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Diclazuril preventive therapy of gamma interferon knockout mice fed Sarcocystis neurona sporocysts
Veterinary Parasitology 94 (2001) 257–264
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Diclazuril preventive therapy of gamma interferon knockout mice fed Sarcocystis neurona sporocysts J.P. Dubey a,∗ , D. Fritz b , D.S. Lindsay c , S.K. Shen a , O.C.H. Kwok a , K.C. Thompson d a United States Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, Parasite Biology, Epidemiology and Systematics Laboratory, Beltsville Agricultural Research Center, Building 1001, Beltsville, MD 20705-2305, USA b Pathology Division, USAMRIID, 1425 Porter Street, Ft. Detrick, MD 21702, USA c Center of Molecular and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, 1410 Prices Fork Road, Blacksburg, VA 24061-0342, USA d Schering-Plough Animal Health Corp., Pharmaceutical Research, 1095 Morris Avenue, Union, NJ 07083, USA
Received 23 August 2000; accepted 20 September 2000
Abstract Gamma interferon knockout (KO) mice (n = 74) were fed a lethal dose of ∼1000 sporocysts of the SN15-OP isolate of Sarcocystis neurona. Groups of mice were given pelleted rodent feed containing 50 ppm of diclazuril at different times before and after feeding sporocysts. All mice were examined at necropsy and their tissues were examined immunohistochemically for S. neurona infection. Twenty mice were fed sporocysts and given diclazuril starting 5 days before feeding sporocysts and continuing 30–39 days post-infection (p.i.). One mouse died of causes unrelated to S. neurona with no demonstrable parasites; the remaining 19 mice remained clinically normal and S. neurona organisms were not found in their tissues. Sarcocystis neurona organisms were not demonstrable by bioassay of the brains of these 19 mice in uninfected KO mice. Sarcocystis neurona organisms were not found in tissues of five mice treated with diclazuril, starting 7 days after feeding sporocysts and continuing up to 39 days p.i. Therapy was less efficient when diclazuril was given 10 days p.i. Sarcocystis neurona organisms were found in two of 19 mice treated with diclazuril starting 10 days after feeding sporocysts, in two of five mice starting therapy 12 days p.i., and in 10 of 10 mice when treatment was delayed until 15 days p.i. All 15 mice fed S. neurona, but not given diclazuril, developed neural sarcocystosis and were euthanized 22–30 days after feeding
∗ Corresponding author. Tel.: +1-301-504-8128; fax: +1-301-504-9222. E-mail address: [email protected] (J.P. Dubey).
0304-4017/01/$ – see front matter Published by Elsevier Science B.V. PII: S 0 3 0 4 - 4 0 1 7 ( 0 0 ) 0 0 3 7 6 - 9
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sporocysts. Six mice not fed S. neurona, but given diclazuril for 44 days, remained clinically normal. Results indicate that diclazuril can kill the early stages of S. neurona. Published by Elsevier Science B.V. Keywords: Sarcocystis neurona; Sporocysts; Oral; Equine protozoal myeloencephalitis; EPM; Knockout mice; Diclazuril; Treatment
1. Introduction Equine protozoal myeloencephalitis (EPM) is the most commonly diagnosed neurologic disorder in horses in the Americas and Sarcocystis neurona is the most common cause of EPM (Dubey et al., 1991; MacKay et al., 1992; Dubey et al., 2001). To date, there is no reproducible method to induce clinical EPM in horses with demonstrable parasites, and only a small percentage of naturally infected horses develop clinical EPM (Dubey et al., 2001). The life cycle of S. neurona is not completely known. Opossums (Didelphis virginiana) are the definitive hosts and horses are considered an aberrant intermediate host. The natural intermediate host, harboring the sarcocyst stage, is unknown. Recently, gamma interferon knockout (KO) mice were found to be susceptible to S. neurona infection; KO mice fed sporocysts developed neurologic disease, similar to EPM in horses, and S. neurona was demonstrated in their tissues (Dubey and Lindsay, 1998). Although several anti-protozoal drugs have been used empirically to treat EPM there is no reliable method to evaluate therapy for EPM in animals. Diclazuril (C17 H9 Cl3 N4 O2 ) is a triazine derivative anticoccidial (Clinacox, ScheringPlough Animal Health Corporation, Union, NJ, USA) approved in the US for use in broiler chickens. It has anti-protozoal activity in vivo and in vitro against Toxoplasma gondii (Lindsay and Blagburn, 1994) and in vitro against N. caninum and S. neurona (Lindsay et al., 1995; Lindsay and Dubey, 2000, 2001). It has been used to treat EPM in horses (Granstrom et al., 1997; Dirikolu et al., 1999). In the present paper, we report anti-S. neurona activity of diclazuril in KO mice fed S. neurona sporocysts. 