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The emergence of SARS-CoV-2 and the subsequent COVID-19 pandemic has resulted in significant global impact. However, COVID-19 is just one of several high-impact infectious diseases that emerged from wildlife and are linked to the human relationship with nature. The rate of emergence of new zoonoses (diseases of animal origin) is increasing, driven by human-induced environmental changes that threaten biodiversity on a global scale. This increase is directly linked to environmental drivers including biodiversity loss, climate change and unsustainable resource extraction. Australia is a biodiversity hotspot and is subject to sustained and significant environmental change, increasing the risk of it being a location for pandemic origin. Moreover, the global integration of markets means that consumption trends in Australia contributes to the risk of disease spill-over in our regional neighbours in Asia-Pacific, and beyond. Despite the clear causal link between anthropogenic pressures on the environment and increasing pandemic risks, Australia’s response to the COVID-19 pandemic, like most of the world, has centred largely on public health strategies, with a clear focus on reactive management. Yet, the span of expertise and evidence relevant to the governance of pandemic risk management is much wider than public health and epidemiology. It involves animal/wildlife health, biosecurity, conservation sciences, social sciences, behavioural psychology, law, policy and economic analyses to name just a few. The authors are a team of multidisciplinary practitioners and researchers who have worked together to analyse, synthesise, and harmonise the links between pandemic risk management approaches and issues in different disciplines to provide a holistic overview of current practice, and conclude the need for reform in Australia. We discuss the adoption of a comprehensive and interdisciplinary ‘One Health’ approach to pandemic risk management in Australia. A key goal of the One Heal

The emergence of SARS-CoV-2 and the subsequent COVID-19 pandemic has resulted in significant global impact. However, COVID-19 is just one of several high-impact infectious diseases that emerged from wildlife and are linked to the human relationship with nature. The rate of emergence of new zoonoses (diseases of animal origin) is increasing, driven by human-induced environmental changes that threaten biodiversity on a global scale. This increase is directly linked to environmental drivers including biodiversity loss, climate change and unsustainable resource extraction. Australia is a biodiversity hotspot and is subject to sustained and significant environmental change, increasing the risk of it being a location for pandemic origin. Moreover, the global integration of markets means that consumption trends in Australia contributes to the risk of disease spill-over in our regional neighbours in Asia-Pacific, and beyond. Despite the clear causal link between anthropogenic pressures on the environment and increasing pandemic risks, Australia's response to the COVID-19 pandemic, like most of the world, has centred largely on public health strategies, with a clear focus on reactive management. Yet, the span of expertise and evidence relevant to the governance of pandemic risk management is much wider than public health and epidemiology. It involves animal/wildlife health, biosecurity, conservation sciences, social sciences, behavioural psychology, law, policy and economic analyses to name just a few.The authors are a team of multidisciplinary practitioners and researchers who have worked together to analyse, synthesise, and harmonise the links between pandemic risk management approaches and issues in different disciplines to provide a holistic overview of current practice, and conclude the need for reform in Australia. We discuss the adoption of a comprehensive and interdisciplinary 'One Health' approach to pandemic risk management in Australia. A key goal of the One Healt

Cryptosporidium is an important protozoan parasite and due to its resistance to chlorine is a major cause of swimming pool-associated gastroenteritis outbreaks. The present study combined contact tracing and molecular techniques to analyse cryptosporidiosis cases and outbreaks in Western Australia in 2019 and 2020. In the 2019 outbreak, subtyping at the 60 kDa glycoprotein (gp60) gene identified 89.0% (16/18) of samples were caused by the C. hominis IdA15G1 subtype. Amplicon next generation sequencing (NGS) at the gp60 locus identified five C. hominis IdA15G1 subtype samples that also had C. hominis IdA14 subtype DNA, while multi locus sequence typing (MLST) analysis on a subset (n = 14) of C. hominis samples identified three IdA15G1 samples with a 6 bp insertion at the end of the trinucleotide repeat region of the cp47 gene. In 2020, 88.0% (73/83) of samples typed were caused by the relatively rare C. hominis subtype IbA12G3. Four mixed infections were observed by NGS with three IdA15G1/ IdA14 mixtures and one C. parvum IIaA18G3R1 sample mixed with IIaA16G3R1. No genetic diversity using MLST was detected. Epidemiological and molecular data indicates that the outbreaks in 2019 and 2020 were each potentially from swimming pool point sources and a new C. hominis subtype IbA12G3 is emerging in Australia. The findings of the present study are important for understanding the introduction and transmission of rare Cryptosporidium subtypes to vulnerable populations.

