hanoi Province（14）and Yen Bai Province（5）
Incidence and intensity: No information
Incidence and intensity: No information
Comment: Mackerras［75］described two onchocercid
Comment: The species was originally described
filariae of the subfamily Oswaldofilariinae in Australian
from the green-bellied lizard Darevskia chlorogaster
varanid lizards and newly named them as Piratuba
（syn. Lacerta chlorogaster）（Sauria: Lacertidae）in
queenslandensis and Piratuba varanicola. Bain and
Azerbaijan. Referring to the absence of caudal alae in
Sulahian［11］moved them from the genus Piratuba
the description of‘Spauligodon azerbajdzanicus’by
（equal spicules in size and shape, a short and simple
Sharpio［111］, Bursey and Goldberg［24］reassigned
ovejector, and numerous caudal papillae）to a new
the species as Skrjabinodon azerbajdzanicus.
genus, Piratuboides, characterized as having subequal
spicules, a long but simple ovejector, and a smooth
3-3-16．Pharyngodon duci Tran et al., 2007
female tail or one with small terminal elevations. The
Host and location: Gekko badenii, large intestine［119］
type species of the genus is Piratuboides zeae（Bain,
Locality: Gia Lai Province（36）and Tay Ninh Province
1974）from skinks of Scincidae in Central America,
（46）
and Piratuboides huambensis was described from blue-
Incidence and intensity: 90.0%（9/10）with no intensity
tailed skink Trachylepis quinquetaeniata（syn. Mabuya
information（Gia Lai Province）
quinquetaeniata）in Angola［97］.
Comment: As with the genera Spauligodon Skrjabin,
Schikhobalova et Lagodovskaja, 1960, and Skrjabinodon
3-3-14．Spauligodon vietnamensis Tran et al. 2007
, the genus Pharyngodon Diesing, 1861,
Inglis, 1968［98］
Host and location: Gekko badenii （syn. Gekko
is confined to reptile and amphibian hosts. Although
ulikovskii）
, large intestine［119］
members of these three genera resemble each other,
Locality: Gia Lai Province（36）and Tay Ninh Province
major differences lie in the presence（Pharyngodon and
（46）
Spauligodon）or absence（Skrjabinodon）of caudal alae.
Incidence and intensity: 90.0%（9/10）with no intensity
Caudal alae of Pharyngodon spp. are supported by all
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Endoparasites of Vietnamese lizards recorded in the last 50 years（1966 − 2015）
three pairs of genital papillae, whereas those of Spauligodon
3-4．Acanthocephala
spp. are supported by the two anterior pairs only. The
N g u y e n ［ 8 9 ］r e c e n t l y p r o v i d e d a l i s t o f 7 6
genus Pharyngodon currently contains 37 species, of
acanthocephalan species recorded in Vietnam up to the
which five were recorded from amphibians［25, 26,
year 2015. This list comprised 13 spp. from freshwater
43］. Pharyngodon duci recorded in golden geckos from
fish, 21 spp. from marine fish, three spp. from amphibians,
Vietnam was the 35th species assigned to the genus
five spp. from reptiles, 29 spp. from birds and five spp.
［119］.
from mammals. One recorded species in Vietnamese
lizards was classified in the family Echinorhynchidae
3-3-17．Thelandros vietnamensis Bui et al., 2009
Cobbold, 1876（Pseudoacanthocephalus Petrochenko,
Host and location: Leiolepis reevesii, intestine［22］
1956）.
Locality: Ha Tinh Province（28）
Incidence and intensity: 85.0% （17/20） with an
3-4-1．Pseudoacanthocephalus nguyenthileae Amin
et al., 2008
average of 25.8 worms/host
Comment: The genus Thelandros is closely related
Syn. Acanthocephalus sp. sensu Nguyen et al., 2005
to the aforementioned pharyngodonid genera. Parasites
Host and location: Hemidactylus frenatus, small
of this genus have three, sometimes four, pairs of
intestine［6, 85］
genital papillae clearly separated into an anterior
Locality: Bac Kan Province（7）
group（two pairs）around the cloaca and one posterior
Incidence and intensity: 4.0%（1/25）with 2 worms/
pair. There is often a fringed membrane covering the
host
cloaca［98］. Petter and Quentin［98］synonymized
Comment: This species was dedicated to Prof. Nguyen
Parapharyngodon Chatterji, 1933, with Thelandros
Thi Le, a parasitologist of IEBR, VAST［6］. It was
Wedl, 1862, but Bursey and Goldberg［25］disagreed
also found in amphibians and other reptiles in northern
with this view based on several morphological
Vietnam, i.e. Hylarana guentheri, Hylarana taipehensis,
differences. According to Bursey and Goldberg［25］, 31
Duttaphrynus melanostictus, Quasipaa verrucospinosa
species are currently assigned to the genus Thelandros,
（syn. Paa verrucospinosa）
, Polypedates mutus and Naja
with only two being described from the Oriental
. Amin et al.［6］provided an identification
atra［6, 89］
region. In the case of the genus Parapharyngodon, 41
key for 11 valid Pseudoacanthocephalus spp. in the
species are assigned, with five being described from
world.
the Oriental region. After Bursey and Goldberg’s 2005
article ［25］, T. vietnamensis recorded in Reeves’
3-5．Arthropoda
butterfly lizard（L. reevesii）from Vietnam has become
The subclass Pentastomida Diesing, 1836（phylum
the 32nd（and 3rd Oriental）species assigned to the
Arthropoda: subphylum Crustacea: class Maxillopoda
genus［22］.
Dahl, 1956）is commonly known as tongue worms,
parasitizing the respiratory tracts of vertebrates［5,
3-3-18．Abbreviata deschiensi Le et Nguyen, 1966
105］. It is divided into four orders: Cephalobaenida
Host and location: Calotes versicolor, stomach［68］
Heymons, 1935（one family）; Porocephalida Heymons,
Locality: Binh Thuan Province（43）
1935 （four families）; Raillietiellida Almeida et
Incidence and intensity: No information
Christoffersen, 1999（one family）; and Reighardiida
Comment: The physalopterid genus Abbreviata
Almeida et Christoffersen, 1999（one family）. The
Travassos, 1920, has an internolateral tooth and
worms have a segmented body covered by a chitinous
externolateral tooth, and two double pairs of submedian
cuticle and the anterior end bears five appendages, i.e.
teeth on each pseudolabium［29］. Multiple Abbreviata
one mouth and two pairs of hooks for attachment to
spp. have been recorded in varanid lizards and snakes
the host.
［54-59］.
