The aim of this study was to identify uterine pluripotent cells both in bovine uterine tissues as well in epithelial, stromal, and myometrial uterine cell populations. Moreover, the relationship of pluripotent markers expression with age and the uterine horn side was considered. Uterine tissue was collected from ipsilateral and contralateral horns (days 8-10 of the estrous cycle). Immunohistostaining for C-KIT, OCT3/4, NANOG, and SOX2 in uterine tissue was determined. mRNA expression of C-KIT, OCT3/4, NANOG and SOX2 was evaluated in uterine tissue relative to the age of the cow and uterine horn side. Gene and protein expression of these markers in the uterine luminal epithelial, stromal, and myometrial cells was evaluated by real-time PCR and western blotting respectively. The expression of pluripotent cell markers OCT3/4, NANOG, and SOX2 was identified by flow cytometry assay in epithelial, stromal, and myometrial cells. Multilineage differentiation of the bovine uterine cells was performed. mRNA expression of OCT3/4, NANOG, and SOX2 in uterine tissue was higher in the ipsilateral horn than in the contralateral horn. Flow cytometry assay revealed positive fluorescence for OCT3/4, NANOG, and SOX2 in all uterine cell types. Results showed the age-dependent expression of pluripotent markers in uterine tissue. Beside, the different expression of pluripotent cells in each horn of uterus suggests the influence of ovarian hormones on these characteristics. The highest mRNA and protein expression for pluripotent markers was observed in stromal cells among uterine cells, which indicates this population of cells as the main site of pluripotent cells in the cow uterus.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's Disease in ruminants. This enteritis has significant economic impact and worldwide distribution. Vaccination is one of the most cost effective infectious disease control measures. Unfortunately, current vaccines reduce clinical disease and shedding, but are of limited efficacy and do not provide long-term protective immunity. Several strategies have been followed to mine the MAP genome for virulence determinants that could be applied to vaccine and diagnostic assay development. In this study, a comprehensive mutant bank of 13,536 MAP K-10 Tn5367 mutants (P > 95%) was constructed and screened in vitro for phenotypes related to virulence. This strategy was designated to maximize identification of genes important to MAP pathogenesis without relying on studies of other mycobacterial species that may not translate into similar effects in MAP. This bank was screened for mutants with colony morphology alterations, susceptibility to D-cycloserine, impairment in siderophore production or secretion, reduced cell association, and decreased biofilm and clump formation. Mutants with interesting phenotypes were analyzed by PCR, Southern blotting and DNA sequencing to determine transposon insertion sites. These insertion sites mapped upstream from the MAP1152-MAP1156 cluster, internal to either the Mod operon gene MAP1566 or within the coding sequence of lsr2, and several intergenic regions. Growth curves in broth cultures, invasion assays and kinetics of survival and replication in primary bovine macrophages were also determined. The ability of vectors carrying Tn5370 to generate stable MAP mutants was also investigated.

Mastitis-inflammation of the mammary gland is an important disease affecting dairy animals worldwide. The disease is caused by mammary pathogenic bacteria, and Escherichia coli is frequently implicated. Intramammary challenge with bacterial LPS is sufficient to elicit the disease. However, using toll-like receptor (TLR) 4-deficient mice, we previously found that mammary pathogenic E. coli is still able to elicit neutrophil recruitment, indicating the presence of bacterial virulence factors other than LPS. To date, no specific virulence factors have been identified in E. coli isolates associated with mastitis, and other microbe-associated molecular patterns (MAMPs), such as bacterial lipoproteins, are prime candidates. The synthetic analog of bacterial lipopeptides, Pam3CSK4, is recognized by TLR2 and mimics the proinflammatory properties of triacylated lipoproteins of Gram-negative bacteria. The aim of the present work was to determine the role of bacterial lipoproteins recognized by TLR2 on mammary cells as virulence factors in the mammary gland. Using the murine mastitis model, we previously showed that following intramammary LPS challenge, neutrophil recruitment is strictly dependent on alveolar macrophages. Thus, the role of alveolar macrophages in the response to intramammary bacterial lipoprotein challenge was also studied. Here, Pam3CSK4 infusion induced mastitis in wild-type mice, but not in TLR2-deficient mice. The wild-type phenotype was not restored by adoptive transfer of TLR2-expressing macrophages into the alveolar milk space of TLR2-deficient mice, indicating that cells other than alveolar macrophages are essential for Pam3CSK4/TLR2 signaling. In contrast to the Pam3CSK4 treatment, infection with E. coli P4 resulted in inflammation, even in the absence of TLR2 signaling, indicating that lipoproteins are sufficient, but not essential virulence factors in the pathogenesis of the intact bacteria. However, in the absence of TLR2, the infecting E. coli P4 invaded the alveolar epithelial cells and formed intracellular bacterial communities, indicating that intact lipoprotein/TLR2 signaling is essential to restricting bacterial invasion.

