Granzyme B Is an Essential Mediator in CD8+ T Cell Killing of Theileria parva-Infected Cells.

There is established evidence that cytotoxic CD8+ T cells are important mediators of immunity against the bovine intracellular protozoan parasite Theileria parva However, the mechanism by which the specific CD8+ T cells kill parasitized cells is not understood. Although the predominant pathway used by human and murine CD8+ T cells to kill pathogen-infected cells is granule exocytosis, involving the release of perforin and granzyme B, there is to date a lack of published information on the biological activities of bovine granzyme B. The present study set out to define the functional activities of bovine granzyme B and determine its role in mediating the killing of T. parva-parasitized cells. DNA constructs encoding functional and nonfunctional forms of bovine granzyme B were produced, and the proteins expressed in Cos-7 cells were used to establish an enzymatic assay to detect and quantify the expression of functional granzyme B protein. Using this assay, the levels of killing of different T. parva-specific CD8+ T cell clones were found to be significantly correlated with the levels of granzyme B protein but not the levels of mRNA transcript expression. Experiments using inhibitors specific for perforin and granzyme B confirmed that CD8+ T cell killing of parasitized cells is dependent on granule exocytosis and, specifically, granzyme B. Further studies showed that the granzyme B-mediated death of parasitized cells is independent of caspases and that granzyme B activates the proapoptotic molecule Bid.

Proteomic Profiling of Cranial (Superior) Cervical Ganglia Reveals Beta-Amyloid and Ubiquitin Proteasome System Perturbations in an Equine Multiple System Neuropathy.

Equine grass sickness (EGS) is an acute, predominantly fatal, multiple system neuropathy of grazing horses with reported incidence rates of ∼2%. An apparently identical disease occurs in multiple species, including but not limited to cats, dogs, and rabbits. Although the precise etiology remains unclear, ultrastructural findings have suggested that the primary lesion lies in the glycoprotein biosynthetic pathway of specific neuronal populations. The goal of this study was therefore to identify the molecular processes underpinning neurodegeneration in EGS. Here, we use a bottom-up approach beginning with the application of modern proteomic tools to the analysis of cranial (superior) cervical ganglion (CCG, a consistently affected tissue) from EGS-affected patients and appropriate control cases postmortem. In what appears to be the proteomic application of modern proteomic tools to equine neuronal tissues and/or to an inherent neurodegenerative disease of large animals (not a model of human disease), we identified 2,311 proteins in CCG extracts, with 320 proteins increased and 186 decreased by greater than 20% relative to controls. Further examination of selected proteomic candidates by quantitative fluorescent Western blotting (QFWB) and subcellular expression profiling by immunohistochemistry highlighted a previously unreported dysregulation in proteins commonly associated with protein misfolding/aggregation responses seen in a myriad of human neurodegenerative conditions, including but not limited to amyloid precursor protein (APP), microtubule associated protein (Tau), and multiple components of the ubiquitin proteasome system (UPS). Differentially expressed proteins eligible for in silico pathway analysis clustered predominantly into the following biofunctions: (1) diseases and disorders, including; neurological disease and skeletal and muscular disorders and (2) molecular and cellular functions, including cellular assembly and organization, cell-to-cell signaling and interaction (including epinephrine, dopamine, and adrenergic signaling and receptor function), and small molecule biochemistry. Interestingly, while the biofunctions identified in this study may represent pathways underpinning EGS-induced neurodegeneration, this is also the first demonstration of potential molecular conservation (including previously unreported dysregulation of the UPS and APP) spanning the degenerative cascades from an apparently unrelated condition of large animals, to small animal models with altered neuronal vulnerability, and human neurological conditions. Importantly, this study highlights the feasibility and benefits of applying modern proteomic techniques to veterinary investigations of neurodegenerative processes in diseases of large animals.

Ethanol-induced mast cell-mediated inflammation leads to increased susceptibility of intestinal tumorigenesis in the APC Δ468 min mouse model of colon cancer.

