イベント・セミナー詳細

専門家

水曜会(第680回)

情報公開日:2013年9月17日 (火曜日)

日時

平成25年9月25日(水曜日)13時30分~

場所

(独)農業・食品産業技術総合研究機構 動物衛生研究所 講堂
茨城県つくば市観音台3-1-5 (交通案内/会場案内(建物3))
Tel: 029-838-7707(企画管理部 業務推進室 交流チーム)
Fax: 029-838-7907

今回はJICA研修員 ナジールさん、アリマさん、バチカさんによる発表です。
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内容

座長:中村 義男(動衛研)

Recovery rates of Eimeria oocysts from cattle feces by Wisconsin sugar flotation method (20min)

○Nazir Ahmad Tookhy1, Batchuluun Damdinjav2, Alimaa Tsagaan3, Yoshio Nakamura4 (1Herat University, Afghanistan, 2State Central Veterinary Laboratory, Mongolia, 3Institute of Veterinary Medicine, Mongolia, 4National Institute of Animal Health, Japan)

Wisconsin sugar flotation method (WSFM) is the most popular method for detection of parasite oocysts and eggs in fecal samples. The purpose of this study was to know the recovery rates of Eimeria oocysts through the course of flotation in sugar solution by WSFM. A fecal sample was collected from a calf infected with Eimeria bovis. The sample contained 7.8 oocysts per 0.01g counted by a direct method. Stage one of this study involved recovering oocyts from surface of sugar solution in a test tube (1.5cm in diameter, 10.5 cm in length) with cover slips replaced at time points of 10, 20, 30, 40, 50, 60, 90 min, 2, 3, 4, 5, 6, 24, 48 hr, and 5 days. Oocysts were continuously detected up to 48 hr, with a linear increase in recovery counts for the first 2 hr, and the average total count was 953 per gram. Recovery rates of oocysts were 8.1, 13.3 and 16.4% at 10, 20 and 30 min, respectively. Stage 2 involved a set of 3 tubes. Cover slips were put at intervals of 10, 20 and 30 min on the first tube, while one cover slip was kept for 20 min on the second tube and one for 30 min on the third tube. No significant differences were detected in oocyst counts at 20 min between the first and second tubes, and at 30 min between the first and third tubes. Stage 3 involved the comparison of recovery rates at 10 min between sugar solutions with specific gravity of 1.200 and 1.266. There was no significant difference in oocyst counts between the two solutions. These results indicated that it takes time to recover all Eimeria oocysts from cattle fecal samples by WSFM, but total oocyst counts per gram can be estimated by counting oocysts detected at any sampling time points, such as 10, 20 or 30 min using one cover slip. No improvement of recovery rates was seen by using a higher concentrated sugar solution.

座長:中村 義男(動衛研)

Evaluation of commercially available ELISA kits for the serodiagnosis of Neospora caninum infection in cattle (20min)

○Alimaa Tsagaan1, Batchuluun Damdinjav2, Nazir Ahmad Tookhy3, Yoshio Nakamura4 (1Institute of Veterinary Medicine, Mongolia, 2State Central Veterinary Laboratory, Mongolia, 3Herat University, Afghanistan, 4National Institute of Animal Health, Japan)