2. Materials and methods 2.1. Sarcocystis neurona Sarcocystis neurona (isolate SN15-OP) sporocysts were obtained from intestinal scrapings of an opossum (no. 9108) as described by Dubey (2000). This isolate was selected for the present study because it was used previously to study migration and development of S. neurona in KO mice (Dubey, 2001). There are sporocysts from three or more species of Sarcocystis in opossum feces (Dubey and Lindsay, 1999). Although S. falcatula sporocysts were present in this opossum, S. falcatula is not infective to KO mice; S. speeri sporocysts were not present in opossum 9108 (Dubey, 2000). The KO mice used in the present study were bred in-house from stock mice obtained from the Jackson Laboratory, Bar Harbor, Maine, USA. Most mice were males, weighed 15–20 g and were 5–8 weeks old at the time of infection. Ten-fold dilutions (10−1 –10−6 ) of sporocysts from stock inoculum were made
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in antibiotic saline and stored at 4◦ C as described by Dubey (2001). A 10−6 dilution was not infective to two mice, a 10−5 dilution of the inoculum was infective to three of 10 mice, and a 10−4 dilution was infective to two of two mice (Dubey, 2001). In the present study, a 10−2 dilution of the inoculum was fed to each KO mice. Thus, there were between 100 and 1000 mouse infective sporocysts in the inoculum given to each mouse. Mice were fed sporocysts by a stomach tube and housed in groups of four or six mice per cage. For 7 days after feeding sporocysts, all bedding and feces were incinerated to kill sporocysts that might have passed unexcysted in mouse feces (Box, 1983). 2.2. Diclazuril Diclazuril was administerd to mice in pelleted rodent feed. For this, diclazuril powder was mixed in rodent feed (NIH-07 rat and mouse diet) powder at 50 ppm, and then mixed feed was pelleted by Zeigler Brothers, Gardners, Pennsylvania, USA. Our objective was to deliver 10 mg of diclazuril per kg body weight of the mouse, based on a 20 g mouse consuming 4 g of rodent feed per day. Control mice were fed unmedicated NIH-07 diet. Mice were given water and feed ad libitum. Groups of mice were given medicated feed at different time periods before and after feeding sporocysts (Table 1). Two trials were performed. In trial 1, 55 KO mice (groups 1–12) were used; 49 mice were fed sporocysts and six mice (group 1) were not fed sporocysts. Twenty mice (groups 2–5) fed sporocysts were given diclazuril medicated feed starting 5 days before feeding S. neurona and continuing for 30–39 days after feeding S. neurona; these mice were killed 4–10 days after cessation of therapy (Table 1). Five mice (group 6) were given drug starting 7 days after feeding S. neurona and therapy was continued for a total of 32 days. Fourteen mice (groups 7–9) were given diclazuril starting 10, 12, and 20 days after feeding S. neurona, respectively, and medication was continued for 18–20 days (Table 1). Ten mice (groups 10, 11) fed S. neurona were not given diclazuril. The six mice in group 1, not fed S. neurona, were given diclazuril starting 5 days before the start of the experiment (counting starting date as the day sporocysts were fed) and the drug was continued for a total of 44 days; these mice were killed on day 44 from the start of the drug feeding (data not shown in Table 1). In trial 2, 25 KO mice were used. Five mice (group 12) fed sporocysts were not given diclazuril. Twenty mice (groups 13–16) were fed sporocysts and were given diclazuril starting 10 or 15 days after feeding sporocysts and therapy was continued for 18–30 days (Table 1). 2.3. Histologic examination For histologic studies, all mice were examined at necropsy. Portions of brain, eyes, heart, lungs, liver, spleen, mesenteric lymph nodes, small intestine, kidneys, adrenals, uterus, testes, abdominal muscle, urinary bladder, skeletal muscle from legs, and tongue were fixed in 10% neutral buffered formalin (NBF). Tissue sections from each mouse were embedded in paraffin blocks, sections were cut at 5 m, and tissues examined microscopically after staining with hematoxylin and eosin (H&E). The entire vertebral column including muscles was fixed in NBF, decalcified in ethylene diamino tetra acetic acid (EDTA), and longitudinal and cross sections of spinal column with bone marrow were prepared as above.