BACKGROUND: Chronic and frequent alcohol (ethanol [EtOH]) intake has been associated with an increased incidence of several types of cancers including breast, mouth, throat, esophageal, stomach, and colorectal (CRC). The underlying mechanism of this deleterious carcinogenic effect of alcohol has not been clearly established but inflammation may be 1 unifying feature of these cancers. We have recently shown that intestinal mast cells play a central role in intestinal carcinogenesis. In this study, we tested our hypothesis that mast cell-mediated inflammation is 1 underlying mechanism by which chronic alcohol promotes intestinal tumorigenesis. METHODS: APC(Δ468) mice were fed either an alcohol-containing Nanji liquid diet or isocaloric dextrose-containing Nanji diet for 10 weeks and then sacrificed to collect small and large intestine samples. Assessments of tumor number and size as well as mast cell number and mast cell activity and histology score for invasion were compared between Control (dextrose-fed) and alcohol-fed APC(∆468) mice. The effect of alcohol on mast cell-mediated tumor migration was also assessed using an in vitro migration assay. RESULTS: Alcohol feeding increased both polyp number and size within both the small and the large intestines of APC(∆468) mice. Only alcohol-fed mice showed evidence of tumor invasion. Chronic alcohol feeding also resulted in an increased mast cell number and activity in tumor stroma and invading borders. In vitro migration assay showed that alcohol significantly increases mast cell-mediated tumor migration in vitro. CONCLUSIONS: Our data show that chronic alcohol intake promotes: (i) intestinal tumorigenesis and tumor invasion in genetically susceptible mice; (ii) increases in polyp-associated mast cells; and (iii) mast cell-mediated tumor migration in vitro. Both our in vivo and in vitro studies suggest that mast cell-mediated inflammation could be 1 mechanism by which alcohol promotes carcinogenesis.

The goblet cell is the cellular source of the anti-microbial angiogenin 4 in the large intestine post Trichuris muris infection.

BACKGROUND: Mouse angiogenin 4 (Ang4) has previously been described as a Paneth cell-derived antimicrobial peptide important in epithelial host defence in the small intestine. However, a source for Ang4 in the large intestine, which is devoid of Paneth cells, has not been defined. METHODOLOGY/PRINCIPAL FINDINGS: Analysis was performed on Ang4 expression in colonic tissue by qPCR and immunohistochemistry following infection with the large intestine dwelling helminth parasite Trichuris muris. This demonstrated an increase in expression of the peptide following infection of resistant BALB/c mice. Further, histological analysis of colonic tissue revealed the cellular source of this Ang4 to be goblet cells. To elucidate the mechanism of Ang4 expression immunohistochemistry and qPCR for Ang4 was performed on colonic tissue from T. muris infected mouse mutants. Experiments comparing C3H/HeN and C3H/HeJ mice, which have a natural inactivating mutation of TLR4, revealed that Ang4 expression is TLR4 independent. Subsequent experiments with IL-13 and IL-4 receptor alpha deficient mice demonstrated that goblet cell expression of Ang4 is controlled either directly or indirectly by IL-13. CONCLUSIONS: The cellular source of mouse Ang4 in the colon following T. muris infection is the goblet cell and expression is under the control of IL-13.

Changes in protein expression in the sheep abomasum following trickle infection with Teladorsagia circumcincta.

Continual low-level exposure of sheep to the helminth Teladorsagia circumcincta elicits a temporary protective immunity, where factors in the immune abomasal mucosa prevent penetration of infective larvae, but which is essentially lost within 6 weeks of cessation of parasite challenge. Here, a proteomic approach was used to identify proteins that are differentially regulated in immune compared to naïve sheep, as potential key mediators of immunity. Six naïve sheep and 12 sheep trickle-infected with T. circumcincta were treated with anthelmintic, and the naïve (control) and 6 immune sheep were killed 7 days later. The remaining 6 sheep (immune waning) were killed 42 days after anthelmintic treatment. Abomasal tissue samples were subjected to 2D-gel electrophoresis and densitometric analysis. Selected spots (n=73) were identified by peptide mass fingerprinting and confirmatory Western blotting was carried out for 10 proteins. Spots selectively up-regulated in immune versus control, but not immune waning versus control sheep, included galectin-15 and thioredoxin, which were confirmed by Western blotting. In immune sheep, serum albumin was significantly down-regulated and albumin proteolytic cleavage fragments were increased compared to controls. Unexpectedly, albumin mRNA was relatively highly expressed in control mucosa, down-regulated in immune, and was immunolocalized to mucus-producing epithelial cells. Thus we have identified differential expression of a number of proteins following T. circumcincta trickle infection that may play a role in host protection and inhibition of parasite establishment.

Genome-wide transcriptomic analysis of intestinal tissue to assess the impact of nutrition and a secondary nematode challenge in lactating rats.

