Interobserver Variation in the Diagnosis of Neurologic Abnormalities in the Horse.

BACKGROUND: The diagnosis of equine protozoal myeloencephalitis (EPM) relies heavily on the clinical examination. The accurate identification of neurologic signs during a clinical examination is critical to the interpretation of laboratory results. OBJECTIVE: To investigate the level of agreement between board-certified veterinary internists when performing neurologic examinations in horses. ANIMALS: Ninety-seven horses admitted to the Veterinary Teaching Hospital at The Ohio State University from December 1997 to June 1998. METHODS: A prospective epidemiologic research design was used. Horses enrolled in the study were examined by the internist responsible for care of the horse, and later by an internist who was not aware of the presenting complaint or other patient history. Data were analyzed by descriptive statistics, and kappa (K) statistics were calculated to assess interobserver agreement. RESULTS: Ninety-seven horses were enrolled in the study. Overall, examiners, also referred to as observers, agreed that 60/97 (61.9%) were clinically abnormal, 21/97 (21.6%) were clinically normal, and the status of 16/97 (16.5%) of horses was contested. There was complete agreement among the examiners with regard to cranial nerve signs and involuntary movements. Disagreement involving severity of clinical signs occurred in 31 horses, and 25 of those horses (80.6%) were considered either normal or mildly affected by the primary observer. When examining the results of all paired clinical examinations for 11 different categories, there was wide variability in the results. When examiners rated the presence or absence of any neurologic abnormalities, lameness, or ataxia, the agreement among observers was either good or excellent for 80% of horses. When assessing truncal sway, the agreement among observers was good or excellent for 60% of the horses. When examining the horses for asymmetry of deficits, agreement was either good or excellent for 40% of the horses. Agreement among observers was excellent or good for only 20% of the horses when assessing muscle atrophy, spasticity (hypermetria), and overall assessment of the severity of neurologic abnormalities. CONCLUSIONS AND CLINICAL IMPORTANCE: This study underscores the subjectivity of the neurologic examination and demonstrates a reasonable level of agreement that may be achieved when different clinicians examine the same horse.

Sarcocyst Development in Raccoons (Procyon lotor) Inoculated with Different Strains of Sarcocystis neurona Culture-Derived Merozoites.

Sarcocystis neurona is considered the major etiologic agent of equine protozoal myeloencephalitis (EPM), a neurological disease in horses. Raccoon ( Procyon lotor ) is considered the most important intermediate host in the life cycle of S. neurona in the United States; S. neurona sarcocysts do mature in raccoon muscles, and raccoons also develop clinical signs simulating EPM. The focus of this study was to determine if sarcocysts would develop in raccoons experimentally inoculated with different host-derived strains of in vitro-cultivated S. neurona merozoites. Four raccoons were inoculated with strains derived from a raccoon, a sea otter, a cat, and a horse. Raccoon tissues were fed to laboratory-raised opossums ( Didelphis virginiana ), the definitive host of S. neurona . Intestinal scraping revealed sporocysts in opossums who received muscle tissue from raccoons inoculated with the raccoon-derived or the sea otter-derived isolates. These results demonstrate that sarcocysts can mature in raccoons inoculated with in vitro-derived S. neurona merozoites. In contrast, the horse and cat-derived isolates did not produce microscopically or biologically detected sarcocysts. Immunoblot analysis revealed both antigenic and antibody differences when testing the inoculated raccoons. Immunohistochemical staining indicated differences in staining between the merozoite and sarcocyst stages. The successful infections achieved in this study indicates that the life cycle can be manipulated in the laboratory without affecting subsequent stage development, thereby allowing further purification of strains and artificial maintenance of the life cycle.

An update on Sarcocystis neurona infections in animals and equine protozoal myeloencephalitis (EPM).

Equine protozoal myeloencephalitis (EPM) is a serious disease of horses, and its management continues to be a challenge for veterinarians. The protozoan Sarcocystis neurona is most commonly associated with EPM. S. neurona has emerged as a common cause of mortality in marine mammals, especially sea otters (Enhydra lutris). EPM-like illness has also been recorded in several other mammals, including domestic dogs and cats. This paper updates S. neurona and EPM information from the last 15 years on the advances regarding life cycle, molecular biology, epidemiology, clinical signs, diagnosis, treatment and control.

