Periplasmic protein EipA determines envelope stress resistance and virulence in Brucella abortus.

Molecular components of the Brucella abortus cell envelope play a major role in its ability to infect, colonize and survive inside mammalian host cells. In this study, we have defined a role for a conserved gene of unknown function in B. abortus envelope stress resistance and infection. Expression of this gene, which we name eipA, is directly activated by the essential cell cycle regulator, CtrA. eipA encodes a soluble periplasmic protein that adopts an unusual eight-stranded ß-barrel fold. Deletion of eipA attenuates replication and survival in macrophage and mouse infection models, and results in sensitivity to treatments that compromise the cell envelope integrity. Transposon disruption of genes required for LPS O-polysaccharide biosynthesis is synthetically lethal with eipA deletion. This genetic connection between O-polysaccharide and eipA is corroborated by our discovery that eipA is essential in Brucella ovis, a naturally rough species that harbors mutations in several genes required for O-polysaccharide production. Conditional depletion of eipA expression in B. ovis results in a cell chaining phenotype, providing evidence that eipA directly or indirectly influences cell division in Brucella. We conclude that EipA is a molecular determinant of Brucella virulence that functions to maintain cell envelope integrity and influences cell division.

The virB-encoded type IV secretion system is critical for establishment of infection and persistence of Brucella ovis infection in mice.

Brucella spp. are gram-negative intracellular bacterial pathogens that cause chronic infections. Brucella virulence factors include a type IV secretion system (T4SS) and its lipopolysaccharide (LPS), which are essential for persistence. However, the role of the virB-encoded T4SS has not been investigated in naturally rough Brucella species such as Brucella ovis. In this study, male 6-week old BALBc mice were infected with B. ovis, Brucella abortus, and their respective ΔvirB2 mutant strains. During early infection, B. ovis and B. abortus wild type strains were similarly recovered from spleen. Interestingly, in contrast to ΔvirB2 B. abortus that was recovered at similar levels when compared to the wild type strain, the ΔvirB2 B. ovis was markedly attenuated as early as 24h post infection (hpi). The ΔvirB2 B. ovis was unable to survive and multiply in murine peritoneal macrophages and extracellularly within the peritoneal cavity at 12 and 24 hpi with lower splenic colonization than the parental strain at 6, 12 and 24 hpi. In contrast, wild type B. abortus and ΔvirB2 B. abortus had a similar kinetics of infection in this model. As expected, the T4SS was essential for intracellular replication of smooth and rough strains in RAW macrophages at 48 hpi. These results suggest that T4SS is important for survival of B. ovis in murine model, and that a T4SS deficient B. ovis strain is cleared at earlier stages of infection when compared to a similar B. abortus mutant.

Cinética de la infección por Brucella ovis en carneros durante una época de empadre / Kinetics of Brucella ovis infection in rams during breeding season

Con la finalidad de estudiar la evolución de la infección a Brucella ovis en condiciones de campo, una punta de carneros de plantel (n=250) de una empresa ovejera de la sierra central del país fueron examinados clínica y serológicamente durante el período de empadre (2 meses). Al inicio del empadre ningún animal tenía evidencias clínicas de la enfermedad (epididimitis), pero el 58.7 por ciento era positivo a la prueba de ELISA indirecta. Al término del empadre el 28.4 por ciento de los reproductores tuvo que ser eliminado por lesiones testiculares evidentes, en tanto que los exámenes de ELISA demostraron que 74.4 por ciento de los carneros eran positivos a la prueba. El estudio evidenció una rápida evolución de un estado de animal seropositivo a animal clínicamente enfermo. En estas condiciones de campo, los exámenes clínicos no son suficientes medidas para controlar la infección, por lo que es fundamental realizar exámenes serológicos y eliminar a todo reactor positivo. Si no se practica estas recomendaciones, es mejor buscar otras alternativas de control incluyendo el uso de vacunas.

