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Sarcocystis neurona: parasitemia in a severe combined immunodeficient (SCID) horse fed sporocysts
Experimental Parasitology 100 (2002) 150–154 www.academicpress.com
Sarcocystis neurona: parasitemia in a severe combined immunodeficient (SCID) horse fed sporocysts Maureen T. Long,a Melissa T. Mines,b Donald P. Knowles,c Susan M. Tanhauser,a John B. Dame,d Timothy J. Cutler,a Robert J. MacKay,a and Debra C. Sellonb,* a
Department of Large Animal Clinical Sciences, University of Florida, Gainesville, FL 32610, USA Department of Veterinary Clinical Sciences, Washington State University, Pullman, WA 99164, USA Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164, USA d Department of Pathobiology, University of Florida, Gainesville, FL 32610, USA b
c
Received 23 April 2001; accepted 7 March 2002
Abstract Sarcocystis neurona was isolated from the blood of a 5-month-old Arabian foal with severe combined immunodeficiency. The foal had been inoculated approximately 3 weeks previously with 5 105 sporocysts that were isolated from the intestines of an opossum and identified by restriction enzyme analysis of PCR products as S. neurona. The isolate obtained from the blood of this foal was characterized by genetic, serologic, and morphologic methods and identified as S. neurona (WSU1). This represents the first time that S. neurona has been isolated from any tissue after experimental infection of a horse. This is also the first time a parasitemia has been detected during either natural or experimental infection. The severe combined immunodeficiency foal model provides a unique opportunity to study the pathogenesis of S. neurona infection in horses and to determine the role of the immune system in the control of infection with and development of neurologic disease. Index Descriptors and Abbreviations. Sarcocystis neurona; protozoa; horse; equine; EPM, equine protozoal myeloencephalitis; SCID, severe combined immunodeficiency; CNS, central nervous system; PCR, polymerase chain reaction; IFN-c, interferon gamma; KO, knockout; DNA, deoxyribonucleic acid; PID, post-infection day; DMEM, Dulbecco’s modified essential media; SC, subcutaneous; IP, intraperitoneal; M, molar; CSF, cerebrospinal fluid. Ó 2002 Elsevier Science (USA). All rights reserved.
EPM is a progressive neurologic disease of horses caused by a protozoal infection of the CNS. The most commonly identified parasite associated with this disease is the apicomplexan protozoa, Sarcocystis neurona (Davis et al., 1991; Dubey et al., 2001). There is considerable evidence to show that the opossum is the definitive host in the life cycle of this parasite and that horses are ‘‘accidental’’ hosts after ingestion of sporocysts passed in opossum feces (Fenger et al., 1995). A high percentage of horses in many areas of the United States have serum antibodies to S. neurona but few develop neurologic diseases (Mackay, 1997). Experimental oral infection of immunocompetent horses with S. neurona sporocysts shed by opossums does not consistently result in clinical signs (Dubey *
Corresponding author. Fax: +509-335-0880. E-mail address: [email protected] (D.C. Sellon).
et al., 2001). Similarly, the infection of immunocompetent Balb/c mice with S. neurona sporocysts or merozoites does not result in neurologic abnormalities (Marsh et al., 1997). However, an infection of IFN-k KO mice by oral or parenteral routes consistently results in a fulminant neurologic disease (Dubey and Lindsay, 1998). Intraperitoneal infection of nude mice with culture-derived merozoites of S. neurona induced encephalitis, but two similarly infected SCID mice did not develop neurologic abnormalities (Marsh et al., 1997). Arabian horses with SCID lack specific B and T cell responses because of a mutation in the DNA-dependent kinase catalytic subunit (Shin et al., 1997), offering the opportunity to characterize S. neurona infection in the immunodeficient horse. We hypothesized that S. neurona infection of a SCID foal will result in CNS infection and neuropathology.
