Amy J. Asher

Evaluation of a PCR protocol for sensitive detection of Giardia intestinalis in human faeces

Giardia intestinalis is a protozoan parasite and a human pathogen. It is a leading cause of human diarrheal disease and a significant cause of morbidity worldwide. At the molecular level, G. intestinalis is a species complex, consisting of genetic assemblages (A to G) and sub-assemblage strains. The genotypes that cause human disease have been characterised to assemblages A and B, and include strains AI, AII, BIII and BIV. PCR amplification of diagnostic loci is used to genotype samples and is required to understand different transmission cycles within communities. A multi-locus approach is required for validation of Giardia genotyping and molecular diagnostic techniques that are efficient across numerous loci have not been established. This study evaluated several published protocols for the 18S small subunit ribosomal RNA (18S rRNA) and glutamate dehydrogenase genes (gdh) genes. Assays were compared using spiked faecal samples and by measuring the concentration of DNA generated following DNA extraction and PCR amplification. An optimal molecular method for G. intestinalis identification was established from direct DNA extraction of faecal material and GC-rich PCR chemistry. The protocol was applied to 50 clinical samples and produced PCR success rates of 90% and 94% at the 18S rRNA and gdh loci. Cyst concentration prior to DNA extraction was not necessary, and the optimal protocol was highly sensitive and an efficient method for testing clinical samples.

Giardia duodenalis and Cryptosporidium occurrence in Australian sea lions (Neophoca cinerea) exposed to varied levels of human interaction

Graphical Abstract

Evolution of class 1 integrons: Mobilization and dispersal via food-borne bacteria

Class 1 integrons have played a major role in the global dissemination of antibiotic resistance. Reconstructing the history of class 1 integrons might help us control further spread of antibiotic resistance by understanding how human activities influence microbial evolution. Here we describe a class 1 integron that represents an intermediate stage in the evolutionary history of clinical integrons. It was embedded in a series of nested transposons, carried on an IncP plasmid resident in Enterobacter, isolated from the surface of baby spinach leaves. Based on the structure of this integron, we present a modified hypothesis for integron assembly, where the ancestral clinical class 1 integron was captured from a betaproteobacterial chromosome to form a Tn402-like transposon. This transposon then inserted into a plasmid-borne Tn21-like ancestor while in an environmental setting, possibly a bacterium resident in the phyllosphere. We suggest that the qacE gene cassette, conferring resistance to biocides, together with the mercury resistance operon carried by Tn21, provided a selective advantage when this bacterium made its way into the human commensal flora via food. The integron characterized here was located in Tn6007, which along with Tn6008, forms part of the larger Tn6006 transposon, itself inserted into another transposable element to form the Tn21-like transposon, Tn6005. This element has previously been described from the human microbiota, but with a promoter mutation that upregulates integron cassette expression. This element we describe here is from an environmental bacterium, and supports the hypothesis that the ancestral class 1 integron migrated into anthropogenic settings via foodstuffs. Selection pressures brought about by early antimicrobial agents, including mercury, arsenic and disinfectants, promoted its initial fixation, the acquisition of promoter mutations, and subsequent dissemination into various species and pathogens.

Giardiasis in NSW: Identification of Giardia duodenalis assemblages contributing to human and cattle cases, and an epidemiological assessment of sporadic human giardiasis.

Two genetic assemblages (A and B) of the protozoan parasite species, Giardia duodenalis, infect humans, domestic animals and wildlife. In New South Wales, Australia, over 2000 sporadic human giardiasis cases are reported annually, but parasite sources and links between sporadic cases are unknown. This study describes G. duodenalis assemblages contributing to human and cattle cases in NSW, and examines demographic, spatial, and temporal distributions of NSW human infections and G. duodenalis assemblages. Genotyping by PCR-restriction fragment length polymorphism of the glutamate dehydrogenase (gdh) gene identified G. duodenalis assemblage B as the most common (86%) cause of infection among human cases (n=165). Approximately 37% of cattle DNA samples were PCR positive (18S rRNA, gdh), and G. duodenalis assemblages E (69%) or B (31%) were identified from these samples. Human assemblage A was more common among older age groups, and seasonality in the geographic dispersal of human assemblage A was observed. The results of this study indicate G. duodenalis assemblage B is highly prevalent among humans in NSW, and the potential for cross-species transmission exists between humans and cattle in this region. Spatio-temporal and demographic distributions of human assemblage A and B are highlighted, and risk factors associated with these dispersal patterns warrants further research.

Distribution of giardia duodenalis assemblages A and B among children living in a remote Indigenous Community of the Northern Territory, Australia

Giardiasis is a communicable gastrointestinal disease caused by Giardia duodenalis and two genetic assemblages, A and B, cause human infection. In remote Indigenous communities of Australia, giardiasis is highly prevalent among children but disease transmission is poorly understood. This study investigated the prevalence of Giardia and genetic subtypes contributing to human disease in a remote Indigenous community, in the Northern Territory of Australia. Eighty-seven faecal samples were collected from 74 children (<15 years) over an 18 month period, and the distribution of positive cases relative to participant age and gender were examined. Screening by microscopy and 18S rRNA PCR amplification showed 66.7% (58/87) of faecal samples were positive for Giardia. Both males and females were equally affected and high detection rates were obtained for participants aged 0–<5 years and 5–<10 years (66.0 and 60.0% respectively). For 58.6% of the positive samples, Giardia was only detected by 18S rRNA PCR. Approximately 75% of cases were assemblage B, and subassemblage analyses using terminal restriction fragment length polymorphism of the glutamate dehydrogenase gene demonstrated that a variety of genetic variants were present. The high proportion of positive cases that were not detectable by microscopy, and dominance of assemblage B cases highlights the need for further research in this community, to assess the contribution of Giardia to chronic gastrointestinal disease among children, and to understand conditions conductive to assemblage B transmission.

