ORIGINAL PAPER Reliable molecular differentiation of Trichuris ovis and Trichuris discolor from sheep (Ovis orientalis aries) and roe deer (Capreolus capreolus) and morphological characterisation of their females: morphology does not work sufficiently Pavel Vejl 1 & Stanislava Nechybová 2 & Pavla Peřinková 2 & Martina Melounová 1 & Vladimíra Sedláková 1 & Jakub Vašek 1 & Daniela Čílová 1 & Kateřina Rylková 1 & Ivana Jankovská 2 & Jaroslav Vadlejch 2 & Iva Langrová 2 Received: 24 January 2017 /Accepted: 26 May 2017 # Springer-Verlag Berlin Heidelberg 2017 Abstract The main aim of the study was to evaluate associ- ations between morphological variability of Trichuris females from sheep and roe deer and their rDNA polymorphism in whipworm populations from the Czech Republic. The results introduced the use of new molecular markers based on the internal transcribed spacer (ITS)1-5.8S RNA-ITS2 region polymorphisms, as useful tools for the unambiguous differen- tiation of congeners Trichuris ovis and Trichuris discolor . These markers revealed both parasites in roe deer and in sheep; however, T. ovis females predominated in sheep while T. discolor females occurred mostly in roe deer. Additional analysis of ITS1-5.8 rRNA-ITS2 discovered the genetic uni- formity of the analysed T. discolor but high haplotype varia- tion of T. ovis. Simultaneously, molecularly designated female individuals of both species were categorised into four morphotypes (MT) on the basis of morphology of genital pore area. MT1 and MT4 (vulvar opening on everted vaginal appendage/on visible cuticular bulge) occurred only in T. ovis, MT2 (uneverted vagina—vulvar opening without any elevation) was identified only in T. discolor and MT3 (transient type of vulvar opening on a small swelling) was observed in both species. Statistical analysis of biometric data confirmed that morphology of vulva is not a reliable marker for the species determination. On the basis of the ITS1-5.8S RNA-ITS2 region variability, we carried out a phylogenetic analysis (maximum likelihood method, Hasegawa-Kishino- Yano model) which showed that T. ovis haplotypes from the Czech Republic and Ireland and T. discolor haplotypes from the Czech Republic, Spain, Iran and Japan are sister OTUs. Keywords Trichuris discolor . Trichuris ovis . ITS1-5.8S rRNA-ITS2 region . Molecular markers . Morphotypes Introduction Trichuris nematodes are common endoparasites of both do- mestic and wild ruminants. Individual species differ from each other based on host preference, body length, length of the anterior oesophageal portion and body width. However, they differ mainly in the shape and size of their reproductive or- gans. Spicule length is considered the main criterion used to identify Trichuris males. Female trichurids are more difficult to identify than are males (Tenora et al. 1993). The determi- nation of females is based primarily on vulvar characteristics (Skrjabin et al. 1957; Knight 1971; Baruš et al. 1978; Rickard and Bishop 1991; Lanfredi et al. 1995; Cutillas et al. 2009), vagina structure and lining and, alternatively, the distance from the vulva to the uterine sphincter (Knight 1971; Callejón et al. 2012, 2015). However, the shape and size of this structure is variable (Zaman 1984; Tenora et al. 1988, 1993). Trichuris ovis, Trichuris globulosa and Trichuris skrjabini are common endoparasites of sheep (Ovis orientalis aries) (Makovcová et al. 2008; Khan et al. 2010; Khalafalla et al. 2011). The vagina in T. globulosa females never appears * Pavel Vejl vejl@af.czu.cz 1 Department of Genetics and Breeding, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences in Prague, Prague, Czech Republic 2 Department of Zoology and Fisheries, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences in Prague, Prague, Czech Republic Parasitol Res DOI 10.1007/s00436-017-5524-9