Three recorded pentastomid species from Vietnamese
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Binh Thi TRAN, Son Truong NGUYEN, Tao Thien NGUYEN, Pham Van LUC, Eliakunda MAFIE, Fatema Hashem RUPA, Hiroshi SATO
lizards were classified in Raillietiellidae Sambon, 1922
（Raillietiella Sambon, 1910）
.
hosts of Colubridae, Elapidae, Viperidae and Boidae
in Southeast Asia, India, Philippines, Taiwan, Japan
and China［3, 4, 30］. Kelehear et al.［63］reported
3-5-1．Raillietiella frenatus Ali, Riley et Self, 1981
high prevalences of this Asian pentastomid species
Host and location: Hemidactylus frenatus, lungs［2, 85］
in wild snakes native to the Australian tropics such
Locality: Hanoi（14）, Yen Bai（5）, Tuyen Quang（6）,
as Tropidonophis mairii（Colubridae）, Acanthophis
Bac Kan（7）Provinces and southern Vietnam（not
praelongus（Elapidae）
, Demansia vestigiata（Elapidae）
specified）
and Liasis fuscus（Pythonidae）
. They considered these
Incidence and intensity: 30.9%（46/149）with 1–14
records as a recent translocation of alien parasites via
worms/host
an unknown pathway. In Vietnam, R. orientalis causes
Comment: Members of the genus Raillietiella are
outbreaks of serious infection in farmed snakes such
parasites in the respiratory tract of carnivorous lizards.
as Naja naja and Ptyas mucosus. Dang［33］conducted
This species was recorded from H. frenatus in Malaysia,
epidemiological surveys of R. orientalis in Indian cobras
Thailand, Vietnam, Indonesia, Philippines and Taiwan,
（Naja naja）and Asian common toads（Duttaphrynus
Japalura swinhonis and Eutropis longicaudata in Taiwan
melanostictus） in the field of Vietnam and found
［1, 2, 78］,and Hemidactylus platyurus（syn. Cosymbotus
infections at an incidence of 39.6%（44/111）with an
platyurus）and Gehyra mutilata in Indonesia［1, 78］.
average intensity of 6.2 worms/snake（range 1–50）
Barton［13］reported R. frenatus from invasive Asian
and 0.4%（2/500）with 3 worms/toad, respectively.
house geckos（H. frenatus）as well as native geckos,
Furthermore, he demonstrated a direct life cycle of
Gehyra australis, in northern Australia, suggesting the
this pentastomid species by an experimental infection.
possible spread of alien parasites through introduced
Nguyen et al.［84］recorded the species from two
hosts. Furthermore, Kelehear et al.［63］collected R.
varanid species in Vietnam; however, no detailed
frenatus not only from invasive Asian house geckos（H.
description was given. In addition, they reported the
frenatus）but also from invasive cane toads（Rhinella
recovery of possibly another pentastomid species in
marina）and native tree frogs（Litoria caerulea）in
these two varanid species.
tropical Australia. Riley et al.［105］and Goldberg
and Bursey［46］reported a similar problem, i.e. the
3-5-3．Raillietiella affinis Bovien, 1927
invasion of alien parasites through introduced hosts, in
Host and location: Eutropis longicaudata, lungs［85］
Texas and Hawaii, respectively.
Locality: Hanoi（14）, Yen Bai（5）, Tuyen Quang（6）
,
Kelehear et al.［62］emphasized the importance
Bac Kan（7）Provinces
of molecular analyses of pentastomes in addition to
Incidence and intensity: 30.9%（46/149）with 1–14
morphological characterization for valid descriptions
worms/host
of new species, because often the same species adopts
Comment: This species was first collected from the
different morphological phenotypes of taxonomic
lungs of Gekko gecko（syn. Gekko verticillatus）in Java,
importance in different host species.
Indonesia, then noted in geckos and skinks from Egypt,
Sudan or Hawaii［30］. As an invasive species, Dervin
3-5-2．Raillietiella orientalis（Hett, 1915）Sambon,
et al.［38］reported R. affinis from the Madagascar
1922
giant day gecko, Phelsuma grandis. Human cases of R.
Host and location: Varanus salvator and Varanus
affinis infection are also known.
nebulosus, lungs［84］
In Vietnam, Nguyen et al.［85］recorded a parasitism
Locality: Vietnam（not specified）
of the same gecko species with possibly another
Incidence and intensity: 12.8%（2/20 V. salvator and
Raillietiella sp., different from R. frenatus and R.
3/19 V. nebulosus）with no intensity information
affinis.
Comment: This species has a wide spectrum of snake
― 49 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Endoparasites of Vietnamese lizards recorded in the last 50 years（1966 − 2015）
to other scientists for future research. As mentioned
４．DISCUSSION
above, Kelehear et al.［62］emphasized the taxonomic
In this article, we present a list of 45 species of
importance of molecular analyses of pentastomes
endoparasite of Vietnamese lizards. Specifically, 11
in addition to morphological characterization for
cestode, 12 trematode, 18 nematode, one acanthocephalan
accurate species differentiation. Furthermore, recent
and three pentastomid species have been recorded
worldwide spreads of invasive parasites accompanying
from 10 host species. As shown in Table 1, the majority
hosts beyond geographical borders make molecular
of endoparasite species（55.6%, i.e. 25/45）was recorded
characterization of isolated parasites a key technology
from two varanid lizards, V. nebulosus and V. salvator.
in understanding their exact taxonomic situation, i.e.
These lizard species are widely endemic in Vietnam
invasive species or native species.
as well as other Southeast and South Asian countries
At the beginning of this review, we highlighted the
such as Myanmar, Laos, Thailand, Cambodia, Malaysia
rich herpetofauna diversity of Vietnam（385 reptiles
and Indonesia for the former species, and India, Sri
and 181 amphibians）
. However, only a fraction of
Lanka, Bangladesh, South China, Myanmar, Laos,
the parasites of reptiles has been recorded in this
Thailand, Cambodia, Malaysia, Singapore, Indonesia
richly biodiverse territory. Additionally, most of the
and Philippines for the latter species［92］. Table 1 also
specimens recorded in the past are no longer available.
shows that 14 of the 45 endoparasite species（31.1%）
Parasitological surveys, if actually possible, on multiple
were detected in two gecko species, Hemidactylus
lizard hosts in a territory with rich herpetofauna
frenatus and Eutropis longicaudata（149 and 38 geckos
diversity may disclose the ecological relationships
. The remaining six endoparasite
examined, respectively）
among different categories of host lizards in Vietnam
species（13.3%）were nematodes described from five
or clarify the taxonomic relationship of parasites from
agamid species and one gecko species. The reports
Vietnamese lizards with those from lizards of other
on these six endoparasite species include five new
neighbouring or remote regions. In this sense, it is
species descriptions, concentrating on one or two
again recommended that parasites are characterized
targeted parasite（s）. Therefore, it would appear that
both morphologically and genetically.
rather than these lizard host species having only
Research on parasite diversity in Vietnam, as having
a few endoparasites, more parasites remain to be
been conducted as large-scale surveys in the country,
recorded from them. Indeed, we collected at least two
exclusively used a classical descriptional approach on
Meteterakis spp. from scale-bellied tree lizards, but due
parasites based on morphological criteria. With this
to difficulties with taxonomic differentiation, specific
approach, it is often difficult to evaluate morphological
identification has yet to be completed.
variation, as having been experienced in many categories
Except for two species, Cosmocercoides tonkinensis
of parasites. Consequently, more recent parasitological
and Strongyluris calotis from agamid lizards, no
research has applied molecular genetic technologies
molecular analyses accompanied the taxonomical
to surveys and parasite characterization, enabling
characterization of collected parasites ［120, 122］.
synonymization of morphological variants or detection
Strongyluris calotis collected from two different agamid
of cryptic species with an identical morphological
species showed two morphotypes with different numbers
manifestation. Furthermore, with the latest taxonomic
or arrangements of caudal papillae, which are believed
approaches, we can determine the phylogenetic position
to be of taxonomic importance to separate Strongyluris
of observed parasites or evolutional relationships with
species ［121, 122］. When the geographical and/or
related taxa. Usability of these advanced molecular
ecological isolation of host species are distinct and
approaches is dependent on the calibre of the background
multiple morphotypes of parasites from them are noted,
genetic data of the targeted species as well as related
molecular characterization of parasites can support
species. In contrast to parasites of medical and veterinary
our specific differentiation or leave invaluable clues
importance, the depository of molecular genetic data
― 50 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Binh Thi TRAN, Son Truong NGUYEN, Tao Thien NGUYEN, Pham Van LUC, Eliakunda MAFIE, Fatema Hashem RUPA, Hiroshi SATO
of lizard or amphibian parasites is sparse at present.
genus Raillietiella Sambon, 1910 with a description
General interest in the biodiversity of local nature or
of a new species. Syst. Parasitol. 7：111-123.
worldwide spread of invasive parasites via translocation
5. Almeida, W. O., Christoffersen, M. L., Amorim, D. S.,
of vertebrates beyond natural borders may enhance
Amorim, D. S. and Eloy, E. C. C. 2008. Morphological
our particular understanding of all organisms including
support for the phylogenetic positioning of
parasites. We are still a long way from disclosing the
Pentastomida and related fossils. Biotemas 21：81-
full repertoire of endoparasite fauna of lizards from
90.
