Journal Articles
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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 Historical translocations by Māori may explain the distribution and genetic structure of a threatened surf clam in Aotearoa (New Zealand)(Springer Nature Ltd, 2018-11-22) Ross PM; Knox MA; Smith S; Smith H; Williams J; Hogg IDThe population genetic structure of toheroa (Paphies ventricosa), an Aotearoa (New Zealand) endemic surf clam, was assessed to determine levels of inter-population connectivity and test hypotheses regarding life history, habitat distribution and connectivity in coastal vs. estuarine taxa. Ninety-eight toheroa from populations across the length of New Zealand were sequenced for the mitochondrial cytochrome c oxidase I gene with analyses suggesting a population genetic structure unique among New Zealand marine invertebrates. Toheroa genetic diversity was high in Te Ika-a Māui (the North Island of New Zealand) but completely lacking in the south of Te Waipounamu (the South Island), an indication of recent isolation. Changes in habitat availability, long distance dispersal events or translocation of toheroa to southern New Zealand by Māori could explain the observed geographic distribution of toheroa and their genetic diversity. Given that early-Māori and their ancestors, were adept at food cultivation and relocation, the toheroa translocation hypothesis is plausible and may explain the disjointed modern distribution of this species. Translocation would also explain the limited success in restoring what may in some cases be ecologically isolated populations located outside their natural distributions and preferred nichesItem 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 Abundant dsRNA picobirnaviruses show little geographic or host association in terrestrial systems.(Elsevier, 2023-08) Knox MA; Wierenga J; Biggs PJ; Gedye K; Almeida V; Hall R; Kalema-Zikusoka G; Rubanga S; Ngabirano A; Valdivia-Granda W; Hayman DTSPicobirnaviruses are double-stranded RNA viruses known from a wide range of host species and locations but with unknown pathogenicity and host relationships. Here, we examined the diversity of picobirnaviruses from cattle and gorillas within and around Bwindi Impenetrable Forest National Park (BIFNP), Uganda, where wild and domesticated animals and humans live in relatively close contact. We use metagenomic sequencing with bioinformatic analyses to examine genetic diversity. We compared our findings to global Picobirnavirus diversity using clustering-based analyses. Picobirnavirus diversity at Bwindi was high, with 14 near-complete RdRp and 15 capsid protein sequences, and 497 new partial viral sequences recovered from 44 gorilla samples and 664 from 16 cattle samples. Sequences were distributed throughout a phylogenetic tree of globally derived picobirnaviruses. The relationship with Picobirnavirus diversity and host taxonomy follows a similar pattern to the global dataset, generally lacking pattern with either host or geography.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.Item Community health and human-animal contacts on the edges of Bwindi Impenetrable National Park, Uganda(PLOS, 2021-11-24) Muylaert RL; Davidson B; Ngabirano A; Kalema-Zikusoka G; MacGregor H; Lloyd-Smith JO; Fayaz A; Knox MA; Hayman DTS; Fèvre ECross-species transmission of pathogens is intimately linked to human and environmental health. With limited healthcare and challenging living conditions, people living in poverty may be particularly susceptible to endemic and emerging diseases. Similarly, wildlife is impacted by human influences, including pathogen sharing, especially for species in close contact with people and domesticated animals. Here we investigate human and animal contacts and human health in a community living around the Bwindi Impenetrable National Park (BINP), Uganda. We used contact and health survey data to identify opportunities for cross-species pathogen transmission, focusing mostly on people and the endangered mountain gorilla. We conducted a survey with background questions and self-reported diaries to investigate 100 participants' health, such as symptoms and behaviours, and contact patterns, including direct contacts and sightings over a week. Contacts were revealed through networks, including humans, domestic, peri-domestic, and wild animal groups for 1) contacts seen in the week of background questionnaire completion, and 2) contacts seen during the diary week. Participants frequently felt unwell during the study, reporting from one to 10 disease symptoms at different intensity levels, with severe symptoms comprising 6.4% of the diary records and tiredness and headaches the most common symptoms. After human-human contacts, direct contact with livestock and peri-domestic animals were the most common. The contact networks were moderately connected and revealed a preference in contacts within the same taxon and within their taxa groups. Sightings of wildlife were much more common than touching. However, despite contact with wildlife being the rarest of all contact types, one direct contact with a gorilla with a timeline including concerning participant health symptoms was reported. When considering all interaction types, gorillas mostly exhibited intra-species contact, but were found to interact with five other species, including people and domestic animals. Our findings reveal a local human population with recurrent symptoms of illness in a location with intense exposure to factors that can increase pathogen transmission, such as direct contact with domestic and wild animals and proximity among animal species. Despite significant biases and study limitations, the information generated here can guide future studies, such as models for disease spread and One Health interventions.Item Uncovering the genetic diversity of Giardia intestinalis in isolates from outbreaks in New Zealand(BioMed Central Ltd, 2022-12) Ogbuigwe P; Biggs PJ; Garcia-Ramirez JC; Knox MA; Pita A; Velathanthiri N; French NP; Hayman DTSBACKGROUND: Giardia intestinalis is one of the most common causes of diarrhoea worldwide. Molecular techniques have greatly improved our understanding of the taxonomy and epidemiology of this parasite. Co-infection with mixed (sub-) assemblages has been reported, however, Sanger sequencing is sometimes unable to identify shared subtypes between samples involved in the same epidemiologically linked event, due to samples showing multiple dominant subtypes within the same outbreak. Here, we aimed to use a metabarcoding approach to uncover the genetic diversity within samples from sporadic and outbreak cases of giardiasis to characterise the subtype diversity, and determine if there are common sequences shared by epidemiologically linked cases that are missed by Sanger sequencing. METHODS: We built a database with 1109 unique glutamate dehydrogenase (gdh) locus sequences covering most of the assemblages of G. intestinalis and used gdh metabarcoding to analyse 16 samples from sporadic and outbreak cases of giardiasis that occurred in New Zealand between 2010 and 2018. RESULTS: There is considerable diversity of subtypes of G. intestinalis present in each sample. The utilisation of metabarcoding enabled the identification of shared subtypes between samples from the same outbreak. Multiple variants were identified in 13 of 16 samples, with Assemblage B variants most common, and Assemblages E and A present in mixed infections. CONCLUSIONS: This study showed that G. intestinalis infections in humans are frequently mixed, with multiple subtypes present in each host. Shared sequences among epidemiologically linked cases not identified through Sanger sequencing were detected. Considering the variation in symptoms observed in cases of giardiasis, and the potential link between symptoms and (sub-) assemblages, the frequency of mixed infections could have implications for our understanding of host-pathogen interactions.