Distribution, clearance, and mortality of environmental pseudomonads in mice upon intranasal exposure.

When introduced intranasally, P. cepacia AC1100 (approximately 10(8) CFU/animal) and P. aeruginosa AC869 (approximately 10(3) CFU/animal) were readily cleared from the mouse. However, a approximately 10(7)-CFU dose of AC869 persisted for 14 days. Strain AC869 had a 50% lethal dose of 2.7 x 10(7) CFU. Slight morbidity occurred in animals treated with approximately 10(7) CFU of AC869 or approximately 10(8) CFU of AC1100.     

Pulmonary clearance and inflammatory response in C3H/HeJ mice after intranasal exposure to Pseudomonas spp.

The environmental release of engineered microorganisms has caused health and environmental concerns. In this study, an animal model was used to examine health effects following pulmonary exposure to environmental and clinical isolates. In order to rule out the possibility that an adverse response was caused by endotoxin, 50% lethal doses (LD50) were determined, when possible, with endotoxin-sensitive (C3HeB/FeJ) and endotoxin-resistant (C3H/HeJ) mice by using both environmental isolates (Pseudomonas aeruginosa BC16, BC17, BC18, and AC869 and Pseudomonas maltophilia BC6) and clinical isolates (P. aeruginosa PAO1 and DG1). The LD50 of strains AC869, DG1, and PAO1 are 1.05 x 10(7), 6.56 x 10(6), and 1.02 x 10(7) CFU, respectively, in C3HeB/FeJ mice and 1.05 x 10(7), 1.00 x 10(7), and 2.75 x 10(6) CFU, respectively, in C3H/HeJ mice. Strains BC17 and BC18 were not lethal to the animals. On the basis of the LD50 data, an appropriate sublethal dose (approximately 10(6) CFU) was selected. Animals were challenged intranasally with microorganisms, and clearance from the lungs and nasal cavity was determined. Strains BC17, BC18, and AC869 were not detected in lungs or nasal washes 14 days following treatment. Strains BC6, BC16, and DG1 were recovered from the nasal cavities at the end of the experiment. Only strain PAO1 was detected in lungs and in nasal cavities 14 days after treatment. At selected intervals following treatment, the percentages of polymorphonuclear leukocytes and lymphocytes in bronchoalveolar lavage samples were determined. P. aeruginosa AC869, PAO1, and DG1 elicited a relatively strong inflammatory response which was indirectly related to lung clearance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Blockade of tissue factor-factor X binding attenuates sepsis-induced respiratory and renal failure

Tissue factor expression in sepsis activates coagulation in the lung, which potentiates inflammation and leads to fibrin deposition. We hypothesized that blockade of factor X binding to the tissue factor-factor VIIa complex would prevent sepsis-induced damage to the lungs and other organs. Acute lung injury was produced in 15 adult baboons primed with killed Escherichia coli [1 x 10(9) colony-forming units (CFU)/kg], and then 12 h later, they were given 1 x 10(10) CFU/kg live E. coli by infusion. Two hours after live E. coli, animals received antibiotics with or without monoclonal antibody to tissue factor intravenously to block tissue factor-factor X binding. The animals were monitored physiologically for 34 h before being killed and their tissue harvested. The antibody treatment attenuated abnormalities in gas exchange and lung compliance, preserved renal function, and prevented tissue neutrophil influx and bowel edema relative to antibiotics alone (all P < 0.05). It also attenuated fibrinogen depletion (P < 0.01) and decreased proinflammatory cytokines, e.g., IL-6 and -8 (P < 0.01), in systemic and alveolar compartments. Similar protective effects of the antibody on IL-6 and -8 expression and permeability were found in lipopolysaccharide-stimulated endothelial cells. Blockade of factor X binding to the tissue factor-factor VIIa complex attenuates lung and organ injuries in established E. coli sepsis by attenuating the neutrophilic response and inflammatory pathways.     