In the present study, a 37‐year‐old immunosuppressed female in Western Australia (WA) was identified as positive for Cryptosporidium by microscopy and treated with nitazoxanide. Molecular analyses at the 18S ribosomal RNA (18S) and 60 kDa glycoprotein (gp60) loci identified C. fayeri subtype IVgA10G1T1R1, which had previously been identified in western grey kangaroos (Macropus fuliginosus) in WA. Next generation sequencing (NGS) of the gp60 locus confirmed the absence of mixed infections with other Cryptosporidium species. This is only the second report of C. fayeri in a human host highlighting the zoonotic potential of this wildlife‐associated species. Routine diagnosis using molecular methods in laboratories is required to better understand the diversity and epidemiology of Cryptosporidium parasite.

Purpose There is a dearth of research conducted on the Knowledge, Attitude and Practices (KAP) of swimming pool patrons and staff to determine their understanding of the importance of Cryptosporidium and its transmission in swimming pools. Methods We conducted a KAP survey of public swimming pool patrons (n = 380) and staff (n = 40) attending five public swimming pools in Western Australia (WA). Results Knowledge, attitudes and practices (KAP) of Cryptosporidium varied between patrons and staff but were generally limited. Only 26.1% and 25.0% of patrons and staff had heard of Cryptosporidium, while 17.4% and 10.0% knew that it causes diarrhoea, respectively. Thirty-one percent of patrons were aware of their pool policy concerning gastroenteritis and Cryptosporidium, compared to 62.5% of staff. Less than 50% of patrons demonstrated awareness of how features within the pool environment were relevant to the control of Cryptosporidium. Only about a third of patrons (35%) and staff (37.5%) were aware that showering before swimming reduced the risk of gastroenteritis. Conclusion Raising awareness about hygiene-related practices through the delivery of targeted health education messages to the general public is essential to reduce the burden of Cryptosporidium infections in aquatic environments.

Context: Ecologists need robust and effective methods to quantify the diet of animals. However, assessing dietary composition can be challenging because most animals are seldom observed eating, especially when studying rare or cryptic species. Aims: Morphological analysis of scats has been extensively used previously, and recent advances in the accessibility of DNA barcoding techniques have also made molecular approaches a viable alternative for diet analysis from scats. We compared the results from two methods of scat analysis, to trial the use of contemporary approaches in scat analysis. Methods: In the present study, we used morphological analysis and DNA barcoding of matter in scats to catalogue the diet of a generalist omnivore, the greater bilby (Macrotis lagotis Thylacomyidae), in the West Kimberley. The composition and diversity of diet items, as well as the taxonomic identification level, were compared between methods. Key results: Each method provided complimentary results; morphological analysis uncovered the type of matter consumed (e.g. root, seed) and relative proportion of the total undigested content, whereas DNA barcoding could assign such matter to a taxon. Even though dietary DNA could be extracted from only 38% of scats, DNA barcoding identified a greater diversity of taxa in scats than did morphological analyses. Barcoding could detect the presence of highly-digestible items such as cossid moths (Cossidae) and spiders (Araneae). Conclusions: Morphological analysis was useful for quantifying relative abundance of diet categories; however, DNA barcoding detected a greater diversity of dietary items within scats. Despite the expense of DNA barcoding, the method can more accurately identify the taxa consumed, whereas morphology can greatly underestimate dietary species diversity. However, the technical requirements for performing DNA analysis make it expensive, while resource-limited field ecologists can generally perform morphological analysis with

The species name Cryptosporidium bollandi n. sp. is proposed for Cryptosporidium piscine genotype 2 based on morphological, biological and molecular characterisation. Phylogenetic analyses of 18S rRNA (18S) sequences revealed that C. bollandi n. sp. was most closely related to piscine genotype 4 (5.1% genetic distance) and exhibited genetic distances of 10.0%, 12.2% and 25.2% from Cryptosporidium molnari, Cryptosporidium huwi and Cryptosporidium scophthtalmi, respectively. At the actin locus, C. bollandi n. sp. was again most closely related to piscine genotype 4 (6.8% genetic distance) and exhibited 15.5% (C. molnari), 18.4% (C. huwi), 22.9% (C. scophthalmi) and up to 27.5% genetic distance from other Cryptosporidium spp. (Cryptosporidium felis). Phylogenetic analysis of concatenated 18S and actin sequences showed that C. bollandi n. sp. exhibited 12.9% (C. molnari) to 21.1% (C. canis) genetic distance from all other Cryptosporidium spp. Genetic data as well as previous histological analysis clearly supports the validity of C. bollandi n. sp. as a separate species.

Rejection (nomen rejiciendum) of the name Borreliella and all new combinations therein is being requested on grounds of risk to human health and patient safety (Principle 1, subprinciple 2 and Rule 56a) and violation to aim for stability of names, to avoid useless creation of names (Principle 1, subprinciple 1 and 3) and that names should not be changed without sufficient reason (Principle 9 of the International Code of Nomenclature of Prokaryotes).

