CONTROL OF EQUINE GASTROINTESTINAL PARASITES: IMMUNOLOGY, HOST GENETICS, AND DRUG RESISTANCE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0230714
• Project Start Date: Oct 1, 2012
• Project End Date: Sep 30, 2017
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF KENTUCKY
500 S LIMESTONE 109 KINKEAD HALL
LEXINGTON,KY 40526-0001

Performing Department
Veterinary Science

Non Technical Summary
Both deworming and vaccination are carried out on a routine basis in all managed equine establishments, and it is not unusual to deworm and vaccinate horses on the same day. Until recently, this was considered to be an unproblematic approach, but recent findings have raised concerns about possible interactions between deworming and vaccination. Deworming causes a mild and transient inflammatory reaction in the horse, which appear to depend on the type of dewormer used. This has been found to cause disease in newly dewormed horses, but usually the inflammatory reaction is mild with no apparent symptoms. However, it is unknown whether this reaction can possibly affect the result of vaccination. Our hypothesis is that deworming causes an inflammatory reaction that affects vaccination. This one year study will evaluate this by exposing three groups of ponies (8 per group) to three different types of vaccine administered at the same time (one with adjuvant, one being a live virus vaccine, and one with no adjuvant). Two of the groups will be dewormed with two different dewormers (pyrantel pamoate and ivermectin) at the time of vaccination. This allows us to measure the effectiveness of the deworming, the antibody response to the vaccine, and the inflammatory reaction to vaccination with and without the accompanying deworming. Ponies will be followed for a total of 60 days with collection of fecal and blood samples to evaluate deworming effectiveness, inflammatory response and generation of vaccine-specific antibody levels. The study will generate useful information about the interaction between deworming and vaccination and the possible influence on vaccination effects. In addition, the study allows evaluation of possible differences between two types of dewormer in their effect upon vaccination responses.

Animal Health Component

100%

Research Effort Categories

Basic

(N/A)

Applied

100%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3130	1110	25%
311	3810	1110	25%
313	3130	1110	25%
313	3810	1110	25%

Knowledge Area
311 - Animal Diseases; 313 - Internal Parasites in Animals;

Subject Of Investigation
3130 - Nematodes; 3810 - Horses, ponies, and mules;

Field Of Science
1110 - Parasitology;

Keywords

parasite

control

drug resistance

immunity

Goals / Objectives
Objectives: The objectives are to characterize the immunological and inflammatory reaction to equine gastrointestinal infection as well as anthelmintic treatment of these, and to evaluate whether these processes affect other important immune functions. The hypothesisis as follows: Intestinal parasites modulates immune responses and inflammatory reactions in the horse