Natural cellular resistance of beige mice against Cryptococcus neoformans

Previous reports have demonstrated that natural killer (NK) cells are capable of inhibiting the growth of Cryptococcus neoformans in vitro, and recent studies indicate that adoptively transferred NK cell-enriched spleen cell populations enhance clearance of cryptococci from the tissues of cyclophosphamide-pretreated recipients. The primary objective of these studies was to confirm that NK cells participate in early clearance of C. neoformans in vivo. Secondarily, the anti-cryptococcal activities of polymorphonuclear leukocytes and macrophages were examined. Seven-week-old C57BL/6 bg/+ mice, which have normal levels of NK cell activity, were compared with their bg/bg littermates, which have impaired NK cell function. One and 3 days after injecting both groups of mice i.v. with 2 X 10(4) cryptococci, we assessed the NK cell activities in spleens, lungs, and livers and clearance of the organism from corresponding tissues as determined by the mean log10 numbers of cryptococcal colony-forming units (CFU) per organ. Three days postinfection, the mean numbers of cryptococcal CFU in lungs and spleens of bg/+ mice were significantly lower than in the corresponding organs of bg/bg mice. NK cell activities in spleens and lungs of bg/+ mice were significantly higher than were the NK cell activities in similar cell populations from bg/bg mice. In contrast, the mean numbers of cryptococcal CFU in livers of the two groups of animals were nearly equivalent, a situation not unexpected, since liver NK cell activities were extremely low and similar in both groups of animals. Although these data indicated a correlation between early clearance of cryptococci from tissues and levels of NK cell activities in the corresponding tissues, it was also possible that differences in phagocytic cell function between the bg/+ and bg/bg animals could account for the observed differences in clearance of cryptococci from the tissues. Therefore, phagocytic cells from the two groups of animals were compared with respect to their abilities to phagocytize and inhibit the growth of cryptococci and to their abilities to respond to chemotactic stimuli in vivo. Peritoneal PMNL from bg/+ and bg/bg mice were similar in their abilities to phagocytize and inhibit the growth of cryptococci, as well as in their chemotactic responses to viable cryptococci or sodium caseinate. In addition, there were no differences in splenic macrophage functions between the two groups of mice.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pathogenesis of Pulmonary Cryptococcus gattii Infection: A Rat Model

A model of pulmonary cryptococcosis in immunocompetent rats was developed to better understand the virulence of Cryptococcus gattii. Six isolates were studied, representing four molecular genotypes (VGI-MATα, VGIIa-MATα, VGIIa-MAT a, VGIIb-MATα), obtained from Australia, Vancouver (Canada) and Colombia. These originated from human patients, a cat and the environment and were administered intratracheally (i.t.) or transthoracically into Fischer 344 or Wistar-Furth rats in doses varying from 10⁴ to 10⁷ colony-forming units (CFU) in 0.1 ml of saline. With the exception of animals given the VGIIa-MAT a isolate, rats consistently became ill or died of progressive cryptococcal pneumonia following i.t. doses exceeding 10⁷ CFU. Affected lungs increased in weight up to tenfold and contained numerous circumscribed, gelatinous lesions. These became larger and more extensive, progressing from limited hilar and/or tracheal lesions, to virtually confluent gelatinous masses. Disease was localized to the lungs for at least 3–4 weeks, with dissemination to the brain occurring in some animals after day 29. The dose–response relationship was steep for two VGI isolates studied (human WM179, environmental WM276); doses up to 10⁶ CFU i.t. did not produce lesions, while 10⁷ or more yeast cells produced progressive pneumonia. Intratracheal inoculation of rats with C. gattii provides an excellent model of human pulmonary cryptococcosis in healthy hosts, mimicking natural infections. Disease produced by C. gattii in rats is distinct from that caused by C. neoformans in that infections are progressive and ultimately fatal.     

New beta-lactamase-resistant cephem treatment of guinea pigs infected with Legionella pneumophila

The in vivo antimicrobial effect of seven new beta-lactamase-resistant cephems (cefotaxime, latamoxef, ceftazidime, ceftriaxone, cefotiam, cefbuperazone, and MT-141) on Legionella pneumophila (strain 81-066, serogroup IV) in guinea pigs was compared with that of erythromycin. As the minimal LD100 within one week was about 4.0 X 10(9) CFU/ml by intraperitoneal injection of the strain, the animals were inoculated with 2.0 ml of twofold dilutions of a suspension of this bacterium. The animals developed purulent peritonitis and systemic involvement demonstrated by the development of periangitis, pneumonia and pleuritis in the lungs. Three different doses of antibiotics were administered intraperitoneally immediately after the rectal temperature reached more than 40 C. Erythromycin had a significant therapeutic effect but none of the new cephems tested death of the infected guinea pigs.     

