SUMMARY. Anaerobic intestinal spirochetes (genus Brachyspira) include several species that are recognized as pathogens of poultry. Surveys undertaken in Europe and Australia have shown that layer and breeder flocks are often colonized by the pathogenic species Brachyspira intermedia and Brachyspira pilosicoli, but similar surveys have not been conducted in the United States. In the current study, DNA was extracted from fecal samples (n = 50) collected from each of 21 flocks of laying hens >40 wk of age in Pennsylvania, and this material was tested for B. intermedia and B. pilosicoli using a duplex PCR. Negative samples also were tested using a Brachyspira genus-specific PCR. The consistency of the feces was observed, and manure handling systems and medication histories were recorded. Brachyspira intermedia was detected in 662 (63.1%) samples from 17 (81%) flocks, with a within-flock prevalence of 10%-100%. Brachyspira pilosicoli was detected in 112 (10.7%) samples from 5 flocks (23.8%), with a within-flock prevalence of 8%-82%. Four of the flocks had both pathogenic species present, three had no pathogenic species detected, and two had no Brachyspira species detected. Nine flocks had many fecal samples with a wet appearance and/or a caramel color, and all of these were colonized with one or the other of the two pathogenic species. Nine of 12 flocks with manure that was mainly dry also were colonized. Differences in colonization rates between flocks with or without wet manure were not significant. Colonization with pathogenic Brachyspira species, and particularly B. intermedia, occurs very commonly in layer flocks >40 wk of age in Pennsylvania. The significance of this high rate of colonization requires further investigation.RESUMEN. Las espiroquetas Brachyspira intermedia y Brachyspira pilosicoli se encuentran comunmente en parvadas de aves de postura de mayor edad en el estado de Pennsylvania.Las espiroquetas intestinales anaerobicas del genero Brachyspira incluyen varias especies que estan reconocidas como patogenos en la avicultura. Los muestreos llevados a cabo en Europa y Australia ha demostrado que las parvadas de postura y de reproductoras estan colonizadas por las especies patogenas Brachyspira intermedia y Brachyspira pilosicoli, pero no se han realizados muestreos similares en los Estados Unidos. En el presente estudio, se extrajo ADN de muestras de excretas (n = 50) recolectadas de 21 parvadas de aves de postura mayores de 40 semanas de edad en Pennsylvania, este material se analizo para detectar la presencia de B. intermedia y B. pilosicoli mediante el uso de un metodo PCR para detectar simultaneamente ambas especies. Las muestras negativas tambien fueron analizadas usando un metodo de PCR especifico para el genero Brachyspira. Se observo la consistencia de las excretas, y se registraron los sistemas de manejo de la cama y la historia de medicaciones. Se detecto Brachyspira intermedia en 662 muestras (63.2%) de 17 (81%) parvadas, con prevalencia dentro de las parvadas y entre las parvadas de 10% a 10...