Salmonella Typhimurium: an extracellular parasite in porcine tonsils and gut-associated lymph nodes

Salmonellosis is one of the most important bacterial zoonotic diseases in humans. In Belgium, Salmonella enterica subspecies enterica serovar Typhimurium (Salmonella Typhimurium) was most frequently associated with human salmonellosis in 2006 due to the consumption of contaminated pork. Pig carcass contamination with Salmonella Typhimurium can largely be attributed to persistently infected pigs. Tonsils are a predilection site for Salmonella persistence in pigs1. The mechanism used by Salmonella Typhimurium to persist in the tonsil is not yet understood but apparently differs from that used for intestinal colonization2. Virulence mechanisms, necessary for cell invasion and intracellular survival, do not contribute to tonsil colonization, suggesting that Salmonella Typhimurium resides extracellularly in porcine tonsils. It was the aim of the present study to determine whether or not Salmonella Typhimurium persists intracellularly in porcine tonsils. Four-week-old piglets were inoculated 7 days after arrival with a nalidixic acid resistant Salmonella Typhimurium strain 112910a, phage type 120/ad. On days 5 and 28 post inoculation (pi), 9 and 4 piglets respectively were euthanized. Samples of tonsils and mesenteric and ileocaecal lymph nodes were collected to prepare single cell suspensions and cryosections. Single cell suspensions were obtained by treating tissue samples with collagenase. One ml of this suspension was plated on brilliant green agar containing nalidixic acid (BGANAL) to assess the total number of intra- and extracellular salmonellae. The remaining suspension was treated with gentamicin, killing all extracellular salmonellae. The tissue cells were then lysed and again 1 ml of the suspension was plated on BGANAL to determine the number of intracellular salmonellae. The cryosections were treated with a primary antibody mixture containing rabbit antibody to the Salmonella O4 antigen and mouse antibody to tissue cell receptor CD172α. Sections were then incubated with a secondary antibody mixture containing goat anti-rabbit Alexa Fluor 488 and goat anti-mouse Alexa Fluor 568. Sections were evaluated using a Leica DMRB fluorescence microscope. Five as well as 28 days pi, only a minority of the Salmonella bacteria (respectively 90%) and 28 days (>85%) pi. The results obtained by single cell suspensions and fluorescence microscopy show that Salmonella Typhimurium resides largely extracellularly in porcine tonsils and gut-associated lymph nodes in the early and late phase of infection. These results suggest that control measures interfering with the persistence of Salmonella Typhimurium in pigs should focus primarily on the extracellular bacteria