Daniel J. Houlihan1, Maria E. Prado 1,2 and Sharon Patton3 Techniques for determining efficacy of different anti-coccidia drugs on the growth and development of the camelid parasite, Eimeria macusaniensis, in cell cultures Daniel J. Houlihan1, Maria E. Prado 1,2 and Sharon Patton3 1Department of Animal Science, 2Department of Large Animal Clinical Sciences and 3Department of Comparative Medicine, College of Veterinary Medicine, The University of Tennessee, Knoxville, TN Abstract Eimeria macusaniensis, a coccidian parasite, continues to be a threat to New World Camelids (NWCs) in the United States because the efficacy of anti-coccidia drugs has not been determined in these species. Currently used treatment protocols for E.macusaniensis infection in llamas and alpacas have been extracted from those used to treat non-camelid species against coccidiosis. The objective of the research project reported here was to optimize various protocols associated with the detection, concentration and sporulation of E. macusaniensis. We successfully induced sporulation of E. macusaniensis under in vitro conditions. In addition, we were able to concentrate E. macusaniensis using a simplified version of the concentration protocol developed for other coccidias. The protocols developed for this project will serve to complete phase two of our research in which infectious sporozoites will be inoculated in cell-cultures in vitro to test the efficacy of various anti-protozoal drugs. Introduction Eimeria macusaniensis is a protozoal parasite of the Phylum Apicomplexa that causes fatal enteritis in NWCs worldwide1. Oocysts, which are shed in the feces of infected NWCs, become infectious to others following sporulation. In contrast to other Eimeria species, E. macusaniensis oocysts are quite large (93.6 μm by 67.4 μm) and once ingested, invade the intestinal cell lining and deeper cells associated with the intestine. Thus, E. macusaniensis is more likely to cause damage which may lead to secondary bacterial and viral infections2. Clinical signs, which appear more often in young or immuno-compromised NWCs include lethargy, anorexia, hemorrhagic diarrhea and sudden death3. Detection of oocysts in the feces of infected animals is not always successful and might go undetected. In addition, there is limited information regarding the life cycle of E. macusaniensis (Fig. 1). Therefore, development of an in vitro system that will help understand the life cycle of this parasite and the efficacy of traditional anti-coccidia drugs is imperative. Figure 1 – Eimeria macusaniensis prospective life cycle. Materials & Methods Detection of E. macusaniensis: Multiple NWCs (approximately 100) in Tennessee and surrounding states were screened for the presence of E. macusaniensis in fecal samples. A local farm had one positive llama, “Larry,” with a pre-existing infection. Feces from “Larry” were collected every day and stored in zip-lock bags at 3-4°C until processed. Fecal floatations were performed per UTCVM’s Parasitology Diagnostic Laboratory protocol using Sheather’s sugar solution (SG 1.275) to float the oocysts. An oocyst count per gram of feces was performed using the Wisconsin-double cover slip method on positive samples for quantification. Concentration of E. macusaniensis oocysts: Several methods were tested to determine which protocol was the most efficacious. The various protocols involved different degrees of filtration combined with dissimilar flotation techniques (both sugar and salt floatations were compared). Briefly, E. macusaniensis positive samples of 600 grams or more were mixed with approximately 1.5 liters of water and filtered through 3 layers of grade #50 cheesecloth and a standard #400 sieve. Sediment from the sieve was then collected, washed and centrifuged at 1500 rpm for 5 minutes. This step was repeated until the sample was clean. The concentrated oocysts were floated to the top using Sheather’s Sugar Solution without preservative (SG 1.275). Image 1: Sporulated E. macusaniensis after 19 days in 4% potassium dichromate Sporulation of E. macusaniensis: Induction of sporulation was tested through various protocols to find the most successful and efficient method. Samples were subjected to different mediums (4% potassium dichromate vs. 2% sulfuric acid) and different incubation temperatures (room temperature vs. 27.3°C). In addition, we used different types of containers (cell culture flasks and glass beakers and/or flasks) with or without continuous airflow. B C D A E Figure 2 – 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 Results & Discussion 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 Center4. In addition, replacing the #400 sieve with a #270 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. It was also found that the method using a concentrated sodium chloride soultion5 was not as efficient in this project as the method described previously. We were able to observe changes occurring to E. macusaniensis occysts at various times during the sporulation process (Figure 2). We successfully sporulated E. macusaniensis (image 1) 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. Conclusions In summary, there is obviously a need to continue research on E. macusaniensis, a NWC parasite, since little information currently exists on its life cycle and effective treatment. This paper, which reviewed and modified protocols needed to concentrate and sporulate E. macusaniensis will serve as a basis for future research projects in which the infectious sporozoites will be inoculated into vero cell cultures for drug testing. The information obtained, combined with the better understanding of the life cycle of E. macusaniensis builds a strong base for future study of this devastating coccidia. Acknowledgements This research was partially supported by a grant from the Chancellor’s Honors Program and CASNR. I would like to acknowledge my advisor Dr. Maria E. Prado as well as Mrs. Aly Chapman, Mrs. Amanda Fanning Widner, Dr. Sharon Patton, Dr. Charles Faulkner and Dr. Craig Reinemeyer for their support and assistance with this project. References Rickard, L.G. 1994 Vet Clin NA Food Animal. Cafrune, M et al. 2009 J Vet Parasitology. Ballweber 2009 Vet Clin NA Food Animal. Dr. Craig Reinemeyer personal communication. Hofmann, J. et al. 1990 Parasitol.