Evidence of intrathecally-derived antibodies against Toxoplasma gondii in horses suspected of neurological disease consistent with equine protozoal myeloencephalitis.

Among the recognized neurologic diseases in horses, equine protozoal myeloencephalitis (EPM) has been reported around the world and still presents challenges in diagnosis and treatment. Horses can present with clinical neurologic signs consistent with EPM while testing negative for the two main causative agents, Sarcocystis neurona or Neospora hughesi, and may still be clinically responsive to anti-parasitic drug therapy. This context led to our hypothesis that another protozoal parasite, Toxoplasma gondii, which is known to cause toxoplasmosis in other mammalian species, is a potential pathogen to cause neurologic disease in horses. To evaluate this hypothesis, serum and cerebrospinal fluid (CSF) were collected from 210 horses presenting with clinical signs compatible with EPM, and the indirect immunofluorescent antibody test (IFAT) was used to detect antibody titers for T. gondii, S. neurona, and N. hughesi. Additionally, the serum to CSF titer ratio was calculated for T. gondii, S. neurona, and N. hughesi infections, suggesting intrathecally-derived antibodies for each of the three agents if the serum:CSF ratio was ≤ 64. There were 133 (63.3%) horses positive for serum T. gondii antibodies using a cutoff titer of 160, and 31 (14.8%) positive for CSF T. gondii antibodies using a cutoff titer of 5. Overall, 21 (10.0%) of EPM-suspect horses had a serum:CSF ratio ≤ 64 for antibodies for T. gondii, while 43 (20.5%) and 8 (3.8%) horses had a serum to CSF ratio ≤ 64 for antibodies for S. neurona and N. hughesi, respectively. A total of 6 (2.9%) animals presented evidence of concurrent intrathecally-derived antibodies for T. gondii and at least one other apicomplexan parasite in this study. Signalment and clinical signs were not different across the groups aforementioned. These data provide evidence of intrathecal production of anti-T. gondii antibodies, indicative of T. gondii infection in the brain and/or spinal cord of horses with EPM-like disease.

Molecular detection of Sarcocystis neurona in cerebrospinal fluid from 210 horses with suspected neurologic disease.

An ante-mortem diagnosis of equine protozoal myeloencephalitis (EPM) is presently based on clinical presentation, immunodiagnostics performed on serum and cerebrospinal fluid (CSF), and ruling out other neurological disorders. Molecular techniques introduce a novel and promising approach for the detection of protozoal agents in CSF. Hypothesizing that real-time PCR (rtPCR) can be a useful complement to EPM diagnostics, 210 CSF samples from horses suspected of neurological disease with EPM included as a differential diagnosis were tested using rtPCR to detect Sarcocystis neurona DNA and immunodiagnostics targeting antibodies against the same pathogen, performed on serum and CSF samples. Molecular and immunological results were compared with respect to origin of the horse, time of the year, signalment, clinical signs and treatment history. Twenty-five horses tested positive in CSF for S. neurona by rtPCR only, while 30 horses had intrathecally-derived antibodies to S. neurona only (serum to CSF ratio ≤ 64 by indirect fluorescent antibody test - IFAT), and 13 horses tested rtPCR-positive in CSF with evidence of intrathecally-derived antibodies to S. neurona. Previous treatment for EPM was the only variable presenting statistical difference between the two testing modalities, highlighting that animals with history of anti-protozoal treatment were more likely to test positive solely in IFAT, while horses without treatment were more likely to test positive by rtPCR only. The results support the use of molecular diagnosis for EPM caused by S. neurona as a complement to immunodiagnostics. The use of rtPCR in CSF for the detection of S. neurona may improve the diagnostic work-up of neurologic disease suspected horses, especially in animals without previous anti-protozoal treatment.

Non-lethal diagnostic methods for koi herpesvirus in koi Cyprinus carpio.

