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For the first time we have defined naïve, central memory, effector memory and differentiated effector porcine CD8 T cells and analyzed their distribution in lymphoid and respiratory tissues after influenza infection or immunization, using peptide-MHC tetramers of three influenza nucleoprotein (NP) epitopes. The hierarchy of response to the three epitopes changes during the response in different tissues. Most NP-specific CD8 T cells in broncho-alveolar lavage (BAL) and lung are tissue resident memory cells (TRM) that express CD69 and downregulate CD45RA and CCR7. NP-specific cells isolated from BAL express genes characteristic of TRM, but gene expression differs at 7, 21 and 63 days post infection. In all tissues the frequency of NP-specific CD8 cells declines over 63 days almost to background levels but is best maintained in BAL. The kinetic of influenza specific memory CD8 T cell in this natural host species differs from that in small animal models.

Ovine pulmonary adenocarcinoma (OPA) is an infectious neoplastic lung disease of sheep caused by jaagsiekte sheep retrovirus. OPA is an important veterinary problem and is also a valuable large animal model for human lung adenocarcinoma. JSRV infects type 2 alveolar epithelial cells in the lung and induces the growth of tumors, but little is known about the molecular events that lead to the activation of oncogenic pathways in infected cells. MicroRNAs (miRNAs) are small RNA molecules of approximately 22 nucleotides with important roles in regulating gene expression in eukaryotes and with well-established roles in cancer. Here we used small-RNA sequencing to investigate the changes in miRNA expression that occur in JSRV-infected ovine lung. After filtering out low abundance miRNAs, we identified expression of 405 miRNAs, 32 of which were differentially expressed in JSRV-infected lung compared to mock-inoculated control lung. Highly upregulated miRNAs included miR-182, miR-183, miR-96 and miR-135b, which have also been associated with oncogenic changes in human lung cancer. Network analysis of genes potentially targeted by the deregulated miRNAs identified their involvement in pathways known to be dysregulated in OPA. We found no evidence to support the existence of miRNAs encoded by JSRV. This study provides the first information on miRNA expression in OPA and identifies a number of targets for future studies into the role of these molecules in the pathogenesis of this unique veterinary model for human lung adenocarcinoma.IMPORTANCEOvine pulmonary adenocarcinoma is a neoplastic lung disease of sheep caused by jaagsiekte sheep retrovirus (JSRV). OPA is a significant welfare and economic concern for sheep producers and is a valuable large animal model for human lung adenocarcinoma. MicroRNAs are small RNA molecules of approximately 22 nucleotides with important functions in regulating gene expression in eukaryotes and with well-established roles in cancer. In this study, we examined the changes in microRNA expression that occur in the lung in response to JSRV infection. We identified differential expression of a number of host-encoded microRNAs in infected tissue, including microRNAs with roles in human cancer. We found no evidence that JSRV encodes a microRNA. This study provides new insights on the cellular response to JSRV infection in the ovine lung, which will inform future studies into the pathogenesis of OPA in sheep and its use as a model for human lung adenocarcinoma.

We defined naïve, central memory, effector memory and terminally differentiated porcine CD8 T cells and analyzed their phenotype in lymphoid and respiratory tissues after influenza infection or immunization using peptide-MHC tetramers of three influenza nucleoprotein (NP) epitopes. The hierarchy of response to the three epitopes changes during the response in different tissues. Most NP-specific CD8 T cells in broncho-alveolar lavage (BAL) and lung are tissue resident memory cells (TRM), that express CD69 and have an effector memory or terminally differentiated phenotype. NP-specific cells isolated from BAL express genes characteristic of TRM, but gene expression differs at 7, 21 and 63 days post infection. The frequency of NP-specific cells declines over 63 days in all tissues but is best maintained in BAL. The pig is a powerful model for understanding how best to induce and harness local immunity to respiratory viruses.One sentence summaryInfluenza NP-specific porcine tissue resident memory CD8 T cells persist in the lung with major changes in gene expression.

