Proliferation and transmission patterns of Pasteurella multocida B:2 in goats.

This report describes the proliferation and transmission patterns of Pasteurella multocida B:2 among stressful goats, created through dexamethasone injections. Thirty seven clinically healthy adult goats were divided into three groups consisted of 15 goats in group A, 11 goats in group B and the remaining 11 in group C. At the start of the study, all goats of group A were exposed intranasally to 1.97 x 10(10) CFU/ml of live P multocida B:2. Dexamethasone was immediately administered intramuscularly for 3 consecutive days at a dosage rate of 1 mg/kg. The exposed goats were observed for signs of HS for a period of 1 month. At the end of the 1-month period, 11 goats from group B were introduced into and commingled with the surviving goats of group A before all goats from both groups were immediately injected intramuscularly with dexamethasone for 3 consecutive days. The treatment with dexamethasone was then carried out at monthly interval throughout the 3-month study period. Goats of group C were kept separately as negative control. Three surviving goats from each group were killed at 2-week interval for a complete post-mortem examination. Two (13%) goats of group A were killed within 24 hours after intranasal exposure to P multocida B:2 while another two (13%) goats from the same group were killed on day 40, approximately 10 days after the second dexamethasone injection. All four goats showed signs and lesions typical of haemorrhagic septicaemia. Bacteraemia was detected in 3 goats of group A that were having rectal temperature higher than 41degrees C. The P. multocida B:2 isolation pattern was closely associated with dexamethasone injections when significantly (p < 0.05) higher rate of isolations from both groups were observed after each dexamethasone injection. Transmission of P multocida B:2 from goats of group A to group B was successful when P multocida B:2 was isolated from goats of group B for a period of 28 days. There was a strong correlation between dexamethasone injections, rate of bacterial isolation and serum cortisol level. The IgG level showed an increasing trend 2 weeks after exposure to P multocida B:2 and remained high throughout the study period.

Social stress increases fecal shedding of Salmonella typhimurium by early weaned piglets.

"Segregated early weaning" (SEW) of pigs reduces exposure to pathogenic bacteria, but upon arrival at grower facilities pigs may be co-mingled regardless of farm of origin. The present study was designed to examine the effect of mixing (social) stress on populations of Salmonella enterica Typhimurium in SEW pigs. Piglets (7 days old; n = 28 in each of 2 replicates) were separated into 2 treatments (control and mixed groups) of 2 pens per treatment (7 piglets/pen). One (n = 1) "seeder" pig/pen was inoculated with 10(9) CFU of S. Typhimurium. Each seeder was placed with non-inoculated "contact" piglets (n = 6). A"contact" piglet was swapped each day between the "mixed" pens for 5 days; pigs in control pens were not exchanged. On day 5, the incidence of fecal Salmonella shedding was higher in the mixed contact pigs (P < 0.05). Rectal Salmonella and cecal coliform populations in mixed pigs were significantly (P < 0.05) greater than in control pigs but cecal Salmonella populations were not different. Mixed pigs were more susceptible to tissue invasiveness (i.e., Salmonella-positive tonsils and lymph nodes) than control pigs. These results indicate that social stress of weaned pigs may increase susceptibility to and/or fecal shedding of Salmonella. Food-borne Salmonella infections in the United States are estimated to cost the economy dollar 2.4 billion annually (ERS/USDA, 2001). Approximately 6-9% of human salmonellosis is associated with the consumption of pork products (Frenzen et al., 1999). Salmonella is relatively common on swine farms and has been isolated from all stages of the pork production chain (Davies et al., 1999; Fedorka-Cray et al., 1997b; Rostagno et al., 2003). Salmonella is a threat to the pork industry not only from a food-safety perspective as a public health concern, but some Salmonella serotypes can cause clinical illnesses in swine, negatively impacting production efficiency and profitability (Schwartz, 1991).

Stress induces the translocation of cutaneous and gastrointestinal microflora to secondary lymphoid organs of C57BL/6 mice.

