Molecular detection of two major gastrointestinal parasite genera in cattle using a novel droplet digital PCR approach

• Published in: Parasitology research (1987) Vol. 118; no. 10; pp. 2901 - 2907
• Main Authors: Baltrušis, Paulius, Halvarsson, Peter, Höglund, Johan
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01-10-2019; Springer; Springer Nature B.V
• Subjects: Animals; Anthelmintic agents; Anthelmintics - therapeutic use; Beef cattle; Biochemistry and Molecular Biology; Biokemi och molekylärbiologi; Biomedical and Life Sciences; Biomedicine; Cattle; Cattle Diseases - diagnosis; Cattle Diseases - drug therapy; Cattle Diseases - parasitology; Clinical Science; Cooperia; Deoxyribonucleic acid; DNA; DNA probes; DNA sequencing; DNA, Helminth - genetics; Feces - parasitology; Fluorophores; Gastrointestinal Diseases; Health aspects; Helminthology - Original Paper; Immunology; Intestinal Diseases, Parasitic - diagnosis; Intestinal Diseases, Parasitic - drug therapy; Intestinal Diseases, Parasitic - veterinary; Klinisk vetenskap; Medical Microbiology; Microbiology; Nematoda; Nematodes; Nucleotide sequence; Nucleotide sequencing; Ostertagia - genetics; Parasite Egg Count - veterinary; Parasites; Pasture; Pest resistance; Polymerase chain reaction; Polymerase Chain Reaction - methods; Trichostrongyloidea - genetics; ddPCR; ITS2; Strongyles; Ostertagia; Cooperia
• ISSN: 0932-0113; 1432-1955; 1432-1955
• DOI: 10.1007/s00436-019-06414-7

Cooperia
sp. and
Ostertagia
sp
.
are two cosmopolitan parasitic nematodes often found in mixed gastrointestinal infections in cattle across temperate regions. In light of the recent increase in the emergence of anthelmintic resistance in these and other nematodes derived from cattle around the globe, and their negative impact on animal health and productivity, novel molecular assays need to be put forth in order to facilitate the monitoring of parasite burden in infected herds, using pasture and/or fecal samples. Here, we describe a novel droplet digital PCR platform–based concept for precise identification and quantification of the two most abundant and important parasite genera in grazing western European cattle. By exploiting a single nucleotide difference in the two parasites’ ITS2 sequence regions, we have developed two specific hydrolysis probes labeled with FAM™ or HEX™ fluorophores, which can not only distinguish between the DNA sequences of the two, but also quantify them in mixed DNA samples. A third, newly developed universal probe was also tested along the genus-specific probes to provide a robust and accurate reference. It was evident that the universal probe displayed congruent results to those obtained by the genus-specific probes when used with DNA from both parasites in a single sample. All in all, the results of our assay suggest that this novel protocol could be used to distinguish and quantify cattle parasites belonging to the two most important genera (i.e.,
Cooperia
and
Ostertagia
) in a single mixed DNA sample.