Flagellate Protozoa in the Digestive Tract of the

Japanese Field Vole, Microtus montebelli

Soichi IMAI and Keiji OGIMOTO*

Department of Parasitology, Nippon Veterinary and Zootechnical College, Musashino-shi 180 *Department of Animal Science , Tohoku University, Sendai-shi 980

(Received October 15, 1987)

Abstract Protozoal fauna of the gastrointestinal tract of Japanese field vole, Microtus montebelli was examined. As the result of survey, no ciliate protozoa such as those in rumen or large intestine of horse were detected, but many trichomonad flagellate protozoa from all the parts of the digestive tract including the fore-stomach. In the cecum and rectum, two types of flagellates were recog- nized and they were identified as Tritrichomonas muris and Tetratrichomonas microti, respectively. In the fore-stomach and stomach, only one type of flagel- lates was detected, and they had no anterior flagella. From the comparison of morphology and measurement, they were concluded as the transit stage of T. muris. Jpn. J. Zootech. Sci., 59 (4): 351-356, 1988 Key words: Japanese field vole, Tetratrichomonas microti, Tritrichomonas muris, transit stage

It is well known that many and various protozoa inhabit the digestive tracts of large herviborous animals including ruminants1-3). Recently, similar protozoa to those in rumen protozoa were found in the capybara, Hydrochoerus hydrocaeris, one of the rodents having herviborous behavior4). On the other hand, an attempt has been done to use the Japanese field voles which have plural stomachs in their body, as the experimental animals for the model of ruminants5,6). Regarding the intestinal micro- biological examinations on the voles, KUDO7,8) isolated and identified some bacterial species as same as those isolated from the rumen. However, no reports have been published on the protozoal fauna, thus we examined whether the protozoa similar to that in capybaraor in ruminants are inhabiting the digestive tract of the Japanese field voles or not. As a result, many flagellate protozoa were detected in various parts of the gastrointestinal tract including fore-stomach, then their species and distribution in the digestive tract were surveyed.

Materials and Methods

Thirteen Japanese field voles were captured at Sugadaira, Nagano Pref. For taxonomical and morphological examinations, the animals were immediately sucrified by cervical dislocation. After laparotomy the contents of fore-stomach, stomach, Jpn. J. Zootech. Sci., 59 (4): 351-356 351 1988 I MAI and OGIMOTO

cecum and rectum were sampled. A part of each content was smeared on a slideglass and treated with Giemsa's staining method. The other part of content was fixed in 1 (V/V) glutaraldehyde solution buffered with 0.1 M phosphate buffer (pH 7.2) and 2% % (W/V) potassium permanganate aqueous solution, dehydrated with ethanol series, dried in a Hitachi model HCP-2 critical-point-dryer, coated with thin layer of gold, and observed with a Hitachi-Akashi model MSM-4 scanning electron microscope. For counting protozoa, the intestinal contents obtained were weighed and fixed in 5 ml of 10% (V/V) buffered formalin, then protozoal number was calculated using a plankton counter slideglass1).

Results and Discussion

As the result of observation, no ciliate protozoa such as those in rumen or large intestine of horse or capybara were detected, but flagellate protozoa from all the parts of the digestive tract surveyed. Table 1 shows the frequency of appearance of the flagellates in each part of the digestive tract. In cecum and rectum, the flagellates were found from all the voles examined, but the frequency of appearance in the fore-stomach and stomach were 69.2% and 30.8%, respectively. Fig.1 shows the number of flagellates in the gastrointestinal tract of the voles. The number of

Table 1. Frequency of appearance of protozoa in the gastrointestinal tract of Microtus montebelli

: Above 105/g protozoa. + : Under 103/g protozoa. : 104-105/g protozoa.++ - : Not observed .

Fig.1. Number of protozoa in the gastro- intestinal tract of Microtus montebelli

352

+++ Protozoa in the Japanese Field Vole flagellates was the highest in the cecum, which value was about 106/g, in contrast, the value in the stomach was the lowest, about 102/g. Two types of flagellates were detected from the cecum and rectum (Figs. 3,4,6 and 7), and both protozoa had apparent undulating membrane, anterior flagella and costa. Therefore, these flagellates were considered to be belonging to Trichostomatida. Trichomonad flagellates have widely been detected from the lower intestine such as cecum and colon of various animals including rodents, such as mice, rats, hamsters and guinea pigs9). The two types of trichomonad species detected in the present exami- nation were identified as Tritrichomonas muris (Grassi, 1879) and Tetratrichomonas microti (Wenrich & Saxe, 1950), respectively, from the number of anterior flagella, which were three in T. muris and four in T. microti, and the body sizes as shown in Table 2. Tritrichomonas muris has been very commonly found from various rodents10) and T. microti has also been reported from rats, guinea pigs, hamsters and one of the field voles, Microtus pennsylvanicus11). On the other hand, the flagellates detected from the fore-stomach and stomach of the Japanese field voles were only one type, and morphologically different from the two types of trichomonad species in the cecum and rectum. These flagellates were oval in shape with about 10μm in length and 7μm in width (Table 2). A spherical nucleus at the anterior part of body, an undulating membrane at the side of body, and a costa (Figs. 2 and 5) were recognized in their body. However, no anterior flagella which number has been one of the most important characteristics for identification of the trichomonad species, were observed. Regarding such flagellates, KUROKI et al.12) observed 4 kinds of shape, pseudocyst, trophozoite, and transit stages to pseudocyst and trophozoite, of Tritrichomonas muris in various parts of gastrointestinal tract of hamsters and mice, and reported that the flagellates of transit stage to the trophozoite lost their anterior flagella in the stomach of the hosts. The flagellates obtained from the fore-stomach and stomach of the Japanese field voles had very similar morphology to that reported by KUROKI et al.12), and similar body size to that of Tritrichomonas muris obtained from the cecum (Table 2), so that, these flagellates were decided to be a transit stage to the trophozoite of T. muris as reported by KUROKI et al.12) on the hamsters.

