title: Multiple sampling and discriminatory fingerprinting reveals clonally complex and compartmentalized infections by M. bovis in cattle.
abstract: The combination of new genotyping tools and a more exhaustive sampling policy in the analysis of infection by Mycobacterium tuberculosis has shown that infection by this pathogen is more complex than initially expected. Mixed infections, coexistence of clonal variants from a parental strain, and compartmentalized infections are all different modalities of this clonal complexity. Until recently, genotyping of Mycobacterium bovis in animal populations was based on spoligotyping and analysis of a single isolate per infection; therefore, clonal complexity is probably underdetected. We used multiple sampling combined with highly discriminatory MIRU-VNTR to study compartmentalized infections by M. bovis in a low-tuberculosis prevalence setting. We spoligotyped the M. bovis isolates from two or more anatomic locations sampled from 55 animals on 39 independent farms. Compartmentalized infections, with two different strains infecting independent lymph nodes in the same animal, were found in six cases (10.9%). MIRU-VNTR analysis confirmed that the compartmentalization was strict and that only one strain was present in each infected node. MIRU-VNTR analysis of additional infected animals on one of the farms confirmed that the compartmentalized infection was a consequence of superinfection, since the two strains were independently infecting other animals. This same analysis revealed the emergence of a microevolved clonal variant in one of the lymph nodes of the compartmentalized animal. Clonal complexity must also be taken into consideration in M. bovis infection, even in low-prevalence settings, and analyses must be adapted to detect it and increase the accuracy of molecular epidemiology studies.
authors.affiliation: Servicio Microbiología Clínica y Enfermedades Infecciosas, Hospital General Universitario Gregorio Marañón, Madrid, Spain; Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain; CIBER Enfermedades respiratorias CIBERES, Spain; CEI Campus Moncloa, UCM-UPM, Madrid, Spain; Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Universidad Complutense Madrid, 28040 Madrid, Spain.
authors.forename: Yurena
authors.initials: Y
authors.lastname: Navarro
authors.affiliation: CEI Campus Moncloa, UCM-UPM, Madrid, Spain; Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Universidad Complutense Madrid, 28040 Madrid, Spain.
authors.forename: Beatriz
authors.initials: B
authors.lastname: Romero
authors.affiliation: Laboratorio de Sanidad Animal del Principado de Asturias, 33299 Gijón, Spain.
authors.forename: María Francisca
authors.initials: MF
authors.lastname: Copano
authors.affiliation: Servicio Microbiología Clínica y Enfermedades Infecciosas, Hospital General Universitario Gregorio Marañón, Madrid, Spain; Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain; CIBER Enfermedades respiratorias CIBERES, Spain; Departamento de Medicina, Facultad de Medicina, Universidad Complutense de Madrid, Madrid, Spain.
authors.forename: Emilio
authors.initials: E
authors.lastname: Bouza
authors.affiliation: CEI Campus Moncloa, UCM-UPM, Madrid, Spain; Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Universidad Complutense Madrid, 28040 Madrid, Spain; Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense Madrid, 28040 Madrid, Spain.
authors.forename: Lucas
authors.initials: L
authors.lastname: Domínguez
authors.affiliation: CEI Campus Moncloa, UCM-UPM, Madrid, Spain; Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Universidad Complutense Madrid, 28040 Madrid, Spain; Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense Madrid, 28040 Madrid, Spain.
authors.forename: Lucía
authors.initials: L
authors.lastname: de Juan
authors.affiliation: Servicio Microbiología Clínica y Enfermedades Infecciosas, Hospital General Universitario Gregorio Marañón, Madrid, Spain; Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain; CIBER Enfermedades respiratorias CIBERES, Spain; CEI Campus Moncloa, UCM-UPM, Madrid, Spain. Electronic address: dgviedma2@gmail.com.
authors.forename: Darío
authors.initials: D
authors.lastname: García-de-Viedma
mesh_terms: D000818:Animals; D015373:Bacterial Typing Techniques / Q000662:veterinary; D002417:Cattle; D002418:Cattle Diseases / Q000382:microbiology*; D060965:Clonal Evolution; D060085:Coinfection; D005838:Genotype; D017720:Molecular Epidemiology; D009163:Mycobacterium bovis / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000662:veterinary; D013048:Specimen Handling; D014380:Tuberculosis, Bovine / Q000382:microbiology*
keywords: Clonal complexity; Coinfection; Compartmentalization; M. bovis; Microevolution