Ly6C(hi) monocytes seed the healthy intestinal lamina propria to give rise to resident CX(3)CR1(+) macrophages that contribute to the maintenance of gut homeostasis. Here we report on two alternative monocyte fates in the inflamed colon. We showed that CCR2 expression is essential to the recruitment of Ly6C(hi) monocytes to the inflamed gut to become the dominant mononuclear cell type in the lamina propria during settings of acute colitis. In the inflammatory microenvironment, monocytes upregulated TLR2 and NOD2, rendering them responsive to bacterial products to become proinflammatory effector cells. Ablation of Ly6C(hi) monocytes ameliorated acute gut inflammation. With time, monocytes differentiated into migratory antigen-presenting cells capable of priming naive T cells, thus acquiring hallmarks reminiscent of dendritic cells. Collectively, our results highlight cellular dynamics in the inflamed colon and the plasticity of Ly6C(hi) monocytes, marking them as potential targets for inflammatory bowel disease (IBD) therapy.

Lumpy skin disease (LSD) is a severe viral disease of cattle. Circumstantial evidence suggests that the virus is transmitted mechanically by blood-feeding arthropods. We compared the importance of transmission via direct and indirect contact in field conditions by using mathematical tools. We analyzed a dataset collected during the LSD outbreak in 2006 in a large dairy herd, which included ten separated cattle groups. Outbreak dynamics and risk factors for LSD were assessed by a transmission model. Transmission by three contact modes was modelled; indirect contact between the groups within a herd, direct contact or contact via common drinking water within the groups and transmission by contact during milking procedure. Indirect transmission was the only parameter that could solely explain the entire outbreak dynamics and was estimated to have an overall effect that was over 5 times larger than all other possible routes of transmission, combined. The R0 value induced by indirect transmission per the presence of an infectious cow for 1 day in the herd was 15.7, while the R0 induced by direct transmission was 0.36. Sensitivity analysis showed that this result is robust to a wide range of assumptions regarding mean and standard deviation of incubation period and regarding the existence of sub-clinically infected cattle. These results indicate that LSD virus spread within the affected herd could hardly be attributed to direct contact between cattle or contact through the milking procedure. It is therefore concluded that transmission mostly occurs by indirect contact, probably by flying, blood-sucking insects. This has important implications for control of LSD.

An outbreak of epizootic haemorrhagic disease virus (EHDV) in cattle in Israel in 2006 enabled a comparison of the spatial distribution of epidemic exposure to EHDV with that of exposure to bluetongue virus (BTV), which is endemic in the country. The seroprevalence of both viruses was examined in 1650 serum samples collected from 139 farms representative of the spatial distribution of dairy cattle in Israel. A significant association between exposure to EHDV and BTV was demonstrated in both univariate and multivariate analyses. Recent exposure to BTV and EHDV (demonstrated by seroprevalence in calves) was clustered in different geographical locations, indicating that the two viruses had different patterns of spread, that of EHDV being influenced by winds and terrain barriers and that of BTV by herd immunity.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important cause of diarrhea, hemorrhagic colitis and hemolytic uremic syndrome in humans worldwide. The two major virulence determinants of EHEC are the Shiga toxins (Stx) and the type III secretion system (T3SS), including the injected effectors. Lack of a good model system hinders the study of EHEC virulence. Here, we investigated whether bovine and human intestinal xenografts in SCID mice can be useful for studying EHEC and host tissue interactions. Fully developed, germ-free human and bovine small intestine and colon were established by subcutaneous transplantation of human and bovine fetal gut into SCID mice. Xenografts were allowed to develop for 3-4 months and thereafter were infected by direct intraluminal inoculation of Stx-negative derivatives of EHEC O157:H7, strain EDL933. The small intestine and colon xenografts closely mimicked the respective native tissues. Upon infection, EHEC induced formation of typical attaching and effacing lesions and tissue damage that resembled hemorrhagic colitis in colon xenografts. By contrast, xenografts infected with an EHEC mutant deficient in T3SS remained undamaged. Furthermore, EHEC did not attach to or damage the epithelium of small intestinal tissue, and these xenografts remained intact. EHEC damaged the colon in a T3SS-dependent manner, and this model is therefore useful for studying the molecular details of EHEC interactions with live human and bovine intestinal tissue. Furthermore, we demonstrate that Stx and gut microflora are not essential for EHEC virulence in the human gut.

