Scalable cryopreservation of infectious Cryptosporidium hominis oocysts by vitrification.

Cryptosporidium hominis is a serious cause of childhood diarrhea in developing countries. The development of therapeutics is impeded by major technical roadblocks including lack of cryopreservation and simple culturing methods. This impacts the availability of optimized/standardized singular sources of infectious parasite oocysts for research and human challenge studies. The human C. hominis TU502 isolate is currently propagated in gnotobiotic piglets in only one laboratory, which limits access to oocysts. Streamlined cryopreservation could enable creation of a biobank to serve as an oocyst source for research and distribution to other investigators requiring C. hominis. Here, we report cryopreservation of C. hominis TU502 oocysts by vitrification using specially designed specimen containers scaled to 100 µL volume. Thawed oocysts exhibit ~70% viability with robust excystation and 100% infection rate in gnotobiotic piglets. The availability of optimized/standardized sources of oocysts may streamline drug and vaccine evaluation by enabling wider access to biological specimens.

Pregnant sows immunized with Cryptosporidium parvum significantly reduced infection in newborn piglets challenged with C. parvum but not with C. hominis.

BACKGROUND: The piglet is the only model to investigate the immunogenic relationship between Cryptosporidium hominis and C. parvum, the species responsible for diarrhea in humans. Despite being indistinguishable antigenically, and high genetic homology between them, they are only moderately cross protective after an active infection. METHODOLOGY/PRINCIPAL FINDINGS: Here we examined the degree of passive protection conferred to piglets suckling sows immunized during pregnancy with C. parvum. After birth suckling piglets were challenged orally with either C. parvum or C. hominis at age 5 days. Animals challenged with C. parvum had significant reduction of infection rate, while piglets challenged with C. hominis showed no reduction despite high C. parvum serum and colostrum IgG and IgA antibody. CONCLUSIONS/SIGNIFICANCE: We add these data to earlier studies where we described that infection derived immunity provides partial cross-protection. Together, it appears that for full protection, vaccines against human cryptosporidiosis must contain antigenic elements derived from both species.

The Impact of Actotoxumab Treatment of Gnotobiotic Piglets Infected With Different Clostridium difficile Isogenic Mutants.

Nosocomial infections with Clostridium difficile are on the rise in the Unites States, attributed to emergence of antibiotic-resistant and hypervirulent strains associated with greater likelihood of recurrent infections. In addition to antibiotics, treatment with Merck anti-toxin B (TcdB) antibody bezlotoxumab is reported to reduce recurrent infections. However, treatment with anti-toxin A (TcdA) antibody actotoxumab was associated with dramatically increased disease severity and mortality rates in humans and gnotobiotic piglets. Using isogenic mutants of C. difficile strain NAPI/BI/027 deficient in TcdA (A-B+) or TcdB (A+B-), and the wild type, we investigated how and why treatment of infected animals with anti-TcdA dramatically increased disease severity. Contrary to the hypothesis, among piglets treated with anti-TcdA, those with A+B- infection were disease free, in contrast to the disease enhancement seen in those with wild-type or A-B+ infection. It seems that the lack of TcdA, through either deletion or neutralization with anti-TcdA, reduces a competitive pressure, allowing TcdB to freely exert its profound effect, leading to increased mucosal injury and disease severity.

Therapeutic Efficacy of Bumped Kinase Inhibitor 1369 in a Pig Model of Acute Diarrhea Caused by Cryptosporidium hominis.

Recent reports highlighting the global significance of cryptosporidiosis among children have renewed efforts to develop control measures. We evaluated the efficacy of bumped kinase inhibitor (BKI) 1369 in the gnotobiotic piglet model of acute diarrhea caused by Cryptosporidium hominis, the species responsible for most human cases. Five-day treatment with BKI 1369 reduced signs of disease early during treatment compared to those of untreated animals. Piglets treated with BKI 1369 exhibited significant reductions of oocyst excretion, mucosal colonization by C. hominis, and mucosal lesions, which resulted in considerable symptomatic improvement. BKI 1369 reduced the parasite burden and disease severity in the gnotobiotic pig model. Together these data suggest that a BKI-mediated therapeutic may be an effective treatment against cryptosporidiosis.

Infection with anthroponotic Cryptosporidium parvum does not fully protect the host against a subsequent challenge with C. hominis.

Cryptosporidium hominis and Cryptosporidium parvum are the major Cryptosporidium species that infect humans. Earlier studies in gnotobiotic piglets, model susceptible to both, showed that piglets recovered from infection with C. hominis were fully protected against challenge with same species but incompletely protected against C. parvum challenge. In the present study, piglets were infected with C. parvum first, and after recovery were re-challenged with C. parvum or C. hominis. Again, full protection was only observed when piglets were challenged with the homologous parasite strain. Although the two species are genetically/antigenically almost identical, they do not confer complete protection against each other.

Failure to propagate Cryptosporidium spp. in cell-free culture.

The successful propagation of Cryptosporidium parvum in cell-free culture medium was recently reported. To investigate whether this phenomenon could be broadened to include other C. parvum isolates, as well as Cryptosporidium hominis, we attempted to propagate 3 isolates in cell-free medium under reported culture conditions. Cryptosporidium oocysts from C. parvum strains Moredun (MD) or IOWA or C. hominis strain TU502 were added to media containing coagulated newborn calf serum. The cultures were sampled at various times throughout a 45 (IOWA) or 78 (MD, TU502)-day period and were microscopically examined for various life stages of Cryptosporidium. Cell-free cultures harvested on days 45 and 68 postinoculation were tested for in vitro infectivity on Madrin-Darby bovine kidney cells. In vivo infectivity testing was performed using either infant or 2-wk-old immunosuppressed C57BL mice with cell-free cultures harvested on days 52 and 78. Fecal and gut samples collected from mice were examined by modified acid-fast staining. Data from wet mounts, electron microscopy, and in vitro and in vivo infectivity testing showed that the original oocysts did not complete their life cycle and produce new, viable, infectious oocysts in cell-free culture. Thus, we conclude that this is not a universal phenomenon or readily accomplished.