Identification of the Causative Agents of Ehrlichia canis and Anaplasma phagocytophilum in Dogs in Taiwan by Nested PCR, Indirect Immunofluorescent-Antibody Assay, and Sequence Analysis of the 16S rRNA Gene

以巢式PCR、間接免疫螢光抗體法及16S rRNA基因序列分析鑑定台灣地區犬隻感染Ehrlichia canis及Anaplasma phagocytophilum

劉宏仁(Hung J. Liu) ； 殷嘉諄(Chia C. Yin) ； 謝耀清(Yao C. Hsieh) ； 江友中(Yu C. Chiang) ； 張清棟(Ching D. Chang) ； 廖明輝(Ming H. Liao) ； 江強華(Chiang H. Chiang) ； 吳永惠(Yeong H. Wu) ； 林孫權(Suen C. Lin)

《臺灣獸醫學雜誌》 32卷2期 (2006/06) Pp. 76-87

https://doi.org/10.7009/TVJ.200606.0076

摘要

為了鑑定台灣地區犬隻感染18Ehrlich canis及Anaplasma phagocyrophilum，於1999年6月至2000年3年間，在國立屏東科技大學附設教學動物醫院門診病例隨機採取血液並以血液塗抹片檢查法、間接免疫螢光抗體法及巢式PCR等三種技術鑑定110個臨床血液樣品．以巢式PCR增幅Ehrlichia cains及Anaplasma phagocytophilum之165 rRNA基因，可擴增預期PCR產物大小為520 bp及925 bp。巢式PCR偵測Ehrlichia canis呈陽性反應者為20%（22/110）。其中只有兩個血液樣品可偵測到Anaplasma phagocytophilum佔1.82%（2/110）。PCR產物經定序比對分析證實PCR之特異性，與標準株（M73221）序列相似度達97%以上。親源開係樹狀圖分析亦證實台灣地區犬隻感染Ehrlichia canis及Anaplasma phagocytophilum與國外分離株之親源關係相近。110個犬病例中隨機取樣60個血清樣本進行間接免疫螢光抗體試驗，結果呈陽性反應者為40%（24/60），而巢式PCR陽性反應者僅為28.3%（17/60）。間接免疫螢光抗體試驗與巢式PCR結果不一致，推測犬隻可能曾感染其它Ehrlichia species．在22個Ehrlichia canis PCR陽性犬隻．只有5個病例在周邊淋巴球可發現艾利希體菌包涵體（morulae），而66個PCR陰性病例，細胞質含偶氮顆粒樣包涵體之淋巴球出現率小於20%者佔93.94%（62/86），其絕對數低於600/μL者佔95.52%（64/66）。本研究發現，以血液作為檢測樣本時，以間接免疫螢光抗體法之檢出率最高，巢式PCR次之。異性方面，以巢式PCR較高，間接免疫螢光抗體法次之，血液塗抹片法最低。因此偵測犬隻急性或慢性艾利希體症之最佳之方式是合併使用巢式PCR及間接免疫螢光抗體法。

關鍵字

艾利希體症 ； 親源關係分析 ； 間接免疫螢光抗體試驗 ； 巢式PCR

並列摘要

To determine the prevalence of Ehrlichia canis and Anaplasma phagocytophilum (formerly known as E. equi) in dogs in Taiwan, we collected canine blood samples randomly from clinical cases at Department of Teaching Animal Hospital, National Pingtung University of Science and Technology from June, 1999 to March 2000. These samples were examined by blood smear examination, indirect immunofluorescent-antibody (IFA) assay, and nested polymerase chain reaction (PCR) were used to examine one hundred-and-ten blood samples from dogs. Two sets of the nested PCR were developed for detecting Ehrlichia species in dogs, and predicted nested PCR products from E. canis and Anaplasma phagocytophilum were 520 bp and 925 bp, respectively. Twenty two of 110 dogs (20%) showed positive results of E. canis in nested PCR, while only two of 110 dogs (1.82%) showed positive results of Anaplasma phagocytophilum in nested PCR. The PCR products from E. canis and Anaplasma phagocytophilum were sequenced and compared with the previously published sequences. The sequence similarities of the 16S rRNA gene among them were above 97%. Phylogenetic tree were inferred from nucleotide sequences of the 16S rRNA gene, showing that the Taiwanese species were closely related to other Ehrlichia species. By IFA test, 24 of 60 (40%) dogs were seroreactive against E. canis antigens while only 17 of 60 (28.3% ) dogs were nested PCR positive. The discordant IFA and nested PCR results obtained in this study were unexpected and may have been related to exposure of dogs to an Ehrlichia species other than E. canis. Ehrlichial morulae in peripheral lymphocytes were found in 5 of 22 cases of PCR positive dogs. The peripheral lymphocytes containing elementary bodies (EB) and/or initial bodies (IB) less than 20% was found in 93.94% (62/66) of PCR negative dogs, and the absolute number of similar lymphocytes less than 600/μL was found in 95.52% (64/66) of PCR negative dogs. Although the positive rate was highest in IFA test, the nested PCR exhibited the higher specificity than IFA test in the detection of canine ehrlichiosis using blood samples. The results suggested that nested PCR in combination with IFA test is the best suited for the detection of canine ehrlichiosis while acute or chronic phase serum samples were tested.

並列關鍵字

Ehrlichiosis ； phylogenetic analysis ； indirect immunofluorescent-antibody assay ； nested PCR