Search

Introduction: Avian typhoid and pulorosis, diseases caused by Salmonella enterica subsp. enterica serotype Gallinarum biotype Gallinarum and biotype Pullorum, respectively, are responsible for high mortality in poultry, generating significant economic losses for poultry farmers. Objective: To strengthen the detection of the causative agents of Fowl typhoid and pullorum disease by optimizing molecular techniques, such as endpoint PCR and real-time PCR (qPCR). Methods: Bacterial strains control groups, isolates, and tissue from poultry infected with Salmonella Gallinarum were used to standardize the detection by endpoint PCR and qPCR. Results: For the endpoint PCR, a repeatability, specificity, and sensitivity of 100% was obtained, and a Kappa value of 0.98 for reproducibility. With the qPCR, an efficiency of 103% was obtained, while the coefficients of variation for repeatability and reproducibility were lower than 6%. The detection limit of genomic DNA was 6.4 pg/μL, and the number of viable cells was 3x102 CFU/mL for endpoint PCR and 10 copies of DNA per reaction for qPCR. Through sequencing and analysis of the molecular taxonomy of the PCR products, the strains in this study were positioned together with those of serotype S. Gallinarum, corroborating their identity. Conclusion: It was possible to optimize a molecular technique that allows a rapid, reliable, and sensitive detection of Salmonella Gallinarum /Pullorum, which can reduce the waiting time to take action in cases of clinical suspicion and possible outbreaks. Introducción: La tifosis aviar y la pulorosis, enfermedades ocasionadas por Salmonella enterica subsp. enterica serotipo Gallinarum biotipo Gallinarum y biotipo Pullorum respectivamente, son responsables de una elevada mortalidad en aves de corral generando grandes pérdidas económicas para los avicultores. Objetivo: Fortalecer la detección de los agentes causales de la tifosis aviar y la pulorosis mediante la optimización de técnicas moleculares, tales como la PCR punto final y la PCR tiempo real (qPCR). Métodos: Se emplearon cepas bacterianas control, aislamientos y tejidos de aves de corral infectadas con Salmonella Gallinarum para estandarizar la detección mediante PCR punto final y qPCR. Resultados: Para la PCR punto final se obtuvo una repetibilidad, especificidad y sensibilidad del 100%, y un valor Kappa de 0.98 para la reproducibilidad. Mientras que, con la qPCR se obtuvo una eficiencia del 103% y valores de repetibilidad y reproducibilidad con coeficientes de variación menores al 6%. El límite de detección de ADN genómico fue de 6.4 pg/μL y el del número de células viables de 3x102 UFC/mL para la PCR punto final y de 10 copias de ADN por reacción para la qPCR. Mediante la secuenciación y análisis de los productos de PCR, la topología de taxonomía molecular posicionó a las cepas de este estudio junto con las del serotipo S. Gallinarum, corroborando su identidad. Conclusión: Se logra optimizar una técnica molecular que permite una detección rápida, confiable y sensible de Salmonella Gallinarum/Pullorum, lo cual puede reducir el tiempo de espera para tomar acción en casos de sospecha clínica y posibles brotes.

