The Development of 3D Bovine Intestinal Organoid Derived Models to Investigate Mycobacterium Avium ssp Paratuberculosis Pathogenesis.

Mycobacterium avium subspecies paratuberculosis (MAP) is the etiological agent of Johne's Disease, a chronic enteritis of ruminants prevalent across the world. It is estimated that approximately 50% of UK dairy herds are infected with MAP, but this is likely an underestimate of the true prevalence. Infection can result in reduced milk yield, infertility and premature culling of the animal, leading to significant losses to the farming economy and negatively affecting animal welfare. Understanding the initial interaction between MAP and the host is critical to develop improved diagnostic tools and novel vaccines. Here we describe the characterisation of three different multicellular in vitro models derived from bovine intestinal tissue, and their use for the study of cellular interactions with MAP. In addition to the previously described basal-out 3D bovine enteroids, we have established viable 2D monolayers and 3D apical-out organoids. The apical-out enteroids differ from previously described bovine enteroids as the apical surface is exposed on the exterior surface of the 3D structure, enabling study of host-pathogen interactions at the epithelial surface without the need for microinjection. We have characterised the cell types present in each model system using RT-qPCR to detect predicted cell type-specific gene expression, and confocal microscopy for cell type-specific protein expression. Each model contained the cells present in the original bovine intestinal tissue, confirming they were representative of the bovine gut. Exposure of the three model systems to the K10 reference strain of MAP K10, and a recent Scottish isolate referred to as C49, led to the observation of intracellular bacteria by confocal microscopy. Enumeration of the bacteria by quantification of genome copy number, indicated that K10 was less invasive than C49 at early time points in infection in all model systems. This study shows that bovine enteroid-based models are permissive to infection with MAP and that these models may be useful in investigating early stages of MAP pathogenesis in a physiologically relevant in vitro system, whilst reducing the use of animals in scientific research. Bos taurus: urn:lsid:zoobank.org:act:4C90C4FA-6296-4972-BE6A-5EF578677D64.

Susceptibility to disease (tropical theileriosis) is associated with differential expression of host genes that possess motifs recognised by a pathogen DNA binding protein.

BACKGROUND: Knowledge of factors that influence the outcome of infection are crucial for determining the risk of severe disease and requires the characterisation of pathogen-host interactions that have evolved to confer variable susceptibility to infection. Cattle infected by Theileria annulata show a wide range in disease severity. Native (Bos indicus) Sahiwal cattle are tolerant to infection, whereas exotic (Bos taurus) Holstein cattle are susceptible to acute disease. METHODOLOGY/PRINCIPAL FINDINGS: We used RNA-seq to assess whether Theileria infected cell lines from Sahiwal cattle display a different transcriptome profile compared to Holstein and screened for altered expression of parasite factors that could generate differences in host cell gene expression. Significant differences (<0.1 FDR) in the expression level of a large number (2211) of bovine genes were identified, with enrichment of genes associated with Type I IFN, cholesterol biosynthesis, oncogenesis and parasite infection. A screen for parasite factors found limited evidence for differential expression. However, the number and location of DNA motifs bound by the TashAT2 factor (TA20095) were found to differ between the genomes of B. indicus vs. B. taurus, and divergent motif patterns were identified in infection-associated genes differentially expressed between Sahiwal and Holstein infected cells. CONCLUSIONS/SIGNIFICANCE: We conclude that divergent pathogen-host molecular interactions that influence chromatin architecture of the infected cell are a major determinant in the generation of gene expression differences linked to disease susceptibility.

Inflammasome Activation in Bovine Peripheral Blood-Derived Macrophages Is Associated with Actin Rearrangement.