East Coast fever (ECF) in cattle is caused by the Apicomplexan protozoan parasiteTheileria parva, transmitted by the three-host tickRhipicephalus appendiculatus. The African buffalo (Syncerus caffer) is the natural host forT. parvabut does not suffer disease, whereas ECF is often fatal in cattle. The genetic relationship betweenT. parvapopulations circulating in cattle and buffalo is poorly understood, and has not been studied in sympatric buffalo and cattle. This study aimed to determine the genetic diversity ofT. parvapopulations in cattle and buffalo, in an area where livestock co-exist with buffalo adjacent to the Serengeti National Park, Tanzania. ThreeT. parvaantigens (Tp1, Tp4, and Tp16), known to be recognized by CD8+and CD4+T cells in immunized cattle, were used to characterize genetic diversity ofT. parvain cattle (n= 126) and buffalo samples (n= 22). Long read (PacBio) sequencing was used to generate full or near-full length allelic sequences. Patterns of diversity were similar across all three antigens, with allelic diversity being significantly greater in buffalo-derived parasites compared to cattle-derived (e.g., for Tp1 median cattle allele count was 9, and 81.5 for buffalo), with very few alleles shared between species (8 of 651 alleles were shared for Tp1). Most alleles were unique to buffalo with a smaller proportion unique to cattle (412 buffalo unique vs. 231 cattle-unique for Tp1). There were indications of population substructuring, with one allelic cluster of Tp1 representing alleles found in both cattle and buffalo (including the TpM reference genome allele), and another containing predominantly only alleles deriving from buffalo. These data illustrate the complex interplay betweenT. parvapopulations in buffalo and cattle, revealing the significant genetic diversity in the buffaloT. parvapopulation, the limited sharing of parasite genotypes between the host species, and highlight that a subpopulation ofT. parvais maintained by transmission within cattle. The data indicate that fuller understanding of buffaloT. parvapopulation dynamics is needed, as only a comprehensive appreciation of the population genetics ofT. parvapopulations will enable assessment of buffalo-derived infection risk in cattle, and how this may impact upon control measures such as vaccination.

Animal African Trypanosomiasis (AAT) is a debilitating livestock disease prevalent across sub-Saharan Africa, a main cause of which is the protozoan parasite Trypanosoma congolense. In comparison to the well-studied T. brucei, there is a major paucity of knowledge regarding the biology of T. congolense. Here, we use a combination of omics technologies and novel genetic tools to characterise core metabolism in T. congolense mammalian-infective bloodstream-form parasites, and test whether metabolic differences compared to T. brucei impact upon sensitivity to metabolic inhibition. Like the bloodstream stage of T. brucei, glycolysis plays a major part in T. congolense energy metabolism. However, the rate of glucose uptake is significantly lower in bloodstream stage T. congolense, with cells remaining viable when cultured in concentrations as low as 2 mM. Instead of pyruvate, the primary glycolytic endpoints are succinate, malate and acetate. Transcriptomics analysis showed higher levels of transcripts associated with the mitochondrial pyruvate dehydrogenase complex, acetate generation, and the glycosomal succinate shunt in T. congolense, compared to T. brucei. Stable-isotope labelling of glucose enabled the comparison of carbon usage between T. brucei and T. congolense, highlighting differences in nucleotide and saturated fatty acid metabolism. To validate the metabolic similarities and differences, both species were treated with metabolic inhibitors, confirming that electron transport chain activity is not essential in T. congolense. However, the parasite exhibits increased sensitivity to inhibition of mitochondrial pyruvate import, compared to T. brucei. Strikingly, T. congolense exhibited significant resistance to inhibitors of fatty acid synthesis, including a 780-fold higher EC50 for the lipase and fatty acid synthase inhibitor Orlistat, compared to T. brucei. These data highlight that bloodstream form T. congolense diverges from T. brucei in key areas of metabolism, with several features that are intermediate between bloodstream- and insect-stage T. brucei. These results have implications for drug development, mechanisms of drug resistance and host-pathogen interactions., Author summary Animal African Trypanosomiasis (AAT), also known as Nagana, is a devastating disease affecting livestock across sub-Saharan Africa. AAT is primarily caused by the parasite Trypanosoma congolense, yet our biological knowledge about this pathogen is poor, especially compared to the related species T. brucei, subspecies of which cause the human disease Sleeping Sickness. Understanding the core metabolism of T. congolense is crucial in order to gain insights into the infection biology of this important pathogen, as well as providing the potential to identify new drug targets. In this work, we addressed the lack of knowledge concerning T. congolense by carrying out a comprehensive analysis of core metabolism, and comparing the data to T. brucei. We then used the findings of metabolic differences to predict differential sensitivity to inhibitors of metabolic function. We show that unlike T. brucei, where glucose metabolism leads to high levels of pyruvate excretion, T. congolense metabolises glucose to other end-products, namely succinate, malate and acetate. Moreover, there are pronounced differences in the way T. congolense uses glucose to feed into other areas of metabolism. Further analysis also suggests that T. congolense mostly scavenges lipids and fatty acids, rather than synthesising them de novo. To validate these findings, we confirm that T. congolense is differentially susceptible to metabolic inhibitors compared to T. brucei, and that, in particular, T. congolense is significantly less sensitive to inhibitors of fatty acid synthesis. Our study provides a foundation of functional metabolic knowledge on T. congolense, with insights into how this parasite fundamentally differs from T. brucei.

