應用創傷模式分析及去氧核糖核酸檢驗於遭犬貓攻擊的野生動物之動物法醫調查

Abstract

遊蕩犬貓的數量日趨增加，襲擊當地的野生動物的事件也逐年增加。此現象對於野生動物的生存有著負面的影響，進而對臺灣生態系統構成重大的威脅，亦是野生動物被送往救援中心的主要原因之一。然而，辨識攻擊野生動物之攻擊物種，目前僅依賴臨床獸醫師的經驗，搭配目擊史或其他監視影像證據；對於未有目擊史的攻擊案件，除了臨床檢查外，尚缺乏其他實證方法。近期的研究探討了利用傷口拭子上所殘留的唾液進行DNA分析以區分攻擊物種，這種方法已被證實是可行並有價值的。但運用傳統聚合酶連鎖反應方法（Polymerase chain reaction，PCR）檢驗大量傷口檢體之DNA將花費較多時間及成本，因此發展更有效益的檢驗方法是必要的。在多篇已發表的研究中，分析遭攻擊之野生動物的創傷模式，例如成對穿透傷彼此間的的距離，並與潛在的掠食物種之犬齒距離比較，以判斷攻擊物種，可以作為另一種識別手段。然而，僅使用穿透傷辨別攻擊物種在應用上將有所侷限，例如在被攻擊的動物身上若未見成對穿透傷、因皮下空間導致齒距量測誤差，或多個攻擊者物種的齒距相近等情況下，就無法使用此方法來判定攻擊者物種，因此更全面的創傷分析是必要的，包含常見到創傷的解剖位置、常見到的創傷類型，以及在不同解剖位置最常見到的創傷類型，都有助於提供更多有用的訊息。 本研究旨在分析遭犬貓攻擊之臺灣不同原生物種的創傷模式，並結合DNA檢測，以確定被害動物與犬貓間的接觸事實。本研究所涵蓋的物種多為非保育類哺乳類及鳥類物種、非狂犬病疫區之非保育類食肉目動物，以及少量保育類哺乳類及鳥類物種。送檢單位會採集疑似遭受攻擊的活體野生動物之傷口檢體，而如有被害動物之大體亦會送檢以進行法醫解剖來獲得全面的創傷分析，並從其傷口表面與內緣收集拭子、傷口表面毛髮或羽毛以採集殘留於傷口的犬或貓之唾液檢體，而後進行DNA提取，接著使用其他文獻所建立的犬隻引子與本研究設計的貓隻引子進行多重聚合酶連鎖反應，以偵測是否有犬貓DNA於傷口檢體上。其次，針對大體所收集的數據包括物種資訊、性別、年齡、相關目擊攻擊史、其他病史、創傷分佈與創傷種類，並根據核酸檢測結果與解剖過程中觀察到的傷害模式進行交叉驗證。除創傷模式分析，亦會針對傷口表面與內緣拭子之DNA檢驗陽性率差異、傷口拭子與毛髮或羽毛之DNA檢驗陽性率差異。 本研究自全台灣多個單位共收集219例懷疑遭犬或貓攻擊之野生動物檢體，其中包含111例大體。樣本有120例來自13種哺乳類動物的傷口檢體及大體，並以山羌及白鼻心為大宗，亦有99例來自32種鳥類動物的傷口檢體及大體，並以珠頸斑鳩及金背鳩為大宗。在傷口樣本之PCR結果中，呈現犬陽性、貓陽性或犬貓雙陽性之總陽性率為60.3%（132/219），其中哺乳類傷口檢體的總陽性率為69.2%，鳥類傷口檢體的總陽性率為49.5%。在219例檢體中，共有91例（91/219；41.6%）呈現犬之核酸陽性訊號，多在哺乳類動物之檢體上發現；其次有38例（38/219；17.4%）呈現貓之核酸陽性訊號，多為鳥類動物之檢體，另在鳥類動物的檢體上，有3例（3/219；1.4%）呈現犬貓核酸雙陽性訊號。傷口表面拭子的陽性率（54.9%）與傷口內緣拭子的陽性率（57.7%）雖無顯著差異（P=0.632），但其表明傷口內緣的區域也能採集到攻擊者的DNA，因此建議同時採集傷口表面及內緣的區域，以增加所採樣的DNA含量。另一方面，傷口拭子（47.7%）與鳥類羽毛的陽性率（59.1%）略有差異，但並無顯著差異，而在哺乳類毛髮的陽性率則與傷口拭子的陽性率相同，此結果顯示羽毛與毛髮亦是另一個可獲取到犬貓DNA樣本的來源之一，並在總陽性率較低的鳥類族群中，同時採集傷口拭子與羽毛可增加獲取DNA的來源。創傷分析上，在傷口呈現犬陽性的山羌以在臀部區域之撕裂傷，與在胸廓背側及體幹背側的線性擦傷為犬攻擊的特點；在傷口呈現犬陽性的白鼻心則以落在肩胛及胸廓背側、背部及體幹外側的穿透傷為常見的犬攻擊的創傷形式。在傷口呈現貓陽性訊號、呈現犬陽性訊號以及呈現犬貓雙陽性的3個鳥類組別中，傷勢皆主要集中在體幹背側、腹側與翅膀及體幹外側上，並且撕裂傷與瘀傷的發生率較穿透傷高，因此要單從創傷分佈去辨別攻擊鳥類的物種較為困難，因其常見的創傷模式相似。但在多數貓陽性的鳥類案件之傷勢數量較少、傷勢較為輕微，以及穿透傷的直徑約為0.1至0.5公分，相較於犬陽性的案件之傷勢來的輕微。因此在疑似遭受犬貓攻擊的鳥類族群上，建議進行全面的創傷分析，並透過DNA檢驗去確認攻擊者的物種。而在本研究中，少數案件具有與目擊史不相符的DNA檢驗結果或是其他較出乎意料的陽性結果，例如目擊貓咬但呈現犬陽性之翠翼鳩、目擊犬咬但呈現貓陽性之珠頸斑鳩，以及呈現犬貓雙陽性之鳥類案件，其說明這些動物可能在被目擊前就與其他犬或貓有接觸，或是在被發現者救援後，與當時周遭環境的犬或貓有接觸，因此被攻擊的鳥類動物即使具有目擊史，仍然建議採集傷口拭子或羽毛來進行DNA檢驗，而因犬陽性、其他特殊陽性的鳥類案件數較稀少，因此還未能得到完整的創傷分析比較。 本研究證明核酸分析在臺灣野生動物創傷原因之鑑別方面為有價值的法醫工具，並且結合創傷模式分析與DNA分析更有助於判定攻擊野生動物之物種。