Mammals are colonized by a vast array of bacteria that reside as part of the host's microflora. Despite their enormous levels, these microorganisms tend to be restricted to cutaneous and mucosal surfaces. In the current experiment, only a small percentage of non-stressed mice exhibited detectable levels of bacteria in their inguinal lymph nodes (ILN), spleen, liver, or mesenteric lymph nodes (MLN). However, after experiencing repeated social disruption (SDR), a significant increase in the number of animals having bacteria in their ILN and MLN was found. Since SDR involves fighting in which bite wounds on the skin could provide a portal of entry into the host, it was determined whether experimental wounding (full-thickness skin biopsy), chronic restraint (which is a potent stressor that does not disrupt the skin barrier), or wounding combined with restraint would increase the occurrence of bacteria in secondary lymphoid tissues and liver. Wounding did not significantly increase the prevalence of bacteria in the ILN, MLN, or liver. Interestingly, a larger percentage of restrained and restrained plus wounded mice, in comparison to controls, had bacteria in the ILN, MLN, and liver. Although the stressors increased the number of animals that became colonized, the levels of bacteria in the stressed mice were similar to the levels found in the few non-stressed mice that did become colonized. Our results indicate that psychological components of social stress facilitate the translocation of indigenous bacteria into the host, thus identifying an additional facet through which stressors may impact health.

Enterocyte cytoskeleton changes are crucial for enhanced translocation of nonpathogenic Escherichia coli across metabolically stressed gut epithelia.

Substantial data implicate the commensal flora as triggers for the initiation of enteric inflammation or inflammatory disease relapse. We have shown that enteric epithelia under metabolic stress respond to nonpathogenic bacteria by increases in epithelial paracellular permeability and bacterial translocation. Here we assessed the structural basis of these findings. Confluent filter-grown monolayers of the human colonic T84 epithelial cell line were treated with 0.1 mM dinitrophenol (which uncouples oxidative phosphorylation) and noninvasive, nonpathogenic Escherichia coli (strain HB101, 10(6) CFU) with or without pretreatment with various pharmacological agents. At 24 h later, apoptosis, tight-junction protein expression, transepithelial resistance (TER; a marker of paracellular permeability), and bacterial internalization and translocation were assessed. Treatment with stabilizers of microtubules (i.e., colchicine), microfilaments (i.e., jasplakinolide) and clathrin-coated pit endocytosis (i.e., phenylarsine oxide) all failed to block DNP+E. coli HB101-induced reductions in TER but effectively prevented bacterial internalization and translocation. Neither the TER defect nor the enhanced bacterial translocations were a consequence of increased apoptosis. These data show that epithelial paracellular and transcellular (i.e., bacterial internalization) permeation pathways are controlled by different mechanisms. Thus, epithelia under metabolic stress increase their endocytotic activity that can result in a microtubule-, microfilament-dependent internalization and transcytosis of bacteria. We speculate that similar events in vivo would allow excess unprocessed antigen and bacteria into the mucosa and could evoke an inflammatory response by, for example, the activation of resident or recruited immune cells.

Experimental handling stress as infection-facilitating factor for the goldfish ulcerative disease.

Experimental handling stress (EHS) was applied to clinically asymptomatic farmed goldfish (Carassius auratus L.). EHS affected the gills and skin integrity of the fish and was accompanied by increased levels of plasma glucose, cortisol and interleukin-10 (IL-10). EHS application was followed by highly significant enhancement of the rate of infection with a virulent Aeromonas salmonicida isolate. Cumulative ulceration at the initial phase of the ensuing goldfish ulcerative disease (GUD) evidenced a facilitating role of EHS in the onset of GUD. Host susceptibility to the pathogen increased from 40% in unstressed fish to 90% in the stressed fish. A. salmonicida could be reisolated from the early-stage skin lesions only, whereas opportunistic strains, other than A. salmonicida (A. sobria and A. hydrophila), were recovered from progressive-stage ulcers. The implication of these findings in fish aquaculture is discussed.

Intestinal translocation of Streptococcus suis type 2 EF+ in pigs.

Sepsis with subsequent multisystem organ failure after translocation of bacteria from the gut is a serious risk associated with stress situations. We showed that intestinal bacterial translocation could be one of the pathways for pathogenic Streptococcus suis infections in the pig. In 24 piglets weighing 10-14 kg, free of the extracellular factor (EF+) producing phenotype of S. suis serotype 2, a silicon canula was placed in the proximal jejunum to enable intestinal inoculation and bypassing the upper alimentary tract. The pigs were individually housed. After stress induction in 18 pigs by means of a truck drive in individual cages for 1h, pigs were inoculated through the intestinal canula either with S. suis type 2 EF+ or with growth medium only, and put back in their original housing. The six not transported pigs were also inoculated with the same strain. To prevent oral self-infection, faeces were collected in a bag that was glued around the anus. Clinical and behavioral symptoms were recorded for 72 h post inoculation, and then the animals were sacrificed for pathological and bacteriological examination. In three animals, the inoculation strain was re-isolated from mesenterial lymph nodes and typically affected organs. No S. suis type 2 EF+ was detected by specific polymerase chain reaction (PCR) in any of the tonsil-swabs and -homogenates. We concluded that infection of the organs had taken place after bacterial translocation out of the gut and that the intestinal tract can be a porte d'entree for S. suis type 2 EF+.