Efficacy of a commercial bacterin in protecting strain 13 guineapigs against Bordetella bronchiseptica pneumonia

Bordetella bronchiseptica is known to be endemic in many guineapig (Cavia porcellus) colonies, and periodically is the aetiological agent of fatal epizootics of bronchopneumonia. A commercial, non-adjuvant B. bronchiseptica bacterin, which is approved for use in canines, was evaluated for induction of a protective immune response in Strain 13/N guineapigs against an airborne challenge of virulent B. bronchispeptica in small-particle aerosol. Seronegative animals were vaccinated on days 0 and 21 with intramuscular injections of 0.2 ml of bacterin. Humoral antibody titres of the vaccinated animals, as determined by ELISA, ranged from 128-1024 on day 49. On day 30 following the second dose of bacterin (study day 51), 12 vaccinated and 12 PBS sham-vaccinated animals were exposed to an inhaled dose of 4.3 x 10(5) CFU of B. bronchiseptica (325 LD50). Vaccinated, challenged animals remained clinically normal, although each guineapig did develop a localized upper respiratory infection. The rate of weight gain as well as rectal temperature of these animals were analogous to those exhibited by the control groups. Examination of 4 of the vaccinated, challenged animals on day 7 after exposure showed bacteria present in moderate to high numbers in the larynx and trachea but only minimally detectable in the lungs; by 30 days after exposure, the numbers of bacteria in the larynx and trachea were diminished, with none being detected in the lungs. Pathological alterations induced by B. bronchiseptica were not detected at either day 7 or day 30 after challenge in any of the vaccinated, challenged animals. Protection induced in Strain 13/N guineapigs by the commercial canine bacterin was sufficient to preclude the development of pulmonary disease, even in animals presented with a massive challenge of virulent bacteria in a small-particle aerosol.     

Combination Efficacy of Voriconazole and Amphotericin B in the Experimental Disease in Immunodeficient Mice Caused by Fluconazole-resistant <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

The therapeutic efficacy of amphotericin B and voriconazole alone and in combination with one another were evaluated in immunodeficient mice (BALB/c-SCID) infected with a fluconazole-resistant strain of Cryptococcus neoformans var. grubii. The animals were infected intravenously with 3 × 10⁵ cells and intraperitoneally treated with amphotericin B (1.5 mg/kg/day) in combination with voriconazole (40 mg/kg/days). Treatment began 1 day after inoculation and continued for 7 and 15 days post-inoculation. The treatments were evaluated by survival curves and yeast quantification (CFUs) in brain and lung tissues. Treatments for 15 days significantly promoted the survival of the animals compared to the control groups. Our results indicated that amphotericin B was effective in assuring longest-term survival of infected animals, but these animals still harbored the highest CFU of C. neoformans in lungs and brain at the end of the experiment. Voriconazole was not as effective alone, but in combination with amphotericin B, it prolonged survival for the second-longest time period and provided the lowest colonization of target organs by the fungus. None of the treatments were effective in complete eradication of the fungus in mice lungs and brain at the end of the experiment.     

自然感染途径建立肺炎链球菌感染性肺炎小鼠模型

目的探索自然感染途径制备肺炎链球菌感染性肺炎小鼠模型的方法,为肺炎链球菌性肺炎的相关研究提供实验基础。方法肺炎链球菌标准菌株(CMCC 31203)经血平板培养18 h后配成2麦氏浊度。小鼠浅麻醉状态下,破损鼻黏膜,滴注一定量肺炎链球菌菌液。于3、7、14和21 d分别处死小鼠,取肺脏进行组织病理学观察,确定肺炎小鼠模型制备的最佳方式。结果鼻黏膜破损组小鼠3 d时肺泡间隔内毛细血管扩张,肺泡腔内以红细胞和纤维素渗出为主;7 d时病变以纤维素和中性粒细胞浸润为主;14 d时肺泡腔内渗出减少,炎症开始减轻;21 d时肺脏外观趋于正常,肺泡腔内渗出物质基本溶解吸收。结论以3×107CFU的肺炎链球菌菌液通过破损的鼻黏膜感染7 d时可以制备出比较典型、稳定的肺炎小鼠模型。     