Somatotropic axis resistance and ghrelin in critically ill foals.

REASONS FOR PERFORMING STUDY: Resistance to the somatotropic axis and increases in ghrelin concentrations have been documented in critically ill human patients, but limited information exists in healthy or sick foals. OBJECTIVES: To investigate components of the somatotropic axis (ghrelin, growth hormone and insulin-like growth factor-1 [IGF-1]) with regard to energy metabolism (glucose and triglycerides), severity of disease and survival in critically ill equine neonates. It was hypothesised that ghrelin and growth hormone would increase and IGF-1 would decrease in proportion to severity of disease, supporting somatotropic axis resistance, which would be associated with severity of disease and mortality in sick foals. STUDY DESIGN: Prospective multicentre cross-sectional study. METHODS: Blood samples were collected at admission from 44 septic, 62 sick nonseptic (SNS) and 19 healthy foals, all aged <7 days. Foals with positive blood cultures or sepsis scores ≥12 were considered septic, foals with sepsis scores of 5-11 were classified as SNS. Data were analysed by nonparametric methods and multivariate logistic regression. RESULTS: Septic foals had higher ghrelin, growth hormone and triglyceride and lower IGF-1 and glucose concentrations than healthy foals (P<0.01). Sick nonseptic foals had higher growth hormone and triglycerides and lower IGF-1 concentrations than healthy foals (P<0.05). Growth hormone:IGF-1 ratio was higher in septic and SNS foals than healthy foals (P<0.05). Hormone concentrations were not different between septic nonsurvivors (n = 14) and survivors (n = 30), but the growth hormone:IGF-1 ratio was lower in nonsurvivors (P = 0.043). CONCLUSIONS: Somatotropic axis resistance, characterised by a high growth hormone:IGF-1 ratio, was frequent in sick foals, associated with the energy status (hypoglycaemia, hypertriglyceridaemia) and with mortality in septic foals. POTENTIAL RELEVANCE: A functional somatotropic axis appears to be important for foal survival during sepsis. Somatotropic resistance is likely to contribute to severity of disease, a catabolic state and likelihood of recovery.

Accurate antemortem diagnosis of equine protozoal myeloencephalitis (EPM) based on detecting intrathecal antibodies against Sarcocystis neurona using the SnSAG2 and SnSAG4/3 ELISAs.

BACKGROUND: Recent work demonstrated the value of antigen-specific antibody indices (AI and C-value) to detect intrathecal antibody production against Sarcocystis neurona for antemortem diagnosis of equine protozoal myeloencephalitis (EPM). OBJECTIVES: The study was conducted to assess whether the antigen-specific antibody indices can be reduced to a simple serum : cerebrospinal fluid (CSF) titer ratio to achieve accurate EPM diagnosis. ANIMALS: Paired serum and CSF samples from 128 horses diagnosed by postmortem examination. The sample set included 44 EPM cases, 35 cervical-vertebral malformation (CVM) cases, 39 neurologic cases other than EPM or CVM, and 10 non-neurologic cases. METHODS: Antibodies against S. neurona were measured in serum and CSF pairs using the SnSAG2 and SnSAG4/3 (SnSAG2, 4/3) ELISAs, and the ratio of each respective serum titer to CSF titer was determined. Likelihood ratios and diagnostic sensitivity and specificity were calculated based on serum titers, CSF titers, and serum : CSF titer ratios. RESULTS: Excellent diagnostic sensitivity and specificity was obtained from the SnSAG2, 4/3 serum : CSF titer ratio. Sensitivity and specificity of 93.2 and 81.1%, respectively, were achieved using a ratio cutoff of ≤100, whereas sensitivity and specificity were 86.4 and 95.9%, respectively, if a more rigorous cutoff of ≤50 was used. Antibody titers in CSF also provided good diagnostic accuracy. Serum antibody titers alone yielded much lower sensitivity and specificity. CONCLUSIONS AND CLINICAL IMPORTANCE: The study confirms the value of detecting intrathecal antibody production for antemortem diagnosis of EPM, and they further show that the antigen-specific antibody indices can be reduced in practice to a simple serum : CSF titer ratio.