Splenic norepinephrine and serum corticosterone level fluctuations associated with bacteria-induced stress.

Corticosterone (CORT) and norepinephrine (NE), two effector molecules of the hypothalamic-pituitary-adrenal (HPA) and the sympathetic-lymphoid (SL) axes, respectively, differentially influence murine host resistance to Listeria monocytogenes (LM). Serum CORT and splenic NE levels early (< or =24 h) after infection correlated positively with host resistance, as long as the LM burden did not exceed approximately 10(6) cfu LM per spleen. As previously reported, mice with right-circling preference (R-mice) have significantly greater host resistance to LM than those with left-circling preference (L-mice) and early after infection, R-mice had significantly higher serum CORT levels than L-mice. However, rapid pathogenesis with a high bacterial burden induced high activation of the HPA and SL axes, which prevented observable differences in the defense against LM, especially later in infection. With the high bacterial inoculum (10(5) LM), the splenic NE levels significantly increased, but no differences among R- and L-mice were discernible. We suggest that endogenous asymmetry of neuroimmune circuits contributes to differential host resistance, but the level of stress (bacterial inoculum) is critical. With regard to the neuroendocrine factors assessed, CORT, but not NE, levels significantly correlated with the enhanced defenses of R-mice in comparison to L-mice. The differential host resistance based on brain laterality seems to be more a function of the HPA axis and possibly other CNS effects on peripheral immunity than neurotransmitter release by the sympathetic innervation of the spleen.

Postnatal microbial colonization programs the hypothalamic-pituitary-adrenal system for stress response in mice.

Indigenous microbiota have several beneficial effects on host physiological functions; however, little is known about whether or not postnatal microbial colonization can affect the development of brain plasticity and a subsequent physiological system response. To test the idea that such microbes may affect the development of neural systems that govern the endocrine response to stress, we investigated hypothalamic-pituitary-adrenal (HPA) reaction to stress by comparing germfree (GF), specific pathogen free (SPF) and gnotobiotic mice. Plasma ACTH and corticosterone elevation in response to restraint stress was substantially higher in GF mice than in SPF mice, but not in response to stimulation with ether. Moreover, GF mice also exhibited reduced brain-derived neurotrophic factor expression levels in the cortex and hippocampus relative to SPF mice. The exaggerated HPA stress response by GF mice was reversed by reconstitution with Bifidobacterium infantis. In contrast, monoassociation with enteropathogenic Escherichia coli, but not with its mutant strain devoid of the translocated intimin receptor gene, enhanced the response to stress. Importantly, the enhanced HPA response of GF mice was partly corrected by reconstitution with SPF faeces at an early stage, but not by any reconstitution exerted at a later stage, which therefore indicates that exposure to microbes at an early developmental stage is required for the HPA system to become fully susceptible to inhibitory neural regulation. These results suggest that commensal microbiota can affect the postnatal development of the HPA stress response in mice.

Effect of 2-deoxy-d-glucose induced stress on Salmonella choleraesuis shedding and persistence in swine.

A glucose analog, 2-deoxy-d-glucose (2DG), previously shown in swine to induce many of the hallmark parameters of stress, was administered to Salmonella choleraesuis carrier-swine and the effects on Salmonella fecal shedding and tissue colonization were evaluated. Initially, pigs were divided into two groups, one that received 1 x 10 (6) S. choleraesuis and one group that received saline. At 3 or 6 weeks post inoculation (PI), half of each group received an injection of 2DG and the other half received saline. Throughout the study, individual fecal samples were collected and quantitatively cultured for Salmonella, tonsil and nasal swabs were qualitatively cultured, clinical signs were monitored, temperatures were measured and whole blood collected. Pigs were necropsied 8-18 days after 2DG treatment. The experimental stress induced by 2DG was not sufficient to cause recrudescence of Salmonella fecal shedding even when tissues were culture positive for Salmonella. In addition, persistent shedding was not affected by 2DG administration. Although the complex set of parameters that constitute the stress phenomenon is still relatively unknown, it is now apparent that the stressful event(s) sufficient to trigger Salmonella recrudescence involves more than just increased blood glucose, increased cortisol, and inhibition of lymphocyte proliferation.

