Evaluation of LAMP for Fasciola hepatica detection from faecal samples of experimentally and naturally infected cattle.

Fasciola hepatica causes liver fluke disease in production animals and humans worldwide. Faecal egg counts (FEC) are the most common diagnostic tool for the diagnosis of liver fluke disease. However, FEC has low sensitivity and is often unreliable for the detection of patent infection. In this study, loop-mediated isothermal amplification (LAMP) was optimised and evaluated for the detection of Fasciola hepatica infection, with the aim of increased sensitivity and making it suitable for on-farm application. LAMP was initially conducted under laboratory conditions, optimised to enable visual detection using calcein dye. DNA extraction based on bead-beating was developed to enable on-farm application. LAMP results were compared to FEC and polymerase chain reaction (PCR). Under laboratory conditions, LAMP was conducted using two incubation methods: a conventional PCR thermocycler and a field-deployable LAMP instrument. When compared to a 'rigorous' FEC protocol consisting of multiple counts using a comparatively large volume of faeces and with infection confirmed post-mortem, LAMP was highly sensitive and specific (using silica membrane DNA extraction sensitivity 88 %, specificity 100 %; using sieving and beat-beating DNA extraction sensitivity 98.9 %, specificity 100 %). When applied on-farm, LAMP was compared to conventional FEC, which suggested high sensitivity but low specificity (sensitivity 97 %, specificity 37.5 %). However, further analysis, comparing field LAMP results to laboratory PCR, suggested that the low specificity was likely the outcome of the inability of conventional FEC to detect all true F. hepatica positive samples. Based on the high sensitivity and specificity of LAMP compared to a 'rigorous' FEC protocol and its ability to be used in field settings, the study demonstrates the potential of LAMP for diagnosing F. hepatica infection in agriculture.

Global diversity and antimicrobial resistance of typhoid fever pathogens: Insights from a meta-analysis of 13,000 Salmonella Typhi genomes.

Background: The Global Typhoid Genomics Consortium was established to bring together the typhoid research community to aggregate and analyse Salmonella enterica serovar Typhi (Typhi) genomic data to inform public health action. This analysis, which marks 22 years since the publication of the first Typhi genome, represents the largest Typhi genome sequence collection to date (n=13,000). Methods: This is a meta-analysis of global genotype and antimicrobial resistance (AMR) determinants extracted from previously sequenced genome data and analysed using consistent methods implemented in open analysis platforms GenoTyphi and Pathogenwatch. Results: Compared with previous global snapshots, the data highlight that genotype 4.3.1 (H58) has not spread beyond Asia and Eastern/Southern Africa; in other regions, distinct genotypes dominate and have independently evolved AMR. Data gaps remain in many parts of the world, and we show the potential of travel-associated sequences to provide informal 'sentinel' surveillance for such locations. The data indicate that ciprofloxacin non-susceptibility (>1 resistance determinant) is widespread across geographies and genotypes, with high-level ciprofloxacin resistance (≥3 determinants) reaching 20% prevalence in South Asia. Extensively drug-resistant (XDR) typhoid has become dominant in Pakistan (70% in 2020) but has not yet become established elsewhere. Ceftriaxone resistance has emerged in eight non-XDR genotypes, including a ciprofloxacin-resistant lineage (4.3.1.2.1) in India. Azithromycin resistance mutations were detected at low prevalence in South Asia, including in two common ciprofloxacin-resistant genotypes. Conclusions: The consortium's aim is to encourage continued data sharing and collaboration to monitor the emergence and global spread of AMR Typhi, and to inform decision-making around the introduction of typhoid conjugate vaccines (TCVs) and other prevention and control strategies. Funding: No specific funding was awarded for this meta-analysis. Coordinators were supported by fellowships from the European Union (ZAD received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 845681), the Wellcome Trust (SB, Wellcome Trust Senior Fellowship), and the National Health and Medical Research Council (DJI is supported by an NHMRC Investigator Grant [GNT1195210]).

