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Sporocyst size of isolates of Sarcocystis shed by the Virginia opossum (Didelphis virginiana)
Veterinary Parasitology 95 (2001) 305–311
Sporocyst size of isolates of Sarcocystis shed by the Virginia opossum (Didelphis virginiana) M.A. Cheadle∗ , J.B. Dame, E.C. Greiner Department of Pathobiology, College of Veterinary Medicine, University of Florida, PO Box 110880, 2015 SW 16th Avenue, Gainesville, FL 32610-0880, USA
Abstract The Virginia opossum (Didelphis virginiana) is a definitive host for multiple Sarcocystis species including Sarcocystis neurona, one of the causative agents of equine protozoal myeloencephalitis (EPM), a severe, neuromuscular disease of horses. Size and morphologic characteristics of isolates of Sarcocystis shed by the opossum were examined to determine if differences were useful in discriminating between the isolates and/or species. Collections of sporocysts from 17 opossums were molecularly characterized and measured using an ocular micrometer. The mean sporocyst size of isolates of S. neurona was 10.7 m × 7.0 m, Sarcocystis falcatula 11.0 m × 7.1 m, Sarcocystis speeri 12.2 m × 8.8 m, 1085-like isolate 10.9 m × 6.8 m, and 3344-like isolate 19.4 m × 10.5 m. The length and width of S. speeri were statistically different (p < 0.05) from the sporocysts of other types. The length of S. neurona and S. falcatula sporocysts were statistically different (p < 0.05) from each other and the width of S. falcatula and 1085 differed (p < 0.05). The fifth sporocyst type (3344) was observed, but due to pronounced morphological characteristics, statistical analysis was not performed. There was no consistent difference between the taxa based on internal structure of the sporocyst. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Sarcocystis; Opossum; Sporocyst; Size
1. Introduction Equine protozoal myeloencephalitis (EPM) is a severe neuromuscular disease affecting many horses in North and South America (Dubey et al., 1991; Granstrom et al., 1992; Masri et al., 1992; MacKay, 1997). While the disease has been diagnosed for several years, only recently have causative agents been identified. Two different organisms have been implicated as causative agents of EPM, Sarcocystis neurona and Neospora hughesi (Dubey et al., 1991; Marsh et al., 1998; Cheadle et al., 1999). While information on N. hughesi is ∗ Corresponding author. Tel.: +1-352-392-4700, ext: 5825; fax: +1-352-392-9704. E-mail address: [email protected] (M.A. Cheadle).
0304-4017/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 0 ) 0 0 3 9 6 - 4
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emerging, the contribution of this organism to EPM is unknown. The main focus of research regarding EPM has been centered on S. neurona. The definitive host of S. neurona, the Virginia opossum (Didelphis virginiana), sheds sporocysts of several types of Sarcocystis spp. in its feces. The current method used to differentiate these organisms is DNA-based marker analysis developed in this laboratory (Tanhauser et al., 1999). In our paper, we assess the potential to differentiate the five types of sporocysts based on light microscopy.
