隱孢子蟲在臺灣高屏地區新生仔山羊下痢原因中之角色

Title of Thesis: Role of Cryptosporidium in the Etiology of Neonatal Diarrhea in Goat Kid in Kaoshiung and Pingtung Areas of Taiwan

夏魁山(Kuei - Shan Hsia)

指導教授 ： 吳永惠

國立屏東科技大學/農學院/獸醫學系所/碩士(2012年)

https://doi.org/10.6346/NPUST.2012.00015

摘要

下痢是世界各地引起仔山羊損失相當常見之原因，其發生是多因子性 ( multi-factors )，隱孢子蟲（Cryptosporidium）在世界各國被認為是1個月齡內仔山羊下痢最普遍之原因，但在臺灣有關隱孢子蟲在新生仔山羊下痢原因中之角色之文獻則尚相當不足。因此，本研究針對臺灣地區山羊隱孢子蟲之發生情形及隱孢子蟲在新生仔山羊下痢原因中之角色進行探討研究。首先，為調查在南臺灣高雄、屏東2縣市山羊隱孢子蟲感染之發生情形，於2008年10月至2009年3月間隨機自兩縣市7場採集280隻1-12月齡山羊糞便，以改良式抗酸染色法 ( modified acid-fast Ziehl-Neelsen method, MZN法 ) 及巢式聚合酶鏈鎖反應 ( nested PCR法 )，來檢查隱孢子蟲卵囊；並任選3場MZN陽性場之3株nested PCR陽性產物進行定序，來與GenBank所發表之隱孢子蟲基因型別比對。其次，為探討隱孢子蟲感染在新生仔山羊下痢原因中之角色，於2011年8月至2012年6月間，對高屏2縣市24場採集119隻生後1個月齡內下痢之仔羊，採其糞便以可測定rotavirus、coronavirus、Cryptosporidium 和 E. coli F5抗原之ELISA套組 ( Digestive ELISA Kit Antigenic，Bio-X Diagnostics, Belgique ) 進行病原檢查，並評估下痢便性狀與隱孢子蟲感染間之關係。結果逢機由7場280隻1-12月齡山羊糞便之隱孢子蟲感染率在MZN法為9.3% ( 26/280 )，在PCR法為16.8% ( 47/280 )，PCR法之檢出率顯著 ( P<0.05 ) 高於MZN法；隱孢子蟲感染率在高、屏間無論就MZN法 ( 10.8%，13/120 vs 8.1%，13/160 )，或就PCR法 ( 14.2% ，17/120 vs 18.8 %，30/160 )，均無顯著差異。在MZN法檢查，小於3個月齡仔羊之隱孢子蟲感染率 ( 15.7%, 22/140 ) 顯著 ( P<0.05 ) 高於較老之3-12月齡羊隻 ( 2.9%, 4/140 )。3株nested PCR陽性產物之核酸定序比對，其相似性與GenBank 之小隱孢子蟲 ( C. parvum ) 和牛隱孢子蟲 ( C. bovis ) 分別達93.6- 94.1 %和90.7- 95.0 %。另方面，在119隻生後1個月齡內下痢之仔羊之ELISA檢查，隱孢子蟲感染率則高達52.1% ( 62/119 )，其中隱孢子蟲單獨感染者有29.4% ( 35/119 )，隱孢子蟲與其他病原併發感染者有22.7% ( 27/119 )。屏東地區之隱孢子蟲感染率 ( 75.0%， 24/32 ) 顯著 ( P<0.05 ) 高於高雄地區 ( 43.7%，38/87 )，屏東地區隱孢子蟲單獨感染之比例 ( 46.9%, 15/32 ) 亦顯著 ( P<0.05 ) 高於高雄地區 ( 23.0%, 20/87 )。有隱孢子蟲感染之場數比率為91.7% ( 22/24 )，此種比率在高雄 ( 88.2%，15/17 ) 與屏東 ( 100%，7/7 ) 間並無顯著差異。大多數的場 ( 75.0%，18/24 ) 有3種或4種病原混合感染。呈現水樣下痢便之比率，在單獨感染隱孢子蟲者 ( 22.8%, 8/35 ) 顯著 ( P<0.05 ) 高於單獨感染其他三種病原者 ( 3.8%, 1/26 )。由以上結果我們結論認為隱孢子蟲感染在台灣地區新生仔山羊下痢之病因上佔有很重要之病原性角色，而大多數新生仔羊下痢在台灣地區亦是由多種病原感染之複合性感染病 ( complex infection )。

