Histopathological Analysis of <i>Salmonella</i> Chronic Carriage in the Mouse Hepatopancreatobiliary System

<div><p><i>Salmonella</i> Typhi asymptomatic chronic carriage represents a challenge for the diagnosis and prevention of typhoid fever in endemic areas. Such carriers are thought to be reservoirs for further spread of the disease. Gallbladder carriage has been demonstrated to be mediated by biofilm formation on gallstones and by intracellular persistence in the gallbladder epithelium of mice. In addition, both gallstones and chronic carriage have been associated with chronic inflammation and the development of gallbladder carcinoma. However, the pathogenic relationship between typhoid carriage and the development of pre-malignant and/or malignant lesions in the hepatopancreatobiliary system as well as the host-pathogen interactions occurring during chronic carriage remains unclear. In this study, we monitored the histopathological features of chronic carriage up to 1 year post-infection. Chronic cholecystitis and hepatitis ranging from mild to severe were present in infected mice regardless of the presence of gallstones. Biliary epithelial hyperplasia was observed more commonly in the gallbladder of mice with gallstones (uninfected or infected). However, pre-malignant lesions, atypical hyperplasia and metaplasia of the gallbladder and exocrine pancreas, respectively, were only associated with chronic <i>Salmonella</i> carriage. This study has implications regarding the role of <i>Salmonella</i> chronic infection and inflammation in the development of pre-malignant lesions in the epithelium of the gallbladder and pancreas that could lead to oncogenesis.</p> </div

Chronic hepatitis is exacerbated in infected mice at 3 and 6 months post-infection.

<p>(A) Inflammation grades in the liver at all time points post-infection (± bacteria, ± gallstones). Means of each time point values were compared by a Student’s <i>t</i> test (*, <i>p</i><0.05; ***, <i>p</i><0.001). (B) Representative HE images of each group at 3 and 6 months post-infection. Inflammation scores are as follows: B1 (0); B2 (1); B3 and B6 (2); B4 and B5 (4). Infected mice have scattered inflammatory cells distributed randomly within the lobule (B3-B5) and/or in portal areas (B6) composed of neutrophils, lymphocytes, plasma cells and histiocytes. Inflammation was often associated with necrosis of individual hepatocytes (B3) or large confluent areas of hepatocellular necrosis (B4-B5) (noted as N). 20x. </p

Chronic cholecystitis occurs as a result of both gallstone disease and <i>Salmonella</i> carriage.

<p>(A) Inflammation grades in the gallbladder at all time points post-infection (± bacteria, ± gallstones). Means of each time point values were compared by a Student’s <i>t</i> test. No statistical significant difference was observed between values of each time point (<i>p</i><0.5). (B) Representative HE images of each group at 3 and 9 months post-infection. B1 has an inflammation score of 0; B2, B3, B5 and B6 have a inflammation score of 2; whereas B4 has a score of 3. Hyperplasia was only evident in mice harboring gallstones and with concurrent epithelial hyalinosis (*) (B2, B5 and B6). Black arrows indicate scattered inflammatory cells within the lamina propria and muscularis, they were mainly composed of lymphocytes, plasma cells and fewer neutrophils. 40x.</p

<i>Salmonella</i> LPS staining was detected in the liver regardless of the presence of gallstones.

<p>Representative IHC images of mouse liver sections at 3 and 9 months post-infection using an anti-<i>Salmonella</i> LPS antibody. Panel C shows a typhoid nodule. Black arrows indicate the presence of <i>Salmonella</i> inside inflammatory macrophages (Panel C) or Kupffer cells (Panel F) or intracellularly in hepatocytes (Panels D, E). DAB and hematoxylin counterstain. 40x.</p

Pancreatic mucinous metaplasia was only evident in infected mice regardless of the presence of gallstones.

<p>Representative HE images of pancreas 6 months post-infection. Black arrows indicate mucinous metaplasia associated with exocrine pancreatic acini at the periphery of marked chronic necrosuppurative inflammation. 20x. </p

Chronic pancreatitis is exacerbated in infected mice at 3 and 6 months post-infection.

<p>(A) Inflammation grades in the pancreas at all time points post-infection (± bacteria, ± gallstones). Means of each time point values were compared by a Student’s <i>t</i> test (*, <i>p</i><0.05; **, <i>p</i><0.01 ***, <i>p</i><0.001). (B) Representative HE images of each group at 3 and 6 months post-infection. Inflammation scores are as follows: B1 (0); B2 (1); B3 (2); B4, B5 and B6 (3). Inflammatory cells composed of scattered neutrophils, lymphocytes and plasma cells were occasionally distributed within the interstitium of the exocrine pancreas between acini (circle). In more severe cases, inflammation was centered upon and typically obliterated large ducts (B4-B6) and was characterized by necrotic debris (*) centrally, and lymphoplasmacytic aggregates and mucous metaplasia (black arrows) peripherally. 20x. </p

<i>Salmonella</i> LPS staining was frequently detected in the gallbladder of infected mice regardless of the presence of gallstones.

<p>Representative IHC images of mouse gallbladder tissue at 6 and 12 months post-infection using an anti-<i>Salmonella</i> LPS antibody. Such bacterial staining occurs at time points in which no CFU were detected from the gallbladder of cage mates. Black arrows show <i>Salmonella</i> LPS staining in the muscularis (Panel C and D), in the epithelium (Panel E) or in the lumen (Panel F). DAB and hematoxylin counterstain. 40x.</p

<i>Salmonella</i> detection in organs and bodily fluids of chronically infected mice up to 1 year post-infection.

<p>Bacterial CFU enumeration of <i>Salmonella</i> at various times post-infection in the absence and presence of gallstones. nd, not detected. Limit of detection was 100 CFU /mL. No statistically significant difference was observed between infected mice with or without gallstones at any time point (Student’s <i>t</i> test). nd, not detected.</p

<i>Salmonella</i> LPS staining was detected in the exocrine pancreas regardless of the presence of gallstones.

<p>Representative IHC images of mouse pancreas sections at 3 and 9 months post-infection using an anti-<i>Salmonella</i> LPS antibody. <i>Salmonella</i> was detected in the interstitium around acini or ductular cells (Panels D, E and F). DAB and hematoxylin counterstain. 40x.</p

Atypical hyperplasia in the absence of marked necrosuppurative inflammation and/or epithelial hyalinosis was observed in the gallbladder of infected mice, regardless of the presence of gallstones.

<p>Representative HE images of gallbladder 3 months post-infection. Black arrows indicate tall columnar biliary epithelial cells with large crowded, basophilic to vesicular nuclei and loss of polarity.40x.</p