0014-4894/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 1 4 - 4 8 9 4 ( 0 2 ) 0 0 0 1 2 - 7
M.T. Long et al. / Experimental Parasitology 100 (2002) 150–154
A 33-day-old male Arabian horse homozygous for the SCID trait was infected via nasogastric tube with 5 105 S. neurona sporocysts retrieved from the intestines of wild opossums (Cutler et al., 1999). Sporocysts were identified as S. neurona by a PCR protocol that differentiates S. neurona from the closely related opossum isolates, S. falcatula, S. speeri, and 1085 (Tanhauser et al., 1999). Immediately after birth, the foal suckled colostrum from his EPM seronegative dam. The foal was immediately placed in isolation with his dam and removed from his dam at approximately 21 days of age. The day before infection, day 0, and everyday during the study period complete physical and neurologic examinations were performed. Hemogram and serum biochemical profile were evaluated at intervals. Peripheral blood for buffy coat PCR and culture were collected prior to infection and on days 1–8, 10, 14, 17, 21, 24, 28, and 31. Baseline bloodwork (hemogram and serum biochemical profile) and physical and neurologic examination parameters were within normal limits for the SCID foal (with the exception of the absence of lymphocytes). After infection, the foal was intermittently neutropenic, hyperfibrinogenemic, and febrile with mild diarrhea and/or a cough and increased lung sounds. These findings were attributed to secondary infections common in SCID foals (Perryman et al., 1978) and the foal was treated with ceftiofur and gentamicin. At PID 39 the foal demonstrated a mild knuckling of the right hind limb that was intermittently apparent until euthanasia. On PID 53 the foal was euthanized and CSF and appropriate tissues were obtained for culture and PCR analysis. Buffy coat samples or homogenized tissue samples were suspended in DMEM and inoculated over a monolayer of bovine turbinate cells passaged 24 h previously. Cultures were examined microscopically twice a week for evidence of cytopathology and/or parasites. At 12–18 weeks of culture, cultures were assessed by PCR and immunohistochemistry. PCR positive cultures were maintained. Cultures of buffy coat samples obtained from the SCID foal on PID 21, 24, and 31 were positive by PCR for S. neurona at 12 weeks of culture, as were cultures of tissues from the brain of the SCID foal. At approximately 14 weeks of culture, an apicomplexan parasite was identified only in the PID 21 buffy coat culture from the SCID foal (Fig. 1). This organism was designated the WSU1 isolate of S. neurona. Protozoal organisms were not isolated from any other PCR positive culture. Development of the protozoal organism in culture was asynchronous with organisms dividing by endopologeny (Fig. 1). For electron microscopy, cells were pelleted by centrifugation, washed in cold PBS, and then fixed in cold 2.5% glutaraldehyde in 1% cacodylate buffer (pH 7.2). The pellet was post-fixed in 2% w/v osmium tetroxide,
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Fig. 1. Photomicrograph of a cytospin preparation of the cell culture supernatant of an isolate consistent with S. neurona. This isolate was cultured from the blood of an SCID foal 21 days after oral infection. This is a photomicrograph of a cell with nucleus (a) and ruptured cytoplasm, which contained schizonts composed of several merozoites 4–5 lm (b). Cell free merozoites are present throughout preparation. These forms are generally larger than the intracellular forms, measuring 4–6 lm.
dehydrated in ethanol, passed through propylene oxide and embedded in Epon/Araldite (Polysciences, Warrington, PA). Thin sections were stained in uranyl acetate and lead citrate and examined using a transmission electron microscope operating at 80 kV. Electron microscopic evaluation revealed mature cell-free merozoites that ranged from approximately 4 to 6 lm in length; intracellular forms consisted of schizonts containing merozoites of approximately 4–5 lm in length. Merozoites isolated from the day 21 buffy coat culture were morphologically indistinguishable from S. neurona by light or electron microscopy. The observed morphology was consistent with S. neurona with single or clustered merozoites that contained a single nucleus, conoid, anterior placement of micronemes, and no rhoptries. Three isolates of S. neurona obtained from the spinal cord of clinically neurologic horses were used for comparison to the WSU1 isolate for morphologic, genetic, and antigenic analyses. The isolate UCD1 was isolated from a horse in California (Marsh et al., 1996). The isolate SN6, provided by S. Tornquist of Oregon State University, was isolated from a horse in Oregon (Dubey et al., 1999). The isolate FL1 was isolated from a horse in Florida. S. falcatula was purchased from ATCC (Beltsville, MD). All isolates were passaged as previously described in bovine turbinate cells (Davis et al., 1991). For PCR analysis, DNA was extracted from merozoites freshly blown from cell cultures of UCD1, SN6, FL1, S. falcatula, and WSU1. These samples were amplified by PCR using S. neurona specific primers (Tanhauser et al., 1999). Controls consisted of negative tissue controls (either uninfected equine tissues or noninoculated cell cultures) and a no DNA control. The
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products of the primers were cut by restriction enzymes to confirm the identity of WSU1. Amplification products were sequenced by the ABI prism method and compared to known DNA sequences (GenBank #AF093158 and GenBank #AF093153) of S. neurona and related species (Marsh et al., 1996). WSU1 was confirmed to be S. neurona by PCR and restriction enzyme analysis (Fig. 2) and DNA sequencing of PCR amplified DNA (Fig. 3). PCR was performed on all isolates simultaneously. A normal adult horse seronegative to S. neurona was inoculated intramuscularly with 1 106 –1 108 merozoites of the WSU1 isolate every two weeks for four doses. Serum was collected by jugular venipuncture on the day of the first inoculation and weekly thereafter. During the study period the horse was maintained in a pasture that was fenced and electronically wired to prevent the entering of small, wild mammals. Seroconversion to S. neurona in this horse infected with WSU1 was independently verified by two commercial laboratories. CSF obtained from the atlanto-occipital space of
Fig. 2. (a)–(e) Ethidium bromide-stained agarose gel (1.8%) demonstrating PCR products differentiating all of the S. neurona isolates from S. falcatula. Lane 1 contains a 100 bp ladder with the brightest visible band equal to approximately 500 bp (Promega), lanes 2–7 on all gels consist of S. neurona (UCD1), S. neurona (SN6), S. falcatula (ATCC #), S. neurona (WSU1) S. neurona (FL1), and water control, respectively. (a) Top-PCR products from primer pair JNB25/JD396 demonstrating a 334 base pair sequence for both S. neurona and S. falcatula; bottom-primers JNB63/JNB65 generate a 1202 bp product for S. neurona and a 959 bp primer for S. falcatula. (b) Top-PCR primers JNB48/JNB50 generate a 870 bp product for both S. neurona and S. falcatula; bottom-PCR primers JNB33/JNB54 generate a 1100 bp product for both S. neurona and S. falcatula. (c) Top-restriction enzyme digestion with HindIII cuts the products of JNB25/JD396 primers into 180 and 154 bp fragments for the S. falcatula isolate and did not cut the S. neurona product; bottom-restriction enzyme digestion with HinfI cuts the same primer products into 64, 108, and 62 bp fragments for S. neurona isolates and 170 and 164 bp fragments for S. falcatula. (d) Top-restriction enzyme digest with DraI of the JNB33/ JNB54 fragments resulted in 884 and 216 bp fragments for S. neurona and did not cut S. falcatula; bottom-restriction enzyme digest with HinfI of the JNB33/JNB54 PCR product does not cut the S. neurona fragment and generates a 745 and 355 bp for S. falcatula. (e) Top-restriction enzyme digest with RsaI of the JNB48/JNB50 PCR product resulted in a 665 bp fragment for S. neurona and cuts S. falcatula into 428 and 237 bp fragments.