Vietnam.
6. Amin, O. M., Nguyen, V. H. and Heckmann, R. A.
2008. New and already known acanthocephalans
from amphibians and reptiles in Vietnam, with keys
ACKNOWLEDGEMENT
to species of Pseudoacanthocephalus Petrochenko,
This study was supported in part by a Grant-in-Aid
1956（Echinorhynchidae）and Sphaerechinorhynchus
for International Collaboration Research in Asia 2016
Johnston and Deland, 1929（Plagiorhychidae）. J.
from the Heiwa Nakajima Foundation and also by JSPS
Parasitol. 94：181-189.
KAKENHI Grant Number 26291080. We are greatly
7. Amin, O. M., Düşen, S. and Oğuz, M. C. 2012. Review
indebted to Prof. Hideo Hasegawa（Oita University,
of the helminth parasites of Turkish anurans
Japan）, Prof. Yuzaburo Oku （Tottori University,
（Amphibia）
. Sci. Parasitol. 13：1-16.
Japan）and Dr. Kayoko Matsuo（Gifu Prefectural Office,
8. Arora, S. and Agarwal, S. M. 1960. Studies on some
Japan）for their helpful discussions and encouragement
intra-specific variations in Paradistomum orientalis
regarding the present work. We would also like to thank
Narain and Das collected from the gall-bladder of
Seiho Sakaguchi, Laboratory of Veterinary Parasitology,
Calotes versicolor Daud: Part I.（Dicrocoeliidae:
Yamaguchi University, Japan, for preparing the
Trematoda）.Bull. Zool. Soc. India 3：43-52.
illustrations for the figures.
9. Arora, S., Agarwal, M. M. and Agarwal, S. M.
1962. Studies on some intra-specific variations in
Paradistomum orientalis Narain and Das collected
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Dalsgaard, A., Chai, J. Y., Sohn, W. M. and
114.Sommerville, C. 1982. The life history of Haporchis
Murrell, K. D. 2007. Fishborne zoonotic intestinal
pumilio（Looss, 1896）from cultured tilapias. J.
trematodes, Vietnam. Emerg. Infect. Dis. 13：1828-
Fish Dis. 5：233-241.
1833.
115.Sprent, J. F. A. 1985. Ascaridoid nematodes of
124.Tubangui, M. A. 1938. Pseudophyllidean cestodes
amphibians and reptiles: Raillietascaris n. g. Ann.
occurring in the Philippines. Livro Jubilar Lauro
Parasitol. Hum. Comp. 60：601-611.
Travassos 1938：489-494.
116.Tkach, V. V. 2008. Family Encyclometridae Mehra,
125.Vlnová, I. 2014. Revize diphyllobothriidních tasemnic
1931. pp.349-352. In: Keys to the Trematoda,
plazů （Eucestoda: Solenophoridae）. Jihočeská
Volume 3（Bray, R. A., Gibson, D. I. and Jones,
univerzita v Českých Budějovicích, Přírodovědecká
A. eds.）, CAB International and Natural History
fakulta. Master thesis, 61 pp.
Museum, London, UK.
126.Woodland, W. N. F. 1940. A revision of African and
117.Tran, B. T. 2009. Study of parasitic cestodes in
Asiatic forms of the genus Duthiersia（Cestoda）.
some reptiles from Northern Vietnam. pp.41-
Proc. Zool. Soc. London B110：207-218.
45. In: Proceedings of the 3rd National Scientific
127.Yamaguti, S. 1952. Parasitic worms mainly from
Conference on Ecology and Biological Resources,
Celebes. Part 7. Cestodes of reptiles. Acta Med.
Hanoi, October 22, 2009. （in Vietnamese with
Okayama 8：1-12.
English summary）
128.Yamaguti, S. 1954. Systema Helminthum, Volume
118.Tran, B. T., Nguyen, T. L. and Nguyen, V. H. 2005.
II. The cestodes of vertebrates. Interscience
Cestode parasite in house gecko（Hemydactylus
Publishers, Inc., New York, U.S.A., 860 pp.
renatus）. pp.412-414. In: Proceedings of the 2005
129.Yamaguti, S. 1958. Systema Helminthum, Volume
National Conference on Life Sciences, Hanoi
I. The digenetic trematodes of vertebrates.
Medical University, November 03, 2005. （in
Interscience Publishers, Inc., New York, U.S.A.,
Vietnamese with English summary）
1575 pp.
119.Tran, B. T., Bursey, C. R., Goldberg, S. R. 2007.
130.Yamaguti, S. 1961. Systema Helminthum, Volume
Two new species of Pharyngodonidae（Nematoda,
Ⅲ. The nematodes of vertebrates. Interscience
Oxyuroidea）in Gekko ulikovskii from Vietnam. Publishers, Inc., New York, U.S.A., 1261 pp.
― 57 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Endoparasites of Vietnamese lizards recorded in the last 50 years（1966 − 2015）
131.Yildirimhan, H. S., Bursey, C. R. and Altunel, F.
Province, China. Acta Parasitol. 56：48-53.
N. 2011. Helminth parasites of the Balkan green
133.Ziegler, T. and Nguyen, T. Q. 2010. New discoveries
lizard, Lacerta trilineata Bedriaga 1886, from Bursa,
of amphibians and reptiles from Vietnam. Bonn
Turkey. Turk. J. Zool. 35：519-535.
Zool. Bull. 57：137-147.
132.Zhang, H., Zhang, S. and Zhang, L. 2011. Two species
of the genus Kalicephalus Molin, 1861（Nematoda,
Diaphanocephaloidea）from the water monitor,
Varanus salvator（Laurenti, 1768）in Guangdong
Correspondence：Hiroshi SATO, Laboratory of Parasitology, Joint Faculty of Veterinary Medicine, 1677-1 Yoshida,
Yamaguchi 753-8515, Japan.
