1. 2 3 4 1 A 1 2 3 4 B Study Design During March-July 2008 we examination fecal samples of sheltered dogs from different locations in Nebraska. Animal shelters were chosen based on distinct precipitation zones in the state. The shelters that were chosen were: 1) North Platte Animal Shelter (Lincoln Co.); 2) Kearney Area Humane Society (Buffalo Co.); 3) Capital Humane Society in Lincoln (Lancaster Co.); and 4) Beatrice Humane Society (Gage Co) (Fig. 1). Additionally, our data was compared to similar studies in 3 different locations in North America that differ in their average yearly precipitation to each other and Nebraska (Fig 1) These location included: 1) New Mexico; 3) Wisconsin; and 4) New Jersey (Fig. 1). Companion animals such as dogs are important reservoirs of a number of human zoonotic protozoan and helminth parasites. However, environmental conditions such as precipitation, soil moisture and temperature directly influence free living life cycle stages of different types of parasites. Nebraska is an ideal location to study parasitism throughout different environment conditions across the state. Average yearly precipitation varies from 28-34 inches in the eastern part of the state, to 20-24 inches in the central part of the state, and 16-18 inches in the western part of the state. In this study we document the first survey of intestinal parasites of sheltered dogs in the state of Nebraska, and examine the role of precipitation, dog age and sex on the distribution of intestinal protozoan and helminth parasites of dogs in Nebraska. Additionally, we compare our data to similar studies in other locations in North America that vary in precipitation. Ashlee Hartman* and Matthew Bolek, *Department of Biology, University of Nebraska at Kearney, Kearney, NE 68849; and Department of Zoology, Oklahoma State University, Stillwater, OK, 74078 ABSTRACT INTRODUCTION MATERIAL AND METHODS ACKNOWLEDGMENTS We would like to thank members of the Kearney Area Humane Society, North Platte Animal Shelter, Capital Humane Society, and Beatrice Humane Society for their efforts to help collect specimens. Without their cooperation, this project would not have been possible. We would also like to thank the Department of Biology at UNK for use of facilities. Last, we would like to thank Heather Tracy for helping us obtain samples from North Platte Animal Shelter. Most parasites use multiple hosts throughout their life cycle and the conditions outside of their internal environment must be adequate for their survival and development. We examined dog fecal samples from four animal shelters in different areas of Nebraska that differ in their average annual precipitation to evaluate the effects of precipitation on parasite recruitment. We found six species of protozoan and helminth parasites infecting sheltered dogs from Nebraska including: Cystoisospora canis, Toxocara canis, Toxocara leonine, Trichuris vulpis, Ancylostoma caninum, and Taenia type eggs. We did not find any statistically significant difference in individual and overall parasite prevalence or species richness among dogs sampled from different locations in the state. However we did find a statistically significant difference in overall parasite prevalence and parasite species richness when comparing our data to other locations across the United States. Both overall parasite prevalence and species richness increased from west to east with average annual precipitation across the United States. Our data suggests that either the precipitation gradient in Nebraska is not large enough to effect parasite development in the external environment or other factors may be important in effecting parasite development in the external environment. Collections Fresh fecal samples (1-4 hrs old) were collected from individual untreated dogs. Dog location, sex and age was recorded for each animal and all samples were stored on ice or in the refrigerator in individual Ziplock plastic bags before processing. Dogs less then 1 year old were classified as puppies, whereas dogs 1 year or older were classified as adults. Fecal Processing and Parasite Identification Approximately 5 g