Development of a multiplex droplet digital PCR assay for simultaneous detection and quantification of Escherichia coli, E. marmotae, and E. ruysiae in water samples.

Escherichia coli are widely used by water quality managers as Fecal Indicator Bacteria, but current quantification methods do not differentiate them from benign, environmental Escherichia species such as E. marmotae (formerly named cryptic clade V) or E. ruysiae (cryptic clades III and IV). Reliable and specific techniques for their identification are required to avoid confounding microbial water quality assessments. To address this, a multiplex droplet digital PCR (ddPCR) assay targeting lipB (E. coli and E. ruysiae) and bglC (E. marmotae) was designed. The ddPCR performance was assessed using in silico analysis; genomic DNA from 40 local, international, and reference strains of target and non-target coliforms; and spiked water samples in a range relevant to water quality managers (1 to 1000 cells/100 mL). Results were compared to an analogous quantitative PCR (qPCR) and the Colilert method. Both PCR assays showed excellent sensitivity with a limit of detection of 0.05 pg/µL and 0.005 pg/µl for ddPCR and qPCR respectively, and of quantification of 0.5 pg/µL of genomic DNA. The ddPCR allowed differentiation and quantification of three Escherichia species per run by amplitude multiplexing and showed a high concordance with concentrations measured by Colilert once proportional bias was accounted for. In silico specificity testing underlined the possibility to further detect and distinguish Escherichia cryptic clade VI. Finally, the applicability of the ddPCR was successfully tested on environmental water samples where E. marmotae and E. ruysiae potentially confound E. coli counts based on the Most Probable Number method, highlighting the utility of this novel ddPCR as an efficient and rapid discriminatory test to improve water quality assessments.

Draft genome sequences of Escherichia spp. isolates from New Zealand environmental sources.

Escherichia coli is often used as a fecal indicator bacterium for water quality monitoring. We report the draft genome sequences of 500 Escherichia isolates including newly described Escherichia species, namely Escherichia marmotae, Escherichia ruysiae, and Escherichia whittamii, obtained from diverse environmental sources to assist with improved public health risk assessments.

High-resolution genomic analysis to investigate the impact of the invasive brushtail possum (Trichosurus vulpecula) and other wildlife on microbial water quality assessments.

Escherichia coli are routine indicators of fecal contamination in water quality assessments. Contrary to livestock and human activities, brushtail possums (Trichosurus vulpecula), common invasive marsupials in Aotearoa/New Zealand, have not been thoroughly studied as a source of fecal contamination in freshwater. To investigate their potential role, Escherichia spp. isolates (n = 420) were recovered from possum gut contents and feces and were compared to those from water, soil, sediment, and periphyton samples, and from birds and other introduced mammals collected within the Makirikiri Reserve, Dannevirke. Isolates were characterized using E. coli-specific real-time PCR targeting the uidA gene, Sanger sequencing of a partial gnd PCR product to generate a gnd sequence type (gST), and for 101 isolates, whole genome sequencing. Escherichia populations from 106 animal and environmental sample enrichments were analyzed using gnd metabarcoding. The alpha diversity of Escherichia gSTs was significantly lower in possums and animals compared with aquatic environmental samples, and some gSTs were shared between sample types, e.g., gST535 (in 85% of samples) and gST258 (71%). Forty percent of isolates gnd-typed and 75% of reads obtained by metabarcoding had gSTs shared between possums, other animals, and the environment. Core-genome single nucleotide polymorphism (SNP) analysis showed limited variation between several animal and environmental isolates (<10 SNPs). Our data show at an unprecedented scale that Escherichia clones are shared between possums, other wildlife, water, and the wider environment. These findings support the potential role of possums as contributors to fecal contamination in Aotearoa/New Zealand freshwater. Our study deepens the current knowledge of Escherichia populations in under-sampled wildlife. It presents a successful application of high-resolution genomic methods for fecal source tracking, thereby broadening the analytical toolbox available to water quality managers. Phylogenetic analysis of isolates and profiling of Escherichia populations provided useful information on the source(s) of fecal contamination and suggest that comprehensive invasive species management strategies may assist in restoring not only ecosystem health but also water health where microbial water quality is compromised.