Project Methods
This study will examine the potential interaction between deworming and vaccination. Twenty-four ponies aged approximately 8 months will be allocated to the study. Ponies will not have received anthelmintic treatment prior to the study. Strongyle parasite fecal egg counts will be used to assign the ponies to three cohort groups, such that variance in egg counts is equivalent among the groups. All groups will undergo the same vaccination regimen with three different vaccines given simultaneously, but two of the groups will also receive two different anthelmintic treatments at the time of vaccination. Anthelmintic efficacy will be monitored with fecal egg counts. Vaccine specific antibodies will be measured for all three vaccines in the three groups, and the isotype profiles of antibodies will be determined. Cytokines representing Th1, Th2, and Treg responses will be evaluated based on mRNA expression over the days following each vaccine/antihelmintic treatment, using the PAXgeneTM blood RNA collection system. Vaccines: All ponies will be vaccinated with three different vaccines: A non-adjuvanted heterologous protein, keyhole limpet hemocyanin (KLH). A commercially available live recombinant vaccine for West Nile Virus (antigen expressed by recombinant canarypox). A commercially available adjuvanted killed herpes rhinopneumonitis virus vaccine. All ponies will receive all three vaccinations simultaneously. All vaccines will be applied twice, 30 days apart. In group 1, all ponies will be dewormed with ivermectin paste (200 µg/kg) on both vaccination occasions. In group 2, all ponies will be dewormed with pyrantel pamoate (6.6 mg/kg) on both vaccination occasions. Group 3 will not receive anthelmintic treatments but will follow the same vaccination protocol as the two other groups. Fecal egg counts Fecal strongyle egg counts will be generated using a recently developed egg counting technique, the FLOTAC, which is characterized by low variability between counts and a detection limit of5 egg per gram (EPG) of feces. Egg counts will be used to monitor anthelmintic treatment efficacy by use of the fecal egg count reduction test (FECRT). Samples will be collected and analyzed weekly until egg reappearance. Acute phase proteins The acute phase inflammatory markers Serum Amyloid A, Haptoglobin, and Fibrinogen will be measured on the same days as for the hematology mentioned above. Serum iron will be measured as well. Vaccine-specific antibodies All vaccines will be administered at the same time, by the intramuscular route in different sites in the right and left brachiocephalicus/serratus cervicus muscles of the neck and the pectoral muscles, after appropriate cleaning of the vaccination area with ethanol. Vaccinations will be repeated 30 days later. Serum from each pony will be collected by jugular venipuncture immediately prior to each vaccination and also 2 weeks after the second vaccination (days 0, 30, and 45). KLH ELISA: Immulon I - B microtiter plates (Fisher Scientific, Hanover Park, IL) will be coated overnight at 4Ë™C with 300 μg KLH per well. Coated plates will then be washed with PBS/0.05% TWEEN®-20 (PBS-T; Sigma) and blocked with PBS containing 0.5% polyvinyl alcohol (Sigma) for 1 h. The serum samples will be diluted 1:300 in PBS-T. Plates are washed with PBS-T and serum is added to wells in triplicate. Plates will be incubated at 37Ë™C for 1 h. Plates will be washed with PBS-T, secondary antibody (Peroxidase-conjugated AffiniPure Goat Anti-Horse IgG; Jackson Immuno Research, West Grove, PA) is added to each well at 1:2500 dilution, and plates will be incubated again at 37Ë™C for 1 h. After washing plates with PBS-T, substrate (3,3´ 5,5´,-tetramethylbenzidine; Sureblue TMB 1, KPL, Gaithersburg, MD) will be added and A standard curve developed from the serum of a previously vaccinated animal shown to have a strong response to the vaccine will be used to create a standard curve on each plate for the determination of relative ELISA units. EHV-specific and WNV-specific antibody ELISAs are essentially similar except for the antigens used to coat the wells. For EHV-specific ELISAs, we will use whole EHV-1 virus, T953 strain, as antigen. Virus will be cultured to high titer in RK13 cells and purified from cellular contaminants by sucrose density gradient ultracentrifugation. Virus protein content will be measured by using the Bradford protein assay (Pierce). ELISA plates will be coated with purified EHV-1 particles (5 µg/ml, 200 µl/well). For WNV-specific ELISAs we will use E.coli derived 18kda recombinant protein containing the WNV Envelope (E) protein N-terminal immunodominant regions (Feldan Inc., Quebec). This protein is fused with 6xHis tag and chromatography-purified to >95%. We will use 0.5 µg/well to coat wells as recommended by the manufacturer. IgG isotype quantitation will be done by ELISA . For these, wells are coated with the different antigens as above and equine serum samples will be added at different dilutions (1:50, 1:250, 1:1250, 1:6250). Primary detection antibodies consisting of mouse monoclonal antibodies CVS 39, CVS 40, and CVS 45, recognizing equine IgG4/7 (IgGb), IgG3/5 (IgG(T)), and IgG1 (IgGa), respectively will be added and plates incubated for 1 hr. The secondary antibody of HRP-conjugated goat anti-mouse IgG(H&L) (1:2000 dilution) (Jackson ImmunoResearch) and substrate will follow. Plates will be read in an ELISA reader at 450nm. Real-time PCR analysis of the cytokine gene expression PAXgene™ blood RNA tubes (Qiagen/PreAnalytiX, Valencia, CA) will be used to collect approximately 2.5 ml of blood from the jugular vein at the day of each vaccination and at one, three, and five days post vaccination.Following collection, these tubes will be immediately inverted 8-10 times, incubated at room temperature overnight and then stored at -20ºC. For analysis, the PAXgene™ will be allowed to thaw, and processed according to the manufacturer's protocol for the PAXgene™ blood RNA kit for total RNA isolation. Reverse transcription reactions will be completed by combining total RNA (one microgram from each sample) and reverse transcription master mix. The master mix will be prepared by using the following reagents: 0.5 μl (20 U/μl) avian myeloblastosis virus [AMV] reverse transcriptase (Promega, Madison, WI), 1 μl oligo dT primer (0.5 μg/μl; Promega), 1 μl RNAsin (40 U/μl, Promega), 4 μl dNTP (10 mM; Promega), 16 μl AMV buffer (Promega), and 16 μl MgCl2 (25 mM, Promega). The one microgram of total RNA will be brought up to 41.5 μl in nuclease-free water (Qiagen, Valencia, CA), after which 38.5 μl of master mix is added to each sample. The reactions will be incubated in a thermocycler for 15 minutes at 42 °C and then for 5 minutes at 95 °C. The cDNA is diluted with an equivalent volume of RNase-free water (Qiagen) to ensure that the volume was sufficient for detection of multiple cytokines. We will measure the cytokines IL-1β, IL-4, IL-6, IL-10, TNF-α, and IFN-γ. Induction of IL-1β, IL-6, and IFN-γ are characteristic of Th1-like responses; IL-4 induction is characteristic of Th2-like responses, and IL-10 induction is characteristic of Treg responses. Beta-glucuronidase mRNA will be used as the endogenous control. The gene sequence encoding these equine cytokines are known and primers and probes for their detection using the RT-PCR method have been developed and used by our group. Personnel performing all lab assays will be blinded as to the anthelmintic group to which the individual ponies belong.