Mastitis, the inflammation of the mammary gland, is an important disease affecting dairy animals worldwide. The disease is caused by mammary pathogenic bacteria and Escherichia coli are frequently implicated. Virulence factors of mammary pathogenic E. coli are only partially known and intramammary challenge with LPS elicits neutrophil recruitment in experimental bovine and murine mastitis models. We have previously shown that neutrophil recruitment in LPS-induced murine mastitis is strictly dependent on mammary alveolar macrophages. However, the relative role of alveolar macrophages and blood neutrophils in E. coli mastitis is not well defined. To this end, we selectively depleted mammary alveolar macrophages or blood neutrophils before intramammary challenge with E. coli strain P4 (ECP4). Mice depleted of alveolar macrophages prior to intramammary challenge recruited neutrophils normally and restricted bacterial growth and interstitial invasion. Importantly however, upon depletion of alveolar macrophages, ECP4 invaded the mammary alveolar epithelial cells and formed intracellular bacterial communities. In contrast, neutrophil depletion prior to intramammary infection with ECP4 was associated with unrestricted bacterial growth, tissue damage, severe sepsis and mortality. This study suggests that neutrophils but not alveolar macrophages provide essential antimicrobial defense against mammary pathogenic E. coli. Furthermore, we show here similar invasion after depletion of alveolar macrophages as in our previous studies showing that LPS/TLR4 signaling on alveolar macrophages abrogates ECP4 invasion of the mammary epithelium. Interestingly, similar ECP4 invasion and formation of intracellular communities were also observed following intramammary infection of either iNOS gene-deficient or IL-1 receptor type 1 gene-deficient mice.

Mastitis, inflammation of the mammary tissue, is a common disease in dairy animals and mammary pathogenic Escherichia coli (MPEC) is a leading cause of the disease. Lipopolysaccharide (LPS) is an important virulence factor of MPEC and inoculation of the mammary glands with bacterial LPS is sufficient to induce an inflammatory response. We previously showed using adoptive transfer of normal macrophages into the mammary gland of TLR4-deficient C3H/HeJ mice that LPS/TLR4 signaling on mammary alveolar macrophages is sufficient to elicit neutrophil recruitment into the alveolar space. Here we show that TLR4-normal C3H/HeN mice, depleted of alveolar macrophages, were completely refractory to LPS intramammary challenge. These results indicate that alveolar macrophages are both sufficient and essential for neutrophil recruitment elicited by LPS/TLR4 signaling in the mammary gland. Using TNFalpha gene-knockout mice and adoptive transfer of wild-type macrophages, we show here that TNFalpha produced by mammary alveolar macrophages in response to LPS/TLR4 signaling is an essential mediator eliciting blood neutrophil recruitment into the milk spaces. Furthermore, using the IL8 receptor or IL1 receptor gene-knockout mice we observed abrogated recruitment of neutrophils into the mammary gland and their entrapment on the basal side of the alveolar epithelium in response to intramammary LPS challenge. Adoptive transfer of wild-type neutrophils to IL1 receptor knockout mice, just before LPS challenge, restored normal neutrophil recruitment into the milk spaces. We conclude that neutrophil recruitment to the milk spaces is: (i) mediated through TNFalpha, which is produced by alveolar macrophages in response to LPS/ TLR4 signaling and (ii) is dependent on IL8 and IL1beta signaling and regulated by iNOS-derived NO.