Fig. 3. Four rows of sequence data spanning approximately 250 bp of the PCR target from JNB25/JD396 (TGF). The sequences (top to bottom) from UCD1, WSU1, S. falcatula, 1085, respectively, highlight the single and multiple base pair differences between the isolates. The isolate, WSU1, obtained from an SCID is completely identical to UCD1 (GenBank #AF093158 and GenBank #AF093153).
this horse at approximately six months post-inoculation was positive by Western blot for antibody to S. neurona. Detailed neurologic examination of this horse remained within normal limits. Sera obtained from two horses experimentally infected with sporocysts from opossum feces identified by PCR as S. neurona were compared to the serum obtained from the horse infected with WSU1 in a Western blot format with WSU1 as antigen. For immunoblot analyses, aliquots of WSU1 isolate were mixed with PBS and SDS–PAGE buffer, boiled for 5 min and electrophoresed in a 7.5–17.5% SDS–polyacrylamide gradient gel and 4% stacking gel using the one-dimensional discontinuous method of Laemmli. Biotin-conjugated mol wt standards (Bio-Rad Laboratories, Hercules, CA) were used. After SDS–PAGE electrophoresis, the antigens were electrophoretically transferred to nitrocellulose filters (Bio-Rad Laboratories, Hercules, CA). Serum from sample horses was diluted in blocking buffer and incubated with filters at room temperature. After incubation and washing in buffer, bound antibodies were detected by incubation with peroxidase-conjugated rabbit anti-horse IgG1 (Pierce, Rockford, IL). All bound antibodies and mol wt standards were visualized by chemiluminescence (ECL, Amersham Life Sciences). Reactivity post-inoculation was consistent with exposure to S. neurona (data not shown). Culture supernatant from bovine turbinate cells infected with the WSU1 isolate from the SCID foal was harvested, centrifuged, and resuspended in cell culture media. Eight 6-week-old IFN-c KO mice (BALB/cIfngtmits ) were infected with harvested organisms. Two
M.T. Long et al. / Experimental Parasitology 100 (2002) 150–154
mice each were inoculated with either 105 organisms SC, 106 organisms SC, 106 organisms IP, or 107 organisms IP. One mouse from each KO group was sacrificed at 30 days post-infection; the second mouse from each KO group was sacrificed at 60 days post-infection or at the onset of clinical signs. Tissues including lung, liver, kidney, cardiac muscle, brain, and spinal cord were harvested for histopathology. Representative samples of each tissue were extracted for DNA and analyzed by PCR. One mouse infected subcutaneously with 105 merozoites of the WSU1 isolate of S. neurona developed severe neurologic signs at approximately 48 days postinfection and was euthanized at that time. The mouse had a right head tilt and circled compulsively to the left. No other mice exhibited recognizable neurologic abnormalities. All other mice were euthanized at 30 or 60 days post-infection. Polymerase chain reaction analysis and culture results of brain tissue for S. neurona were positive in the mice both at 30 and 60 days postinfection. Brain and spinal cord from the mouse that developed neurologic signs were PCR positive for S. neurona at the time of euthanasia and histopathological analysis revealed pathological abnormalities consistent with S. neurona infection. These data represent the first isolation of S. neurona obtained after experimental infection of a horse. This study is also the first time that S. neurona has been isolated after infection of the horse from a site other than the CNS. Confirmation that this isolate is indeed S. neurona is based upon the morphology at both light and electron microscopic levels. Genetic sequencing reveals an organism identical to that described previously for S. neurona and neither S. falcatula nor 1085 (Tanhauser et al., 1999). Antibody responses in horses inoculated with WSU1 were indistinguishable by Western blot from antibody responses of horses infected with other isolates of S. neurona. Phenotypically, the WSU1 isolate is neuropathogenic in gamma interferon knockout mice (Dubey and Lindsay, 1998). It is surprising that only one mouse developed obvious neurologic deficits. However, the majority of these mice were PCR positive for S. neurona in their brain and spinal cord. The absence of obvious neurologic deficits in most mice may be a result of euthanasia prior to the development of signs, a decreased pathogenicity of the WSU1 isolate because of passage through a SCID foal or multiple cell passages, or the difficulties in accurately detecting subtle neurologic deficits in mice. S. neurona likely undergoes excystation and intestinal invasion after oral infection with sporocysts. Before neural invasion takes place, presumably a parasitemia occurs. This has not been characterized previously by culture or PCR. The isolation of the organism at PID 21 appears to be appropriately timed for parasitemia, leading to infection within the CNS. Horses seroconvert
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at approximately 28 days post-inoculation, presumably corresponding to the time after peak parasitemia (Fenger et al., 1997). The immunodeficient state of this foal likely permitted a high enough parasitemia to allow for isolation of the organism from the blood of this foal. The SCID foal offers tremendous potential for the development of a model of S. neurona infection in the horse for characterization of the kinetics of equine infection. The lack of development of abnormal neurologic signs in this foal was surprising. This could be the result of infection with a strain of S. neurona that is not neurovirulent or it could indicate a role of specific immune responses in the neuropathogenesis of infection. Infection of additional SCID hosts will be necessary to develop this model to consistently induce CNS infection, disease, and parasite isolation from the nervous tissue. This model has been used successfully in other equine diseases including equine infectious anemia and babesiosis to define the role of the immune system in disease pathogenesis or protection from infection (Knowles et al., 1994; Perryman et al., 1988). Infection of SCID foals and full development of this model will allow this type of investigation into the immunopathogenesis of S. neurona.