Impact of systemic antimicrobial therapy on the faecal microbiome in symptomatic dairy cows.

Antimicrobial resistance is a global threat to human and animal health, with the misuse and overuse of antimicrobials suggested as the main drivers of resistance. Antimicrobial therapy can alter the bacterial community composition and the faecal resistome in cattle. Little is known about the impact of systemic antimicrobial therapy on the faecal microbiome in dairy cows in the presence of disease. Therefore, this study aimed to assess the impact of systemic antimicrobial therapy on the faecal microbiome in dairy cows in the pastoral farm environment, by analysing faecal samples from cattle impacted by several different clinically-defined conditions and corresponding antimicrobial treatments. Analysis at the individual animal level showed a decrease in bacterial diversity and richness during antimicrobial treatment but, in many cases, the microbiome diversity recovered post-treatment when the cow re-entered the milking herd. Perturbations in the microbiome composition and the ability of the microbiome to recover were specific at the individual animal level, highlighting that the animal is the main driver of variation. Other factors such as disease severity, the type and duration of antimicrobial treatment and changes in environmental factors may also impact the bovine faecal microbiome. AmpC-producing Escherichia coli were isolated from faeces collected during and post-treatment with ceftiofur from one cow while no third-generation cephalosporin resistant E. coli were isolated from the untreated cow samples. This isolation of genetically similar plasmid-mediated AmpC-producing E. coli has implications for the development and dissemination of antibiotic resistant bacteria and supports the reduction in the use of critically important antimicrobials.

Pangenome graphs in infectious disease: a comprehensive genetic variation analysis of Neisseria meningitidis leveraging Oxford Nanopore long reads.

Whole genome sequencing has revolutionized infectious disease surveillance for tracking and monitoring the spread and evolution of pathogens. However, using a linear reference genome for genomic analyses may introduce biases, especially when studies are conducted on highly variable bacterial genomes of the same species. Pangenome graphs provide an efficient model for representing and analyzing multiple genomes and their variants as a graph structure that includes all types of variations. In this study, we present a practical bioinformatics pipeline that employs the PanGenome Graph Builder and the Variation Graph toolkit to build pangenomes from assembled genomes, align whole genome sequencing data and call variants against a graph reference. The pangenome graph enables the identification of structural variants, rearrangements, and small variants (e.g., single nucleotide polymorphisms and insertions/deletions) simultaneously. We demonstrate that using a pangenome graph, instead of a single linear reference genome, improves mapping rates and variant calling for both simulated and real datasets of the pathogen Neisseria meningitidis. Overall, pangenome graphs offer a promising approach for comparative genomics and comprehensive genetic variation analysis in infectious disease. Moreover, this innovative pipeline, leveraging pangenome graphs, can bridge variant analysis, genome assembly, population genetics, and evolutionary biology, expanding the reach of genomic understanding and applications.

Species-wide genomics of kakapo provides tools to accelerate recovery.

The kakapo is a critically endangered, intensively managed, long-lived nocturnal parrot endemic to Aotearoa New Zealand. We generated and analysed whole-genome sequence data for nearly all individuals living in early 2018 (169 individuals) to generate a high-quality species-wide genetic variant callset. We leverage extensive long-term metadata to quantify genome-wide diversity of the species over time and present new approaches using probabilistic programming, combined with a phenotype dataset spanning five decades, to disentangle phenotypic variance into environmental and genetic effects while quantifying uncertainty in small populations. We find associations for growth, disease susceptibility, clutch size and egg fertility within genic regions previously shown to influence these traits in other species. Finally, we generate breeding values to predict phenotype and illustrate that active management over the past 45 years has maintained both genome-wide diversity and diversity in breeding values and, hence, evolutionary potential. We provide new pathways for informing future conservation management decisions for kakapo, including prioritizing individuals for translocation and monitoring individuals with poor growth or high disease risk. Overall, by explicitly addressing the challenge of the small sample size, we provide a template for the inclusion of genomic data that will be transformational for species recovery efforts around the globe.

