台北都會區動物園溫血動物及路死貓 弓蟲感染的調查

Abstract

弓蟲（T. gondii）是一種絕對細胞內寄生的原生動物，會引起人畜共通傳染疾病，分佈於世界各地；貓科動物是這種寄生蟲的最終宿主，而包括鳥類和人類在內的幾乎所有溫血動物，都是其中間宿主。在2019年6月，台北市立動物園有四隻環尾狐猴（Lemur catta）死於急性弓蟲感染；此後，在對死亡動物進行病理解剖時，常發現動物有弓蟲感染的情形，且後續送檢的血液檢體，也常於PCR（Polymerase chain reaction）檢測呈現陽性反應；因此，對動物園圈養動物進行大規模篩查，以了解弓蟲感染情形似乎非常急迫和重要。本研究首先收集2019年1月至2021年5月間，動物園75種動物的326份血清樣本，使用多物種ELISA（Enzyme-linked immunosorbent assay）試劑，進行弓蟲感染的篩查，檢測結果顯示動物感染率為27 % (88/326) ；接著應用LAT(Latex agglutination test)試驗以確(118/326)，且建議此多物種ELISA試劑僅適用於我們研究的75 個物種中的31 個。其次，我們應用針對弓蟲ITS1(Internal transcribed spacer1)及GRA7(Dense granule protein 7)基因的巢式PCR(nPCR)，分別篩檢8隻動物身上採集的10 個肝臟或血液DNA檢體，結果有8個檢體驗出GRA7基因，但僅有4個檢體驗出ITS1基因，推測GRA7基因似乎是檢測弓蟲感染較好的分子靶標，且依據nPCR陽性產物序列分析，推測台北動物園的動物感染了至少三種不同的弓蟲變異株。為了進一步了解台北都會區弓蟲感染的情形，將nPCR應用於檢測路死貓各種組織的弓蟲感染情形。由46隻路死貓身上收集的224個包括肝臟、肌肉、小腸、脾臟和/或血液的檢體，以GRA7 nRCR檢測貓的陽性率是93.5%（43/46），而檢體總陽性率為58.5%（131/224)，各臟器陽性率分別是肝臟58.7%（27/46）、肌肉65.2%（30/46）、小腸58.7%（27/46）、脾臟56.5%（26/46）和血液55%（22/40），而ITS1 nPCR檢測中僅有10個(4.5%)檢體呈陽性，分別是2個肝臟、4個肌肉、1個小腸、2脾臟和1個血液檢體。後續，根據GRA7 nPCR陽性產物的序列分析，推測路死貓感染了至少14種不同的弓蟲變異株(包含動物園發現的兩株)，且有約37%(17/46)同時被兩種以上的蟲株感染，這些蟲株應多為第三型弓蟲，且與中國大陸流行的蟲株較為相近。

Abstract

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan causing an important zoonotic disease with worldwide distribution. Felids are the definitive hosts of this parasite, while virtually all warm-blooded animals including birds and human serve as the intermediate hosts. At June 2019, four Ring-tailed lemur (Lemur catta) in Taipei Zoo died of acute T. gondii infection. Since then, T. gondii infection was identified frequently during regular blood examines or the necropsy of death animals. Therefore, a general survey of T. gondii infection for captive animals in Taipei Zoo is urgent and needed. In this study, an indirect multi-species ELISA (Enzyme-linked immunosorbent assay) kit was firstly applied to screen T. gondii infection in 326 serum samples collected from 75 species of animals between January, 2019 and May, 2021. The infection rate of captive animals in Taipei Zoo was 27% (88/326). Later, a LAT (Latex agglutination test) assay was applied to reconfirm the results of ELISA and the infection rate was adjusted to 36.2% (118/326) according to the results of LAT assay. The ELISA kit we used appeares to be applicable to 31 of 75 species included in this study. Next, nested PCR (nPCR) targeting the ITS1 gene and GRA7 (dense granule protein 7) gene were also used to detect T. gondii in DNA samples extracted from 10 (liver or blood) samples collected from 8 animals. Since 8 and 4 of 10 samples were respectively identified T. gondii infection by using GRA7 and ITS1 nPCR, the GRA7 gene seemed to be the better molecular target for the detection of T. gondii infection. According to the analysis of nPCR product sequences, captive animals in Taipei Zoo were speculatively infected by at least three different T. gondii variants. To further investigate the prevalence and variants of T. gondii infection in Taipei metropolitan area, nPCR was applied to detect T. gondii from various tissues or organs of road mortality cats. Total 224 samples including liver, muscle, small intestine, spleen, and/or blood were collected from 46 road mortality cats in Taipei metropolitan area. By using nPCR to detect T. gondii infection, 93.5 %(43/46) cats 131 of 224 samples including liver 58.7%(27/46), muscle 65.2%(30/46), small intestine 58.7%(27/46), spleen 56.5%(26/46) and blood 55%(22/40) were positive for GRA7 gene, while only 10 samples (2 livers, 4 muscles, one small intestine, 2 spleens and 1 blood) were positive for ITS1 gene. At last, according to the sequence analysis of GRA7 nPCR product, at least 14 strains (including two strains found in the animals’ DNA of Taipei Zoo) of T. gondii can be found in road mortality cats. Thirty-seven percent of RMCs were infected by at least two strains. Those strains should be type III T. gondii and similar to the strains found in the mainland China.