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2.4. Immunohistochemical examination For immunohistochemical (IHC) staining, sections from each mouse were reacted with anti-S. neurona serum prepared in a rabbit injected with culture-derived merozoites of S. neurona as described by Dubey and Lindsay (1999). The specificity of the S. neurona antiserum has been described (Dubey and Hamir, 2000). Positive and negative controls were included for IHC staining. The positive control consisted of brain tissue from a mouse experimentally infected with S. neurona (Dubey and Lindsay, 1998). Negative controls included liver and skeletal muscles from a mouse infected with S. speeri (Dubey and Lindsay, 1999). A Dako EnVision Peroxidase (Dako, Carpinteria, California, USA) rabbit kit was used for staining, and the procedure recommended by the manufacturer was followed. Briefly, after deparaffinization, sections were treated with quenching solutions (3% hydrogen peroxide in absolute methanol), rinsed with water, digested in 0.4% pepsin, rinsed with BRIJ-PBS (0.75% BRIJ 35 in phosphate buffered saline, Sigma, St. Louis, Missouri, USA), rinsed in blocking solution (0.5% casein in BRIJ-PBS), treated with primary antibody at 37◦ C for 30 min (S. neurona serum diluted 1:5000 in 1% casein in BRIJ-PBS ), rinsed and treated for 30 min with Dako EnVision kit labeled Polymer HRP Solution at 37◦ C, rinsed with buffer, treated with substrate 3-amino-9-ethylcarbazole (AEC), rinsed with buffer, counter-stained with Mayer’s hematoxylin, covered with Crystal Mount, air dried, covered with Permount and coverslips, and examined microscopically. 2.5. Bioassays of S. neurona infection To determine the presence of viable S. neurona in mice treated with diclazuril, brains from seven groups of KO mice given diclazuril for different time periods (Table 1) were subinoculated into unifected KO mice. For this, one-half of all brains in the group was homogenized in saline (0.85% NaCl) and inoculated subcutaneously (s.c.) into 28 (two or four mice per group) KO mice (Table 1). The recipient mice that did not become ill were bled and killed 55–65 days after s.c. inoculation with brains of mice fed sporocysts, and their tissues were examined immunohistochemically for S. neurona infection. 2.6. Serologic examination for S. neurona Mice sera were tested for antibodies to S. neurona using a newly developed S. neurona agglutination test (SAT) as described by Lindsay and Dubey (2001).
3. Results 3.1. Trial 1 The six KO mice (group 1) fed diclazuril, but not given S. neurona, remained clinically normal and there were no histopathologic lesions (data not shown in Table 1). All 10 mice
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(groups 10, 11) fed sporocysts, but not given diclazuril, developed neurologic signs and were euthanized between 22 and 30 days post-infection (p.i.); all had severe encephalomyelitis with histologically demonstrable S. neurona (Table 1). Of the 20 mice (group 2–5) given diclazuril starting 5 days before feeding sporocysts, one mouse in group 3 became ill during the third week after feeding sporocysts but S. neurona was not demonstrable in its tissues; it had suppurative meningitis, probably of bacterial origin. Lesions or S. neurona were not seen in tissues of the remaining 19 mice given diclazuril starting 5 days before feeding sporocysts and continuing for 30 and 39 days after feeding sporocysts; S. neurona was not demonstrable in their brains by subinoculation into KO mice (Table 1). All four mice (group 7) given drug 10 days after feeding sporocysts survived and were killed 15 days after cessation of therapy; S. neurona was found in tissues of two of these four mice (Table 1). Sarcocystis neurona was found in tissues of one of five mice when diclazuril (group 8) was fed 12 days after feeding sporocysts; S. neurona was not found in the remaining four mice histologically or passage in KO mice. Sarcocystis neurona was found in all five mice when diclazuril (group 9) was given 20 days after feeding sporocysts but the lesions were less severe than those in mice not given diclazuril. 3.2. Trial 2 In this trial diclazuril was given starting 10 or 15 days after feeding S. neurona. All 15 mice (groups 13–15) given drug starting 10 days after sporocyst feeding recovered and had no demonstrable parasites by immunohistochemistry. Sarcocystis neurona was found in tissues of all five mice (group 16) fed diclazuril starting 15 days after sporocyst feeding. All five untreated mice (group 12) fed sporocysts developed neurologic signs and were euthanized 25 or 26 days p.i.; all had S. neurona in their tissues.
