臺灣五種瀕危龜類之白血球型態學及超微結構研究

Abstract

研究背景：瀕危龜種的保育亟需其在醫學診斷及處置上的進展，然而過去針對海龜白血球分類之研究文獻存在不一致性且多有缺漏，而柴棺龜(Mauremys mutica)及食蛇龜(Cuora flavomarginata)更無已發表之相關資料可供參考。此外，異嗜球的毒性變化廣泛被臨床獸醫採用作為全身性炎症反應以及預後的重要指標，但在海龜仍缺乏相關文獻，因此目前對於此一變化的臨床意義了解非常有限。 研究目的：本研究目標為辨識5個台灣瀕危龜種血循中白血球的特徵並清楚分類，比較三種常用血片染色方法的效果，並且增進對於海龜異嗜球毒性變化臨床上應用及限制之了解。 研究方法：本研究中的血液樣本來自2017年一月至2018年九月的30隻救傷海龜(包含21隻綠蠵龜Chelonia mydas、6隻欖蠵龜Lepidochelys olivacea及3隻玳瑁Eretmochelys imbricata)、8隻柴棺龜及7隻食蛇龜。血球型態學檢驗及穿透式電子顯微鏡超微結構檢驗均依標準流程進行。 研究結果：在各研究物種，均可將白血球分類為異嗜球、嗜酸性球、嗜鹼性球、淋巴球以及單核球五種血球，其型態學特徵大致與文獻中相近物種類似。海龜具有兩種嗜酸性球(大嗜酸性球及小嗜酸性球)，且發現其嗜鹼性球型態與其他爬蟲及鳥類物種相似。在穿透式電子顯微鏡下，海龜的不同種異嗜球顆粒在細胞質當中分布不均，而在嗜酸性球當中，並沒有觀察到具有晶體結構之顆粒。柴棺龜及食蛇龜的顆粒球超微結構與黃腹彩龜（Trachemys scripta scripta）及廟龜（Heosemys annandalii）相似，但在食蛇龜嗜鹼性球顆粒中觀察到的多角形晶體並沒有在其他淡水龜有記錄。有毒性變化的海龜異嗜球之形態特徵包含細胞質顆粒染色不均、細胞質嗜鹼性增加、脫顆粒以及出現嗜鹼性顆粒，而在穿透式電子顯微鏡下則可發現毒性異嗜球具有平滑的細胞膜、細胞顆粒數量顯著減少、顆粒變小或大小不一、細胞質中膜性胞器及聚集的深色多聚核糖體數量顯著增加。Wright Giemsa染劑、Diff-Quik染劑以及劉氏染劑在白血球分類上之效果並無顯著差異，但Wright Giemsa染劑及劉氏染劑在用於判讀異嗜球毒性變化上顯著的較Diff-Quik染劑可靠。在異嗜球毒性變化的檢測上，血片鏡檢與穿透式電子顯微鏡檢驗相當，但異嗜球毒性變化與臨床上炎症狀態及治療預後的相關性均弱且不顯著。 結論：本研究明確分類出5個瀕危龜種血循中之白血球，並提供詳細型態學特徵敘述及高品質的顯微照片，同時，首次描述了海龜異嗜球毒性變化之型態學特徵，亦為首次關於爬蟲類異嗜球毒性變化的超微結構研究。本研究提供了瀕危龜種的基礎血液學資訊，以及未來這些物種進一步在臨床病理及免疫學研究上的方向。

Background: Advances in medical diagnosis and management is warranted for the conservation of endangered chelonian species. Disagreement and deficiency exist among past studies on sea turtles’ leukocytes differentiation, and no published morphologic or ultrastructural study on Mauremys mutica and Cuora flavomarginata was available. Widely used as an important indication of systemic inflammation and prognosis in many species, heterophil toxic change is, however, poorly understood in sea turtles. Objectives: This study aims to identify and characterize the circulating leukocytes in 5 endangered turtle species in Taiwan, compare the efficacy of accessible staining methods, and provide better understanding of the clinical applications and limitations of toxic change examination in sea turtles. Methods: Blood samples were collected from 30 rescued sea turtles (21 Chelonia mydas, 6 Lepidochelys olivacea and 3 Eretmochelys imbricata), 8 captive Mauremys mutica and 7 captive Cuora flavomarginata from January 2017 to September 2018. Morphologic and ultrastructural examination were performed using standard methods. Results: 5 types of leukocytes were identified in each species: heterophils, eosinophils, basophils, lymphocytes and monocytes. Morphologic features were generally comparable to similar species. Two types of eosinophils (large and small) were seen in sea turtles. Basophils of sea turtles were similar to those of other reptile and avian species. Ultrastructurally, heterophil granules were unevenly distributed. No crystalloid granulations were observed in eosinophils of sea turtles. The granulocytes ultrastructure of Mauremys mutica and Cuora flavomarginata were similar to those of Trachemys scripta scripta and Hieremys annandalii, but the polygonal crystalloid structures seen in the basophil granules of Cuora flavomarginata have not been reported in other species of freshwater turtles. Toxic heterophils in sea turtles were characterized with uneven staining of cytoplasmic granules, increased cytoplasmic basophilia, degranulation, and the presence of prominent basophilic cytoplasmic granules. Ultrastructurally, toxic heterophils had smooth cellular membrane, reduced cytoplasmic granules that were smaller or more variable in size, and cytoplasm containing much greater amount of membranous organelles and clusters of dark polyribosomes. There was no significant difference among Wright Giemsa stain, Diff-Quik stain and Liu’s stain on the efficacy of leukocyte differentiation, however Wright-Giemsa’s stain and Liu’s stain were significantly superior than Diff-Quik stain for assessing toxic change. Microscopic examination of toxic change is comparable to TEM examination, however the correlation of the presence toxic change with both clinical inflammatory state and the treatment outcome is both weak and insignificant in sea turtles. Conclusions: The differentiation and characteristics of circulating leukocytes of 5 endangered chelonian species was clarified and described in details, each complemented with high quality micrographs. Toxic change morphology in sea turtles and toxic change ultrastructure in reptile species were described for the first time in this study. This study provided fundamental hematologic information for endangered turtle species and insights to further investigations on clinical pathology and immunity of these species.