2. Materials and methods 2.1. Sporocyst collection and storage Opossums killed by automobiles on roadways were collected and feces and/or gut scrapings were examined for the presence of Sarcocystis sporocysts using sugar flotation with centrifugation. For samples found to be positive for the presence of sporocysts, intestines were removed and the mucosa was scraped using the edge of a glass slide to collect sporocysts contained within the mucosa. The collection was then placed into a solution containing 50% sodium hypochlorite and water, mixed thoroughly, and incubated on ice for 30 min. After filtration through gauze, the sporocysts were washed by repetitive centrifugation and resuspension in distilled water to remove residual amounts of bleach. Once the bleach was removed, storage media (Hank’s balanced salt solution (500 ml) plus antibiotics (10×104 IU penicillin and 10 × 104 g streptomycin (Mediatech, Herndon, VA)) and fungicide (250 g amphotericin (Mediatech, Herndon, VA)) was placed in the tube containing the sporocyst mixture. The sporocysts were stored at 4◦ C until use. 2.2. Sporocyst typing Sporocyst type was elucidated using restriction enzyme analysis as described by Tanhauser et al. (1999). Sarcocystis speeri was differentiated based upon sequence analysis (Dame, personal communication). 2.3. Observation and measurement Sporocyst isolates used from each taxa are as follows: S. neurona (3027, 3113, 3120, 3063); 1085 type (3013, 1114, 1086, 1085); S. falcatula (3114, 3106, 3105, 3101, 3021); S. speeri (2226, 2079, 2046); 3344 type (3344). Tubes containing stored sporocysts of each isolate were removed from refrigeration and an aliquot of approximately 10 l was placed on the center of a glass slide. To prevent deformation of the sporocysts caused by the weight of the coverslip, four small dots of glycerine jelly (Fisher, Fair Lawn, NJ) were applied to each corner of a glass coverslip. The coverslip was then placed over the 10 l of media forming a hanging drop. The slide was then observed on a light microscope under oil at 1000× magnification. Twenty sporocysts of each isolate from the five taxa were measured using a calibrated ocular micrometer. Sporocysts were observed for differences in internal structure as well.
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Table 1 Comparison of Sarcocystis sporocysts sizes (S.E.: standard error of the mean)a Taxa
Length (m) Mean (S.E.)
S. neurona 1085 type S. falcatula S. speeri 3344 type
(0.09)a
10.65 10.85 (0.07)a,b 10.95 (0.13)b 12.23 (0.14)c 19.43 (0.20)∗
Width (m) Range 9.7–11.4 9.7–11.9 8.8–11.9 11.0–13.2 17.6–20.7
Mean (S.E.) (0.09)a,b
7.03 6.81 (0.07)a 7.05 (0.10)b 8.81 (0.09)c 10.49 (0.20)∗
Range 6.2–8.4 6.2–7.9 6.6–7.9 7.5–9.7 9.2–11.9
a Isolates with the same superscript (a, b, c) within columns designate no significant differences between isolates. ∗ Statistical analysis not performed on this isolate.
2.4. Data analysis Because length and width data was not normally distributed (p < 0.001 by Kolmogorov– Smirnov test for normality), we analyzed our findings by the Kruskal–Wallis one way analysis of variance on rank (p < 0.05). Where a significant difference was found, pairwise multiple comparisons were made using Dunn’s method. All statistical calculations were carried out using SigmaPlot® and SigmaStat® software for windows version 2.03 (SPSS, Illinois).
3. Results Results of sporocyst measurements are presented in Table 1. The length and width of S. speeri (Fig. 1) was statistically different (p < 0.05) from the other sporocyst isolates. The length of S. neurona (Fig. 2) and S. falcatula (Fig. 3) sporocysts were statistically different
Fig. 1. Brightfield micrographs of sporocysts of Sarcocystis spp. shed by opossums. 1. Sporocyst of S. speeri (2079 isolate). Note the sporozoite (arrow). Bar = 6 m.
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M.A. Cheadle et al. / Veterinary Parasitology 95 (2001) 305–311
Fig. 2. Brightfield micrographs of sporocysts of Sarcocystis spp. shed by opossums. 2. Sporocysts of S. neurona (3063 isolate). Note the sporozoite (arrow) and residual body (arrowhead). Bar = 5.5 m.
(p < 0.05) from each other and the width of S. falcatula and 1085 type (Fig. 4.) differed (p < 0.05). Due to the obvious size difference of the 3344 type (Fig. 5) sporocyst, statistical data were not compared between it and the other isolates. This type has been found in the feces of 10 opossums. This sporocyst is morphologically different from other isolates because it is much larger and has more pointed ends with plug-like structures (Fig. 6). Although other sporocyst isolates have a more rounded shape and are devoid of the plug-like structures (Figs. 1–4), they do exhibit a similar refractory quality when observed using light microscopy. Diagnosis
Fig. 3. Brightfield micrographs of sporocysts of Sarcocystis spp. shed by opossums. 3. Sporocyst of S. falcatula (3106 isolate). Note the sporozoites (arrow). Bar = 5.5 m.