Letter to the editor

The main research question is: ‘What is the relation between certifications and sustainable business practices?’. The aim of this study is to identify a relationship between certifications and sustainable business practices. Literature and practical examples of B Corps were compared with each other in order to answer this question.

Cryptosporidium species are a major cause of diarrhoea worldwide. In the present study, a retrospective analysis of 109 microscopically Cryptosporidium-positive faecal specimens from Western Australian patients, collected between 2015 and 2018 was conducted. Sequence analysis of the 18S rRNA and the 60 kDa glycoprotein (gp60) gene loci identified four Cryptosporidium species: C. hominis (86.2%, 94/109), C. parvum (11.0%, 12/109), C. meleagridis (1.8%, 2/109) and C. viatorum (0.9%, 1/109). Subtyping at the gp60 locus identified a total of 11 subtypes including the emergence of the previously rare C. hominis IfA12G1R5 subtype in 2017 as the dominant subtype (46.7%, 21/45). This subtype has also recently emerged as the dominant subtype in the United States but the reasons for its emergence are unknown. This is also the first report of C. viatorum in humans in Australia and a novel subtype (XVaA3g) was identified in the one positive patient.

No abstract available

No abstract available

The genus Borrelia, originally described by Swellengrebel in 1907, contains tick- or louse-transmitted spirochetes belonging to the relapsing fever (RF) group of spirochetes, the Lyme borreliosis (LB) group of spirochetes and spirochetes that form intermittent clades. In 2014 it was proposed that the genus Borrelia should be separated into two genera; Borrelia Swellengrebel 1907 emend. Adeolu and Gupta 2014 containing RF spirochetes and Borreliella Adeolu and Gupta 2014 containing LB group of spirochetes. In this study we conducted an analysis based on a method that is suitable for bacterial genus demarcation, the percentage of conserved proteins (POCP). We included RF group species, LB group species and two species belonging to intermittent clades, Borrelia turcica Güner et al. 2004 and Candidatus Borrelia tachyglossi Loh et al. 2017. These analyses convincingly showed that all groups of spirochetes belong into one genus and we propose to emend, and re-unite all groups in, the genus Borrelia.

Coxiellaburnetii is the causative agent of coxiellosis in animals and Q fever in humans. Despite being a vaccine preventable disease, Q fever remains a frequently reported zoonotic infection in Australia. Recently, a Coxiella species was identified in brown dog ticks (Rhipicephalus sanguineus) in urban and rural regions of Australia. Further molecular characterisation revealed that it is genetically identical to ‘Candidatus Coxiella massiliensis’ (KM079627) described in R. sanguineus ticks removed from humans with eschars in France and serologic cross-reactivity among ‘Ca. Coxiella massiliensis’ and C.burnetii may occur. This report highlights the need for molecular testing of seropositive companion animals and humans to determine which species of Coxiella they are infected with, in order to further assess Coxiella species associated with Coxiella infections in Australia.

It may seem perplexing that there is any uncertainty in Australia about the existence of zoonotic tick-associated infections1–3. Outside this country, particularly in the northern hemisphere, tick-borne diseases such as human granulocytic anaplasmosis, babesiosis, Boutonneuse fever, ehrlichiosis, Lyme borreliosis, and tick-borne encephalitis, have well documented aetiologies, epidemiology, diagnostic methods, and treatments. Why is Australia different and what research is being conducted to address this issue? This article briefly addresses these questions and explains how high-throughput metagenomic analysis has started to shed light on bacterial microbiomes in Australian ticks, providing new data on the presence and distribution of potentially zoonotic microbial taxa.

Wastewater recycling is an increasingly popular option in worldwide to reduce pressure on water supplies due to population growth and climate change. Cryptosporidium spp. are among the most common parasites found in wastewater and understanding the prevalence of human-infectious species is essential for accurate quantitative microbial risk assessment (QMRA) and cost-effective management of wastewater. The present study conducted next generation sequencing (NGS) to determine the prevalence and diversity of Cryptosporidium species in 730 raw influent samples from 25 Australian wastewater treatment plants (WWTPs) across three states: New South Wales (NSW), Queensland (QLD) and Western Australia (WA), between 2014 and 2015. All samples were screened for the presence of Cryptosporidium at the 18S rRNA (18S) locus using quantitative PCR (qPCR), oocyst numbers were determined directly from the qPCR data using DNA standards calibrated by droplet digital PCR, and positives were characterized using NGS of 18S amplicons. Positives were also screened using C. parvum and C. hominis specific qPCRs. The overall Cryptosporidium prevalence was 11.4% (83/730): 14.3% (3/21) in NSW; 10.8% (51/470) in QLD; and 12.1% (29/239) in WA. A total of 17 Cryptosporidium species and six genotypes were detected by NGS. In NSW, C. hominis and Cryptosporidium rat genotype III were the most prevalent species (9.5% each). In QLD, C. galli, C. muris and C. parvum were the three most prevalent species (7.7%, 5.7%, and 4.5%, respectively), while in WA, C. meleagridis was the most prevalent species (6.3%). The oocyst load/Litre ranged from 70 to 18,055 oocysts/L (overall mean of 3426 oocysts/L: 4746 oocysts/L in NSW; 3578 oocysts/L in QLD; and 3292 oocysts/L in WA). NGS-based profiling demonstrated that Cryptosporidium is prevalent in the raw influent across Australia and revealed a large diversity of Cryptosporidium species and genotypes, which indicates the potential contribution of livestock, wildlife