Sarcocystis neurona infection in gamma interferon gene knockout (KO) mice: comparative infectivity of sporocysts in two strains of KO mice, effect of trypsin digestion on merozoite viability, and infectivity of bradyzoites to KO mice and cell culture.

The protozoan Sarcocystis neurona is the primary cause of Equine Protozoal Myeloencephalitis (EPM). EPM or EPM-like illness has been reported in horses, sea otters, and several other mammals. The gamma interferon gene knockout (KO) mouse is often used as a model to study biology and discovery of new therapies against S. neurona because it is difficult to induce clinical EPM in other hosts, including horses. In the present study, infectivity of three life cycle stages (merozoites, bradyzoites, sporozoites) to KO mice and cell culture was studied. Two strains of KO mice (C57-black, and BALB/c-derived, referred here as black or white) were inoculated orally graded doses of S. neurona sporocysts; 12 sporocysts were infective to both strains of mice and all infected mice died or became ill within 70 days post-inoculation. Although there was no difference in infectivity of sporocysts to the two strains of KO mice, the disease was more severe in black mice. S. neurona bradyzoites were not infectious to KO mice and cell culture. S. neurona merozoites survived 120 min incubation in 0.25% trypsin, indicating that trypsin digestion can be used to recover S. neurona from tissues of acutely infected animals.

Prevalence of Toxoplasma gondii from free-range chickens (Gallus domesticus) from Addis Ababa, Ethiopia.

Prevalence of Toxoplasma gondii in free-range chickens (Gallus domesticus) is a good indicator of the environmental contamination with oocysts because chickens become infected mainly by feeding from ground, feed, or soil contaminated with oocysts. The seroprevalence of T. gondii antibodies in 125 free-range chickens from the Addis Ababa, Ethiopia, was determined. Antibodies to T. gondii were assayed by the modified agglutination test; 48 of 125 (38.4%) chickens were seropositive, with titers of 1:5 in 14, 1:10 in 12, 1:20 in 14, 1: 40 in 3, 1: 80 in 1, 1:160 in 1, 1:320 in 1, and ≥1:640 in 2 chickens. The hearts of 115 chickens were bioassayed for T. gondii infection. Hearts of 72 seronegative (modified agglutination test [MAT] < 1:5) chickens were pooled in 4 groups (20 + 18 + 19 + 15) and fed to 4 T. gondii -free cats; none of these 4 cats shed oocysts in their feces examined 3-21 days after feeding chicken tissues. Hearts of 43 seropositive chickens (MAT ≥ 1:5) were bioassayed individually in mice. Toxoplasma gondii was isolated from only 1 chicken, with a MAT titer of 1:80. This isolate was designated TgCKEt1 and was not pathogenic for outbred mice. Restricted fragment length polymorphism (RFLP) genotyping using 10 loci indicated the TgCKEt1 was ToxoDB polymerase chain reaction-RFLP genotype #1 (Type II clonal). Results of this study indicate very low environmental contamination with T. gondii oocysts around Addis Ababa.

An investigation into the seroprevalence of Toxoplasma gondii, Bartonella spp., feline immunodeficiency virus (FIV), and feline leukaemia virus (FeLV) in cats in Addis Ababa, Ethiopia.

Toxoplasma gondii and Bartonella spp. are zoonotic pathogens of cats. Feline immunodeficiency virus (FIV), and feline leukaemia virus (FeLV) are immunosuppressive viruses of cats that can affect T. gondii oocyst shedding. In this study, the prevalence of antibodies to T. gondii, Bartonella spp., FIV, as well as FeLV antigens were determined in sera from feral cats (Felis catus) from Addis Ababa, Ethiopia. Using the modified agglutination test, IgG antibodies to T. gondii were found in 41 (85.4%) of the 48 cats with titres of 1:25 in one, 1:50 in one, 1:200 in six, 1:400 in six, 1:800 in six, 1:1600 in eight, and 1:3200 in 13 cats. Toxoplasma gondii IgM antibodies were found in 11/46 cats tested by ELISA, suggesting recent infection. Antibodies to Bartonella spp. were found in five (11%) of 46 cats tested. Antibodies to FIV or FeLV antigen were not detected in any of the 41 cats tested. The results indicate a high prevalence of T. gondii and a low prevalence of Bartonella spp. infection in cats in Ethiopia.

