Fatal toxoplasmosis in Little Penguins (Eudyptula minor) from Penguin Island, Western Australia.

Routine post mortems of deceased penguins from Penguin Island, Western Australia, found that a temporal cluster of cases presented with characteristic gross and microscopic changes, namely birds in good body condition with hepatomegaly and splenomegaly, multifocal hepatic and splenic necrosis and numerous, 1-2 µm diameter protozoan parasites within the necrotic foci. Electron microscopy identified the protozoa as belonging to the phylum Apicomplexa. Molecular investigations by PCR gave inconsistent results. PCR performed by an external laboratory identified a novel Haemoproteus spp. organism in samples from 4 of 10 cases from this group, while PCR at Murdoch University identified Toxoplasma gondii in 12 of 13 cases (including 9 of the 10 assayed at the external laboratory). Immunohistochemistry of formalin fixed tissues also identified Toxoplasma in the hepatic and splenic lesions. The distinctive mortalities which were observed in this group of penguins are attributed to a fulminant toxoplasmosis, with a concurrent Haemoproteus infection in some cases. Though the clinical signs of infection are unknown, the gross and microscopic appearance at post mortem is sufficiently characteristic to allow a diagnosis to be made on these features. Definitive confirmation of Toxoplasma infection can be made by immunohistochemistry or PCR.

Early and sustained Lactobacillus plantarum probiotic therapy in critical illness: the randomised, placebo-controlled, restoration of gut microflora in critical illness trial (ROCIT).

PURPOSE: In adults requiring treatment in an intensive care unit, probiotic therapy using Lactobacillus plantarum 299v may reduce nosocomial infection. The aim of this study was to determine whether early and sustained L. plantarum 299v therapy administered to adult ICU patients increased days alive and at home. METHODS: A multicentre, parallel group, placebo-controlled, randomised clinical trial was conducted. Adult patients within 48 h of intensive care admission and expected to require intensive care beyond the day after recruitment were eligible to participate. L plantarum 299v or placebo were administered immediately after enrolment and continued for 60 days. The primary outcome was days alive and out of hospital to Day 60 (DAOH60). Secondary outcomes included nosocomial infections. RESULTS: The median [interquartile range (IQR)] number of DAOH60 in the probiotic (n = 110) and placebo group (n = 108) was 49.5 (IQR 37.0-53.0) and 49.0 (IQR 43.8-53.0) respectively, between-group difference of 0.0 [95% confidence interval (CI) - 6.10 to 7.1, P = 0.55]. Nosocomial infection occurred in 8 (7.3%) and 5 (4.6%) of the probiotic and placebo group participants, respectively, odds ratio 1.62 (95% CI 0.51-5.10), P = 0.57. There were no serious, or probiotic-associated adverse events. CONCLUSION: Early and sustained untargeted administration of probiotic therapy with Lactobacillus plantarum 299v to adult patients admitted to the ICU is safe, but not associated with improved patient outcomes.

Population structure and genetic diversity of Trichomonas vaginalis clinical isolates in Australia and Ghana.

Population genetic studies of Trichomonas vaginalis have detected high genetic diversity associated with phenotypic differences in clinical presentations. In this study, microscopy and next generation-multi-locus sequence typing (NG-MLST) were used to identify and genetically characterise T. vaginalis isolates from patients in Australia and Ghana. Seventy-one polymorphic nucleotide sites, 36 different alleles, 48 sequence types, 24 of which were novel, were identified among 178 isolates, revealing a geneticallly diverse T. vaginalis population. Polymorphism was found at most loci, clustering genotypes into eight groups among both Australian and Ghanaian isolates, although there was some variation between countries. The number of alleles for each locus ranged from two to nine. Study results confirmed geographic expansion and diversity of the T. vaginalis population. Two-type populations in almost equal frequencies and a third unassigned group were identified in this study. Linkage disequilibrium was observed, suggesting T. vaginalis population is highly clonal. Multillocus disequilibrium was observed even when analysing clades separately, as well as widespread clonal genotypes, suggesting that there is no evidence of recent recombination. A more comprehensive study to assess the extent of genetic diversity and population structure of T. vaginalis and their potential impact on varied pathology observed among infected individuals is recommended.