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Fig. 4. Brightfield micrographs of sporocysts of Sarcocystis spp. shed by opossums. 4. Sporocysts of 1085 type (3013 isolate). Note the sporozoite (arrow) and residual bodies (arrowhead). Bar = 5 m.
and collection of this sporocyst type was similar to that of the other taxa, however, it was very difficult to store due to the trait of collapsing after collection. As seen in Fig. 7, the sporocyst collapses within minutes of processing as compared to other taxa which maintain wall integrity throughout processing and storage. After collection and storage, intact sporocysts were not observed and thus the isolate has yet to be molecularly characterized. The internal morphology of the sporocysts was observed. All sporocysts contained four sporozoites and a residuum. Although there was no consistent difference between internal morphology of isolates, there appeared to be many forms. Forms ranged from a single, large, ball-like residual body approximately one third the size of the sporocyst to upwards of 20 small, ball-like residual bodies. Multiple, small bodies were found in compact areas
Fig. 5. Brightfield micrographs of sporocysts of Sarcocystis spp. shed by opossums. 5. Sporocyst of 3344 type (3344 isolate). Note the sporozoite (arrow). Bar = 6.6 m.
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Fig. 6. Brightfield micrographs of sporocysts of Sarcocystis spp. shed by opossums. 6. Sporocyst of 3344 type (3344 isolate). Note the polar plug-like structures (open arrowhead), sporozoite (arrow), and residual body (arrowhead). Bar = 6.3 m.
Fig. 7. Brightfield micrograph of 3344 type sprocyst. 7. Collapsed sporocyst of 3344 type (isolate 3395). Photographed 10 min post-flotation. Bar = 6.5 m.
and diffuse throughout the inside of the sporocyst. There did not appear to be any trend that would allow the differentiation of type based on the makeup of the sporocyst residuum.
4. Discussion Based on gross visual observations by light microscopy, only S. speeri and 3344 type sporocysts were different from the other similar isolates found in the feces of the opossum. Single dimension differences such as S. falcatula vs. 1085 (width) and S. falcatula vs.
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S. neurona (length), were not considered sufficient to differentiate between the species/types. The 3344 type of sporocyst was originally thought not to be a sporocyst, but after close examination, four sporozoites and a distinct residuum were observed (Fig. 6). Dubey et al. (1989) did not describe sporocysts of this size being found in the feces of the opossum or any other definitive host. This type composed <5% of infections with sporocysts found in the feces and/or gut scrapings. Because this sporocyst collapses soon after flotation, fecal flotations of this preparation should be read immediately after the flotation procedure is finished. We have been unable to determine why this sporocyst type exhibits this characteristic and whether this type utilizes the opossum as a host or simply is contained within a food item that is being passed through the gut during the time of sampling. The isolates in this study cannot be proven to be pure isolates. There is no way to insure that there is only a single sporocyst species/type present in the primary isolates collected from opossums used in this study. However, based on molecular data showing that the isolate is of one species, the authors feel that if there is a mixed infection in the opossum, the number of sporocysts of the second species/type would be low and should not influence the outcome of this analysis.