Bovine neosporosis is recognized as a major cause of abortion, neonatal mortality and economic loss in the dairy industry throughout the world including Japan. An indirect fluorescent antibody test (IFAT) is routinely used as a reliable test to find animals infected with Neospora caninum, however, it takes time to screen many samples and the determination becomes subjective. The aim of this study was to evaluate two ELISA kits commercially available in comparison with IFAT. Serum samples were obtained from 22 cattle reared in farms where neosporosis frequently occurred, and from 41 cattle in farms where neosporosis was never recorded. One sample from a positive farm was serially diluted two-fold 16 times with one sample from a clean farm. These samples were assayed using the #192 indirect and #218 competitive ELISA kits (Bio-X Diagnostics) and IFA Substrate Slide (VMRD). Three positive and two negative reference samples of the kits were used as standards. Antibody levels of the serially diluted samples were determined positive up to the 5th dilution in the IFAT and #192 ELISA, and up to the 15th dilution in the #218 ELISA. In the #218 ELISA, values decreased in a linear fashion up to the 7th dilution, followed by remaining constant with a level ranging 33-40%, which was equal to or slightly higher than the cut-off value of 33%, like a background. Screening of the 68 field and reference samples showed that results from 64 (94%) or 55 (81%) samples accorded between the #192 or #218 ELISA and IFAT, respectively. In the #192 ELISA, 4 samples from positive farms were determined positive, but negative in the IFAT. In the #218 ELISA, 7 and 4 samples from positive and clean farms, respectively, were determined positive, but negative in the IFAT. Of the 11 samples, 4 samples each from positive and clean farms showed values of 33-38% suggesting low reliability in a range close to the cut-off value. Such low reliability may have resulted from the single-well determination, with no control well to check background, using high concentrated samples diluted only to double in this kit. In addition, two positive reference samples for the #192 ELISA and IFAT were determined negative in this kit. Costs were 458, 417 and 833 yen per sample for the #192, #218 ELISAs and IFAT, respectively. These results suggested that the #192 ELISA is the better choice for the screening of N. caninum infection in cattle.

座長:中村 義男(動衛研)

Analysis of amplicons from Babesia ovata strains isolated in Japan by b-tubulin and B. ovata-specific PCRs (20min)

○Batchuluun Damdinjav1, Alimaa Tsagaan2, Nazir Ahmad Tookhy3, Yoshio Nakamura4 (1State Central Veterinary Laboratory, Mongolia, 2Institute of Veterinary Medicine, Mongolia, 3Herat University, Afghanistan, 4National Institute of Animal Health, Japan)

Babesia ovata is a hemoparasite of cattle widely distributed in Japan, which is different from B. bigemina in virulence and vector ticks. Ota et al. (1995) isolated a similar parasite in Hokkaido and proposed a variety name as B. ovata oshimensis. PCR assays have been developed for the detection of b-tubulin gene from Babesia and Theileria spp. (Caccio et al. 2000), and for the specific detection of unknown gene from B. ovata or B. o. oshimensis (Ota et al. 1995, 2000). However, B. ovata has not been tested in the b-tubulin PCR, and only two strains have been tested in the specific PCRs. The purpose of this study was to analyze the structure of target fragments generated from B. ovata (including B. o. oshimensis) strains isolated in Japan by b-tubulin and B. ovata-specific PCRs. DNA was extracted from blood stabilates of B. ovata Miyake (type strain), Akashima, Aso, B. o. oshimensis Oshima and B. bigemina Kochinda strains collected from experimentally infected cattle after isolation. Target fragments of amplification were purified and directly sequenced. Phylogenetic analysis was performed using the MEGA4 software. In b-tubulin PCR, a target fragment of 457 or 458 bp was amplified from all the B. ovata strains or B. bigemina Kochinda, respectively. Phylogenetic analysis of the sequences except for primer binding sites revealed that B. ovata Miyake/Akashima and B. ovata Aso/B. o. oshimensis Oshima were in separate clusters which were different from a cluster of B. bigemina. Homology of the sequences was 100% between Miyake and Akashima strains, 98% between Aso and Oshima strains, 91-92% between Miyake/Akashima and Aso/Oshima strains, and 82-84% between the four B. ovata strains and B. bigemina Kochinda. Corresponding amino acid sequences were identical among the B. ovata and B. bigemina strains, even though variation of 3-7% and 16-19% was found in codons of exsons among B. ovata strains, and between B. ovata and B. bigemina strains, respectively. In B. ovata-specific PCR, only B. ovata Miyake showed the target band of 835 bp. In B. o. oshimensis-specific PCR, only B. o. oshimensis Oshima showed the target band of 670 bp. These results suggested some variation among populations of B. ovata distributed in Japan. Specific PCR for universal detection of any B. ovata strains should be established.

法人番号 7050005005207