4. Discussion Diclazuril was most effective when administered to mice early in S. neurona infection. One reason for this may be related to the life cycle of the parasite in the KO mouse. The life cycle of S. neurona in the horse is unknown. Schizonts and merozoites are the only stages known and they have been found only in the central nervous system (CNS). The route of migration of S. neurona from the time of ingestion of sporocysts to its final destination is unknown. Recently, the life cycle of S. neurona has been elucidated in KO mice fed sporocysts (Dubey, 2001). Within 6 h of feeding sporocysts, sporozoites excyst in the small intestine and sporozoites can be found in enterocytes and in the lamina propria cells throughout the intestine, including the cecum. Some sporozoites are also found in cells in Peyers patches within 6 h of ingesting sporocysts. Parasitemia can occur within 24 h and parasites have been isolated from the brain as early as 4 days after feeding sporocysts (Dubey, unpublished). The first generation of schizonts of S. neurona occurs in tissues such as kidneys, lungs, and heart up to 11 days after ingesting sporocysts. Although a few organisms may be found in the brain earlier than 10 days, S. neurona does not multiply appreciably in the CNS until day 13 p.i. KO mice generally do not develop clinical signs of infection until day 14 p.i., irrespective of the dose (Dubey, unpublished). Failure to find
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S. neurona in mice given diclazuril as late as 7 days after feeding sporocysts suggest that the drug is most effective against the first generation of schizonts or that enough time was needed for the diclazuril to attain a parasiticidal concentration in the CNS. Finding antibodies to S. neurona in mice given diclazuril suggests multiplication of S. neurona in tissues before parasiticidal effect. In the present study, chemotherapy was not effective in eliminating S. neurona once KO mice developed clinical signs. Since we did not measure the feed intake, we are uncertain of the amount of drug ingested by mice with neurologic signs. Daily dosing of a known amount of diclazuril might have been more effective than the administration of the drug in mouse feed. Diclazuril is insoluble in water therefore, it was not given in the drinking water. Diclazuril given orally once daily at 10 mg/kg on 1 day prior to infection and then daily for 10 days prevents clinical disease and death in mice inoculated with the virulent RH strain T. gondii (Lindsay and Blagburn, 1994). These mice do not develop protective immunity as evidenced by their development of fatal toxoplasmosis following challenge with tachyzoites. This indicates that diclazuril worked so well that little replication and anitgenic stimulation occurred. Mice treated similarly with 1 mg/kg diclazuril, had a 20% mortality but the surviving mice developed immunity to challenge (Lindsay and Blagburn, 1994). Diclazuril is also highly effective for therapeutic treatment of acute toxoplasmosis in mice (Lindsay et al., 1995). Oral treatment with 10 mg/kg diclazuril prevented death in 90% of mice with acute toxoplasmosis when given once daily for 10 days beginning on day 6 when mice are clinically ill. Treatment of mice with 1 mg/kg diclazuril prevented death in only 20% of mice (Lindsay et al., 1995). Similar experiments with diclazuril in KO mice may be worth investigating. References Box, E.D., 1983. Recovery of Sarcocystis sporocysts from feces after oral administration. Proc. Helminthol. Soc. Wash. 50, 348–350. Dirikolu, L., Lehner, F., Nattrass, C., Bentz, B.G., Woods, W.E., Carter, W.G., Karpiesiuk, W., Jacobs, J., Boyles, J., Harkins, J.D., Granstrom, D.E., Tobin, T., 1999. Diclazuril in the horse: its identification and detection and preliminary pharmacokinetics. J. Vet. Pharmacol. Therap. 22, 374–379. Dubey, J.P., 2000. Prevalence of Sarcocystis species sporocysts in wild caught opossums (Didelphis virginiana). J. Parasitol. 86, 705–710. Dubey, J.P., 2001. Migration and development of Sarcocystis neurona in tissues of mice fed sporocysts from naturally infected opossums. Vet. Parasitol, in press. Dubey, J.P., Hamir, A.N., 2000. Immunohistochemical confirmation of Sarcocystis neurona infections in raccoons, mink, cat, skunk and pony. J. Parasitol. 86, 1150–1152. Dubey, J.P., Lindsay, D.S., 1998. Isolations in immunodeficient mice of Sarcocystis neurona from opossum (Didelphis virginiana) faeces, and its differentiation from Sarcocystis falcatula. Int. J. Parasitol. 28, 1823– 1828. Dubey, J.P., Lindsay, D.S., 1999. Sarcocystis speeri n. sp. (Protozoa: Sarcocystidae) from the opossum (Didelphis virginiana). J. Parasitol. 85, 903–909. 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. Dubey, J.P., Lindsay, D.S., Saville, W.J.A., Reed, S.M., Granstrom, D.E., Speer, C.A., 2001. A review of Sarcocystis neurona and equine protozoal myeloencephalitis (EPM). Vet. Parasitol., in press.