Isolation of viable Toxoplasma gondii from tissues and feces of cats from Addis Ababa, Ethiopia.

Cats are important in the epidemiology of Toxoplasma gondii because they are the only hosts that excrete environmentally resistant oocysts in feces. In the present study, hearts, serum, and feces from 36 feral cats from Addis Ababa area, Ethiopia, were examined for T. gondii infection. Antibodies to T. gondii were determined with the modified agglutination test (MAT, cutoff 1:25); 33 cats were seropositive. Hearts of all 36 cats were homogenized, digested in pepsin, and bioassayed in mice. Feces were examined for T. gondii oocysts by bioassay in mice. Viable T. gondii was isolated from heart of 26 by bioassay in mice and from 25 seropositive and 1 seronegative cats. Toxoplasma gondii was isolated from feces (oocysts) by bioassay in mice. In total, viable T. gondii was isolated from 27 of the 36 cats, and these isolates were designated TgCatEt1 to TgCatEt27. The high prevalence of T. gondii oocysts in feces of 8 (19.4%) of 36 cats is of high epidemiologic significance. This is the first report of isolation of viable T. gondii from any host in Ethiopia.

The SnSAG merozoite surface antigens of Sarcocystis neurona are expressed differentially during the bradyzoite and sporozoite life cycle stages.

Sarcocystis neurona is a two-host coccidian parasite whose complex life cycle progresses through multiple developmental stages differing at morphological and molecular levels. The S. neurona merozoite surface is covered by multiple, related glycosylphosphatidylinositol-linked proteins, which are orthologous to the surface antigen (SAG)/SAG1-related sequence (SRS) gene family of Toxoplasma gondii. Expression of the SAG/SRS proteins in T. gondii and another related parasite Neospora caninum is life-cycle stage specific and seems necessary for parasite transmission and persistence of infection. In the present study, the expression of S. neurona merozoite surface antigens (SnSAGs) was evaluated in the sporozoite and bradyzoite stages. Western blot analysis was used to compare SnSAG expression in merozoites versus sporozoites, while immunocytochemistry was performed to examine expression of the SnSAGs in merozoites versus bradyzoites. These analyses revealed that SnSAG2, SnSAG3 and SnSAG4 are expressed in sporozoites, while SnSAG5 was appeared to be downregulated in this life cycle stage. In S. neurona bradyzoites, it was found that SnSAG2, SnSAG3, SnSAG4 and SnSAG5 were either absent or expression was greatly reduced. As shown for T. gondii, stage-specific expression of the SnSAGs may be important for the parasite to progress through its developmental stages and complete its life cycle successfully. Thus, it is possible that the SAG switching mechanism by these parasites could be exploited as a point of intervention. As well, the alterations in surface antigen expression during different life cycle stages may need to be considered when designing prospective approaches for protective vaccination.

Insulin, glucagon, and leptin in critically ill foals.

BACKGROUND: Endocrine dysregulation of hormones of energy metabolism is well documented in critically ill humans, but limited information exists in septic foals. The purpose of this study was to provide information on the hormonal response to energy metabolism in critically ill foals, focusing on insulin, glucagon, and leptin. HYPOTHESIS: Concentrations of insulin, glucagon, leptin, and triglycerides will be higher, whereas glucose concentration will be lower in septic foals than in healthy and sick nonseptic foals. The magnitude of these differences will be associated with severity of disease and nonsurvival. ANIMALS: Forty-four septic, 62 sick nonseptic, and 19 healthy foals <7 days of age. METHODS: In this prospective multicenter cross-sectional study, blood samples were collected at admission. Foals with positive blood culture or sepsis score ≥12 were considered septic. RESULTS: Septic foals had lower glucose and insulin and higher triglyceride and glucagon concentrations than did healthy foals. Glucagon concentrations were not different between septic foals that died (n = 14) or survived (n = 30). Higher insulin and lower leptin concentrations were associated with mortality. Quantitative insulin-sensitivity check index was higher in septic foals. CONCLUSIONS AND CLINICAL IMPORTANCE: Energy metabolism and the endocrine response of related hormones in septic foals are characterized by hypoglycemia, hypertriglyceridemia, low insulin concentration, and high glucagon concentration. Leptin and insulin may have prognostic value for nonsurvival in septic foals. The hormonal response related to energy metabolism in critical illness differs between foals and humans.