Establishment of Coral-Bacteria Symbioses Reveal Changes in the Core Bacterial Community With Host Ontogeny.

Bacterial communities are fundamental symbionts of corals. However, the process by which bacterial communities are acquired across the life history of corals, particularly in larval and early juvenile stages, is still poorly characterized. Here, transfer of bacteria of the Scleractinian coral Acropora digitifera from adults to spawned egg-sperm bundles was analyzed, as well as acquisition across early developmental stages (larvae and newly settled spat), and 6-month-old juveniles. Larvae were reared under manipulated environmental conditions to determine the source (maternal, seawater, or sediment) of bacteria likely to establish symbiotic relationships with the host using amplicon sequencing of the 16S rRNA gene. Maternal colonies directly transferred bacteria from the families Rhodobacteraceae, Cryomorphaceae, and Endozoicimonaceae to egg-sperm bundles. Furthermore, significant differences in the microbial community structure were identified across generations, yet the structure of the coral bacterial community across early life history stages was not impacted by different environmental rearing conditions. These data indicate that the uptake and structure of bacterial communities is developmentally, rather than environmentally, regulated. Both maternal coral colonies and ubiquitous bacteria found across environmental substrates represent a potential source of symbionts important in establishing the coral microbiome. Uniquely, we report the presence of variation with ontogeny of both the core and resident bacterial communities, supporting the hypothesis that microbial communities are likely to play specific roles within the distinct life history stages of the coral host.

No evidence for widespread Babesia microti transmission in Australia.

BACKGROUND: A fatal case of autochthonous Babesia microti infection was reported in Australia in 2012. This has implications for Australian public health and, given that babesiosis is transfusion transmissible, has possible implications for Australian blood transfusion recipients. We investigated the seroprevalence of antibodies to B. microti in Australian blood donors and in patients with clinically suspected babesiosis. STUDY DESIGN AND METHODS: Plasma samples (n = 7,000) from donors donating in at-risk areas and clinical specimens from patients with clinically suspected babesiosis (n = 29) were tested for B. microti IgG by immunofluorescence assay (IFA). IFA initially reactive samples were tested for B. microti IgG and IgM by immunoblot and B. microti DNA by polymerase chain reaction. RESULTS: Although five donors were initially reactive for B. microti IgG by IFA, none was confirmed for B. microti IgG (zero estimate; 95% confidence interval, 0%-0.05%) and all were negative for B. microti DNA. None of the patient samples had B. microti IgG, IgM, or DNA. CONCLUSIONS: This study does not provide evidence for widespread exposure to B. microti in Australian blood donors at local theoretical risk, nor does it provide evidence of B. microti infection in Australian patients with clinically suspected babesiosis. Given that confirmed evidence of previous exposure to B. microti was not seen, these data suggest that transmission of this pathogen is currently uncommon in Australia and unlikely to pose a risk to transfusion safety at present.

Identification of eukaryotic microorganisms with 18S rRNA next-generation sequencing in wastewater treatment plants, with a more targeted NGS approach required for Cryptosporidium detection.

While some microbial eukaryotes can improve effluent quality in wastewater treatment plants (WWTPs), eukaryotic waterborne pathogens are a threat to public health. This study aimed to identify Eukarya, particularly faecal pathogens including Cryptosporidium, in different treatment stages (influent, intermediate and effluent) from four WWTPs in Western Australia (WA). Three WWTPs that utilise stabilisation ponds and one WWTP that uses activated sludge (oxidation ditch) treatment technologies were sampled. Eukaryotic 18S rRNA (18S) was targeted in the wastewater samples (n = 26) for next-generation sequencing (NGS), and a mammalian-blocking primer was used to reduce the amplification of mammalian DNA. Overall, bioinformatics analyses revealed 49 eukaryotic phyla in WWTP samples, and three of these phyla contained human intestinal parasites, which were primarily detected in the influent. These human intestinal parasites either had a low percent sequence composition or were not detected in the intermediate and effluent stages and included the amoebozoans Endolimax sp., Entamoeba sp. and Iodamoeba sp., the human pinworm Enterobius vermicularis (Nematoda), and Blastocystis sp. subtypes (Sarcomastigophora). Six Blastocystis subtypes and four Entamoeba species were identified by eukaryotic 18S NGS, however, Cryptosporidium sp. and Giardia sp. were not detected. Real-time polymerase chain reaction (PCR) also failed to detect Giardia, but Cryptosporidium-specific NGS detected Cryptosporidium in all WWTPs, and a total of nine species were identified, including five zoonotic pathogens. Although eukaryotic 18S NGS was able to identify some faecal pathogens, this study has demonstrated that more specific NGS approaches for pathogen detection are more sensitive and should be applied to future wastewater pathogen assessments.

