Investigation of the transcriptome of the rumen fluke Fischoederius elongatus

Fischoederius elongatus, a pouched amphistomes in the family Gastrothylacidae, is a neglected food-borne parasite that infects a large number of cattle in Thailand. The fluke causes economic losses by massive infection in the rumen of ruminants. This causes obstruction of food absorption and results in decreased milk and meat production. Also, acute infection with the fluke causes severe mucosal damage and significantly increases mortality in young animals. Importantly, Fischoederius elongatus was reported as a case of zoonosis trematode in a woman who had severe vomiting in Guangdong, China (1991). At present, routine diagnosis of the fluke infection is based on microscopic examination by using fecal egg count and morphological egg identification. It is difficult to distinguish Fischoederius elongatus from other amphistomes by egg morphology. In addition, the rumen fluke eggs are similar to eggs of the liver fluke Fasciola spp. Misdiagnosis may lead to incorrect drug treatment and development of resistance. The aim of the present study was to characterize Fischoederius elongatus in Thailand by observing the fluke morphology, and by analysis of molecular biology, taxonomy, physiology and pathology using mitochondrial genome characterization and transcriptomics. The morphology of Fischoederius specimens collected in this study was found following the classic literature description of Fischoederius elongatus by Sey (1991). Fischoederius specimens collected in this study during the years 2014–2019 had either large or small testes. Specimens with large testes were mature and contained often intrauterine eggs. Specimens with small testes were immature and also had an undeveloped ovary and only a small number of vitellaria. The developmental stage was confirmed by transcriptome data which lacked for example vitelline protein encoding transcripts. Moreover, molecular studies of Fischoederius will provide basic knowledge usable for diagnosis. A PCR-amplified fragment of ribosomal ITS2 originating from one of the collected specimens showed significant identity (99%, E-value: 0.0) to closely related trematodes in the database; however, this region has high sequence conservation making it not suitable for species identification. For this reason, mtCOX1, a universal marker for species identification, was investigated. PCR products of mtCOX1 from 48 single flukes were used for restriction analysis with MseI. Surprisingly, nine restriction pattern (A–I) were observed and sequence analysis of mtCOX1 pattern A–I showed 0.7–9.3% sequence difference. Together with a phylogenetic analysis these findings suggested that the collected specimens represented five distinct species A, [BEG], [CFH], D, and I. Hence, Fischoederius elongatus might represent a cryptic species, a complex of morphologically indistinguishable species. Furthermore, the mitochondrial genome of Fischoederius mtCOX1 MseI pattern A was characterized. The complete genome with 14,780 bp in length contains twelve protein-coding genes (cox3 > cytb > nad4L > nad4 > atp6 > nad2 > nad1 > nad3 > cox1 > cox2 > nad6 > nad5), 23 tRNA genes, two rRNA genes (rrnS and rrnL), and two non-coding regions (SNR and LNR). All genes are transcribed in the same direction. Interestingly, inverted repeats causing hairpin-like structures were detected in the mitochondrial genomes of Fischoederius mtCOX1 MseI pattern A, [BE], C, D, and I that might have originated from transposable elements and possibly affect DNA recombination, replication and transcription. Moreover, the obtained transcriptome data of Fischoederious mtCOX1 MseI pattern A, C, and E showed similar expression profiles. Highly abundant genes with complete sequence and known function included cathepsin-like cysteine protease (B, C, L), fatty acid-binding protein, thioredoxin, and tegumental calcium-binding protein. Polyclonal mouse Fischoederius anti‐ES antibody detected a predominant 35 kDa antigen in an immunoblot of Fischoederius antigens. This antigen might be Fischoederius cathepsin B by its molecular weight in accordance with the high abundance of cathepsin B transcripts in the transcriptome data. Cysteine proteases play important roles in parasite feeding, host tissue invasion, and immune evasion. These proteins should be further investigated for application in diagnosis and vaccine innovation.

