For six months, Martin K. Nielsen, DVM, was a visiting scientist at the Gluck Equine Research Center from the University of Copenhagen in Denmark through the Albert and Lorraine Clay Research Fellowship award. Nielsen, an assistant professor in the Department of Large Animal Sciences at the University of Copenhagen, began his stint at the Gluck Center in November 2008 and returned home in May 2009, but he built a bridge of cooperation between the two universities.

“The Clay Fellowship plays an important role in bringing scientists together, which allows for generating new ideas and building networks,” Nielsen said. “I am deeply grateful for the opportunities I was given because of the Fellowship.

Dr. Martin Krarup Nielsen

“Upon my return to Denmark, it has become clear that my time in Kentucky has been exceptionally fruitful,” Nielsen added. “My own project is only a part of this. What strikes me is the wonderful research environment at the Gluck, where research ideas easily arise and develop.”

After returning to Denmark in May, Nielsen submitted an article highlighting the research projects he conducted during his time at the Gluck Center, including a two-part project on polymerase chain reaction (PCR) detection of DNA from the bloodworm Strongylus vulgaris in the bloodstream of horses and an on-farm survey of S. vulgaris and other strongyles in horses in Denmark and the United States. This research, designed to simplify detection of bloodworms via a blood test, was performed in collaboration with UK’s Dan Howe, PhD.

PCR Detection of S. Vulgaris DNA in the Bloodstream
S. vulgaris spends about four months in the bloodstream of horses, and the intention was to attempt to detect DNA from this parasite in blood samples from naturally infected horses.

Necropsies were performed on two horses from the herd of Gene Lyons, PhD; this herd, kept on the university farm, has not been treated for parasites. Both of these horses had high numbers of arterial larvae of S. vulgaris. In addition, blood samples were collected from other horses where the S. vulgaris prevalence is known to be 100%. DNA was extracted from serum and whole blood using a Qiagen blood and tissue kit.

Straight PCRs did not yield positive signals from any of the horses tested. Therefore, a nested PCR approach was attempted. For that, strongyle DNA was first amplified using a broad primer set targeting the whole ITS-2 region. In the second round, real-time PCR was performed with a S. vulgaris specific primer/probe set previously developed. With this approach, S. vulgaris DNA could be detected from heavily infected animals, but unfortunately repeated analyses from the same horse were not consistent and other horses in the herd remained negative on testing.

In conclusion, DNA of S. vulgaris cannot reliably be detected by PCR in blood samples. The reason for this is most likely that S. vulgaris is not a circulating pathogen and DNA released by this parasite becomes very dilute in the large blood volume of horses.

Based on the experience generated from this endeavor, a new research approach was decided with Howe. The new project involves creation of cDNA libraries and immunoscreening for identification of candidates for diagnostic targets in an antigen-capture approach. At present, RNA has been extracted from both migrating L4 and adult stages of both S. vulgaris and S. edentatus. These preparations are kept on stock in Howe’s laboratory for further processing.

“The intention is to recruit a graduate student for this project,” Nielsen said. “Separate funding will be applied for. The student will be based at the University of Copenhagen, but will spend a longer period in Dr. Howe’s laboratory to receive the training needed.”

Several publications are expected to result from the research project.

Survey of Worms on U.S., Denmark, and Sweden Farms
The second part of the project was to collect fecal samples from horses on farms around Lexington, Ky. The criteria for inclusion were that horses had not been treated for at least six weeks prior to sampling. Fecal egg counts were generated from every horse, and eggs were isolated for subsequent DNA extraction and PCR analysis. In addition, information about the deworming schedule was obtained on each farm. With help from Lyons, about 500 samples were collected. Similar numbers of samples will be collected in Denmark and Sweden.

All samples will be analyzed with a real-time PCR assay Nielsen previously developed and validated for detection and quantification of S. vulgaris DNA in fecal samples. The samples will be shipped to Denmark, where the PCR analysis will be performed.

The end results of the two-part project will be published in scientific journals.

Future projects also arose during Nielsen’s visit with researchers at the Gluck Center and other universities. Research projects include generating a spreadsheet of data on the relation between worm and egg counts, analyzing data evaluating cytokine expression and fecal egg counts in response to anthelmintic treatment, a study evaluating the potential genetic background for horses consistently shedding low parasite egg numbers, and the use of novel DNA/RNA sequencing techniques for studying mechanisms of anthelmintic resistance in equine nematodes