of feces from each dog along with 20 ml Sheather’s sugar solution (specific gravity 1.30) was placed in individual Dixie cups, and mixed thoroughly with an applicator stick. Samples were strained through cheesecloth into 15 ml test tubes and centrifuged at 1,500 rpm for 10 min. Slides were scanned on a compound microscope at 100x magnification for protozoan cysts and helminth eggs and larvae. All slides were scanned three times to minimize the possibility of missing low infections. Data Analysis Overall and individual parasite prevalence and mean species richness was calculated for location, age and sex of dogs from Nebraska, whereas overall prevalence and species richness was calculated from the published literature for different locations in North America. The chi-square test for independence was calculated to compare differences in overall and individual parasite prevalence among different locations, and age and sex of dogs. Because variances were heteroscedastic, the Kruskal–Wallis test and the Kolmogorov–Smirnov 2-sample test were used to compare differences in parasite species richness among different locations in Nebraska and North America. FIGURE 1. Precipitation maps of Nebraska and North America and location of shelters where fecal samples were obtained. (A) 1) North Platte, Lincoln Co., 2) Kearney Buffalo C., 3) Lincoln, Lancaster Co., and 4) Beatrice, Gage Co. (B) 1) New Mexico, 2) Nebraska (pooled data), 3) Wisconsin, and 4) New Jersey. Maps from USDA-NRCS National Water and Climate Center RESULTS Of the 57 dogs examined, 11 (19.3%) were infected with six intestinal parasites including 1 coccidian, 4 nematodes, and 1 tapeworm species (Fig 2). Individual parasite species prevalence was low ranging from 0-17% (Table I). The overall mean species richness was 0.21 + 0.45. Forty six dogs had 0 parasite species, 10 dogs had 1 parasite species and 1 dog had 2 parasites species. There were no statistically significant differences in overall or individual parasite prevalence or species richness among different locations, or age and sex of dogs from Nebraska (P > 0.05). However, statistically significant differences were observed in overall prevalence and mean parasite species richness among intestinal protozoan and helminth parasites from dogs examined from New Mexico, Nebraska, Wisconsin and New Jersey (χ2 = 236.71; P < 0.0001; H corrected = 269.569; P < 0.0001; Fig. 3). BeatriceLincolnKearneyN. PlatteTotal Cystoisospora canis 0% (0/6)0 %(0/20)5%(1/20)0 %(0/11)1.75%(1/57) Toxocara canis0% (0/6)0 %(0/20)15%(3/20)0 %(0/11)5.26%(3/57) Toxascaris leonine 0% (0/6)0 %(0/20) 9% (1/11)1.75%(1/57) Trichuris vulpis0% (0/6)5%(1/20)10%(2/20)0 %(0/11)5.26%(3/57) Ancylostoma caninum 17% (1/6)0 %(0/20) 9% (1/11)3.50%(2/57) Taenia type eggs0% (0/6)0 %(0/20)5%(1/20)9% (1/11)3.50%(2/57) Total17% (1/6)5%(1/20)30%(6/20)27%(3/11)19.30%(11/57) Table I. Prevalence (No. infected/No. examined) of intestinal parasites in shelter dogs from four locations in Nebraska. The Effects of Precipitation Zones on the Occurrence of Parasitism in Dogs at Different Locations in Nebraska and North America FIGURE 3. (A) Overall prevalence (given as %) of intestinal protozoan and helminths parasites from dogs in the United States surveyed by flotation technique. (B) Mean species richness + 1SD of intestinal protozoan and helminth parasites from dogs in the United States surveyed by flotation techniques. The states represented are New Mexico, Nebraska, Wisconsin, and New Jersey (from left to right). Kolmogorov-Smirnov 2-sample test: a from b, P < 0.05; a from c, and b from c, all P < 0.0001. FIGURE 2. Photomicrographs of eggs and cysts of intestinal helminth and protozoan parasites recovered from shelter dogs from four locations in Nebraska. A) Trichuris vulpis egg. Scale bar = 38 µm. B) Toxascaris leonina egg. Scale bar = 40 µm. C) Toxocara canis egg. Scale bar = 40 µm. D) Ancylostoma caninum egg. Scale bar = 30 µm. E) Cystoisospora canis oocyst. Scale bar = 18 µm. F) Taenia type egg. Scale bar = 18.5 µm. A BC DEF Ave. Species Richness + 1SD Overall Prevalence (%) A B χ 2 = 236.71; P < 0.0001 H corrected = 269.569; P < 0.0001 a a b c