Progress 10/01/12 to 09/30/17

Outputs
Target Audience:In the reporting period, we have presented our work at the American Association for Veterinary Parasitology Conference, which is a scientific meetingas well as Mid-American Veterinary Conference, which is a continuing education conference. The target audiences at these venues are other scientists and veterinarians in practice. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has involved training of several undergraduate and graduate students. One paper was published by an undergraduate student, Faith Miller, while another had Dr. Nielsen's PhD student Jennifer Bellaw as first author. Currently,another of Dr. Nielsen'sPhd-students, Ashley Steuer, is engaged in this line of work and has just published her first publication on aspects of intestinal mucosal reactions to parasite infection. How have the results been disseminated to communities of interest?Theaforementioned works involvingDr. Nielsen's students have all been presented at scientific conferences. Furthermore,Dr. Nielsenhave presented at acontinuing veterinary education conference in the reporting period (Mid-American Veterinary Conference, 2017) and again at the Veterinary Meeting & Expo in 2018, which is the world's largest continuing education conference for veterinarians. A recent departmental seminar given by Dr. Nielsen in February, 2018will be posted online as a webinar and contained a summary of the work described here. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We reached a deeper understanding of the inflammatory reaction associated with intestinal cyathostomin parasite infection in horses, and an additional paper has just been accepted for publication in 2018 (i.e., outside the reporting period). Specifically, we have shown that inflammatory reactions to anthelmintic treatment are minimal, and that goblet cell hyperplasia may be driving spontaneous worm expulsion in naturally infected horses. We are currently seeking funding to investigate the phenomenon of worm expulsionfurther.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Nielsen MK, Lyons ET. Encysted cyathostomin larvae in foals - progression of stages and the effect of seasonality. Veterinary Parasitology 2017, 236, 108-112.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Miller, F.L., Bellaw, J.L., Lyons, E.T., Nielsen, M.K. Strongyloides westeri worm and egg counts in naturally infected young horses. Veterinary Parasitology 2017, 15, 1-3.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Salas-Romero J, Gomez-Cabrera K, Molento MB, Lyons ET, Delgado A, Gonzales L, Arenal A, Nielsen MK. Efficacy of two extra-label anthelmintic formulations against equine strongyles in Cuba. Veterinary Parasitology: Regional Studies and Reports 2017, 8, 39-42.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Bellaw, J.L., Krebs, K., Reinemeyer, C.R., Norris, J.K., Scare, J.A., Pagano, S., Nielsen, M.K. Anthelmintic therapy of equine cyathostomin nematodes  larvicidal efficacy, egg reappearance period, and drug resistance. International Journal for Parasitology 2018, 48, 97-105.
• Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Steuer, A., Loynachan, A., Nielsen, M.K. Evaluation of the mucosal inflammatory responses to larvicidal treatment of equine cyathostomins in response to anthelmintic treatment. Veterinary Immunology and Immunopathology  in press.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:In the reporting period, we have presented our work at several scientific conferences as well as continuing education conferences. The target audiences at these venues are other scientists and veterinarians in practice. Furthermore, we have disseminated information to horse owners via presentations made available on a large equine news portal (www.thehorse.com). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This work has enabled us to make efforts to investigate this issue further. Local responses within the large intestinal mucosal walls are currently being characterized in an on-going study. Preliminary findings indicate that some inflammatory reactions do occur locally and a histopathology study is currently aiming at further characterization. This work involves graduate students so there is a clear training/educational aspect as well. How have the results been disseminated to communities of interest?In 2016, we have disseminated this knowledge to veterinarians at continuing education conferences (Equine Infectious Disease Conference in Argentina, British Equine Veterinary Association Conference in England, and European College for Equine Internal Medicine Conference in Finland). What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we plan to finish the current study described above, and to disseminate the information generated in research publications and at scientific conferences.