Rapid identification of Giardia duodenalis assemblages in NSW using terminal-restriction fragment length polymorphism.

Humans are infected by 2 genetic assemblages (A and B) of Giardia duodenalis, a protozoan parasite that causes gastro-intestinal disease. Sub-assemblages AI, AII, BIII and BIV are commonly identified in human cases. Detection requires amplification of G. duodenalis loci. Subsequent DNA sequencing or restriction fragment length polymorphism (RFLP) identifies sub-assemblages but is expensive (DNA sequencing) or insensitive (RFLP). This study investigated a fluorescence-based detection method, using terminal-restriction fragment length polymorphism (T-RFLP) of the glutamate dehydrogenase gene to characterize human infections. Clinical samples (n=73), positive for Giardia were collected in New South Wales, Australia, and were used to evaluate T-RFLP detection. The accuracy and sensitivity of T-RFLP detection was established by comparison to DNA sequencing and RFLP. Sub-assemblage assignment by T-RFLP identified BIV as the common subtype in N.S.W cases, whilst AI, AII and BIII were also detected. When compared to DNA sequencing and RFLP, analysis by T-RFLP was a reliable and reproducible method. Automated fluorescent detection enabled accurate sizing of restriction fragments and provided a sensitive alternative to RFLP. Discrimination of sub-assemblages by T-RFLP was comparable to DNA sequencing, but was efficient and inexpensive. The protocol described here provides a rapid and sensitive diagnostic tool for routine sample screenings in epidemiological research.

Chemical, biological, and DNA markers for tracing slaughterhouse effluent

ABSTRACT Agricultural practices, if not managed correctly, can have a negative impact on receiving environments via waste disposal and discharge. In this study, a chicken slaughter facility on the rural outskirts of Sydney, Australia, has been identified as a possible source of persistent effluent discharge into a peri‐urban catchment. Questions surrounding the facilitys environmental management practices go back more than four decades. Despite there having never been a definitive determination of the facilitys impact on local stream water quality, the New South Wales Environment Protection Authority (NSW EPA) has implemented numerous pollution reduction requirements to manage noise and water pollution at the slaughter facility. However, assessment of compliance remains complicated by potential additional sources of pollution in the catchment. To unravel this long‐standing conundrum related to water pollution we apply a forensic, multiple lines of evidence approach to delineate the origin of the likely pollution source(s). Water samples collected between 2014 and 2016 from irrigation pipes and a watercourse exiting the slaughter facility had elevated concentrations of ammonia (max: 63,000 &mgr;g/L), nitrogen (max: 67,000 &mgr;g/L) and phosphorus (max: 39,000 &mgr;g/L), which were significantly higher than samples from adjacent streams that did not receive direct runoff from the facility. Arsenic, sometimes utilised in growth promoting compounds, was detected in water discharging from the facility up to ˜4 times (max 3.84 &mgr;g/L) local background values (<0.5 &mgr;g/L), with inorganic As(∑V+III) being the dominant species. The spatial association of elevated water pollution to the facility could not unequivocally distinguish a source and consequently DNA analysis of a suspected pollution discharge event was undertaken. Analysis of catchment runoff from several local streams showed that only water sampled at the downstream boundary of the facility tested positive for chicken DNA, with traces of duck DNA being absent, which was a potential confounder given that wild ducks are present in the area. Further, PCR analysis showed that only the discharge water emanating from the slaughter facility tested positive for a generalized marker of anthropogenic pollution, the clinical class 1 integron‐integrase gene. The environmental data collected over a three‐year period demonstrates that the slaughter facility is indisputably the primary source of water‐borne pollution in the catchment. Moreover, application of DNA and PCR for confirming pollution sources demonstrates its potential for application by regulators in fingerprinting pollution sources. HighlightsMultiple lines of evidence approach to delineate the origin of water pollution.Arsenic speciation and clinical class 1 integron‐integrase gene used as indicators.Multidisciplinary approach for addressing global concerns about agricultural waste.

Corrigendum to “Chemical, biological, and DNA markers for tracing slaughterhouse effluent” [Environ. Res. 156 (2017) 534–541]

a Department of Earth and Planetary Sciences, Faculty of Science and Engineering, Macquarie University, Sydney, NSW 2109, Australia b Department of Environmental Sciences, Faculty of Science and Engineering, Macquarie University, Sydney, NSW 2109, Australia c Ecochemistry Laboratory, Institute for Applied Ecology, University of Canberra, Bruce, ACT 2601, Australia d Department of Biological Sciences, Faculty of Science and Engineering, Macquarie University, Sydney, NSW 2109, Australia