Evaluation of 16S next-generation sequencing of hypervariable region 4 in wastewater samples: An unsuitable approach for bacterial enteric pathogen identification.

Recycled wastewater can carry human-infectious microbial pathogens and therefore wastewater treatment strategies must effectively eliminate pathogens before recycled wastewater is used to supplement drinking and agricultural water supplies. This study characterised the bacterial composition of four wastewater treatment plants (WWTPs) (three waste stabilisation ponds and one oxidation ditch WWTP using activated sludge treatment) in Western Australia. The hypervariable region 4 (V4) of the bacterial 16S rRNA (16S) gene was sequenced using next-generation sequencing (NGS) on the Illumina MiSeq platform. Sequences were pre-processed in USEARCH v10.0 and denoised into zero-radius taxonomic units (ZOTUs) with UNOISE3. Taxonomy was assigned to the ZOTUs using QIIME 2 and the Greengenes database and cross-checked with the NCBI nr/nt database. Bacterial composition of all WWTPs and treatment stages (influent, intermediate and effluent) were dominated by Proteobacteria (29.0-87.4%), particularly Betaproteobacteria (9.0-53.5%) and Gammaproteobacteria (8.6-34.6%). Nitrifying bacteria (Nitrospira spp.) were found only in the intermediate and effluent of the oxidation ditch WWTP, and denitrifying and floc-forming bacteria were detected in all WWTPs, particularly from the families Comamonadaceae and Rhodocyclales. Twelve pathogens were assigned taxonomy by the Greengenes database, but comparison of sequences from genera and families known to contain pathogens to the NCBI nr/nt database showed that only three pathogens (Arcobacter venerupis, Laribacter hongkongensis and Neisseria canis) could be identified in the dataset at the V4 region. Importantly, Enterobacteriaceae genera could not be differentiated. Family level taxa assigned by Greengenes database agreed with NCBI nr/nt in most cases, however, BLAST analyses revealed erroneous taxa in Greengenes database. This study highlights the importance of validating taxonomy of NGS sequences with databases such as NCBI nr/nt, and recommends including the V3 region of 16S in future short amplicon NGS studies that aim to identify bacterial enteric pathogens, as this will improve taxonomic resolution of most, but not all, Enterobacteriaceae species.

An Australian dog diagnosed with an exotic tick-borne infection: should Australia still be considered free from Hepatozoon canis?

Recent molecular and sero-surveillance studies of the tick-borne pathogen Hepatozoon canis have identified new hosts, potential vector species, and have revealed that H. canis is more widespread than previously thought. We report the first diagnosed case of canine hepatozoonosis in Australia from a Maremma Sheepdog in Sarina, Queensland. Hepatozoon canis was detected with blood smear examination and 18S rRNA sequencing. It is unknown when or how the organism was introduced into Australia, which raises questions about border biosecurity policies and the H. canis infection status of its potential vectors and hosts in Australia. Surveillance for this pathogen is required to determine whether H. canis has established in Australia.

Direct oxygen uptake from air by novel glycogen accumulating organism dominated biofilm minimizes excess sludge production.

