Table 1.

Comparison of methods for cyathostomin species identification.

Identification method		Life stage	Usefulness and limitations	References
MORPHOLOGICAL IDENTIFICATION
		Eggs	No species differentiation possible	Lichtenfels, 1975, 2008; Dvojnos and Kharchenko, 1994; Tolliver, 2000; Lichtenfels et al., 2008; Kharchenko et al., 2009; Kornaś et al., 2009; Santos et al., 2016
		Larvae	L3 can be allocated to different larvae types, but not to individual species
		Adults	Identification keys published but species identification is difficult for inexperienced workers
MOLECULAR METHODS
Marker	Method
ITS-1 and ITS-2	PCR and sequencing	All	Species identification and phylogenetic analysis, identification of cryptic species Sometimes only small differences between closely related species Not applicable for mixed samples, isolation of DNA from individual specimen necessary	Campbell et al., 1995; Chilton et al., 1997; Hung et al., 1999a, 2000; Bu et al., 2013
	SSCP-PCR	All	Delineation of 14 Strongylida species (9 Cyathostomin species), Isolation of DNA from individual specimen necessary	Gasser and Monti, 1997
	PCR-RFLP	All	Distinction of Strongylinae eggs, Distinction of two Cyathstomin species (C.ashworthi, C.nassatus). Isolation of DNA from individual specimen necessary, established for larvae	Campbell et al., 1995; Hung et al., 1997
IGS	PCR-ELISA	All	Screening for 6 cyathostomin species in mixed samples possible. Established for eggs and larvae	Hodgkinson et al., 2003, 2005
	RLB	All	Differentiation of up to 18 common species, less time consuming and costly than other molecular methods, screening of strongyle population before and after anthelmintic treatment possible, mixed samples possible, but more viable for individual worms; semi-quantitative approach possible	Traversa et al., 2007; Ionita et al., 2010; Cwiklinski et al., 2012; Kooyman et al., 2016
COI	PCR and sequencing	All	Investigation of intraspecies genetic variability, identification of cryptic species, not applicable for mixed samples, isolation of DNA from individual specimen necessary	Hung et al., 1999a; Traversa et al., 2008
SEROLOGICAL METHODS
Protein-based ELISA		Larvae	Pre-patent detection of four common cyathostomin species possible from serum, no cyathostomin species differentiation possible	Mitchell et al., 2016
PROTEMICS METHOD
		Potentially all	Method is established for bacteria, fungi, several species of arthropods; only one study on nematodes (Trichinella spp.), protocols and reference spectra data base have to be established	Karger et al., 2012; Yssouf et al., 2013, 2016; Mayer-Scholl et al., 2016; Singhal et al., 2016;