Impacts
What was accomplished under these goals? Work under this goal is ongoing. We have reached a deeper understanding of inflammatory and immunologic responses to equine strongyle infection and to anthelmintic treatment of these infections. We have documented that systemic responses are very subtle if not negligible. the data generated in our laboratory do support the hypothesis that worms modulate the immune response.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Bellaw JL, Pagan J, Cadell S, Phethean E, Donecker JM, Nielsen MK. Objective evaluation of two deworming regimens in young Thoroughbreds using parasitological and performance parameters. Veterinary Parasitology 2016, 221, 69-75.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Nielsen MK, Jacobsen S, Olsen S, Bousquet E, Pihl TH. Non-strangulating intestinal infarction associated with Strongylus vulgaris in referred Danish equine patients. Equine Veterinary Journal 2016, 48, 376-379.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Nielsen MK, Donoghue EM, Stephens ML, Stowe CJ, Donecker JM, Fenger CK. An ultrasonographic scoring method for transabdominal monitoring of ascarid burdens in foals. Equine Veterinary Journal 2016, 48, 380-386.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Slusarewicz P, Pagano S, Mills C, Popa G, Chow M, Mendenhall M, Rodgers DW, Nielsen MK. Automated parasite fecal egg counting using fluorescence labeling, smartphone image capture and computational image analysis. International Journal for Parasitology 2016 46, 485-493.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Leathwick DM, Donecker JM, Nielsen MK. A model for the development and growth of the parasitic stages of Parascaris spp. in the horse. Veterinary Parasitology 2016, 228, 108-115.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Fabiani JV, Lyons ET, Nielsen MK. Dynamics of Parascaris and Strongylus spp. parasites in untreated juvenile horses. Veterinary Parasitology 30, 62-66.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Noel ML, Scare JA, Bellaw JL, Nielsen MK. Accuracy and precision of Mini-FLOTAC and McMaster techniques for determining equine strongyle egg counts. Journal of Equine Veterinary Science  in press.

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:The target audience is horse owners, horse farm managers, and equine veterinarians. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has involved 6 undergraduate students and 2 graduate level students. Furthermore, we have had two graduate students visiting from the University of Georgia to learn about some of the procedures involved. How have the results been disseminated to communities of interest?The work has been presented at the Nordic Equine Veterinary Conference, American Association for Veterinary Parasitology Conference, and the American Veterinary Medicine Association Conference in 2015. Furthermore, news articles have been published in EQUUS Magazine, EquiManagement Magazine and The Horse Magazine in 2015. Finally, the work has been published in a scientific journals. What do you plan to do during the next reporting period to accomplish the goals?We recently finished another study evaluating the larvicidal efficacy in naturally infected ponies. Tissue and blood samples have been collected to further study the local immunologic and inflammatory reactions in the mucosal walls. We have submitted one proposal for research funding to a potential sponsor and hope to get to do this work in 2016