E-mail：sato7dp4@yamaguchi-u.ac.jp
ベトナム産トカゲの内部寄生虫 : 過去 50 年間（1966−2015）の記録
トラン・ビンティ 1、グエン・ソントゥルオン 2、グエン・タオティエン 3、ルック・ファンヴァン 3、
マフィエ・エリアクンダ 4、ルパ・ファテムハシム 4、佐藤　宏 4
1
ベトナム科学技術院（VAST）生態・生物資源研究所（IEBR）寄生虫学部門、2 脊椎動物学部門、
3
ベトナム国立自然史博物館、4 山口大学大学院 連合獣医学研究科
要　約
ベトナムの両生類、爬虫類の寄生虫について現在までに得られた知見は限られている。これまでに 45 種の内部寄
生虫がベトナム産トカゲから記録されている。その内訳は、条虫 11 種、吸虫 12 種、線虫 18 種、鉤頭虫 1 種、舌虫
類 3 種で、宿主となるトカゲは 10 種を数える。ベトナムが両生類・爬虫類相が世界屈指の豊富さを誇ることを考え
ると（最近の報告によると、ベトナム国内から記録された爬虫類は 385 種、両生類は 181 種とされている）、宿主の
生物多様性に比して寄生虫相の研究は遠く及んでいないと考えざるを得ない。今後も正確な寄生虫種の同定を行うと
ともに、ベトナム国内産トカゲから得た種と近隣地域あるいは世界から報告された種との異同や類縁性を明確にする
研究が取り組まれねばならないが、その実施に際しては形態学的種同定とともに系統分類学的特徴づけが必要である。
Key words：ベトナム、トカゲ、寄生虫、蠕虫、舌虫、記録
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Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Essay
Veterinary Education in Nepal
Gokarna GAUTAM 1 and Ishwari Prasad DHAKAL 1,2
1
Faculty of Animal Science, Veterinary Science and Fisheries, Agriculture and Forestry University, Nepal
2
Vice Chancellor of Agriculture and Forestry University
ABSTRACCT
Nepal is an agricultural country with livestock being an integral component of the agriculture sector. Due to
Nepal’s agrarian economy and livestock product-based enterprises being one of the major businesses in both rural
and urban areas of the country, veterinarians presently available in the country are not sufficient to take care the
livestock and poultry health, thus, the number of institutions providing veterinary education has recently increased.
The university level undergraduate degree of veterinary science in Nepal is BVSc&AH（Bachelor of Veterinary
Science and Animal Husbandry）. There are currently four academic institutes in Nepal that offer this bachelor
degree of veterinary science. Two of these institutes are government funded, while the other two are private
institutions. To date, the Nepal Veterinary Council has registered 856 veterinarians in the country. Maintenance
of the quality of education that the institutes provide following the guidelines of the Nepal Veterinary Council is
of great importance. For this purpose, the institutions require infrastructure development, adequate numbers of
highly qualified academic staff, enhanced performances of faculty supporting staffs, upgraded laboratories and the
provision of research facilities
Key words：Veterinary education, Nepal, livestock.
in South Asia and has been stagnant in recent years.
１．INTRODUCTION
One of the main reasons for this low productivity is
Nepal is a landlocked and mountainous country with
the failure to achieve adequate productivity from the
a population of around 27.5 million. It is predominantly
animals, maintain their health and ensure access to
an agricultural country; about 65.6% of the Nepalese
resources for farmers raising livestock［4］.
people depend on agriculture for their livelihood. The
There are approximately 7.2 million cattle, 5.2 million
agriculture sector in Nepal contributes approximately
buffaloes, 0.8 million sheep, 10 million goats, 1.2 million
35% to the national gross domestic product（GDP）
pigs and 48.1 million poultry in Nepal. Of these, only
［1］. Livestock is an integral and important component
20% are commercially managed, while the rest are
of the Nepalese agriculture sector, contributing
owned by small farmers ［5］. In livestock rearing,
approximately 24% to the agricultural GDP ［2］.
various diseases cause an estimated annual loss of
It also plays important roles in human food and
845 million NPR（Nepalese rupee）to the cattle and
nutritional security, livelihood, regional balance, gender
buffalo industry of the country［6］. Nevertheless, the
mainstreaming and rural poverty alleviation ［3］.
livestock subsector has been one of the fastest growing
Approximately 87% of the total Nepalese people keep
subsectors in agriculture in Nepal. The Agriculture
some form of livestock. With 5.8 heads of livestock
Perspective Plan 1995–2015 identified livestock
and poultry per household, Nepal has one of the
production and productivity as an engine for growth in
highest ratios of livestock to humans in Asia. However,
agriculture［7］.
agricultural productivity in Nepal is one of the lowest
There are currently four academic institutes in
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Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Veterinary Education in Nepal
Table 1. Status of Veterinary Institutions in Nepal.
Establishment（Year）
Student number
enrolled per year
Remarks
Himalayan College of Agricultural Sciences and
Technology（HICAST）
2001
48
Private
Nepal Polytechnic Institute（NPI）
2010
48
Private
Faculty of Animal Science, Veterinary Science and
Fisheries（FAVF）
2012
50
Government
Institute of Agriculture and Animal Science (IAAS)
2013
40
Government
University
Agriculture and Forestry
University（AFU）
Tribhuvan University（TU）
⎫
︱
︱
︱
⎬
︱
︱
︱
⎭
Purbanchal University（PU）
Institution
Nepal that offer courses for the attainment of bachelor
faculties within the university; FAVF, Faculty of
degree of veterinary science（Table 1）. These are:
Agriculture, and Faculty of Forestry.
（i）Faculty of Animal Science, Veterinary Science
The university has a total of 110 faculty members.
and Fisheries（FAVF）at Agriculture and Forestry
The current university student population consists of
University（AFU）, Rampur, Chitwan;（ii）Institute
around 1,300 undergraduates, 200 postgraduates and
of Agriculture and Animal Science（IAAS）under
50 PhD scholars in different disciplines of agriculture,
Tribhuvan University（TU）at Paklihawa, Rupandehi;
animal science, veterinary science, fisheries and
（iii） Himalayan College of Agricultural Sciences
forestry. The Rampur academic complex occupies
and Technology（HICAST）affiliated to Purbanchal
an area of 280 ha and the Hetauda Forestry Campus
University（PU）at Kalanki, Kathmandu; and（iv）Nepal
has an area of 95 ha. The faculty members of the
Polytechnic Institute（NPI）affiliated to PU at Bharatpur,
university are involved in different research and
Chitwan. BVSc&AH（Bachelor of Veterinary Science
developmental activities with the support of national
and Animal Husbandry）is a five-year programme
and international institutions and agencies. At present,
comprising 10 semesters – nine semesters of study
various classrooms, farm buildings, laboratories, student
at college and a six-month internship at various
dormitories and residential buildings are in need of
institutions related to the veterinary profession.
renovation（Figs. 1 and 2）. The university requires
more support both nationally and internationally
２．
SCHOOLS FOR VETERINARY
EDUCATION IN NEPAL
for human resources development. As it is a new
university, the faculties and students also need to learn
2-1．FAVF at AFU
AFU was established in Rampur, Chitwan, Nepal,
in June 2010. This is the first technical university
in the country. It was founded upon the merger
of the Rampur Agriculture Campus of the IAAS
and the Hetauda Forestry Campus of the Institute
of Forestry of TU, thus inheriting undergraduate,
graduate and PhD programmes in agriculture and
veterinary sciences and fisheries from the Rampur
Campus and undergraduate and Master programmes
in forestry from the Hetauda Campus. The educational
programmes are supported by quality research and
developmental activities. Presently, there are three
Fig. 1.BVSc&AH classroom building, AFU, Rampur,
Chitwan.
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Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Gokarna GAUTAM, Ishwari Prasad DHAKAL
Around 50 students enrol each year in the BVSc&AH
programme. In 2015, the faculty enrolled 56 students
in the BVSc&AH programme and 16 students in the
BSc Fisheries programme. Additionally, 37 Masters
students and nine PhD students were enrolled in
different disciplines of FAVF. As it is a new university,
the first intake of BVSc&AH students is currently in
its sixth semester［8］
.