Free-roaming dogs and cats are increasingly preying on native wildlife, posing a severe threat to the ecosystem in Taiwan. The adverse impact of dog and cat attacks on wild animals is evident, as it remains the primary cause for their admission to rescue centers. However, identifying the attacking species relies solely on the clinician experience or witness account, lacking validated methods on the cases without other attack evidence. Recent studies have explored the potential of DNA analysis from saliva found on wound swabs to differentiate the attacking species. However, conventional PCR testing of DNA from a large number of wound samples would be time-consuming and costly, necessitating the development of more efficient testing methods. Furthermore, analysis of injury patterns can serve as an additional means of identification, as another research focusing on applying the canine distance of different attacking species to distinguish the distance of paired puncture wounds on the wild animals. However, relying solely on puncture wounds for species identification has limitations, such as when paired puncture wounds are absent or when the canine distances among multiple attacking species are similar. Therefore, a comprehensive injury analysis is necessary. This study aims to analyze the patterns of trauma in various native wildlife species fallen victim to attacks by dogs and cats. Additionally, DNA detection techniques are employed to confirm the contact between the affected animals and dogs or cats. The species covered in this study were mostly non-protected mammal and bird species, non-protected carnivores from non-rabies endemic areas. Specimens of suspected attacked wild animals were collected by the reporting units, and if available, the carcasses were also conducted forensic necropsy for comprehensive trauma analysis. Swabs from the surface and inner rim of wounds, turfed hair, or feathers on the wound surface were collected to gather saliva residues from dogs or cats, followed by DNA extraction. Multiplex polymerase chain reaction (PCR) using established canine primers and feline primers designed in this study were then employed to detect the presence of canine or feline DNA. Cross-validation was conducted based on the PCR results and observed patterns of damage during the necropsy process. In addition to trauma pattern analysis, differences in the PCR positive rates between swabs from the wound surface and edges, as well as between wound swabs and hair or feathers, were also investigated. This study collected a total of 219 cases of wound samples, including 111 carcasses. The samples consisted of 120 cases of wound samples or carcasses from 13 mammalian species, with a predominant representation from Formosan muntjac and masked palm civet. Additionally, there were 99 cases of wound samples or carcasses from 32 avian species, with a majority represented by spotted dove and oriental turtle dove. In the PCR results of wound samples, the total positivity rate for dog-positive, cat-positive, or dog-cat double-positive was 60.3% (132/219), with a total positivity rate of 69.2% for mammal wound samples and 49.5% for avian wound samples. Among the 219 samples, there were a total of 91 cases (91/219; 41.6%) showing positive signals against canine DNA, followed by 38 cases (38/219; 17.4%) showing positive signals against feline DNA. Additionally, there were 3 cases (3/219; 1.4%) showing double-positive signals of canine and feline DNA on avian specimens. Although there was no significant difference between the positivity rates of swabs from the wound surface (54.9%) and swabs from the inner rim (57.7%) (P=0.632), it indicated that DNA from attackers could also be collected from the inner rim of the wound. Therefore, it is recommended to collect samples from both sites to increase the DNA yield. On the other hand, the positivity rates of wound swabs (47.7%) and bird feathers (59.1%)were not significant (P=0.285). The results suggested that feathers and hair were another source of dog or cat DNA samples, simultaneously collected to increase the DNA yield. In terms of injury analysis, Formosan muntjac with dog-positive wounds typically exhibited lacerations in the hip area. Masked palm civet with dog-positive wounds commonly showed puncture wounds accompanied by bruises on the shoulder to dorsal thorax, and back and flank. In the three bird groups where wounds were cat-positive, dog-positive, or dog-cat double-positive, injuries were mainly concentrated on the dorsal and ventral flank, wings and lateral flank, with a higher occurrence of lacerations and bruises than puncture wounds. Therefore, distinguishing attacking species based solely on the distribution of injuries is difficult due to the similarity in common trauma patterns. However, in most cat-positive bird cases, the diameter of puncture wounds range from 0.1 to 0.5 cm compared to dog-positive cases. Thus, for suspected bird populations attacked by dogs or cats, comprehensive trauma analysis is recommended, and DNA testing is suggested to confirm the species of the attacker. In this study, a few cases had DNA test results that did not match witness accounts or had other unexpected positive results, which indicates that these animals might have had contact with other dogs or cats before being witnessed or might have had contact with dogs or cats in the surrounding environment after being rescued, suggesting that even if birds have witness accounts, it is still recommended to collect samples for DNA detection. DNA analysis thus emerges as a valuable forensic tool for identifying the causes of wildlife trauma in Taiwan. The integration of injury patterns and DNA analysis facilitates the species identification of attackers. This study provides valuable insights into injury patterns observed in various endemic mammals and birds in Taiwan.