A novel, stain-free, natural auto-fluorescent signal, Sig M, identified from cytometric and transcriptomic analysis of infectivity of Cryptosporidium hominis and Cryptosporidium parvum.

Cryptosporidiosis is a worldwide diarrheal disease caused by the protozoan Cryptosporidium. The primary symptom is diarrhea, but patients may exhibit different symptoms based on the species of the Cryptosporidium parasite they are infected with. Furthermore, some genotypes within species are more transmissible and apparently virulent than others. The mechanisms underpinning these differences are not understood, and an effective in vitro system for Cryptosporidium culture would help advance our understanding of these differences. Using COLO-680N cells, we employed flow cytometry and microscopy along with the C. parvum-specific antibody Sporo-Glo™ to characterize infected cells 48 h following an infection with C. parvum or C. hominis. The Cryptosporidium parvum-infected cells showed higher levels of signal using Sporo-Glo™ than C. hominis-infected cells, which was likely because Sporo-Glo™ was generated against C. parvum. We found a subset of cells from infected cultures that expressed a novel, dose-dependent auto-fluorescent signal that was detectable across a range of wavelengths. The population of cells that expressed this signal increased proportionately to the multiplicity of infection. The spectral cytometry results confirmed that the signature of this subset of host cells closely matched that of oocysts present in the infectious ecosystem, pointing to a parasitic origin. Present in both C. parvum and C. hominis cultures, we named this Sig M, and due to its distinct profile in cells from both infections, it could be a better marker for assessing Cryptosporidium infection in COLO-680N cells than Sporo-Glo™. We also noted Sig M's impact on Sporo-Glo™ detection as Sporo-Glo™ uses fluoroscein-isothiocynate, which is detected where Sig M also fluoresces. Lastly, we used NanoString nCounter® analysis to investigate the transcriptomic landscape for the two Cryptosporidium species, assessing the gene expression of 144 host and parasite genes. Despite the host gene expression being at high levels, the levels of putative intracellular Cryptosporidium gene expression were low, with no significant difference from controls, which could be, in part, explained by the abundance of uninfected cells present as determined by both Sporo-Glo™ and Sig M analyses. This study shows for the first time that a natural auto-fluorescent signal, Sig M, linked to Cryptosporidium infection can be detected in infected host cells without any fluorescent labeling strategies and that the COLO-680N cell line and spectral cytometry could be useful tools to advance the understanding of Cryptosporidium infectivity.

Investigating the genetic components of tuber bruising in a breeding population of tetraploid potatoes.

BACKGROUND: Tuber bruising in tetraploid potatoes (Solanum tuberosum) is a trait of economic importance, as it affects tubers' fitness for sale. Understanding the genetic components affecting tuber bruising is a key step in developing potato lines with increased resistance to bruising. As the tetraploid setting renders genetic analyses more complex, there is still much to learn about this complex phenotype. Here, we used capture sequencing data on a panel of half-sibling populations from a breeding programme to perform a genome-wide association analysis (GWAS) for tuber bruising. In addition, we collected transcriptomic data to enrich the GWAS results. However, there is currently no satisfactory method to represent both GWAS and transcriptomics analysis results in a single visualisation and to compare them with existing knowledge about the biological system under study. RESULTS: When investigating population structure, we found that the STRUCTURE algorithm yielded greater insights than discriminant analysis of principal components (DAPC). Importantly, we found that markers with the highest (though non-significant) association scores were consistent with previous findings on tuber bruising. In addition, new genomic regions were found to be associated with tuber bruising. The GWAS results were backed by the transcriptomics differential expression analysis. The differential expression notably highlighted for the first time the role of two genes involved in cellular strength and mechanical force sensing in tuber resistance to bruising. We proposed a new visualisation, the HIDECAN plot, to integrate the results from the genomics and transcriptomics analyses, along with previous knowledge about genomic regions and candidate genes associated with the trait. CONCLUSION: This study offers a unique genome-wide exploration of the genetic components of tuber bruising. The role of genetic components affecting cellular strength and resistance to physical force, as well as mechanosensing mechanisms, was highlighted for the first time in the context of tuber bruising. We showcase the usefulness of genomic data from breeding programmes in identifying genomic regions whose association with the trait of interest merit further investigation. We demonstrate how confidence in these discoveries and their biological relevance can be increased by integrating results from transcriptomics analyses. The newly proposed visualisation provides a clear framework to summarise of both genomics and transcriptomics analyses, and places them in the context of previous knowledge on the trait of interest.