Impacts
What was accomplished under these goals? We have generated new knowledge about the immunological response to larvicidal anthelmintic treatment in naturally infected horses. Through histopathology, we have characterized the cellular reponse in the mucosal walls of the cecum and ventral colon in horses treated with either moxidectin (0.4 g/kg) or fenbendazole (five days of 10 mg/kg). We found subtle reactions to moxidectin treatment, which had not been reported before. No clear reactions could be seen to fenbendazole treatment, but this was probably due to the very low effiacy of this drug. Furthermore, there were no clear differences in systemic responses between treatment groups.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Nielsen MK, Scare J, Gravatte HS, Bellaw JL, Prado JC, Reinemeyer CR. Changes in serum Strongylus vulgaris-specific antibody concentrations in response to anthelmintic treatment of experimentally infected foals. Frontiers in Veterinary Science 2015, 2, 17.
• Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Nielsen MK, Donoghue EM, Stephens ML, Stowe CJ, Donecker JM, Fenger CK. An ultrasonographic scoring method for transabdominal monitoring of ascarid burdens in foals. Equine Veterinary Journal  in press.
• Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Donoghue EM, Lyons ET, Bellaw JL, Nielsen MK, 2015. Biphasic appearance of corticated and decorticated ascarid egg shedding in untreated horse foals. Veterinary Parasitology  in press.
• Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Nielsen MK, Loynachan AT, Jacobsen S, Stewart JC, Reinemeyer CR, Horohov DW. Local and systemic inflammatory and immunologic reactions to cyathostomin larvicidal therapy in horses. Veterinary Immunology and Immunopathology  in press.

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Equine veterinarians, horse owners and farm managers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? We have had two graduate students and two undergradaduate students enrolled during the evaluation period. These have all received training in veterinary science in general and equine parasitology in particular. Further, the laboratory currently has one visiting scholar assigned to a project related to the present Hatch project. How have the results been disseminated to communities of interest? We have presented our work at two scientific conferences this summer: AMCOP and AAVP. Further, it will be presented at an additional three conferences in December and January What do you plan to do during the next reporting period to accomplish the goals? We have one more study ongoing and aiming at accomplishing these goals. We continue to conduct a number of studies related to this Hatch project

Impacts
What was accomplished under these goals? The interaction between deworming and vaccination in equids has been extensively evaluated with a large number of inflammatory and immunological parameters. Results indicate that deworming does interact with the inflammatory reaction to vaccination, but this does not significantly impact the vaccine efficacy.A paper has been submitted for publication and is currently under review.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Andersen UV, Reinemeyer CR, Toft N, Olsen SN, Jacobsen S, Nielsen MK. Physiologic and systemic acute phase inflammatory responses in young horses repeatedly infected with cyathostomins and Strongylus vulgaris. Veterinary Parasitology 2014, 201, 67-74.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Nielsen MK, Reist M, Kaplan RM, Pfister K, van Doorn DCK, Becher A. Equine parasite control under prescription-only conditions  awareness, knowledge, perception, and strategies applied. Veterinary Parasitology 2014, 204, 64-72.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Reinemeyer CR, Prado JC, Andersen UV, Nielsen MK, Schricker B, Kennedy T. Effects of daily pyrantel tartrate on strongylid population dynamics and performance parameters of young horses repeatedly infected with cyathostomins and Strongylus vulgaris. Veterinary Parasitology 2014, 204, 229-237.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Xu J, Vidyashankar AN, Nielsen MK. Drug Resistance or Re-emergence? Simulating Equine Parasites. Transactions on Modeling and Computer Simulation, 2014, 24, Article 20.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Nielsen MK, Wang J, Davis R, Bellaw JL, Lyons ET, Lear TL, Goday C. Parascaris univalens  a victim of large-scale misidentification? Parasitology Research 2014, 113, 4485-4490.
• Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Robert M, Hu W, Nielsen MK, Stowe CJ. Attitudes towards implementation of surveillance-based parasite control on Kentucky Thoroughbred farms  current strategies, awareness, and willingness-to-pay. Equine Veterinary Journal  in press.
• Type: Journal Articles Status: Under Review Year Published: 2014 Citation: Nielsen, M.K., Rubinson, E.F., Chambers, T.M., Horohov, D.W., Wagner, B., Betancourt, A., Reedy, S.E., Jacobsen, J. Interaction between anthelmintic treatment and vaccine responses in ponies naturally infected with cyathostomins. Veterinary Immunology and Immunopathology
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Nielsen MK, Vidyashankar AN, Gravatte HS, Bellaw J, Lyons ET, Andersen UV. Development of Strongylus vulgaris-specific serum antibodies in naturally infected foals. Veterinary Parasitology 2014, 200, 233-304.