Effects of niacin on bleomycin-induced increases in myeloperoxidase,prolyl hydroxylase,and superoxide dismutase activities and collagen accumulation in the lungs of hamsters

It has been shown that lung nicotinamide adenine dinucleotide (NAD) depletion accompanies bleomycin (BL)-induced lung fibrosis in the hamster and that treatment with niacin (NA), a precursor of NAD, was found to attenuate lung fibrosis caused by this agent. Niacin was used in the present study to investigate changes in some biochemical parameters and enzymes involved in the development of BL-induced lung fibrosis in the hamster. Niacin (500 mg/kg, IP), or an equivalent volume of saline (SA, IP), was given daily 2 days prior to intratracheal instillation of BL (7.5 U/5 mL/ kg) or SA and everyday thereafter throughout the study. Hamsters were killed at 1, 4, 7, 10, and 14 days after the BL or SA instillation and their lungs processed for various biochemical assays. Hydroxyproline content and superoxide dismutase (SOD) activity in SABL treated animals were significantly (P ± 0.05) elevated at 7 and 10 days, peaking at 14 days to 161 ± 11% and 159 ± 11% of the SASA treated animals, respectively. Although the hydroxyproline level of NABL treated animals was significantly elevated at 7 and 10 days and peaked at 14 days to 123 ± 8% of the NASA control, these values were significantly lower than the SABL treated animals at the corresponding times. The lung SOD activity of NABL groups was significantly higher at 4 days but significantly lower at 10 and 14 days than the SABL groups at the corresponding times. Prolyl hydroxylase (PH) activity and total lung calcium in SABL treated groups were significantly elevated compared to SASA treated groups starting at 4 days, with PH peaking at 10 days to 163 ± 13% and calcium peaking at 7 days to 148 ± 8% of SASA treated groups. The NABL treated animals displayed a significant elevation in PH activity at 4 days only (132 ± 15%), while the calcium content in this group was significantly increased at 4 and 14 days compared to NASA treated animals. However, the activity of PH in the NABL treated animals was significantly lower than the SABL treated animals at 7, 10, and 14 days. The calcium content of the NABL group was significantly lower than the SABL group at 7 and 10 days. The thiobarbituric acid reactive substance equivalents (TBARS) content and myeloperoxidase (MPO) activity were significantly elevated at all time points in SABL groups as compared to SASA groups, with peak elevation of TBARS to 160 ± 9% at 4 days and MPO to 268 ± 40% at 1 day. The TBARS content of NABL groups was significantly elevated above NASA groups at 1, 4, 7, and 10 days. The MPO activity in NABL groups was significantly elevated above NASA groups at 1, 4, 7, and 14 days, but the activity was significantly lower than SABL groups at 4 and 14 days. The data suggest that NA inhibits the extent of oxidative damage or enhances processes involved in the repair of BL-induced alveolar epithelial damage.     

Comparative study of four antifungal drugs in an experimental model of murine cryptococcosis

A comparative study among amphotericin B, 5-fluorocytosine, itraconazole and fluconazole in the treatment of experimental cryptococcosis in mice, was carried out.Seventy male Balb C mice were inoculated intraperitoneally with 10⁷ cells of Cryptococcus neoformans var. neoformans. They were divided in 7 groups of 10 animals each one: 1) treated with fluconazole by gavage at a daily dose of 16 mg/kg; 2) treated with itraconazole by gavage at a daily dose of 16 mg/kg; 3) treated with 5-fluorocytosine by gavage at a daily dose of 300 mg/kg; 4) treated with amphotericin B intraperitoneally at a dose of 6 mg/kg every other day; 5) control animals receiving polietilenglicol 200 by gavage; 6) control animals receiving distilled water by gavage and 7) control animals receiving sterile distilled water by intraperitoneal route. All the treatments started 5 days after the challenge inoculation and they were given for 2 weeks.The following parameters were taken into account: survival time, macroscopic aspect of the organ after the complete autopsy, microscopic investigation of yeasts in brain, lungs, spleen and liver, histopathology studies of these organs, the colony forming units per gram and massive seeding of brain and lungs.The survival index of the different groups was the most efficient method to measure the antifungal compounds activity. Amphotericin B increased significantly the animals survival and modified the histopathologic response in the studied organs. The colony forming units and the massive seeding in brain and lung showed that this antifungal agent is unable of producing the biological cure of this experimental model.The remaining drugs assayed did not promote important modifications in the histopathologic picture as well as in the tissues cultures. However, the three drugs increased the animals survival. In this aspect, fluconazole proved to be slightly superior.     