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Granstrom, D.E., McCrillis, S., Wulff-Strobel, C., Baker, C.B., Carter, W., Harkins, J.D., Tobin, T., Saville, W.J., 1997. Diclazuril and equine protozoal myeloencephalitis. In: AAEP Proceedings. Phoenix, Arizona, December 7–10, 1999, Vol. 43, pp. 13–14. Lindsay, D.S., Blagburn, B.L., 1994. Activity of diclazuril against Toxoplasma gondii in cultured cells and mice. Am. J. Vet. Res. 55, 530–533. Lindsay, D.S., Dubey, J.P., 2000. Determination of the activity of diclazuril against Sarcocystis neurona and Sarcocystis falcatula in cell cultures. J. Parasitol. 86, 164–166. Lindsay, D.S., Dubey, J.P., 2001. Direct agglutination test for the detection of antibodies to Sarcocystis neurona in experimentally infected animals. Vet. Parasitol., in press. Lindsay, D.S., Rippey, N.S., Toivio-Kinnucan, M.A., Blagburn, B.L., 1995. Ultrastructural effects of diclazuril against Toxoplasma gondii and investigation of a diclazuril-resistant mutant. J. Parasitol. 81, 459–466. MacKay, R.J., Davis, S.W., Dubey, J.P., 1992. Equine protozoal myeloencephalitis. Comp. Cont. Educ. Pract. Vet. 14, 1359–1367.
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Diclazuril preventive therapy of gamma interferon knockout mice fed Sarcocystis neurona sporocysts J.P. Dubey a,∗ , D. Fritz b , D.S. Lindsay c , S.K. Shen a , O.C.H. Kwok a , K.C. Thompson d a United States Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, Parasite Biology, Epidemiology and Systematics Laboratory, Beltsville Agricultural Research Center, Building 1001, Beltsville, MD 20705-2305, USA b Pathology Division, USAMRIID, 1425 Porter Street, Ft. Detrick, MD 21702, USA c Center of Molecular and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, 1410 Prices Fork Road, Blacksburg, VA 24061-0342, USA d Schering-Plough Animal Health Corp., Pharmaceutical Research, 1095 Morris Avenue, Union, NJ 07083, USA
Received 23 August 2000; accepted 20 September 2000
Abstract Gamma interferon knockout (KO) mice (n = 74) were fed a lethal dose of ∼1000 sporocysts of the SN15-OP isolate of Sarcocystis neurona. Groups of mice were given pelleted rodent feed containing 50 ppm of diclazuril at different times before and after feeding sporocysts. All mice were examined at necropsy and their tissues were examined immunohistochemically for S. neurona infection. Twenty mice were fed sporocysts and given diclazuril starting 5 days before feeding sporocysts and continuing 30–39 days post-infection (p.i.). One mouse died of causes unrelated to S. neurona with no demonstrable parasites; the remaining 19 mice remained clinically normal and S. neurona organisms were not found in their tissues. Sarcocystis neurona organisms were not demonstrable by bioassay of the brains of these 19 mice in uninfected KO mice. Sarcocystis neurona organisms were not found in tissues of five mice treated with diclazuril, starting 7 days after feeding sporocysts and continuing up to 39 days p.i. Therapy was less efficient when diclazuril was given 10 days p.i. Sarcocystis neurona organisms were found in two of 19 mice treated with diclazuril starting 10 days after feeding sporocysts, in two of five mice starting therapy 12 days p.i., and in 10 of 10 mice when treatment was delayed until 15 days p.i. All 15 mice fed S. neurona, but not given diclazuril, developed neural sarcocystosis and were euthanized 22–30 days after feeding
∗ Corresponding author. Tel.: +1-301-504-8128; fax: +1-301-504-9222. E-mail address: [email protected] (J.P. Dubey).
0304-4017/01/$ – see front matter Published by Elsevier Science B.V. PII: S 0 3 0 4 - 4 0 1 7 ( 0 0 ) 0 0 3 7 6 - 9
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sporocysts. Six mice not fed S. neurona, but given diclazuril for 44 days, remained clinically normal. Results indicate that diclazuril can kill the early stages of S. neurona. Published by Elsevier Science B.V. Keywords: Sarcocystis neurona; Sporocysts; Oral; Equine protozoal myeloencephalitis; EPM; Knockout mice; Diclazuril; Treatment
1. Introduction Equine protozoal myeloencephalitis (EPM) is the most commonly diagnosed neurologic disorder in horses in the Americas and Sarcocystis neurona is the most common cause of EPM (Dubey et al., 1991; MacKay et al., 1992; Dubey et al., 2001). To date, there is no reproducible method to induce clinical EPM in horses with demonstrable parasites, and only a small percentage of naturally infected horses develop clinical EPM (Dubey et al., 2001). The life cycle of S. neurona is not completely known. Opossums (Didelphis virginiana) are the definitive hosts and horses are considered an aberrant intermediate host. The natural intermediate host, harboring the sarcocyst stage, is unknown. Recently, gamma interferon knockout (KO) mice were found to be susceptible to S. neurona infection; KO mice fed sporocysts developed neurologic disease, similar to EPM in horses, and S. neurona was demonstrated in their tissues (Dubey and Lindsay, 1998). Although several anti-protozoal drugs have been used empirically to treat EPM there is no reliable method to evaluate therapy for EPM in animals. Diclazuril (C17 H9 Cl3 N4 O2 ) is a triazine derivative anticoccidial (Clinacox, ScheringPlough Animal Health Corporation, Union, NJ, USA) approved in the US for use in broiler chickens. It has anti-protozoal activity in vivo and in vitro against Toxoplasma gondii (Lindsay and Blagburn, 1994) and in vitro against N. caninum and S. neurona (Lindsay et al., 1995; Lindsay and Dubey, 2000, 2001). It has been used to treat EPM in horses (Granstrom et al., 1997; Dirikolu et al., 1999). In the present paper, we report anti-S. neurona activity of diclazuril in KO mice fed S. neurona sporocysts. 