生物晶片應用於乳牛乳房炎病原菌之診斷與中草藥對乳房炎及產後子宮復舊效果之探討

Abstract

乳牛乳房炎與繁殖障礙為台灣乳牛之兩大疾病，造成酪農與乳業的重大經濟損失，因此本論文以此為研究課題，首先專研乳房炎病原菌之快速診斷方法，接續探討中草藥對乳房炎與產後子宮復舊之效果，因此將實驗分成三大部分，第一部分為研發快速診斷乳房炎病原菌之生物晶片檢測法，達成在六小時內快速檢出生乳中之金黃色葡萄球菌（Staphylococcus aureus）與無乳鏈球菌（Streptococcus agalactiae）兩種傳染性病原菌，以及大腸桿菌（Escherichia coli）、異乳鏈球菌（Streptococcus dysgalactiae）、乳房鏈球菌（Streptococcus uberis）及牛鏈球菌（Streptococcus bovis）等四種環境性病原菌之目的，進而提供快速防治乳房炎之對策，讓經濟損失降至最低。首先進行第一代乳房炎生物晶片之臨床試驗，檢測步驟為：採集1 ml的生乳，經去乳脂、萃取微生物DNA，經PCR反應、DNA雜合反應與呈色反應後，根據晶片上具專一性之細菌核酸探針的點陣排列圖直接判讀。結果計篩檢 207戶泌乳牛群之總乳檢體，以牛鏈球菌陽性檢出率最高為163戶（78.7%），其次依序為乳房鏈球菌（60.4%）、大腸桿菌（45.4%）、異乳鏈球菌（30.0%）、無乳鏈球菌（27.5%）及金黃色葡萄球菌（9.2%）。接續進行第二代乳房炎生物晶片之研發，相較第一代晶片則新增牛棒狀桿菌（Corynebacterium bovis）與牛黴漿菌（Mycoplasma bovis）兩項傳染性病原菌，但刪減大腸桿菌，計可快速偵測7種細菌，並與傳統微生物培養法進行臨床乳樣檢測之比較，結果得知第二代乳房炎生物晶片與傳統微生物培養法之準確性相同，顯示乳房炎生物晶片是一快速檢測乳房炎病原菌的新方法，未來應用於乳房炎防治工作深具潛力。 第二部分為中草藥對乳房炎效果之探討，首先調配以蒲公英（Taraxacum mongolicum Hand.-Mazz.）為主之複方蒲公英散，進行降低牛乳體細胞數之臨床試驗，挑選生乳體細胞數大於50×104 cells /ml之泌乳牛（n = 20），分別餵食複方蒲公英散（每頭牛每天餵食260 g）之試驗組（n = 10）與不餵食中草藥之對照組（n = 10），為期14天，於試驗前一天及第15天，採集乳樣進行體細胞數與乳成分分析，以及血清之臨床生化值分析。結果顯示，試驗組之試驗前平均體細胞數為196.6 ± 48.7 ×104 cells/ml，試驗後平均體細胞數為91.1 ± 71.9 ×104 cells/ml，下降105.5 ± 87.7 ×104 cells/ml。而對照組由215.6 ± 59.0 ×104 cells/ml升至317.1 ± 175.2 ×104 cells/ml。在試驗前後血清生化值平均天門冬氨酸轉氨酶（asparatate aminotransferase, AST）與血中尿素氮（blood urea nitrogen, BUN）值，試驗組與對照組皆在正常值範圍內，顯示食用複方蒲公英散不影響牛之肝腎功能，且具有降低生乳體細胞數的效果。 延伸研究單一蒲公英的抗發炎效果，進行蒲公英萃取物（Taraxacum mongolicum extract, TME）及蒲公英藥粉分別對荷蘭牛在體外（in vitro）及體內（in vivo）之抗發炎試驗。in vitro，牛乳體細胞先加入各種濃度的TME （31 to 500, μg/ml）作用後再加入細菌脂多醣（Lipopolysaccharide, LPS, 1 μg/ml）刺激，結果顯示TME對細胞無毒害性，不影響細胞的存活率（viability）。而且TME能顯著降低一氧化氮（nitric oxide, NO）、間白素（interleukin, IL）-8與間白素-1β和腫瘤壞死因子（tumor necrosis factor, TNF）-α之產生。in vivo，14頭非臨床性乳房炎牛隻隨機分為兩組，試驗組（n = 7, 150 g 餵食蒲公英藥粉/頭/天）及對照組（n = 7）不餵食蒲公英，為期14天，結果得知吃蒲公英的試驗組比對照組有顯著（P<0.05）降低牛乳中之體細胞數、總生菌數和間白素-8濃度。結論得知，蒲公英之抗發炎功效是經由調降NO及IL-8與IL-1β和TNF-α等細胞激素所協同產生，另將蒲公英餵牛可降低非臨床性乳房炎之發炎作用。 再將中草藥應用於乳牛繁殖之研究，即為本論文之第三部分，採用複方中草藥生化湯（Sheng Hua Tang, SHT）探討其對乳牛產後子宮復舊及卵巢活性之影響，試驗組牛隻（n = 10）於產後第1天開始灌服SHT（70 g）連續5天，對照組牛隻（n = 10）則否。從產後第7天開始每隔三天進行超音波及血液檢查，為期4週。結果顯示，於產後第7天，在子宮內膜面積及直徑方面，服用SHT的試驗組比對照組顯著較小（P<0.01）。於產後第13天，在子宮積液容量方面，試驗組平均為1.2 ± 0.6 cm3 明顯低於對照組平均之2.3 ± 0.8 cm3 （P<0.01）。此外，於產後第19天，在子宮張力分數方面，試驗組平均為1.0 ± 0.0 明顯低於對照組平均之1.5 ± 0.5 （P<0.01）。綜合結果得知，生化湯能提升乳牛產後之子宮復舊與卵巢活性。