Salmonella Typhi (Typhi) is a type of bacteria that causes typhoid fever. More than 110,000 people die from this disease each year, predominantly in areas of sub-Saharan Africa and South Asia with limited access to safe water and sanitation. Clinicians use antibiotics to treat typhoid fever, but scientists worry that the spread of antimicrobial-resistant Typhi could render the drugs ineffective, leading to increased typhoid fever mortality. The World Health Organization has prequalified two vaccines that are highly effective in preventing typhoid fever and may also help limit the emergence and spread of resistant Typhi. In low resource settings, public health officials must make difficult trade-off decisions about which new vaccines to introduce into already crowded immunization schedules. Understanding the local burden of antimicrobial-resistant Typhi and how it is spreading could help inform their actions. The Global Typhoid Genomics Consortium analyzed 13,000 Typhi genomes from 110 countries to provide a global overview of genetic diversity and antimicrobial-resistant patterns. The analysis showed great genetic diversity of the different strains between countries and regions. For example, the H58 Typhi variant, which is often drug-resistant, has spread rapidly through Asia and Eastern and Southern Africa, but is less common in other regions. However, distinct strains of other drug-resistant Typhi have emerged in other parts of the world. Resistance to the antibiotic ciprofloxacin was widespread and accounted for over 85% of cases in South Africa. Around 70% of Typhi from Pakistan were extensively drug-resistant in 2020, but these hard-to-treat variants have not yet become established elsewhere. Variants that are resistant to both ciprofloxacin and ceftriaxone have been identified, and azithromycin resistance has also appeared in several different variants across South Asia. The Consortium's analyses provide valuable insights into the global distribution and transmission patterns of drug-resistant Typhi. Limited genetic data were available fromseveral regions, but data from travel-associated cases helped fill some regional gaps. These findings may help serve as a starting point for collective sharing and analyses of genetic data to inform local public health action. Funders need to provide ongoing supportto help fill global surveillance data gaps.

Pneumococcal carriage, serotype distribution, and antimicrobial susceptibility in Papua New Guinean children vaccinated with PCV10 or PCV13 in a head-to-head trial.

BACKGROUND: Children in Papua New Guinea (PNG) are at high risk of pneumococcal infections. We investigated pneumococcal carriage rates, serotype distribution, and antimicrobial susceptibility in PNG children after vaccination with 10-valent or 13-valent pneumococcal conjugate vaccines (PCV10; PCV13). METHODS: Infants (N = 262) were randomized to receive 3 doses of PCV10 or PCV13 at 1-2-3 months of age, followed by pneumococcal polysaccharide vaccination (PPV) or no PPV at 9 months of age. Nasopharyngeal swabs (NPS) collected at ages 1, 4, 9, 10, 23 and 24 months were cultured using standard bacteriological procedures. Morphologically distinct Streptococcus pneumoniae colonies were serotyped by the Quellung reaction. Antimicrobial susceptibility was determined by Kirby-Bauer disc diffusion and minimum inhibitory concentration (MIC). RESULTS: S. pneumoniae was isolated from 883/1063 NPS collected at 1-23 months of age, including 820 serotypeable (64 different serotypes) and 144 non-serotypeable isolates. At age 23 months, 93.6% (95%CI 86.6-97.6%) of PCV10 recipients and 88.6% (95%CI 80.1-94.4%) of PCV13 recipients were pneumococcal carriers, with higher carriage of PCV10 serotypes by PCV10 recipients (19.8%, 95%CI 12.2-29.5) than PCV13 recipients (9.3%, 95%CI 4.1-17.3) (p = 0.049). There were no other statistically significant differences between PCV10 and PCV13 recipients and children receiving PPV or no PPV. Nearly half (45.6%) of carried pneumococci were non-susceptible to penicillin based on the meningitis breakpoint (MIC ≥ 0.12 µg/mL), but resistance was rare (1.1%) using the non-meningitis cut-off (MIC ≥ 8 µg/mL). Non-susceptibility to trimethoprim-sulfamethoxazole (SXT) was common: 23.2% of isolates showed intermediate resistance (MIC 1/19-2/38 µg/mL) and 16.9% full resistance (MIC ≥ 4/76 µg/mL). PCV serotypes 14 and 19A were commonly non-susceptible to both penicillin (14, 97%; 19A, 70%) and SXT (14, 97%; 19A, 87%). CONCLUSION: Even after PCV10 or PCV13 vaccination, children living in a high-risk setting such as PNG continue to experience high levels of pneumococcal colonization, including carriage of highly antimicrobial-resistant PCV serotypes. The study is registered with ClinicalTrials.gov (CTN NCT01619462).

Phylodynamic signatures in the emergence of community-associated MRSA.