BACKGROUND: Gastrointestinal nematode infection is a major challenge to the health and welfare of mammals. Although mammals eventually acquire immunity to nematodes, this breaks down around parturition, which renders periparturient mammals susceptible to re-infection and an infection source for their offspring. Nutrient supplementation reduces the extent of periparturient parasitism, but the underlying mechanisms remain unclear. Here, we use a genome wide approach to assess the effects of protein supplementation on gene expression in the small intestine of periparturient rats following nematode re-infection. METHODOLOGY/PRINCIPAL FINDINGS: The use of a rat whole genome expression microarray (Affymetrix Gene 1.0ST) showed significant differential regulation of 91 genes in the small intestine of lactating rats, re-infected with Nippostrongylus brasiliensis compared to controls; affected functions included immune cell trafficking, cell-mediated responses and antigen presentation. Genes with a previously described role in immune response to nematodes, such as mast cell proteases, and intelectin, and others newly associated with nematode expulsion, such as anterior gradient homolog 2 were identified. Protein supplementation resulted in significant differential regulation of 64 genes; affected functions included protein synthesis, cellular function and maintenance. It increased cell metabolism, evident from the high number of non-coding RNA and the increased synthesis of ribosomal proteins. It regulated immune responses, through T-cell activation and proliferation. The up-regulation of transcription factor forkhead box P1 in unsupplemented, parasitised hosts may be indicative of a delayed immune response in these animals. CONCLUSIONS/SIGNIFICANCE: This study provides the first evidence for nutritional regulation of genes related to immunity to nematodes at the site of parasitism, during expulsion. Additionally it reveals genes induced following secondary parasite challenge in lactating mammals, not previously associated with parasite expulsion. This work is a first step towards defining disease predisposition, identifying markers for nutritional imbalance and developing sustainable measures for parasite control in domestic mammals.

Oral tolerance induction does not resolve gastrointestinal inflammation in a mouse model of food allergy.

SCOPE: Oral immunotherapy (OIT) involving continuous oral administration of allergenic foods has gained attention as a therapy for food allergies. To study the influence of oral administration of allergenic foods on gastrointestinal symptoms including inflammation, we established a mouse model of food-induced intestinal allergy. METHODS AND RESULTS: BALB/c mice were fed an egg white (EW) diet containing ovalbumin (OVA, a major EW allergen) after intraperitoneal sensitisation with OVA and Alum. The mice on the EW diet for one wk presented gastrointestinal symptoms (i.e. weight loss and soft stools) and inflammation in the small intestines (i.e. duodenum, jejunum and ileum). Further continuous EW diet resolved the weight loss but not the soft stools. Splenic CD4(+) T-cells of EW diet-fed mice on the continuous diet showed less proliferation and cytokine production compared with those of control mice, suggesting tolerance induction by the diet. The continuous EW diet reduced levels of OVA-specific IgE antibodies, but significantly aggravated the inflammation in the jejunum. CONCLUSION: Our mouse model would be useful to investigate inflammatory and regulatory mechanisms in food-induced intestinal allergies. Our results suggest potential gastrointestinal inflammation in patients undergoing OIT as continuous administration of allergenic foods, even though the therapy may induce clinical tolerance.

Novel gene expression responses in the ovine abomasal mucosa to infection with the gastric nematode Teladorsagia circumcincta.

Infection of sheep with the gastric nematode Teladorsagia circumcincta results in distinct Th2-type changes in the mucosa, including mucous neck cell and mast cell hyperplasia, eosinophilia, recruitment of IgA/IgE producing cells and neutrophils, altered T-cell subsets and mucosal hypertrophy. To address the protective mechanisms generated in animals on previous exposure to this parasite, gene expression profiling was carried out using samples of abomasal mucosa collected pre- and post- challenge from animals of differing immune status, using an experimental model of T. circumcincta infection. Recently developed ovine cDNA arrays were used to compare the abomasal responses of sheep immunised by trickle infection with worm-naïve sheep, following a single oral challenge of 50 000 T. circumcincta L3. Key changes were validated using qRT-PCR techniques. Immune animals demonstrated highly significant increases in levels of transcripts normally associated with cytotoxicity such as granulysin and granzymes A, B and H, as well as mucous-cell derived transcripts, predominantly calcium-activated chloride channel 1 (CLCA1). Challenge infection also induced up-regulation of transcripts potentially involved in initiating or modulating the immune response, such as heat shock proteins, complement factors and the chemokine CCL2. In contrast, there was marked infection-associated down-regulation of gene expression of members of the gastric lysozyme family. The changes in gene expression levels described here may reflect roles in direct anti-parasitic effects, immuno-modulation or tissue repair.