Inflammation is critical for infection control and acts as an arsenal defense mechanism against invading microbes through activation of the host immune system. It works via its inflammasome components to sense the dangerous invading microorganism and send messages to the immune system to destroy them. To date, the function of bovine macrophage inflammasome and its relationship with actin has not been identified. This study aimed to investigate the activation of bovine inflammasome by phase one flagellin from Salmonella typhimurium and its interaction with actin. Bovine monocyte-derived macrophages were prepared and challenged with S. typhimurium SL1344 phase one flagellin. The results demonstrated the relationship between the flagellin-based activation of inflammasome and actin rearrangement. The flagellin-based activation of inflammasome promoted the activation and co-localization of F-actin and the inflammasome complex. Actin was remodeled to different degrees according to the stage of inflammasome activation. The actin redistribution varied from polymerization to filopodia, while at the stage of pyroptotic cell death, actin was broken down and interacted with activated inflammasome complexes. In conclusion, flagellin-dependent inflammasome activation and actin localization to the inflammasome at the stage of pyroptotic cell death may be of importance for appropriate immune responses, pending further studies to explore the exact cross-linking between the inflammasome complex and actin.

Quantifying Mycobacterium avium subspecies paratuberculosis infection of bovine monocyte derived macrophages by confocal microscopy.

Quantification of Mycobacterium avium subspecies paratuberculosis (MAP) during in vitro infection experiments is challenging due to limitations of currently utilised methods, such as colony counting. Here we describe quantifying MAP infection of bovine macrophages (Mφ) using confocal microscopy. Bovine monocyte derived macrophages were infected with MAP at a high or low dose and the number of intracellular bacteria calculated at 2 h post infection using confocal microscopy. Bacteria within simultaneously infected Mφ were quantified by colony counting in order to compare confocal microscopy results with results obtained by an established method. Confocal microscopy provided a robust alternative quantification method that allowed for assessment of the infection at the individual Mφ level. This demonstrated that MAP infection was not homogeneous, and that there were higher numbers of both infected Mφ and intracellular bacteria and bacterial aggregates at the high dose compared to the low dose, potentially impacting the Mφ response to infection. Confocal microscopy can therefore provide a level of detail regarding the infection unobtainable by other quantification methods.

Interleukin 10 knock-down in bovine monocyte-derived macrophages has distinct effects during infection with two divergent strains of Mycobacterium bovis.

Mycobacterium bovis is the causative agent of bovine tuberculosis (TB), a cattle disease of global importance. M. bovis infects bovine macrophages (Mø) and subverts the host cell response to generate a suitable niche for survival and replication. We investigated the role of the anti-inflammatory cytokine interleukin (IL) 10 during in vitro infection of bovine monocyte-derived Mø (bMDM) with two divergent UK strains of M. bovis, which differentially modulate expression of IL10. The use of IL10-targeting siRNA revealed that IL10 inhibited the production of IL1B, IL6, tumour necrosis factor (TNF) and interferon gamma (IFNG) during infection of bMDM with the M. bovis strain G18. In contrast, IL10 only regulated a subset of these genes; TNF and IFNG, during infection with the M. bovis reference strain AF2122/97. Furthermore, nitric oxide (NO) production was modulated by IL10 during AF2122/97 infection, but not at the nitric oxide synthase 2 (NOS2) mRNA level, as observed during G18 infection. However, IL10 was found to promote survival of both M. bovis strains during early bMDM infection, but this effect disappeared after 24 h. The role of IL10-induced modulation of TNF, IFNG and NO production in M. bovis survival was investigated using siRNA targeting TNF, IFNG receptor 1 (IFNGR1) and NOS2. Knock-down of these genes individually did not promote survival of either M. bovis strain and therefore modulation of these genes does not account for the effect of IL10 on M. bovis survival. However, TNF knock-down was found to be detrimental to the survival of the M. bovis strain G18 during early infection. The results provide further evidence for the importance of IL10 during M. bovis infection of Mø. Furthermore, they highlight M. bovis strain specific differences in the interaction with the infected bMDM, which may influence the course of infection and progression of bovine TB.

Variation in the Early Host-Pathogen Interaction of Bovine Macrophages with Divergent Mycobacterium bovis Strains in the United Kingdom.

