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Item Mammal-related Cryptosporidium infections in endemic reptiles of New Zealand.(Springer Nature, 2023-05-01) Garcia-R JC; Pita AB; Velathanthiri N; Pas A; Hayman DTSNew Zealand's endemic reptile fauna is highly threatened and pathogens causing infectious diseases may be a significant risk to already endangered species. Here, we investigate Cryptosporidium infection in captive endemic New Zealand reptiles. We found two mammal-related Cryptosporidium species (C. hominis and C. parvum) and six subtypes from three gp60 families (Ib, Ig and IIa) in 12 individuals of captive endemic Tuatara, Otago and Grand skinks, and Jewelled and Rough geckos. Cryptosporidium serpentis was identified in two Jewelled geckos using 18S. In New Zealand, C. hominis and C. parvum are associated with infections in humans and introduced domestic animals but have also been recently found in wildlife. Our finding of Cryptosporidium infection in endemic reptiles can help inform strategies to monitor the conservation of species and manage potential introductions of pathogens to in-situ and ex-situ populations.Item Quantifying Replication Slippage Error in Cryptosporidium Metabarcoding Studies(Oxford University Press, 2024-07-15) Knox MA; Biggs PJ; Garcia-R JC; Hayman DTSGenetic variation in Cryptosporidium, a common protozoan gut parasite in humans, is often based on marker genes containing trinucleotide repeats, which differentiate subtypes and track outbreaks. However, repeat regions have high replication slippage rates, making it difficult to discern biological diversity from error. Here, we synthesized Cryptosporidium DNA in clonal plasmid vectors, amplified them in different mock community ratios, and sequenced them using next-generation sequencing to determine the rate of replication slippage with dada2. Our results indicate that slippage rates increase with the length of the repeat region and can contribute to error rates of up to 20%.Item A novel, stain-free, natural auto-fluorescent signal, Sig M, identified from cytometric and transcriptomic analysis of infectivity of Cryptosporidium hominis and Cryptosporidium parvum.(Frontiers Media S.A., 2023-05-22) Ogbuigwe P; Roberts JM; Knox MA; Heiser A; Pita A; Haack NA; Garcia-Ramirez JC; Velathanthiri N; Biggs PJ; French NP; Hayman DTS; Xu RCryptosporidiosis is a worldwide diarrheal disease caused by the protozoan Cryptosporidium. The primary symptom is diarrhea, but patients may exhibit different symptoms based on the species of the Cryptosporidium parasite they are infected with. Furthermore, some genotypes within species are more transmissible and apparently virulent than others. The mechanisms underpinning these differences are not understood, and an effective in vitro system for Cryptosporidium culture would help advance our understanding of these differences. Using COLO-680N cells, we employed flow cytometry and microscopy along with the C. parvum-specific antibody Sporo-Glo™ to characterize infected cells 48 h following an infection with C. parvum or C. hominis. The Cryptosporidium parvum-infected cells showed higher levels of signal using Sporo-Glo™ than C. hominis-infected cells, which was likely because Sporo-Glo™ was generated against C. parvum. We found a subset of cells from infected cultures that expressed a novel, dose-dependent auto-fluorescent signal that was detectable across a range of wavelengths. The population of cells that expressed this signal increased proportionately to the multiplicity of infection. The spectral cytometry results confirmed that the signature of this subset of host cells closely matched that of oocysts present in the infectious ecosystem, pointing to a parasitic origin. Present in both C. parvum and C. hominis cultures, we named this Sig M, and due to its distinct profile in cells from both infections, it could be a better marker for assessing Cryptosporidium infection in COLO-680N cells than Sporo-Glo™. We also noted Sig M's impact on Sporo-Glo™ detection as Sporo-Glo™ uses fluoroscein-isothiocynate, which is detected where Sig M also fluoresces. Lastly, we used NanoString nCounter® analysis to investigate the transcriptomic landscape for the two Cryptosporidium species, assessing the gene expression of 144 host and parasite genes. Despite the host gene expression being at high levels, the levels of putative intracellular Cryptosporidium gene expression were low, with no significant difference from controls, which could be, in part, explained by the abundance of uninfected cells present as determined by both Sporo-Glo™ and Sig M analyses. This study shows for the first time that a natural auto-fluorescent signal, Sig M, linked to Cryptosporidium infection can be detected in infected host cells without any fluorescent labeling strategies and that the COLO-680N cell line and spectral cytometry could be useful tools to advance the understanding of Cryptosporidium infectivity.Item Quantifying replication slippage error in Cryptosporidium metabarcoding studies (accepted manuscript)(Oxford University Press on behalf of the Infectious Diseases Society of America, 2024-02-08) Knox MA; Biggs PJ; Garcia-R JC; Hayman DTSGenetic variation in Cryptosporidium, a common protozoan gut parasite in humans, is often based on marker genes containing trinucleotide repeats, which differentiate subtypes and track outbreaks. However, repeat regions have high replication slippage rates, making it difficult to discern biological diversity from error. Here, we synthesised Cryptosporidium DNA in clonal plasmid vectors, amplified them in different mock community ratios and sequenced them using next generation sequencing to determine the rate of replication slippage with dada2. Our results indicate that slippage rates increase with the length of the repeat region and can contribute to error rates of up to 20%.Item Epidemiological studies of cryptosporidiosis : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Pathology, Massey University, Palmerston North, New Zealand(Massey University, 2009) Grinberg, AlejandroAn interpretive overview of the literature on intestinal cryptosporidiosis in humans and domestic mammals (Chapter 1) is followed by two studies of the population genetic structure of the protozoan parasites Cryptosporidium parvum and Cryptosporidium hominis (Chapter 2), five epidemiological studies of cryptosporidiosis in foals, calves and humans in New Zealand (Chapter 3), and an investigation of a serendipitous outbreak of cryptosporidiosis among a class of veterinary students, which occurred at the end of 2006 (Chapter 4). The analysis of the population genetic structure of C. parvum and C. hominis indicates the existence of a significant genetic segregation of geographically separated parasite populations, consistent with allopatry. The results do not conform to a simplistic model that considers all C. parvum as multi-host anthropozoonotic agents, and provide statistical support to the idea of the occurrence of anthroponotic cycles that do not involve cattle. Rather than conforming to a rigid paradigm of either a clonal or a panmictic species, data are consistent with the co-occurrence of clonal and recombinatorial diversification in C. hominis, and perhaps C. parvum. The results of the epidemiological studies in New Zealand suggest cryptosporidiosis caused by C. parvum is relatively common in young foals and calves. In the time and space frames underlying these studies, humans, calves, and foals were infected with a genetically homogeneous C. parvum population. This feature is in accordance with previous reports that have indicated C. parvum as the dominant species in humans during the peaks of incidence of cryptosporidiosis in winter and spring, and support the view that the peaks are in large part attributable to direct and/or indirect zoonotic transmission of C. parvum. Finally, the outbreak of cryptosporidiosis among a class of veterinary students highlighted the potential hazard for explosive large-scale outbreaks in New Zealand. The results of the investigation were consistent with point-source exposure and zoonotic transmission of a rare C. parvum subtype through direct contact with calves during a practicum.