Piroplasms, including the genera Babesia and Theileria, are intra-erythrocytic protozoa that are generally transmitted by ticks and are the aetiological agents for piroplasmosis in animals, as well as humans, worldwide. In Australia, numerous studies have been conducted on piroplasms in domestic animals; however, less is known about these protozoa in ticks from native wildlife. The present study characterised piroplasms in Ixodes australiensis (n = 119) and Amblyomma triguttatum (n = 35) ticks collected from kangaroos in Western Australia (WA). Approximately 7.6% (9/119) (95% CI 2.8–12.2) of the I. australiensis ticks were positive for piroplasms using nested-PCR at the 18S rRNA locus, whereas no piroplasm 18S rDNA was detected in the A. triguttatum ticks. All sequences from I. australiensis ticks were identical. Using a 852 bp multiple nucleotide alignment at the 18S rRNA variable region, sequences shared 97.6%, 94.3%, 93.5% and 93.4% pairwise identity with Theileria fuliginosa, Theileria brachyuri, Theileria penicillata, and a Theileria sp. (K1), derived from a burrowing bettong or boodie (Bettongia lesueur), respectively. Phylogenetic analysis revealed that the Theileria sp. from I. australiensis clustered together in the marsupial-associated Theileria group, with T. fuliginosa as closest sister species. Hence, we conclude that this is the first observation of T. fuliginosa-like species in I. australiensis ticks parasitising kangaroos in WA.

No abstract available

The extent of within-host genetic diversity of parasites has implications for our understanding of the epidemiology, disease severity and evolution of parasite virulence. As with many other species, our understanding of the within-host diversity of the enteric parasite Cryptosporidium is changing. The present study compared Sanger and Next Generation Sequencing of glycoprotein 60 (gp60) amplicons from Cryptosporidium hominis (n = 11), Cryptosporidium parvum (n = 22) and Cryptosporidium cuniculus (n = 8) DNA samples from Australia and China. Sanger sequencing identified only one gp60 subtype in each DNA sample: one C. hominis subtype (IbA10G2) (n = 11), four C. parvum subtypes belonging to IIa (n = 3) and IId (n = 19) and one C. cuniculus subtype (VbA23) (n = 8). Next Generation Sequencing identified the same subtypes initially identified by Sanger sequencing, but also identified additional gp60 subtypes in C. parvum and C. cuniculus but not in C. hominis, DNA samples. The number of C. parvum and C. cuniculus subtypes identified by Next Generation Sequencing within individual DNA samples ranged from two to four, and both C. parvum IIa and IId subtype families were identified within the one host in two samples. The finding of the present study has important implications for Cryptosporidium transmission tracking as well as vaccine and drug studies.

The genus term Isospora is now applied specifically to parasites of birds, with the term Cystoisospora preferred for parasites which infect mammals. Isospora is a common parasitic coccidian in birds worldwide, especially in passerine birds, in which it can cause systemic coccidiosis. The complete mitochondrial genome sequences from two recently identified Isospora species; Isospora serinuse in a domestic canary and Isospora manorinae in a yellow-throated miner, were sequenced and compared with those of other closely related coccidian species. The complete mitochondrial genome sequence for Isospora serinuse is 6260. bp in size and 6223. bp for Isospora manorinae. The mitochondrial genomes of Isospora serinuse and Isospora manorinae include three protein-coding genes (COI, COIII and CytB), 19 LSU and 14 SSU rDNA fragments, including one newly identified putative LSU fragment in Isospora sp. The arrangement of coding regions in these two Isospora species were identical to that of available Isospora sp. and Eimeria spp. mitochondrial genomes and the start codon usage for protein coding genes was conservative. Phylogenetic analysis of the mt genome of the two Isospora species based on the three coding regions further support that the monophyletic nature of avian Isospora.