Bovine tuberculosis has been an escalating animal health issue in the United Kingdom since the 1980s, even though control policies have been in place for over 60 years. The importance of the genetics of the etiological agent, Mycobacterium bovis, in the reemergence of the disease has been largely overlooked. We compared the interaction between bovine monocyte-derived macrophages (bMDM) and two M. bovis strains, AF2122/97 and G18, representing distinct genotypes currently circulating in the United Kingdom. These M. bovis strains exhibited differences in survival and growth in bMDM. Although uptake was similar, the number of viable intracellular AF2122/97 organisms increased rapidly, while G18 growth was constrained for the first 24 h. AF2122/97 infection induced a greater transcriptional response by bMDM than G18 infection with respect to the number of differentially expressed genes and the fold changes measured. AF2122/97 infection induced more bMDM cell death, with characteristics of necrosis and apoptosis, more inflammasome activation, and a greater type I interferon response than G18. In conclusion, the two investigated M. bovis strains interact in significantly different ways with the host macrophage. In contrast to the relatively silent infection by G18, AF2122/97 induces greater signaling to attract other immune cells and induces host cell death, which may promote secondary infections of naive macrophages. These differences may affect early events in the host-pathogen interaction, including granuloma development, which could in turn alter the progression of the disease. Therefore, the potential involvement of M. bovis genotypes in the reemergence of bovine tuberculosis in the United Kingdom warrants further investigation.

Host species adaptation of TLR5 signalling and flagellin recognition.

Toll-like receptor 5 (TLR5) recognition of flagellin instigates inflammatory signalling. Significant sequence variation in TLR5 exists between animal species but its impact on activity is less well understood. Building on our previous research that bovine TLR5 (bTLR5) is functional, we compared human and bovine TLR5 activity and signalling in cognate cell lines. bTLR5 induced higher levels of CXCL8 when expressed in bovine cells and reciprocal results were found for human TLR5 (hTLR5) in human cells, indicative of host cell specificity in this response. Analysis of Toll/interleukin-1 receptor (TIR) sequences indicated that these differential responses involve cognate MyD88 recognition. siRNA knockdowns and inhibitor experiments demonstrated that there are some host differences in signalling. Although, PI3K activation is required for bTLR5 signalling, mutating bTLR5 F798 to hTLR5 Y798 within a putative PI3K motif resulted in a significantly reduced response. All ruminants have F798 in contrast to most other species, suggesting that TLR5 signalling has evolved differently in ruminants. Evolutionary divergence between bovine and human TLR5 was also apparent in relation to responses measured to diverse bacterial flagellins. Our results underscore the importance of species specific studies and how differences may alter efficacy of TLR-based vaccine adjuvants.

Live and inactivated Salmonella enterica serovar Typhimurium stimulate similar but distinct transcriptome profiles in bovine macrophages and dendritic cells.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major cause of gastroenteritis in cattle and humans. Dendritic cells (DC) and macrophages (Mø) are major players in early immunity to Salmonella, and their response could influence the course of infection. Therefore, the global transcriptional response of bovine monocyte-derived DC and Mø to stimulation with live and inactivated S. Typhimurium was compared. Both cell types mount a major response 2 h post infection, with a core common response conserved across cell-type and stimuli. However, three of the most affected pathways; inflammatory response, regulation of transcription and regulation of programmed cell death, exhibited cell-type and stimuli-specific differences. The expression of a subset of genes associated with these pathways was investigated further. The inflammatory response was greater in Mø than DC, in the number of genes and the enhanced expression of common genes, e.g., interleukin (IL) 1B and IL6, while the opposite pattern was observed with interferon gamma. Furthermore, a large proportion of the investigated genes exhibited stimuli-specific differential expression, e.g., Mediterranean fever. Two-thirds of the investigated transcription factors were significantly differentially expressed in response to live and inactivated Salmonella. Therefore the transcriptional responses of bovine DC and Mø during early S. Typhimurium infection are similar but distinct, potentially due to the overall function of these cell-types. The differences in response of the host cell will influence down-stream events, thus impacting on the subsequent immune response generated during the course of the infection.

Phenotypic and functional analysis of monocyte populations in cattle peripheral blood identifies a subset with high endocytic and allogeneic T-cell stimulatory capacity.