The cost associated with treatment and disposal of excess sludge produced is one of the greatest operational expenses in wastewater treatment plants. In this study, we quantify and explain greatly reduced excess sludge production in the novel glycogen accumulating organism (GAO) dominated drained biofilm system previously shown to be capable of extremely energy efficient removal of organic carbon (biological oxygen demand or BOD) from wastewater. The average excess sludge production rate was 0.05 g VSS g-1 BOD (acetate) removed, which is about 9-times lower than that of comparative studies using the same acetate based synthetic wastewater. The substantially lower sludge yield was attributed to a number of features such as the high oxygen consumption facilitated by direct oxygen uptake from air, high biomass content (21.41 g VSS L-1 of reactor), the predominance of the GAO (Candidatus competibacter) with a low growth yield and the overwhelming presence of the predatory protozoa (Tetramitus) in the biofilm. Overall, the combination of low-energy requirement for air supply (no compressed air supply) and the low excess sludge production rate, could make this novel "GAO drained biofilm" process one of the most economical ways of biological organic carbon removal from wastewater.

Identification of Theileria fuliginosa-like species in Ixodes australiensis ticks from western grey kangaroos (Macropus fuliginosus) in Western Australia.

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.

Recent insights into the tick microbiome gained through next-generation sequencing.

The tick microbiome comprises communities of microorganisms, including viruses, bacteria and eukaryotes, and is being elucidated through modern molecular techniques. The advent of next-generation sequencing (NGS) technologies has enabled the genes and genomes within these microbial communities to be explored in a rapid and cost-effective manner. The advantages of using NGS to investigate microbiomes surpass the traditional non-molecular methods that are limited in their sensitivity, and conventional molecular approaches that are limited in their scalability. In recent years the number of studies using NGS to investigate the microbial diversity and composition of ticks has expanded. Here, we provide a review of NGS strategies for tick microbiome studies and discuss the recent findings from tick NGS investigations, including the bacterial diversity and composition, influential factors, and implications of the tick microbiome.

A novel Ehrlichia species in blood and Ixodes ornithorhynchi ticks from platypuses (Ornithorhynchus anatinus) in Queensland and Tasmania, Australia.

Worldwide, Ehrlichia spp. are emerging infectious organisms of domestic animals and people, however, most Ehrlichia spp. naturally infect wildlife reservoirs causing mainly asymptomatic infections. Australian ecosystems have been under-explored for these potentially pathogenic organisms, and recent studies have identified a range of novel Ehrlichia, and their sister genera, Anaplasma and 'Candidatus Neoehrlichia' species, from native Australian ticks. We used bacterial 16S rRNA (16S) next-generation sequencing and genus-specific PCR to profile the bacterial communities in platypus (Ornithorhynchus anatinus) blood samples and platypus ticks (Ixodes ornithorhynchi), and identified a high prevalence of Ehrlichia sequences. We also observed Ehrlichia-like intra-neutrophilic inclusions (morulae) in PCR-positive stained platypus blood films that were consistent in morphology with other Ehrlichia spp. Bayesian phylogenetic analysis of 16S (1343 bp), gltA (1004 bp), and groEL (1074 bp) gene sequences group the platypus Ehrlichia with 'Candidatus Ehrlichia khabarensis' from far-eastern Russia, and demonstrate that the platypus Ehrlichia is clearly distinct from all other Ehrlichia spp. Enough genetic divergence exists to delineate this platypus Ehrlichia as a separate species that we propose to designate 'Candidatus Ehrlichia ornithorhynchi'. There is no evidence that 'Candidatus Ehrlichia ornithorhynchi' causes disease in wild platypuses, however, the organism does seem to be widespread in Australia, being found in both Queensland and Tasmania. 'Candidatus Ehrlichia ornithorhynchi' is the second native Australian Ehrlichia described and adds to the rapidly growing diversity of recently described native Australian tick-borne bacteria.

Cryptosporidium in fish: alternative sequencing approaches and analyses at multiple loci to resolve mixed infections.