Enteric parasites are major contributors to the global diarrhoeal disease load, infecting >67.2 million people. Their prevalence and clinical impact, however, are underestimated due to lack of adequate detection, which is largely still based on microscopy, particularly in developing countries. New commercially available enteric panel assays, which detect parasites (as well as bacteria and/or viruses) using multiplex PCR, offer enhanced sensitivity and specificity as well as the ability to detect mixed infections, and will play an important role in epidemiological surveillance and outbreak investigations. A major limitation of these technologies, however, particularly for developing countries, is the costs involved. Emerging technologies for low-resource, point-of-care (POC) settings have the potential to dramatically improve the cost and accuracy of enteric parasite detection in the future. Accurate diagnosis of diarrhoea caused by enteric parasites requires rapid, cost-effective, scalable, high-throughput, reproducible, and sensitive detection methods.Mixed infections are common, requiring clinicians and the detection methods they employ to screen for multiple pathogens.Current detection methods are labour-intensive, time-consuming, lack sensitivity and specificity, and fail to detect mixed infections.Gastrointestinal panel (GIP) assays based on multiplex PCR afford the opportunity to detect mixed infections and adhere to the requirements for diarrhoeal disease diagnosis.Costs associated with GIP assays are still unaffordable for laboratories, particularly in developing countries.More cost-effective, field-deployable technologies are required.

Cryptosporidium is one of the most common zoonotic waterborne parasitic diseases worldwide and represents a major public health concern of water utilities in developed nations. As animals in catchments can shed human-infectious Cryptosporidium oocysts, determining the potential role of animals in dissemination of zoonotic Cryptosporidium to drinking water sources is crucial. In the present study, a total of 952 animal faecal samples from four dominant species (kangaroos, rabbits, cattle and sheep) inhabiting Sydney's drinking water catchments were screened for the presence of Cryptosporidium using a quantitative PCR (qPCR) and positives sequenced at multiple loci. Cryptosporidium species were detected in 3.6% (21/576) of kangaroos, 7.0% (10/142) of cattle, 2.3% (3/128) of sheep and 13.2% (14/106) of rabbit samples screened. Sequence analysis of a region of the 18S rRNA locus identified C. macropodum and C. hominis in 4 and 17 isolates from kangaroos respectively, C. hominis and C. parvum in 6 and 4 isolates respectively each from cattle, C. ubiquitum in 3 isolates from sheep and C. cuniculus in 14 isolates from rabbits. All the Cryptosporidium species identified were zoonotic species with the exception of C. macropodum. Subtyping using the 5' half of gp60 identified C. hominis IbA10G2 (n = 12) and IdA15G1 (n = 2) in kangaroo faecal samples; C. hominis IbA10G2 (n = 4) and C. parvum IIaA18G3R1 (n = 4) in cattle faecal samples, C. ubiquitum subtype XIIa (n = 1) in sheep and C. cuniculus VbA23 (n = 9) in rabbits. Additional analysis of a subset of samples using primers targeting conserved regions of the MIC1 gene and the 3' end of gp60 suggests that the C. hominis detected in these animals represent substantial variants that failed to amplify as expected. The significance of this finding requires further investigation but might be reflective of the ability of this C. hominis variant to infect animals. The finding of zoonotic Cryptosporidium species in these animals may

[No abstract available]

Background: The Australian paralysis tick (Ixodes holocyclus) is of significant medical and veterinary importance as a cause of dermatological and neurological disease, yet there is currently limited information about the bacterial communities harboured by these ticks and the risk of infectious disease transmission to humans and domestic animals. Ongoing controversy about the presence of Borrelia burgdorferi sensu lato (the aetiological agent of Lyme disease) in Australia increases the need to accurately identify and characterise bacteria harboured by I. holocyclus ticks. Methods: Universal PCR primers were used to amplify the V1-2 hyper-variable region of bacterial 16S rRNA genes present in DNA samples from I. holocyclus and I. ricinus ticks, collected in Australia and Germany respectively. The 16S amplicons were purified, sequenced on the Ion Torrent platform, and analysed in USEARCH, QIIME, and BLAST to assign genus and species-level taxonomy. Initial analysis of I. holocyclus and I. ricinus identified that > 95 % of the 16S sequences recovered belonged to the tick intracellular endosymbiont “Candidatus Midichloria mitochondrii” (CMM). A CMM-specific blocking primer was designed that decreased CMM sequences by approximately 96 % in both tick species and significantly increased the total detectable bacterial diversity, allowing identification of medically important bacterial pathogens that were previously masked by CMM.Results: Borrelia burgdorferi sensu lato was identified in German I. ricinus, but not in Australian I. holocyclus ticks. However, bacteria of medical significance were detected in I. holocyclus ticks, including a Borrelia relapsing fever group sp., Bartonella henselae, novel “Candidatus Neoehrlichia” spp., Clostridium histolyticum, Rickettsia spp., and Leptospira inadai. Conclusions: Abundant bacterial endosymbionts, such as CMM, limit the effectiveness of next-generation 16S bacterial community profiling in arthropods by masking less abundant bacte

