DOI: 10.14457/tu.the.2019.1657
title: Investigation of the transcriptome of the rumen fluke Fischoederius elongatus

description: 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 &gt; cytb &gt; nad4L &gt; nad4 &gt; atp6 &gt; nad2 &gt; nad1 &gt; nad3 &gt; cox1 &gt; cox2 &gt; nad6 &gt; 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.