RESUMEN
Infection by Cryptosporidium serpentis occurs in reptiles, particularly in snakes. This disease is characterized by chronic infection with the presence of hypertrophic gastritis. The objectives of this study were to use real-time polymerase chain reaction (PCR) targeting the heat shock protein 70 (Hsp70) gene for the detection of C. serpentis in fecal samples from snakes and to determine the analytical and epidemiological specificity and sensitivity of this approach relative to the gold standard of nested PCR for the amplification of a fragment of the 18S subunit of the ribosomal RNA (18S rRNA) gene followed by the sequencing of amplified fragments (nPCR/S). Individual fecal samples were collected on a single occasion from 503 asymptomatic adult snakes housed in the serpentarium of the Butantan Institute in São Paulo, Brazil. The nested PCR revealed that 60 samples (11.98%) were positive for Cryptosporidium sp. The sequencing of amplified fragments, which was possible for 38 samples, resulted in the identification of Cryptosporidium tyzzeri (7), Cryptosporidium muris (4), Cryptosporidium varanii (12) and C. serpentis (15) in fecal samples from several snake species. The real-time PCR approach indicated that 17 samples (3.37%) were positive for C. serpentis, whereas the nPCR/S indicated that 15 samples (2.98%) were positive for C. serpentis. The epidemiological sensitivity and specificity of real-time PCR were 93.8% and 99.5%, respectively. Thus, we conclude that real-time PCR targeting the Hsp70 gene is a sensitive and specific method for the detection of C. serpentis in snake fecal samples.
Asunto(s)
Criptosporidiosis/epidemiología , Cryptosporidium/aislamiento & purificación , Proteínas HSP70 de Choque Térmico/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Serpientes/parasitología , Animales , Secuencia de Bases , Brasil/epidemiología , Criptosporidiosis/parasitología , Cryptosporidium/genética , ADN Protozoario/química , ADN Protozoario/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Heces/parasitología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/veterinaria , Proteínas Protozoarias/genética , ARN Ribosómico 18S/genética , Sensibilidad y Especificidad , Análisis de Secuencia de ADN/veterinariaRESUMEN
Infection by Cryptosporidium serpentis is one of the most important diseases in reptiles and is characterized by chronic clinical or subclinical infection and the presence of hypertrophic gastritis, food regurgitation, progressive weight loss, mortality, and intermittent or continuous shedding of oocysts in the feces. The objectives of this study were to standardize an indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies against C. serpentis and to evaluate the clinical, parasitological, and humoral immune response in snakes naturally infected with C. serpentis. Twenty-one snakes naturally infected with C. serpentis and housed at the Butantan Institute, São Paulo, Brazil, underwent clinical and parasitological analyses for C. serpentis infection through daily records of clinical signs and a monthly survey of fecal shedding of oocysts using the Kinyoun's acid-fast staining. The serological evaluation was performed monthly by indirect ELISA using crude total antigen from oocysts of C. serpentis to detect anti-C. serpentis antibodies. Clinical symptoms consisted of food regurgitation, inappetence, and progressive weight loss. The parasitological analysis revealed intermittent fecal shedding of a variable number of oocysts in all snakes, with positivity in 85.32% (157/184) of the samples. The indirect ELISA was positive in 68.25% (86/126) of the samples. A humoral immune response was observed in most animals; however, fluctuating antibodies levels, leading to alternating positive and negative results, were observed in most snakes.