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Item Metabarcoding captures genetic diversity and links cases in outbreaks of cryptosporidiosis in New Zealand.(Elsevier Ltd on behalf of The British Infection Association, 2025-01-30) Ogbuigwe P; Biggs PJ; Garcia-Ramirez JC; Knox MA; Pita A; Velathanthir N; French NP; Hayman DTSCryptosporidiosis is a disease caused by the parasite Cryptosporidium. Globally, it is a leading cause of diarrhoea and a notifiable disease in New Zealand. Molecular analyses of Cryptosporidium isolated from notified cases do not always provide support for epidemiological links between individuals. We hypothesised this could be due to undetected diversity and the use of consensus Sanger sequence analyses. Here, we analysed 105 Cryptosporidium samples from outbreaks and sporadic cases occurring between 2010 and 2018 in New Zealand using both Next-Generation Sequencing (NGS) and Sanger sequencing of the glycoprotein 60 (gp60) locus. NGS metabarcoding at the gp60 locus uncovered significant intra- and inter-sample genotypic diversity in outbreaks and identified subtypes shared by epidemiologically linked cases, along with rare subtypes, suggesting it may be a useful tool for epidemiological investigations.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 review and analysis of cryptosporidiosis outbreaks in New Zealand.(Cambridge University Press, 2023-06-01) Garcia-R JC; Hayman DTSCryptosporidium is a leading global cause of diarrhoea with many reported outbreaks related to water and zoonotic transmission. This study summarizes data from Public Health Surveillance reports since 2010 in New Zealand to describe exposures associated with human diarrhoea outbreaks caused by Cryptosporidium. We investigate the species and subtypes of cases involved in some of the outbreaks to elucidate transmission routes and the predominant aetiological agents of cryptosporidiosis. For the period 2010–2017, 318 cryptosporidiosis outbreaks were reported in New Zealand resulting in 1634 cases and 20 hospitalizations. The most important mode of transmission was person-to-person (primary infections and secondary or close contacts infections), relating to 260 outbreaks and 1320 cases, followed by 113 outbreaks associated with animals, resulting in 436 human cases. From 2018 to 2021, there were 37 cryptosporidiosis outbreaks associated with 324 cases. We identified the subtypes by using polymerase chain reaction targeting the gp60 gene and the likelihood of mixed subtype infections with the Tracking of Indels by DEcomposition (TIDE) algorithm. Subtype families Ib and Ig of Cryptosporidium hominis and IIa and IId of Cryptosporidium parvum were found among cases; however, C. hominis subtypes occurred in 8 of the 11 outbreaks reviewed where molecular data were available. Examination of the chromatograms showed no mixed subtype infections in the samples assessed. Subtyping data need to be routinely incorporated into national surveillance programmes to better understand the epidemiology, sources, transmission and extent of cryptosporidiosis outbreaks in New Zealand. Our study highlights the value of integrating epidemiological information and molecular typing to investigate and manage clusters of cryptosporidiosis cases.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 Absence of Cryptosporidium hominis and dominance of zoonotic Cryptosporidium species in patients after Covid-19 restrictions in Auckland, New Zealand(Cambridge University Press, 2021-09) Knox MA; Garcia-R JC; Ogbuigwe P; Pita A; Velathanthiri N; Hayman DTSCoronavirus disease-2019 (Covid-19) nonpharmaceutical interventions have proven effective control measures for a range of respiratory illnesses throughout the world. These measures, which include isolation, stringent border controls, physical distancing and improved hygiene also have effects on other human pathogens, including parasitic enteric diseases such as cryptosporidiosis. Cryptosporidium infections in humans are almost entirely caused by two species: C. hominis, which is primarily transmitted from human to human, and Cryptosporidium parvum, which is mainly zoonotic. By monitoring Cryptosporidium species and subtype families in human cases of cryptosporidiosis before and after the introduction of Covid-19 control measures in New Zealand, we found C. hominis was completely absent after the first months of 2020 and has remained so until the beginning of 2021. Nevertheless, C. parvum has followed its typical transmission pattern and continues to be widely reported. We conclude that ~7 weeks of isolation during level 3 and 4 lockdown period interrupted the human to human transmission of C. hominis leaving only the primarily zoonotic transmission pathway used by C. parvum. Secondary anthroponotic transmission of C. parvum remains possible among close contacts of zoonotic cases. Ongoing 14-day quarantine measures for new arrivals to New Zealand have likely suppressed new incursions of C. hominis from overseas. Our findings suggest that C. hominis may be controlled or even eradicated through nonpharmaceutical interventions.