Impact of confinement housing on study end-points in the calf model of cryptosporidiosis

Background Diarrhea is the second leading cause of death in children < 5 years globally and the parasite genus Cryptosporidium is a leading cause of that diarrhea. The global disease burden attributable to cryptosporidiosis is substantial and the only approved chemotherapeutic, nitazoxanide, has poor efficacy in HIV positive children. Chemotherapeutic development is dependent on the calf model of cryptosporidiosis, which is the best approximation of human disease. However, the model is not consistently applied across research studies. Data collection commonly occurs using two different methods: Complete Fecal Collection (CFC), which requires use of confinement housing, and Interval Collection (IC), which permits use of box stalls. CFC mimics human challenge model methodology but it is unknown if confinement housing impacts study end-points and if data gathered via this method is suitable for generalization to human populations. Methods Using a modified crossover study design we compared CFC and IC and evaluated the impact of housing on study end-points. At birth, calves were randomly assigned to confinement (n = 14) or box stall housing (n = 9), or were challenged with 5 x 107 C. parvum oocysts, and followed for 10 days. Study end-points included fecal oocyst shedding, severity of diarrhea, degree of dehydration, and plasma cortisol. Findings Calves in confinement had no significant differences in mean log oocysts enumerated per gram of fecal dry matter between CFC and IC samples (P = 0.6), nor were there diurnal variations in oocyst shedding (P = 0.1). Confinement housed calves shed significantly more oocysts (P = 0.05), had higher plasma cortisol (P = 0.001), and required more supportive care (P = 0.0009) than calves in box stalls. Conclusion Housing method confounds study end-points in the calf model of cryptosporidiosis. Due to increased stress data collected from calves in confinement housing may not accurately estimate the efficacy of chemotherapeutics targeting C. parvum.
Author summary After respiratory disease, diarrhea is the leading cause of death in children < 5 years worldwide. Recent multicenter studies have identified the parasite genus, Cryptosporidium to be one of the most common causes of pediatric diarrhea in resource-poor settings. In industrialized nations the parasite is well controlled through centralized water treatment facilities. Consequently, drug development targeting this neglected tropical disease has been limited. There are currently no vaccines to prevent cryptosporidiosis. Nitazoxanide is the only approved drug targeting Cryptosporidium, but it has poor efficacy in HIV positive children. Owing to the recently published multicenter findings there have been renewed Cryptosporidium drug development efforts. The calf model of cryptosporidiosis is widely recognized as the best approximation of human disease but it is difficult to execute resulting in varied experimental approaches. Here, we describe the two most common methods of executing the calf model of cryptosporidiosis and investigate how these methods impact study end-points used to inform identification of drug candidates and clinical trials. We find that confinement housing significantly increases plasma cortisol in calves and impacts key study end-points. Data collected via confinement housing may not accurately estimate drug efficacy, underscoring the need for model harmonization. The results of this study should be considered when interpreting chemotherapeutic data derived from the calf model of cryptosporidiosis.