2. Materials and methods 2.1. Sarcocystis neurona Sarcocystis neurona (isolate SN15-OP) sporocysts were obtained from intestinal scrapings of an opossum (no. 9108) as described by Dubey (2000). This isolate was selected for the present study because it was used previously to study migration and development of S. neurona in KO mice (Dubey, 2001). There are sporocysts from three or more species of Sarcocystis in opossum feces (Dubey and Lindsay, 1999). Although S. falcatula sporocysts were present in this opossum, S. falcatula is not infective to KO mice; S. speeri sporocysts were not present in opossum 9108 (Dubey, 2000). The KO mice used in the present study were bred in-house from stock mice obtained from the Jackson Laboratory, Bar Harbor, Maine, USA. Most mice were males, weighed 15–20 g and were 5–8 weeks old at the time of infection. Ten-fold dilutions (10−1 –10−6 ) of sporocysts from stock inoculum were made
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in antibiotic saline and stored at 4◦ C as described by Dubey (2001). A 10−6 dilution was not infective to two mice, a 10−5 dilution of the inoculum was infective to three of 10 mice, and a 10−4 dilution was infective to two of two mice (Dubey, 2001). In the present study, a 10−2 dilution of the inoculum was fed to each KO mice. Thus, there were between 100 and 1000 mouse infective sporocysts in the inoculum given to each mouse. Mice were fed sporocysts by a stomach tube and housed in groups of four or six mice per cage. For 7 days after feeding sporocysts, all bedding and feces were incinerated to kill sporocysts that might have passed unexcysted in mouse feces (Box, 1983). 2.2. Diclazuril Diclazuril was administerd to mice in pelleted rodent feed. For this, diclazuril powder was mixed in rodent feed (NIH-07 rat and mouse diet) powder at 50 ppm, and then mixed feed was pelleted by Zeigler Brothers, Gardners, Pennsylvania, USA. Our objective was to deliver 10 mg of diclazuril per kg body weight of the mouse, based on a 20 g mouse consuming 4 g of rodent feed per day. Control mice were fed unmedicated NIH-07 diet. Mice were given water and feed ad libitum. Groups of mice were given medicated feed at different time periods before and after feeding sporocysts (Table 1). Two trials were performed. In trial 1, 55 KO mice (groups 1–12) were used; 49 mice were fed sporocysts and six mice (group 1) were not fed sporocysts. Twenty mice (groups 2–5) fed sporocysts were given diclazuril medicated feed starting 5 days before feeding S. neurona and continuing for 30–39 days after feeding S. neurona; these mice were killed 4–10 days after cessation of therapy (Table 1). Five mice (group 6) were given drug starting 7 days after feeding S. neurona and therapy was continued for a total of 32 days. Fourteen mice (groups 7–9) were given diclazuril starting 10, 12, and 20 days after feeding S. neurona, respectively, and medication was continued for 18–20 days (Table 1). Ten mice (groups 10, 11) fed S. neurona were not given diclazuril. The six mice in group 1, not fed S. neurona, were given diclazuril starting 5 days before the start of the experiment (counting starting date as the day sporocysts were fed) and the drug was continued for a total of 44 days; these mice were killed on day 44 from the start of the drug feeding (data not shown in Table 1). In trial 2, 25 KO mice were used. Five mice (group 12) fed sporocysts were not given diclazuril. Twenty mice (groups 13–16) were fed sporocysts and were given diclazuril starting 10 or 15 days after feeding sporocysts and therapy was continued for 18–30 days (Table 1). 2.3. Histologic examination For histologic studies, all mice were examined at necropsy. Portions of brain, eyes, heart, lungs, liver, spleen, mesenteric lymph nodes, small intestine, kidneys, adrenals, uterus, testes, abdominal muscle, urinary bladder, skeletal muscle from legs, and tongue were fixed in 10% neutral buffered formalin (NBF). Tissue sections from each mouse were embedded in paraffin blocks, sections were cut at 5 m, and tissues examined microscopically after staining with hematoxylin and eosin (H&E). The entire vertebral column including muscles was fixed in NBF, decalcified in ethylene diamino tetra acetic acid (EDTA), and longitudinal and cross sections of spinal column with bone marrow were prepared as above.