Sheep intelectin-2 co-purifies with the mucin Muc5ac from gastric mucus.

Secretion of gastric mucins plays an essential role in host protection, and modifications in mucus properties are characteristic of the protective immune responses to pathogens. This study describes the purification and characterisation of sheep gastric mucins, and identification of those proteins that co-purify with mucins, with the potential to modify mucus properties. Gastric mucus was collected and pooled from four abattoir sheep and separated by CsCl density gradient centrifugation. Proteomic analysis of the mucin-containing fraction indicated the presence of gastric mucin (Muc5ac) and several co-purifying proteins, including intelectin-2 (Itln2). Further experimentation indicated that a combination of denaturation and reduction was required to fully release Itln2 from gastric mucin. A putative correlation was found between mucin-bound intelectin concentration and rheological properties in further sheep gastric mucus samples. In conclusion, this study provides the first characterisation of sheep gastric mucins and their purification partners, revealing potentially important mucin-intelectin interactions.

Strain-specific copy number variation in the intelectin locus on the 129 mouse chromosome 1.

BACKGROUND: C57BL/6J mice possess a single intelectin (Itln) gene on chromosome 1. The function of intelectins is not well understood, but roles have been postulated in insulin sensitivity, bacterial recognition, intestinal lactoferrin uptake and response to parasites and allergens. In contrast to C57BL/6J mice, there is evidence for expansion of the Itln locus in other strains and at least one additional mouse Itln gene product has been described. The aim of this study was to sequence and characterise the Itln locus in the 129S7 strain, to determine the nature of the chromosomal expansion and to inform possible future gene deletion strategies. RESULTS: Six 129S7 BAC clones were sequenced and assembled to generate 600 kbp of chromosomal sequence, including the entire Itln locus of approximately 500 kbp. The locus contained six distinct Itln genes, two CD244 genes and several Itln- and CD244-related pseudogenes. It was approximately 433 kbp larger than the corresponding C57BL/6J locus. The expansion of the Itln locus appears to have occurred through multiple duplications of a segment consisting of a full-length Itln gene, a CD244 (pseudo)gene and an Itln pseudogene fragment. Strong evidence for tissue-specific distribution of Itln variants was found, indicating that Itln duplication contributes more than a simple gene dosage effect. CONCLUSIONS: We have characterised the Itln locus in 129S7 mice to reveal six Itln genes with distinct sequence and expression characteristics. Since C57BL/6J mice possess only a single Itln gene, this is likely to contribute to functional differences between C57BL/6J and other mouse strains.

NKp46 defines ovine cells that have characteristics corresponding to NK cells.

Natural killer (NK) cells are well recognized as playing a key role in innate immune defence through cytokine production and cytotoxic activity; additionally recent studies have identified several novel NK cell functions. The ability to study NK cells in the sheep has been restricted due to a lack of specific reagents. We report the generation of a monoclonal antibody specific for ovine NKp46, a receptor which in a number of mammals is expressed exclusively in NK cells. Ovine NKp46+ cells represent a population that is distinct from CD4+ and γδ+ T-cells, B-cells and cells of the monocytic lineage. The NKp46+ cells are heterogenous with respect to expression of CD2 and CD8 and most, but not all, express CD16--characteristics consistent with NK cell populations in other species. We demonstrate that in addition to populations in peripheral blood and secondary lymphoid organs, ovine NKp46+ populations are also situated at the mucosal surfaces of the lung, gastro-intestinal tract and non-gravid uterus. Furthermore, we show that purified ovine NKp46+ populations cultured in IL-2 and IL-15 have cytotoxic activity that could be enhanced by ligation of NKp46 in re-directed lysis assays. Therefore we conclude that ovine NKp46+ cells represent a population that by phenotype, tissue distribution and function correspond to NK cells and that NKp46 is an activating receptor in sheep as in other species.

The role of Intelectin-2 in resistance to Ascaris suum lung larval burdens in susceptible and resistant mouse strains.