Epithelia under metabolic stress perceive commensal bacteria as a threat.

The normal gut flora has been implicated in the pathophysiology of inflammatory bowel disease and there is increased interest in the role that stress can play in gut disease. The chemical stressor dinitrophenol (DNP, uncouples oxidative phosphorylation) was injected into the ileum of laparotomized rats and mitochondria structure, epithelial permeability, and inflammatory cell infiltrate were examined 6 and 24 hours later. Monolayers of human colonic epithelial cells (T84, HT-29) were treated with DNP +/- commensal Escherichia coli, followed by assessment of epithelial permeability, bacterial translocation, and chemokine (ie, interleukin-8) synthesis. Delivery of DNP into rat distal ileum resulted in disruption of epithelial mitochondria; similar changes were noted in mildly inflamed ileal resections from patients with Crohn's disease. Also, DNP-treated ileum displayed increased gut permeability and immune cell recruitment. Subsequent studies revealed deceased barrier function, increased bacterial translocation, increased production of interleukin-8, and enhanced mobilization of the transcription factor AP-1 in the model epithelial cell lines exposed to commensal bacteria (E. coli strains HB101 or C25), but only when the monolayers were pretreated with DNP (0.1 mmol/L). These data suggest that enteric epithelia under metabolic stress perceive a normally innocuous bacterium as threatening, resulting in loss of barrier function, increased penetration of bacteria into the mucosa, and increased chemokine synthesis. Such responses could precipitate an inflammatory episode and contribute to existing enteric inflammatory disorders.

Pseudomonas aeruginosa expresses a lethal virulence determinant, the PA-I lectin/adhesin, in the intestinal tract of a stressed host: the role of epithelia cell contact and molecules of the Quorum Sensing Signaling System.

OBJECTIVE: We have previously demonstrated that P. aeruginosa can have profound effects on the intestinal epithelial barrier via one of its virulence factors, the PA-I lectin/adhesin. The aims of the present study were to further characterize the interaction of P. aeruginosa and the intestinal epithelium using both in vitro and in vivo approaches. METHODS: In vitro assays examining the effect of bacterial growth phase, epithelial cell contact, and butanoyl homoserine lactone (C4-HSL), a quorum sensing signaling molecule know to affect various extracellular virulence factors in P. aeruginosa, on PA-I expression in P. aeruginosa were performed. In vivo studies were carried out by modeling catabolic stress in mice using a 30% surgical hepatectomy and direct introduction of P. aeruginosa and various virulence components into the cecum. The effect of this model on PA-I expression in P. aeruginosa was determined. RESULTS: Results demonstrated that PA-I expression in P. aeruginosa is affected by its phase of growth, its contact to the intestinal epithelium, and its exposure to the quorum sensing molecule, C4-HSL. Furthermore, data from the present study suggest that the PA-I lectin/adhesin of P. aeruginosa may be increased in vivo by local factors within the cecum of mice in response to surgical stress. CONCLUSIONS: These data indicate that multiple factors present in the intestinal microenvironment of a stressed host may induce certain opportunistic pathogens to express key virulence factors leading to a state of lethal gut-derived sepsis.

Immune changes during acute cold/restraint stress-induced inhibition of host resistance to Listeria.