Circulating monocytes in several mammalian species can be subdivided into functionally distinct subpopulations based on differential expression of surface molecules. We confirm that bovine monocytes express CD172a and MHC class II with two distinct populations of CD14(+)CD16(low/-)CD163(+) and CD14(-)CD16(++)CD163(low-) cells, and a more diffuse population of CD14(+)CD16(+)CD163(+) cells. In contrast, ovine monocytes consisted of only a major CD14(+)CD16(+) subset and a very low percentage of CD14(-)CD16(++)cells. The bovine subsets expressed similar levels of CD80, CD40 and CD11c molecules and mRNA encoding CD115. However, further mRNA analyses revealed that the CD14(-)CD16(++) monocytes were CX3CR1(high)CCR2(low) whereas the major CD14(+) subset was CX3CR1(low)CCR2(high). The former were positive for CD1b and had lower levels of CD11b and CD86 than the CD14(+) monocytes. The more diffuse CD14(+)CD16(+) population generally expressed intermediate levels of these molecules. All three populations responded to stimulation with phenol-extracted lipopolysaccharide (LPS) by producing interleukin (IL)-1ß, with the CD16(++) subset expressing higher levels of IL-12 and lower levels of IL-10. The CD14(-)CD16(++) cells were more endocytic and induced greater allogeneic T cell responses compared to the other monocyte populations. Taken together the data show both similarities and differences between the classical, intermediate and non-classical definitions of monocytes as described for other mammalian species, with additional potential subpopulations. Further functional analyses of these monocyte populations may help explain inter-animal and inter-species variations to infection, inflammation and vaccination in ruminant livestock.

Functional analysis of bovine TLR5 and association with IgA responses of cattle following systemic immunisation with H7 flagella.

Flagellin subunits are important inducers of host immune responses through activation of TLR5 when extracellular and the inflammasome if cytosolic. Our previous work demonstrated that systemic immunization of cattle with flagella generates systemic and mucosal IgA responses. The IgA response in mice is TLR5-dependent and TLR5 can impact on the general magnitude of the adaptive response. However, due to sequence differences between bovine and human/murine TLR5 sequences, it is not clear whether bovine TLR5 (bTLR5) is able to stimulate an inflammatory response following interaction with flagellin. To address this we have examined the innate responses of both human and bovine cells containing bTLR5 to H7 flagellin from E. coli O157:H7. Both HEK293 (human origin) and embryonic bovine lung (EBL) cells transfected with bTLR5 responded to addition of H7 flagellin compared to non-transfected controls. Responses were significantly reduced when mutations were introduced into the TLR5-binding regions of H7 flagellin, including an R90T substitution. In bovine primary macrophages, flagellin-stimulated CXCL8 mRNA and secreted protein levels were significantly reduced when TLR5 transcript levels were suppressed by specific siRNAs and stimulation was reduced with the R90T-H7 variant. While these results indicate that the bTLR5 sequence produces a functional flagellin-recognition receptor, cattle immunized with R90T-H7 flagella also demonstrated systemic IgA responses to the flagellin in comparison to adjuvant only controls. This presumably either reflects our findings that R90T-H7 still activates bTLR5, albeit with reduced efficiency compared to WT H7 flagellin, or that other flagellin recognition pathways may play a role in this mucosal response.

Comparison of small interfering RNA (siRNA) delivery into bovine monocyte-derived macrophages by transfection and electroporation.

