Specialized Diagnostics: Polymerase Chain Reaction (PCR) and Nucleic Acid Sequencing

This technique, which depends on the detection of parasite nucleic acid, can be applied to a variety of samples, including fresh and fixed tissues, blood, faeces, and the parasites themselves. The technique can be used for detection, identification, and quantification. PCR techniques and equipment are evolving rapidly. PCR can also be used to tease apart taxonomic relationships, for example for the various species/genotypes of Giardia and Cryptosporidium. The major issues to be dealt with are the identification of unique parts of the nucleic acid of each genus or species of parasite that can be used for reliable diagnosis, the standardization of PCR laboratory procedures, and optimization of DNA extraction methods to minimize the effects of a number of inhibitors that interfere with the technique.

PCR assays have been developed as research and diagnostic tools for several parasites including:

• Several gastro-intestinal nematodes of ruminants
• Dictyocaulus (ruminant lungworm)
• Dorsal-spined larvae of protostrongylid parasites of free-ranging ruminants
• Some hookworms and ascarids
• Echinococcus multilocularis (alveolar hydatid)
• Some intestinal protozoa including: Giardia, Cryptosporidium, Entamoeba histolytica, some Eimeria (coccidian), Toxoplasma, Neospora, Sarcocystis, and some microsporidia – e.g. Encephalitozoon)
• Distinguishing between the various biotypes of Trichinella
• Detection of a variety of parasites in tissues of both definitive and intermediate hosts

Nucleic Acid Sequencing:

This technique is generally more time-consuming and expensive than PCR alone, but does have applications in veterinary parasitology. For example, we have recently used DNA sequencing applied to first-stage larvae recovered from faeces to map the North American distribution of the muscle worm Parelaphostrongylus odocoilei in its various free-ranging ungulate hosts. Public reference sequence databases such as Genbank (hosted by the National Center for Biotechnology Information, National Institutes of Health) are growing rapidly, increasing the utility of sequence-based approaches.