The use of sedatives, analgesic and anaesthetic drugs in the horse: an electronic survey of members of the American Association of Equine Practitioners (AAEP).

REASONS FOR PERFORMING STUDY: To determine the sedative, analgesic and anaesthetic drugs and techniques that are used by equine veterinarians. HYPOTHESIS OR OBJECTIVES: To provide equine veterinarians with information concerning veterinary use of anaesthetic techniques, a reflection of the collective experiences of the profession. METHODS: A survey was conducted of those members of the American Association of Equine Practitioners (AAEP) with an electronic mail address on file with the organisation using proprietary, web-based software. The survey was comprised of 30 questions divided into 8 sections: nonsteroidal anti-inflammatory drugs; local anaesthesia; alternative techniques; standing chemical restraint; epidural anaesthesia; short-term anaesthesia; long-term anaesthesia; and a place for the respondent to make comments. RESULTS: The response rate was 13.8% (952/6911) AAEP member veterinarians primarily use phenylbutazone and flunixin as anti-inflammatory drugs, and lidocaine and mepivacaine for local anaesthesia. Combinations of drugs are preferred for standing chemical restraint. While many veterinarians frequently utilise short-term anaesthesia, longer anaesthesia is less frequently performed. CONCLUSIONS: Most AAEP member veterinarians use sedatives in combination to provide standing chemical restraint. Extra-label use of drugs is a core component of current equine sedation and anaesthetic practice. POTENTIAL RELEVANCE: Equine veterinarians can compare their choices of anaesthetic drugs with others practising equine medicine and surgery and may be stimulated to investigate alternative methods of providing comfort to horses.

Calcium regulating hormones and serum calcium and magnesium concentrations in septic and critically ill foals and their association with survival.

BACKGROUND: Disorders of calcium regulation are frequently found in humans with critical illness, yet limited information exists in foals with similar conditions including septicemia. The purpose of this study was to determine whether disorders of calcium exist in septic foals, and to determine any association with survival. HYPOTHESIS: Blood concentrations of ionized calcium (Ca(2+)) and magnesium (Mg(2+)) will be lower in septic foals with concomitant increases in parathyroid hormone (PTH), calcitonin (CT), and parathyroid-related peptide (PTHrP) compared with healthy foals. The magnitude of these differences will be negatively associated with survival. ANIMALS: Eighty-two septic, 40 sick nonseptic, and 24 healthy foals of or=14 were considered septic. Foals with disease other than sepsis and healthy foals were used as controls. Hormone concentrations were measured with validated immunoassays. RESULTS: Septic foals had decreased Ca(2+) (5.6 versus 6.1 mg/dL, P < .01) and increased serum PTH (16.2 versus 3.2 pmol/L, P < .05), and phosphorus concentrations (7.1 versus 6.3 mg/dL, P < .01). No differences in serum Mg(2+), PTHrP, and CT concentrations were found. Nonsurviving septic foals (n = 42/82) had higher PTH concentrations (41.1 versus 10.7 pmol/L, P < .01) than survivors (n = 40/82). CONCLUSIONS AND CLINICAL IMPORTANCE: Septic foals were more likely to have disorders of calcium regulation compared with healthy foals, where hyperparathyroidemia was associated with nonsurvival.

Seroepidemiology of Toxoplasma gondii in zoo animals in selected zoos in the midwestern United States.