Our research divided into three parts. In the first study, to efficiently prevent and treat bovine mastitis and minimize its impact on dairy industry in Taiwan, a sensitive, rapid, and specific test is required for identifying the mastitis-causing pathogens. We used biochip to examine the distribution of mastitis-causing pathogens in Taiwan. The biochip is capable of detecting 6 common species of mastitis-causing pathogens within 6 hours, including Streptococcus bovis, Streptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae, Escherichia coli and Staphylococcus aureus. The technique is based on DNA amplification of genes specific to the target pathogens and consists of 4 basic steps: DNA extraction of bacteria, PCR reaction, DNA hybridization, colorimetric reaction. The Biochip was used for detecting bacteria in bulk tank milk samples from 207 DHI-participating dairy farms. The results show that S. bovis, detected in samples from 163 (78.7%) farms, was the most prevalent species, followed by S. uberis (60.4%), E. coli (45.4%), S. dysgalactiae (30.0%), S. agalactiae (27.5%), and Staph. aureus (9.2%). In the next study, a new biochip capable of detecting 7 species of mastitis-causing pathogens, including Corynebacterium bovis, Mycoplasma bovis, Staphylococcus aureus, S. agalactiae, S. bovis, S. uberis and S. dysgalactiae, within 6 hr was developed. To examine the accuracy and specificity of this biochip, a preliminary test with 82 random quarter milk samples were analyzed and compared to results from conventional microbiological methods conducted simultaneously. Results from all but one sample analyzed by the biochip were in agreement with those analyzed by bacteriology. The biochip could be a feasible tool for rapidly diagnosing mastitis-causing pathogens in milk and providing information for a more effective treatment to cure mastitis. The second study was conducted to treat dairy cows with high somatic cell count (SCC) with Chinese herbal medicine-Pu-Gong-Ying Shan. This formula composed of Taraxacum mongolicum 100 g, Viola patrinu 50 g, Lonicera japonica Thunb. 50 g, Citri reticulatae viride pericarpium 30 g, and Glycyrrhiza uralensis Fischer 30 g per cow. SCCs in raw milk of the cows were all over 50×104 cells/ml. Twenty dairy cows were divided into treatment (n=10) group and control (n=10) group. In treatment group, each cow was fed 260 g complex Chinese herb everyday. Each experiment lasted 14 days. At the beginning and end of the experiments, the SCC in raw milk was measured. The results showed that the mean of SCC in raw milk were 196.6 ± 48.7 × 104 cells/ml and 91.1 ± 71.9 × 104 cells /ml for treatment group at the beginning and end of experiment, respectively. The means of SCC in raw milk were 215.6 ± 59.0 × 104 cells/ml and 317.1 ± 175.2 × 104 cells/ml for control group at the beginning and end of experiment, respectively. There was significant difference between them (P<0.05). In blood chemistry assay, the concentration of asparatate aminotransferase (AST) and blood urea nitrogen (BUN) showed no significant difference between treatment group and control group. The results showed that Pu-Gong-Ying Shan did decrease SCC in raw milk of dairy cow and it had a good potential for preventing and treating bovine mastitis. The anti-inflammatory effects of Taraxacum mongolicum (TM) were investigated in Holstein-Friesian dairy cows, in vitro and in vivo. In vitro: Isolated milk somatic cells were pretreated with various concentrations (31 to 500, μg/ml) of TM extract (TME) and subsequently incubated with lipopolysaccharide (LPS, 1 μg/ml). The results showed that TME treatment had no effect on cell viability; however, it significantly suppressed LPS-induced expression of nitric oxide (NO), interleukin(IL)-8, IL-1β, and tumor necrosis factor (TNF)-α in milk somatic cells, in a dose-dependent manner. In vivo, 14 lactating cows, with subclinical mastitis, were randomly assigned to two groups and fed a diet with (treatment group, n=7, 150 g TM powder per head per day) or without (control group, n=7) TM supplementation for 14 days. Cows fed with TM powder had a significantly (P<0.05) reduced SCC, total bacteria count and IL-8 in milk compared to the control group. In conclusion, the anti-inflammatory effects of TM were associated with down-regulation of NO and pro-inflammatory cytokines. Addition of TM as a dietary supplement might minimize the impact of subclinical bovine mastitis. The third study, the effects of Sheng Hua Tang (SHT) on uterine involution and ovarian activity were investigated in postpartum dairy cows. SHT (70 g) was given to dairy cows (n=10) to evaluate its effects for five days from the first postpartum day. Postpartum cows fed with a basal diet without SHT were used as the control group (n=10). Ultrasounds and blood tests were recorded for four weeks from postpartum day seven with a 3-day interval. The results showed that the areas and diameters of endometria were significantly (P<0.01) reduced in the group that received SHT compared to the control group on the seventh postpartum day. The group that received SHT had an intrauterine fluid volume mean of 1.2 ± 0.6 cm3, which was significantly lower than that of the control group, 2.3 ± 0.8 cm3 (P<0.01) on the 13th postpartum day. In addition, the uterine tension score was a mean of 1.0 ± 0.0 in the group that received SHT, which was also significantly lower than that of the control group, 1.5 ± 0.5 (P<0.01) on the 19th postpartum day. Taken together, the SHT promoted uterine involution and ovarian activity in postpartum dairy cows.