长菲策吸虫（Fischoederius elongatus）是隶属于胃叶科（Gastrothylacidae）的囊状前后盘吸虫，是一种长期被忽视的食源性寄生虫，在泰国广泛感染大量牛只。该吸虫寄生于反刍动物瘤胃并引发重度感染，进而造成经济损失：其会阻碍食物吸收，导致乳肉产量下降。此外，该吸虫的急性感染会造成严重的黏膜损伤，并显著提高幼龄动物的死亡率。值得注意的是，1991年中国广东曾报道一例女性严重呕吐病例，其病因即为长菲策吸虫所致的人畜共患吸虫病。

目前，该吸虫感染的常规诊断手段仍基于粪便虫卵计数与虫卵形态鉴定的显微镜检查方法。但通过虫卵形态难以将长菲策吸虫与其他前后盘吸虫区分开来，且瘤胃吸虫虫卵与片形吸虫属（Fasciola spp.）虫卵形态相似，误诊可能导致不合理的药物治疗，进而催生耐药性。

本研究旨在通过观察吸虫形态，并结合线粒体基因组特征分析与转录组学开展分子生物学、分类学、生理学及病理学研究，对泰国境内的长菲策吸虫进行系统鉴定与特征描述。

本研究采集的菲策吸虫标本形态符合Sey（1991）对长菲策吸虫的经典文献描述。2014-2019年间采集的菲策吸虫标本可分为睾丸大小两种类型：睾丸较大的标本为成熟个体，通常携带子宫内虫卵；睾丸较小的标本未成熟，同时卵巢发育不全，卵黄腺数量稀少。转录组数据证实了这一发育阶段差异——例如后者缺失卵黄蛋白编码转录本。

此外，对菲策吸虫的分子研究可为诊断提供基础理论依据。本研究从一份采集标本中扩增得到的核糖体ITS2片段，与数据库中近缘吸虫的同源序列具有极高一致性（99%，E值：0.0），但该区域序列保守性过高，不适用于物种鉴定。因此，本研究选用物种鉴定通用标记线粒体COX1（mtCOX1）开展研究。

对48份单个吸虫的mtCOX1 PCR扩增产物进行MseI限制性酶切分析，意外观察到9种酶切图谱（A-I）。对这9种图谱的mtCOX1序列分析显示，其序列差异为0.7%-9.3%。结合系统发育分析结果，这些发现表明所采集的标本可分为5个独立物种：A、[BEG]、[CFH]、D及I。由此推测，长菲策吸虫可能代表一个隐存种复合体，即一组形态上无法区分的物种。

进一步对mtCOX1 MseI酶切图谱为A型的菲策吸虫进行线粒体基因组特征分析。该完整基因组全长14780 bp，包含12个蛋白质编码基因（cox3 > cytb > nad4L > nad4 > atp6 > nad2 > nad1 > nad3 > cox1 > cox2 > nad6 > nad5）、23个转运RNA（tRNA）基因、2个核糖体RNA（rRNA）基因（rrnS与rrnL）以及2个非编码区（SNR与LNR）。所有基因均沿同一方向转录。

有趣的是，在mtCOX1 MseI酶切图谱为A型、[BE]、C、D及I的菲策吸虫线粒体基因组中，均检测到源自转座因子的反向重复序列，其可形成发夹样结构，可能对DNA重组、复制及转录产生影响。

此外，对mtCOX1 MseI酶切图谱为A型、C型及E型的菲策吸虫转录组数据进行分析，发现其表达谱相似。高丰度表达且功能明确的基因包括组织蛋白酶样半胱氨酸蛋白酶（B、C、L型）、脂肪酸结合蛋白、硫氧还蛋白以及皮层钙结合蛋白。

多克隆小鼠抗菲策吸虫排泄分泌（ES）抗体在菲策吸虫抗原的免疫印迹实验中，检测到一条主要的35 kDa抗原条带。结合转录组数据中组织蛋白酶B转录本的高丰度表达，该抗原可能为菲策吸虫组织蛋白酶B。半胱氨酸蛋白酶在寄生虫摄食、宿主组织侵袭及免疫逃逸过程中发挥重要作用，此类蛋白有望进一步开发用于诊断与疫苗研发。