Virulence of invasive <i>Salmonella</i> Typhimurium ST313 in animal models of infection

<div><p><i>Salmonella</i> 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 <i>in vivo</i> virulence of ST313 strains have been reported. To resolve these differences, we tested clinical <i>Salmonella</i> Typhimurium ST313 and ST19 strains in murine and rhesus macaque infection models. The 50% lethal dose (LD₅₀) was determined for three <i>Salmonella</i> 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 <i>Salmonella</i> 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.</p></div

Qualitative and quantitative detection of <i>Salmonella</i> Typhimurium in blood, ileum, colon and mesenteric lymph nodes (MLN) of rhesus macaques.

<p>Qualitative and quantitative detection of <i>Salmonella</i> Typhimurium in blood, ileum, colon and mesenteric lymph nodes (MLN) of rhesus macaques.</p

Histopathology of tissues infected with <i>Salmonella</i> Typhimurium.

<p>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 <i>Salmonella</i> Typhimurium I77 (ST19). Images on the right (20X magnification) are from animals infected with <i>Salmonella</i> 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.</p

Bacterial load of <i>Salmonella</i> Typhimurium ST313 and ST19 in mice.

<p>CD-1 mice were infected perorally with 3 strains of <i>Salmonella</i> 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 <i>Salmonella</i> 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.</p

Diarrhea, lethargy and dysentery elicited by <i>Salmonella</i> Typhimurium I77 (ST19) and D65 (ST313) in Indian rhesus macaques.

<p>Diarrhea, lethargy and dysentery elicited by <i>Salmonella</i> Typhimurium I77 (ST19) and D65 (ST313) in Indian rhesus macaques.</p

Shedding of <i>Salmonella</i> Typhimurium in the feces of rhesus macaques.

<p>Following infection with <i>Salmonella</i> 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.</p

50% lethal dose of ST19 and ST313 strains in juvenile BALB/c and adult BALB/c and CD-1 mice.

<p>50% lethal dose of ST19 and ST313 strains in juvenile BALB/c and adult BALB/c and CD-1 mice.</p

Clinical signs in rhesus macaques.

<p>Rhesus macaques infected with <i>Salmonella</i> 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.</p

Differential Response of the Cynomolgus Macaque Gut Microbiota to <i>Shigella</i> Infection

<div><p>Little is known about the role of gut microbiota in response to live oral vaccines against enteric pathogens. We examined the effect of immunization with an oral live-attenuated <i>Shigella dysenteriae</i> 1 vaccine and challenge with wild-type <i>S. dysenteriae</i> 1 on the fecal microbiota of cynomolgus macaques using 16 S rRNA analysis of fecal samples. Multi-dimensional cluster analysis identified different bacterial community types within macaques from geographically distinct locations. The fecal microbiota of Mauritian macaques, observed to be genetically distinct, harbored a high-diversity community and responded differently to <i>Shigella</i> immunization, as well as challenge compared to the microbiota in non-Mauritian macaques. While both macaque populations exhibited anti-<i>Shigella</i> antibody responses, clinical shigellosis was observed only among non-Mauritian macaques. These studies highlight the importance of further investigation into the possible protective role of the microbiota against enteric pathogens and consideration of host genetic backgrounds in conducting vaccine studies.</p></div

Community types within the fecal microbiota of cynomolgus macaques.

<p>(<b>A–C</b>) Principal coordinates analysis (PCoA) of axes 1 vs. 2 (<b>A</b>), 2 vs. 3 (<b>B</b>), and a three-dimensional plot of axes 1–3 (<b>C</b>) based on Jensen-Shannon divergence of all samples (n = 374), color-coded by community type clusters identified by the partitioning around medoids (pam) clustering algorithm. Percent variance is shown for each axis. (<b>D</b>) Boxplots of the median relative abundance (y-axis) of dominant genera and (<b>E</b>) Shannon diversity index (y-axis), observed in each community type (x-axis). Error bars indicate the interquartile range between the first and third quartiles.</p