1. Daniel Houlihan Project Mentor: Dr. Maria Prado

2.  This project explored the procedures necessary to locate, concentrate and sporulate Eimeria macusaniensis in a laboratory setting  Eimeria macusaniensis (E.mac) is a dangerous coccidial parasite that affects New World Camelids (NWCs)  Since there are currently no proven methods of treatment for coccidiosis caused by E.mac, the protocols developed in this project will be used to test the efficacy of anti-coccidial drugs in a future research project

3.  Find an infected animal to shed E.mac for use in the lab ▪ Must have E.mac to work with it!  Amend protocols used to locate, concentrate and sporulate E.mac  Sporulate E.mac in a laboratory setting  Observe the life cycle during the sporulation process  Establish efficient protocols for later use in research with E.mac

4.  E.mac is a protozoal parasite of the Phylum Apicomplexa that causes fatal enteritis in New World camelids (Rickard 1994; Lenghaus, O’Callaghan et al. 2004; Cebra, Valentine et al. 2007)  It is a coccidial parasite which causes an intracellular infestation of the cells lining the small intestine  E.mac may measure up to twice the size of other NWC specific Eimeria, measuring 93.6 µm by 67.4 µm compared to a range of 30-60 µm by 30-40 µm of other Eimeria animals (Cafrune et al. 2009)  The large size of E.mac compared to other Eimeria causes substantial damage to the cell lining of the intestine Figure : E.macusaniensis compared to a typical gastro-intestinal nematode egg (GIN)

5.  Because of its large size, E.mac increases the chances of secondary bacterial and viral infections of the small intestine  Coccidiosis from E.mac can lead to lethargy, anorexia, weight loss, hemorrhagic diarrhea and sudden death (Ballweber 2009)  Typically, healthy adults will not demonstrate clinical signs. However, young, elderly, or immuno-compromised animals can be greatly harmed by this parasite

6.  Due to the destructive nature of intracellular parasites, the animal may demonstrate clinical signs prior to oocysts being found in the feces  Often, the number of occysts found in feces is not correlated to the severity of the disease  Animals can exhibit clinical signs with very few oocysts found in feces and vise versa

7.  Oocycts are shed by infected animals  Oocysts sporulate in the environment to become infectious (approximately 30 days)  Infectious sporozoites (found in sproblasts) are ingested  Infectious sporozoites invade host cells and form merozoites through an internal fission process called schizogony  Oocysts are released by rupture of the host cell and pass into the feces (Bowman) Figure 1 – Eimeria macusaniensis prospective life cycle.

8.  Locate E.mac  Find efficient method for concentrating the sample down  Find efficient method for sporulation of oocysts

9.  Initially, fecal samples were collected from local herd owners (in Virginia and Tennessee (Kinser and Dreamland Farms) over the years 2008-2009  Samples were shipped chilled (4°C ) to the University of Tennessee College of Veterinary Medicine Parasitology Diagnostics Laboratory  Samples were stored at similar conditions  Fecal floats were preformed per Parasitology Diagnostics protocol using Sheather’s Sugar Solution (SG 1.275) to test for the presence of E.mac  Results  Only a few cases of E.mac were found  Those that were found, quickly treated their animals, limiting the amount of collectable E.mac for research purposes  This quickly became one of the largest hurdles of the project- must have E.mac to work with it

10. With grant money, we were able to obtain two llamas Larry “Como se llama” (white) and Peter “M Nyte Llamalan” (brown) Both were housed at the East Tennessee Clinical Research Center under Dr. Craig Reinemeyer Upon fecal examinations, it was found that Larry (pictured left) had a naturally occurring E.mac infection Samples were collected daily and stored at (4°C) to be used for the project

11. Collecting initial samples from both Larry and Peter

12.  Due to the little amount of research available for Eimeria macusaniensis, many of the protocols had to be amended from other species of Eimeria  Protocols for both concentrating the parasite down and sporulation were changed

13.  E.mac must first be concentrated down from large quantities of feces to be able to work with it  Various methods of concentration were looked at  Different sized sieves were used (Cheese Cloth, #80, #400, and #330) as well as different solutions to float the feces (Sheather’s Sugar with no preservative SG 1.275 and concentrated Sodium Chloride SG 1.195)

14. Feces must first be mixed with water to form a homologous solution Typically 600-750 grams were mixed with 1.5 liters of luke-warm water