The underlying mechanism of predisposition to Ascaris infection is not yet understood but host genetics are thought to play a fundamental role. We investigated the association between the Intelectin-2 gene and resistance in F2 mice derived from mouse strains known to be susceptible and resistant to infection. Ascaris larvae were isolated from murine lungs and the number of copies of the Intelectin-2 gene was determined in F2 mice. Intelectin-2 gene copy number was not significantly linked to larval burden. In a pilot experiment, the response to infection in parental mice of both sexes was observed in order to address the suitability of female F2 mice. No overall significant sex effect was detected. However, a divergence in resistance/susceptibility status was observed between male and, female hybrid offspring. The responsiveness to Ascaris in mice is likely to be controlled by multiple genes and, despite a unique absence from the susceptible C57BL/6j strain, the Intelectin-2 gene does not play a significant role in resistance. The observed intra-strain variation in larval burden requires further investigation but we hypothesize that it stems from social/dominance hierarchies created by the presence of female mice and possibly subsequent hormonal perturbations that modify the intensity of the immune response.

Chronic allergen challenge induces bronchial mast cell accumulation in BALB/c but not C57BL/6 mice and is independent of IL-9.

As genetically engineered mutant mice deficient in single genes are usually generated on a C57BL/6 background, to study mast cell trafficking in mutant mice, we initially investigated whether mast cells accumulated in bronchi in C57BL/6 mice challenged with OVA allergen acutely or chronically for 1 to 3 months. The total number of bronchial mast cells were quantitated using toluidine blue staining in airways of different sizes, i.e. , small (<90 microm), medium (90-155 microm), or large (>150 microm) airways. Non-OVA challenged and acute OVA challenged mice (C57BL/6 and BALB/c) had no detectable bronchial mast cells. Chronic OVA challenge in BALB/c mice for 1 or 3 months induced a significant increase in the number of bronchial mast cells in small-, medium-, and large-sized airways but minimal change in the number of bronchial mast cells in C57BL/6 mice. Both BALB/c and C57BL/6 mice developed significant lung eosinophilia following acute or chronic OVA challenge. Studies of IL-9-deficient mice on a BALB/c background demonstrated a significant increase in the number of bronchial mast cells in IL-9-deficient mice suggesting that IL-9 was not required for the bronchial accumulation of mast cells. Overall, these studies demonstrate that the chronic OVA challenge protocol we have utilized in BALB/c mice provides a model to study the mechanism of bronchial mast cell accumulation and that bronchial mast cell accumulation in chronic OVA challenged mice is independent of IL-9 in this model.

In vitro interactions of extracellular histones with LDL suggest a potential pro-atherogenic role.

BACKGROUND: Nuclear histones have previously been shown to aggregate LDL in vitro, suggestive of a possible pro-atherogenic role. Recent studies indicate that histones are released during acute inflammation, and therefore might interact with circulating lipoproteins in vivo. In view of the associative link between inflammation and cardiovascular disease, the behaviour of histones was investigated using in vitro models of LDL retention and foam cell formation. METHODOLOGY/PRINCIPAL FINDINGS: Heparin agarose beads were used as a model of a matrix rich in sulphated glycosaminoglycans, to which histones bind strongly. Histone-modified beads were observed to pull down more LDL from solution than untreated beads, indicating that histones can function as bridging molecules, enhancing LDL retention. Furthermore, addition of heparin inhibited histone-induced aggregation of LDL. To model foam cell formation, murine RAW 264.7 macrophages were incubated for 24 h in the presence of LDL, histones, LDL plus histones or vehicle control. Cells incubated with LDL in the presence of histones accumulated significantly more intracellular lipid than with LDL or histone alone. CONCLUSIONS/SIGNIFICANCE: These results are consistent with a potential pro-atherogenic role for extracellular histones, which should be investigated further.

Proteomic identification of interactions between histones and plasma proteins: implications for cytoprotection.

Extracellular histones released from cells during acute inflammation contribute to organ failure and death in a mouse model of sepsis, and histones are known to exert in vitro cytotoxicity in the absence of serum. Since addition of histones to serum and plasma is known to induce protein aggregation, we reasoned that plasma proteins may afford protection from cytotoxicity. We found that MODE-K mouse small intestinal epithelial cells were protected from histone-induced toxicity in the presence of 10% FCS. Therefore, the main aim of this study was to identify histone-interacting plasma proteins that might be involved in cytoprotection. The precipitate formed following addition of calf thymus histones to human EDTA plasma was characterised by shotgun proteomics, identifying a total of 36 protein subunits, including complement components, coagulation factors, protease inhibitors and apolipoproteins. The highly sulphated glycosaminoglycan heparin inhibited histone-induced plasma protein aggregation. Moreover, histones bound to heparin agarose were capable of pulling down plasma proteins from solution, indicating their effective cross-linking properties. It was particularly notable that inter-alpha-trypsin inhibitor was prominent among the histone-precipitated proteins, since it contains a chondroitin sulphate glycan chain, and suggests a potential role for this protein in histone sequestration during acute inflammation in vivo.