Acknowledgments This work was supported by funds from the State of Washington, Grayson-Jockey Club Research Foundation, and the State of Florida Division of Pari-mutuel Wagering. We gratefully acknowledge the expert technical assistance of Ms. Tressa Hochstatter, Karen Gillis, Francesca Griffen, and Susan Tornquist.
References Cutler, T.J., Mackay, R.J., Ginn, P.E., Greiner, E.C., Porter, R., Yowell, C.A., Dame, J.B., 1999. Are Sarcocystis neurona and Sarcocystis falcatula synonymous? A horse infection challenge. Journal of Parasitology 85, 301–305. Davis, S.W., Speer, C.A., Dubey, J.P., 1991. In vitro cultivation of Sarcocystis neurona from the spinal cord of a horse with equine protozoal myelitis. Journal of Parasitology 77, 789–792. Dubey, J.P., Lindsay, D.S., 1998. Isolation in immunodeficient mice of Sarcocystis neurona from opossum (Didelphis virginiana) faeces, and its differentiation from Sarcocystis falcatula. International Journal of Parasitology 28, 1823–1828. Dubey, J.P., Lindsay, D.S., Saville, W.J., Reed, S.M., Granstrom, D.E., Speer, C.A., 2001. A review of Sarcocystis neurona and equine protozoal myeloencephalitis (EPM). Veterinary Parsitology 95, 89– 131. Dubey, J.P., Mattson, D.E., Speer, C.A., Baker, R.J., Mulrooney, D.M., Tornquist, S.J., Hamir, A.N., Gerros, T.C., 1999. Characterization of a Sarcocystis neurona isolate SN6 from a naturally infected horse from Oregon. Journal of Eukaryotic Microbiology 46, 500–506.
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Fenger, C.K., Granstrom, D.E., Gajadhar, A.A., Williams, N.M., Mccrillis, S.A., Stamper, S., Langemeier, J.L., Dubey, J.P., 1997. Experimental induction of equine protozoal myeloencephalitis in horses using Sarcocystis sp. sporocysts from the opossum (Didelphis virginiana). Veterinary Parasitology 68, 199–213. Fenger, C.K., Granstrom, D.E., Langemeier, J.L., Stamper, S., Donahue, J.M., Patterson, J.S., Gajadhar, A.A., Marteniuk, J.V., Xiaomin, Z., Dubey, J.P., 1995. Identification of opossums (Didelphis virginiana) as the putative definitive host of Sarcocystis neurona. Journal of Parasitology 81, 916–919. Knowles Jr., D.P., Kappmeyer, L.S., Perryman, L.E., 1994. Specific immune responses are required to control parasitemia in Babesia equi infection. Infection and Immunity 62, 1909–1913. Mackay, R.J., 1997. Equine protozoal myeloencephalitis. Veterinary Clinics of North America, Equine Practice 13, 79–96. Marsh, A.E., Barr, B.C., Lakritz, J., Nordhausen, R., Madigan, J.E., Conrad, P.A., 1997. Experimental infection of nude mice as a model for Sarcocystis neurona-associated encephalitis. Parasitology Research 83, 706–711.
Marsh, A.E., Barr, B.C., Madigan, J., Lakritz, J., Conrad, P.A., 1996. Sequence analysis and polymerase chain reaction amplification of small subunit ribosomal DNA from Sarcocystis neurona. American Journal of Veterinary Research 57, 975–981. Perryman, L.E., Mcguire, T.C., Crawford, T.B., 1978. Maintenance of foals with combined immunodeficiency: causes and control of secondary infections. American Journal of Veterinary Research 39, 1043–1047. Perryman, L.E., O’rourke, K.I., Mcguire, T.C., 1988. Immune responses are required to terminate viremia in equine infectious anemia lentivirus infection. Journal of Virology 62, 3073–3076. Shin, E.K., Perryman, L.E., Meek, K., 1997. A kinase-negative mutation of DNA-PK(CS) in equine SCID results in defective coding and signal joint formation. Journal of Immunology 158, 3565–3569. Tanhauser, S.M., Yowell, C.A., Cutler, T.J., Griner, E.C., Mackay, R.J., Dame, J.B., 1999. Multiple DNA markers differentiate Sarcocystis neurona and Sarcocystis falcatula. Journal of Parasitology 85, 221–228.