Community-associated, methicillin-resistant Staphylococcus aureus (MRSA) lineages have emerged in many geographically distinct regions around the world during the past 30 y. Here, we apply consistent phylodynamic methods across multiple community-associated MRSA lineages to describe and contrast their patterns of emergence and dissemination. We generated whole-genome sequencing data for the Australian sequence type (ST) ST93-MRSA-IV from remote communities in Far North Queensland and Papua New Guinea, and the Bengal Bay ST772-MRSA-V clone from metropolitan communities in Pakistan. Increases in the effective reproduction number (Re) and sustained transmission (Re > 1) coincided with spread of progenitor methicillin-susceptible S. aureus (MSSA) in remote northern Australian populations, dissemination of the ST93-MRSA-IV genotype into population centers on the Australian East Coast, and subsequent importation into the highlands of Papua New Guinea and Far North Queensland. Applying the same phylodynamic methods to existing lineage datasets, we identified common signatures of epidemic growth in the emergence and epidemiological trajectory of community-associated S. aureus lineages from America, Asia, Australasia, and Europe. Surges in Re were observed at the divergence of antibiotic-resistant strains, coinciding with their establishment in regional population centers. Epidemic growth was also observed among drug-resistant MSSA clades in Africa and northern Australia. Our data suggest that the emergence of community-associated MRSA in the late 20th century was driven by a combination of antibiotic-resistant genotypes and host epidemiology, leading to abrupt changes in lineage-wide transmission dynamics and sustained transmission in regional population centers.

Distinct Streptococcus pneumoniae cause invasive disease in Papua New Guinea.

Streptococcus pneumoniae is a key contributor to childhood morbidity and mortality in Papua New Guinea (PNG). For the first time, whole genome sequencing of 174 isolates has enabled detailed characterisation of diverse S. pneumoniae causing invasive disease in young children in PNG, 1989-2014. This study captures the baseline S. pneumoniae population prior to the introduction of 13-valent pneumococcal conjugate vaccine (PCV13) into the national childhood immunisation programme in 2014. Relationships amongst lineages, serotypes and antimicrobial resistance traits were characterised, and the population was viewed in the context of a global collection of isolates. The analyses highlighted adiverse S. pneumoniae population associated with invasive disease in PNG, with 45 unique Global Pneumococcal Sequence Clusters (GPSCs) observed amongst the 174 isolates reflecting multiple lineages observed in PNG that have not been identified in other geographic locations. The majority of isolates were from children with meningitis, of which 52% (n=72) expressed non-PCV13 serotypes. Over a third of isolates were predicted to be resistant to at least one antimicrobial. PCV13 serotype isolates had 10.1 times the odds of being multidrug-resistant (MDR) compared to non-vaccine serotype isolates, and no isolates with GPSCs unique to PNG were MDR. Serotype 2 was the most commonly identified serotype; we identified a highly clonal cluster of serotype 2 isolates unique to PNG, and a distinct second cluster indicative of long-distance transmission. Ongoing surveillance, including whole-genome sequencing, is needed to ascertain the impact of the national PCV13 programme upon the S. pneumoniae population, including serotype replacement and antimicrobial resistance traits.

Presence and antimicrobial resistance profiles of Escherichia coli, Enterococcusspp. and Salmonellasp. in 12 species of Australian shorebirds and terns.

Antibiotic resistance is an ongoing threat to both human and animal health. Migratory birds are a potential vector for the spread of novel pathogens and antibiotic resistance genes. To date, there has been no comprehensive study investigating the presence of antibiotic resistance (AMR) in the bacteria of Australian shorebirds or terns. In the current study, 1022 individual birds representing 12 species were sampled across three states of Australia (Victoria, South Australia, and Western Australia) and tested for the presence of phenotypically resistant strains of three bacteria with potential to be zoonotic pathogens; Escherichia coli, Enterococcusspp., and Salmonellasp. In total, 206 E. coli, 266 Enterococcusspp., and 20 Salmonellasp. isolates were recovered, with AMR detected in 42% of E. coli, 85% of Enterococcusspp., and 10% of Salmonellasp. Phenotypic resistance was commonly detected to erythromycin (79% of Enterococcusspp.), ciprofloxacin (31% of Enterococcusspp.) and streptomycin (21% of E. coli). Resident birds were more likely to carry AMR bacteria than migratory birds (p ≤ .001). Bacteria isolated from shorebirds and terns are commonly resistant to at least one antibiotic, suggesting that wild bird populations serve as a potential reservoir and vector for AMR bacteria. However, globally emerging phenotypes of multidrug-resistant bacteria were not detected in Australian shorebirds. This study provides baseline data of the carriage of AMR bacteria in Australian shorebirds and terns.