Aristaeella hokkaidonensis gen. nov. sp. nov. and Aristaeella lactis sp. nov., two rumen bacterial species of a novel proposed family, Aristaeellaceae fam. nov.

Two strains of Gram-negative, anaerobic, rod-shaped bacteria, from an abundant but uncharacterized rumen bacterial group of the order 'Christensenellales', were phylogenetically and phenotypically characterized. These strains, designated R-7T and WTE2008T, shared 98.6-99.0 % sequence identity between their 16S rRNA gene sequences. R-7T and WTE2008T clustered together on a distinct branch from other Christensenellaceae strains and had <88.1 % sequence identity to the closest type-strain sequence from Luoshenia tenuis NSJ-44T. The genome sequences of R-7T and WTE2008T had 83.6 % average nucleotide identity to each other, and taxonomic assignment using the Genome Taxonomy Database indicates these are separate species within a novel family of the order 'Christensenellales'. Cells of R-7T and WTE2008T lacked any obvious appendages and their cell wall ultra-structures were characteristic of Gram-negative bacteria. The five most abundant cellular fatty acids of both strains were C16 : 0, C16 : 0 iso, C17 : 0 anteiso, C18 : 0 and C15 : 0 anteiso. The strains used a wide range of the 23 soluble carbon sources tested, and grew best on cellobiose, but not on sugar-alcohols. Xylan and pectin were fermented by both strains, but not cellulose. Acetate, hydrogen, ethanol and lactate were the major fermentation end products. R-7T produced considerably more hydrogen than WTE2008T, which produced more lactate. Based on these analyses, Aristaeellaceae fam. nov. and Aristaeella gen. nov., with type species Aristaeella hokkaidonensis sp. nov., are proposed. Strains R-7T (=DSM 112795T=JCM 34733T) and WTE2008T (=DSM 112788T=JCM 34734T) are the proposed type strains for Aristaeella hokkaidonensis sp. nov. and Aristaeella lactis sp. nov., respectively.

Abundant dsRNA picobirnaviruses show little geographic or host association in terrestrial systems.

Picobirnaviruses are double-stranded RNA viruses known from a wide range of host species and locations but with unknown pathogenicity and host relationships. Here, we examined the diversity of picobirnaviruses from cattle and gorillas within and around Bwindi Impenetrable Forest National Park (BIFNP), Uganda, where wild and domesticated animals and humans live in relatively close contact. We use metagenomic sequencing with bioinformatic analyses to examine genetic diversity. We compared our findings to global Picobirnavirus diversity using clustering-based analyses. Picobirnavirus diversity at Bwindi was high, with 14 near-complete RdRp and 15 capsid protein sequences, and 497 new partial viral sequences recovered from 44 gorilla samples and 664 from 16 cattle samples. Sequences were distributed throughout a phylogenetic tree of globally derived picobirnaviruses. The relationship with Picobirnavirus diversity and host taxonomy follows a similar pattern to the global dataset, generally lacking pattern with either host or geography.

Co-Selection of Bacterial Metal and Antibiotic Resistance in Soil Laboratory Microcosms.

Accumulation of heavy metals (HMs) in agricultural soil following the application of superphosphate fertilisers seems to induce resistance of soil bacteria to HMs and appears to co-select for resistance to antibiotics (Ab). This study aimed to investigate the selection of co-resistance of soil bacteria to HMs and Ab in uncontaminated soil incubated for 6 weeks at 25 °C in laboratory microcosms spiked with ranges of concentrations of cadmium (Cd), zinc (Zn) and mercury (Hg). Co-selection of HM and Ab resistance was assessed using plate culture on media with a range of HM and Ab concentrations, and pollution-induced community tolerance (PICT) assays. Bacterial diversity was profiled via terminal restriction fragment length polymorphism (TRFLP) assay and 16S rDNA sequencing of genomic DNA isolated from selected microcosms. Based on sequence data, the microbial communities exposed to HMs were found to differ significantly compared to control microcosms with no added HM across a range of taxonomic levels.