Animal African Trypanosomiasis (AAT) is a debilitating livestock disease prevalent across sub-Saharan Africa, a main cause of which is the protozoan parasiteTrypanosoma congolense. In comparison to the well-studiedT. brucei, there is a major paucity of knowledge regarding the biology ofT. congolense. Here, we use a combination of omics technologies and novel genetic tools to characterise core metabolism inT. congolensemammalian-infective bloodstream-form parasites, and test whether metabolic differences compared toT. bruceiimpact upon drug sensitivity. LikeT. brucei, glycolysis plays a major part inT. congolenseenergy metabolism. However, the rate of glucose uptake is significantly reduced inT. congolense, with cells remaining viable when cultured in concentrations as low as 2 mM. Instead of pyruvate, the primary glycolytic endpoints are succinate, malate and acetate. Comparative transcriptomics analysis showed higher levels of activity associated with the mitochondrial pyruvate dehydrogenase complex, acetate generation and the succinate shunt inT. congolense. However, based on omics analysis and chemical inhibition, there does not appear to be significant levels of oxidative phosphorylation. Stable-isotope labelling of glucose enabled the comparison of carbon usage betweenT. bruceiandT. congolense, highlighting differences in nucleotide and fatty acid metabolism. To validate the metabolic similarities and differences, both species were treated with pharmacological inhibitors, confirming a lack of essential electron transport chain activity inT. congolense,but increased sensitivity to inhibition of mitochondrial pyruvate import. Strikingly,T. congolenseexhibited significant resistance to inhibitors of fatty acid synthesis, including a 780-fold greater EC50against the lipase and fatty acid synthase inhibitor Orlistat, compared toT. brucei. These data highlight that bloodstream formT. congolensediverges fromT. bruceiin key areas of metabolism, with several features that are intermediate between bloodstream- and insect-stageT. brucei. These results have implications for drug development, mechanisms of drug resistance and host-pathogen interactions.