Whole genome sequence analysis of Salmonella Typhi in Papua New Guinea reveals an established population of genotype 2.1.7 sensitive to antimicrobials.

BACKGROUND: Typhoid fever, a systemic infection caused by Salmonella enterica serovar Typhi, remains a considerable public health threat in impoverished regions within many low- and middle-income settings. However, we still lack a detailed understanding of the emergence, population structure, molecular mechanisms of antimicrobial resistance (AMR), and transmission dynamics of S. Typhi across many settings, particularly throughout the Asia-Pacific islands. Here we present a comprehensive whole genome sequence (WGS) based overview of S. Typhi populations circulating in Papua New Guinea (PNG) over 30 years. PRINCIPLE FINDINGS: Bioinformatic analysis of 86 S. Typhi isolates collected between 1980-2010 demonstrated that the population structure of PNG is dominated by a single genotype (2.1.7) that appears to have emerged in the Indonesian archipelago in the mid-twentieth century with minimal evidence of inter-country transmission. Genotypic and phenotypic data demonstrated that the PNG S. Typhi population appears to be susceptible to former first line drugs for treating typhoid fever (chloramphenicol, ampicillin and co-trimoxazole), as well as fluoroquinolones, third generation cephalosporins, and macrolides. PNG genotype 2.1.7 was genetically conserved, with very few deletions, and no evidence of plasmid or prophage acquisition. Genetic variation among this population was attributed to either single point mutations, or homologous recombination adjacent to repetitive ribosomal RNA operons. SIGNIFICANCE: Antimicrobials remain an effective option for the treatment of typhoid fever in PNG, along with other intervention strategies including improvements to water, sanitation and hygiene (WaSH) related infrastructure and potentially the introduction of Vi-conjugate vaccines. However, continued genomic surveillance is warranted to monitor for the emergence of AMR within local populations, or the introduction of AMR associated genotypes of S. Typhi in this setting.

Phylodynamic Inference of Bacterial Outbreak Parameters Using Nanopore Sequencing.

Nanopore sequencing and phylodynamic modeling have been used to reconstruct the transmission dynamics of viral epidemics, but their application to bacterial pathogens has remained challenging. Cost-effective bacterial genome sequencing and variant calling on nanopore platforms would greatly enhance surveillance and outbreak response in communities without access to sequencing infrastructure. Here, we adapt random forest models for single nucleotide polymorphism (SNP) polishing developed by Sanderson and colleagues (2020. High precision Neisseria gonorrhoeae variant and antimicrobial resistance calling from metagenomic nanopore sequencing. Genome Res. 30(9):1354-1363) to estimate divergence and effective reproduction numbers (Re) of two methicillin-resistant Staphylococcus aureus (MRSA) outbreaks from remote communities in Far North Queensland and Papua New Guinea (PNG; n = 159). Successive barcoded panels of S. aureus isolates (2 × 12 per MinION) sequenced at low coverage (>5× to 10×) provided sufficient data to accurately infer genotypes with high recall when compared with Illumina references. Random forest models achieved high resolution on ST93 outbreak sequence types (>90% accuracy and precision) and enabled phylodynamic inference of epidemiological parameters using birth-death skyline models. Our method reproduced phylogenetic topology, origin of the outbreaks, and indications of epidemic growth (Re > 1). Nextflow pipelines implement SNP polisher training, evaluation, and outbreak alignments, enabling reconstruction of within-lineage transmission dynamics for infection control of bacterial disease outbreaks on portable nanopore platforms. Our study shows that nanopore technology can be used for bacterial outbreak reconstruction at competitive costs, providing opportunities for infection control in hospitals and communities without access to sequencing infrastructure, such as in remote northern Australia and PNG.

Lack of effectiveness of 13-valent pneumococcal conjugate vaccination against pneumococcal carriage density in Papua New Guinean infants.