The objective was to investigate the associations between body condition scores (BCS) and daily body weight (BW) in the first 150 d of lactation (DIM) and reproductive performance in high-producing dairy cows. Data included automated daily BW measurements and BCS of 2,020 Israeli Holstein cows from 7 commercial farms. Individual BW series were smoothed using penalized cubic splines, and variables representing BW patterns were generated. The presence of 7- and 21-d cycles in BW was determined using time-series analysis. Associations between BW and BCS and conception at first artificial insemination (AI) were analyzed using generalized estimating equations. Multivariate survival analysis was used for associations between BW and BCS and the calving-to-first AI interval, first AI-to-conception interval, and calving-to-conception interval. First-parity cows that lost >or=12% and second-parity cows that lost >or=15% of their BW from calving to nadir BW were less likely to conceive at first AI. Cows without 7-d cycles in BW were 1.48 times more likely to conceive at first AI relative to cows with 7-d cycles. The odds of conceiving at first AI increased by 53% for each additional unit in BCS from 40 to 60 DIM. In the multivariate survival analysis, a BCS of or=7% from calving to 10 DIM were associated with reduced reproductive performance. The presence of 21-d cycles in BW was associated with high reproductive performance in first-parity [odds ratio (OR) = 1.18] and second-parity cows (OR = 1.22). The presence of 7-d cycles in BW was associated with low reproductive performance in first-parity cows (OR = 0.77), but not in older cows. Based on previous findings and on the associations found in this study, we postulate that 21-d cycles are probably related to the sexual cycle and could be used as a proxy for assessing ovarian activity. Variables representing relative BW loss (%) were better predictors for impaired reproductive performance than those representing absolute BW loss (kg) and may be more suitable for estimating individual adaptation to negative energy balance in herds for which automated daily BW is available.

The objective of this study was to determine associations between body weight (BW) and body condition score (BCS) variables indicating a more severe negative energy balance in early lactation and events of somatic cell counts (SCC) >250,000 cells/mL and SCC >400,000 cells/mL in dairy cows. We studied lactations from 634 primiparous and 1,086 multiparous Israeli Holstein dairy cows originating from 7 commercial dairy farms. Generalized mixed models with a random herd effect were used to quantify the effects of BW and BCS variables in early lactation on events of elevated SCC. Data were analyzed using 2 different approaches. In the first approach, only first events in a lactation were taken into account, whereas in the second approach, all events in a lactation were analyzed and repeated events from the same cow were accounted for. Although no associations were found between the different BW and BCS variables and first events of elevated SCC, associations were present between these variables and events of elevated SCC when all events were analyzed. The cumulative incidence of a lactation with multiple events of SCC >250,000 cells/mL was 8.8 and 27.7% for primiparous and multiparous cows, respectively. The odds of an event of SCC >250,000 cells/mL were 25% greater for cows belonging to the upper quartile in relative BW loss from calving to nadir BW (loss >12.3, 15.0, and 15.7% for first-, second-, and third- parity and greater cows, respectively) compared with cows losing less relative BW. Odds of an event were 44% greater for cows with ketosis when compared with cows without. The cumulative incidence of a lactation with multiple events of SCC >400,000 cells/mL was 4.1 and 14.3% for primiparous and multiparous cows, respectively. The odds of an event of SCC >400,000 cells/mL were 43% greater for cows belonging to the upper quartile in relative BW loss from calving to nadir BW compared with cows losing less relative BW. Odds of an event were 33% greater for cows with ketosis when compared with cows without. Assuming that extreme BW loss and ketosis in early lactation indicate a more severe negative energy balance, our findings support the hypothesis that greater negative energy balance in early lactation predisposes dairy cows to udder inflammation. Considering the fact that many of the events were recurring, we stress the importance of including all events in the analysis and postulate the possibility of long-term detrimental effects of negative energy balance on udder health.

Crohn disease is a chronic inflammatory bowel disease of unknown etiology. Mycobacterium avium paratuberculosis (MAP) was found in the gut of patients with Crohn disease, but causality was not established. Fully developed, germ-free human small intestine and colon were established by subcutaneous transplantation of fetal gut into SCID (severe combined immunodeficiency) mice thereafter infected by direct intraluminal inoculation of MAP. We have found that MAP actively invades the human gut epithelial goblet cells of the small intestine, inducing severe tissue damage and inflammation. These observations indicate that MAP can specifically colonize the normal human small intestine and can elicit inflammation and severe mucosal damage.