Genomic epidemiology of bovine mastitis-causing Staphylococcus aureus in New Zealand.

We analysed the genomes of 188 bovine-mastitis-causing S. aureus isolates obtained over a 17-year period from more than 65 dairy farms across New Zealand. The analysis revealed a unique pattern of dominance over the entire period of study, of clonal complex 1, sequence type 1 (CC1/ST1), which accounted for ∼75% of the isolates. CC1/ST1 was also the commonest lineage infecting humans in New Zealand in the same period, but most bovine CC1/ST1 analysed in this study carried the genes coding for the bovine-adaptive bicomponent leucocidin lukF and lukM and lacked the corresponding human-adaptive lukF-PV and lukS-PV genes. Typical ruminant-associated lineages, such as ST97, ST151 and CC133 were also observed. Cluster analyses of the core and accessory genomes revealed genomic segregations according to the CCs, but lack of segregation based on the geographical location or collection year, suggesting a stable population in space and time. To our knowledge, this is the first identification of genomic markers of host adaptation to cattle in S. aureus CC1/ST1, a lineage commonly associated with humans, worldwide. The temporal clonal stability observed would enable the development of a S. aureus vaccine for New Zealand cattle, which is unlikely to undergo substantial reduction of efficacy due to clonal drifts or shifts.

Urinary MicroRNA Analysis Indicates an Epigenetic Regulation of Chronic Kidney Disease of Unknown Etiology in Sri Lanka.

BACKGROUND: Chronic kidney disease of unknown etiology (CKDu) is reported among male paddy farmers in the dry zone of Sri Lanka. The exact cause of this disease remains undetermined. Genetic susceptibility is identified as a major risk factor for CKDu Objectives: In this study, small urinary RNAs were characterized in CKDu patients, healthy endemic and non-endemic controls. Differently expressed urinary miRNAs and their associated pathways were identified in the study population. METHODS: Healthy and diseased male volunteers (n = 9) were recruited from Girandurukotte (endemic) and Mawanella (non-endemic) districts. Urinary small RNAs were purified and sequenced using Illumina MiSeqTM. The sequence trace files were assembled and analyzed. Differentially ex-pressed miRNAs among these three groups were identified and pathway analysis was conducted. RESULTS: The urine samples contained 130,623 sequence reads identified as non-coding RNAs, PIWI-interacting RNAs (piRNA), and miRNAs. Approximately four percent of the total small RNA reads represented miRNA, and 29% represented piRNA. A total of 409 miRNA species were ex-pressed in urine. Interestingly, both diseased and endemic controls population showed significantly low expression of miRNA and piRNA. Regardless of the health status, the endemic population ex-pressed significantly low levels of miR-10a, miR-21, miR-148a, and miR-30a which have been linked with several environmental toxins Conclusion: Significant downregulation of miRNA and piRNA expression in both diseased and healthy endemic samples indicates an epigenetic regulation of CKDu involving genetic and environmental interaction. Further studies of specific miRNA species are required to develop a miRNA panel to identify individuals susceptible to CKDu.

Molecular characterisation and additional morphological descriptions of Eimeria spp. (Apicomplexa: Eimeriidae) from brown kiwi (Apteryx mantelli Bartlett).

Brown kiwi (Apteryx mantelli Bartlett), a ratite endemic to New Zealand, is currently listed as "Vulnerable" under the IUCN classification system due to predation by introduced mammals. Operation Nest Egg (ONE) raises chicks and juveniles in predator-proof enclosures until they are large enough to defend themselves. These facilities experience an environmental accumulation of coccidial oocysts, which leads to severe morbidity and mortality of these kiwi. Four species of coccidia have been morphologically described from sporulated oocysts with additional opportunistic descriptions of endogenous stages. This research continues the morphological descriptions of these species of Eimeria with an additional novel morphotype also morphologically described. It also provides the first genetic characterisation targeting the mitochondrial cytochrome c oxidase I (COI) gene. Based on these findings, it was determined there are at least five morphotypes of Eimeria that infect brown kiwi and co-infections are common at the ONE facilities surveyed. The COI amplicon targeted for this study was sufficient to provide differentiation from other members of this genus. Sanger sequencing yielded ambiguous bases, indicating the need for more in-depth sequencing.