FAVF has linked up with universities abroad
for capacity building, enhancement of research and
laboratory capacity, faculty and student exchange
Fig. 2.Veterinary Teaching Hospital, AFU, Rampur,
Chitwan.
research collaboration, etc. The faculty is fully
committed to sharing samples and reagents in order to
produce quality peer-reviewed publications and reports.
how successful universities work and improve their
FAVF is happy to provide space and accommodation
standing in society. Faculty and student exchange
for the conduct of workshop and related research
would benefit both parties in the long term［8］.
activities by participating faculty members from
As mentioned above, FAVF is one of three faculties
South Asia and abroad. The faculty has very good
within AFU. The FAVF fosters student self-development,
working relationships with local organizations such
commitment and responsibility for the welfare of
as Department of Livestock Service（DLS）, Nepal
Nepalese society. Chitwan District is one of the areas
, Department of
Agricultural Research Council（NARC）
with the most potential for agriculture, livestock, poultry
National Parks and Wildlife Conservation（DNPWC）,
and aquaculture farming. Presently, the livestock-
Himalayan Animal Rescue Trust （HART） Nepal,
raising pattern is changing from household level to
Local Initiatives for Biodiversity Research and
commercialization. This is mainly occurring in poultry
Development（LI-BIRD）, National Zoonoses and Food
and dairy farming. It is greatly facilitated by committed
Hygiene Research Centre（NZFHRC）, Asia Network
farmers and other allied entrepreneurs, the presence of
of Sustainable Agriculture and Bioresource（ANSAB）,
adequate technical support from AFU, the suitability of
etc. FAVF also has links with foreign institutions such
the environment for farming and adequate marketing
as The United Graduate School of Veterinary Science,
facilities［8］.
Yamaguchi University, Japan（Fig. 3）
, HART UK,
Currently, there are 10 departments in the veterinary
science programme. These are: Veterinary Anatomy,
Physiology and Biochemistry; Veterinary Microbiology
and Parasitology; Veterinary Pathology and Clinics;
Veterinary Pharmacology and Surgery; Theriogenology;
Veterinary Medicine and Public Health; Animal Breeding
and Biotechnology; Animal Nutrition and Fodder
Production; Livestock Production and Management;
and Aquaculture and Fisheries. There are 38 faculty
members, 42 supporting staff and nine adjunct faculties
at FAVF. Two academic programmes are provided,
namely a five-year BVSc&AH and a four-year BSc.
Fisheries. In 2013, the faculty incorporated BSc.
Fisheries programme for the first time in the country.
Fig. 3.Ceremony for the renewed MOU（Memorandum
of Understanding）between AFU and UVY（The
United Graduate School of Veterinary Science,
Yamaguchi University）on 3 March, 2015.
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Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Veterinary Education in Nepal
University of Veterinary Medicine, Vienna, Austria,
of bachelor students who were enrolled at the Rampur
St. George’s University, Grenada, West Indies, Anglia
Campus prior to the establishment of AFU. Bachelor
Ruskin University, Cambridge, UK, etc［8］.
programmes are now conducted at its Lamjung
Campus（BScAg）and Paklihawa Campus（BScAg and
2-2．IAAS under TU at Paklihawa, Rupandehi
BVSc&AH）
（Fig. 5）
［9］.
IAAS is an agricultural institute under TU in
The veterinary science programme of IAAS was
Nepal for college level education in agriculture and
initiated in 1992 at the Rampur Campus, Chitwan,
veterinary sciences. The IAAS originated as a School
by converting its four-year BSc Animal Science
of Agriculture established in 1957 in Kathmandu to
programme into a five-and-a-half-year BVSc&AH
train Junior Technical Assistants. In 1968, the school
degree. This programme conversion made it the first
was upgraded to a College of Agriculture. In 1972,
veterinary school in the country and approximately 300
the college was recognized as IAAS under TU and
veterinarians have been trained at IAAS to date.
moved to Rampur, Chitwan, in 1974. It offers a BSc in
Following the acquisition of the facilities of IAAS,
Agriculture（BScAg）
, BVSc&AH, Master of Science
Rampur, by AFU, the Veterinary School of TU moved
（Agriculture）, Master of Science（Animal Science）,
to the Paklihawa Campus, Rupandehi, in 2013. At
Master of Veterinary Science, Master of Science
present, it enrols approximately 40 students each
（Aquaculture）and PhD programmes. Following the
year in the BVSc&AH programme. There are four
government of Nepal’s policy in 2010 to establish
departments in the veterinary science programme:
AFU at Rampur, Chitwan, the physical properties
Veterinary Anatomy, Physiology and Biochemistry;
and other assets of IAAS, Rampur, were acquired
Veterinary Medicine, Pharmacology and Surgery;
by AFU. Consequently, the office of the IAAS Dean
Veterinary Microbiology, Parasitology and Epidemiology;
was moved to Kathmandu in 2011 and directs its
and Veterinary Pathology, Theriogenology and Clinic.
programmes and activities through the Dean’s contact
Additionally, the three departments of Animal Breeding
offices in Sanepa, Lalitpur and Kirtipur, Kathmandu.
and Biotechnology, Animal Nutrition and Fodder
Although AFU was established in the facilities of
Production, and Livestock Production and Management
IAAS, some administration offices of IAAS remain in
work closely with the four aforementioned departments
Rampur（Fig. 4）. IAAS academic programmes are not
for a broader perspective of animal science study and
conducted there, with the exception of the final years
research［10］.
2-3．HICAST
HICAST was established in 2000 （Fig. 6）. It is
affiliated to PU and is the first private veterinary
school in Nepal. It recently moved to Kalanki,
Kathmandu, from its original site in Bhaktapur. The
college aims to teach and contribute to all fields of
Fig. 4. Joint office of AFU and IAAS, TU at Rampur,
Chitwan, during the transition phase.
knowledge in agricultural and applied sciences, from
the farmer’s intuitive knowledge to the sciences and
from the theoretical to the applied. It offers BScAg and
BVSc&AH programmes. The veterinary programme
（BVSc&AH）at HICAST was initiated in 2001. For
the last few years, it has enrolled 48 students per year
in the BVSc&AH programme. To date, it has trained
nearly 200 veterinarians［11］.
Fig. 5. Paklihawa Campus, Rupandehi, IAAS, TU.
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Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Gokarna GAUTAM, Ishwari Prasad DHAKAL
Fig. 7. NPI, Bharatpur, Chitwan.
three schools that conduct a three-year veterinary
Fig. 6. HICAST, PU at Kalanki, Kathmandu.
diploma programme. There are more than 7,500 paravets （JT/JTA） in the country. Because the small
2-4．NPI
number of veterinarians in Nepal cannot personally
NPI Ltd. was established in Bharatpur, Chitwan,
attend all the farms, these para-vets serve the livestock
in 1994 as a training institute to conduct short-term
sector of our country to a great extent.
training in various technical fields（Fig. 7）
. Subsequently,
university level undergraduate programmes were also
launched at the institute. In 2010, an undergraduate
４. CONCLUSION
level veterinary programme（BVSc&AH）was introduced.