Agradecemos al Servicio Nacional de Salud Animal (SENASA) por permitir la ejecución de este trabajo, en especial por el acceso a recursos, banco de aislamientos y tejidos en sus instalaciones. Al Laboratorio de Biotecnología de Plantas (LBP) de la Escuela de Ciencias Biológicas, UNA, por facilitar el equipo LightCycler® 96 Instrument (Roche). A la Vicerrectoría de Investigación de la Universidad Nacional por el fondo económico brindado a través del Fondo para el Fortalecimiento de las Capacidades Estudiantiles en Investigación (FOCAES). Introducción: La tifosis aviar y la pulorosis, enfermedades ocasionadas por Salmonella enterica subsp. enterica (serotipo Gallinarum, biotipos Gallinarum y Pullorum), son responsables de una elevada mortalidad en aves de corral y generan grandes pérdidas económicas. Objetivo: Optimizar técnicas moleculares, como la PCR punto final y la PCR de tiempo real (qPCR), para detectar tifosis aviar y pulorosis. Métodos: Se emplearon cepas bacterianas control, aislamientos y tejidos de aves de infectadas con Salmonella Gallinarum para estandarizar la detección con ambas técnicas. Resultados: Para la PCR de punto final se obtuvo una repetibilidad, especificidad y sensibilidad del 100%, y un valor Kappa de 0,98 para la reproducibilidad; para qPCR una eficiencia del 103% y variación menor al 6% en repetibilidad y reproducibilidad. El límite de detección de ADN genómico fue de 6,4 pg/μL y el del número de células viables de 3x102 UFC/mL para la PCR punto final, y de 10 copias de ADN por reacción para la qPCR. Confirmamos la identidad de S. Gallinarum. Y se redujo el tiempo de detección a unas 48 horas, Conclusión: Logramos optimizar una técnica molecular para detección rápida, confiable y sensible de Salmonella Gallinarum/Pullorum, la cual reduce el tiempo de espera para tomar acción en casos de sospecha clínica y posibles brotes. "Optimization of PCR techniques optimization for detection of Salmonella enterica (serotype: Gallinarum) in Costa Rican poultry". Introduction: Avian typhoid and pullorosis, diseases caused by Salmonella enterica subsp. enterica (Gallinarum serotype, Gallinarum and Pullorum biotypes), cause high mortality in poultry and generate large economic losses. Objective: To optimize molecular techniques, such as endpoint PCR and real-time PCR (qPCR), to detect avian typhoid and pullorosis. Methods: We used control bacterial strains, isolates and tissues from birds infected with Salmonella Gallinarum to standardize detection with both techniques. Results: For the endpoint PCR, we obtained 100% repeatability, specificity and sensitivity, and a Kappa value of 0.98 for reproducibility; for qPCR, 103% efficiency with a variation under 6% in repeatability and reproducibility. The detection limit for genomic DNA was 6.4 pg/μL and the limit for the number of viable cells was 3x102 CFU/mL for endpoint PCR, and 10 DNA copies per reaction for qPCR. We also confirmed the identity of S. Gallinarum, and was reduced. We reduced the detection time to about 48 hours. Conclusion: We optimized a molecular technique for rapid, reliable and sensitive detection of Salmonella Gallinarum/Pullorum, which reduces the waiting time to take action in cases of clinical suspicion and possible outbreaks. Universidad Nacional, Costa Rica Escuela de Medicina Veterinaria

Rhodococcus equi pneumonia is an important cause of morbidity and mortality in foals all over the world. Prevalence and/or virulence of bacteria is apparently low in Costa Rica since no outbreaks have been reported and suspicious cases show only mild to moderate clinical signs. However, there has never been a definitive diagnosis of the disease in Costa Rican horses. R. equi is a G (+) facultative intracellular bacteria causing pneumonia in young foals (up to 4-5 months of age). This paper refers to a 4-week old Friesian foal with a humerus fracture and omphalophlebitis that was referred to the Equine Hospital, National University, Costa Rica, for surgery. In the postoperatory period, the patient showed signs of moderate bilateral nasal discharge, cough, fever, lymphocytosis, and neutrophilia. The radiological evaluation of the thorax showed a bronchoalveolar pattern in the ventro cranial region of the lungs. The transtracheal aspirate cytology confirmed the presence of suppurative bronchopneumonia. The samples were aerobically cultured in Columbia agar + 5% blood at 37°C for 24h. The macroscopic, microscopic, and biochemical characteristics of the isolated bacteria, in addition to the