Acknowledgements We would like to thank Dr. Susan Tanhauser for her assistance with DNA-based marker analysis and Dr. Charles Courtney for his assistance performing statistical analysis. We would also like to thank Kristin Munsterman, Taj Ryland, Kimberly Baird, Randa Antar, and Melissa Perez Velasco for their help with opossum collection, sporocyst detection, and sporocyst purification. This study was supported by United States Department of Agriculture (USDA) grant No. 98-35204-6487. References Cheadle, M.A., Lindsay, D.S., Rowe, S., Dykstra, C.C., Williams, M.A., Spencer, J.A., Toivio-Kinnucan, M.A., Lenz, S.D., Newton, J.C., Rolsma, M.D., Blagburn, B.L., 1999. Serosurvey of antibodies to Neospora hughesi in horses from Alabama and biological characterization of an isolate recovered from a naturally infected horse. Int. J. Parasitol. 29, 1537–1543. Dubey, J.P., Speer, C.A., Fayer, R., 1989. General biology. In: Sarcocystosis of Animals and Man. CRC Press, Boca Raton, FL, pp. 1–91. Dubey, J.P., Davis, S.W., Speer, C.A., Bowman, D.D., de Lahunta, A., Granstrom, D.E., Topper, M.J., Hamir, A.N., Cummings, J.F., Suter, M.M., 1991. Sarcocystis neurona n. sp. (Protozoa: Apicomplexa), the etiologic agent of equine protozoal myeloencephalitis. J. Parasitol. 77, 212–218. Granstrom, D.E., Alvarez Jr., O., Dubey, J.P., Comer, P.F., Williams, N.M., 1992. Equine protozoal myelitis in Panamanian horses and isolation of Sarcocystis neurona. J. Parasitol. 78, 909–912. MacKay, R.J., 1997. Equine protozoal myeloencephalitis. Vet. Clin. N. Am. Equine Pract. 13, 79–96. Marsh, A.E., Barr, B.C., Packham, A.E., Conrad, P.A., 1998. Description of a new Neospora species (Protozoa: Apicomplexa: Sarcocystidae). J. Parasitol. 84, 983–991. Masri, M.D., Alda, J.L., Dubey, J.P., 1992. Sarcocystis neurona-associated ataxia in horses in Brazil. Vet. Parasitol. 44, 311–314. Tanhauser, S.M., Yowell, C.A., Cutler, T.J., Greiner, E.C., Mackay, R.J., Dame, J.B., 1999. Multiple DNA markers differentiate Sarcocystis neurona and Sarcocystis falcatula. J. Parasitol. 85, 221–228.
Sporocyst size of isolates of Sarcocystis shed by the Virginia opossum (Didelphis virginiana) M.A. Cheadle∗ , J.B. Dame, E.C. Greiner Department of Pathobiology, College of Veterinary Medicine, University of Florida, PO Box 110880, 2015 SW 16th Avenue, Gainesville, FL 32610-0880, USA
Abstract The Virginia opossum (Didelphis virginiana) is a definitive host for multiple Sarcocystis species including Sarcocystis neurona, one of the causative agents of equine protozoal myeloencephalitis (EPM), a severe, neuromuscular disease of horses. Size and morphologic characteristics of isolates of Sarcocystis shed by the opossum were examined to determine if differences were useful in discriminating between the isolates and/or species. Collections of sporocysts from 17 opossums were molecularly characterized and measured using an ocular micrometer. The mean sporocyst size of isolates of S. neurona was 10.7 m × 7.0 m, Sarcocystis falcatula 11.0 m × 7.1 m, Sarcocystis speeri 12.2 m × 8.8 m, 1085-like isolate 10.9 m × 6.8 m, and 3344-like isolate 19.4 m × 10.5 m. The length and width of S. speeri were statistically different (p < 0.05) from the sporocysts of other types. The length of S. neurona and S. falcatula sporocysts were statistically different (p < 0.05) from each other and the width of S. falcatula and 1085 differed (p < 0.05). The fifth sporocyst type (3344) was observed, but due to pronounced morphological characteristics, statistical analysis was not performed. There was no consistent difference between the taxa based on internal structure of the sporocyst. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Sarcocystis; Opossum; Sporocyst; Size
1. Introduction Equine protozoal myeloencephalitis (EPM) is a severe neuromuscular disease affecting many horses in North and South America (Dubey et al., 1991; Granstrom et al., 1992; Masri et al., 1992; MacKay, 1997). While the disease has been diagnosed for several years, only recently have causative agents been identified. Two different organisms have been implicated as causative agents of EPM, Sarcocystis neurona and Neospora hughesi (Dubey et al., 1991; Marsh et al., 1998; Cheadle et al., 1999). While information on N. hughesi is ∗ Corresponding author. Tel.: +1-352-392-4700, ext: 5825; fax: +1-352-392-9704. E-mail address: [email protected] (M.A. Cheadle).