Mucosal-associated invariant T (MAIT) cells are a population of innate-like T cells that utilise a semi-invariant T cell receptor (TCR) α chain and are restricted by the highly conserved antigen presenting molecule MR1. MR1 presents microbial riboflavin biosynthesis derived metabolites produced by bacteria and fungi. Consistent with their ability to sense ligands derived from bacterial sources, MAIT cells have been associated with the immune response to a variety of bacterial infections, such as Mycobacterium spp., Salmonella spp. and Escherichia coli. To date, MAIT cells have been studied in humans, non-human primates and mice. However, they have only been putatively identified in cattle by PCR based methods; no phenotypic or functional analyses have been performed. Here, we identified a MAIT cell population in cattle utilising MR1 tetramers and high-throughput TCR sequencing. Phenotypic analysis of cattle MAIT cells revealed features highly analogous to those of MAIT cells in humans and mice, including expression of an orthologous TRAV1-TRAJ33 TCR α chain, an effector memory phenotype irrespective of tissue localisation, and expression of the transcription factors PLZF and EOMES. We determined the frequency of MAIT cells in peripheral blood and multiple tissues, finding that cattle MAIT cells are enriched in mucosal tissues as well as in the mesenteric lymph node. Cattle MAIT cells were responsive to stimulation by 5-OP-RU and riboflavin biosynthesis competent bacteria in vitro. Furthermore, MAIT cells in milk increased in frequency in cows with mastitis. Following challenge with virulent Mycobacterium bovis, a causative agent of bovine tuberculosis and a zoonosis, peripheral blood MAIT cells expressed higher levels of perforin. Thus MAIT cells are implicated in the immune response to two major bacterial infections in cattle. These data suggest that MAIT cells are functionally highly conserved and that cattle are an excellent large animal model to study the role of MAIT cells in important zoonotic infections.

More people globally depend on the water buffalo than any other domesticated species, and as the most closely related domesticated species to cattle they can provide important insights into the shared evolutionary basis of domestication. Here, we sequence the genomes of 79 water buffalo across seven breeds and compare patterns of between breed selective sweeps with those seen for 294 cattle genomes representing 13 global breeds. The genomic regions under selection between cattle breeds significantly overlap regions linked to stature in human genetic studies, with a disproportionate number of these loci also shown to be under selection between water buffalo breeds. Investigation of potential functional variants in the water buffalo genome identifies a rare example of convergent domestication down to the same mutation having independently occurred and been selected for across domesticated species. Cross-species comparisons of recent selective sweeps can consequently help identify and refine important loci linked to domestication., The comparative genomics of domesticated lineages can yield insights into the signatures of artificial selection. This study sequences 79 water buffalo genomes from 7 breeds and reveals examples of convergent domestication at the genetic level between water buffalo and cattle.

Trypanosoma congolense is one of the principal agents causing livestock trypanosomiasis in sub-Saharan Africa. This wasting disease is costing these developing economies billions of dollars and undermining food security. Only two old drugs, the diamidine diminazene and the phenanthridine isometamidium are regularly used, and resistance is widespread but poorly understood. We induce diminazene resistance in T. congolense laboratory strain IL3000 in vitro. Resistance was stable and not deleterious to in vitro growth. There was no cross-resistance with the phenanthridine drugs, with melaminophenyl arsenicals, with two promising new oxaborole trypanocides, nor with other diamidine trypanocides such as pentamidine, except the close structural analogues DB829 and DB75. Fluorescence microscopy showed that accumulation of DB75 was inhibited by folate. Uptake of [3H]-diminazene was also partly inhibited by folate, as well as by competing diamidine drugs, albeit at quite high concentrations, and uptake of tritiated diminazene and pentamidine was slow and low affinity. Uptake of [3H]-folate was in turn partly inhibited by diminazene, and inhibition of diminazene uptake by folate and pentamidine appeared to be additive, indicating multiple low affinity transport mechanisms for the drug. Expression of the T. congolense folate transporters TcoFT1-3 in diminazene-resistant T. b. brucei significantly sensitized the cells to diminazene and DB829, but not to oxaborole AN7973. However, [3H]-diminazene uptake was not different in T. congolense IL3000 and its diminazene resistant clones and RNAseq and whole-genome sequencing of multiple resistant clones did not reveal major changes in folate transporter sequence or expression. Instead, flow cytometry revealed a strong and stable reduction in the mitochondrial membrane potential Ψm in all resistant clones. We conclude that diminazene uptake in T. congolense proceed via multiple low affinity mechanisms including folate transporters and that resistance is the result of a reduction in Ψm that limits mitochondrial accumulation of the drug.