Cyprinid herpesvirus 3, also known as koi herpesvirus (KHV), is a viral pathogen responsible for mass mortalities of carp worldwide. In this study, we compared the sensitivity and specificity of ELISA and quantitative PCR (qPCR) methods for the diagnosis of KHV in experimentally infected koi Cyprinus carpio over an 11 mo period. Koi were exposed to KHV at 18 ± 1°C (permissive temperatures for KHV disease) in laboratory-controlled conditions. At 21 d post challenge, the temperature in the system was decreased to <15°C (non-permissive temperature for KHV disease), and fish were monitored for the following 11 mo. At different time points throughout the study, samples of blood and gills were collected from exposed and control koi and subjected to qPCR and ELISA. Survival proportions of 53.3 and 98.8% in exposed and control treatments, respectively, were recorded at the end of the challenge. Traditional receiver-operating characteristic analysis was used to compare the sensitivity of the ELISA and blood and gill qPCR during permissive and non-permissive temperatures. ELISA was superior to qPCR of gills and whole-blood samples in detecting previous exposure to KHV. Similar results were obtained in a second experiment exposing koi to KHV and inducing persistent infection at >30°C (non-permissive temperature for KHV disease). Finally, KHV ELISA specificity was confirmed using cyprinid herpesvirus 1-exposed koi through a period of 3 mo. This study demonstrates that the combination of ELISA and gill qPCR should be recommended in the diagnosis of KHV exposure of suspected carrier-state fish.

Comparison of prevalence factors in horses with and without seropositivity to Neospora hughesi and/or Sarcocystis neurona.

Equine protozoal myeloencephalitis is a commonly diagnosed neurological disease of horses in North America and is caused by infection with Sarcocystis neurona or Neospora hughesi. The aim of this study was to compare prevalence factors among horses seropositive or seronegative to N. hughesi and/or S. neurona. A total of 3123 submissions were included in the study, with horses originating from 49 States. Thirty-eight animals from 21 States tested seropositive for N. hughesi only, 840 horses from 40 States were seropositive for S. neurona only, 25 horses from 14 States were seropositive for both protozoa, and 2220 horses from 49 States tested seronegative for both parasites. Significant associations were found between geographical location (State), month of submission, breed and serological status.

Determinación de la velocidad de tránsito intestinal en el ratón BALB/c / Determinaton of the velocity of intestinal transit in the BALB/c mouse

La determinación de la velocidad del tránsito intestinal en un modelo animal es de primordial importancia, sobre todo cuando se pretende utilizar la vía digestiva del mismo como sitio para la presentación de antígenos. En el presente trabajo se determinó la velocidad del tránsito intestinal en el ratón BALB/c, uno de los animales de laboratorio frecuentemente utilizado en la investigación de la respuesta inmune. Se administró un colorante vegetal por medio de una sonda orogástrica, a 45 ratones, mayores de 8 semanas de edad. Posteriormente se sacrificaron los animales a diferentes tiempos en un rango de 10 min a 24 h. El tracto intestinal se dividió de manera arbitraria, el intestino delgado en tres tercios y el intestino grueso, en dos partes. Las determinaciones mostraron que 25 min después de administrado el colorante, éste se localizó a nivel del primer tercio y en menor proporción, a nivel del segundo tercio del intestino delgado. A los 90 min, el colorante alcanzó la primera mitad del intestino grueso, y a las 2 h comenzó a ser eliminado en las heces. Sin embargo, 3 h después de la administración aún se encontró colorante en el estómago, con lo anterior se demostró que el colorante se emilinó de manera intermitente a partir del estómago hasta las 3 h. A las 10 h se encontró colorante en intestino grueso, pero no en intestino delgado ni en estómago, a las 24 h se había eliminado por completo. Se concluye que en el intestino, el tránsito del colorante es constante, aunque la liberación a partir del estómago es intermitente, y que la totalidad del proceso de tránsito intestinal se lleva a cabo antes de 24 h