The manipulation of the RNA interference pathway using small interfering RNA (siRNA) has become the most frequently used gene silencing method. However, siRNA delivery into primary cells, especially primary macrophages, is often considered challenging. Here we report the investigation of the suitability of two methodologies: transient transfection and electroporation, to deliver siRNA targeted against the putative immunomodulatory gene Mediterranean fever (MEFV) into primary bovine monocyte-derived macrophages (bMDM). Eleven commercial transfection reagents were investigated with variable results with respect to siRNA uptake, target gene knock-down, cell toxicity and type I interferon (IFN) response induction. Three transfection reagents: Lipofectamine 2000, Lipofectamine RNAiMAX and DharmaFECT 3, were found to consistently give the best results. However, all the transfection reagents tested induced an IFN response in the absence of siRNA, which could be minimized by reducing the transfection reagent incubation period. In addition, optimized siRNA delivery into bMDM by electroporation achieved comparable levels of target gene knock-down as transient transfection, without a detectable IFN response, but with higher levels of cell toxicity. The optimized transient transfection and electroporation methodologies may provide a starting point for optimizing siRNA delivery into macrophages derived from other species or other cells considered difficult to investigate with siRNA.

The level of H2O2 type oxidative stress regulates virulence of Theileria-transformed leukocytes.

Theileria annulata infects predominantly macrophages, and to a lesser extent B cells, and causes a widespread disease of cattle called tropical theileriosis. Disease-causing infected macrophages are aggressively invasive, but this virulence trait can be attenuated by long-term culture. Attenuated macrophages are used as live vaccines against tropical theileriosis and via their characterization one gains insights into what host cell trait is altered concomitant with loss of virulence. We established that sporozoite infection of monocytes rapidly induces hif1-α transcription and that constitutive induction of HIF-1α in transformed leukocytes is parasite-dependent. In both infected macrophages and B cells induction of HIF-1α activates transcription of its target genes that drive host cells to perform Warburg-like glycolysis. We propose that Theileria-infected leukocytes maintain a HIF-1α-driven transcriptional programme typical of Warburg glycolysis in order to reduce as much as possible host cell H2 O2 type oxidative stress. However, in attenuated macrophages H2O2 production increases and HIF-1α levels consequently remained high, even though adhesion and aggressive invasiveness diminished. This indicates that Theileria infection generates a host leukocytes hypoxic response that if not properly controlled leads to loss of virulence.

Differential response of bovine mammary epithelial cells to Staphylococcus aureus or Escherichia coli agonists of the innate immune system.

Mastitis caused by Escherichia coli and Staphylococcus aureus is a major pathology of dairy cows. To better understand the differential response of the mammary gland to these two pathogens, we stimulated bovine mammary epithelial cells (bMEC) with either E. coli crude lipopolysaccharide (LPS) or with S. aureus culture supernatant (SaS) to compare the transcriptomic profiles of the initial bMEC response. By using HEK 293 reporter cells for pattern recognition receptors, the LPS preparation was found to stimulate TLR2 and TLR4 but not TLR5, Nod1 or Nod2, whereas SaS stimulated TLR2. Biochemical analysis revealed that lipoteichoic acid, protein A and α-hemolysin were all present in SaS, and bMEC were found to be responsive to each of these molecules. Transcriptome profiling revealed a core innate immune response partly shared by LPS and SaS. However, LPS induced expression of a significant higher number of genes and the fold changes were of greater magnitude than those induced by SaS. Microarray data analysis suggests that the activation pathways and the early chemokine and cytokine production preceded the defense and stress responses. A major differential response was the activation of the type I IFN pathway by LPS but not by SaS. The higher upregulation of chemokines (Cxcl10, Ccl2, Ccl5 and Ccl20) that target mononuclear leucocytes by LPS than by SaS is likely to be related to the differential activation of the type I IFN pathway, and could induce a different profile of the initial recruitment of leucocytes. The MEC responses to the two stimuli were different, as LPS was associated with NF-κB and Fas signaling pathways, whereas SaS was associated with AP-1 and IL-17A signaling pathways. It is noteworthy that at the protein level secretion of TNF-α and IL-1ß was not induced by either stimulus. These results suggest that the response of MEC to diffusible stimuli from E. coli and S. aureus contributes to the onset of the response with differential leucocyte recruitment and distinct inflammatory and innate immune reactions of the mammary gland to infection.

Escherichia coli- and Staphylococcus aureus-induced mastitis differentially modulate transcriptional responses in neighbouring uninfected bovine mammary gland quarters.

