人工飼養玉米蛇致死性下痢關連菌及其毒力因子研究

Abstract

臺灣人工養殖用作寵物的玉米蛇（Pantherophis guttatus）常見發生致死性的下痢，患病蛇隻出現下痢、水樣狀下痢、拒食、脫水等症狀，最終導致死亡。此病好發於孵化後六月齡內的玉米蛇，造成飼主醫療負擔，並引致養殖業者造成經濟損失。為探討人工飼養玉米蛇致死性下痢關連菌種，本研究前期蒐集四個田間發生場內蛇隻直腸拭子檢體，調查動物下痢及腸道發炎常見菌種與玉米蛇下痢之間的關聯性，每場採集正常與下痢蛇隻直腸拭子各10支進行菌種分離，比較正常與下痢蛇隻直腸菌種檢出率及菌落數的差異性，藉以找出差異菌種。後期調查納入另外8個下痢發生場內下痢與正常蛇隻前期差異菌種各8株，含前期四場共24株差異菌種進行該菌株毒力因子分析比較。前期直腸拭子菌種分離結果顯示，每場正常蛇隻均可分離出沙門氏菌（Salmonella spp.）、大腸桿菌（Escherichia coli）及產氣莢膜梭菌（Clostridium perfringens），場檢出率分別為100%（4/4）、100%（4/4）及100%（4/4），樣本檢出率為50%（20/40）、45%（18/40）及75%（30/40）；下痢蛇隻沙門氏菌、大腸桿菌及產氣莢膜梭菌場檢出率依序為100%（4/4）、100%（4/4）及100%（4/4），樣本檢出率為30%（12/40）、37.5%（15/40）及100%（40/40），統計分析發現三個標的菌種於正常與下痢蛇隻場檢出率雖無統計差異，下痢蛇隻直腸中產氣莢膜梭菌樣本檢出率與樣本分離菌量顯著高於正常蛇隻（p = 0.0007；p < 0.0001）。 後期由12個下痢發生場正常與下痢蛇隻各自分離的12株產氣莢膜梭菌（n=24）以聚合酶鏈鎖反應（polymerase chain reaction；PCR）進行毒素分型，共檢出A、B、D及G四種毒素型（toxinotype），A毒素型佔比最高（54.17%；16/24）。後以cadA、cna、nanI、netB及tpeL等5個毒力因子以即時定量聚合酶連鎖反應（real time PCR）進行基因攜帶數絕對定量分析比較，下痢蛇隻產氣莢膜梭菌的cadA基因攜帶量顯著低於正常蛇隻分離株（p=0.0343），cna、nanI、netB及tpeL等毒力基因攜帶數於兩者間則未有統計差異（cna, p=0.5927；nanI, p=0.1970；netB, p=0.7553；tpeL, p=0.3778）。綜上，下痢玉米蛇與正常蛇隻相比，直腸內具有顯著高量的產氣莢膜梭菌，並以A毒素型為主要菌型，直腸中產氣莢膜梭菌檢出率對玉米蛇是否下痢具有顯著影響。下痢蛇隻產氣莢膜梭菌中cadA基因攜帶量顯著低於正常蛇隻分離株，顯示cadA基因於該菌致病機制中可能扮演重要角色，值得就其功能性探究該基因攜帶量少是否有助菌株長期附著腸道，造成持續性的下痢結果。

Corn snakes (Pantherophis guttatus) are commonly bred as exotic pets in Taiwan, where outbreaks of fatal diarrhea are frequently observed, particularly in juveniles less than six months of age. Affected snakes typically present with profuse watery diarrhea, anorexia, dehydration, and progressive deterioration, often culminating in death. These disease episodes impose medical costs on pet owners and result in economic losses for breeders. To identify bacterial pathogens associated with diarrhea in captive corn snakes, we conducted a two-phase investigation. In the initial phase, rectal swab samples were collected from four affected captive facilities, including 10 clinically healthy and 10 diarrheic snakes from each facility (n = 80). Bacterial cultures were performed to isolate and identify common enteric pathogens. Salmonella spp., Escherichia coli, and Clostridium perfringens were isolated from all facilities, with farm-level detection rates of 100% (4/4) for each species. At the individual level, C. perfringens was significantly more frequently detected in diarrheic snakes than in healthy ones (100% vs. 75%; p = 0.0007), with markedly higher bacterial loads (p < 0.0001). No significant differences were observed for Salmonella spp. or E. coli detection rates between groups. In the second phase, a total of 24 C. perfringens isolates—12 from healthy snakes and 12 from diarrheic snakes across 12 facilities—were subjected to toxinotyping by PCR. Four toxinotypes (A, B, D, and G) were identified, with type A being predominant (54.17%; 16/24). Quantitative real-time PCR was subsequently used to assess the absolute copy numbers of five virulence-associated genes (cadA, cna, nanI, netB, and tpeL). Among these, the cadA gene copy number was significantly lower in isolates from diarrheic snakes compared to those from healthy individuals (p = 0.0343), while no significant differences were observed for the remaining genes. Collectively, our findings indicate that C. perfringens, particularly toxinotype A, is strongly associated with diarrhea in captive corn snakes. The elevated prevalence and bacterial load of C. perfringens in diarrheic individuals suggest a potential pathogenic role. Furthermore, the lower cadA gene copy number in isolates from diarrheic snakes may reflect an adaptive mechanism facilitating prolonged intestinal colonization and chronic disease, warranting further functional studies.