Currently, the systematics, biology and epidemiology of piscine Cryptosporidium species are poorly understood. Here, we compared Sanger ‒ and next-generation ‒ sequencing (NGS), of piscine Cryptosporidium, at the 18S rRNA and actin genes. The hosts comprised 11 ornamental fish species, spanning four orders and eight families. The objectives were: to (i) confirm the rich genetic diversity of the parasite and the high frequency of mixed infections; and (ii) explore the potential of NGS in the presence of complex genetic mixtures. By Sanger sequencing, four main genotypes were obtained at the actin locus, while for the 18S locus, seven genotypes were identified. At both loci, NGS revealed frequent mixed infections, consisting of one highly dominant variant plus substantially rarer genotypes. Both sequencing methods detected novel Cryptosporidium genotypes at both loci, including a novel and highly abundant actin genotype that was identified by both Sanger sequencing and NGS. Importantly, this genotype accounted for 68·9% of all NGS reads from all samples (249 585/362 372). The present study confirms that aquarium fish can harbour a large and unexplored Cryptosporidium genetic diversity. Although commonly used in molecular parasitology studies, nested PCR prevents quantitative comparisons and thwarts the advantages of NGS, when this latter approach is used to investigate multiple infections.

Increased genetic diversity and prevalence of co-infection with Trypanosoma spp. in koalas (Phascolarctos cinereus) and their ticks identified using next-generation sequencing (NGS).

Infections with Trypanosoma spp. have been associated with poor health and decreased survival of koalas (Phascolarctos cinereus), particularly in the presence of concurrent pathogens such as Chlamydia and koala retrovirus. The present study describes the application of a next-generation sequencing (NGS)-based assay to characterise the prevalence and genetic diversity of trypanosome communities in koalas and two native species of ticks (Ixodes holocyclus and I. tasmani) removed from koala hosts. Among 168 koalas tested, 32.2% (95% CI: 25.2-39.8%) were positive for at least one Trypanosoma sp. Previously described Trypanosoma spp. from koalas were identified, including T. irwini (32.1%, 95% CI: 25.2-39.8%), T. gilletti (25%, 95% CI: 18.7-32.3%), T. copemani (27.4%, 95% CI: 20.8-34.8%) and T. vegrandis (10.1%, 95% CI: 6.0-15.7%). Trypanosoma noyesi was detected for the first time in koalas, although at a low prevalence (0.6% 95% CI: 0-3.3%), and a novel species (Trypanosoma sp. AB-2017) was identified at a prevalence of 4.8% (95% CI: 2.1-9.2%). Mixed infections with up to five species were present in 27.4% (95% CI: 21-35%) of the koalas, which was significantly higher than the prevalence of single infections 4.8% (95% CI: 2-9%). Overall, a considerably higher proportion (79.7%) of the Trypanosoma sequences isolated from koala blood samples were identified as T. irwini, suggesting this is the dominant species. Co-infections involving T. gilletti, T. irwini, T. copemani, T. vegrandis and Trypanosoma sp. AB-2017 were also detected in ticks, with T. gilletti and T. copemani being the dominant species within the invertebrate hosts. Direct Sanger sequencing of Trypanosoma 18S rRNA gene amplicons was also performed and results revealed that this method was only able to identify the genotypes with greater amount of reads (according to NGS) within koala samples, which highlights the advantages of NGS in detecting mixed infections. The present study provides new insights on the natural genetic diversity of Trypanosoma communities infecting koalas and constitutes a benchmark for future clinical and epidemiological studies required to quantify the contribution of trypanosome infections on koala survival rates.

Next Generation Sequencing uncovers within-host differences in the genetic diversity of Cryptosporidium gp60 subtypes.

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.

New Technologies for Detection of Enteric Parasites.

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.

Prevalence, genetic diversity and potential clinical impact of blood-borne and enteric protozoan parasites in native mammals from northern Australia.