15. Solution was then filtered through various sieves to filter out some of the larger sediment

16.  Sediment was then collected from the #400 Sieve (38 µm) knowing that E.mac was too large to filter through the mesh  Sediment was then washed via centrifugation (1500 rpm for 5 minutes)  Oocysts were floated to the top and collected (using both Sheather’s Sugar Solution (SG 1.275) and Concentrated Saline Solution (SG 1.195)

17.  Once concentrated, the oocysts must be sporulated to become infectious  The sporulation process was attempted in two different solutions (2% Sulfuric acid and 4% Potassium Dichromate) at two different temperatures (room temperature and incubated at 27.3 °C )

18. Sporulation process in 4% potassium dicrhomate

19.  Over the course of sporulation (which took approximately 19-21 days), the process involved in sporulation was closely monitored  Pictures were taken on various days to observe the changing oocysts as they went through the sporulation process (to become infectious)

21. We successfully optimized different protocols that will aid in detection, concentration and sporulation of E.macusaniensis in vitro We successfully optimized different protocols that will aid in detection, concentration and sporulation of E.macusaniensis in vitro Among the protocols used to concentrate E.macusaniensis oocysts, we found that the most successful protocol for concentrating oocysts was a modified version from East Tennessee Clinical Research Center 5 Among the protocols used to concentrate E.macusaniensis oocysts, we found that the most successful protocol for concentrating oocysts was a modified version from East Tennessee Clinical Research Center 5 In addition, replacing the #400 sieve with a #270/#330 sieve will decrease the time required to run the sample through In addition, replacing the #400 sieve with a #270/#330 sieve will decrease the time required to run the sample through Similarly, allowing the concentrated oocysts to float overnight in Sheather’s Sugar Solution without preservative increases collection rates from approximately 45% to 90% of estimated oocysts Similarly, allowing the concentrated oocysts to float overnight in Sheather’s Sugar Solution without preservative increases collection rates from approximately 45% to 90% of estimated oocysts It was also found that the method using a concentrated sodium chloride soultion was not as efficient in this project as the method described previously It was also found that the method using a concentrated sodium chloride soultion was not as efficient in this project as the method described previously

22. We successfully sporulated E.macusaniensis in both 2% sulfuric acid and 4% potassium dichromate. The most efficient method included a flask containing 4% potassium dichromate with a constant air supply provided by a common fish aquarium pump which led to sporulation in 19 days We successfully sporulated E.macusaniensis in both 2% sulfuric acid and 4% potassium dichromate. The most efficient method included a flask containing 4% potassium dichromate with a constant air supply provided by a common fish aquarium pump which led to sporulation in 19 days

23. Sporulated E.macusaniensis after 19 days in 4% potassium dichromate

24. We were able to observe changes occurring to E.macusaniensis occysts at various times during the sporulation process (Figure on next slide) We were able to observe changes occurring to E.macusaniensis occysts at various times during the sporulation process (Figure on next slide)

25. B C D A E Changes during sporulation of E.macusaniensis oocysts. A) “Larry” sheds oocysts, B) Unsporulated E.macusaniensis oocyst, C) Oocyst demonstrating signs of invagination after 7 days in potassium dichromate, D) Oocyst showing 4 distinct sporoblasts after day 9 of sporulation and E) fully sporulated E.macusaniensis in the infectious form at day 19 of sporulation. At this point, if ingested, it is likely to cause infection

26.  This project has laid the foundation for efficacy testing of various drugs on the destruction of E.mac  Using the protocols developed in this project, sporulated E.mac will be used to inoculate vero cell cultures  The cultures will then be subjected to varying amounts of anti-coccidial drugs to demonstrate proper dosing amounts and success of each drug in NWCs on the destruction of E.mac  By following the protocols laid out in this project, one will be able to locate, concentrate and sporulate E.macusaniensis in an efficient and timely manner for use in further research and study of this coccida

27.  A special thanks Dr. Maria Prado for mentoring and overseeing this research  To Dr. Sharon Patton for use of her lab and wisdom  Thanks to Aly Chapman and Amanda Fanning Widner for all of their supportive help  To Dr. Craig Reinemeyer at the East Tennessee Clinical Research Center for care and housing of Peter “M. Nyte Llamalan” and Larry “Como se Llama”  To CASNR and Chancellor’s Honors Program for funding this project

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