The nine-banded armadillo (Dasypus novemcinctus) is an intermediate host for Sarcocystis neurona

The nine-banded armadillo (Dasypus novemcinctus) is an intermediate host of at least three species of Sarcocystis, Sarcocystis dasypi, Sarcocystis diminuta, and an unidentified species; however, life cycles of these species have not been determined. Following feeding of armadillo muscles containing sarcocysts to the Virginia opossum (Didelphis virginiana), the opossums shed sporulated Sarcocystis sporocysts in their faeces. Mean dimensions for sporocysts were 11.0x7.5 microm and each contained four sporozoites and a residual body. Sporocysts were identified as Sarcocystis neurona using PCR and DNA sequencing. A 2-month-old foal that was negative for S. neurona antibodies in the CSF was orally inoculated with 5x10(5) sporocysts. At 4 weeks post-infection, the foal had a 'low positive' result by immunoblot for CSF antibodies to S. neurona and by week 6 had a 'strong positive' CSF result and developed an abnormal gait with proprioceptive deficits and ataxia in all four limbs. Based on the results of this study, the nine-banded armadillo is an intermediate host of S. neurona.     

The striped skunk (Mephitis mephitis) is an intermediate host for Sarcocystis neurona

Striped skunks, initially negative for antibodies to Sarcocystis neurona, formed sarcocysts in skeletal muscles after inoculation with S. neurona sporocysts collected from a naturally infected Virginia opossum (Didelphis virginiana). Skunks developed antibodies to S. neurona by immunoblot and muscles containing sarcocysts were fed to laboratory-reared opossums which then shed sporulated Sarcocystis sporocysts in their faeces. Mean dimensions for sporocysts were 11.0 x 7.5 microm and each contained four sporozoites and a residuum. Sarcocysts from skunks and sporocysts from opossums fed infected skunk muscle were identified as S. neurona using PCR and DNA sequence analysis. A 2-month-old, S. neurona-naive pony foal was orally inoculated with 5 x 10(5) sporocysts. Commercial immunoblot for antibodies to S. neurona performed using CSF collected from the inoculated pony was low positive at 4 weeks p.i., positive at 6 weeks p.i., and strong positive at 8 weeks p.i. Gamma-interferon gene knockout mice inoculated with skunk/opossum derived sporocysts developed serum antibodies to S. neurona and clinical neurologic disease. Merozoites of S. neurona present in the lung, cerebrum, and cerebellum of mice were detected by immunohistochemistry using polyclonal antibodies to S. neurona. Based on the results of this study, the striped skunk is an intermediate host of S. neurona.     

Early migration of Sarcocystis neurona in ponies fed sporocysts

Sarcocystis neurona is the most important cause of equine protozoal myeloencephalitis (EPM), a neurologic disease of the horse. In the present work, the kinetics of S. neurona invasion is determined in the equine model. Six ponies were orally inoculated with 250 x 10(6) S. neurona sporocysts via nasogastric intubation and killed on days 1, 2, 3, 5, 7, and 9 postinoculation (PI). At necropsy, tissue samples were examined for S. neurona infection. The parasite was isolated from the mesenteric lymph nodes at 1, 2, and 7 days PI; the liver at 2, 5, and 7 days PI; and the lungs at 5, 7, and 9 days PI by bioassays in interferon gamma gene knock out mice (KO) and from cell culture. Microscopic lesions consistent with an EPM infection were observed in brain and spinal cord of ponies killed 7 and 9 days PI. Results suggest that S. neurona disseminates quickly in tissue of naive ponies.     

Isolation and Identification of 5′-Methylthioadenosine Sulfoxide from Human Urine

Abstract

An adenine nucleoside isolated from human urine has been identified by mass spectra and other techniques as 5′-deoxy-5′-methyl-thioadenosine sulfoxide. Elevated levels (3–5 nmols/μmol creatinine) were noted in two children with severe combined immunodeficiency.     