The evolution of Brucella ovis infection was documented clinically and serologically in a flock of 250 field reared rams from a large sheep production company in the central sierra of Peru. Although no clinical evidence of Brucellosis (epididymitis) was detected at the start of a 2-month breeding campaign, 58.7 of the animals were positive to the indirect ELISA test. At the end of the breeding season, 74.4 percent tested positive using indirect ELISA and 28.4 percent of the animals had to be eliminated due to testicular lesions. These results demonstrate rapid evolution of the bacterial infection from seropositive to clinical manifestation, indicating that reliance on clinical observation alone is insufficient to control the infection under field conditions. Serological testing is necessary in order to identify and eliminate all positive reactors. If this procedure is not followed, alternative control alternatives such as vaccines should be employed to control the disease.

Uso de la REV-1 en el control de la brucelosis ovina en una empresa ovejera del Perú / Using the REV-1 in the control of ovine brucellosis in sheep company in Peru

Se evaluó el efecto de la vacuna Rev-1 en el control de la brucelosis ovina causado por Brucella ovis en una empresa lanar de la sierra central del Perú. El uso de esta vacuna fue reintroducida en 1996, después de un lapso de 5 años. Al momento de la evaluación, la empresa mantenía el 86.3 por ciento de carneros vacunados (3,284/3,804) y el 100 por ciento de carnerillos vacunados (n=1,811). La evaluación consistió en exámenes clínicos testiculares a toda la población de reproductores machos (n=5,615) durante la campaña de esquila del año 2000. Paralelamente, se evaluó niveles de infección en 320 muestras sanguíneas (214 de carneros y 106 de carnerillos), detectando anticuerpos específicos para la Brucella ovis mediante la prueba de inmunodifusión en gel de agarosa (AGID). Los exámenes testiculares revelaron prevalencias de lesiones de epididimitis en tasas de 89.4 x 10 mil en la población adulta (carneros) y 38.6 x 10 mil en la población de jóvenes (carnerillos). Estas prevalencias son significativamente inferiores a las encontradas antes de la reintroducción de la vacuna, en la que se detectaron prevalencias de 817 x 10 mil en carneros y 241 x 10 mil en carnerillos, encontrándose una asociación directa entre el uso de la vacuna y la disminución progresiva de la prevalencia de epididimitis. Asimismo, la prevalencia de la infección global en la población de machos disminuyó significativamente desde 1,186.4 x 10 mil en 1996 a 531.2 x 10 mil en el año 2000; pero con niveles altos de infección en la población de carneros vacunados (635.8 x 10 mil). La población de carneros vacunados muestra una relativa alta prevalencia de la enfermedad clínica (97.4 x 10 mil) comparada con la población no vacunada (38.5 x 10 mil), pero con menor tasa de infección (635.8 x 10 mil) que los no vacunados (1,219.5 x 10 mil).

In 1996, after a 5 year hiatus, the use of the Rev-1 vaccine was reintroduced to control ovine brucellosis (Brucella ovis) in a large sheep company of the central Peruvian Andes, and by the year 2000, 86.3 percent of the rams (3,284 of 3,804) and 100 percent of the young males (n=1,811) were vaccinated. During the shearing campaign for year 2000, testicles of the entire male breeding population (n=5,615) were examined manually and 320 blood samples (214 rams and 106 yearlings) were taken for AGID testing to determine the presence of Brucella ovis antibodies. Epididymitis lesions were found in 89.4 x 10,000 of the rams and 38.6 x 10,000 of the yearlings, compared to 817 x 10,000 for rams and 214 x 10,000 for yearlings prior to reintroduction of Rev-1 vaccination. The progressive reduction in epididymitis was directly related to vaccination. The level of infection was found 1,186.4 x 10,000 in 1996 decreasing to 531.2 x 10,000 in 2000, but infection levels remained high in the vaccinated population (635.8 x 10,000). Clinical expression of the disease was 38.5 x 10,000 in unvaccinated males compared to 97.4 x 10,000 in vaccinated animals, but infection rates were considerably lower in vaccinated (635.8 x 10,000) than in unvaccinated (1,219 x 10,000) males. These results clearly demonstrate the efficaciousness of Rev-1 in controlling epididymitis in rams.