BACKGROUND: The most important disease of dairy cattle is mastitis, caused by the infection of the mammary gland by various micro-organisms. Although the transcriptional response of bovine mammary gland cells to in vitro infection has been studied, the interplay and consequences of these responses in the in vivo environment of the mammary gland are less clear. Previously mammary gland quarters were considered to be unaffected by events occurring in neighbouring quarters. More recently infection of individual quarters with mastitis causing pathogens, especially Escherichia coli, has been shown to influence the physiology of neighbouring uninfected quarters. Therefore, the transcriptional responses of uninfected mammary gland quarters adjacent to quarters infected with two major mastitis causing pathogens, E. coli and Staphylococcus aureus, were compared. RESULTS: The bacteriologically sterile, within-animal control quarters exhibited a transcriptional response to the infection of neighbouring quarters. The greatest response was associated with E. coli infection, while a weaker, yet significant, response occurred during S. aureus infection. The transcriptional responses of these uninfected quarters included the enhanced expression of many genes previously associated with mammary gland infections. Comparison of the transcriptional response of uninfected quarters to S. aureus and E. coli infection identified 187 differentially expressed genes, which were particularly associated with cellular responses, e.g. response to stress. The most affected network identified by Ingenuity Pathway analysis has the immunosuppressor transforming growth factor beta 1 (TGFB1) at its hub and largely consists of genes more highly expressed in control quarters from S. aureus infected cows. CONCLUSIONS: Uninfected mammary gland quarters reacted to the infection of neighbouring quarters and the responses were dependent on pathogen type. Therefore, bovine udder quarters exhibit interdependence and should not be considered as separate functional entities. This suggests that mastitis pathogens not only interact directly with host mammary cells, but also influence discrete sites some distance away, which will affect their response to the subsequent spread of the infection. Understanding the underlying mechanisms may provide further clues for ways to control mammary gland infections. These results also have implications for the design of experimental studies investigating immune regulatory mechanisms in the bovine mammary gland.

Living with the enemy or uninvited guests: functional genomics approaches to investigating host resistance or tolerance traits to a protozoan parasite, Theileria annulata, in cattle.

Many breeds of cattle with long histories of living in areas of endemic disease have evolved mechanisms that enable them to co-exist with specific pathogens. Understanding the genes that control tolerance and resistance could provide new strategies to improve the health and welfare of livestock. Around one sixth of the world cattle population is estimated to be at risk from one of the most debilitating tick-borne diseases of cattle, caused by the protozoan parasite, Theileria annulata. The parasite mainly infects cells of the myeloid lineage which are also the main producers of inflammatory cytokines. If an infectious or inflammatory insult is sufficiently great, inflammatory cytokines produced by macrophages enter the circulation and induce an acute phase proteins (APP) response. The Bos taurus Holstein breed produces higher and more prolonged levels of inflammatory cytokine induced APP than the Bos indicus Sahiwal breed in response to experimental infection with T. annulata. The Sahiwal exhibits significantly less pathology and survives infection, unlike the Holstein breed. Therefore, we hypothesised that the causal genes were likely to be expressed in macrophages and control the production of inflammatory cytokines. A functional genomics approach revealed that the transcriptome profile of the B. taurus macrophages was more associated with an inflammatory programme than the B. indicus macrophages. In particular the most differentially expressed gene was a member of the signal regulatory protein (SIRP) family. These are mainly expressed on myeloid cell surfaces and control inflammatory responses. Other differentially expressed genes included bovine major histocompatibility complex (MHC) (BoLA) class II genes, particularly BoLA DQ, and transforming growth factor (TGF)B2. We are now exploring whether sequence and functional differences in the bovine SIRP family may underlie the resistance or tolerance to T. annulata between the breeds. Potentially, our research may also have more general implications for the control of inflammatory processes against other pathogens. Genes controlling the balance between pathology and protection may determine how livestock can survive in the face of infectious onslaught. Next generation sequencing and RNAi methodologies for livestock species will bring new opportunities to link diversity at the genome level to functional differences in health traits in livestock species.