The objective of this study was to investigate, describe, and quantify daily body weight (BW) changes in the first 120 d of lactation in high-producing dairy cows. Data included 255,287 daily BW measurements from 2,167 Israeli Holstein dairy cows originating from 7 commercial dairy farms. Individual series of measurements were first smoothed using cubic splines for generating variables representing BW changes in early lactation and further analysis of the data. To construct standard BW curves stratified by parity and adjusted for farm, mixed models for repeated measurements were fit to the smoothed data, and least squares means for day in lactation were plotted. Time-series analysis techniques using polynomial functions of day in lactation and pairs of sine and cosine functions representing 7- and 21-d cycles were performed separately on each individual series of measurements. Additionally, generalized estimating equations were used to perform similar analysis on the data set as a whole. Mean days from calving to nadir BW increased significantly from first to later parities, as did mean BW loss from calving to nadir. The first-parity cow lost 6.5% of her BW from calving to d 29 in lactation, and second-parity and greater-parity cows lost 8.5 and 8.4% of their BW to d 34 and 38 in lactation, respectively. After nadir BW was reached, first-parity cows regained relative BW at a greater rate than did older parity cows. The trend in BW was nonlinear. A 7-d cycle was present in 247 cows (11.4%) and a 21-d cycle was present in 715 cows (33.0%). Presence of a 21-d cycle was associated with a 33% reduction in the risk of being diagnosed with inactive ovaries. Fewer days from calving to nadir BW and smaller BW loss from calving to nadir, coupled with a faster post-nadir increase in relative BW in first-parity cows compared with older cows indicated a smaller energy deficit in early lactation. Association between 21-d cycles in BW and ovarian activity suggest that these cycles were physiological and related to the estrous cycle. Therefore, monitoring them could be useful for indirectly assessing ovarian activity in a herd.

Escherichia coli is an important bacterial species isolated from bovine mastitis. The rate of neutrophil recruitment into the mammary gland and their bactericidal activity largely affect the severity and outcome of the disease. Ketosis is a common metabolic disease, and affected dairy cows are known to have increased risk for mastitis and other infectious conditions. The disease is associated with high blood and milk levels of beta-hydroxybutyrate (BHBA), previously shown to negatively affect neutrophil function by unknown mechanisms. We show here that the mammary pathogenic E. coli strain P4 activates normal bovine neutrophils to form neutrophil extracellular traps (NETs), which are highly bactericidal against this organism. Preincubation of these neutrophils with increasing concentrations (0.1 to 8 mmol/liter) of BHBA caused a fivefold decrease of E. coli P4 phagocytosis, though intracellular killing was unaffected. Furthermore, BHBA caused a 10-fold decrease in the NETs formed by E. coli P4-activated neutrophils and a similar decrease in NET bactericidal activity against this organism. These negative effects of BHBA on bovine neutrophils might explain the increased susceptibility of ketotic cows to mastitis and other infectious conditions.

Mastitis, inflammation of the mammary gland, is a common and economically important disease in dairy animals. Mammary pathogenic organisms, such as Escherichia coli, invade the teat canal,milk ducts, and mammary alveolar space, replicate in mammary secretions, and elicit a local inflammatory response characterized by massive recruitment of blood polymorphonuclear neutrophil leukocytes (PMN) into the alveoli and milk ducts. CD44 is a trans-membrane glycoprotein previously shown to play a role in mediation and control of blood PMN recruitment in response to inflammatory signals. Here we show, for the first time, increased expression of CD44 on recruited milk PMN in bovine mastitis and the expression of a CD44 variant, CD44v10, on these PMN. Furthermore, we demonstrate that CD44 mediates specific adhesion of bovine blood PMN to hyaluronic acid and mammary epithelial cells. Our results suggest that in mastitis CD44 plays a role in recruiting blood PMN into the mammary glands, the exact nature of this role needs to be elucidated.

Pathogenic Escherichia coli can be classified into several pathotypes, and it is believed that each pathotype carries one or more specific gene repertoire (or virulence factors combination) that distinguishes them from non-pathogenic E. coli strains and from other pathotypes. In contrast to this notion, it was proposed that this is not the case for E. coli mastitis, a common disease in farm animals and that any given E. coli isolate can cause this disease, even strains that are considered non-pathogenic. In this review we will re-examine this latter concept and recent advances in the study E. coli mastitis.