Antigenic variation is employed by many pathogens to evade the host immune response, andTrypanosoma bruceihas evolved a complex system to achieve this phenotype, involving sequential use of variant surface glycoprotein (VSG) genes encoded from a large repertoire of ∼2,000 alleles.T. bruceiexpress multiple, sometimes closely related, VSGs in a population at any one time, and the ability to resolve and analyse this diversity has been limited. We applied long read sequencing (PacBio) to VSG amplicons generated from blood extracted from batches of mice sacrificed at time points (days 3, 6, 10 and 12) post-infection with T. brucei TREU927. The data showed that long read sequencing is reliable for resolving allelic differences between VSGs, and demonstrated that there is significant expressed diversity (449 VSGs detected across 20 mice) and across the timeframe of study there was a clear semi-reproducible pattern of expressed diversity (median of 27 VSGs per sample at day 3 post infection (p.i.), 82 VSGs at day 6 p.i., 187 VSGs at day 10 p.i. and 132 VSGs by day 12 p.i.). There was also consistent detection of one VSG dominating expression across replicates at days 3 and 6, and emergence of a second dominant VSG across replicates by day 12. The innovative application of ecological diversity analysis to VSG reads enabled characterisation of hierarchical VSG expression in the dataset, and resulted in a novel method for analysing such patterns of variation. Additionally, the long read approach allowed detection of mosaic VSG expression from very few reads – this was observed as early as day 3, the earliest that such events have been detected. Therefore, our results indicate that long read analysis is a reliable tool for resolving diverse allele expression profiles, and provides novel insights into the complexity and nature of VSG expression in trypanosomes, revealing significantly higher diversity than previously shown and identifying mosaic gene formation unprecedentedly early during the infection process.

Cattle are an economically important domestic animal species. In vitro 2D cultures of intestinal epithelial cells or epithelial cell lines have been widely used to study cell function and host–pathogen interactions in the bovine intestine. However, these cultures lack the cellular diversity encountered in the intestinal epithelium, and the physiological relevance of monocultures of transformed cell lines is uncertain. Little is also known of the factors that influence cell differentiation and homeostasis in the bovine intestinal epithelium, and few cell-specific markers that can distinguish the different intestinal epithelial cell lineages have been reported. Here we describe a simple and reliable procedure to establish in vitro 3D enteroid, or “mini gut”, cultures from bovine small intestinal (ileal) crypts. These enteroids contained a continuous central lumen lined with a single layer of polarized enterocytes, bound by tight junctions with abundant microvilli on their apical surfaces. Histological and transcriptional analyses suggested that the enteroids comprised a mixed population of intestinal epithelial cell lineages including intestinal stem cells, enterocytes, Paneth cells, goblet cells and enteroendocrine cells. We show that bovine enteroids can be successfully maintained long-term through multiple serial passages without observable changes to their growth characteristics, morphology or transcriptome. Furthermore, the bovine enteroids can be cryopreserved and viable cultures recovered from frozen stocks. Our data suggest that these 3D bovine enteroid cultures represent a novel, physiologically-relevant and tractable in vitro system in which epithelial cell differentiation and function, and host–pathogen interactions in the bovine small intestine can be studied. Electronic supplementary material The online version of this article (10.1186/s13567-018-0547-5) contains supplementary material, which is available to authorized users.

Additional file 3. Mean expression profiles of the genes in each of the largest 50 co-expression clusters. Individual mean expression profiles of the genes in each of the largest 50 co-expression clusters derived from the network graph. The x axis shows the samples ordered as follows: C, isolated intestinal crypts; P0, freshly prepared enteroids; P1, passage 1 enteroids, etc. The y axis shows the mean expression intensity (transcripts/million reads, TPM) for the cluster.

Pathogens target phytohormone signalling pathways to promote disease. Plants deploy salicylic acid (SA)-mediated defences against biotrophs. Pathogens antagonize SA immunity by activating jasmonate signalling, for example Pseudomonas syringae pv. tomato DC3000 produces coronatine (COR), a jasmonic acid (JA) mimic. This study found unexpected dynamics between SA, JA and COR and co-operation between JAZ jasmonate repressor proteins during DC3000 infection. We used a systems-based approach involving targeted hormone profiling, high-temporal-resolution micro-array analysis, reverse genetics and mRNA-seq. Unexpectedly, foliar JA did not accumulate until late in the infection process and was higher in leaves challenged with COR-deficient P. syringae or in the more resistant JA receptor mutant coi1. JAZ regulation was complex and COR alone was insufficient to sustainably induce JAZs. JAZs contribute to early basal and subsequent secondary plant defence responses. We showed that JAZ5 and JAZ10 specifically co-operate to restrict COR cytotoxicity and pathogen growth through a complex transcriptional reprogramming that does not involve the basic helix-loop-helix transcription factors MYC2 and related MYC3 and MYC4 previously shown to restrict pathogen growth. mRNA-seq predicts compromised SA signalling in a jaz5/10 mutant and rapid suppression of JA-related components on bacterial infection.