Experiments were conducted to delineate the cellular changes modulated by acute cold/restraint stress (ACRS), a physical and psychological stressor, in response to a Listeria monocytogenes(LM) infection. In addition to wild type (WT) BALB/c mice, CD4-deficient (CD4-/-) BALB/c mice, which have no effective adaptive immunity, were used to determine the involvement of adaptive versus innate immunity. ACRS-induced suppression of host resistance to LM was not observed in CD4-/- mice, suggesting the involvement of CD4+T cells in the acute cold/restraint stress (ACRS)-induced inhibition. The in vivo splenic leukocyte phenotypes and activities of WT BALB/c mice after infection and in vitro lymphocyte responses to heat-killed LM (HKLM) also were examined. There were no significant differences in the numbers of splenic T and B lymphocytes, natural killer cells, macrophages, or neutrophils between nonstressed and ACRS-treated WT mice. However, higher levels of activated T cells and non-T lymphocytes were observed in the ACRS-treated mice; beta-adrenergic receptor (beta-ADR) antagonists (propranolol and atenolol) eliminated these elevated levels of activation, as well as the ACRS-induced suppression of host resistance. ACRS and control mice also had equivalent activation of macrophages. With in vitro HKLM stimulation, splenocytes from ACRS-treated mice produced significantly higher levels of IFNgamma and slightly higher levels of IL-6 in comparison with the nonstressed mice, although equivalent levels of lymphocyte proliferation were obtained. Additionally, ACRS-treated mice showed comparable elevation of serum nitric oxide after infection, indicating macrophage bactericidal activity similar to nonstressed mice. Thus, it appears that ACRS inhibits host resistance through regulatory CD4+ T cells and/or effector cell functions downstream of CD4+ T cell activation, as well as through beta-ADR signaling, in that blockage of these receptors appears to aid host defenses by means other than elevation of helper T cell activity. Because CD4 T cell deficiency and beta-ADR blockage produced equivalent effects, beta-ADR+ CD4+ T cells may have a negative role on host defenses after ACRS.

Immunocompetence of macrophages in rats exposed to Candida albicans infection and stress.

The integration of innate and adaptive immune responses is required for efficient control of Candida albicans. The present work aimed to assess, at the local site of the infection, the immunocompetence of macrophages in rats infected intraperitoneally with C. albicans and exposed simultaneously to stress during 3 days (CaS group). We studied the 1) ability to remove and kill C. albicans, 2) tumor necrosis factor-alpha (TNF-alpha) release, 3) balance of the inducible enzymes NO synthase (iNOS) and arginase, and 4) expression of interleukin (IL)-1beta and IL-1 receptor antagonist (ra) mRNA. Compared with only infected animals (Ca group), the number of colony-forming units was significantly higher in CaS rats (P < 0.01), and the macrophage candidicidal activity was approximately 2.5-fold lower (P < 0.01). Release of TNF-alpha was diminished in both unstimulated and heat-killed C. albicans restimulated macrophages of the CaS group (Ca vs. CaS, P < 0.03 and P < 0.05, respectively). In Ca- and CaS-group rats, the rates for both the arginase activity and the NO synthesis were significantly enhanced. However, the stress exposure downregulated the activity of both enzymes (CaS vs. Ca, P < 0.05). After in vitro restimulation, the IL-1ra/IL-1beta ratio was significantly diminished in CaS-group rats (P < 0.05). Our results indicate that a correlation exists between early impairment of macrophage function and stress exposure.

Suppression of host resistance to Listeria monocytogenes by acute cold/restraint stress: lack of direct IL-6 involvement.

We conducted kinetic studies to evaluate the effects of acute cold/restraint stress (ACRS) on both primary and secondary host resistance to Listeria monocytogenes (LM). The involvement of IL-6 also was investigated using IL-6 knockout (KO) mice on the BALB/c background. ACRS dramatically increased the serum corticosterone levels, indicating that ACRS activated the hypothalamic-pituitary-adrenal (HPA) axis. ACRS significantly inhibited host resistance to LM during a primary but not a secondary LM infection. During the primary infection, ACRS caused a significant delay in clearance of LM, loss of body weight, reduced food/water intake, and elevated levels of pro-inflammatory cytokines (IL-6, IL-1beta, and TNFalpha) and IFNgamma. ACRS IL-6 KO mice showed higher LM burdens than did IL-6 KO controls, suggesting that IL-6 is not required for the ACRS-impairment of host resistance. Elevated levels of IL-1beta and TNFalpha may compensate for the absence of IL-6 and maintain the ACRS-induced impairment, in that the serum and splenic IL-1beta and TNFalpha levels were significantly higher in infected ACRS IL-6 KO mice, but not in control IL-6 KO mice, as compared to respective wild type controls. ACRS appears to inhibit IL-6 independent mechanisms associated with innate immunity and/or the development of adaptive immunity, but these reactions are unable to modulate the more efficient secondary immune responses.

Stress and the periodontal diseases: effects of catecholamines on the growth of periodontal bacteria in vitro.