Toxoplasma gondii infections in zoo animals are of interest because many captive animals die of clinical toxoplasmosis and because of the potential risk of exposure of children and elderly to T. gondii oocysts excreted by cats in the zoos. Seroprevalence of T. gondii antibodies in wild zoo felids, highly susceptible zoo species, and feral cats from 8 zoos of the midwestern United States was determined by using the modified agglutination test (MAT). A titer of 1:25 was considered indicative of T. gondii exposure. Among wild felids, antibodies to T. gondii were found in 6 (27.3%) of 22 cheetahs (Acynonyx jubatus jubatus), 2 of 4 African lynx (Caracal caracal), 1 of 7 clouded leopards (Neofelis nebulosa), 1 of 5 Pallas cats (Otocolobus manul), 12 (54.5%) of 22 African lions (Panthera leo), 1 of 1 jaguar (Panthera onca), 1 of 1 Amur leopard (Panthera pardus orientalis), 1 of 1 Persian leopard (Panthera pardus saxicolor), 5 (27.8%) of 18 Amur tigers (Panthera tigris altaica), 1 of 4 fishing cats (Prionailurus viverrinus), 3 of 6 pumas (Puma concolor), 2 of 2 Texas pumas (Puma concolor stanleyana), and 5 (35.7%) of 14 snow leopards (Uncia uncia). Antibodies were found in 10 of 34 feral domestic cats (Felis domesticus) trapped in 3 zoos. Toxoplasma gondii oocysts were not found in any of the 78 fecal samples from wild and domestic cats. Among the macropods, antibodies were detected in 1 of 3 Dama wallabies (Macropus eugenii), 1 of 1 western grey kangaroo (Macropus fuliginosus), 1 of 2 wallaroos (Macropus robustus), 6 of 8 Bennett's wallabies (Macropus rufogriseus), 21 (61.8%) of 34 red kangaroos (Macropus rufus), and 1 of 1 dusky pademelon (Thylogale brunii). Among prosimians, antibodies were detected in 1 of 3 blue-eyed black lemurs (Eulemur macaco flavifrons), 1 of 21 ring-tailed lemurs (Lemur catta), 2 of 9 red-ruffed lemurs (Varecia variegata rubra), and 2 of 4 black- and white-ruffed lemurs (Varecia variegata variegata). Among the avian species tested, 2 of 3 bald eagles (Haliaeetus leucocephalus) were seropositive. Among 7 possible risk factors, sex, freezing meat temperature (above -13 C vs. below -13 C), washing vegetables thoroughly, frequency of feral cat sightings on zoo grounds (occasionally vs. frequently), frequency of feral cat control programs, capability of feral cats to enter hay/grain barn, and type of animal exhibit, exhibiting animals in open enclosures was the only factor identified as a significant risk (OR 3.22, P = 0.00).

Sarcocystis neurona: molecular characterization of enolase domain I region and a comparison to other protozoa.

Sarcocystis neurona causes protozoal myeloencephalitis and has the ability to infect a wide host range in contrast to other Sarcocystis species. In the current study, five S. neurona isolates from a variety of sources, three Sarcocystis falcatula, one Sarcocystis dasypi/S. neurona-like isolate, and one Besnoitia darlingi isolate were used to compare the enolase 2 gene segment containing the domain I region to previously sequenced enolase genes from Neospora caninum, Neospora hughesi, Toxoplasma gondii, Plasmodium falciparum, and Trypanosoma cruzi; enolase 2 segment containing domain I region is highly conserved amongst these parasites of veterinary and medical importance. Immunohistochemistry results indicates reactivity of T. gondii enolase 1 and 2 antibodies to S. neurona merozoites and metrocytes, but no reactivity of anti-enolase 1 to the S. neurona bradyzoite stage despite reactivity to T. gondii bradyzoites, suggesting expression differences between organisms.

Risk factors for owner-reported occurrence of equine protozoal myeloencephalitis in the US equine population.

BACKGROUND: Equine protozoal myeloencephalitis (EPM) is a serious and often fatal neurologic disease of horses, but few studies have investigated risk factors. OBJECTIVES: To evaluate operation- and individual-level factors associated with likelihood of the occurrence of EPM. ANIMALS: Data were collected as part of a study of the US equine industry from 1,178 operations representing 83.9% of horses and 51.6% of operations with > or =3 horses in 28 states. METHODS: Probability-based sampling was used to enroll representative operations in a cross-sectional study. Interviews were conducted to collect information regarding health and management of horses. A nested case-control study was used to investigate risk factors among individual horses. Interview data were combined with climate data, human population density, and opossum regional ecology categories. Data were analyzed using logistic regression to identify risk factors for the occurrence of EPM. RESULTS: Owners reported that 95% of EPM cases included in this study were diagnosed by veterinarians. Variables associated with EPM occurrence on premises included opossum regional ecology, reported exposure to small wildlife, climate, terrain, housing, choice of bedding material, method of storing feeds, equine stocking density, and primary use of horses. Among individual horses, age was most strongly associated with disease risk. Associations also were identified with sex, breed, primary use, and participation in competitions. CONCLUSIONS AND CLINICAL IMPORTANCE: Because the risk of EPM occurrence on operations is closely tied to factors that impact exposure to opossums, their feces, and their environment, controlling these exposures may be important in preventing the occurrence of EPM.