Insect Freeze-Tolerance Downunder: The Microbial Connection.

Insects that are freeze-tolerant start freezing at high sub-zero temperatures and produce small ice crystals. They do this using ice-nucleating agents that facilitate intercellular ice growth and prevent formation of large crystals where they can damage tissues. In Aotearoa/New Zealand the majority of cold adapted invertebrates studied survive freezing at any time of year, with ice formation beginning in the rich microbiome of the gut. Some freeze-tolerant insects are known to host symbiotic bacteria and/or fungi that produce ice-nucleating agents and we speculate that gut microbes of many New Zealand insects may provide ice-nucleating active compounds that moderate freezing. We consider too the possibility that evolutionary disparate freeze-tolerant insect species share gut microbes that are a source of ice-nucleating agents and so we describe potential transmission pathways of shared gut fauna. Despite more than 30 years of research into the freeze-tolerant mechanisms of Southern Hemisphere insects, the role of exogenous ice-nucleating agents has been neglected. Key traits of three New Zealand freeze-tolerant lineages are considered in light of the supercooling point (temperature of ice crystal formation) of microbial ice-nucleating particles, the initiation site of freezing, and the implications for invertebrate parasites. We outline approaches that could be used to investigate potential sources of ice-nucleating agents in freeze-tolerant insects and the tools employed to study insect microbiomes.

Extended-spectrum ß-lactamase- and AmpC ß-lactamase-producing Enterobacterales associated with urinary tract infections in the New Zealand community: a case-control study.

OBJECTIVES: To assess whether having a pet in the home is a risk factor for community-acquired urinary tract infections associated with extended-spectrum ß-lactamase (ESBL)- or AmpC ß-lactamase (ACBL)- producing Enterobacterales. METHODS: An unmatched case-control study was conducted between August 2015 and September 2017. Cases (n = 141) were people with community-acquired urinary tract infection (UTI) caused by ESBL- or ACBL-producing Enterobacterales. Controls (n = 525) were recruited from the community. A telephone questionnaire on pet ownership and other factors was administered, and associations were assessed using logistic regression. RESULTS: Pet ownership was not associated with ESBL- or ACBL-producing Enterobacterales-related human UTIs. A positive association was observed for recent antimicrobial treatment, travel to Asia in the previous year, and a doctor's visit in the last 6 months. Among isolates with an ESBL-/ACBL-producing phenotype, 126/134 (94%) were Escherichia coli, with sequence type 131 being the most common (47/126). CONCLUSIONS: Companion animals in the home were not found to be associated with ESBL- or ACBL-producing Enterobacterales-related community-acquired UTIs in New Zealand. Risk factors included overseas travel, recent antibiotic use, and doctor visits.

The impact of primary and secondary processing steps on Campylobacter concentrations on chicken carcasses and portions.

Campylobacteriosis is the most commonly notified foodborne disease in New Zealand and poultry meat is the major source for human infection. Carcasses and portions were sampled from key points along primary and secondary processing chains of three New Zealand poultry processors to determine the impact of processing steps on Campylobacter concentrations. Primary processing reduced Campylobacter concentrations on carcasses by almost 6-log; the biggest reduction was achieved by the spinchill, followed by the scald step. Significant plant differences in the degree of Campylobacter reduction were also observed at these steps. The spinchill and final acidified sodium chlorite wash resulted in carcasses with low-to-no levels of Campylobacter regardless of concentrations at prior steps. A similar study was conducted at primary processing for one plant in 2013; significant improvements in Campylobacter mitigation since 2013 were noted. Campylobacter concentrations from final product from secondary processing were higher than concentrations at the end of primary processing. Drumsticks had lower Campylobacter concentrations than other portion types. Skin removal from product did not consistently result in product with lower Campylobacter concentrations. Results identify key areas to target for further reduction of Campylobacter on poultry meat, and provide a benchmark to compare the efficacy of future interventions.