Are Sarcocystis neurona and Sarcocystis falcatula synonymous? A horse infection challenge

Equine protozoal myeloencephalitis (EPM) is a debilitating neurologic disease of the horse. The causative agent. Sarcocystis neurona, has been suggested to be synonymous with Sarcocystis falcatula, implying a role for birds as intermediate hosts. To test this hypothesis, opossums (Didelphis virginiana) were fed muscles containing S. falcatula sarcocysts from naturally infected brown-headed cowbirds (Molothrus ater). Ten horses were tested extensively to ensure no previous exposure to S. neurona and were quarantined for 14 days, and then 5 of the horses were each administered 10(6) S. falcatula sporocysts collected from laboratory opossums. Over a 12-wk period, 4 challenged horses remained clinically normal and all tests for S. neurona antibody and DNA in serum and cerebrospinal fluid were negative. Rechallenge of the 4 seronegative horses had identical results. Although 1 horse developed EPM, presence of S. neurona antibody prior to challenge strongly indicated that infection occurred before sporocyst administration. Viability of sporocysts was confirmed by observing excystation in equine bile in vitro and by successful infection of naive brown-headed cowbirds. These data suggest that S. falcatula and S. neurona are not synonymous. One defining distinction is the apparent inability of S. falcatula to infect horses, in contrast to S. neurona, which was named when cultured from equine spinal cord.     

Biological characterisation of Sarcocystis neurona isolated from a Southern sea otter (Enhydra lutris nereis)

Sarcocystis neurona was isolated from the brain of a juvenile, male southern sea otter (Enhydra lutris nereis) suffering from CNS disease. Schizonts and merozoites in tissue sections of the otter's brain reacted with anti-S. neurona antiserum immunohistochemically. Development in cell culture was by endopolyogeny and mature schizonts were first observed at 3 days postinoculation. PCR of merozoite DNA using primer pairs JNB33/JNB54 and restriction enzyme digestion of the 1100 bp product with Dra I indicated the organism was S. neurona. Four of four interferon-gamma gene knockout mice inoculated with merozoites developed S. neurona-associated encephalitis. Antibodies to S. neurona but not Sarcocystis falcatula, Toxoplasma gondii, or Neospora caninum were present in the serum of inoculated mice. This is the first isolation of S. neurona from the brain of a non-equine host.     

Naturally occurring Sarcocystis infection in domestic cats (Felis catus)

Equine protozoal myeloencephalitis is an important neurological disease of horses in the United States. Consequently, there is an active research effort to identify hosts associated with the primary causative agent, Sarcocystis neurona. The purpose of this study was to determine whether the domestic cat (Felis catus) is a natural host for S. neurona. Muscle sections from 50 primarily free-roaming domestic cats were examined for the presence of sarcocysts. Serum from cats in this group and another group of 50 free-roaming cats were evaluated for the presence of S. neurona antibody. Sarcocysts were found in five of 50 (10%) cats, and S. neurona antibody in five of 100 (5%) cats. Morphological, molecular (including ribosomal RNA genes), and biological characterisation of these sarcocysts showed that they were not S. neurona or S. neurona-like. Sarcocysts found in the cats were identified morphologically as Sarcocystis felis, a common parasite of wild felids. The life cycle of S. felis is not known, and prior to this study, no molecular marker for S. felis existed. Although cats were found to be infected with S. felis sarcocysts, serological data provided evidence of possible infection with S. neurona as well. Further work is needed to determine the role of the domestic cat in the life cycle of S. neurona.     

Sarcocystis neurona infections in sea otter (Enhydra lutris): evidence for natural infections with sarcocysts and transmission of infection to opossums (Didelphis virginiana).