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.

Immunohistochemical properties of ocular adenomas, adenocarcinomas and medulloepitheliomas.

Ocular medulloepitheliomas, adenomas and adenocarcinomas share a common phenotype and originate from the optic cup neuroectoderm. This can make it very difficult to differentiate between these tumors histopathologically. Therefore, this study focused on identifying a combination of immunologic markers that might be used in the diagnosis of these tumors. These markers included AE1/AE3, CK7, CK20, and telomerase reverse transcriptase (TERT). Routine immunohistochemical staining was performed on 27 whole globes diagnosed with one of these tumors. The tumors that immunostained for TERT showed increasing immunoreactivity as the tumor types increased in aggressiveness. None of the tumor types were immunopositive for CK7. CK20 immunostaining was found in the adenomas but not in the adenocarcinomas or medulloepitheliomas. AE1/AE3 expression was present more consistently in the adenocarcinomas and less frequently in the adenomas. AE1/AE3 expression was present in only one of six medulloepitheliomas. Furthermore, CK20 and TERT showed inverse expression patterns, i.e. TERT increased in expression and CK20 decreased in expression with increasing aggressiveness. These results may be important diagnostic and prognostic indicators for these tumors.

Prophylactic administration of ponazuril reduces clinical signs and delays seroconversion in horses challenged with Sarcocystis neurona.

The ability of ponazuril to prevent or limit clinical signs of equine protozoal myeloencephalitis (EPM) after infection with Sarcocystis neurona was evaluated. Eighteen horses were assigned to 1 of 3 groups: no treatment, 2.5 mg/kg ponazuril, or 5.0 mg/kg ponazuril. Horses were administered ponazuril, once per day, beginning 7 days before infection (study day 0) and continuing for 28 days postinfection. On day 0, horses were stressed by transport and challenged with 1 million S. neurona sporocysts per horse. Sequential neurologic examinations were performed, and serum and cerebrospinal fluid were collected and assayed for antibodies to S. neurona. All horses in the control group developed neurologic signs, whereas only 71 and 40% of horses in the 2.5 and 5.0 mg/kg ponazuril groups, respectively, developed neurologic abnormalities. This was significant at P = 0.034 by using Fisher exact test. In addition, seroconversion was decreased in the 5.0 mg/kg group compared with the control horses (100 vs. 40%; P = 0.028). Horses with neurologic signs were killed, and a post-mortem examination was performed. Mild-to-moderate, multifocal signs of neuroinflammation were observed. These results confirm that treatment with ponazuril at 5.0 mg/kg minimizes, but does not eliminate, infection and clinical signs of EPM in horses.

Development and evaluation of a Sarcocystis neurona-specific IgM capture enzyme-linked immunosorbent assay.

Equine protozoal myeloencephalitis (EPM) is a serious neurologic disease of horses caused primarily by the protozoal parasite Sarcocystis neurona. Currently available antemortem diagnostic testing has low specificity. The hypothesis of this study was that serum and cerebrospinal fluid (CSF) of horses experimentally challenged with S neurona would have an increased S neurona-specific IgM (Sn-IgM) concentration after infection, as determined by an IgM capture enzyme linked immunoassay (ELISA). The ELISA was based on the S neurona low molecular weight protein SNUCD-1 antigen and the monoclonal antibody 2G5 labeled with horseradish peroxidase. The test was evaluated using serum and CSF from 12 horses experimentally infected with 1.5 million S neurona sporocysts and 16 horses experimentally infected with varying doses (100 to 100,000) of S neurona sporocysts, for which results of histopathologic examination of the central nervous system were available. For horses challenged with 1.5 million sporocysts, there was a significant increase in serum Sn-IgM concentrations compared with values before infection at weeks 2-6 after inoculation (P < .0001). For horses inoculated with lower doses of S neurona, there were significant increases in serum Sn-IgM concentration at various points in time after inoculation, depending on the challenge dose (P < .01). In addition, there was a significant increase between the CSF Sn-IgM concentrations before and after inoculation (P < .0001). These results support further evaluation of the assay as a diagnostic test during the acute phase of EPM.