Strengthening insights into host responses to mastitis infection in ruminants by combining heterogeneous microarray data sources.

BACKGROUND: Gene expression profiling studies of mastitis in ruminants have provided key but fragmented knowledge for the understanding of the disease. A systematic combination of different expression profiling studies via meta-analysis techniques has the potential to test the extensibility of conclusions based on single studies. Using the program Pointillist, we performed meta-analysis of transcription-profiling data from six independent studies of infections with mammary gland pathogens, including samples from cattle challenged in vivo with S. aureus, E. coli, and S. uberis, samples from goats challenged in vivo with S. aureus, as well as cattle macrophages and ovine dendritic cells infected in vitro with S. aureus. We combined different time points from those studies, testing different responses to mastitis infection: overall (common signature), early stage, late stage, and cattle-specific. RESULTS: Ingenuity Pathway Analysis of affected genes showed that the four meta-analysis combinations share biological functions and pathways (e.g. protein ubiquitination and polyamine regulation) which are intrinsic to the general disease response. In the overall response, pathways related to immune response and inflammation, as well as biological functions related to lipid metabolism were altered. This latter observation is consistent with the milk fat content depression commonly observed during mastitis infection. Complementarities between early and late stage responses were found, with a prominence of metabolic and stress signals in the early stage and of the immune response related to the lipid metabolism in the late stage; both mechanisms apparently modulated by few genes, including XBP1 and SREBF1.The cattle-specific response was characterized by alteration of the immune response and by modification of lipid metabolism. Comparison of E. coli and S. aureus infections in cattle in vivo revealed that affected genes showing opposite regulation had the same altered biological functions and provided evidence that E. coli caused a stronger host response. CONCLUSIONS: This meta-analysis approach reinforces previous findings but also reveals several novel themes, including the involvement of genes, biological functions, and pathways that were not identified in individual studies. As such, it provides an interesting proof of principle for future studies combining information from diverse heterogeneous sources.

TGF-b2 induction regulates invasiveness of Theileria-transformed leukocytes and disease susceptibility.

Theileria parasites invade and transform bovine leukocytes causing either East Coast fever (T. parva), or tropical theileriosis (T. annulata). Susceptible animals usually die within weeks of infection, but indigenous infected cattle show markedly reduced pathology, suggesting that host genetic factors may cause disease susceptibility. Attenuated live vaccines are widely used to control tropical theileriosis and attenuation is associated with reduced invasiveness of infected macrophages in vitro. Disease pathogenesis is therefore linked to aggressive invasiveness, rather than uncontrolled proliferation of Theileria-infected leukocytes. We show that the invasive potential of Theileria-transformed leukocytes involves TGF-b signalling. Attenuated live vaccine lines express reduced TGF-b2 and their invasiveness can be rescued with exogenous TGF-b. Importantly, infected macrophages from disease susceptible Holstein-Friesian (HF) cows express more TGF-b2 and traverse Matrigel with great efficiency compared to those from disease-resistant Sahiwal cattle. Thus, TGF-b2 levels correlate with disease susceptibility. Using fluorescence and time-lapse video microscopy we show that Theileria-infected, disease-susceptible HF macrophages exhibit increased actin dynamics in their lamellipodia and podosomal adhesion structures and develop more membrane blebs. TGF-b2-associated invasiveness in HF macrophages has a transcription-independent element that relies on cytoskeleton remodelling via activation of Rho kinase (ROCK). We propose that a TGF-b autocrine loop confers an amoeboid-like motility on Theileria-infected leukocytes, which combines with MMP-dependent motility to drive invasiveness and virulence.

Comparative genomics of Toll-like receptor signalling in five species.