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: We are targeting horse owners, managers, equine professionals and veterinary practitioners Changes/Problems: Despite several grant submissions we have not been able to get funding for studies addressing genetic regulation of worm burden and ivermectin resistance in cyathostomins. As mentioned on the previous page, we have now revised the project description accordingly. What opportunities for training and professional development has the project provided? I currently have two graduate students enrolled: Jennifer Bellaw and Emily Rubinson. They are both working with aspects of this project. We are collaborating with Dr. Stine Jacobsen, University of Copenhagen who is a world authority within inflammatory markers in horses. We have participated in one national and two international conferences in the past year. How have the results been disseminated to communities of interest? I have given several seminars to veterinarians in the past year: in the US, in Denmark, and in the United Kingdom. What do you plan to do during the next reporting period to accomplish the goals? We are currently working with one funded project, and will launch another funded project in a couple ofmonths. These are both addressing the interaction between parasites and the immune system. We expect to be presenting data from these studies at conferences in 2014, as well as submitting research articles for peer-review.

Impacts
What was accomplished under these goals? We have published a paper on the immunologic and inflammatory reaction to deworming. We have received a grant from Grayson-Jockey Club Research Foundation to study immunologic reactions to vaccination and deworming, and the study is ongoing. The other two legs of the program have not been pursued further due to lack of funding. We have revised the project accordingly.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: Andersen UV, Howe DK, Olsen SN, Nielsen MK. Recent advances in diagnosing pathogenic equine gastrointestinal helminths: The challenge of prepatent detection. Veterinary Parasitology 2013, 192, 1-9.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Nielsen MK, Vidyashankar AN, Hanlon BM, Diao G, Petersen SL, Kaplan RM. Hierarchical model for evaluating pyrantel efficacy against strongyle parasites in horses. Veterinary Parasitology 2013, 197, 614-622.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Carstensen H, Larsen L, Ritz C, Nielsen MK. Daily variability of strongyle fecal egg counts in horses. Journal of Equine Veterinary Science 2013, 33, 161-164.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Andersen UV, Howe DK, Dangoudoubiyam S, Toft N, Reinemeyer CR, Lyons ET, Olsen SN, Monrad J, Nejsum P, Nielsen MK. rSvSXP: A Strongylus vulgaris antigen with potential for prepatent diagnosis. Parasites & Vectors 2013, 6: 84.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Cao X, Vidyashankar AN, Nielsen MK. Association between large strongyle genera in larval cultures  using rare-event Poisson regression. Parasitology 2013, 140, 1246-1251.
• Type: Journal Articles Status: Accepted Year Published: 2013 Citation: horses. Veterinary Parasitology 2013, 197, 614-622. 33. Nielsen MK, Betancourt A, Lyons ET, Horohov DW, Jacobsen S. Characterization of the inflammatory response to anthelmintic treatment in ponies naturally infected with cyathostomin parasites. Veterinary Journal, in press.
• Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Nielsen MK, Reinemeyer CR, Donecker JM, Leathwick DM, Marchiondo AA, Kaplan RM. Anthelmintic resistance in equine parasites - current evidence and knowledge gaps. Veterinary Parasitology, in press.
• Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Nielsen MK, Reist M, Kaplan RM, Pfister K, van Doorn DCK, Becher A. Equine parasite control under prescription-only conditions  awareness, knowledge, perception, and strategies applied. Veterinary Parasitology, in press.