In Nepal, the current veterinary education is recognized
This programme is affiliated to PU. It is the second
as a major determinant for the all-round development
private veterinary school in Nepal. NPI enrols 48
of the country. The quality of the education has to be
students each year in the BVSc&AH programme. The
enhanced to produce able, productive, disciplined and
first intake of BVSc&AH students will graduate in
socially responsible citizens and to create a workforce
July/August 2016［12］.
capable of facing the challenges of the 21st century. The
immense potential of the livestock sector in enhancing
３．JUNIOR TECHNICIANS AND
JUNIOR TECHNICAL ASSISTANTS
（PARA-VETS）FOR VETERINARY
SERVICE
the income of the poor is becoming increasingly
apparent. Livestock distribution is more equitable than
land distribution. Thus, the livestock sector provides a
natural entry point for poverty alleviation programmes.
However, a major issue affecting the livestock economy
Apart from these veterinary colleges, there are
is the quality of the education and the effectiveness
various technical training schools affiliated to the
of the delivery of livestock services to communities.
Council for Technical Education and Vocational
At present, a good number of institutions provide
Training （CTEVT） that educate students to the
veterinary education in Nepal. However, it is important
levels of Junior Technician（JT）and Junior Technical
to maintain the quality of the education they provide.
Assistant（JTA）in the field of veterinary science.
For this purpose, the institutions require infrastructure
After passing the School Leaving Certificate（SLC）
development, adequate numbers of highly qualified
examination, students can join JTA and JT level
academic staff, enhanced performances of faculty
courses. There are about three dozen training centres
supporting staffs, upgraded laboratories and the
that conduct 15-month veterinary JTA training and
provision of research facilities.
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Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Veterinary Education in Nepal
on the Social Dimension of Globalization: Geneva,
ACKNOWLEDGEMENT
Switzerland, 2004; p. 143.
We thank Prof. Hiroshi Sato, Dean of The United
4. iRIN. 2013. Why livestock matters in Nepal.
Graduate School of Veterinary Science, Yamaguchi
［Available online: http://www.irinnews.org/report/
University, Japan, for his guidance and encouragement
in the preparation of this manuscript. We also thank
98463/analysis-why-livestock-matters-nepal］
5. DLS. 2015. Department of Livestock Services,
Dr. Subir Singh, Department of Veterinary Medicine
Ministry of Livestock Development, Nepal.
and Public Health, AFU, Rampur, Chitwan, and
6. Lohani, M.N. and Rasali, D.P. 1992. Economic
Dr. Shambhu Shah, Faculty of Veterinary Science,
analysis of animal diseases in Nepal, Proceedings of
Paklihawa Campus, IAAS, TU, for providing valuable
the 4th Nepal Veterinary Conference, Nov 17–19.
information.
7. MoAC. 1995. Agriculture Perspective Plan（App）
,
1995–2015; Government of Nepal: Kathmandu,
Nepal.
REFERENCES
8. http://www.afu.edu.np/about-us.
1. Statistical information on Nepalese agriculture
9. https://en.wikipedia.org/wiki/Institute_of_
（2013/14）
. 2014. Government of Nepal, Ministry
Agriculture_and_Animal_Science.
of Agricultural Development.［Available online:
10. http://www.iaas.edu.np.
http://www.moad.gov.np/uploads/files/Year%20
11. https://en.wikipedia.org/wiki/Himalayan_College_
book%202014.pdf］
of_Agricultural_Sciences_%26_Technology_
2. ADS. 2012. Ads Assessment Report, Agricultural
Development Strategy Assessment; Government
）.
（HICAST）
12. http://www.npibharatpur.org.np/.
of Nepal, ADB, IFAD, EU, FAO, SDC, JICA,
WFP, USAID, DANIDA, DfID and World Bank:
Kathmandu, Nepal.
3. I L O . 2 0 0 4 . A F a i r G l o b a l i z a t i o n : C r e a t i n g
Opportunities for All; Report of the World Commission
Correspondence：Gokarna GAUTAM, Department of
Theriogenology, Faculty of Animal Science, Veterinary
Science and Fisheries, Agriculture and Forestry University,
Rampur, Chitwan, Nepal.
E-mail：gokarnagautam@gmail.com
ネパール国の獣医学教育の現況
ゴカルナ・ゴータム 1、イシュワリ・プラサド・ダカール 1,2
1
ネパール国農林業大学・畜産獣医水産学部、2 ネパール国農林業大学・副学長
要　約
ネパール国は農業国で、畜産も盛んである。国内の地域を問わず、農業関連産業や畜産関連産業は主要な経済分野
であるため、獣医学教育機関も最近増加してきた。ネパール国で獣医学を学ぶ大学教育を修了すると BVSc&AH（獣
医畜産学士）が授与されるが、この教育は同国内では現在４機関で行われている。そのうちの 2 機関は国立大学、他
の 2 機関は私立大学である。ネパール国獣医師協議会には現在までに 856 名が登録されている。ネパール国獣医師協
議会が定める指針に沿い実施される獣医学教育のレベル維持は極めて重要といえる。そのためには、国内 4 つの獣医
系教育機関の施設整備、教育スタッフの質的・数的充実、教育補助員の技能充実、最新診断技術・解析機器の導入や
研究設備の充実などが今後の課題となっている。
Key words：獣医学教育、ネパール、畜産
― 64 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
施設紹介
大原研究所から馬原アカリ医学研究所へ
―ダニ媒介性感染症研究の断片―
藤　田　博　己 1,2
1
馬原アカリ医学研究所、2 藤田保健衛生大学医学部
要　約
大原研究所は、1925 年に野兎病研究のための専用施設として、当時の福島県福島市にあった私立大原病院内に大
原研究室として開設された。1944 年からの閉鎖期間もあったが 1948 年には再開、1973 年には財団法人大原綜合病院
附属大原研究所に改組され、野兎病研究を主体にダニ媒介性感染症一般にも範囲を広げながら、2012 年の閉鎖まで
活動を続けた。馬原アカリ医学研究所は、2010 年にダニ媒介性疾患研究を目的に、徳島県阿南市の医療法人新心会
馬原医院内に開設された馬原医院アカリ疾患研究センターから始まり、2011 年に馬原医院外の施設での開設に向け
て現在の名称に変更、2012 年から大原研究所の研究材料・資料と研究機能を引き継いで活動を開始した。
Key words：大原研究所、馬原アカリ医学研究所、ダニ媒介性感染症、野兎病、日本紅斑熱、研究活動 .
