A New Murine Model for Gastrointestinal Anthrax Infection

Citation: Xie T, Sun C, Uslu K, Auth RD, Fang H, et al. ( A New Murine Model for Gastrointestinal Anthrax Infection Tao Xie 0 Chen Sun 0 Kadriye Uslu 0 Roger D. Auth 0 Hui Fang 0 Weiming Ouyang 0 David M. Frucht 0 Stefan Bereswill, Charite-University Medicine Berlin, Germany 0 Laboratory of Cell Biology, Division of Monoclonal Antibodies, Office of Biotechnology Products, Center for Drug Evaluation and Research, United States Food and Drug Administration , Bethesda, Maryland , United States of America The scientific community has been restricted by the lack of a practical and informative animal model of gastrointestinal infection with vegetative Bacillus anthracis. We herein report the development of a murine model of gastrointestinal anthrax infection by gavage of vegetative Sterne strain of Bacillus anthracis into the complement-deficient A/J mouse strain. Mice infected in this manner developed lethal infections in a dose-dependent manner and died 30 h-5 d following gavage. Histological findings were consistent with penetration and growth of the bacilli within the intestinal villi, with subsequent dissemination into major organs including the spleen, liver, kidney and lung. Blood cultures confirmed anthrax bacteremia in all moribund animals, with approximately 1/3 showing co-infection with commensal enteric organisms. However, no evidence of immune activation was observed during infection. Time-course experiments revealed early compromise of the intestinal epithelium, characterized by villus blunting and ulceration in the ileum and jejunum. A decrease in body temperature was most predictive of near-term lethality. Antibiotic treatment of infected animals 24 h following high-dose bacterial gavage protected all animals, demonstrating the utility of this animal model in evaluating potential therapeutics. - Recent bioterrorism attacks [1] have focused research on the inhalational route of entry, yet there remains scientific utility in investigating pathogenic mechanisms involved in gastrointestinal anthrax, as it is widely held that it is primarily the enteric route of entry that Bacillus anthracis has evolved to exploit [2,3]. Bacillus anthracis infection is naturally acquired by ruminant herbivores that are exposed to spores when feeding in contaminated fields [2,3]. Ruminants are considered to be the most susceptible group within the mammalian class [3]. However, it has not yet been established when and where spore germination occurs following oral consumption [3]. We have previously shown that anthrax lethal toxin (LT), which is produced by vegetative Bacillus anthracis, elicits rapid breakdown in the gastrointestinal barrier, characterized by villus blunting, hemorrhage, and ulceration [4,5]. We have since been eager to develop an informative animal model that could address the role of anthrax LT, as well as other virulence factors, in mediating host-pathogen interactions during gastrointestinal infection in vivo. Unfortunately, the scientific community has lacked a murine model of gastrointestinal infection that incorporates administration of vegetative Bacillus anthracis, which could be used to investigate pathogenicity via this mode of transmission. Previously reported animal models for anthrax infection have mainly involved the administration of B. anthracis spores via inhalational or parenteral routes [6,7,8,9,10,11,12]. Initial studies with anthrax spores administered via the gastrointestinal route failed to establish anthrax infection models in various animal species [13,14,15]. However, there have been recent reports of the establishment of infections in mice receiving intragastric B. anthracis spores [16,17]. One group administered 108 spores of an encapsulated non-toxigenic strain and reported that B. anthracis expanded in the Peyers patches, eventually disseminating into various organs. However, this model was not capable of assessing the roles of LT in promoting virulence during gastrointestinal infection. Very recently, another model was reported that utilized intragastric administration of spores embedded in a thiobendazole paste [17]. Neither of these models assessed administration of vegetative bacteria. As ruminant animals use bacterial fermentation to facilitate digestion, we considered the possibility that in the setting of natural gastrointestinal infection, the upper gastrointestinal tract would be exposed to large numbers of vegetative bacteria. Bacillus species have been shown to germinate and thrive in the conditions present in the rumen [18]. For this reason, it would seem very likely that B. anthracis spores would germinate and proliferate in the rumen of infected animals prior to establishing infection. Under this scenario, exposure of the gastrointestinal barrier to vegetative bacteria and the toxins they produce would then lead to barrier penetration and subsequent dissemination. We herein report that we have modeled this scenario in A/J mice through gavage of vegetative bacteria from the Bacillus anthracis Sterne strain. Mice infected with toxigenic bacteria via this route develop gastrointestinal disease, which leads to bacteremia and lethal dissemination. Moreover, we demonstrate that this animal model can be used to assess the efficacy of potential therapeutics. Intragastric Administration of Vegetative B. anthracis Sterne Strain (BaS) Results in Systemic Anthrax Infection We hypothesized that we could establish gastrointestinal infections in mice by infecting mice with vegetative bacteria, thereby mimicking conditions that we consider likely to be present during digestion in ruminants, the predominant hosts of infection in nature. To investigate this possibility, we administered increasing concentrations of vegetative Bacillus anthracis Sterne strain (BaS) bacteria via gavage. The percentage of mice that succumbed to this treatment increased in a dose-dependent manner (Figure 1A). At the highest dose (2.36109), 9 of 10 mice died within 4 days of administration. In contrast, mice that received the vehicle alone showed no signs of toxicity and were blood culture negative. The LD50 for infection via this route in this experiment was approximately 2.36107 bacteria. One animal succumbed to infection with the lowest administered dose of 2.36106 bacteria. Interestingly 6/19 mice that were blood culture positive for BaS had mixed infections with commensal bacteria; 4/19 were blood culture positive for Enterococcus faecalis, whereas 2/19 were blood culture positive for enteric Staphylococcus species (Figures 1B and 1C). All mice that died in response to intragastric challenge were confirmed to be blood culture positive for BaS. To confirm that infections were due to vegetative bacteria, and not heat-resistant spores, mice were gavaged with 26108 heat-treated bacteria. None of these mice (0/5) became infected, indicating that bacterial spores were not responsible for gastrointestinal infections in our gavage model. Murine Gastroint (...truncated)