New Zealand moa (Aves: Dinornithiformes) are the only late Quaternary megafauna whose extinction was clearly caused by humans. New Zealand offers the best opportunity to estimate the number of people involved in a megafaunal extinction event because, uniquely, both the Polynesian settlement of New Zealand and moa extinction are recent enough to be dated with a high degree of precision. In addition, the founding human population can be estimated from genetic evidence. Here we show that the Polynesian population of New Zealand would not have exceeded 2,000 individuals before extinction of moa populations in the habitable areas of the eastern South Island. During a brief (<150 years) period and at population densities that never exceeded ~0.01 km-2, Polynesians exterminated viable populations of moa by hunting and removal of habitat. High human population densities are not required in models of megafaunal extinction.

Wairau Bar, New Zealand, is one of the few prehistoric sites in the world that could lay claim to being a site of first human intrusion into a pristine environment. It is certainly one of the best places to study human impact on a hitherto unoccupied land. Its potential status as a colonization phase settlement for New Zealand's Maori population raises questions that require fine-grained chronological resolution. Unfortunately, the simple stratigraphy of the Wairau Bar site has offered little opportunity for the development of high-resolution chronologies. This situation changed recently when new excavations exposed a complex, midden-rich feature which contained a wide range of dateable material, including hundreds of fragments of eggshell of the extinct megaherbivorous moa (Aves: Dinornithiformes). The thick eggshell, with its minimal inbuilt age and high resistance to contamination, is an ideal material for radiocarbon dating. Its refractory properties also allow high-quality preservation of DNA. The moa eggshell yielded radiocarbon that facilitated reconstruction of the chronology of deposition at a fine resolution. Ancient DNA profiling of eggshell fragments was used to ensure that dated fragments were from different individuals. Bayesian analysis of the dated fragments showed that the midden was laid down over a brief period in the early decades of the 14th century CE. This improved chronology provides a benchmark for understanding the duration of site occupation and revises current interpretations of the timing of Polynesian settlement of New Zealand.

The extinction of New Zealand's moa (Aves: Dinornithiformes) followed the arrival of humans in the late 13th century and was the final event of the prehistoric Late Quaternary megafauna extinctions. Determining the state of the moa populations in the pre-extinction period is fundamental to understanding the causes of the event. We sampled 281 moa individuals and combined radiocarbon dating with ancient DNA analyses to help resolve the extinction debate and gain insights into moa biology. The samples, which were predominantly from the last 4,000 years preceding the extinction, represent four sympatric moa species excavated from five adjacent fossil deposits. We characterized the moa assemblage using mitochondrial DNA and nuclear microsatellite markers developed specifically for moa. Although genetic diversity differed significantly among the four species, we found that the millennia preceding the extinction were characterized by a remarkable degree of genetic stability in all species, with no loss of heterozygosity and no shifts in allele frequencies over time. The extinction event itself was too rapid to be manifested in the moa gene pools. Contradicting previous claims of a decline in moa before Polynesian settlement in New Zealand, our findings indicate that the populations were large and stable before suddenly disappearing. This interpretation is supported by approximate Bayesian computation analyses. Our analyses consolidate the disappearance of moa as the most rapid, human-facilitated megafauna extinction documented to date.

Piroplasms belonging to genera Theileria and Babesia (Phylum: Apicomplexa) are vector-borne protozoan haemoparasites with similar phenotype that infect erythrocytes of domesticated mammals and wildlife including birds. In addition, piroplasms of Theileria have an exoerythrocytic life cycle stage within the host’s white blood cells. Observations of intraerythrocytic piroplasms have been made sporadically in peripheral blood films of Australian native mammals since the first report was made nearly a Century ago and the advent of PCR has stimulated renewed research interest and descriptions of the molecular phylogeny of these organisms in a variety of marsupial hosts. A piroplasm (Theileria ornithorhynchi) has been described in previous studies of the platypus (Ornithorhynchus anatinus). As part of a study into the health and ecology of this iconic monotreme, blood samples were collected for haematological and biochemical analysis from wild-trapped platypuses in Tasmania. A subset of these samples, together with their ectoparasites, was evaluated for piroplasm infections by blood film microscopy and molecular analysis of the 18S ribosomal RNA gene, and phylogenetic analysis was performed with NJ trees using BIONJ. Estimates of evolutionary divergence between sequences were calculated by MEGA 5. A total of 27 blood samples and eight ectoparasites (seven ticks, one leech) were examined from 27 platypuses. All ticks were identified as Ixodes ornithorhynchi. Piroplasm infections were highly prevalent in the population studied (27/27; 100% infected); organisms were pleomorphic with infrequent tetrads and intra-leukocytic forms, thought to be Theileria schizonts, were occasionally observed. Anaemia was not detected. Many platypuses were co-infected with trypanosomes. All blood samples and three ectoparasites were positive for piroplasm DNA and phylogenetic analysis suggested the presence of more than one piroplasm species. This study emphasises the importance of combining tr

