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

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

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

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

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

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

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

Percent of total simulation time occupied by the accessible conformations of each STm OPS^a.

<p>Percent of total simulation time occupied by the accessible conformations of each STm OPS<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005493#t003fn001" target="_blank">^a</a>.</p

STm COPS size characterization and the effect of pH on rhamnose and abequose O-acetylation in 1925wzzB-COPS.

<p>(A) SDS-PAGE and Pro-Q staining for LPS from CVD 1925 (lane 1) and CVD 1925 (pSEC10-<i>wzzB</i>) (lane 2). (B) Chromatogram of purified D65 COPS (dashed line) and 1925wzzB-COPS (solid line) assessed by HPLC-SEC with detection by refractive index. (C) ¹H NMR analysis of 1925wzzB-COPS after exposure to different pH levels. Peaks for rhamnose C2/C3 and abequose C2 O-acetyls are indicated. (D) Hestrin analysis of residual O-acetylation of 1925wzzB-COPS after incubation at different pH levels. (E) ELISA reactivity of 1925wzzB-COPS and dOAc-1925wzzB-COPS with monoclonal antibodies against O4 (α-STm O4) and O5 (α-STm-O5) or polyclonal sera recognizing O1,12 and core epitopes (α-SE). Error bars represent s.d. and were derived from technical replicates from one experiment.</p