Therapeutic efficiency of spleen or bone marrow CFU in X-irradiated 89Sr marrow-ablated mice.

Experiments were carried out to compare the therapeutic efficiency (TE: number of CFU required to reduce the mortality from 100 to 50 per cent) of spleen or marrow (BM) stem cells (CFU) grafted into lethally irradiated mice (807 rad) which had been previously treated with 89Sr or splenectomized. It was found that during the reconstitution of the haemopoietic organs, the spleen does not provide more than 10 per cent of the functional cells necessary for survival. Besides, the BM-derived CFU growing in 89Sr marrow-ablated mice remain twice as efficient as the spleen-derived ones. Similarly, spleen-derived CFU transplanted into splenectomized mice are half as efficient as BM-derived ones. It may therefore be assumed that haemopoietic stem cells grafted into a foreign microenvironment retain their original kinetics of growth and differentiation during 7 to 10 days after their transplantation.     

Combined action of antiblastoma preparations on lysozyme activity in animal organs]

The lysozyme activity in the spleen, kidneys and lungs of the mice treated with neocid, sarcolysine or Tio-Fef in therapeutic doses increased or remained at the control level by the 5th or 10th day after the drug administration. The use of sarcolysine per se or in combination with neocid increased the activity of lysozyme in the spleen, kidneys and lungs during the whole period of the experiment as compared to the control. The values of the lysozyme activity in the spleen and lungs of the animals treated with neocid in combination with sarcolysine were higher for 5 days, and in all organs examined were higher by the 10the day as compared to the animals treated with neocid alone. Increased lysozyme activity in the spleen, kidneys and lungs was observed under the effect of neocid in combination with sarcolysine as compared to the lysozyme activity in mice treated with sarcolysine per se (assay on the 10th day). Decreased lysozyme activity was determined in the spleen, kidneys and lungs by the 5th day and in the kidneys by the 10th day in the mice treated with sarcolysine in combination with Tio-Tel and in the spleen and lungs by the 5th and 10th days in the animals treated with neocid in combination with sarcolysine or Tio-Tef as compared to the animals treated with sarcolysine. The lysozyme activity in the kidneys under the effect of sarcolysine combination with Tio-Tef was lower by the 5th days and higher by the 10th day as compared to that under the effect of Tio-Tef.     

Cardiac adenylate cyclase activity in streptozotocin-treated rats after 4 months of diabetes: impairment of epinephrine and glucagon stimulation

Adenylate cyclase (AC) activities of cardiac membranes prepared (a) from rats that had been made diabetic 4 months previously by a single i.v. injection (50 mg/kg) of streptozotocin (STZ), and (b) from diabetic rats which had been treated during the same period by a daily dose of long-acting insulin (2-4 U/animal), were compared with the AC activity of cardiac membranes prepared from age-matched control animals. Basal (Mg++-dependent) and Mn++ (7 mM)-dependent activities, as well as Gpp(NH)p (3 X 10(-7) M) stimulation and Ca++ (pCa = 3.9) inhibition of cardiac AC were not significantly different in the three groups. At the EC50 concentration epinephrine (5 X 10(-7) M) stimulation of AC was reduced in diabetic animals but no change was observed at higher concentrations (10(-4) M). Glucagon stimulation was impaired at both the EC50 concentration (10(-7) M) and at higher concentrations (10(-5) M). Insulin treatment of the diabetic animals partially prevented the impairment of hormone stimulation. These results confirm observations that alterations of cardiac AC activity in STZ-treated rats are indeed due to diabetes and not to STZ-toxicity and suggest that AC-coupled receptors are altered either by an diabetes-induced alteration of cardiac sarcolemma.     