0304-4017/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 0 ) 0 0 3 9 6 - 4
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M.A. Cheadle et al. / Veterinary Parasitology 95 (2001) 305–311
emerging, the contribution of this organism to EPM is unknown. The main focus of research regarding EPM has been centered on S. neurona. The definitive host of S. neurona, the Virginia opossum (Didelphis virginiana), sheds sporocysts of several types of Sarcocystis spp. in its feces. The current method used to differentiate these organisms is DNA-based marker analysis developed in this laboratory (Tanhauser et al., 1999). In our paper, we assess the potential to differentiate the five types of sporocysts based on light microscopy.
2. Materials and methods 2.1. Sporocyst collection and storage Opossums killed by automobiles on roadways were collected and feces and/or gut scrapings were examined for the presence of Sarcocystis sporocysts using sugar flotation with centrifugation. For samples found to be positive for the presence of sporocysts, intestines were removed and the mucosa was scraped using the edge of a glass slide to collect sporocysts contained within the mucosa. The collection was then placed into a solution containing 50% sodium hypochlorite and water, mixed thoroughly, and incubated on ice for 30 min. After filtration through gauze, the sporocysts were washed by repetitive centrifugation and resuspension in distilled water to remove residual amounts of bleach. Once the bleach was removed, storage media (Hank’s balanced salt solution (500 ml) plus antibiotics (10×104 IU penicillin and 10 × 104 g streptomycin (Mediatech, Herndon, VA)) and fungicide (250 g amphotericin (Mediatech, Herndon, VA)) was placed in the tube containing the sporocyst mixture. The sporocysts were stored at 4◦ C until use. 2.2. Sporocyst typing Sporocyst type was elucidated using restriction enzyme analysis as described by Tanhauser et al. (1999). Sarcocystis speeri was differentiated based upon sequence analysis (Dame, personal communication). 2.3. Observation and measurement Sporocyst isolates used from each taxa are as follows: S. neurona (3027, 3113, 3120, 3063); 1085 type (3013, 1114, 1086, 1085); S. falcatula (3114, 3106, 3105, 3101, 3021); S. speeri (2226, 2079, 2046); 3344 type (3344). Tubes containing stored sporocysts of each isolate were removed from refrigeration and an aliquot of approximately 10 l was placed on the center of a glass slide. To prevent deformation of the sporocysts caused by the weight of the coverslip, four small dots of glycerine jelly (Fisher, Fair Lawn, NJ) were applied to each corner of a glass coverslip. The coverslip was then placed over the 10 l of media forming a hanging drop. The slide was then observed on a light microscope under oil at 1000× magnification. Twenty sporocysts of each isolate from the five taxa were measured using a calibrated ocular micrometer. Sporocysts were observed for differences in internal structure as well.
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Table 1 Comparison of Sarcocystis sporocysts sizes (S.E.: standard error of the mean)a Taxa
Length (m) Mean (S.E.)
S. neurona 1085 type S. falcatula S. speeri 3344 type
(0.09)a
10.65 10.85 (0.07)a,b 10.95 (0.13)b 12.23 (0.14)c 19.43 (0.20)∗
Width (m) Range 9.7–11.4 9.7–11.9 8.8–11.9 11.0–13.2 17.6–20.7
Mean (S.E.) (0.09)a,b
7.03 6.81 (0.07)a 7.05 (0.10)b 8.81 (0.09)c 10.49 (0.20)∗
Range 6.2–8.4 6.2–7.9 6.6–7.9 7.5–9.7 9.2–11.9
a Isolates with the same superscript (a, b, c) within columns designate no significant differences between isolates. ∗ Statistical analysis not performed on this isolate.