partial sequencing of the “16SrRNA” gene, were compatible with R. equi. The foal was euthanized due to the humerus re-fracture before the R. equi diagnosis was confirmed. R. equi can potentially cause a severe monetary impact in the equine breeding industry; consequently, accurate diagnosis and treatments are important to avoid the emergence of resistant strains. Further studies are needed to determine the local prevalence of the pathogen and to evaluate its impact. Neumonía por Rhodococcus equi es importante causa de morbilidad y mortalidad en potrillos de todo el mundo. La prevalencia y/o virulencia de la bacteria es, aparentemente, baja en Costa Rica, pues nunca han sido reportados brotes. Los casos sospechosos muestran solamente signos clínicos de leves a moderados. Sin embargo, un diagnóstico definitivo de la enfermedad nunca ha sido hecho anteriormente en caballos costarricenses. R. equi es una bacteria G (+) facultativa intracelular que causa neumonía en potrillos jóvenes (hasta 4-5 meses de edad). Este caso corresponde a un potrillo Frisón de cuatro semanas de edad que fue referido para cirugía al Hospital Equino, Universidad Nacional de Costa Rica, con una fractura de húmero y onfaloflebitis. En el período postoperatorio, el paciente presentó: signos de secreción nasal bilateral moderada, tos, fiebre, linfocitosis y neutrofilia. La evaluación radiológica del tórax mostró un patrón bronquialveolar en el aspecto ventrocraneal de los pulmones. La citología del aspirado transtraqueal confirmó un cuadro de bronconeumonía supurativa. Las muestras fueron cultivadas aeróbicamente en agar Columbia + 5% sangre a 37°C por 24h. Las características macroscópicas, microscópicas, bioquímicas y la identificación, por medio de la secuenciación parcial del gen “16SrRNA”, de la bacteria aislada fueron compatibles con R. equi. El potrillo fue eutanasiado debido a re-fractura del húmero antes de que el diagnóstico de R. equi fuera confirmado. R. equi tiene el potencial de causar un severo impacto monetario a la industria de cría equina, por lo cual, es importante hacer el diagnóstico y tratamiento precisos para evitar el surgimiento de cepas resistentes. Son necesarios más estudios para determinar la prevalencia local del patógeno y evaluar su impacto.

It has been proposed that Helicobacter may play a significant role in the generation of gastritis and cancer in dogs. However, despite several independent efforts, there is still controversy regarding the true ability of Helicobacter to cause gastric lesions in these animals. The foregoing is due in part to the fact that gastritis is a multifactorial disease. Studies focused on naturally acquired infections have been unable to establish a positive correlation between Helicobacter colonization and gastritis. Establishing a clear association between the presence of Helicobacter and gastritis may be difficult due to the following reasons: (i) taxonomy of the genus Helicobacter remains unclear, (ii) virulence factors of Helicobacter species that colonize dogs are not well known, and (iii) simultaneous infections have been reported with several species of Helicobacter. Current knowledge in taxonomy and pathogenesis of Helicobacter species is insufficient to differentiate between pathogenic and non-pathogenic strains. Therefore, no direct association should be made between the detection of Helicobacter and gastritis. It has been proposed that Helicobacter may play a significant role in the generation of gastritis and cancer in dogs. However, despite several independent efforts, there is still controversy regarding the true ability of Helicobacter to cause gastric lesions in these animals. The foregoing is due in part to the fact that gastritis is a multifactorial disease. Studies focused on naturally acquired infections have been unable to establish a positive correlation between Helicobacter colonization and gastritis. Establishing a clear association between the presence of Helicobacter and gastritis may be difficult due to the following reasons: (i) taxonomy of the genus Helicobacter remains unclear, (ii) virulence factors of Helicobacter species that colonize dogs are not well known, and (iii) simultaneous infections have been reported with several species of Helicobacter. Current knowledge in taxonomy and pathogenesis of Helicobacter species is insufficient to differentiate between pathogenic and non-pathogenic strains. Therefore, no direct association should be made between the detection of Helicobacter and gastritis.