で大原八郎によってそれぞれに「再発見」された。国内
１．はじめに
におけるヒト患者の発生は、年間 80 例近くに達した年
大原研究所と馬原アカリ医学研究所は、開設の時期は
もあったが、1970 年代から 1980 年代になると年間発生
大きく異なるが、どちらも民間の医療機関に属し、それ
数は 10 例前後に減少し、最近では発生の見られない年
ぞれに野兎病（やとびょう、tularemia）と日本紅斑熱
も続くようになった。発生が減少しているとはいえ、野
という新疾患との遭遇が開設の契機となっていることで
兎病菌は自然環境内にマダニ類や吸血性昆虫類などを介
共通する。この他にも相互に少なからず関わりを持ち続
して野生動物間に維持されているので、野兎病は現在で
けてきた両施設について、筆者は大原研究所に長年勤務
も常に感染リスクのある疾患である。
し、また現在はアカリ研究所に所属する立場から歴史的
大原研究所の前進の大原研究室は、1924 年に当時の
経過も含めて紹介したい。
大原病院の副院長で外科と耳鼻科を専門とする大原八郎
が不明熱性疾患（後の野兎病）患者に遭遇したことを契
機に開設された。未知の疾患を予感した大原は、1925
２．大原研究所
年に論文を発表するとともに、まだ感染症かどうかも不
大原研究所が 80 年以上の長期にわたって研究を継続
明であったこの疾患の原因究明のために研究室を立ち上
してきた野兎病は、今では発生頻度が低いこともあって、
げた。大原研究室は病院の手術室を改造したもので、16
国内の医療関係者からは忘れ去られた感染症となってし
畳ほどのコンクリート床の大部屋とタイル張りの小部屋
まった。野兎病は野兎病菌の感染による急性の発熱性疾
からなり、当時を知る人によると、まるで穴倉のような
患で、主に北半球の広い地域に発生が知られる人獣共通
研究室だったという。ここには、コッホ式滅菌装置、孵
の感染症である。この疾患のもっとも古い記録が日本に
卵器、遠心器、冷蔵庫、顕微鏡、ミクロトームといった
ある。1837 年の江戸時代に本間棗軒が著した瘍科秘録
機材が設置されてはいたが、研究施設としては必要最小
の第 9 巻に記載された食兎中毒（のちに中兎毒に改訂）
限にも満たないようなものであった。研究スタッフも必
が野兎病に一致する。近代医学の時代以降には、1911
要最小限で、常勤は大原と助手 1 名のみ、ここに研究志
年に米国において、R. A. Pearse によって昆虫媒介性の
望者が 1、2 名訪れては数年間研究生活を送るといった
ヒト疾患として、また同年に G. W. McCoy によってハ
研究方式が続けられた。このように極めて小規模な研究
タリスのペスト様疾患として、次いで、1925 年に日本
施設ながら、ここからは研究成果が次々と発表されてい
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大原研究所から馬原アカリ医学研究所へ ―ダニ媒介性感染症研究の断片―
くことになる。研究開始の 1925 年には、まだ野兎病菌
ロッキー山研究所と旧ソ連モスクワのガマレア研究所の
を安定的に培養できる培地がなかったにもかかわらず、
各野兎病研究部署を主体に各国の野兎病研究者との間で
早くも原因菌を分離・特定して、細菌感染症の 1 種であ
続けられた。
ることを確定した。この時点で野兎病という病名はまだ
大原研究所で徹底されていたことは、「野兎病菌以外
ない。原因菌は大原 - 芳賀球菌と呼称された。病原体確
は雑菌である」ということであった。野兎病菌の分離に
定に向けた検索を主導したのが海軍軍医中佐だった芳賀
は所長からの金一封があった。小動物からの野兎病菌検
竹四郎で、彼は大原にとっては最初の野兎病の共同研究
査の過程でときおり分離されるツツガムシ病リケッチア
者でもあり、大原研究室初の野兎病研究による学位（医
などはまさに雑菌扱いで、上司からは「そんな雑菌は早
学博士）取得者となった。一時閉鎖となる 1944 年まで
く捨てろ」と再三言われた。
に大原研究室からは 145 編の論文発表が続き、芳賀以降
野兎病確定のための病原診断依頼には無料で対応して
にも野兎病研究には各地から 8 名が訪れ、いずれにも学
いた。確定症例については、野兎病調査票の提出をお願
位が授与されている。研究内容は、病型、病理、菌の性
いすることになっていた。検査依頼が大原研究所に集中
状、感染経路、媒介動物、ワクチン、など多方面におよ
していたために、国内の野兎病の発生に関するデータ
んだ。一方では、野兎病研究の本場、米国公衆衛生局の E.
ベースとしてはこの調査票が有力なものとなっている。
Francis や G. W. McCoy らとの情報交換や研究材料のや
野兎病には類似疾患が複数あり、特に 1980 年代から多
り取りを通した交流も行っている。1943 年の大原の死
発傾向になったツツガムシ病を含むリケッチア症との鑑
去にともない 1944 年に研究室は閉鎖となった。
別が必要となった。そこでまずはツツガムシ病を、その
1948 年に大原八郎の長男、甞一郎によってこの穴倉
後は紅斑熱と発疹熱も加えて標準対応とした。研究所に
のような研究室は再開された。大原甞一郎は外科と耳鼻
おけるツツガムシ病やダニへの対応が活性化したため
科を専門とし、野兎病以外の研究テーマに興味を持って
か、大原研究所をツツガムシ病あるいはダニの専門研究
いたものの、その希望は断念せざるを得ない状況であっ
機関との誤解が最近になっても一部にはあるようだが、
た。この時期に野兎病患者が多発し、戦前の大原病院で
本業はあくまでも野兎病研究である。
の野兎病治療を知って受診する患者への対応に迫られた
1999 年、大原研究所は福島市郊外の大原綜合病院附
ためである。ところがすでに父八郎の残した研究材料は
属大原医療センター内への移転に伴い、これまでの野兎
診断用抗原の作成に必須の菌株も含めてほとんど失われ
病研究室、細胞培養室、ダニ研究室を１つの部屋に集約
ていた。そこで、分離株が得られるまでの過渡期には、
させざるを得なくなった。このほかに小さな資料室と動
患者から摘出した病巣部位のリンパ節膿汁から抗原を作
物室が付随した。規模は縮小し研究員も実働 2 名に減少
成して診断に使用したこともあった。
した。2011 年 3 月には東日本大震災に見まわれ、福島
1957 年には新しい施設への移転となった。同じ病院
市内もライフラインは全て止まった。回復には長い日数
内ではあるが、もはや穴倉のような研究室ではない。将
を要したが、研究所のある大原医療センターは早い時点
来の研究所への移行も見据えて、新たな研究室群も用意
で自家発電装置が稼動、また透析施設の維持のため持続
された。まずは細菌室と病理室が稼動し、機材も充実し
的に給水を受けることができた。電源が確保できたこと
始めた。
からフリーザー類は安定していて、研究材料は維持でき
1973 年には大原研究所へ改組となった。筆者が大原
た。もっとも、地震によって物品の多くが散乱した研究
研究所に勤務を始めた 1979 年には、細菌室、病理室、
室の片付け作業は結構大変ではあったし、原発事故に
野兎病研究室、細胞培養室、動物室から構成され、スタッ
よって福島市内でも一時は最大で毎時 24 マイクロシー
フは実働 5 名であった。この年に、野兎病媒介ダニを扱
ベルトの放射線量が記録された。実験室診断は継続でき
うダニ研究室を新設させていただいた。その後、電子顕
ていたものの、交通網は各所で分断されて検体の輸送は
微鏡室が増設された。病院内には小規模ながら野兎病記
滞り、この時期には外部からの検査依頼検体の搬送はほ
念室が設けられていて、この疾患の啓発や研修にも使わ
とんどできなくなった。その中で主治医自らが緊急車両
れていた。病理室は、その後の病理診断の診療科への制
に便乗して、研究所まで検体が届けられたこともあった。
度変更によって、病理部に独立して研究所から分離され
大原研究所の時代にも戦前と同様の研究方式は継続さ
た。
れ、1951 年以降、研究所からの発表論文数は 2011 年ま
戦後の海外との研究交流は、米国モンタナ州にある
でに 350 編を越え、19 名が学位を取得した。
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藤　田　博　己
図１．大原研究所および関係資料．A 大原綜合病院内の研究所入口（1990 年代）; B 研究室で実験中の大原八郎 ; C
研究用ノウサギ捕獲の一行（右端が大原八郎）; D 野兎病の最初の記載者，本間棗軒 ; E 野兎病が記載された
瘍科秘録第九巻（1837 年）表紙の一部 ; F 野兎病（食兎中毒）の記載部分．
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大原研究所から馬原アカリ医学研究所へ ―ダニ媒介性感染症研究の断片―