Sarcocystis neurona: parasitemia in a severe combined immunodeficient (SCID) horse fed sporocysts Maureen T. Long,a Melissa T. Mines,b Donald P. Knowles,c Susan M. Tanhauser,a John B. Dame,d Timothy J. Cutler,a Robert J. MacKay,a and Debra C. Sellonb,* a
Department of Large Animal Clinical Sciences, University of Florida, Gainesville, FL 32610, USA Department of Veterinary Clinical Sciences, Washington State University, Pullman, WA 99164, USA Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164, USA d Department of Pathobiology, University of Florida, Gainesville, FL 32610, USA b
c
Received 23 April 2001; accepted 7 March 2002
Abstract Sarcocystis neurona was isolated from the blood of a 5-month-old Arabian foal with severe combined immunodeficiency. The foal had been inoculated approximately 3 weeks previously with 5 105 sporocysts that were isolated from the intestines of an opossum and identified by restriction enzyme analysis of PCR products as S. neurona. The isolate obtained from the blood of this foal was characterized by genetic, serologic, and morphologic methods and identified as S. neurona (WSU1). This represents the first time that S. neurona has been isolated from any tissue after experimental infection of a horse. This is also the first time a parasitemia has been detected during either natural or experimental infection. The severe combined immunodeficiency foal model provides a unique opportunity to study the pathogenesis of S. neurona infection in horses and to determine the role of the immune system in the control of infection with and development of neurologic disease. Index Descriptors and Abbreviations. Sarcocystis neurona; protozoa; horse; equine; EPM, equine protozoal myeloencephalitis; SCID, severe combined immunodeficiency; CNS, central nervous system; PCR, polymerase chain reaction; IFN-c, interferon gamma; KO, knockout; DNA, deoxyribonucleic acid; PID, post-infection day; DMEM, Dulbecco’s modified essential media; SC, subcutaneous; IP, intraperitoneal; M, molar; CSF, cerebrospinal fluid. Ó 2002 Elsevier Science (USA). All rights reserved.
EPM is a progressive neurologic disease of horses caused by a protozoal infection of the CNS. The most commonly identified parasite associated with this disease is the apicomplexan protozoa, Sarcocystis neurona (Davis et al., 1991; Dubey et al., 2001). There is considerable evidence to show that the opossum is the definitive host in the life cycle of this parasite and that horses are ‘‘accidental’’ hosts after ingestion of sporocysts passed in opossum feces (Fenger et al., 1995). A high percentage of horses in many areas of the United States have serum antibodies to S. neurona but few develop neurologic diseases (Mackay, 1997). Experimental oral infection of immunocompetent horses with S. neurona sporocysts shed by opossums does not consistently result in clinical signs (Dubey *
Corresponding author. Fax: +509-335-0880. E-mail address: [email protected] (D.C. Sellon).
et al., 2001). Similarly, the infection of immunocompetent Balb/c mice with S. neurona sporocysts or merozoites does not result in neurologic abnormalities (Marsh et al., 1997). However, an infection of IFN-k KO mice by oral or parenteral routes consistently results in a fulminant neurologic disease (Dubey and Lindsay, 1998). Intraperitoneal infection of nude mice with culture-derived merozoites of S. neurona induced encephalitis, but two similarly infected SCID mice did not develop neurologic abnormalities (Marsh et al., 1997). Arabian horses with SCID lack specific B and T cell responses because of a mutation in the DNA-dependent kinase catalytic subunit (Shin et al., 1997), offering the opportunity to characterize S. neurona infection in the immunodeficient horse. We hypothesized that S. neurona infection of a SCID foal will result in CNS infection and neuropathology.