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2.4. Immunohistochemical examination For immunohistochemical (IHC) staining, sections from each mouse were reacted with anti-S. neurona serum prepared in a rabbit injected with culture-derived merozoites of S. neurona as described by Dubey and Lindsay (1999). The specificity of the S. neurona antiserum has been described (Dubey and Hamir, 2000). Positive and negative controls were included for IHC staining. The positive control consisted of brain tissue from a mouse experimentally infected with S. neurona (Dubey and Lindsay, 1998). Negative controls included liver and skeletal muscles from a mouse infected with S. speeri (Dubey and Lindsay, 1999). A Dako EnVision Peroxidase (Dako, Carpinteria, California, USA) rabbit kit was used for staining, and the procedure recommended by the manufacturer was followed. Briefly, after deparaffinization, sections were treated with quenching solutions (3% hydrogen peroxide in absolute methanol), rinsed with water, digested in 0.4% pepsin, rinsed with BRIJ-PBS (0.75% BRIJ 35 in phosphate buffered saline, Sigma, St. Louis, Missouri, USA), rinsed in blocking solution (0.5% casein in BRIJ-PBS), treated with primary antibody at 37◦ C for 30 min (S. neurona serum diluted 1:5000 in 1% casein in BRIJ-PBS ), rinsed and treated for 30 min with Dako EnVision kit labeled Polymer HRP Solution at 37◦ C, rinsed with buffer, treated with substrate 3-amino-9-ethylcarbazole (AEC), rinsed with buffer, counter-stained with Mayer’s hematoxylin, covered with Crystal Mount, air dried, covered with Permount and coverslips, and examined microscopically. 2.5. Bioassays of S. neurona infection To determine the presence of viable S. neurona in mice treated with diclazuril, brains from seven groups of KO mice given diclazuril for different time periods (Table 1) were subinoculated into unifected KO mice. For this, one-half of all brains in the group was homogenized in saline (0.85% NaCl) and inoculated subcutaneously (s.c.) into 28 (two or four mice per group) KO mice (Table 1). The recipient mice that did not become ill were bled and killed 55–65 days after s.c. inoculation with brains of mice fed sporocysts, and their tissues were examined immunohistochemically for S. neurona infection. 2.6. Serologic examination for S. neurona Mice sera were tested for antibodies to S. neurona using a newly developed S. neurona agglutination test (SAT) as described by Lindsay and Dubey (2001).
3. Results 3.1. Trial 1 The six KO mice (group 1) fed diclazuril, but not given S. neurona, remained clinically normal and there were no histopathologic lesions (data not shown in Table 1). All 10 mice
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(groups 10, 11) fed sporocysts, but not given diclazuril, developed neurologic signs and were euthanized between 22 and 30 days post-infection (p.i.); all had severe encephalomyelitis with histologically demonstrable S. neurona (Table 1). Of the 20 mice (group 2–5) given diclazuril starting 5 days before feeding sporocysts, one mouse in group 3 became ill during the third week after feeding sporocysts but S. neurona was not demonstrable in its tissues; it had suppurative meningitis, probably of bacterial origin. Lesions or S. neurona were not seen in tissues of the remaining 19 mice given diclazuril starting 5 days before feeding sporocysts and continuing for 30 and 39 days after feeding sporocysts; S. neurona was not demonstrable in their brains by subinoculation into KO mice (Table 1). All four mice (group 7) given drug 10 days after feeding sporocysts survived and were killed 15 days after cessation of therapy; S. neurona was found in tissues of two of these four mice (Table 1). Sarcocystis neurona was found in tissues of one of five mice when diclazuril (group 8) was fed 12 days after feeding sporocysts; S. neurona was not found in the remaining four mice histologically or passage in KO mice. Sarcocystis neurona was found in all five mice when diclazuril (group 9) was given 20 days after feeding sporocysts but the lesions were less severe than those in mice not given diclazuril. 3.2. Trial 2 In this trial diclazuril was given starting 10 or 15 days after feeding S. neurona. All 15 mice (groups 13–15) given drug starting 10 days after sporocyst feeding recovered and had no demonstrable parasites by immunohistochemistry. Sarcocystis neurona was found in tissues of all five mice (group 16) fed diclazuril starting 15 days after sporocyst feeding. All five untreated mice (group 12) fed sporocysts developed neurologic signs and were euthanized 25 or 26 days p.i.; all had S. neurona in their tissues.