Claims of extreme survival of DNA have emphasized the need for reliable models of DNA degradationthrough time. By analysing mitochondrial DNA (mtDNA) from 158 radiocarbon-dated bones of theextinct New Zealand moa, we confirm empirically a long-hypothesized exponential decay relationship.The average DNA half-life within this geographically constrained fossil assemblage was estimated to be521 years for a 242 bp mtDNA sequence, corresponding to a per nucleotide fragmentation rate (k) of5.50 ! 10–6 per year. With an effective burial temperature of 13.18C, the rate is almost 400 timesslower than predicted from published kinetic data of in vitro DNA depurination at pH 5. Althoughbest described by an exponential model (R2 ¼ 0.39), considerable sample-to-sample variance in DNApreservation could not be accounted for by geologic age. This variation likely derives from differencesin taphonomy and bone diagenesis, which have confounded previous, less spatially constrained attempt to study DNA decay kinetics. Lastly, by calculating DNA fragmentation rates on Illumina HiSeq data, we show that nuclear DNA has degraded at least twice as fast as mtDNA. These results provide a baseline for predicting long-term DNA survival in bone.

Quantitative real-time PCR (qPCR) is a technique that is widely used in the fi eld of ancient DNA (aDNA). Quantitative PCR can be used to optimize aDNA extraction methodologies, to detect PCR inhibition, and to quantify aDNA libraries for use in high-throughput sequencing. In this chapter, we outline factors that need to be considered when developing effi cient SYBR Green qPCR assays. We describe how to setup qPCR standards of known copy number and provide some useful tips regarding interpretation of qPCR data generated from aDNA templates.

Avian eggshell fragments recovered from both paleontological and archaeological deposits contain a cache of well-preserved ancient DNA. Here, we describe an extraction protocol that has been optimized to maximize the recovery of ancient DNA from fossil eggshell and minimize the co-purification of PCR inhibitors. In this method, fossil eggshell fragments are powdered, then digested and heated to release DNA from the calcite matrix. The digest then undergoes a concentration step before purification and washing using silica columns. The method has been used to recover aDNA from the eggshell of many avian species including moa, elephant birds, and emu, up to 19,000 years old.

The human colonisation of New Zealand in the late thirteenth century AD led to catastrophic impacts onthe local biota and is among the most compelling examples of human over-exploitation of native fauna,including megafauna. Nearly half of the species in New Zealand’ s pre-human avifauna are now extinct,including all nine species of large, flightless moa (Aves: Dinornithiformes). The abundance of moa inearly archaeological sites demonstrates the significance of these megaherbivores in the diet of the firstNew Zealanders. Combining moa assemblage data, based on DNA identification of eggshell and bone,with morphological identification of bone (literature and museum catalogued specimens), we presentthe most comprehensive audit of moa to date from several significant 13the15th century AD archaeologicaldeposits across the east coast of the South Island. Mitochondrial DNA (mtDNA) was amplifiedfrom 251 of 323 (78%) eggshell fragments and 22 of 27 (88%) bone samples, and the analyses revealed thepresence of four moa species: Anomalopteryx didiformis; Dinornis robustus; Emeus crassus and Euryapteryxcurtus. The mtDNA, along with polymorphic microsatellite markers, enabled an estimate of the minimumnumber of individual eggs consumed at each site. Remarkably, in one deposit over 50 individual eggswere identified e a number that likely represents a considerable proportion of the total reproductiveoutput of moa in the area and emphasises that human predation of all life stages of moa was intense.Molecular sexing was conducted on bones (n ¼ 11). Contrary to previous ancient DNA studies fromnatural sites that consistently report an excess of female moa, we observed an excess of males (2.7:1),suggestive that males were preferential targets. This could be related to different behaviour between thetwo highly size-dimorphic sexes in moa. Lastly, we investigated the moa species from recovered skeletaland eggshell remains from seven Wairau Bar burials, and identified the presence of only