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: Data have been analyzed and an abstract has been submitted for the 2012 Conference of Research Workers in Animal Diseases to be held in Chicago, IL on 12/2-4/2012. The following presentations have been given at scientific conferences: Martin K. Nielsen, Anand N. Vidyashankar, Susanne N. Olsen, Jesper Monrad, Stig M. Thamsborg. Strongylus vulgaris associated with selective therapy on Danish horse farms. Annual meeting, American Association of Veterinary Parasitologists, San Diego, CA, August 4-7, 2012. Ulla V. Andersen, Daniel K. Howe, Susanne N. Olsen, Jesper Monrad, Peter Nejsum, Eugene T. Lyons, Martin K. Nielsen. Serological diagnosis of Strongylus vulgaris infection: use of a recombinant protein. Annual Meeting, American Association of Veterinary Parasitologists, San Diego, CA, August 4-7, 2012. Xin Cao, Anand N. Vidyashankar, Martin K. Nielsen. Evaluating the re-emergence of rare parasites on equine farms. Annual meeting, American Association of Veterinary Parasitologists, San Diego, CA, August 4-7, 2012. Nielsen, M.K. Equine parasitology: the challenge of changing habits. KVMA, Louisville, KY, October 5-7, 2012. Nielsen, M.K. Sustainable Equine Parasite Control: Dos and Don'ts. Equine Infectious Disease Conference, Practitioner Day, Lexington, KY, October 21, 2012. Nielsen, M.K. Vidyashankar, A.N., Olsen, S.N., Monrad, J., Thamsborg, S.M. Selective therapy for strongyle parasite control: Reemergence of Strongylus vulgaris Equine Infectious Disease Conference, Lexington, KY, October 21-26, 2012. Nielsen, M.K., Olsen, S.N., Lyons, E.T., Monrad, J., Thamsborg, S.M. Real-time PCR determination of Strongylus vulgaris in horses on farms with different anthelmintic regimens in Denmark and Central Kentucky. Equine Infectious Disease Conference, Lexington, KY, October 21-26, 2012. Andersen, U.V., Howe, D.K., Olsen, S.N., Monrad, J., Nejsum, P., Lyons, E.T., Nielsen, M.K. Serological diagnosis of Strongylus vulgaris infection. Equine Infectious Disease Conference, Lexington, KY, October 21-26, 2012. Nielsen, M.K. Parasite control in the mare. Hagyard Bluegrass Symposium, Lexington, KY, November 1-4, 2012. Patent application: US Provisional Application "Diagnosis of Strongylus vulgaris" Nielsen, M.K., Andersen, U.V., Howe, D.K. Application serial no. 61/719,762 PARTICIPANTS: PI: Martin Nielsen Collaborators: Eugene T. Lyons, University of Kentucky David W. Horohov, University of Kentucky Dan K. Howe, University of Kentucky Sriveny Dangoudoubiyam, University of Kentucky Stine Jacobsen, University of Copenhagen Students and staff PhD student Ulla Andersen, University of Copenhagen, has worked at University of Kentucky for the past three years. MS student Jennifer Bellaw, University of Kentucky Research analyst Holli Gravatte has worked part time in Dr. Nielsen's laboratory since September 2012. Partner organization: Department of Large Animal Science, University of Copenhagen. Research collaboration regarding Ulla Andersen's project and regarding inflammatory markers (dr. Stine Jacobsen) TARGET AUDIENCES: Target audiences include equine veterinary practitioners, farm managers and horse owners across the world. Outreach activities include several publications in equine magazines such as Equus and The Horse as well as frequent online publication on www.thehorse.com, www.horsetalk.co.nz, and in the monthly e-newsletter Bluegrass Equine Digest. Several online webinars have been delivered to www.thehorse.com. Further, I have recently been appointed chair of a guideline committee within the American Association for Equine Practitioners (AAEP), which is working on a white paper to be published. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
- A diagnostic technique for detecting migrating stages of S. vulgaris has been developed and publications are in preparation. A commercial patent is being pursued, and this assay will become very useful in future studies evaluating equine parasite control programs. - Data generated from the study "Inflammatory reaction to anthelmintic treatment" was included in a recent proposal for the Grayson-Jockey Club. This data set suggested that there are measurable differences of the inflammatory reactions between different drug classes. This may have implication for treatment recommendations given in the future.

Publications

• Bracken MK, Wohlk CBM, Petersen SL, Nielsen MK. Evaluation of conventional PCR for detection of Strongylus vulgaris on horse farms. Veterinary Parasitology 2012, 184, 387-391.
• Nielsen, MK. Equine Parasite Control: perspectives and research needs. Veterinary Parasitology 2012. 185, 32-44.
• Molento MB, Nielsen MK, Kaplan, RM. Avermectin/milbemycin resistance in cyathostomins: current situation. Veterinary Parasitology 2012. 185,16-24.
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• Nielsen MK, Olsen SN, Lyons ET, Monrad J, Thamsborg SM. Real-time PCR evaluation of Strongylus vulgaris in horses on farms in Denmark and Central Kentucky. Veterinary Parasitology 2012, 190, 461-466.
• Andersen UV, Haakansson IT, Roust T, Rhod M, Baptiste KE, Nielsen MK. Developmental stage of strongyle eggs affects the outcome variations of real-time PCR analysis. Veterinary Parasitology 2013,191, 191-196.