關鍵字

新生仔羊下痢 ； 小隱孢子蟲 ( C. parvum ) ； 牛隱孢子蟲( C. bovis ) ； 山羊 ； 臺灣

並列摘要

Diarrhea disease is a very frequent cause of kid loss worldwide, and the incidence is associated with multiple factors. Cryptosporidium is documented as the most commonly diagnosed etiology of diarrhea in kid younger than 1 month of age throughout the world. But the references about the role of Cryptosporidium in the etiology of neonatal kid diarrhea is still very insufficient in Taiwan. Therefore, the incidence of Cryptosporidium infection in goats and the role of cryptosporidia in the etiology of neonatal diarrhea in goat kid in Taiwan was aimed and explored in this study. First, To investigated the incidence of Cryptosporidium infection in goat in Kaoshiung and Pingtung counties of southern Taiwan, 280 goat’s feces of 1-12 month ages randomly obtained from 7 farms in two counties were examined by modified acid-fast Ziehl-Neelsen method ( MZN ) and nested polymerase chain reaction ( PCR ) for Cryptosporidium oocyst from October 2008 to March 2009. Any of the three nested PCR positive samples collected from three MZN positive farms were sequenced and compared with the genotypes of Cryptosporidium reported in Genbank. Then, to evaluate the role of Cryptosporidium in the etiology of neonatal diarrhea in goat kid, feces of 119 diarrheic kids under one month age obtained from 24 farms in that two counties from August 2011 to June 2012 were examinated by the Digestive ELISA Kit Antigenic ( Bio-X Diagnostics, Belgique ), which is a antigenic diagnosis test for rotavirus, coronavirus, E. coli F5 and Cryptosporidium. And the relationship between fecal characters and Cryptosporidium infection were also evaluated. The results were as follows: The detection rates are 9.3% ( 26/280 ) by MZN test and 16.4% ( 46/280 ) by PCR in 280 goats ( 1 to 12 month ages ) randomly obtained from 7 farms. The detection rate of PCR is significantly ( P<0.05 ) higher than MZN. The incidence rates of Cryptosporidium infection between Kaohsiung and Pingtung counties are not significantly different either by MNZ ( 10.8.3%，13/120 vs 8.1%，13/160 ) or by PCR ( 14.2% ，17/120 vs 18.8 %，30/160 ). On the MZN test, the infection rates of Cryptosporidium in kid younger than 3 month of age (15.7%, 22/140 ) are significantly ( P<0.05 ) higher than elder goats ( 3-12month ages, 2.9%, 4/140 ). The similarities of nucleotide sequences of three nested PCR positive products are 93.6-94.1% with C. parvum and 90.7-95.0% with C. bovis in GenBank, respectively. On the other hand, ELISA examination of 119 diarrheic kid younger than 1 month of age, the incidence rates of Cryptosporidium infection is up to 52.1% ( 62/119 ). Among these, out of 29.4% ( 35/119 ) was Cryptosporidium infection alone and 22.7% (27/119 ) was complicated with other pathoens. The incidence rates of Cryptosporidium infection kids in Pingtung county ( 75.0%, 24/32 ) is significantly ( P<0.05 ) higher than Kaohsiung areas ( 43.7%, 38/87 ), and the kid ratios of Cryptosporidium infection alone in Pingtung areas ( 46.9%, 15/32 ) is also significantly ( P<0.05 ) higher than Kaohsiung areas ( 23.0%, 20/87 ). The ratio of farm had Cryptosporidium infection kids is 91.7% (22/24 ). The ratios is not significantly different between Kaohsiung ( 88.2%，15/17 ) and Pingtung ( 100%，7/7 ) areas. The most of farms ( 75.0%, 18/24) had three to four pathogens infection. The rate developing to watery feces in the Cryptosporidium infection alone ( 22.8%, 8/35) is significant higher than other three pathogen alone infection ( 3.8%, 1/26). Base on the above results, we concluded that Cryptosporidium infection plays a very important pathogenic role in the etiology of neonatal diarrhea in goat kid, and the majority of neonatal diarrhea is also a complex infection caused by multi-pathogens in Taiwan.

並列關鍵字

Neonatal kid diarrhea ； Cryptosporidium parvum ； Cryptosporidium bovis ； Goat, Taiwan ； HASH(0xe7a5440)

參考文獻

曾巨羽。臺灣南部地區牛隻隱孢子蟲感染之盛行率、基因型別及病原性角色。碩士論文。國立屏東科技大學獸醫學研究所。1-59，2008。

渡邊裕子。臺灣地區隱孢子蟲症之疫學研究。博士論文，國立中興大學獸醫研究所。1-108，2006。

Abe N, Iseki M. Identification of genotypes of Cryptosporidium parvum isolates from ferrets in Japan. Parasitol Res 89: 400-424, 2003.

Abe N, Matsubayashi M, Kimata I, Iseki M. Subgenotype analysis of Cryptosporidium parvum isolates from humans and animals in Japan using the 60-kDa glycoprotein gene sequences. Parasitol. Res. 99:303-305, 2006.

Andres S, Jimenez A, Sanchez J, Alonso JM, Gomez L, Lopez F, Rey J. Evaluation of some etiological factors predisposing to diarrhoea in lambs in “La Serena” (Southwest Spain). Science Direct 70: 272-275, 2007.