Although Sarcocystis neurona has been identified in an array of terrestrial vertebrates, recent recognition of its capacity to infect marine mammals was unexpected. Here, sarcocysts from 2 naturally infected sea otters (Enhydra lutris) were characterized biologically, ultrastructurally, and genetically. DNA was extracted from frozen muscle of the first of these sea otters and was characterized as S. neurona by polymerase chain reation (PCR) amplification followed by restriction fragment length polymorphism analysis and sequencing. Sarcocysts from sea otter no. 1 were up to 350 microm long, and the villar protrusions on the sarcocyst wall were up to 1.3 microm long and up to 0.25 microm wide. The villar protrusions were tapered towards the villar tip. Ultrastructurally, sarcocysts were similar to S. neurona sarcocysts from the muscles of cats experimentally infected with S. neurona sporocysts. Skeletal muscles from a second sea otter failed to support PCR amplification of markers considered diagnostic for S. neurona but did induce the shedding of sporocysts when fed to a laboratory-raised opossum (Didelphis virginiana). Such sporocysts were subsequently fed to knockout mice for the interferon-gamma gene, resulting in infections with an agent identified as S. neurona on the basis of immunohistochemistry, serum antibodies, and diagnostic sequence detection. Thus, sea otters exposed to S. neurona may support the development of mature sarcocysts that are infectious to competent definitive hosts.     

Lack of Sarcocystis neurona antibody response in Virginia opossums (Didelphis virginiana) fed Sarcocystis neurona-infected muscle tissue

Serum was collected from laboratory-reared Virginia opossums (Didelphis virginiana) to determine whether experimentally infected opossums shedding Sarcocystis neurona sporocysts develop serum antibodies to S. neurona merozoite antigens. Three opossums were fed muscles from nine-banded armadillos (Dasypus novemcinctus), and 5 were fed muscles from striped skunks (Mephitis mephitis). Serum was also collected from 26 automobile-killed opossums to determine whether antibodies to S. neurona were present in these opossums. Serum was analyzed using the S. neurona direct agglutination test (SAT). The SAT was modified for use with a filter paper collection system. Antibodies to S. neurona were not detected in any of the serum samples from opossums, indicating that infection in the opossum is localized in the small intestine. Antibodies to S. neurona were detected in filter-paper-processed serum samples from 2 armadillos naturally infected with S. neurona.     

Horses experimentally infected with Sarcocystis neurona develop altered immune responses in vitro

Equine protozoal myeloencephalitis (EPM) due to Sarcocystis neurona infection is 1 of the most common neurologic diseases in horses in the United States. The mechanisms by which most horses resist disease, as well as the possible mechanisms by which the immune system may be suppressed in horses that develop EPM, are not known. Therefore, the objectives of this study were to determine whether horses experimentally infected with S. neurona developed suppressed immune responses. Thirteen horses that were negative for S. neurona antibodies in serum and cerebrospinal fluid (CSF) were randomly assigned to control (n = 5) or infected (n = 8) treatment groups. Neurologic exams and cerebrospinal fluid analyses were performed prior to, and following, S. neurona infection. Prior to, and at multiple time points following infection, immune parameters were determined. All 8 S. neurona-infected horses developed clinical signs consistent with EPM, and had S. neurona antibodies in the serum and CSF. Both infected and control horses had increased percentages (P < 0.05) of B cells at 28 days postinfection. Infected horses had significantly decreased (P < 0.05) proliferation responses as measured by thymidine incorporation to nonspecific mitogens phorbol myristate acetate (PMA) and ionomycin (I) as soon as 2 days postinfection.     

Life cycle of Sarcocystis neurona in its natural intermediate host,the raccoon,Procyon lotor

Sarcocystis neurona causes encephalomyelitis in many species of mammals and is the most important cause of neurologic disease in the horse. Its complete life cycle is unknown, particularly its development and localization in the intermediate host. Recently, the raccoon (Procyon lotor) was recognized as a natural intermediate host of S. neurona. In the present study, migration and development of S. neurona was studied in 10 raccoons that were fed S. neurona sporocysts from experimentally infected opossums; 4 raccoons served as controls. Raccoons were examined at necropsy 1, 3, 5, 7, 10, 14, 15, 22, 37, and 77 days after feeding on sporocysts (DAFS). Tissue sections of most of the organs were studied histologically and reacted with anti-S. neurona-specific polyclonal rabbit serum in an immunohistochemical test. Parasitemia was demonstrated in peripheral blood of raccoons 3 and 5 DAFS. Individual zoites were seen in histologic sections of intestines of raccoons euthanized 1, 3, and 5 DAFS. Schizonts and merozoites were seen in many tissues 7 to 22 DAFS, particularly in the brain. Sarcocysts were seen in raccoons killed 22 DAFS. Sarcocysts at 22 DAFS were immature and seen only in skeletal muscle. Mature sarcocysts were seen in all skeletal samples, particularly in the tongue of the raccoon 77 DAFS; these sarcocysts were infective to laboratory-raised opossums. This is the first report of the complete development of S. neurona schizonts and sarcocysts in a natural intermediate host.     