BACKGROUND: Over the last decade, several studies have identified quantitative trait loci (QTL) affecting variation of immune related traits in mammals. Recent studies in humans and mice suggest that part of this variation may be caused by polymorphisms in genes involved in Toll-like receptor (TLR) signalling. In this project, we used a comparative approach to investigate the importance of TLR-related genes in comparison with other immunologically relevant genes for resistance traits in five species by associating their genomic location with previously published immune-related QTL regions. RESULTS: We report the genomic localisation of TLR1-10 and ten associated signalling molecules in sheep and pig using in-silico and/or radiation hybrid (RH) mapping techniques and compare their positions with their annotated homologues in the human, cattle and mouse whole genome sequences. We also report medium-density RH maps for porcine chromosomes 8 and 13. A comparative analysis of the positions of previously published relevant QTLs allowed the identification of homologous regions that are associated with similar health traits in several species and which contain TLR related and other immunologically relevant genes. Additional evidence was gathered by examining relevant gene expression and association studies. CONCLUSION: This comparative genomic approach identified eight genes as potentially causative genes for variations of health related traits. These include susceptibility to clinical mastitis in dairy cattle, general disease resistance in sheep, cattle, humans and mice, and tolerance to protozoan infection in cattle and mice. Four TLR-related genes (TLR1, 6, MyD88, IRF3) appear to be the most likely candidate genes underlying QTL regions which control the resistance to the same or similar pathogens in several species. Further studies are required to investigate the potential role of polymorphisms within these genes.

The protozoan parasite Theileria annulata alters the differentiation state of the infected macrophage and suppresses musculoaponeurotic fibrosarcoma oncogene (MAF) transcription factors.

The tick-borne protozoan parasite Theileria annulata causes a debilitating disease of cattle called Tropical Theileriosis. The parasite predominantly invades bovine macrophages (m phi) and induces host cell transformation by a mechanism that has not been fully elucidated. Infection is associated with loss of characteristic m phi functions and phenotypic markers, indicative of host cell de-differentiation. We have investigated the effect of T. annulata infection on the expression of the m phi differentiation marker c-maf. The up-regulation of c-maf mRNA levels observed during bovine monocyte differentiation to m phi was suppressed by T. annulata infection. Furthermore, mRNA levels for c-maf and the closely related transcription factor mafB were significantly lower in established T. annulata-infected cell-lines than in bovine monocyte-derived m phi. Treatment of T. annulata-infected cells with the theileriacidal drug buparvaquone induced up-regulation of c-maf and mafB, which correlated with altered expression of down-stream target genes, e.g. up-regulation of integrin B7 and down-regulation of IL12A. Furthermore, T. annulata infection is associated with the suppression of the transcription factors, Pu.1 and RUNX1, and colony stimulating factor 1 receptor (CSF1R) which are also involved in the regulation of monocyte/m phi differentiation. We believe these results provide the first direct evidence that T. annulata modulates the host m phi differentiation state, which may diminish the defence capabilities of the infected cell and/or promote cell proliferation. Musculoaponeurotic fibrosarcoma oncogene (MAF) transcription factors play an important role in cell proliferation, differentiation and survival; therefore, regulation of these genes may be a major mechanism employed by T. annulata to survive within the infected m phi.

Development and validation of an oligonucleotide microarray for immuno-inflammatory genes of ruminants.

This report describes the development of small DNA microarrays of fully defined genes suitable for projects requiring detailed analysis of gene expression in sheep and/or cattle. Two arrays have been developed; the first is a small reference microarray (RIGRA) that has been used to validate experimental design and methodology; the second, a larger array (RIGUA) containing probes for 516 ruminant immuno-inflammatory genes, each represented by non-overlapping 75mer oligonucleotides. Experiments used to validate this microarray were: (1) a comparison of gene expression profiles from sheep broncho-alveolar macrophages before and after in vitro activation with lipopolysaccharide (LPS), using the RIGRA; (2) the differential gene expression between five in vitro unstimulated sheep keratinocyte cultures; (3) LPS/interferon gamma stimulated and unstimulated blood monocytes purified from Holstein-Friesians (Bos taurus) and Sahiwals (Bos indicus) cattle using the RIGUA. Real-time, quantitative RT-PCR was used to validate the gene expression profiles obtained with the RIGUA microarrays. The potential for using such an immunological tool in understanding the relative gene expression corresponding to immune-inflammatory responses of sheep and cattle is discussed.