Cattle botulism is a food-borne intoxication caused by the ingestion of preformed botulinum neurotoxins (BoNT) of serotypes B, C, or D. Protection in cattle against botulinum intoxication is based on the presence of specific serum neutralizing antibodies upon exposure. Outbreaks in vaccinated cattle have raised concerns about vaccine quality and efficacy. To this end, three different immunization protocols and the effect of maternal anti-BoNT/D antibodies, at the priming dose, were analyzed in 2-month-old dairy calves. Based on previously determined protective anti-BoNT/D antibody levels analyzed in field outbreaks, the immune response to type D toxoids was analyzed using an in-house ELISA system. Here we show that using the current vaccination strategy of using a priming dose in 2-month-old calves followed by booster doses after 4 weeks and annually thereafter, did not result in continuous protective levels of anti-BoNT/D antibodies. As a result of this vaccination protocol, only 15-31% of cattle in parities 1-3 were protected at the time of the annual booster. Vaccination study in calves indicated that adding a 6-month booster dose to the current protocol resulted in continuous protective levels of anti-BoNT/D antibodies well above the cut-off protective levels. The presence of maternally derived anti-BoNT/D antibodies did not interfere with the immune response to toxoids that can be administered to 2-month-old calves.

Serum samples from 35 golden jackals (Canis aureus syriacus), eight wolves (Canis lupus), and four red foxes (Vulpes vulpes) from various regions of Israel were collected during the years 2001-04 and tested for antibodies to Clostridium botulinum neurotoxin (BoNT) types C and D. Antibodies against BoNT types C and D were detected in 10 (29%) and in 3 (9%) of 35 golden jackals, respectively, using enzyme-linked immunosorbent assay. This report describes detection of anti BoNT antibodies in wild canids other than coyotes (Canis latrans) for the first time and demonstrates that C. botulinum type C is prevalent in Israel.

REASONS FOR PERFORMING STUDY: Clostridium botulinum type C is prevalent in Israel and outbreaks recorded in many species, other than horses. Association between levels of anti-BoNT/C antibodies and equine grass sickness (EGS) have been demonstrated but seroprevalence of anti-BoNT/C antibodies in horses has not been reported nor has EGS been reported in Israel. OBJECTIVES: To determine the seroprevalence of specific anti-BoNT/C antibodies in horses in Israel and to determine whether age, breed and gender, or geographical region of farms are potential risk factors for exposure to BoNT/C. HYPOTHESIS: Anti-BoNT/C antibodies are prevalent among horses in Israel and farm and horse-level variables are associated with increased risk for exposure. METHODS: Serum samples from 198 horses were collected and the levels of specific anti-BoNT/C antibodies were determined using enzyme-linked immunosorbent assay (ELISA). For each categorical variable indicator variables were created and the odds ratio (OR) and 95% confidence intervals (95% CI) for the outcome variable were calculated using a univariable and multivariable logistic regression models. RESULTS: A total of 61 (30.8%) horses were ELISA positive for anti-BoNT/C IgG antibodies. The farm and its geographical region were associated significantly with seropositivity, horse-level variables, such as gender and breed, were also associated with seropositivity. Quarter Horse and Warmblood mares placed in the southern region of Israel had the highest odds to be tested positive for anti-BoNT/C IgG antibodies. CONCLUSIONS AND POTENTIAL RELEVANCE: Several farm and various horse-level risk factors for exposure to BoNT/C, found in this study, could be correlated to previously reported risk factors of EGS. Studies are required to determine the predisposing factors that cause EGS, which is apparently not present in Israel.

Mastitis, an inflammatory response of the mammary tissue to invading pathogenic bacteria, is a common disease in breast-feeding women and dairy animals. Escherichia coli is a leading cause of mastitis in dairy animals. During the course of the disease the host mounts a strong inflammatory response, but specific bacterial virulence factors have not yet been identified. Here we report the use of a murine mastitis model to investigate the innate inflammatory reaction of the mammary gland. We show that lipopolysaccharide (LPS) infusion induces mastitis in wild-type mice (C3H/HeN), but not in mice expressing mutated Toll-like receptor 4 (TLR4) (C3H/HeJ). The wild-type phenotype was restored by adoptive transfer of TLR4-expressing macrophages into the alveolar milk space of C3H/HeJ mice. In contrast to the LPS treatment, infection with E. coli P4 (ECP4) resulted in inflammation even in the absence of LPS/TLR4 signalling, indicating that additional factors play a role in the pathogenesis of the intact bacteria. Furthermore, in the absence of functional TLR4 the infecting ECP4 invade the epithelial cells with high efficiency, forming intracellular microcolonies. However, adoptive transfer with TLR4-expressing macrophages drastically reduced the epithelial invasion. Taken together, these results indicate that ECP4 has an invasive potential, which is restricted by alveolar macrophages in response to the LPS/TLR4 signalling.