Microorganisms possess the ability to recognize hormones within the host and utilize them to adapt to their surroundings. Noradrenaline and adrenaline, which are released during human stress responses, may act as environmental cues to alter the growth of individual organisms within subgingival biofilms. The aims of this study were to modify, for anaerobic culture, existing methodology used in determining microorganism catecholamine responses and to investigate the growth responses to noradrenaline and adrenaline of 43 microorganisms found within subgingival microbial complexes. We established initial inocula for each strain using anaerobic culture, re-inoculated into a minimal serum-based medium and grown anaerobically at 35 degrees C. We assessed organism growth by optical density (OD(600nm)) readings, with test and control cultures performed in triplicate. Test cultures were supplemented with 50 microm noradrenaline or 100 microm adrenaline. We observed significant growth effects for supplementation with noradrenaline (20 species responding positively) and adrenaline (27 species responding positively), with differences in growth response observed within bacterial species and within and between microbial complexes. The most pronounced positive growth effects of noradrenaline were demonstrated in Actinomyces naeslundii (+ 49.4%), Actinomyces gerenscseriae (+ 57.2%), Eikenella corrodens (+ 143.3%) and Campylobacter gracilis (+ 79.9%). We also observed inhibitory effects of noradrenaline supplementation for Porphyromonas gingivalis (- 11.9%) and Bacteroides forsythus (- 22.2%). Responses to adrenaline tended to mirror the responses seen with noradrenaline. Individual organisms from different microbial complexes vary in their in vitro growth responses to noradrenaline and adrenaline. Such variation may influence the in vivo composition of the subgingival biofilm in response to stress-induced changes in local catecholamine levels and play a significant role in the aetiology and pathogenesis of the periodontal diseases.

Transport stress--consequences for bacterial translocation, endogenous contamination and bactericidal activity of serum of slaughter pigs.

On transport and at the abattoir animals are confronted with a lot of stressors, such as sound/noise, crowding/mixing, pollutants and infectious agents that act on the organism. After transport stress an endogenous contamination is often seen in slaughter carcasses and presents a hazard for the consumer. These events are often correlated with a rise in endotoxin level (Misawa et al., 1995; Morales et al., 1992) and a modified immune response. Previous own investigations confirm this hypothesis (Zucker and Krüger, 1998, Seidler et al., 2000). The attempt was made to investigate the impact of selected stressors (short term transport (1 h), long term transport (7-8 hrs), high temperature, high humidity and intense handling/moving) on bacterial translocation, endogenous contamination, endotoxin levels and bactericidal activity of body fluids.

Effects of stressors on immune parameters and on the faecal shedding of enterotoxigenic Escherichia coli in piglets following experimental inoculation.

The study examined the effects of stressors on the responses of 3 and a half-week old piglets that had been given an oral dose of enterotoxigenic Escherichia coli (ETEC) and a novel harmless antigen (ovalbumin). Removal from the sow (WEAN), a short-term cold stressor (12;C for 48 hours) (TEMP) and mixing with non-littermates (MIX) were assessed in terms of the effects on faecal shedding of ETEC, immune responses, weight gain and an ACTH stimulation test. WEAN and TEMP reduced weight gain and all stressors increased faecal shedding of ETEC. All stressors increased the IgG responses to F4(K88)ac antigens and WEAN and TEMP increased the IgA responses to the same antigens, probably as a result of increased intestinal proliferation of ETEC. None of the stressors, however, had significant effects on antibody responses to ovalbumin or on lymphocyte proliferation assays. The results indicate that stressors influence the faecal shedding of ETEC in young piglets by a mechanism that may not involve modulation of immune responses.

Gut-derived sepsis occurs when the right pathogen with the right virulence genes meets the right host: evidence for in vivo virulence expression in Pseudomonas aeruginosa.