0014-4894/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 1 4 - 4 8 9 4 ( 0 2 ) 0 0 0 1 2 - 7
M.T. Long et al. / Experimental Parasitology 100 (2002) 150–154
A 33-day-old male Arabian horse homozygous for the SCID trait was infected via nasogastric tube with 5 105 S. neurona sporocysts retrieved from the intestines of wild opossums (Cutler et al., 1999). Sporocysts were identified as S. neurona by a PCR protocol that differentiates S. neurona from the closely related opossum isolates, S. falcatula, S. speeri, and 1085 (Tanhauser et al., 1999). Immediately after birth, the foal suckled colostrum from his EPM seronegative dam. The foal was immediately placed in isolation with his dam and removed from his dam at approximately 21 days of age. The day before infection, day 0, and everyday during the study period complete physical and neurologic examinations were performed. Hemogram and serum biochemical profile were evaluated at intervals. Peripheral blood for buffy coat PCR and culture were collected prior to infection and on days 1–8, 10, 14, 17, 21, 24, 28, and 31. Baseline bloodwork (hemogram and serum biochemical profile) and physical and neurologic examination parameters were within normal limits for the SCID foal (with the exception of the absence of lymphocytes). After infection, the foal was intermittently neutropenic, hyperfibrinogenemic, and febrile with mild diarrhea and/or a cough and increased lung sounds. These findings were attributed to secondary infections common in SCID foals (Perryman et al., 1978) and the foal was treated with ceftiofur and gentamicin. At PID 39 the foal demonstrated a mild knuckling of the right hind limb that was intermittently apparent until euthanasia. On PID 53 the foal was euthanized and CSF and appropriate tissues were obtained for culture and PCR analysis. Buffy coat samples or homogenized tissue samples were suspended in DMEM and inoculated over a monolayer of bovine turbinate cells passaged 24 h previously. Cultures were examined microscopically twice a week for evidence of cytopathology and/or parasites. At 12–18 weeks of culture, cultures were assessed by PCR and immunohistochemistry. PCR positive cultures were maintained. Cultures of buffy coat samples obtained from the SCID foal on PID 21, 24, and 31 were positive by PCR for S. neurona at 12 weeks of culture, as were cultures of tissues from the brain of the SCID foal. At approximately 14 weeks of culture, an apicomplexan parasite was identified only in the PID 21 buffy coat culture from the SCID foal (Fig. 1). This organism was designated the WSU1 isolate of S. neurona. Protozoal organisms were not isolated from any other PCR positive culture. Development of the protozoal organism in culture was asynchronous with organisms dividing by endopologeny (Fig. 1). For electron microscopy, cells were pelleted by centrifugation, washed in cold PBS, and then fixed in cold 2.5% glutaraldehyde in 1% cacodylate buffer (pH 7.2). The pellet was post-fixed in 2% w/v osmium tetroxide,
151
Fig. 1. Photomicrograph of a cytospin preparation of the cell culture supernatant of an isolate consistent with S. neurona. This isolate was cultured from the blood of an SCID foal 21 days after oral infection. This is a photomicrograph of a cell with nucleus (a) and ruptured cytoplasm, which contained schizonts composed of several merozoites 4–5 lm (b). Cell free merozoites are present throughout preparation. These forms are generally larger than the intracellular forms, measuring 4–6 lm.
dehydrated in ethanol, passed through propylene oxide and embedded in Epon/Araldite (Polysciences, Warrington, PA). Thin sections were stained in uranyl acetate and lead citrate and examined using a transmission electron microscope operating at 80 kV. Electron microscopic evaluation revealed mature cell-free merozoites that ranged from approximately 4 to 6 lm in length; intracellular forms consisted of schizonts containing merozoites of approximately 4–5 lm in length. Merozoites isolated from the day 21 buffy coat culture were morphologically indistinguishable from S. neurona by light or electron microscopy. The observed morphology was consistent with S. neurona with single or clustered merozoites that contained a single nucleus, conoid, anterior placement of micronemes, and no rhoptries. Three isolates of S. neurona obtained from the spinal cord of clinically neurologic horses were used for comparison to the WSU1 isolate for morphologic, genetic, and antigenic analyses. The isolate UCD1 was isolated from a horse in California (Marsh et al., 1996). The isolate SN6, provided by S. Tornquist of Oregon State University, was isolated from a horse in Oregon (Dubey et al., 1999). The isolate FL1 was isolated from a horse in Florida. S. falcatula was purchased from ATCC (Beltsville, MD). All isolates were passaged as previously described in bovine turbinate cells (Davis et al., 1991). For PCR analysis, DNA was extracted from merozoites freshly blown from cell cultures of UCD1, SN6, FL1, S. falcatula, and WSU1. These samples were amplified by PCR using S. neurona specific primers (Tanhauser et al., 1999). Controls consisted of negative tissue controls (either uninfected equine tissues or noninoculated cell cultures) and a no DNA control. The
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products of the primers were cut by restriction enzymes to confirm the identity of WSU1. Amplification products were sequenced by the ABI prism method and compared to known DNA sequences (GenBank #AF093158 and GenBank #AF093153) of S. neurona and related species (Marsh et al., 1996). WSU1 was confirmed to be S. neurona by PCR and restriction enzyme analysis (Fig. 2) and DNA sequencing of PCR amplified DNA (Fig. 3). PCR was performed on all isolates simultaneously. A normal adult horse seronegative to S. neurona was inoculated intramuscularly with 1 106 –1 108 merozoites of the WSU1 isolate every two weeks for four doses. Serum was collected by jugular venipuncture on the day of the first inoculation and weekly thereafter. During the study period the horse was maintained in a pasture that was fenced and electronically wired to prevent the entering of small, wild mammals. Seroconversion to S. neurona in this horse infected with WSU1 was independently verified by two commercial laboratories. CSF obtained from the atlanto-occipital space of
Fig. 2. (a)–(e) Ethidium bromide-stained agarose gel (1.8%) demonstrating PCR products differentiating all of the S. neurona isolates from S. falcatula. Lane 1 contains a 100 bp ladder with the brightest visible band equal to approximately 500 bp (Promega), lanes 2–7 on all gels consist of S. neurona (UCD1), S. neurona (SN6), S. falcatula (ATCC #), S. neurona (WSU1) S. neurona (FL1), and water control, respectively. (a) Top-PCR products from primer pair JNB25/JD396 demonstrating a 334 base pair sequence for both S. neurona and S. falcatula; bottom-primers JNB63/JNB65 generate a 1202 bp product for S. neurona and a 959 bp primer for S. falcatula. (b) Top-PCR primers JNB48/JNB50 generate a 870 bp product for both S. neurona and S. falcatula; bottom-PCR primers JNB33/JNB54 generate a 1100 bp product for both S. neurona and S. falcatula. (c) Top-restriction enzyme digestion with HindIII cuts the products of JNB25/JD396 primers into 180 and 154 bp fragments for the S. falcatula isolate and did not cut the S. neurona product; bottom-restriction enzyme digestion with HinfI cuts the same primer products into 64, 108, and 62 bp fragments for S. neurona isolates and 170 and 164 bp fragments for S. falcatula. (d) Top-restriction enzyme digest with DraI of the JNB33/ JNB54 fragments resulted in 884 and 216 bp fragments for S. neurona and did not cut S. falcatula; bottom-restriction enzyme digest with HinfI of the JNB33/JNB54 PCR product does not cut the S. neurona fragment and generates a 745 and 355 bp for S. falcatula. (e) Top-restriction enzyme digest with RsaI of the JNB48/JNB50 PCR product resulted in a 665 bp fragment for S. neurona and cuts S. falcatula into 428 and 237 bp fragments.
