應用原位雜合染色及分子診斷以偵測禽類副黏液病毒及陸龜皰疹病毒

Abstract

圈養動物雖然可能有較好的生長環境而較為長壽，卻也因飼養密度較野外高，而容易在群體間爆發傳染病，傳染病發生原因通常為引入外來個體、開放式場域或與野外個體接觸，甚至是不適當的照養環境。由於目前對於野生動物的認識較少且有特別的棲息環境或生活習性，因此傳染病通常較難以研究，故建立敏感且快速的診斷工具和研究病原的特性將有助於疾病控制。本研究針對發生於金背鳩（Streptopelia Orientalis）的第一型禽類副黏液病毒（Avian paramyxovirus 1）及黃頭陸龜（Indotestudo elongata）的龜科皰疹病毒（Testudinid herpesvirus）進行研究，包含解剖、組織病理學檢查、分子診斷及病原親緣分析，以及原位雜合染色（In situ hybridization, ISH）等。於2021年三月至四月間，於一遊客可進入之封閉式鳥舍發現27隻金背鳩出現神經症狀和死亡。肉眼病變可見個體消瘦，以及胰臟有多發白點。組織病理學檢查可見非特異性的病灶如不等量之淋巴球和漿細胞浸潤於多個臟器，包含心、肝、脾、肺、腎、胰臟、消化道、生殖道及華氏囊。經反轉錄聚合酶鏈鎖分應（reverse transcription polymerase chain reaction , RT-PCR）及病毒分離後，確診為第一型禽類副黏液病毒感染。此外，也利用ISH證實病變中的單核炎症細胞浸潤與病毒感染有關。本分離株NTU/C239/21以融合蛋白（Fusion protein）全長序列進行親緣分析，歸類為第二類(class II)的第六基因型 （genotype VI），亦稱為鴿子副黏液病毒（pigeon paramyxovirus 1），為常見於鳩鴿科之副黏液病毒。進一步與其他已發表之序列進行比較，發現NTU/C239/21與台灣野生鴿子AHRI121/2017分離株極為接近，故推測本次疾病發生可能與野外個體接觸有關。另一傳染病爆發在黃頭陸龜，於2022年三月初發現其中一隻個體出現食慾不佳且有眼、鼻、口腔分泌物，後續接連幾天其他隻個體亦出現相似狀況，並且口腔發生潰瘍、眼鼻紅腫，以及四肢和頸部皮下水腫，雖然給予抗生素、抗病毒藥物及輸液治療，最終仍有十四隻個體死亡。解剖時可見不等量黃白色偽膜附著於呼吸道、消化道黏膜、四肢及頸部嚴重皮下水腫、肝臟腫大和體腔積液等病變。組織病理學檢查可見嚴重炎症反應發生在呼吸道與消化道，混合大量細胞碎片，並且可見嗜伊紅性的核內包含體和融合細胞。經由聚合酶鏈鎖分應（polymerase chain reaction, PCR）檢測確診為第三基因型（genotype 3）陸龜皰疹病毒（Testudinid herpesvirus）感染，利用ISH染色亦可見多數核內包含體均有陽性訊號。以脫氧核糖核酸聚合酶（DNA polymerase）進行親緣分析，發現其與德國4295/7R病毒株最為相似（97.86%）。醣蛋白B在皰疹病毒被認為是一個潛在毒力因子，經由分析胺基酸序列後確定為乙型基因組（genogroup B），即可見嚴重組織病變和包含體會分布在多重臟器。本次發病欄舍內的個體為十多年前就已移入，期間並無其他個體移入，而且過去皆無相關臨床症狀的記錄，因此，此次疾病爆發懷疑為帶原個體從潛伏期（latency）轉為發病期，進而造成該族群全體感染。

In captive environment, although better husbandry may result in longer living span in animals, the higher population density may also cause the increased risk of infectious disease spreading among animals. Contact with free-ranging animals, introduction of new animals, improper husbandry and environment are common causes of the endemics. Due to the limited information about exotic animals, such as habitat or living habits, investigation of infectious diseases is challenging. Therefore, developing sensitive and rapid diagnostic tools and characterizing the pathogens will be helpful for disease control in exotic animals. In the present study, two disease outbreaks in captive animals were investigated, including avian paramyxovirus 1 (APMV-1) infection in oriental turtle doves (Streptopelia orientalis) and testudinid herpesvirus 3 (TeHV-3) infection in elongated tortoises (Indotestudo elongate), by applying series of examinations, such as necropsy, histopathological examination, molecular diagnosis, in situ hybridization (ISH), and phylogenetic analysis. There were 27 oriental turtle doves found showing neuronal clinical signs or dead in a walk-in aviary during March to April in 2021. Grossly, the animals were emaciated and had multiple pale spots at the pancreas. The histopathological findings revealed variable levels of lymphoplasmacytic infiltration in several organs, such as heart, liver, spleen, lungs, kidneys, pancreas, digestive and reproductive tracts and bursa of Fabricius. After reverse transcription polymerase chain reaction (RT-PCR) and virus isolation, avian paramyxovirus 1 (APMV-1) infection was diagnosed. Additionally, the infiltration of mononuclear inflammatory cells was confirmed to be associated with viral infection by ISH. Our NTU/C239/21 strain was categorized to class II and genotype IV, also known as pigeon paramyxovirus 1 (PPMV-1), based on the nucleotide sequence of full-length fusion protein. The result of phylogenetic analysis demonstrated that NTU/C239/21 strain shared high sequence identity with AHRI121/2017 strain, isolated from the free-ranging pigeon in Taiwan, suggesting that the current outbreak may be related to the contact with wild birds. As for the disease outbreak in elongated tortoises, one individual was first found displaying nasal, oral, and ocular discharge and anorexia in early March 2022. After several days, the other individuals in the same population also showed similar clinical signs accompanied with oral ulceration, erythema in the eyelids and nostrils, and subcutaneous oedema at the neck and four limbs. Despite of receiving treatments such as antibiotics, antiviral drugs, and fluid therapy, fourteen tortoises eventually died. During the necropsy, white to yellow pseudomembrane multifocally to diffusely attached to the mucosa of the respiratory and alimentary tracts, with hepatomegaly and coelomic effusions. Microscopically, severe inflammation in the respiratory and alimentary tracts with accumulation of abundant cell debris and the presence of eosinophilic intranuclear inclusion bodies and syncytial cells. Infection of Testudinid herpesvirus genotype 3 was identified by performing polymerase chain reaction (PCR) and most of the inclusion bodies showed positive signals in ISH. Based on the phylogenetic result of DNA polymerase, our isolate shared 97.86% similarity to strain 4295/7R, which was detected in Germany. The glycoprotein B is considered as a potential virulent factor of herpesviruses, and after comparing the ammino acid sequence, the present isolate was further analyzed, and the isolate is classified as genogroup B, which causes more severe lesions and inclusion bodies can be detected in multiple organs. Since the tortoises in the exhibition had been kept for more than 10 years, there were no introduction of new individual and no related clinical signs had been documented, the cause of the outbreak was most likely to be the reactivation of carrier(s) from the latency to the onset stage and consequently transmitted to other tortoises in the same group.