Gut transcriptome reveals differential gene expression and enriched pathways linked to immune activation in response to weaning in pigs.

Weaning represents one of the most critical periods in pig production associated with increase in disease risk, reduction in performance and economic loss. Physiological changes faced by piglets during the weaning period have been well characterised, however little is currently known about the underlying molecular pathways involved in these processes. As pig meat remains one of the most consumed sources of protein worldwide, understanding how these changes are mediated is critical to improve pig production and consequently sustainable food production globally. In this study, we evaluated the effect of weaning on transcriptomic changes in the colon of healthy piglets over time using an RNA-sequencing approach. The findings revealed a complex and coordinated response to weaning with the majority of genes found to be rapidly differentially expressed within 1 day post weaning. Multiple genes and pathways affected by weaning in the colon were associated with immune regulation, cell signalling and bacterial defence. NOD-like receptors, Toll-like receptor and JAK-STAT signalling pathways were amongst the pathways significantly enriched. Immune activation was evidenced by the enrichment of pathways involved in interferon response, cytokines interactions, oxidoreductase activities and response to microbial invasion. Biosynthesis of amino acids, in particular arginine, was also amongst the most enriched KEGG pathways in weaned pigs, reinforcing the critical role of arginine in gut homeostasis under stress conditions. Overall, transcriptomic and physiological results suggest that pigs going through the weaning transition undergo a transient period of inflammatory state with a temporary breakdown of barrier functions in the gut. These findings could provide valuable tools to monitor host response post weaning, and may be of particular relevance for the investigation and development of intervention strategies aimed to reduce antibiotic use and improve pig health and performance.

Differential and defective transcription of koala retrovirus indicates the complexity of host and virus evolution.

Koala retrovirus (KoRV) is unique amongst endogenous (inherited) retroviruses in that its incorporation to the host genome is still active, providing an opportunity to study what drives this fundamental process in vertebrate genome evolution. Animals in the southern part of the natural range of koalas were previously thought to be either virus-free or to have only exogenous variants of KoRV with low rates of KoRV-induced disease. In contrast, animals in the northern part of their range universally have both endogenous and exogenous KoRV with very high rates of KoRV-induced disease such as lymphoma. In this study we use a combination of sequencing technologies, Illumina RNA sequencing of 'southern' (south Australian) and 'northern' (SE QLD) koalas and CRISPR enrichment and nanopore sequencing of DNA of 'southern' (South Australian and Victorian animals) to retrieve full-length loci and intregration sites of KoRV variants. We demonstrate that koalas that tested negative to the KoRV pol gene qPCR, used to detect replication-competent KoRV, are not in fact KoRV-free but harbour defective, presumably endogenous, 'RecKoRV' variants that are not fixed between animals. This indicates that these populations have historically been exposed to KoRV and raises questions as to whether these variants have arisen by chance or whether they provide a protective effect from the infectious forms of KoRV. This latter explanation would offer the intriguing prospect of being able to monitor and selectively breed for disease resistance to protect the wild koala population from KoRV-induced disease.

Pathological Findings in Koala Retrovirus-positive Koalas (Phascolarctos cinereus) from Northern and Southern Australia.

Koala retrovirus (KoRV) infection shows differences in prevalence and load between northern and southern Australian koala populations; however, the effect of this on diseases such as lymphoma and chlamydial disease is unclear. This study compared clinicopathological findings, haematology and splenic lymphoid area of KoRV-positive koalas from northern (Queensland [Qld], n = 67) and southern (South Australia [SA], n = 92) populations in order to provide further insight into KoRV pathogenesis. Blood was collected for routine haematology and for measurement of KoRV proviral load by quantitative polymerase chain reaction (qPCR). Plasma samples were assessed for KoRV viral load by reverse transcriptase qPCR and conjunctival and cloacal swabs were collected for measurement of the load of Chlamydia pecorum (qPCR). During necropsy examination, spleen was collected for lymphoid area analysis. Lymphoma was morphologically similar between the populations and occurred in koalas with the highest KoRV proviral and viral loads. Severe ocular chlamydial disease was observed in both populations, but urinary tract disease was more severe in Qld, despite similar C. pecorum loads. No associations between KoRV and chlamydial disease severity or load were observed, except in SA where viral load correlated positively with chlamydial disease severity. In both populations, proviral and viral loads correlated positively with lymphocyte and metarubricyte counts and correlated negatively with erythrocyte and neutrophil counts. Splenic lymphoid area was correlated positively with viral load. This study has shown further evidence for KoRV-induced oncogenesis and highlighted that lymphocytes and splenic lymphoid tissue may be key sites for KoRV replication. However, KoRV infection appears to be highly complex and continued investigation is required to fully understand its pathogenesis.

Identification of stable reference genes for quantitative PCR in koalas.

To better understand host and immune response to diseases, gene expression studies require identification of reference genes with stable expression for accurate normalisation. This study describes the identification and testing of reference genes with stable expression profiles in koala lymph node tissues across two genetically distinct koala populations. From the 25 most stable genes identified in transcriptome analysis, 11 genes were selected for verification using reverse transcription quantitative PCR, in addition to the commonly used ACTB and GAPDH genes. The expression data were analysed using stable genes statistical software - geNorm, BestKeeper, NormFinder, the comparative ΔCt method and RefFinder. All 13 genes showed relative stability in expression in koala lymph node tissues, however Tmem97 and Hmg20a were identified as the most stable genes across the two koala populations.

Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M) and serine biosynthetic pathway genes are co-ordinately increased during anabolic agent-induced skeletal muscle growth.

We aimed to identify novel molecular mechanisms for muscle growth during administration of anabolic agents. Growing pigs (Duroc/(Landrace/Large-White)) were administered Ractopamine (a beta-adrenergic agonist; BA; 20 ppm in feed) or Reporcin (recombinant growth hormone; GH; 10 mg/48 hours injected) and compared to a control cohort (feed only; no injections) over a 27-day time course (1, 3, 7, 13 or 27-days). Longissimus Dorsi muscle gene expression was analyzed using Agilent porcine transcriptome microarrays and clusters of genes displaying similar expression profiles were identified using a modified maSigPro clustering algorithm. Anabolic agents increased carcass (p = 0.002) and muscle weights (Vastus Lateralis: p < 0.001; Semitendinosus: p = 0.075). Skeletal muscle mRNA expression of serine/one-carbon/glycine biosynthesis pathway genes (Phgdh, Psat1 and Psph) and the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase-M (Pck2/PEPCK-M), increased during treatment with BA, and to a lesser extent GH (p < 0.001, treatment x time interaction). Treatment with BA, but not GH, caused a 2-fold increase in phosphoglycerate dehydrogenase (PHGDH) protein expression at days 3 (p < 0.05) and 7 (p < 0.01), and a 2-fold increase in PEPCK-M protein expression at day 7 (p < 0.01). BA treated pigs exhibit a profound increase in expression of PHGDH and PEPCK-M in skeletal muscle, implicating a role for biosynthetic metabolic pathways in muscle growth.

Molecular Epidemiology of Streptococcus uberis Clinical Mastitis in Dairy Herds: Strain Heterogeneity and Transmission.

Multilocus sequence typing was successfully completed on 494 isolates of Streptococcus uberis from clinical mastitis cases in a study of 52 commercial dairy herds over a 12-month period. In total, 195 sequence types (STs) were identified. S. uberis mastitis cases that occurred in different cows within the same herd and were attributed to a common ST were classified as potential transmission events (PTEs). Clinical cases attributed to 35 of the 195 STs identified in this study were classified PTE. PTEs were identified in 63% of the herds. PTE-associated cases, which include the first recorded occurrence of that ST in that herd (index case) and all persistent infections with that PTE ST, represented 40% of all the clinical mastitis cases and occurred in 63% of the herds. PTE-associated cases accounted for >50% of all S. uberis clinical mastitis cases in 33% of the herds. Nine STs (ST-5, -6, -20, -22, -24, -35, -233, -361, and -512), eight of which were grouped within a clonal complex (sharing at least four alleles), were statistically overrepresented (OVR STs). The findings indicate that 38% of all clinical mastitis cases and 63% of the PTEs attributed to S. uberis in dairy herds may be caused by the nine most prevalent strains. The findings suggest that a small subset of STs is disproportionally important in the epidemiology of S. uberis mastitis in the United Kingdom, with cow-to-cow transmission of S. uberis potentially occurring in the majority of herds in the United Kingdom, and may be the most important route of infection in many herds.

Novel ovine polymorphisms and adaptive evolution in mammalian TLR2 suggest existence of multiple pathogen binding regions.

Toll-like receptors initiate inflammatory responses following the recognition of a wide repertoire of pathogens including bacteria, fungi, protozoa and viruses. They are composed of an extracellular leucine-rich repeat domain responsible for detecting pathogen-associated molecular patterns, a membrane spanning region and an intracellular Toll/Interleukin 1 receptor domain which invokes signal transduction. Toll-like receptor 2 is the most diverse of these receptors as it recognises infectious agents from a range of pathogenic groups. Over 1400 breeds of sheep exist worldwide that inhabit a diverse range of environments, which leads to the potential contact with a wide variety of pathogens likely detected by Toll-like receptor 2. In this study, we evaluated the extent of both long term evolutionary changes, across the mammalian phylogeny of the TLR2 gene, and recent divergence of this same gene in sheep breeds. Evolutionary analyses identified positive selective pressure across the mammalian phylogeny, and differential selection pressure within the artiodactyl and primate lineage. Finally, we identified localised positively-selected sites within two regions of the extracellular domain which suggest that multiple binding regions in TLR2 may be involved in pathogen detection. These results are consistent with the hypothesis that competition between host and pathogen is driving adaptation of Toll-like receptor 2 genes.

Adaptation to the chicken intestine in Salmonella Enteritidis PT4 studied by transcriptional analysis.

