Epidemiology of Sarcocystis neurona infections in domestic cats (Felis domesticus) and its association with equine protozoal myeloencephalitis (EPM) case farms and feral cats from a mobile spay and neuter clinic.

Equine protozoal myeloencephalitis (EPM) is a serious neurologic disease in the horse most commonly caused by Sarcocystis neurona. The domestic cat (Felis domesticus) is an intermediate host for S. neurona. In the present study, nine farms, known to have prior clinically diagnosed cases of EPM and a resident cat population were identified and sampled accordingly. In addition to the farm cats sampled, samples were also collected from a mobile spay and neuter clinic. Overall, serum samples were collected in 2001 from 310 cats, with samples including barn, feral and inside/outside cats. Of these 310 samples, 35 were from nine horse farms. Horse serum samples were also collected and traps were set for opossums at each of the farms. The S. neurona direct agglutination test (SAT) was used for both the horse and cat serum samples (1:25 dilution). Fourteen of 35 (40%) cats sampled from horse farms had circulating S. neurona agglutinating antibodies. Twenty-seven of the 275 (10%) cats from the spay/neuter clinic also had detectable S. neurona antibodies. Overall, 115 of 123 (93%) horses tested positive for anti-S. neurona antibodies, with each farm having greater than a 75% exposure rate among sampled horses. Twenty-one opossums were trapped on seven of the nine farms. Eleven opossums had Sarcocystis sp. sporocysts, six of them were identified as S. neurona sporocysts based on bioassays in gamma-interferon gene knockout mice with each opossum representing a different farm. Demonstration of S. neurona agglutinating antibodies in domestic and feral cats corroborates previous research demonstrating feral cats to be naturally infected, and also suggests that cats can be frequently infected with S. neurona and serve as one of several natural intermediate hosts for S. neurona.

Prevalence of Toxoplasma gondii antibodies in domestic cats from rural Ohio.

Antibodies to Toxoplasma gondii were determined in serum samples from 275 domestic cats from a mobile spay and neuter clinic from 8 counties in Ohio. The modified agglutination test incorporating whole formalinized tachyzoites and mercaptoethanol was used to determine antibodies. Antibodies to T. gondii were found in 133 (48%) out of 275 cats: in titers of 1:25 in 24, 1:50 in 37, and 1:500 or more in 72. The highest prevalence (62% of 78) was in outdoor cats. The prevalence of T. gondii antibodies in 48% of cats suggests wide-spread contamination of the rural environment with oocysts.

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.

Sarcocystis neurona infections in raccoons (Procyon lotor): evidence for natural infection with sarcocysts, transmission of infection to opossums (Didelphis virginiana), and experimental induction of neurologic disease in raccoons.

Equine protozoal myeloencephalitis (EPM) is a serious neurologic disease of horses in the Americas and Sarcocystis neurona is the most common etiologic agent. The distribution of S. neurona infections follows the geographical distributions of its definitive hosts, opossums (Didelphis virginiana, Didelphis albiventris). Recently, cats and skunks were reported as experimental and armadillos as natural intermediate hosts of S. neurona. In the present report, raccoons (Procyon lotor) were identified as a natural intermediate host of S. neurona. Two laboratory-raised opossums were found to shed S. neurona-like sporocysts after ingesting tongues of naturally-infected raccoons. Interferon-gamma gene knockout (KO) mice fed raccoon-opossum-derived sporocysts developed neurologic signs. S. neurona was identified immunohistochemically in tissues of KO mice fed sporocysts and the parasite was isolated in cell cultures inoculated with infected KO mouse tissues. The DNA obtained from the tongue of a naturally-infected raccoon, brains of KO mice that had neurological signs, and from the organisms recovered in cell cultures inoculated with brains of neurologic KO mice, corresponded to that of S. neurona. Two raccoons fed mature S. neurona sarcocysts did not shed sporocysts in their feces, indicating raccoons are not likely to be its definitive host. Two raccoons fed sporocysts from opossum feces developed clinical illness and S. neurona-associated encephalomyelitis was found in raccoons killed 14 and 22 days after feeding sporocysts; schizonts and merozoites were seen in encephalitic lesions.

Utilization of stress in the development of an equine model for equine protozoal myeloencephalitis.

Neurologic disease in horses caused by Sarcocystis neurona is difficult to diagnose, treat, or prevent, due to the lack of knowledge about the pathogenesis of the disease. This in turn is confounded by the lack of a reliable equine model of equine protozoal myeloencephalitis (EPM). Epidemiologic studies have implicated stress as a risk factor for this disease, thus, the role of transport stress was evaluated for incorporation into an equine model for EPM. Sporocysts from feral opossums were bioassayed in interferon-gamma gene knockout (KO) mice to determine minimum number of viable S. neurona sporocysts in the inoculum. A minimum of 80,000 viable S. neurona sporocysts were fed to each of the nine horses. A total of 12 S. neurona antibody negative horses were divided into four groups (1-4). Three horses (group 1) were fed sporocysts on the day of arrival at the study site, three horses were fed sporocysts 14 days after acclimatization (group 2), three horses were given sporocysts and dexamethasone 14 days after acclimatization (group 3) and three horses were controls (group 4). All horses fed sporocysts in the study developed antibodies to S. neurona in serum and cerebrospinal fluid (CSF) and developed clinical signs of neurologic disease. The most severe clinical signs were in horses in group 1 subjected to transport stress. The least severe neurologic signs were in horses treated with dexamethasone (group 3). Clinical signs improved in four horses from two treatment groups by the time of euthanasia (group 1, day 44; group 3, day 47). Post-mortem examinations, and tissues that were collected for light microscopy, immunohistochemistry, tissue cultures, and bioassay in KO mice, revealed no direct evidence of S. neurona infection. However, there were lesions compatible with S. neurona infection in horses. The results of this investigation suggest that stress can play a role in the pathogenesis of EPM. There is also evidence to suggest that horses in nature may clear the organism routinely, which may explain the relatively high number of normal horses with CSF antibodies to S. neurona compared to the prevalence of EPM.

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.

Completion of the life cycle of Sarcocystis neurona.

Sarcocystis neurona is the most important cause of a neurologic disease in horses, equine protozoal myeloencephalitis (EPM). The complete life cycle of S. neurona, including the description of sarcocysts and intermediate hosts, has not been completed until now. Opossums (Didelphis spp.) are definitive hosts, and horses and other mammals are aberrant hosts. In the present study, laboratory-raised domestic cats (Felis domesticus) were fed sporocysts from the intestine of a naturally infected opossum (Didelphis virginiana). Microscopic sarcocysts, with a maximum size of 700 x 50 microm, developed in the muscles of the cats. The DNA of bradyzoites released from sarcocysts was confirmed as S. neurona. Laboratory-raised opossums (D. virginiana) fed cat muscles containing the sarcocysts shed sporocysts in their feces. The sporocysts were approximately 10(-12) x 6.5-8.0 microm in size. Gamma interferon knockout mice fed sporocysts from experimentally infected opossums developed clinical sarcocystosis, and S. neurona was identified in their tissues using S. neurona-specific polyclonal rabbit serum. Two seronegative ponies fed sporocysts from an experimentally-infected opossum developed S. neurona-specific antibodies within 14 days.