A molecular survey was conducted to provide baseline information on the prevalence, genetic diversity and potential clinical impacts of blood-borne and enteric protozoans in native wild mammals from the Northern Territory (NT). A total of 209 blood and 167 faecal samples were collected from four target species; the northern brown bandicoot (Isoodon macrourus), common brushtail possum (Trichosurus vulpecula), northern quoll (Dasyurus hallucatus) and brush-tailed rabbit-rat (Conilurus penicillatus). Blood samples were screened by PCR at the 18S rRNA gene for trypanosomes, piroplasms and haemogregarines, with faecal samples tested for Cryptosporidium spp. at the 18S rRNA locus, and for Giardia spp. at the glutamate dehydrogenase (gdh) and 18S rRNA loci. The potential clinical impact was investigated by associating clinical, haematological and biochemical parameters with presence or absence of infection. Overall, 22.5% (95% CI: 17.0-28.8%) of the animals tested were positive for haemoprotozoans. Trypanosomes were found in 26.6% (95% CI: 18.7-35.7%) of the bandicoots and were identified as Trypanosoma vegrandis G6, except for one unique genotype, most similar to T. vegrandis G3 (genetic distance=7%). The prevalence of trypanosomes in possums was 23.7% (95% CI: 11.4-40.2%), and the genotypes identified clustered within the T. noyesi clade. The presence of Babesia sp. and Hepatozoon sp. was confirmed in bandicoots only, both at a prevalence of 9.7% (95% CI: 2.7-9.2%). The total prevalence of intestinal protozoan parasites observed was relatively low (3%; 95% CI: 1.0-6.9%). No evidence of clinical disease associated with protozoan parasitic infection was observed, however bandicoots positive for Trypanosoma exhibited a significantly lower packed cell volume (PCV) compared to negative bandicoots (p=0.046). To the authors' knowledge, this is the first research conducted in the NT to characterise protozoan parasites in threatened native mammals using both molecular and morphological tools; and to assess the potential clinical impacts of these agents. The absence of clear signs of major morbidity in infected animals seems to exclude a direct association between infections with these agents and possible population decline events in northern Australian native mammals. However until the cause(s) of population decline are ascertained for each individual mammal species, further studies are required. The outcome of the present investigation may be used to inform wildlife conservation and zoonotic disease programs.

Novel Primer Sets for Next Generation Sequencing-Based Analyses of Water Quality.

Next generation sequencing (NGS) has rapidly become an invaluable tool for the detection, identification and relative quantification of environmental microorganisms. Here, we demonstrate two new 16S rDNA primer sets, which are compatible with NGS approaches and are primarily for use in water quality studies. Compared to 16S rRNA gene based universal primers, in silico and experimental analyses demonstrated that the new primers showed increased specificity for the Cyanobacteria and Proteobacteria phyla, allowing increased sensitivity for the detection, identification and relative quantification of toxic bloom-forming microalgae, microbial water quality bioindicators and common pathogens. Significantly, Cyanobacterial and Proteobacterial sequences accounted for ca. 95% of all sequences obtained within NGS runs (when compared to ca. 50% with standard universal NGS primers), providing higher sensitivity and greater phylogenetic resolution of key water quality microbial groups. The increased selectivity of the new primers allow the parallel sequencing of more samples through reduced sequence retrieval levels required to detect target groups, potentially reducing NGS costs by 50% but still guaranteeing optimal coverage and species discrimination.

Rapid adaptation of activated sludge bacteria into a glycogen accumulating biofilm enabling anaerobic BOD uptake.

Glycogen accumulating organisms (GAO) are known to allow anaerobic uptake of biological oxygen demand (BOD) in activated sludge wastewater treatment systems. In this study, we report a rapid transition of suspended activated sludge biomass to a GAO dominated biofilm by selective enrichment using sequences of anaerobic loading followed by aerobic exposure of the biofilm to air. The study showed that within eight weeks, a fully operational, GAO dominated biofilm had developed, enabling complete anaerobic BOD uptake at a rate of 256mg/L/h. The oxygen uptake by the biofilm directly from the atmosphere had been calculated to provide significant energy savings. This study suggests that wastewater treatment plant operators can convert activated sludge systems readily into a "passive aeration" biofilm that avoids costly oxygen transfer to bulk wastewater solution. The described energy efficient BOD removal system provides an opportunity to be coupled with novel nitrogen removal processes such as anammox.

Zoonotic Cryptosporidium Species in Animals Inhabiting Sydney Water Catchments.

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 have important implications for the management of drinking water catchments to minimize risk to public health.