経過は前後するが、2010 年 3 月 10 日、唐突に財団か
おけるこの新しい病気の概念と治療法の確立には特に大
ら 3 月 31 日に研究所を閉鎖するとの通知があった。研
きな難題はなかったものと思われる．国立予防衛生研究
究材料と資料は他の研究機関へ移動するなどして処分す
所（現国立感染症研究所）、秋田大学医学部、福井医科
るようにとのことであった。しかし、その時点で大原研
大学（現福井大学医学部）、徳島大学医学部、愛媛大学
究所が研究分担で参加していた厚生労働省の研究事業が
医学部などからは、随時の研究協力が得られた。大原研
継続中であり、これが終了するまでは研究所を維持すべ
究所も 1986 年以降、主に媒介ダニの野外調査や病原体
きとの交渉の結果、2012 年 3 月末までは継続できるこ
検索、患者の病原診断などで協力関係が続いた。
ととなった。実際には 4 月末までの延長後に閉鎖となっ
2010 年、筆者は馬原院長から研究所立ち上げの協力
た。
要請を受けた。この研究所は、ダニとダニ媒介性疾患の
2012 年 3 月から４月には、実験室診断の受け入れと
研究を目的に馬原医院アカリ疾患研究センターとして、
研究活動を維持しつつ、研究材料と資料は、受け入れに
この年の 1 月に開設となったが、まだ施設の建物はなく、
応じてくれた新設の馬原アカリ医学研究所へと移送作業
馬原医院の関連施設の会議室の一角を間借りして、顕微
が続けられた。同年 4 月末日に退所した最後の研究員 2
鏡 1 台とマダニの採集用具の常備からのスタートとなっ
名は、5 月 1 日に馬原アカリ医学研究所へと移った。
た。正規の研究員は馬原医院の職員 1 名が兼任し、筆者
と他 1 名が外部の客員研究員として所属した。その後，
研究施設候補には馬原医院に近い工場跡地が選定され、
３．馬原アカリ医学研究所
解体されずに残されていた工場従業員用保育所を改装し
主にダニ類媒介性で急性発熱性疾患の紅斑熱群リケッ
て研究所に使用することになった。これに伴い、2011
チア症は、かつて日本には存在しないとされていた。
年に研究所は現在の名称、医療法人新心会馬原アカリ医
1984 年に徳島県阿南市の私立（当時、現在は医療法人）
学研究所に変更された。
馬原医院の馬原文彦院長が同市で発生した不明熱性疾患
2011 年、研究所の建物がほぼ完成した。2012 年の 4
の患者を診察し、これが国内初確認の紅斑熱群リケッチ
月末までには大原研究所からの研究材料や資料の搬入が
ア症、後の日本紅斑熱の発見となった。野兎病の場合に
終了した。リケッチア症や野兎病の病原診断技術と研究
は、患者に初めて遭遇した大原は研究に向けて直ちに研
機能のすべてが大原研究所から引き継がれ、大原研究所
究室を立ち上げたが、馬原は自前の研究施設をすぐには
で実施されてきた病原診断・検査についても、引き続き
作っていない。馬原による紅斑熱研究は、臨床面を主体
こちらで対応できることをこれまでに依頼のあった医療
に続けられたため、医院の施設自体が研究室を兼ねてい
施設や研究機関に通知された。2012 年 5 月 1 日から現
たと見ることもできる。馬原には、確定のための病原診
在の施設での活動が開始された。研究所内にダニとダ
断や媒介ダニ調査などの研究協力者が外部に複数いたこ
ニ関連疾患情報資料の展示室が併設され、資料館とし
とも研究室開設に至らなかった理由だったかもしれな
て 2012 年 7 月から一般公開された。研究所は研修の受
い。外部に確定のための病原診断を随時依頼することが
入れと野外調査の拠点としての利用にも対応している。
できたし、また外部から現地調査の協力や材料提供の依
2016 年現在の人員は、研究員が常勤 2 名と兼任 1 名、
頼があれば共同研究に参画してきた。その意味では当時
外部の客員研究員が 2 名である。
の大原が野兎病について頼るべき外部の研究機関がほぼ
なお、当研究所を国内唯一のダニ専門の民間研究施設
皆無であった状況とは大きく異なっていた。日本紅斑熱
と認識する向きが一部にはあるようだがそれは誤解であ
は新たな独立疾患ではあるが、類似する紅斑熱群リケッ
る。国内には当研究所開設以前からダニ研究を専門とす
チア症は、古くはロッキー山紅斑熱があり、世界各地に
る複数の民間研究施設が存在する。それらの研究から集
発生が既知であったことから、診断法や治療法などの大
積されてきた多くの研究業績と活動に敬意を表したい。
枠については多くの知見が集積されていた。臨床方面に
― 68 ―
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藤　田　博　己
図２．馬原アカリ医学研究所．A 研究所外観 ; B 表札 ; C 資料展示室 ; D 実験室．
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大原研究所から馬原アカリ医学研究所へ ―ダニ媒介性感染症研究の断片―
記
に情報・資料の収集
医療法人新心会 馬原アカリ医学研究所／馬原ダニの資
2． ダニ媒介性疾患の実験室診断、ダニの同定、疫学調
査、その他の研究と医療活動への支援
料館
〒 779-1510 徳島県阿南市新野町是国 56-3
3． ダニ媒介性疾患の資料とダニ類標本の展示公開
Tel/Fax 0884-36-3601
4． その他の関連事項
資料館の開館日：平日 9 時から 17 時。土・日・祝日
は休館。臨時休館日あり。
アクセス：JR 牟岐線「新野」駅下車、徒歩 3 分
事業内容
1． ダニ媒介性疾患と寄生性ダニ類に関する研究ならび
連絡責任者：藤田博己、馬原アカリ医学研究所、〒 779-1510
徳島県阿南市新野町是国 56-3.
E-mail : fujitah7knu@y8.dion.ne.jp
Correspondence：Hiromi FUJITA, Mahara Institute of Medical
Acarology, Korekuni 56-3, Aratano-cho, Anan, Tokushima 7791510, Japan
Brief histories of Ohara Research Laboratoy and
Mahara Institute of Medical Acarology
Hiromi FUJITA 1,2
1
Mahara Institute of Medical Acarology, 2 Fujita Health University
ABSTRACT
Ohara Research Laboratory, formerly The Laboratory of Ohara Hospital（latterly Ohara General Hospital）, in
Fukushima, Japan, was established in 1925 for research on tularemia. Up until 2012, this laboratory conducted
active research on the particular disease caused by Francisella tularensis. Other acari-borne diseases were also
studied at the laboratory in more recent times. In 2010, Mahara Institute of Medical Acarology, formerly Mahara
Hospital Center for Medical Acarology, in Anan, Tokushima, Japan, was established for research on acari-borne
diseases and their vectors. Brief histories of these distinguished institutions are presented here.
Key words：Ohara Research Laboratory, Mahara Institute of Medical Acarology, acari-borne infectious disease,
tularemia, Francisella tularensis, Japanese spotted fever, Rickettsia japonica, research activity.
― 70 ―
Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016

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