4. Discussion Diclazuril was most effective when administered to mice early in S. neurona infection. One reason for this may be related to the life cycle of the parasite in the KO mouse. The life cycle of S. neurona in the horse is unknown. Schizonts and merozoites are the only stages known and they have been found only in the central nervous system (CNS). The route of migration of S. neurona from the time of ingestion of sporocysts to its final destination is unknown. Recently, the life cycle of S. neurona has been elucidated in KO mice fed sporocysts (Dubey, 2001). Within 6 h of feeding sporocysts, sporozoites excyst in the small intestine and sporozoites can be found in enterocytes and in the lamina propria cells throughout the intestine, including the cecum. Some sporozoites are also found in cells in Peyers patches within 6 h of ingesting sporocysts. Parasitemia can occur within 24 h and parasites have been isolated from the brain as early as 4 days after feeding sporocysts (Dubey, unpublished). The first generation of schizonts of S. neurona occurs in tissues such as kidneys, lungs, and heart up to 11 days after ingesting sporocysts. Although a few organisms may be found in the brain earlier than 10 days, S. neurona does not multiply appreciably in the CNS until day 13 p.i. KO mice generally do not develop clinical signs of infection until day 14 p.i., irrespective of the dose (Dubey, unpublished). Failure to find
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S. neurona in mice given diclazuril as late as 7 days after feeding sporocysts suggest that the drug is most effective against the first generation of schizonts or that enough time was needed for the diclazuril to attain a parasiticidal concentration in the CNS. Finding antibodies to S. neurona in mice given diclazuril suggests multiplication of S. neurona in tissues before parasiticidal effect. In the present study, chemotherapy was not effective in eliminating S. neurona once KO mice developed clinical signs. Since we did not measure the feed intake, we are uncertain of the amount of drug ingested by mice with neurologic signs. Daily dosing of a known amount of diclazuril might have been more effective than the administration of the drug in mouse feed. Diclazuril is insoluble in water therefore, it was not given in the drinking water. Diclazuril given orally once daily at 10 mg/kg on 1 day prior to infection and then daily for 10 days prevents clinical disease and death in mice inoculated with the virulent RH strain T. gondii (Lindsay and Blagburn, 1994). These mice do not develop protective immunity as evidenced by their development of fatal toxoplasmosis following challenge with tachyzoites. This indicates that diclazuril worked so well that little replication and anitgenic stimulation occurred. Mice treated similarly with 1 mg/kg diclazuril, had a 20% mortality but the surviving mice developed immunity to challenge (Lindsay and Blagburn, 1994). Diclazuril is also highly effective for therapeutic treatment of acute toxoplasmosis in mice (Lindsay et al., 1995). Oral treatment with 10 mg/kg diclazuril prevented death in 90% of mice with acute toxoplasmosis when given once daily for 10 days beginning on day 6 when mice are clinically ill. Treatment of mice with 1 mg/kg diclazuril prevented death in only 20% of mice (Lindsay et al., 1995). Similar experiments with diclazuril in KO mice may be worth investigating. References Box, E.D., 1983. Recovery of Sarcocystis sporocysts from feces after oral administration. Proc. Helminthol. Soc. Wash. 50, 348–350. Dirikolu, L., Lehner, F., Nattrass, C., Bentz, B.G., Woods, W.E., Carter, W.G., Karpiesiuk, W., Jacobs, J., Boyles, J., Harkins, J.D., Granstrom, D.E., Tobin, T., 1999. Diclazuril in the horse: its identification and detection and preliminary pharmacokinetics. J. Vet. Pharmacol. Therap. 22, 374–379. Dubey, J.P., 2000. Prevalence of Sarcocystis species sporocysts in wild caught opossums (Didelphis virginiana). J. Parasitol. 86, 705–710. Dubey, J.P., 2001. Migration and development of Sarcocystis neurona in tissues of mice fed sporocysts from naturally infected opossums. Vet. Parasitol, in press. Dubey, J.P., Hamir, A.N., 2000. Immunohistochemical confirmation of Sarcocystis neurona infections in raccoons, mink, cat, skunk and pony. J. Parasitol. 86, 1150–1152. Dubey, J.P., Lindsay, D.S., 1998. Isolations in immunodeficient mice of Sarcocystis neurona from opossum (Didelphis virginiana) faeces, and its differentiation from Sarcocystis falcatula. Int. J. Parasitol. 28, 1823– 1828. Dubey, J.P., Lindsay, D.S., 1999. Sarcocystis speeri n. sp. (Protozoa: Sarcocystidae) from the opossum (Didelphis virginiana). J. Parasitol. 85, 903–909. 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. Dubey, J.P., Lindsay, D.S., Saville, W.J.A., Reed, S.M., Granstrom, D.E., Speer, C.A., 2001. A review of Sarcocystis neurona and equine protozoal myeloencephalitis (EPM). Vet. Parasitol., in press.
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