Therapeutic efficacy of moxifloxacin in a murine model of severe systemic mixed infection with Escherichia coli and Bacteroides fragilis

The therapeutic efficacy of moxifloxacin was studied in an experimental murine model of a systemic aerobic/anaerobic mixed infection and compared to therapies with either imipenem or ciprofloxacin plus metronidazole. Groups of 20 mice each were intravenously (iv) infected with approximately 2.5 x 10(6) colony forming units (CFU) of Escherichia coli and 2 x 10(7)CFU of Bacteroides fragilis. Iv therapy was started 24 h post-infection (pi) with either moxifloxacin, imipenem, or ciprofloxacin plus metronidazole, for 3 days. A control group of 20 mice was left untreated. Survival rate at day seven pi was recorded, mice were then sacrificed and bacterial organ contents of livers and kidneys were determined. All mice treated survived at day seven, while six animals of the untreated group died. B. fragilis was not detected in any of the treated mice. E. coli was found in two of the moxifloxacin-treated mice and in two and five of the ciprofloxacin plus metronidazole and imipenem-treated animals, respectively. The results indicate that a therapy of severe mixed aerobic/anaerobic infections with moxifloxacin might be feasible and possibly be as efficacious as current therapy regimens with ciprofloxacin plus metronidazole or imipenem.     

Study on the pathophysiology of experimental Burkholderia pseudomallei infection in mice

Burkholderia pseudomallei is the etiological agent of melioidosis, a potentially fatal disease occurring in man and animals. The aim of this study was to investigate the pathophysiological course of experimental melioidosis, and to identify the target organs, in an animal model. For this purpose SWISS mice were infected intraperitoneally with the virulent strain B. pseudomallei 6068. The bacterial load of various organs was quantified daily by bacteriological analysis and by an enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody specific to B. pseudomallei exopolysaccharide (EPS). Electron microscopic investigation of the spleen was performed to locate the bacteria at the cellular level. In this model of acute melioidosis, B. pseudomallei had a marked organ tropism for liver and spleen, and showed evidence of in vivo growth with a bacterial burden of 1.6x10(9) colony forming units (CFU) per gram of spleen 5 days after infection with 200 CFU. The highest bacterial loads were detected in the spleen at all time points, in a range from 2x10(6) to 2x10(9) CFU g(-1). They were still 50-80 times greater than the load of the liver at the time of peak burden. Other investigated organs such as lungs, kidneys, and bone marrow were 10(2)-10(4)-fold less infected than the spleen, with loads ranging from 3x10(2) to 3x10(6) CFU g(-1). The heart and the brain were sites of a delayed infection, with counts in a range from 10(3) to 10(7) times lower than bacterial counts in the spleen. The EPS-specific ELISA proved to be highly sensitive, particularly at the level of those tissues in which colony counting on agar revealed low contamination. In the blood, EPS was detected at concentrations corresponding to bacterial loads ranging from 8x10(3) to 6x10(4) CFU ml(-1). Electron microscopic examination of the spleen revealed figures of phagocytosis, and the presence of large numbers of intact bacteria, which occurred either as single cells or densely packed into vacuoles. Sparse figures suggesting bacterial replication were also observed. In addition, some bacteria could be seen in vacuoles that seemed to have lost their membrane. These observations provide a basis for further investigations on the pathogenesis of the disease.     

Cultivation of murine B-cell hybridomas in the spleen

The tumorigenic capacity of mouse B-cell hybridomas in both cloned and primary cultures was studied. The cells were selected for inoculation from 24-well plates and introduced into the spleen of syngeneic mice. The cells took in 50% of the animals. The cells of hybridoma tumors induced as the result of intrasplenic inoculation, when subcultured in the second passage following the standard scheme, i.e. inoculated intraperitoneally in a dose of 1 X 10(7) cells into mice previously treated with vaseline oil or pristane, produced tumors in 100% of the animals.     