Identification of a novel polyomavirus from a marsupial host.

We report the identification and analysis of a full sequence of a novel polyomavirus from a brushtail possum (Trichosurus vulpecula) termed possum polyomavirus (PPyV). The sequence was obtained from the next-generation sequencing assembly during an investigation into the aetiological agent for a neurological disease of possums termed wobbly possum disease (WPD), but the virus was not aetiologically involved in WPD. The PPyV genome was 5,224 nt long with the organisation typical for polyomaviruses, including early (large and small T antigens) and late (Viral Protein 1 (VP1), VP2, and VP3) coding regions separated by the non-coding control region of 465 nt. PPyV clustered with betapolyomaviruses in the WUKI clade but showed less than 60 per cent identity to any of the members of this clade. We propose that PPyV is classified within a new species in the genus Betapolyomavirus. These data add to our limited knowledge of marsupial viruses and their evolution.

Mass Spectrometry-Based Lipidomics of Brown Adipose Tissue and Plasma of New-Born Lambs Subjected to Short-Term Cold Exposure.

During cold exposure, brown adipose tissue (BAT) holds the key mechanism in the generation of heat, thus inducing thermogenic adaptation in response to cooler environmental changes. This process can lead to a major lipidome remodelling in BAT, where the increase in abundance of many lipid classes plays a significant role in the thermogenic mechanisms for heat production. This study aimed to identify different types of lipids, through liquid chromatography-mass spectrometry (LC-MS), in BAT and plasma during a short-term cold challenge (2-days), or not, in new-born lambs. Fifteen new-born Romney lambs were selected randomly and divided into three groups: Group 1 (n = 3) with BAT and plasma obtained within 24 h after birth, as a control; Group 2 (n = 6) kept indoors for two days at an ambient temperature (20-22 °C) and Group 3 (n = 6) kept indoors for two days at a cold temperature (4 °C). Significant differences in lipid composition of many lipid categories (such as glycerolipids, glycerophospholipids, sphingolipids and sterol lipids) were observed in BAT and plasma under cold conditions, compared with ambient conditions. Data obtained from the present study suggest that short-term cold exposure induces profound changes in BAT and plasma lipidome composition of new-born lambs, which may enhance lipid metabolism via BAT thermogenic activation and adipocyte survival during cold adaptation. Further analysis on the roles of these lipid changes, validation of potential biomarkers for BAT activity, such as LPC 18:1 and PC 35:6, should contribute to the improvement of new-born lamb survival. Collectively, these observations help broaden the knowledge on the variations of lipid composition during cold exposure.

RNAseq Analysis of Brown Adipose Tissue and Thyroid of Newborn Lambs Subjected to Short-Term Cold Exposure Reveals Signs of Early Whitening of Adipose Tissue.

During the early postnatal period, lambs have the ability to thermoregulate body temperature via non-shivering thermogenesis through brown adipose tissue (BAT), which soon after birth begins to transform into white adipose tissue. An RNA seq approach was used to characterize the transcriptome of BAT and thyroid tissue in newborn lambs exposed to cold conditions. Fifteen newborn Romney lambs were selected and divided into three groups: group 1 (n = 3) was a control, and groups 2 and 3 (n = 6 each) were kept indoors for two days at an ambient temperature (20-22 °C) or at a cold temperature (4 °C), respectively. Sequencing was performed using a paired-end strategy through the BGISEQ-500 platform, followed by the identification of differentially expressed genes using DESeq2 and an enrichment analysis by g:Profiler. This study provides an in-depth expression network of the main characters involved in the thermogenesis and fat-whitening mechanisms that take place in the newborn lamb. Data revealed no significant differential expression of key thermogenic factors such as uncoupling protein 1, suggesting that the heat production peak under cold exposure might occur so rapidly and in such an immediate way that it may seem undetectable in BAT by day three of life. Moreover, these changes in expression might indicate the start of the whitening process of the adipose tissue, concluding the non-shivering thermogenesis period.