Trypanosomosis is one of the most significant infectious threats to cattle in sub-Saharan Africa, and one form has also spread to Asia and South America. The disease is caused by a complex of trypanosome species, and the species and strain of parasite can have a profound influence upon the epidemiology of the host-parasite-vector relationships, the severity and course of infection, and, consequently, the implementation and development of control methods. This review will summarise our current knowledge of the relationship between trypanosome species/genotype and the phenotype of disease in cattle, and the implications that this has for ongoing efforts to develop diagnostics, drugs and vaccines for the control of cattle trypanosomosis.

Transcriptional reprogramming is integral to effective plant defense. Pathogen effectors act transcriptionally and posttranscriptionally to suppress defense responses. A major challenge to understanding disease and defense responses is discriminating between transcriptional reprogramming associated with microbial-associated molecular pattern (MAMP)-triggered immunity (MTI) and that orchestrated by effectors. A high-resolution time course of genome-wide expression changes following challenge with Pseudomonas syringae pv tomato DC3000 and the nonpathogenic mutant strain DC3000hrpA allowed us to establish causal links between the activities of pathogen effectors and suppression of MTI and infer with high confidence a range of processes specifically targeted by effectors. Analysis of this information-rich data set with a range of computational tools provided insights into the earliest transcriptional events triggered by effector delivery, regulatory mechanisms recruited, and biological processes targeted. We show that the majority of genes contributing to disease or defense are induced within 6 h postinfection, significantly before pathogen multiplication. Suppression of chloroplast-associated genes is a rapid MAMP-triggered defense response, and suppression of genes involved in chromatin assembly and induction of ubiquitin-related genes coincide with pathogen-induced abscisic acid accumulation. Specific combinations of promoter motifs are engaged in fine-tuning the MTI response and active transcriptional suppression at specific promoter configurations by P. syringae.

Microbe associated molecular pattern (MAMP) receptors in plants recognize MAMPs and activate basal defences; however a complete understanding of the molecular and physiological mechanisms conferring immunity remains elusive. Pathogens suppress active defence in plants through the combined action of effector proteins. Here we show that the chloroplast is a key component of early immune responses. MAMP perception triggers the rapid, large-scale suppression of nuclear encoded chloroplast-targeted genes (NECGs). Virulent Pseudomonas syringae effectors reprogramme NECG expression in Arabidopsis, target the chloroplast and inhibit photosynthetic CO2 assimilation through disruption of photosystem II. This activity prevents a chloroplastic reactive oxygen burst. These physiological changes precede bacterial multiplication and coincide with pathogen-induced abscisic acid (ABA) accumulation. MAMP pretreatment protects chloroplasts from effector manipulation, whereas application of ABA or the inhibitor of photosynthetic electron transport, DCMU, abolishes the MAMP-induced chloroplastic reactive oxygen burst, and enhances growth of a P. syringae hrpA mutant that fails to secrete effectors.

Trypanosomosis is one of the most significant infectious threats to cattle in sub-Saharan Africa, and one form has also spread to Asia and South America. The disease is caused by a complex of trypanosome species, and the species and strain of parasite can have a profound influence upon the epidemiology of the host-parasite-vector relationships, the severity and course of infection, and, consequently, the implementation and development of control methods. This review will summarise our current knowledge of the relationship between trypanosome species/genotype and the phenotype of disease in cattle, and the implications that this has for ongoing efforts to develop diagnostics, drugs and vaccines for the control of cattle trypanosomosis.