OBJECTIVE: To define the putative role of the PA-I lectin/adhesin, a binding protein of Pseudomonas aeruginosa, on lethal gut-derived sepsis after surgical stress, and to determine if this protein is expressed in vivo in response to physical and chemical changes in the local microenvironment of the intestinal tract after surgical stress. SUMMARY BACKGROUND DATA: Previous work from the authors' laboratory has established that lethal gut-derived sepsis can be induced after the introduction of P. aeruginosa into the cecum of mice after a 30% hepatectomy. This effect does not occur when P. aeruginosa is introduced into the cecum of sham operated control mice. Previous experiments further established that the mechanism of this effect is due to the presence of the PA-I lectin/adhesin of P. aeruginosa, which induces a permeability defect to a lethal cytotoxin of P. aeruginosa, exotoxin A. METHODS: Three strains of P. aeruginosa, one lacking functional PA-I, were tested in two complementary systems to assess virulence. Strains were tested for their ability to adhere to and alter the permeability of cultured human colon epithelial cells, and for their ability to induce mortality when injected into the cecum of mice after a 30% hepatectomy. To determine if PA-I is "in vivo expressed" when present in the cecal environment after hepatectomy, strains were retrieved from the cecum of sham-operated and hepatectomy-treated mice 24 and 48 hours after their introduction into the cecum and their PA-I expression was assessed. RESULTS: Results indicated that PA-I plays a putative role in lethal gut-derived sepsis in the mouse, because strains lacking functional PA-I had an attenuated effect on cultured human epithelial cells, and were nonlethal when injected into the cecum of mice after 30% surgical hepatectomy. Furthermore, surgical stress in the form of hepatectomy significantly altered the intestinal microenvironment, resulting in an increase in luminal norepinephrine associated with an increase in PA-I expression in retrieved strains of P. aeruginosa. Co-incubation of P. aeruginosa with norepinephrine increased PA-I expression in vitro, suggesting that norepinephrine plays a role in the observed response in vivo. CONCLUSIONS: Lethal gut-derived sepsis may occur when intestinal pathogens express virulence determinants in response to environmental signals indicating host stress. In this regard, the PA-I lectin/adhesin of P. aeruginosa appears to be a specific example of in vivo virulence expression in colonizing pathogens in the intestinal tract in response to surgical stress.

In vivo production of neuraminidase by Pasteurella haemolytica in market stressed cattle after natural infection.

Pasteurella haemolytica (Ph) is the most important cause of the bovine acute fibrinohemorrhagic pneumonia that occurs in market stressed calves after shipment to feedyards. Recent characterization of neuraminidase production by these organisms has shown that all 16 serotypes produce an immunologically similar form of the enzyme. Anti-neuraminidase antibody against PhA1 and PhA6 was determined in 101 2- to 5-month-old calves, on their farms of origin, at the order buyer barn (OBB), and through 28 days in the feedyard. Half of the calves were vaccinated with a killed Ph serotype-A1 (PhA1) product. Nasal secretion and tonsil wash specimens were cultured for Ph and Pasteurella multocida (Pm). Serum antibody against PhA1 and PhA6 was measured by indirect hemagglutination (IHA), and anti-neuraminidase antibody was determined by the neutralization assay. At the feedyard, 73 calves had respiratory tract disease. IHA values ranged between 1:2 and 1:1024 for PhA1 and between 1:2 and 1:512 for Ph serotype A6 (PhA6). Forty-two, 24, and 28% of the calves were infected with PhA1, PhA6, and Pm, respectively. Ninety-six percent of the calves experienced an increase in anti-PhA1 neuraminidase antibody when sera drawn on feedyard day 28 were compared with sera drawn on the farm. These data demonstrate that the enzyme neuraminidase is produced in vivo in market stressed cattle after a natural Ph infection.

CD66-mediated phagocytosis of Opa52 Neisseria gonorrhoeae requires a Src-like tyrosine kinase- and Rac1-dependent signalling pathway.

The interaction of Neisseria gonorrhoeae with human phagocytes is a hallmark of gonococcal infections. Recently, CD66 molecules have been characterized as receptors for Opa52-expressing gonococci on human neutrophils. Here we show that Opa52-expressing gonococci or Escherichia coli or F(ab) fragments directed against CD66, respectively, activate a signalling cascade from CD66 via Src-like protein tyrosine kinases, Rac1 and PAK to Jun-N-terminal kinase. The induced signal is distinct from Fcgamma-receptor-mediated signalling and is specific for Opa52, since piliated Opa- gonococci, commensal Neisseria cinerea or E.coli do not stimulate this signalling pathway. Inhibition of Src-like kinases or Rac1 prevents the uptake of Opa52 bacteria, demonstrating the crucial role of this signalling cascade for the opsonin-independent, Opa52/CD66-mediated phagocytosis of pathogenic Neisseria.