Rectal carriage of enterohemorrhagic Escherichia coli O157 in slaughtered cattle

Escherichia coli O157:H7 is an important cause of diarrhea, hemorrhagic colitis, and potentially fatal human illness. Cattle are considered a primary reservoir of infection, and recent experimental evidence has indicated that the terminal rectum is the principal site of bacterial carriage. To test this finding in naturally colonized animals, intact rectum samples from 267 cattle in 24 separate lots were obtained immediately after slaughter, and fecal material and mucosal surfaces were cultured for E. coli O157 by direct and enrichment methods. Two locations, 1 and 15 cm proximal to the recto-anal junction, were tested. In total, 35 animals were positive for E. coli O157 at at least one of the sites and 232 animals were negative as determined by all tests. The frequency of isolation and the numbers of E. coli O157 cells were higher at the site closer to the recto-anal junction, confirming our previous experimental findings. We defined low- and high-level carriers as animals with E. coli O157 levels of <1 x 10(3) CFU g(-1) or <1 x 10(3) CFU ml(-1) and animals with E. coli O157 levels of > or =1 x 10(3) CFU g(-1) or > or =1 x 10(3) CFU ml(-1) in feces or tissues, respectively. High-level carriage was detected in 3.7% of the animals (95% confidence interval, 1.8 to 6.8%), and carriage on the mucosal surface of the terminal rectum was associated with high-level fecal excretion. In summary, our results support previous work demonstrating that the mucosal epithelium in the bovine terminal rectum is an important site for E. coli O157 carriage in cattle. The data also support the hypothesis that high-level fecal shedding (> or =1 x 10(3) CFU g of feces(-1)) of enterohemorrhagic E. coli O157 results from colonization of this site.     

TNF-alpha compensates for the impaired host defense of IL-1 type I receptor-deficient mice during pneumococcal pneumonia.

To determine the role of IL-1 in the host defense against pneumonia, IL-1R type I-deficient (IL-1R(-/-)) and wild-type (Wt) mice were intranasally inoculated with Streptococcus pneumoniae. Pneumonia resulted in elevated IL-1alpha and IL-1beta mRNA and protein levels in the lungs. Survival rates did not differ between IL-1R(-/-) and Wt mice after inoculation with 5 x 10(4) or 2 x 10(5) CFU. At early time points (24 and 48 h) IL-1R(-/-) mice had 2-log more S. pneumoniae CFU in lungs than Wt mice; at 72 h bacterial outgrowth in lungs was similar in both groups. Upon histopathologic examination IL-1R(-/-) mice displayed a reduced capacity to form inflammatory infiltrates at 24 h after the induction of pneumonia. IL-1R(-/-) mice also had significantly less granulocyte influx in bronchoalveolar lavage fluid at 24 h after inoculation. Since TNF is known to enhance host defense during pneumonia, we determined the role of endogenous TNF in the early impairment and subsequent recovery of defense mechanisms in IL-1R(-/-) mice. All IL-1R(-/-) mice treated with anti-TNF rapidly died (no survivors (of 14 mice) after 4 days), while 10-day survival in IL-1R(-/-) mice (control Ab), Wt mice (anti-TNF), and Wt mice (control Ab) was 7 of 13, 3 of 14, and 12 of 13, respectively. These data suggest that TNF is more important for host defense against pneumococcal pneumonia than IL-1, and that the impaired early host defense in IL-1R(-/-) mice is compensated for by TNF at a later phase.     

Minimum bacterial density for bacteriophage replication: implications for significance of bacteriophages in natural ecosystems

Bacteriophage 80 alpha did not increase in number in cultures containing less than about 1.0 X 10(4) to 1.5 X 10(4) CFU of Staphylococcus aureus per ml, but bacteriophage replication did occur when the number of bacteria exceeded this density, either initially or as a result of host cell multiplication. The minimum density of an asporogenous strain of Bacillus subtilis required for an increase in the number of bacteriophage SP beta cI was about 3 X 10(4) CFU/ml. The threshold density of Escherichia coli for the multiplication of bacteriophage T4 was about 7 X 10(3) CFU/ml. In the presence of montmorillonite, bacteriophage T4 did not increase in number until the E. coli population exceeded 10(4) CFU/ml. The mineralization of glucose was not affected in E. coli cultures inoculated with a low number of bacteriophage T4, but it could not be detected in cultures inoculated with a large number of phage. The numbers of bacteriophage T4 and a bacteriophage that lyses Pseudomonas putida declined rapidly after being added to lake water or sewage. We suggest that bacteriophages do not affect the number or activity of bacteria in environments where the density of the host species is below the host cell threshold of about 10(4) CFU/ml.