BACKGROUND: Papua New Guinea (PNG) introduced the 13-valent pneumococcal conjugate vaccine (PCV13) in 2014, with administration at 1, 2, and 3 months of age. PCV13 has reduced or eliminated carriage of vaccine types in populations with low pneumococcal carriage prevalence, carriage density and serotype diversity. This study investigated PCV13 impact on serotype-specific pneumococcal carriage prevalence, density, and serotype diversity in PNG infants, who have some of the highest reported rates of pneumococcal carriage and disease in the world. METHODS: Nasopharyngeal swabs were collected at 1, 4 and 9 months of age from PCV13-vaccinated infants (n = 57) and age-/season-matched, unvaccinated infants (at approximately 1 month, n = 53; 4 months, n = 57; 9 months, n = 52). Serotype-specific pneumococcal carriage density and antimicrobial resistance genes were identified by qPCR and microarray. RESULTS: Pneumococci were present in 89% of swabs, with 60 different serotypes and four non-encapsulated variants detected. Multiple serotype carriage was common (47% of swabs). Vaccine type carriage prevalence was similar between PCV13-vaccinated and unvaccinated infants at 4 and 9 months of age. The prevalence of non-vaccine type carriage was also similar between cohorts, with non-vaccine types present in three-quarters of samples (from both vaccinated and unvaccinated infants) by 4 months of age. The median pneumococcal carriage density was high and similar at each age group (~7.0 log10genome equivalents/mL). PCV13 had no effect on overall pneumococcal carriage density, vaccine type density, non-vaccine type density, or the prevalence of antimicrobial resistance genes. CONCLUSION: PNG infants experience dense and diverse pneumococcal colonisation with concurrent serotypes from 1 month of age. PCV13 had no impact on pneumococcal carriage density, even for vaccine serotypes. The low prevalence of vaccine serotypes, high pneumococcal carriage density and abundance of non-vaccine serotypes likely contribute to the lack of PCV13 impact on carriage in PNG infants. Indirect effects of the infant PCV programs are likely to be limited in PNG. Alternative vaccines with broader coverage should be considered.

Draft Genome Sequences of Four Citrobacter Isolates Recovered from Wild Australian Shorebirds.

Citrobacter is a ubiquitous bacterial genus whose members inhabit a variety of niches. Some species are clinically important for both antimicrobial resistance (AMR) carriage and as the cause of nosocomial infections. Surveillance of Citrobacter species in the environment can provide indicators of the spread of AMR genes outside clinical spaces. In this study, we present draft genome sequences of four Citrobacter isolates obtained from three species of wild Australian shorebirds.

The influences of low protein diet on the intestinal microbiota of mice.

Recent research suggests that protein deficiency symptoms are influenced by the intestinal microbiota. We investigated the influence of low protein diet on composition of the intestinal microbiota through animal experiments. Specific pathogen-free (SPF) mice were fed one of four diets (3, 6, 9, or 12% protein) for 4 weeks (n = 5 per diet). Mice fed the 3% protein diet showed protein deficiency symptoms such as weight loss and low level of blood urea nitrogen concentration in their serum. The intestinal microbiota of mice in the 3% and 12% protein diet groups at day 0, 7, 14, 21 and 28 were investigated by 16S rRNA gene sequencing, which revealed differences in the microbiota. In the 3% protein diet group, a greater abundance of urease producing bacterial species was detected across the duration of the study. In the 12% diet protein group, increases of abundance of Streptococcaceae and Clostridiales families was detected. These results suggest that protein deficiency may be associated with shifts in intestinal microbiota.

Gut microbiota composition in obese and non-obese adult relatives from the highlands of Papua New Guinea.

Obesity is a condition that results from an imbalance between energy intake and expenditure. Recently, obesity has been linked to differences in the composition of gut microbiota. To examine this association in Papua New Guinea (PNG) highlanders, fecal samples were collected from 18 adults; nine obese participants were paired with their non-obese relative. Amplification of the 16S rRNA gene targeting the V1-V2 region was performed on DNA extracts for each participant, with high-quality sequences selected and used for operational taxonomic unit clustering. The data showed Firmicutes and Bacteroidetes were the two dominant phyla, while at genus level Prevotella was the most dominant genus in all of the samples. Nonetheless, statistical evaluation of potential association between nutritional status and bacterial abundance at both phyla and genus levels showed no significant difference. Further studies, ideally in both rural and urban areas, are needed to evaluate the role of the gut microbiome in the occurrence of obesity in PNG and other resource-limited settings.