2.4. Data analysis Because length and width data was not normally distributed (p < 0.001 by Kolmogorov– Smirnov test for normality), we analyzed our findings by the Kruskal–Wallis one way analysis of variance on rank (p < 0.05). Where a significant difference was found, pairwise multiple comparisons were made using Dunn’s method. All statistical calculations were carried out using SigmaPlot® and SigmaStat® software for windows version 2.03 (SPSS, Illinois).
3. Results Results of sporocyst measurements are presented in Table 1. The length and width of S. speeri (Fig. 1) was statistically different (p < 0.05) from the other sporocyst isolates. The length of S. neurona (Fig. 2) and S. falcatula (Fig. 3) sporocysts were statistically different
Fig. 1. Brightfield micrographs of sporocysts of Sarcocystis spp. shed by opossums. 1. Sporocyst of S. speeri (2079 isolate). Note the sporozoite (arrow). Bar = 6 m.
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M.A. Cheadle et al. / Veterinary Parasitology 95 (2001) 305–311
Fig. 2. Brightfield micrographs of sporocysts of Sarcocystis spp. shed by opossums. 2. Sporocysts of S. neurona (3063 isolate). Note the sporozoite (arrow) and residual body (arrowhead). Bar = 5.5 m.
(p < 0.05) from each other and the width of S. falcatula and 1085 type (Fig. 4.) differed (p < 0.05). Due to the obvious size difference of the 3344 type (Fig. 5) sporocyst, statistical data were not compared between it and the other isolates. This type has been found in the feces of 10 opossums. This sporocyst is morphologically different from other isolates because it is much larger and has more pointed ends with plug-like structures (Fig. 6). Although other sporocyst isolates have a more rounded shape and are devoid of the plug-like structures (Figs. 1–4), they do exhibit a similar refractory quality when observed using light microscopy. Diagnosis
Fig. 3. Brightfield micrographs of sporocysts of Sarcocystis spp. shed by opossums. 3. Sporocyst of S. falcatula (3106 isolate). Note the sporozoites (arrow). Bar = 5.5 m.
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Fig. 4. Brightfield micrographs of sporocysts of Sarcocystis spp. shed by opossums. 4. Sporocysts of 1085 type (3013 isolate). Note the sporozoite (arrow) and residual bodies (arrowhead). Bar = 5 m.
and collection of this sporocyst type was similar to that of the other taxa, however, it was very difficult to store due to the trait of collapsing after collection. As seen in Fig. 7, the sporocyst collapses within minutes of processing as compared to other taxa which maintain wall integrity throughout processing and storage. After collection and storage, intact sporocysts were not observed and thus the isolate has yet to be molecularly characterized. The internal morphology of the sporocysts was observed. All sporocysts contained four sporozoites and a residuum. Although there was no consistent difference between internal morphology of isolates, there appeared to be many forms. Forms ranged from a single, large, ball-like residual body approximately one third the size of the sporocyst to upwards of 20 small, ball-like residual bodies. Multiple, small bodies were found in compact areas
Fig. 5. Brightfield micrographs of sporocysts of Sarcocystis spp. shed by opossums. 5. Sporocyst of 3344 type (3344 isolate). Note the sporozoite (arrow). Bar = 6.6 m.
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M.A. Cheadle et al. / Veterinary Parasitology 95 (2001) 305–311
Fig. 6. Brightfield micrographs of sporocysts of Sarcocystis spp. shed by opossums. 6. Sporocyst of 3344 type (3344 isolate). Note the polar plug-like structures (open arrowhead), sporozoite (arrow), and residual body (arrowhead). Bar = 6.3 m.
Fig. 7. Brightfield micrograph of 3344 type sprocyst. 7. Collapsed sporocyst of 3344 type (isolate 3395). Photographed 10 min post-flotation. Bar = 6.5 m.
and diffuse throughout the inside of the sporocyst. There did not appear to be any trend that would allow the differentiation of type based on the makeup of the sporocyst residuum.