Isolation in immunodeficient mice of Sarcocystis neurona from opossum (Didelphis virginiana) faeces, and its differentiation from Sarcocystis falcatula

Sarcocystis neurona was isolated in nude mice and gamma-interferon knockout mice fed sporocysts from faeces of naturally infected opossums (Didelphis virginiana). Mice fed sporocysts became lethargic and developed encephalitis. Protozoa were first found in the brain starting 21 days post-inoculation. Sarcocystis neurona was recovered in cell culture from the homogenate of liver, spleen and brain of a nude mouse 11 days after feeding sporocysts. The protozoa in mouse brain and in cell culture multiplied by schizogony and mature schizonts often had a residual body. Sarcocystis falcatula, which has an avian-opossum cycle, was not infective to nude or knockout mice. Protozoa were not found in tissues of nude mice or knockout mice after subcutaneous injection with culture-derived S. falcatula merozoites and sporocysts from the faeces of opossums presumed to contain only S. falcatula. Results demonstrate that S. neurona is distinct from S. falcatula, and that opossums are hosts for both species.     

Depletion of natural killer cells does not result in neurologic disease due to Sarcocystis neurona in mice with severe combined immunodeficiency

Sarcocystis neurona is an apicomplexan parasite that is the primary etiologic agent of equine protozoal myeloencephalitis in horses. Protective immune responses in horses have not been determined, but interferon-gamma (IFN-gamma) is considered critical for protection from neurologic disease in mice. The role of adaptive and innate immune responses in control of parasites was explored by infecting BALB/c, IFN-gamma knockout (GKO), and severe combined immune deficient (SCID) mice with S. neurona (10(4) sporocysts/mouse). Immune competent BALB/c mice eliminated parasites within 30 days, with no sign of neurologic disease, whereas GKO mice developed fulminant neurologic disease. In contrast, SCID mice remained healthy throughout the experimental period despite the persistence of parasite at low levels in some mice. Treatment with anti-IFN-gamma antibody resulted in neurologic disease in infected SCID mice. Although SCID mice lack adaptive immune responses, they have natural killer (NK) cells capable of producing significant quantities of IFN-gamma. Therefore, SCID mice were infected with sporocysts of S. neurona and treated with anti-asialo GM1. Depletion of NK cells, confirmed by flow cytometry, did not result in neurologic disease in SCID mice. These results indicate that IFN-gamma mediates protection from neurologic disease in SCID mice. Protective levels of IFN-gamma may originate from a low number of nondepleted NK cells or from a non-T cell, non-NK cell population.     

Prevalence of agglutinating antibodies to Sarcocystis neurona in skunks (Mephitis Mephitis), raccoons (Procyon lotor), and opossums (Didelphis Virginiana) from Connecticut

Equine protozoal myeloencephalitis is the most important protozoan disease of horses in North America and is usually caused by Sarcocystis neurona. Natural cases of encephalitis caused by S. neurona have been reported in skunks (Mephitis mephitis) and raccoons (Procyon lotor). Opossums (Didelphis spp.) are the only known definitive host. Sera from 24 striped skunks, 12 raccoons, and 7 opossums (D. virginiana) from Connecticut were examined for agglutinating antibodies to S. neurona using the S. neurona agglutination test (SAT) employing formalin-fixed merozoites as antigen. The SAT was validated for skunk sera using pre- and postinfection serum samples from 2 experimentally infected skunks. Of the 24 (46%) skunks 11 were positive, and all 12 raccoons were positive for S. neurona antibodies. None of the 7 opossums was positive for antibodies to S. neurona. These results suggest that exposure to sporocysts of S. neurona by intermediate hosts is high in Connecticut. The absence of antibodies in opossums collected from the same areas is most likely because of the absence of systemic infection in the definitive host.     