Expulsion of Trichuris muris is associated with increased expression of angiogenin 4 in the gut and increased acidity of mucins within the goblet cell.

BACKGROUND: Trichuris muris in the mouse is an invaluable model for infection of man with the gastrointestinal nematode Trichuris trichiura. Three T. muris isolates have been studied, the Edinburgh (E), the Japan (J) and the Sobreda (S) isolates. The S isolate survives to chronicity within the C57BL/6 host whereas E and J are expelled prior to reaching fecundity. How the S isolate survives so successfully in its host is unclear. RESULTS: Microarray analysis was used as a tool to identify genes whose expression could determine the differences in expulsion kinetics between the E and S T. muris isolates. Clear differences in gene expression profiles were evident as early as day 7 post-infection (p.i.). 43 probe sets associated with immune and defence responses were up-regulated in gut tissue from an E isolate-infected C57BL/6 mouse compared to tissue from an S isolate infection, including the message for the anti-microbial protein, angiogenin 4 (Ang4). This led to the identification of distinct differences in the goblet cell phenotype post-infection with the two isolates. CONCLUSION: Differences in gene expression levels identified between the S and E-infected mice early during infection have furthered our knowledge of how the S isolate persists for longer than the E isolate in the C57BL/6 mouse. Potential new targets for manipulation in order to aid expulsion have been identified. Further we provide evidence for a potential new marker involving the acidity of the mucins within the goblet cell which may predict outcome of infection within days of parasite exposure.

Expression of three intelectins in sheep and response to a Th2 environment.

Sheep intelectin1 and sheep intelectin3 (sITLN1 and sITLN3) were cloned and sequenced. The amino acid sequences of sITLN1 and sITLN3 shared 86% and 91% homology with the previously cloned sheep intelectin2 (sITLN2), respectively. Expression of sITLN1 and sITLN3 transcript was demonstrated in abomasum, lung, colon and gastric lymph node, terminal rectum, skin, jejunum, mesenteric lymph node, ileal peyer's patches, brain, kidney, liver, spleen, skin, ear pinna, heart and ovary in normal sheep tissues. sITLN2 transcript expression was restricted to the abomasal mucosa in normal sheep tissues. Using a non selective chicken anti-intelectin antibody, tissue intelectin protein was demonstrated in mucus neck cells in the abomasum, mucus cells in the colon, free mucus in ileum, goblet cells in the lung, small intestinal epithelium and brush border, epidermal layer of the skin and skin sebaceous glands. The expression of the three sITLN transcripts was examined in two nematode infections in sheep known to induce a Th2 response; a Teladorsagia circumcincta challenge infection model and a Dictyocaulus filaria natural infection. The three sITLN were absent in unchallenged naïve lambs and present in the abomasal mucosa of both naïve and immune lambs following T. circumcincta challenge infection. Upregulation of sITLN2 and sITLN3 was shown in sheep lung following D. filaria natural infection. Intelectins may play an important role in the mucosal response to nematode infections in ruminants.

Bovine intelectins: cDNA sequencing and expression in the bovine intestine.

Intelectins (Itlns) are lectins with potential roles in innate immunity, capable of binding bacteria via galactofuranose residues. Itlns also function as intestinal receptors for the antimicrobial glycoprotein lactoferrin (Lf). Since Lf binds strongly to enterohemorrhagic Escherichia coli O157:H7 (EHEC), we aimed to determine the expression of Lf receptor in terminal rectum, the site of predilection of EHEC in cattle. We sequenced two bovine intelectins (Itln1 and Itln2) and showed that both were expressed in abomasum and rectum, but expression appeared minimal in the jejunum. There was significantly higher expression of Itln2 in terminal rather than proximal rectum. Lactoferrin was expressed in all samples examined. Thus, we have demonstrated two novel bovine Itlns and shown that they are expressed along with Lf in the gastrointestinal tract, where they may interact with microbial pathogens.