4. Discussion Based on gross visual observations by light microscopy, only S. speeri and 3344 type sporocysts were different from the other similar isolates found in the feces of the opossum. Single dimension differences such as S. falcatula vs. 1085 (width) and S. falcatula vs.
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S. neurona (length), were not considered sufficient to differentiate between the species/types. The 3344 type of sporocyst was originally thought not to be a sporocyst, but after close examination, four sporozoites and a distinct residuum were observed (Fig. 6). Dubey et al. (1989) did not describe sporocysts of this size being found in the feces of the opossum or any other definitive host. This type composed <5% of infections with sporocysts found in the feces and/or gut scrapings. Because this sporocyst collapses soon after flotation, fecal flotations of this preparation should be read immediately after the flotation procedure is finished. We have been unable to determine why this sporocyst type exhibits this characteristic and whether this type utilizes the opossum as a host or simply is contained within a food item that is being passed through the gut during the time of sampling. The isolates in this study cannot be proven to be pure isolates. There is no way to insure that there is only a single sporocyst species/type present in the primary isolates collected from opossums used in this study. However, based on molecular data showing that the isolate is of one species, the authors feel that if there is a mixed infection in the opossum, the number of sporocysts of the second species/type would be low and should not influence the outcome of this analysis.
Acknowledgements We would like to thank Dr. Susan Tanhauser for her assistance with DNA-based marker analysis and Dr. Charles Courtney for his assistance performing statistical analysis. We would also like to thank Kristin Munsterman, Taj Ryland, Kimberly Baird, Randa Antar, and Melissa Perez Velasco for their help with opossum collection, sporocyst detection, and sporocyst purification. This study was supported by United States Department of Agriculture (USDA) grant No. 98-35204-6487. References Cheadle, M.A., Lindsay, D.S., Rowe, S., Dykstra, C.C., Williams, M.A., Spencer, J.A., Toivio-Kinnucan, M.A., Lenz, S.D., Newton, J.C., Rolsma, M.D., Blagburn, B.L., 1999. Serosurvey of antibodies to Neospora hughesi in horses from Alabama and biological characterization of an isolate recovered from a naturally infected horse. Int. J. Parasitol. 29, 1537–1543. Dubey, J.P., Speer, C.A., Fayer, R., 1989. General biology. In: Sarcocystosis of Animals and Man. CRC Press, Boca Raton, FL, pp. 1–91. Dubey, J.P., Davis, S.W., Speer, C.A., Bowman, D.D., de Lahunta, A., Granstrom, D.E., Topper, M.J., Hamir, A.N., Cummings, J.F., Suter, M.M., 1991. Sarcocystis neurona n. sp. (Protozoa: Apicomplexa), the etiologic agent of equine protozoal myeloencephalitis. J. Parasitol. 77, 212–218. Granstrom, D.E., Alvarez Jr., O., Dubey, J.P., Comer, P.F., Williams, N.M., 1992. Equine protozoal myelitis in Panamanian horses and isolation of Sarcocystis neurona. J. Parasitol. 78, 909–912. MacKay, R.J., 1997. Equine protozoal myeloencephalitis. Vet. Clin. N. Am. Equine Pract. 13, 79–96. Marsh, A.E., Barr, B.C., Packham, A.E., Conrad, P.A., 1998. Description of a new Neospora species (Protozoa: Apicomplexa: Sarcocystidae). J. Parasitol. 84, 983–991. Masri, M.D., Alda, J.L., Dubey, J.P., 1992. Sarcocystis neurona-associated ataxia in horses in Brazil. Vet. Parasitol. 44, 311–314. Tanhauser, S.M., Yowell, C.A., Cutler, T.J., Greiner, E.C., Mackay, R.J., Dame, J.B., 1999. Multiple DNA markers differentiate Sarcocystis neurona and Sarcocystis falcatula. J. Parasitol. 85, 221–228.