Experimental infection of rhesus monkeys with type D retrovirus.

The naturally occurring immunodeficiency syndrome of macaque monkeys is an important animal model for the acquired immunodeficiency syndrome in humans. A new type D retrovirus, distinct from Mason-Pfizer monkey virus, has been isolated from affected animals at the New England Regional Primate Research Center. We now report the results of experimental infection of macaques with retrovirus D/New England after 13 months of study. Inoculated macaques developed lymphadenopathy without follicular hyperplasia, profound neutropenia, and a transient decrease in peripheral blood lymphocyte blastogenic responsiveness. Despite our varying the strain of virus, the manner in which the virus was grown, the size of the inoculum, and the age of the inoculated animals, infected macaques have not developed opportunistic infections or profound, prolonged loss of T cell function, key features of the macaque immunodeficiency syndrome. Therefore, experimental infection of naive macaques with D/New England has not reproduced the naturally occurring macaque immunodeficiency syndrome.     

Antigenic evaluation of a recombinant baculovirus-expressed Sarcocystis neurona SAG1 antigen

Sarcocystis neurona is the primary parasite associated with equine protozoal myeloencephalitis (EPM). This is a commonly diagnosed neurological disorder in the Americas that infects the central nervous system of horses. Current serologic assays utilize culture-derived parasites as antigen. This method requires large numbers of parasites to be grown in culture, which is labor intensive and time consuming. Also, a culture-derived whole-parasite preparation contains conserved antigens that could cross-react with antibodies against other Sarcocystis species and members of Sarcocystidae such as Neospora spp., Hammondia spp., and Toxoplasma gondii. Therefore, there is a need to develop an improved method for the detection of S. neurona-specific antibodies. The sera of infected horses react strongly to surface antigen 1 (SnSAG1), an approximately 29-kDa protein, in immunoblot analysis, suggesting that it is an immunodominant antigen. The SnSAG1 gene of S. neurona was cloned, and recombinant S. neurona SAG1 protein (rSnSAG1-Bac) was expressed with the use of a baculovirus system. By immunoblot analysis, the rSnSAG1-Bac antigen detected antibodies to S. neurona from naturally infected and experimentally inoculated equids, cats, rabbit, mice, and skunk. This is the first report of a baculovirus-expressed recombinant S. neurona antigen being used to detect anti-S. neurona antibodies in a variety of host species.     

Hepatic sarcocystosis in a horse

Hepatic sarcocystosis was diagnosed in a horse in association with refractory bacterial osteomyelitis and plasma cell tumor of the maxilla and hepatic salmonellosis. Gross lesions included pleural, pericardial, and peritoneal effusions, hepatomegaly, gastric ulceration, colonic edema, and proliferative tissues filling 2 maxillary dental alveoli. Histologically, liver was characterized by severe suppurative, necrotizing, periportal hepatitis, and severe periacinar necrosis. Hepatocytes frequently contained protozoal schizonts in various stages of development. In mature schizonts, merozoites were often arranged radially around a central residual body, consistent with asexual division by endopolygeny. Ultrastructural features of merozoites included an apical conoid and polar ring, anterior micronemes, central nuclei, and absence of rhoptries. These protozoa did not react to antisera raised against Neospora caninum, Sarcocystis neurona, Toxoplasma gondii, or Hammondia hammondi. The microscopic and ultrastructural characteristics and immunoreactivity of this organism are consistent with a Sarcocystis sp. other than S. neurona. This is the first report of Sarcocystis-associated hepatitis in a horse. The life cycle of this organism and source of infection are unknown.