Table 2. Size of protozoa in the gastrointestinal tract of Microtus montebelli1

1: n=12. 2: Mean. 3: Standard deviation.

353 IMAI and OGIMOTO

354 Protozoa in the Japanese Field Vole

Explanation of Figures Figs. 2-4. Light micrographs of the protozoa found in the gastrointestinal tract of Microtus montebelli. Fig. 2. Protozoon from the fore-stomach. Distinct nucleus (N), costa (C) and undulating membrane (UM) are observed, but not anterior flagella. Giemsa's stain. ×1,700.

Fig. 3. Type 1 protozoon (Tritrichomonas muris) from the cecum. Three anterior flagella (AF) in addition to undulating membrane (UM), costa (C) and axostyle (A) are seen. N, nucleus.

Giemsa's stain. ×1,600. Fig. 4. Smaller Type 2 protozoon (Tetratrichomonas microti) from the cecum. Anterior flagella (arrows) are shown. Giema's stain. ×1,500. Figs. 5-8. Scanning

electron micrographs of the protozoa. Fig. 5. An individual from the fore-stomach. Posterior

flagellum (PF) attached to the body by undulating membrane, but no anterior flagella is

shown. ×7,000. Fig. 6. Type 1 protozoon from the cecum. Distinct posterior flagellum (arrow) is observed. ×8,000. Fig. 7. Type 2 protozoon from the cecum. Posterior flagellum (PF) and

anterior flagella (arrow) are shown. ×8,000. Fig. 8. Anterior part of Type 2 protozoon. Four

anterior flagella (arrow) are indicated. ×18,000.

From the results, it was concluded that no peculiar protozoa such as rumen ciliates in ruminants inhabit the fore-stomach or the stomach of the Japanese field voles, and that the flagellates detected from these organs are derived from the cecum by copro- phagy of the voles themselves. In the present examination, no transit type of T. microti was detected. However, further detailed examination may clarify the presence of it. It is not clear on the problem whether these flagellates grow in the fore-stomach or the stomach or not, but it should be an useful hold to clarify the role of these tri- chomonad protozoa in the fore-stomach of the Japanese field voles.

References

1) OGIMOTO, K. and S. IMAI, Atlas of Rumen Microbiology, Japan Scientific Societies Press. Tokyo. 1981. 2) OZEKI, K., S. IMAI and M. KATSUNO, Tohoku J. Agr. Res., 24: 86-101. 1973. 3) VAN HOVEN, W., F. M. C. GILCHRIST and V. L. HAMILTON-ATTWELL,J. Protozool., 34: 338-342. 1987. 4) DEHORITY, B. M., J. Protozool., 34: 143-145. 1987. 5) GOTO, N., Keito Seibutsu, 3: 34-39. 1978 (in Japanese). 6) KUDO, H. and Y. OKI, Exp. Anim., 31: 175-183. 1982 (in Japanese with English summary). 7) KUDO, H., Y. OKI and H. MINATO, Bull. Nippon Vet. Zootech. Coll., 28: 13-19. 1979. 8) KUDO, H., Y. OKI and H. MINATO, Bull. Nippon Vet. Zootech. Coll., 29: 45-53. 1980. 9) LEVINE, N. D., Protozoan Parasites of Domestic Animals and of Man. 2nd ed. 88-110. Burgess Pub. Co. Minneapolis. 1973. 10) FLYNN, R. J., Parasites of Laboratory Animals. 3-35. Iowa State Univ. Press. Ames. 1973. 11) WENRICH, D. H. and L. H. SAXE, J. Parasitol., 36: 261-269. 1950. 12) KUROKI, T., Y. FUKATSU and T. KOYAMA, Jpn. J. Protozool., 14: 14-15. 1981.

355 IMAI and OGIMOTO

ハ タ ネ ズ ミMicrotus montebelliの 消 化 管 内 よ り

見 出 さ れ た 鞭 毛 虫 類

今 井 壮一 ・扇元 敬 司*

日本獣 医畜産大学,武 蔵野市180 *東 北大学農学 部 ,仙 台市980

複 胃 を も ち,反芻 動 物 の モ デ ル動 物 と して有 望 視 され 胃 お よ び後 胃 か らは1種 の み が見 出 さ れ た.本 種 は トリ てい るハ タネ ズ ミの消 化 管 内 に お け る原 虫 相 を検 索 した. コモ ナ ス類 で あ る と考 え られ た もの の,同 定 の 有 力 な特 そ の結 果,反 芻 胃 内 や馬 大 腸 内,ま た カ ピバ ラ の下 部 消 徴 で あ る前鞭 毛 を有 して い な か っ た が,虫 体 の 計 測値 お 化 管 内 か ら報告 され て い るよ うな繊 毛 虫 の存 在 は認 め ら よ び形 態 の比 較 か ら,本 来 下 部 消 化 管 で あ る盲 腸,直 腸 れ な か っ たが,前 胃 を含 む 調査 した全 て の部 位 か ら多 数 に 生 息 し て い るT. murisの 栄 養 体 へ の移 行 型 で あ る の鞭 毛 虫 が 験 出 され た.盲 腸,直 腸 内 か らは2種 の 鞭 毛 と考 え られ た. 虫 が 認 め ら れ,そ れ ぞ れTritrichomonas muris, 日畜 会報,59(4):351-356,1988 Tetratrichomonas microtiと 同 定 さ れ た.一 方,前

日畜 会 報,59(4):351-356 356 1988