ABSTRACT. Differential diagnosis of Mecistocirrus digitatus infection relies on morphological examination of either eggs in faecal samples or L3 larvae developed in vitro. Technical limitations hinder the practicability of these approaches. Hence, in order to develop a specific diagnostic measure for M. digitatus infection, we determined the sequence of the internal transcribed spacer (ITS) of its ribosomal DNA (rDNA) and designed primers for PCR-based species-specific amplification of the ITS to differentiate between M. digitatus and other common gastrointestinal (GI) nematode species. The newly designed primers amplified a single specific 520 base pair (bp) fragment from the M. digitatus ITS, and its detection limit was as low as 0.001 ng. Further, this sensitivity suggested that the specific fragment could be amplified even from a unicellular egg that collected directly from uteri of an adult M. digitatus female. In fact, we designed a method that employs a small piece of a cover slip and a filter paper by which we could differentially amplify a PCR fragment from a unicellular egg. The reliability of the specific PCR assay was also demonstrated with 10 oval samples that collected from bovine faeces by using sugar flotation method. These data suggested that the specific PCR assay of the ITS region of M. digitatus rDNA could be useful for the identification of GI nematodes. Gastrointestinal (GI) nematodes of domestic ruminants are of major veterinary importance due to their high prevalence, ubiquitous distribution and the pathological consequences of infection and the associated economic production losses [1,3,18]. In Japan, the major GI nematode species in cattle are Ostertagia ostertagi, Mecistocirrus digitatus and Trichostrongylus axei which are found in the abomasum; Strongyloides papillosus and Cooperia and Nematodirus species which are found in the small intestine; and Oesophagostomum radiatum found in the large intestine [7,15,16,23]. Of these species, M. digitatus, O. ostertagi and Cooperia oncophora are considered the most prevalent in cattle; in particular, the infection rate of M. digitatus was high at approximately 30-50 % [7,16]. This nematode is commonly described as a large stomach worm, namely, a trichostrongylid nematode, and is an important blood-sucking nematode present in the abomasums of cattle. This parasite may cause mucosal inflammation, haemorrhage, ulcers and necrosis in the abomasums. It appears to be confined mainly to Asian countries; however, it has also been found in Central America, Egypt and Russia [6,7,13,21,22]. The movement of its hosts for agricultural purposes has resulted in the global spread of this parasite.Generally, ruminants are concurrently infected with more than one species of GI nematodes, each having a different pathological effect on the host. It has been a difficult task to eradicate GI nematodes from grazing ruminants due to the variation in host susceptibility to the parasite, the wide distribution of nematodes in nature and the presence of wild ruminants. An...