We describe the response of gnotobiotic piglets orally challenged with C. difficile
in which a spectrum of clinical symptoms and pathological abnormalities largely depending on age of the animal and the size of the infectious dose, were induced. The nature and outcome of the disease in these animals mimic many of the characteristics observed in human patients with CDI. Using the hypervirulent strain 027/BI/NAP1, this animal model offers reproducible results, with 100% colonization within 48 hours of inoculation, and a disease severity that can be tailored according to need. A disease, ranging from profoundly acute and lethal to chronic diarrhea, was readily induced under controlled laboratory settings. The range of clinical signs, including systemic complications, are similar to those observed in human cases [1
], making gnotobiotic piglets an attractive alternative model to the hyperacute hamster model. The piglet model offers flexibility to meet specific research requirements such as studies on pathogenesis, evaluation of virulence attributes, testing the efficacy of therapeutics, and the evaluation of vaccine candidates for eliminating existing infections or protecting against infections. For this purpose, animals can be immunized orally, intranasally, or systemically at one-week of age followed by repeated boosting at ~2 weeks thereafter. Immunized animals can then be monitored for side effects/symptoms in the case of live attenuated vaccines. Sera and feces can be analyzed for bacterial excretion and toxins, protective mucosal and systemic antibodies, T cell responses, cytokine responses, etc. After the immunization, animals can then be challenged orally with wild type strains. To test vaccine candidates the model offers several important parameters for measurements before and after challenge including clinical symptoms, degree of mucosal injury, if any, extent of bacterial colonization in the gut, level of toxin production, cytokine responses, etc. Essentially, the model can provide most of the required information for preclinical evaluation of vaccine candidates.
Though pigs are naturally susceptible to C. difficile
infection and conventional pigs could be used, gnotobiotic piglets were chosen for the models due to several advantages over conventional animals. Gnotobiotic piglets, delivered via Cesarean section, do not nurse their dam, therefore absent are interfering maternal antibodies against C. difficile
in studies involving evaluation of the immune response. They lack normal as well as potentially pathogenic gut flora and consequently do not require starvation and treatment with antibiotics before inoculation to enhance susceptibility, as is required with other animals. The absence of normal gut microflora as a consequence of prolonged antibiotic treatment is the hallmark of CDI, and is considered as one of the most important risk factors for this disease[1
]. Gnotobiotic piglets mimic this state without a need for antibiotic pre-conditioning. Since gnotobiotic piglets are maintained in sterile isolators for the duration of the study, the possibility for introduction of other pathogens from the sow or human caretakers, including C. difficile
which is common in swine, is also eliminated. Because of these traits, inoculation of gnotobiotic piglets produces very consistent and reliable results, which can be modified according to need by manipulating the dose and/or the age of the animal. This is particularly useful in the study of vaccine candidates where several immunizations, with monitoring of immune and clinical responses followed by challenge can be accomplished independently on each animal.
The major objective of this study was to establish a dose and age relationship for the model with the 027/BI/NAP1 strain. In general, younger piglets inoculated with higher doses experience more severe clinical signs of disease. In addition to the range of clinical signs induced by varying the dose and age at inoculation, we also observed that differences in disease severity could be observed even between individuals from the same litter given the same dose at the same age. For the strain used in these experiments, a dose of 105 spores given at 5 days of age induced acute disease in approximately half of the piglets and chronic disease in the other half. Those experiencing acute disease developed systemic lesions of ascites, pleural effusion and lung consolidation, while those with chronic disease developed only gastrointestinal lesions.
The finding that the severity of the disease varies among the piglets resembles the situation in human cases. Humans too develop a range of systemic consequences of C. difficile
infection such as ascites, pleural effusion, cardiopulmonary arrest, liver abscess, and multiple organ dysfunction syndrome[6
], which result in severe and even fatal disease. The reasons for the case differences observed among human patients, as well as those in the piglets in this study, are not well understood. Hopefully, the piglet model will prove to be a useful tool to delineate the relative role of each virulence attribute in contributing to the systemic and to the gastrointestinal disease observed in humans. Immune response is likely to play a deciding role in disease severity, and in this study, we analyzed cytokine levels in the large intestinal contents. IL-8 concentration was significantly elevated in the piglets which developed acute, severe disease compared to the chronically affected or control piglets. IL-8 is a component of the inflammatory response, mediating neutrophil migration, and is also elevated using in vitro experiments with human cells[27
], and in feces of human patients[29
]. Our findings suggest that IL-8 may be a detrimental component of the inflammatory response, as significantly elevated levels of this cytokine correlate with more severe disease.
This study supports the hypothesis that C. difficile
toxins, rather than the bacteria, are responsible for the systemic complications we have observed. The presence of toxin in the serum, pleural effusion and ascites of the systemically affected piglets demonstrates the ability of toxins to reach circulation and to disseminate to extra-intestinal sites and suggests that they play a systemic role, which hopefully the piglet model will help address more precisely in the future. We were unable to culture bacteria from body fluids or sera of severely affected animals, and no bacteria were noted on histologic examination of tissues outside the gastrointestinal tract, indicating that dissemination of bacteria through damaged gut mucosa was not responsible for the systemic effects. The finding of toxin in the serum of systemically affected piglets, but not in the mildly affected or control piglets is especially important, because to our knowledge, toxemia has not been previously documented in human or animal cases. While toxin concentration in feces of infected individuals is quite high and may cause cultured cell rounding in a matter of hours, the concentration in body fluids is much lower and requires a more sensitive assay for detection. The ultrasensitive cytotoxicity assay developed at our laboratory [24
] has increased considerably the ease and speed of toxin detection in body fluids, especially in the serum.