Fig. 3. Four rows of sequence data spanning approximately 250 bp of the PCR target from JNB25/JD396 (TGF). The sequences (top to bottom) from UCD1, WSU1, S. falcatula, 1085, respectively, highlight the single and multiple base pair differences between the isolates. The isolate, WSU1, obtained from an SCID is completely identical to UCD1 (GenBank #AF093158 and GenBank #AF093153).
this horse at approximately six months post-inoculation was positive by Western blot for antibody to S. neurona. Detailed neurologic examination of this horse remained within normal limits. Sera obtained from two horses experimentally infected with sporocysts from opossum feces identified by PCR as S. neurona were compared to the serum obtained from the horse infected with WSU1 in a Western blot format with WSU1 as antigen. For immunoblot analyses, aliquots of WSU1 isolate were mixed with PBS and SDS–PAGE buffer, boiled for 5 min and electrophoresed in a 7.5–17.5% SDS–polyacrylamide gradient gel and 4% stacking gel using the one-dimensional discontinuous method of Laemmli. Biotin-conjugated mol wt standards (Bio-Rad Laboratories, Hercules, CA) were used. After SDS–PAGE electrophoresis, the antigens were electrophoretically transferred to nitrocellulose filters (Bio-Rad Laboratories, Hercules, CA). Serum from sample horses was diluted in blocking buffer and incubated with filters at room temperature. After incubation and washing in buffer, bound antibodies were detected by incubation with peroxidase-conjugated rabbit anti-horse IgG1 (Pierce, Rockford, IL). All bound antibodies and mol wt standards were visualized by chemiluminescence (ECL, Amersham Life Sciences). Reactivity post-inoculation was consistent with exposure to S. neurona (data not shown). Culture supernatant from bovine turbinate cells infected with the WSU1 isolate from the SCID foal was harvested, centrifuged, and resuspended in cell culture media. Eight 6-week-old IFN-c KO mice (BALB/cIfngtmits ) were infected with harvested organisms. Two
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mice each were inoculated with either 105 organisms SC, 106 organisms SC, 106 organisms IP, or 107 organisms IP. One mouse from each KO group was sacrificed at 30 days post-infection; the second mouse from each KO group was sacrificed at 60 days post-infection or at the onset of clinical signs. Tissues including lung, liver, kidney, cardiac muscle, brain, and spinal cord were harvested for histopathology. Representative samples of each tissue were extracted for DNA and analyzed by PCR. One mouse infected subcutaneously with 105 merozoites of the WSU1 isolate of S. neurona developed severe neurologic signs at approximately 48 days postinfection and was euthanized at that time. The mouse had a right head tilt and circled compulsively to the left. No other mice exhibited recognizable neurologic abnormalities. All other mice were euthanized at 30 or 60 days post-infection. Polymerase chain reaction analysis and culture results of brain tissue for S. neurona were positive in the mice both at 30 and 60 days postinfection. Brain and spinal cord from the mouse that developed neurologic signs were PCR positive for S. neurona at the time of euthanasia and histopathological analysis revealed pathological abnormalities consistent with S. neurona infection. These data represent the first isolation of S. neurona obtained after experimental infection of a horse. This study is also the first time that S. neurona has been isolated after infection of the horse from a site other than the CNS. Confirmation that this isolate is indeed S. neurona is based upon the morphology at both light and electron microscopic levels. Genetic sequencing reveals an organism identical to that described previously for S. neurona and neither S. falcatula nor 1085 (Tanhauser et al., 1999). Antibody responses in horses inoculated with WSU1 were indistinguishable by Western blot from antibody responses of horses infected with other isolates of S. neurona. Phenotypically, the WSU1 isolate is neuropathogenic in gamma interferon knockout mice (Dubey and Lindsay, 1998). It is surprising that only one mouse developed obvious neurologic deficits. However, the majority of these mice were PCR positive for S. neurona in their brain and spinal cord. The absence of obvious neurologic deficits in most mice may be a result of euthanasia prior to the development of signs, a decreased pathogenicity of the WSU1 isolate because of passage through a SCID foal or multiple cell passages, or the difficulties in accurately detecting subtle neurologic deficits in mice. S. neurona likely undergoes excystation and intestinal invasion after oral infection with sporocysts. Before neural invasion takes place, presumably a parasitemia occurs. This has not been characterized previously by culture or PCR. The isolation of the organism at PID 21 appears to be appropriately timed for parasitemia, leading to infection within the CNS. Horses seroconvert
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at approximately 28 days post-inoculation, presumably corresponding to the time after peak parasitemia (Fenger et al., 1997). The immunodeficient state of this foal likely permitted a high enough parasitemia to allow for isolation of the organism from the blood of this foal. The SCID foal offers tremendous potential for the development of a model of S. neurona infection in the horse for characterization of the kinetics of equine infection. The lack of development of abnormal neurologic signs in this foal was surprising. This could be the result of infection with a strain of S. neurona that is not neurovirulent or it could indicate a role of specific immune responses in the neuropathogenesis of infection. Infection of additional SCID hosts will be necessary to develop this model to consistently induce CNS infection, disease, and parasite isolation from the nervous tissue. This model has been used successfully in other equine diseases including equine infectious anemia and babesiosis to define the role of the immune system in disease pathogenesis or protection from infection (Knowles et al., 1994; Perryman et al., 1988). Infection of SCID foals and full development of this model will allow this type of investigation into the immunopathogenesis of S. neurona.