Diarrhoeal disease surveillance in Papua New Guinea: findings and challenges.

Diarrhoeal diseases are among the leading causes of morbidity and mortality in the Western Pacific Region. However, data on the major causes of infectious diarrhoea are limited in many countries within the Region, including Papua New Guinea. In 2013-2014, we conducted surveillance for acute diarrhoeal illness in four provinces in Papua New Guinea. One rural health clinic from each province participated in the surveillance activity. Samples were sent to central laboratories and batch analysed for bacterial and viral gastrointestinal pathogens that are commonly associated with diarrhoea. Across the four sites, the most commonly detected pathogens were Shigella spp., Campylobacter spp. and rotavirus. In this paper, we report the results of the surveillance activity and the challenges that we faced. The lessons learnt may be applicable to other parts of the Region with a similar socioeconomic status.

Salmonella enterica Serovar Hvittingfoss in Bar-Tailed Godwits (Limosa lapponica) from Roebuck Bay, Northwestern Australia.

Salmonella enterica serovar Hvittingfoss is an important foodborne serotype of Salmonella, being detected in many countries where surveillance is conducted. Outbreaks can occur, and there was a recent multistate foodborne outbreak in Australia. S Hvittingfoss can be found in animal populations, though a definitive animal host has not been established. Six species of birds were sampled at Roebuck Bay, a designated Ramsar site in northwestern Australia, resulting in 326 cloacal swabs for bacterial culture. Among a single flock of 63 bar-tailed godwits (Limosa lapponica menzbieri) caught at Wader Spit, Roebuck Bay, in 2018, 17 (27%) were culture positive for Salmonella All other birds were negative for Salmonella The isolates were identified as Salmonella enterica serovar Hvittingfoss. Phylogenetic analysis revealed a close relationship between isolates collected from godwits and the S Hvittingfoss strain responsible for a 2016 multistate foodborne outbreak originating from tainted cantaloupes (rock melons) in Australia. While it is not possible to determine how this strain of S Hvittingfoss was introduced into the bar-tailed godwits, these findings show that wild Australian birds are capable of carrying Salmonella strains of public health importance.IMPORTANCESalmonella is a zoonotic pathogen that causes gastroenteritis and other disease presentations in both humans and animals. Serovars of S. enterica commonly cause foodborne disease in Australia and globally. In 2016-2017, S Hvittingfoss was responsible for an outbreak that resulted in 110 clinically confirmed human cases throughout Australia. The origin of the contamination that led to the outbreak was never definitively established. Here, we identify a migratory shorebird, the bar-tailed godwit, as an animal reservoir of S Hvittingfoss. These birds were sampled in northwestern Australia during their nonbreeding period. The presence of a genetically similar S Hvittingfoss strain circulating in a wild bird population, 2 years after the 2016-2017 outbreak and ∼1,500 km from the suspected source of the outbreak, demonstrates a potentially unidentified environmental reservoir of S Hvittingfoss. While the birds cannot be implicated in the outbreak that occurred 2 years prior, this study does demonstrate the potential role for wild birds in the transmission of this important foodborne pathogen.

Variation in gut bacterial composition is associated with Haemonchus contortus parasite infection of sheep.

BACKGROUND: One of the greatest impediments to global small ruminant production is infection with the gastrointestinal parasite, Haemonchus contortus. In recent years there has been considerable interest in the gut microbiota and its impact on health. Relatively little is known about interactions between the gut microbiota and gastrointestinal tract pathogens in sheep. Thus, this study was undertaken to investigate the link between the faecal microbiota of sheep, as a sample representing the gastrointestinal microbiota, and infection with H. contortus. RESULTS: Sheep (n = 28) were experimentally inoculated with 14,000 H. contortus infective larvae. Faecal samples were collected 4 weeks prior to and 4 weeks after infection. Microbial analyses were conducted using automated ribosomal intergenic spacer analysis (ARISA) and 16S rRNA gene sequencing. A comparison of pre-infection microbiota to post-infection microbiota was conducted. A high parasite burden associated with a relatively large change in community composition, including significant (p ≤ 0.001) differences in the relative abundances of Firmicutes and Bacteroidetes following infection. In comparison, low parasite burden associated with a smaller change in community composition, with the relative abundances of the most abundant phyla remaining stable. Interestingly, differences were observed in pre-infection faecal microbiota in sheep that went on to develop a high burden of H. contortus infection (n = 5) to sheep that developed a low burden of infection (n = 5). Differences observed at the community level and also at the taxa level, where significant (p ≤ 0.001) in relative abundance of Bacteroidetes (higher in high parasite burden sheep) and Firmicutes (lower in high parasite burden sheep). CONCLUSIONS: This study reveals associations between faecal microbiota and high or low H. contortus infection in sheep. Further investigation is warranted to investigate causality and the impact of microbiome manipulation.

