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Two genotypes (A and C) of C. psittaci were found in samples from a wider range of both native and introduced species of birds in New Zealand than previously reported. Both genotypes have been globally associated with significant disease in birds and humans. These initial results suggest the host range of C. psittaci in New Zealand birds is under-reported. However, the prevalence of C. psittaci infection in New Zealand, and the associated impact on avian and public health, remains to be determined. There are biosecurity implications associated with the importation of birds to New Zealand if there is a limited diversity of C. psittaci genotypes present.

In molecular biology studies of Anura, nondestructive methods to obtain genetic material are needed as alternatives to toe clipping. This work evaluates a nondestructive method for sampling DNA from blood puncture, comparing the performance of three different extraction protocols (Qiagen Kit, Salting-out and Chelex). We collected 134 individuals of Eleutherodactylus johnstonei, extracting blood via puncture of the medial vein using commercial-grade glucometer lancets. We extracted 100-1880 ng DNA, finding no differences between the extraction protocols. We compared the quality of the resulting DNA through amplification and sequencing of the 16S mitochondrial gene. Amplification was successful for the three extraction protocols, although Chelex showed better performance, making it the most recommendable protocol for extraction of DNA from blood. The resulting sequences corresponded to those registered in the GenBank for this species. Additionally, we found no significant differences in survival or weight change between the individuals that were manipulated and a control group (mean survival 66.7% treated, 62.9% untreated). Data reveal that blood samples obtained by puncture are a convenient alternative to other tissues (phalange, buccal swab, liver) that have traditionally been used as DNA sources for anurans. The technique is applicable to small and large species, covering most anuran diversity, provides enough DNA for many genetic applications and produces no noticeable effect on the survival or performance, given that it does not affect the motor parts or the dexterity of the animals.

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Background Cryptosporidium and Giardia are major food and water-borne causes of diarrhoea globally, and two of the most notified infectious diseases in New Zealand. Diagnosis requires laboratory confirmation carried out mostly via antigen or microscopy-based techniques. However, these methods are increasingly being superseded by molecular techniques for diagnostics. Here we investigate the level of protozoa coinfection identified by molecular methods in Campylobacter positive samples missed through use of antigen-based assays and then investigated different molecular testing protocols. Methods We report the findings of two observational studies; the first among 111 people with diarrhoea during a large Campylobacter outbreak in Havelock North, and the second a study during normal surveillance activities among 158 people presenting with diarrhoea and a positive Campylobacter test, but negative Cryptosporidium and/or Giardia antigen-based diagnostic test result. The molecular methods used for comparison with the antigen-based tests were in-house end-point PCR tests targeting the gp60 gene for Cryptosporidium and gdh gene for Giardia. DNA extraction was performed with and without bead-beating and comparisons with commercial real-time quantitative (qPCR) were made using clinical samples diluted down to 10-5 for Cryptosporidium positive samples. Results The coinfection prevalence was 9% (n= 10, 3–15% 95%CI) for Cryptosporidium and 21% (n=23, 12–29% 95%CI) for Giardia in the 111 Campylobacter patients of the Havelock North outbreak. The coinfection prevalence was 40% (n=62, 32-48% 95%CI) for Cryptosporidium and 1.3% (n=2, 0.2-4.5% 95%CI) for Giardia in the 158 routine surveillance samples. Sequencing identified Cryptosporidium hominis, C. parvum, and Giardia intestinalis assemblages A and B among patients. We found no statistical difference in positive test results between samples using end-point PCR with or without bead-beating prior to DNA extraction, or between the in-house end-point PCR and qPCR. The qPCR Ct value was 36 (35-37 95%CI) for 1 oocyst, suggesting a high limit of detection. Discussion In surveillance and outbreak situations we found diagnostic serology testing substantially underdiagnoses Cryptosporidium and Giardia coinfections in Campylobacter patients. These findings suggest that the impact of protozoa infections may be underestimated, through underdiagnosis, but molecular techniques likely improve detection capabilities. Laboratories need to understand clinical, rather than analytical, test sensitivity, to allow clinicians to better understand the disease aetiologies of patients that enable better health advice.

Cryptosporidiosis 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.

BackgroundGiardia is one of the most common causes of diarrhoea in the world and is a notifiable disease in New Zealand. Recent advances in molecular techniques, such as PCR and Sanger sequencing, have greatly improved our understanding of the taxonomic classification and epidemiology of this parasite. However, there has been an inability to identify shared subtypes between samples from the same epidemiologically linked cases, due to samples showing multiple dominant subtypes within the same outbreak when characterised using Sanger sequencing. MethodsHere, NGS was employed to uncover the genetic diversity within samples from sporadic and outbreak cases of giardiasis that occurred in New Zealand between 2010 and 2018. ResultsThis strategy exposed the significant diversity of subtypes of Giardia present in each sample. The utilisation of NGS and metabarcoding at the glutamate dehydrogenase (gdh) locus enabled the identification of shared subtypes between samples from shared outbreaks, providing a better understanding of the epidemiology of outbreaks of giardiasis in New Zealand.ConclusionsNext-generation sequencing technologies provides a superior tool, when compared to consensus sequencing technologies, for capturing the genetic diversity of Giardia within hosts. This study showed that infections in humans are frequently mixed, with multiple subtypes present in each host.

Cryptosporidium and Giardia are major causes of diarrhoea globally, and two of the most notified infectious diseases in New Zealand. Diagnosis requires laboratory confirmation carried out mostly via antigen or microscopy-based techniques. However, these methods are increasingly being superseded by molecular techniques. Here we investigate the level of protozoa detection by molecular methods in campylobacteriosis cases missed through antigen-based assays and investigate different molecular testing protocols. We report findings from two observational studies; the first among 111 people during a Campylobacter outbreak and the second during normal surveillance activities among 158 people presenting with diarrhoea and a positive Campylobacter test, but negative Cryptosporidium and Giardia antigen-based test results. The molecular methods used for comparison were in-house end-point PCR tests targeting the gp60 gene for Cryptosporidium and gdh gene for Giardia. DNA extraction was performed with and without bead-beating and comparisons with commercial real-time quantitative (qPCR) were made using clinical Cryptosporidium positive sample dilutions down to 10−5. The Cryptosporidium prevalence was 9% (95% CI: 3–15; 10/111) and Giardia prevalence 21% (95% CI: 12–29; 23/111) in the 111 Campylobacter outbreak patients. The Cryptosporidium prevalence was 40% (95% CI: 32–48; 62/158) and Giardia prevalence 1.3% (95% CI: 0.2–4.5; 2/158) in the 158 routine surveillance samples. Sequencing identified Cryptosporidium hominis, C. parvum, and Giardia intestinalis assemblages A and B. We found no statistical difference in positive test results between samples using end-point PCR with or without bead-beating prior to DNA extraction, or between the in-house end-point PCR and qPCR. The qPCR Ct value was 36 (95% CI: 35–37) for 1 oocyst, suggesting a high limit of detection. In conclusion in surveillance and outbreak situations we found diagnostic serology testing underdiagnoses Cryptosporidium and Giardia coinfections in Campylobacter patients, suggesting the impact of protozoa infections may be underestimated through underdiagnosis using antigen-based assays.