Acknowledgments This work was supported by funds from the State of Washington, Grayson-Jockey Club Research Foundation, and the State of Florida Division of Pari-mutuel Wagering. We gratefully acknowledge the expert technical assistance of Ms. Tressa Hochstatter, Karen Gillis, Francesca Griffen, and Susan Tornquist.
References Cutler, T.J., Mackay, R.J., Ginn, P.E., Greiner, E.C., Porter, R., Yowell, C.A., Dame, J.B., 1999. Are Sarcocystis neurona and Sarcocystis falcatula synonymous? A horse infection challenge. Journal of Parasitology 85, 301–305. Davis, S.W., Speer, C.A., Dubey, J.P., 1991. In vitro cultivation of Sarcocystis neurona from the spinal cord of a horse with equine protozoal myelitis. Journal of Parasitology 77, 789–792. Dubey, J.P., Lindsay, D.S., 1998. Isolation in immunodeficient mice of Sarcocystis neurona from opossum (Didelphis virginiana) faeces, and its differentiation from Sarcocystis falcatula. International Journal of Parasitology 28, 1823–1828. Dubey, J.P., Lindsay, D.S., Saville, W.J., Reed, S.M., Granstrom, D.E., Speer, C.A., 2001. A review of Sarcocystis neurona and equine protozoal myeloencephalitis (EPM). Veterinary Parsitology 95, 89– 131. Dubey, J.P., Mattson, D.E., Speer, C.A., Baker, R.J., Mulrooney, D.M., Tornquist, S.J., Hamir, A.N., Gerros, T.C., 1999. Characterization of a Sarcocystis neurona isolate SN6 from a naturally infected horse from Oregon. Journal of Eukaryotic Microbiology 46, 500–506.
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Fenger, C.K., Granstrom, D.E., Gajadhar, A.A., Williams, N.M., Mccrillis, S.A., Stamper, S., Langemeier, J.L., Dubey, J.P., 1997. Experimental induction of equine protozoal myeloencephalitis in horses using Sarcocystis sp. sporocysts from the opossum (Didelphis virginiana). Veterinary Parasitology 68, 199–213. Fenger, C.K., Granstrom, D.E., Langemeier, J.L., Stamper, S., Donahue, J.M., Patterson, J.S., Gajadhar, A.A., Marteniuk, J.V., Xiaomin, Z., Dubey, J.P., 1995. Identification of opossums (Didelphis virginiana) as the putative definitive host of Sarcocystis neurona. Journal of Parasitology 81, 916–919. Knowles Jr., D.P., Kappmeyer, L.S., Perryman, L.E., 1994. Specific immune responses are required to control parasitemia in Babesia equi infection. Infection and Immunity 62, 1909–1913. Mackay, R.J., 1997. Equine protozoal myeloencephalitis. Veterinary Clinics of North America, Equine Practice 13, 79–96. Marsh, A.E., Barr, B.C., Lakritz, J., Nordhausen, R., Madigan, J.E., Conrad, P.A., 1997. Experimental infection of nude mice as a model for Sarcocystis neurona-associated encephalitis. Parasitology Research 83, 706–711.
Marsh, A.E., Barr, B.C., Madigan, J., Lakritz, J., Conrad, P.A., 1996. Sequence analysis and polymerase chain reaction amplification of small subunit ribosomal DNA from Sarcocystis neurona. American Journal of Veterinary Research 57, 975–981. Perryman, L.E., Mcguire, T.C., Crawford, T.B., 1978. Maintenance of foals with combined immunodeficiency: causes and control of secondary infections. American Journal of Veterinary Research 39, 1043–1047. Perryman, L.E., O’rourke, K.I., Mcguire, T.C., 1988. Immune responses are required to terminate viremia in equine infectious anemia lentivirus infection. Journal of Virology 62, 3073–3076. Shin, E.K., Perryman, L.E., Meek, K., 1997. A kinase-negative mutation of DNA-PK(CS) in equine SCID results in defective coding and signal joint formation. Journal of Immunology 158, 3565–3569. Tanhauser, S.M., Yowell, C.A., Cutler, T.J., Griner, E.C., Mackay, R.J., Dame, J.B., 1999. Multiple DNA markers differentiate Sarcocystis neurona and Sarcocystis falcatula. Journal of Parasitology 85, 221–228.