Virulence of invasive Salmonella Typhimurium ST313 in animal models of infection

Abstract

Salmonella Typhimurium sequence type (ST) 313 produces septicemia in infants in sub-Saharan Africa. Although there are known genetic and phenotypic differences between ST313 strains and gastroenteritis-associated ST19 strains, conflicting data about the in vivo virulence of ST313 strains have been reported. To resolve these differences, we tested clinical Salmonella Typhimurium ST313 and ST19 strains in murine and rhesus macaque infection models. The 50% lethal dose (LD50) was determined for three Salmonella Typhimurium ST19 and ST313 strains in mice. For dissemination studies, bacterial burden in organs was determined at various time-points post-challenge. Indian rhesus macaques were infected with one ST19 and one ST313 strain. Animals were monitored for clinical signs and bacterial burden and pathology were determined. The LD50 values for ST19 and ST313 infected mice were not significantly different. However, ST313-infected BALB/c mice had significantly higher bacterial numbers in blood at 24 h than ST19-infected mice. ST19-infected rhesus macaques exhibited moderate-to-severe diarrhea while ST313-infected monkeys showed no-to-mild diarrhea. ST19-infected monkeys had higher bacterial burden and increased inflammation in tissues. Our data suggest that Salmonella Typhimurium ST313 invasiveness may be investigated using mice. The non-human primate results are consistent with clinical data, suggesting that ST313 strains do not cause diarrhea.

Fig 1. Bacterial load of Salmonella Typhimurium ST313 and ST19 in mice.

CD-1 mice were infected perorally with 3 strains of Salmonella Typhimurium ST19 (I77, I41, S52) and ST313 (D65, Q55, S11). The number of CFUs at 3, 24, 72 and 168 h p.i. were determined in blood (A), spleen (B) and liver (C). BALB/c mice were infected perorally with Salmonella Typhimurium I77 and D65 and blood harvested at 3 and 24 h p.i. (D). Results are expressed as mean ± SD. *** represents P < 0.001, Student’s t-test, two-tailed.

Fig 2. Clinical signs in rhesus macaques.

Rhesus macaques infected with Salmonella Typhimurium I77 (ST19) or D65 (ST313) were measured for (A) Percent change in weight, and (B) Temperature (in Fahrenheit) on days 1, 3, 7, 15 and 21/22 days post infection. Blood from infected monkeys was collected on days 0, 7 and 15 and measured for (C) white blood cell (WBC) and (D) neutrophil counts.

Fig 3. Shedding of Salmonella Typhimurium in the feces of rhesus macaques.

Following infection with Salmonella Typhimurium I77 (ST19) or D65 (ST313), fecal samples were collected on days 1 to 7, 9, 11, 15, 18 and 21 or 22. Data is represented as CFU per gram of feces. ** represents P < 0.01, Student’s t-test, two-tailed.

Fig 4. Histopathology of tissues infected with Salmonella Typhimurium.

Hematoxylin and eosin-stained sections of liver, colon, ileum and mesenteric lymph node (MLN) from rhesus macaques. The images in the left (10X magnification) and middle (20X magnification) columns are from animals infected with Salmonella Typhimurium I77 (ST19). Images on the right (20X magnification) are from animals infected with Salmonella Typhimurium D65 (ST313). Areas of necrosis and lymphocytic infiltration of the liver section from a ST19-infected animal are indicated by arrows. Tissue sections from a ST19-infected animal show areas of erosion of mucosal epithelium of the colon and ileum combined with areas of infiltration of lymphocytes and plasma cells. These areas are indicated by arrows. Granulomatous areas consisting of multinucleated giant cells in the mesenteric lymph node of the animal infected with ST19 are indicated by arrows. All sections from D65-infected animals display essentially normal histology.