The transcriptional changes that occurred in Salmonella enterica serovar Enteritidis during colonization of the alimentary tract of newly hatched chickens were studied. A whole genome oligonucleotide microarray was used to compare the expression pattern with that from bacteria cultured in nutrient broth in vitro. Amongst other changes Salmonella Pathogenicity Island (SPI)-1, SPI-2 and SPI-5 genes were up-regulated in vivo suggesting a close association with the mucosa during colonization. Particular attention was paid to genes associated with metabolism of dicarboxylic acids and to responses to high osmolarity. Association between the colonization phenotype and gene mutations indicated that the latter was more important as a contribution to the colonization phenotype.

Hirudo medicinalis: a platform for investigating genes in neural repair.

We have used the nervous system of the medicinal leech as a preparation to study the molecular basis of neural repair. The leech central nervous system, unlike mammalian CNS, can regenerate to restore function, and contains identified nerve cells of known function and connectivity. We have constructed subtractive cDNA probes from whole and regenerating ganglia of the ventral nerve cord and have used these to screen a serotonergic Retzius neuron library. This identifies genes that are regulated as a result of axotomy, and are expressed by the Retzius cell. This approach identifies many genes, both novel and known. Many of the known genes identified have homologues in vertebrates, including man. For example, genes encoding thioredoxin (TRX), Rough Endoplasmic Reticulum Protein 1 (RER-1) and ATP synthase are upregulated at 24 h postinjury in leech nerve cord. To investigate the functional role of regulated genes in neuron regrowth we are using microinjection of antisense oligonucleotides in combination with horseradish peroxidase to knock down expression of a chosen gene and to assess regeneration in single neurons in 3-D ganglion culture. As an example of this approach we describe experiments to microinject antisense oligonucleotide to a leech isoform of the structural protein, Protein 4.1. Our approach thus identifies genes regulated at different times after injury that may underpin the intrinsic ability of leech neurons to survive damage, to initiate regrowth programs and to remake functional connections. It enables us to determine the time course of gene expression in the regenerating nerve cord, and to study the effects of gene knockdown in identified neurons regenerating in defined conditions in culture.

Identifying genes for neuron survival and axon outgrowth in Hirudo medicinalis.

We have studied the molecular basis of nervous system repair in invertebrate (Hirudo medicinalis) nerve cells. Unlike in mammals, neurons in invertebrates survive injury and regrow processes to restore the connections that they held before the damage occurred. To identify genes whose expression is regulated after injury, we have used subtractive probes, constructed from regenerating and non-regenerating ganglia from the leech Hirudo medicinalis, to screen cDNA libraries made from whole leech CNS or from identified microdissected neurons. We have identified genes of known or predicted function as well as novel genes. Known genes up-regulated within hours of injury and that are widely expressed in invertebrate and mammalian cells include thioredoxin and tubulin. Other known genes, e.g. Cysteine Rich Intestinal Protein (CRIP), have previously been identified in mammalian cells though not in regenerating adult neurons. Two regulated genes identified, myohemerythrin and the novel protein ReN3 are exclusively expressed in invertebrates. Thus our approach has enabled us to identify genes, present in a neuron of known function, that are up- and down-regulated within hours of axotomy, and that may underpin the intrinsic ability of invertebrate neurons to survive damage and initiate regrowth programmes.

HmCRIP, a cysteine-rich intestinal protein, is expressed by an identified regenerating nerve cell.

A Hirudo medicinalis cDNA isolated from regenerating CNS tissue at 24 h post-axotomy was identified as a leech homologue of the mammalian cysteine-rich intestinal proteins (CRIPs) and named HmCRIP. HmCRIP is up-regulated within 6 h of axotomy, peaking at 24 h. This is the first demonstration of a CRIP homologue in regenerating CNS and in a serotonergic neurone. In rodents CRIP is an important factor in the regulation of the inflammatory immune response through control of Th1/Th2 differentiation. The role of HmCRIP in the regeneration competent environment of the annelid central nervous system is discussed.

A new sequence motif linking lissencephaly, Treacher Collins and oral-facial-digital type 1 syndromes, microtubule dynamics and cell migration.

A previously unidentified sequence motif has been identified in the products of genes mutated in Miller-Dieker lissencephaly, Treacher Collins, oral-facial-digital type 1 and contiguous syndrome ocular albinism with late onset sensorineural deafness syndromes. An additional homologous motif was detected in a gene product fused to the fibroblast growth factor receptor type 1 in patients with an atypical stem cell myeloproliferative disorder. In total, over 100 eukaryotic intracellular proteins are shown to possess a LIS1 homology (LisH) motif, including several katanin p60 subunits, muskelin, tonneau, LEUNIG, Nopp140, aimless and numerous WD repeat-containing beta-propeller proteins. It is suggested that LisH motifs contribute to the regulation of microtubule dynamics, either by mediating dimerization, or else by binding cytoplasmic dynein heavy chain or microtubules directly. The predicted secondary structure of LisH motifs, and their occurrence in homologues of Gbeta beta-propeller subunits, suggests that they are analogues of Ggamma subunits, and might associate with the periphery of beta-propeller domains. The finding of LisH motifs in both treacle and Nopp140 reinforces previous observations of functional similarities between these nucleolar proteins. Uncharacterized LisH motif-containing proteins represent candidates for other diseases associated with aberrant microtubule dynamics and defects of cell migration, nucleokinesis or chromosome segregation.