We present the first set of microsatellite markers developed exclusively for an extinct taxon. Microsatellite data have been analysed in thousands of genetic studies on extant species but the technology can be problematic when applied to low copy number (LCN) DNA. It is therefore rarely used on substrates more than a few decades old. Now, with the primers and protocols presented here, microsatellite markers are available to study the extinct New Zealand moa (Aves: Dinornithiformes) and, as with single nucleotide polymorphism (SNP) technology, the markers represent a means bywhich the field of ancient DNA can (preservation allowing) move on from its reliance on mitochondrial DNA. Candidate markers were identified using high throughput sequencing technology (GS-FLX) on DNA extracted from fossil moa bone and eggshell. From the ‘shotgun’ reads, .60 primer pairs were designed and tested on DNA from bones of the South Island giant moa (Dinornis robustus). Six polymorphic loci were characterised and used to assess measures of genetic diversity. Because of low template numbers, typical of ancient DNA, allelic dropout was observed in 36–70% of the PCR reactions at each microsatellite marker. However, a comprehensive survey of allelic dropout, combined with supporting quantitative PCR data, allowed us to establish a set of criteria that maximised data fidelity. Finally, we demonstrated the viability of the primers and the protocols, by compiling a full Dinornis microsatellite dataset representing fossils of c. 600–5000 years of age. A multi-locus genotype was obtained from 74 individuals (84% success rate), and the data showed no signs of being compromised by allelic dropout. The methodology presented here provides a framework by which to generate and evaluate microsatellite data from samples of much greater antiquity than attempted before, and opens new opportunities for ancient DNA research.

Using ancient DNA (aDNA) extracted from eggshell of the extinct moa (Aves: Dinornithiformes) wedetermined the species composition and number of eggs found in a late thirteenth century earth ovenfeature at Wairau Bar (South Island, New Zealand) e one of New Zealand’s most significant archaeologicalsites. Mitochondrial and nuclear DNA signatures confirmed this oven feature contained fragmentsof at least 31 moa eggs, representing three moa genera: Emeus; Euryapteryx; Dinornis. We demonstratethrough the genetic identification of 127 moa eggshell fragments that thickness is an unreliable characterfor species assignment. We also present a protocol for assessing the preservation likelihood of DNA inburnt eggshell. This is useful because eggshell fragments found in archaeological contexts have oftenbeen thermally modified, and heat significantly increases DNA fragmentation. Eggshell is widely used inradiocarbon dating and stable isotope research, this study showcases how aDNA can also add to ourknowledge of eggshell in both archaeological and palaeoecological contexts.

Owing to exceptional biomolecule preservation, fossil avian eggshell has been used extensively in geochronologyand palaeodietary studies. Here, we show, to our knowledge, for the first time that fossil eggshell is apreviously unrecognized source of ancientDNA (aDNA).We describe the successful isolation and amplificationofDNAfrom fossil eggshell up to 19 ka old. aDNAwas successfully characterized fromeggshell obtainedfrom New Zealand (extinct moa and ducks), Madagascar (extinct elephant birds) and Australia (emu andowl). Our data demonstrate excellent preservation of the nucleic acids, evidenced by retrieval of both mitochondrialand nuclear DNA from many of the samples. Using confocal microscopy and quantitative PCR,this study critically evaluates approaches to maximizeDNA recovery from powdered eggshell. Our quantitativePCR experiments also demonstrate that moa eggshell has approximately 125 times lower bacterial loadthan bone, making it a highly suitable substrate for high-throughput sequencing approaches. Importantly,the preservation ofDNAin Pleistocene eggshell fromAustralia andHolocene deposits fromMadagascar indicatesthat eggshell is an excellent substrate for the long-term preservation of DNA in warmer climates. Thesuccessful recovery of DNA from this substrate has implications in a number of scientific disciplines; mostnotably archaeology and palaeontology, where genotypes and/or DNA-based species identifications canadd significantly to our understanding of diets, environments, past biodiversity and evolutionary processes.

Genetic variation in microsatellites is rarely examined in the field of ancientDNA (aDNA) due to the low quantity of nuclear DNA in the fossil record together with the lack of characterized nuclear markers in extinct species. 454 sequencing platforms provide a new high-throughput technology capable of generating up to 1 gigabases per run as short (200–400-bp) read lengths. 454 data were generated from the fossil bone of an extinct New Zealand moa (Aves:Dinornithiformes). We identified numerous short tandem repeat (STR) motifs, and here present the successful isolation and characterization of one polymorphic microsatellite (Moa_MS2). Primers designed to flank this locus amplified all three moa species tested here. The presented method proved to be a fast and efficient way of identifying microsatellite markers in ancient DNAtemplates and, depending on biomolecule preservation, has the potential of enabling high-resolution population genetic studies of extinct taxa. As sequence read lengths of the 454 platforms and its competitors (e.g., the SOLEXA and SOLiD platforms) increase, this approach will become increasingly powerful in identifying microsatellites in extinct (and extant) organisms, and will affordnew opportunities to study past biodiversity and extinction processes.