The evolution and genetic diversity of avian influenza A(H9N2) viruses in Cambodia, 2015 - 2016.

Low pathogenic A(H9N2) subtype avian influenza viruses (AIVs) were originally detected in Cambodian poultry in 2013, and now circulate endemically. We sequenced and characterised 64 A(H9N2) AIVs detected in Cambodian poultry (chickens and ducks) from January 2015 to May 2016. All A(H9) viruses collected in 2015 and 2016 belonged to a new BJ/94-like h9-4.2.5 sub-lineage that emerged in the region during or after 2013, and was distinct to previously detected Cambodian viruses. Overall, there was a reduction of genetic diversity of H9N2 since 2013, however two genotypes were detected in circulation, P and V, with extensive reassortment between the viruses. Phylogenetic analysis showed a close relationship between A(H9N2) AIVs detected in Cambodian and Vietnamese poultry, highlighting cross-border trade/movement of live, domestic poultry between the countries. Wild birds may also play a role in A(H9N2) transmission in the region. Some genes of the Cambodian isolates frequently clustered with zoonotic A(H7N9), A(H9N2) and A(H10N8) viruses, suggesting a common ecology. Molecular analysis showed 100% of viruses contained the hemagglutinin (HA) Q226L substitution, which favours mammalian receptor type binding. All viruses were susceptible to the neuraminidase inhibitor antivirals; however, 41% contained the matrix (M2) S31N substitution associated with resistance to adamantanes. Overall, Cambodian A(H9N2) viruses possessed factors known to increase zoonotic potential, and therefore their evolution should be continually monitored.

Diversity of A(H5N1) clade 2.3.2.1c avian influenza viruses with evidence of reassortment in Cambodia, 2014-2016.

In Cambodia, highly pathogenic avian influenza A(H5N1) subtype viruses circulate endemically causing poultry outbreaks and zoonotic human cases. To investigate the genomic diversity and development of endemicity of the predominantly circulating clade 2.3.2.1c A(H5N1) viruses, we characterised 68 AIVs detected in poultry, the environment and from a single human A(H5N1) case from January 2014 to December 2016. Full genomes were generated for 42 A(H5N1) viruses. Phylogenetic analysis shows that five clade 2.3.2.1c genotypes, designated KH1 to KH5, were circulating in Cambodia during this period. The genotypes arose through multiple reassortment events with the neuraminidase (NA) and internal genes belonging to H5N1 clade 2.3.2.1a, clade 2.3.2.1b or A(H9N2) lineages. Phylogenies suggest that the Cambodian AIVs were derived from viruses circulating between Cambodian and Vietnamese poultry. Molecular analyses show that these viruses contained the hemagglutinin (HA) gene substitutions D94N, S133A, S155N, T156A, T188I and K189R known to increase binding to the human-type α2,6-linked sialic acid receptors. Two A(H5N1) viruses displayed the M2 gene S31N or A30T substitutions indicative of adamantane resistance, however, susceptibility testing towards neuraminidase inhibitors (oseltamivir, zanamivir, lananmivir and peramivir) of a subset of thirty clade 2.3.2.1c viruses showed susceptibility to all four drugs. This study shows that A(H5N1) viruses continue to reassort with other A(H5N1) and A(H9N2) viruses that are endemic in the region, highlighting the risk of introduction and emergence of novel A(H5N1) genotypes in Cambodia.

Divergent Barmah Forest Virus from Papua New Guinea.

We report a case of Barmah Forest virus infection in a child from Central Province, Papua New Guinea, who had no previous travel history. Genomic characterization of the virus showed divergent origin compared with viruses previously detected, supporting the hypothesis that the range of Barmah Forest virus extends beyond Australia.