Genomic and virulence analysis of in vitro cultured Cryptosporidium parvum

Abstract

Recent advances in the in vitro cultivation of Cryptosporidium parvum using hollow fiber bioreactor technology (HFB) have permitted continuous growth of parasites that complete all life cycle stages. The method provides access to all stages of the parasite and provides a method for non-animal production of oocysts for use in clinical trials. Here we examined the effect of long-term (>20 months) in vitro culture on virulence-factors, genome conservation, and in vivo pathogenicity of the host by in vitro cultured parasites. We find low-level sequence variation that is consistent with that observed in calf-passaged parasites. Further using a calf model infection, oocysts obtained from the HFB caused diarrhea of the same volume, duration and oocyst shedding intensity as in vivo passaged parasites.

Fig 1. Diagramatic section through the hollow fiber bioreactor.

The cartridge contains a series of hollow fibers through which the host cell growth media, MEM plus supplements and 10% horse serum is pumped through the intracapillary space (ICS). The extracapillary space (ECS) contains the host epithelial cells (HCT-8) that attach to and grow around the outside of the fibers forming a 3D matrix that receives nutrients from the basal surface. C. parvum is inoculated into the ECS after the host cell 3D layer has developed as determined by a drop in the ICS glucose concentration of 50% or more in 24h. C. parvum sporozoites attach to the apical surface of the host epithelial cells as they do in the intestinal tract. (A) Diagrammatic section through the HFB showing the extracapillary space (ECS) and host cells growing around the intracapillary space (ICS). (B) C. parvum oocysts from the HFB stained with Crypt-a-Glo. (C) Sporo-Glo stained merozoites and sporozoites from the HFB. (D) Electron microscope image of the HCT-8 cells grown on the fibers showing the presence of microvilli; obtained by sectioning a 3-month cartridge. (E) Growth of C. parvum based upon qRT-PCR of samples collected from the HFB during the 20-month culture period as described in the methods. (F) Typical C_T plot used to quantitate C. parvum growth.

Fig 2. Strain passage and designation diagram.

Cryptosporidium parvum IOWA oocysts, designated, BGF-T0 were purchased from Bunch Grass Farms in March 2016 and sequenced by BGI. C. parvum IOWA oocysts designated BGF- 2017 were purchased from Bunch Grass Farms and sequenced by the GGBC at the University of Georgia. BGF-T20HF–Originated from BGF-T0 following 20 months of continuous culture in the HFB and sequenced at BGI. IOWA-ATCC–Genomic DNA was ordered from ATCC (catalog number ATCCPRA-67DQ) and sequenced at the WSI as in (20). IOWA-2017 was produced at the Cryptosporidium Production Lab (University of Arizona).

Fig 3. Infectivity and clinical scores for C. parvum isolates BGF-T20HF, compared with calf-passaged isolates BGF-2017 and IOWA-2017.

(A) Average daily fecal volume per calf ± SD of the number of calves in parenthesis BGF-T20HF (4), BGF-2017 (4), IOWA-2017 (5). One HFB (hollow fiber bioreactor derived oocysts) calf was delayed in onset of diarrhea, hence reason for high SDs. (B) Average total fecal volume per calf ± SD of the number of calves in parenthesis BGF-T20HF (4), BGF-2017 (4), IOWA-2017 (5). (C) Daily oocyst numbers shed by BGF-T20HF, BGF-2017, and IOWA-2017. The hollow fiber cultured isolate BGF-T20HF had a similar shedding profile to the parent isolate, BGF-2017 from days 3–10. Non-parametric statistical analysis by the Kruskal-Wallis H-test revealed that there were no significant differences between the groups. (D) Fecal consistency BGF-2017, BGF-T20HF, and IOWA-2017. (E) Daily clinical evaluation. Lower score values indicate healthier calves. Onset of diarrhea was delayed in one BGF-T20HF infected calf. (F) Clinical evaluation score means.

Fig 4. Venn diagram of observed Genome-wide variation found.

(A) All called variants; and (B) Moderate to high-impact variants found in protein coding regions. Data are located in Tables E and F in S1 Table.