Effects of selenium supplementation on virus-induced inflammatory heart disease

The effects of 10 wk of selenium (Se) supplementation (5 ppm) in drinking water on immune responses and resistance to a myocarditic Coxsackie virus B3 (CB3) infection were studied in female Balb/c mice. Se supplementation reduced CB3-induced mortality: at day 14 postinoculation, survival was 58% in the Se-treated group as compared to 25% in the untreated group. Whole-blood glutathione peroxidase (GSH-Px) activity was elevated by 68% (p < 0.001) and Se content in the liver by 24% (p < 0.001). Red (RBC) and white blood cell (WBC) counts, as well as the number of cells in the spleen and thymus, were unaffected. The cellular counts of T-lymphocytes (CD4⁺, CD8⁺) and natural killer (NK⁺) cells in the blood were not affected. However, the CD4⁺/CD8⁺ ratio (5.2) tended to increase after Se supplementation (5.9). The spleen lymphoproliferative response to T and B-cell mitogens were increased by 9 and 43%, respectively (ns), in the Se-supplemented group. The total NK cell activity in blood and spleen showed minor increases, but when the activity in the blood was expressed per cell, the increase amounted to 35% (ns) with Se supplementation. The inflammatory and necrotic lesions in the ventricular myocardium at 7 and 14 d postinoculation were not significantly reduced by Se treatment, probably owing to the increased survival with Se even of mice with the most pronounced heart damage; comparable untreated mice were estimated to have died at day 14. Results indicate that modest doses of Se can improve immune function, which may increase the general resistance to this viral infection.     

Use of a Safe,Reproducible, and Rapid Aerosol Delivery Method to Study Infection by Burkholderia pseudomallei and Burkholderia mallei in Mice

Burkholderia pseudomallei, the etiologic agent of melioidosis, is a saprophytic bacterium readily isolated from wet soils of countries bordering the equator. Burkholderia mallei is a host-adapted clone of B. pseudomallei that does not persist outside of its equine reservoir and causes the zoonosis glanders, which is endemic in Asia, Africa, the Middle East and South America. Infection by these organisms typically occurs via percutaneous inoculation or inhalation of aerosols, and the most common manifestation is severe pneumonia leading to fatal bacteremia. Glanders and melioidosis are difficult to diagnose and require prolonged antibiotic therapy with low success rates. There are no vaccines available to protect against either Burkholderia species, and there is concern regarding their use as biological warfare agents given that B. mallei has previously been utilized in this manner. Hence, experiments were performed to establish a mouse model of aerosol infection to study the organisms and develop countermeasures. Using a hand-held aerosolizer, BALB/c mice were inoculated intratracheally with strains B. pseudomallei 1026b and B. mallei ATCC23344 and growth of the agents in the lungs, as well as dissemination to the spleen, were examined. Mice infected with 10², 10³ and 10⁴ organisms were unable to control growth of B. mallei in the lungs and bacteria rapidly disseminated to the spleen. Though similar results were observed in mice inoculated with 10³ and 10⁴ B. pseudomallei cells, animals infected with 10² organisms controlled bacterial replication in the lungs, dissemination to the spleen, and the extent of bacteremia. Analysis of sera from mice surviving acute infection revealed that animals produced antibodies against antigens known to be targets of the immune response in humans. Taken together, these data show that small volume aerosol inoculation of mice results in acute disease, dose-dependent chronic infection, and immune responses that correlate with those seen in human infections.     

Cellular mechanisms in the protection against infection by Listeria monocytogenes in mice.

Listeria monocytogenes, in doses of 2-0 X 10(3) to 3-0 X 10(3) viable organisms, was injected into athymic nude mice, irradiated mice and mice treated with reticuloendothelial system-blocking agents. Viable counts on liver and spleen homogenates were made at intervals after infection. In both nude mice (nu/nu) and normal littermates (nu/+) of BALB/c background, the bacteria grew rapidly for 24 h but increased only slowly thereafter, to reach a plateau of about 10(5) per organ at 72 h. In nu/+ mice, the number of viable bacteria began to decrease after 6 to 9 days, with complete elimination by day 12. In nude mice, the number of Listeria remained at a stable level of approximately 10(5) per organ during the observation period of 21 days. In lethally irradiated nu/+ mice, bacteria grew progressively and extensively to reach 10(7) per spleen and 10(9) per liver by 72 h. Bacterial growth during the first 72 h was markedly enhanced by treatment with carbon particles, dextran sulphate 500 or silica. These enhancing effects were also observed in nude mice and in AKR, C3H/He and C57BL/6 animals. We conclude that both non-immune phagocytes and T cell-dependent mechanisms contribute to the resistance of mice to Listeria infection.     

Studies with Brugia pahangi. I. Parasitological observations on primary infections of cats (Felis catus)

The large majority of cats given a single inoculation of third stage larvae of Brugia pahangi became microfilaraemic. Some cats had microfilariae in their blood 53 or 54 days after infection and most had become positive before 72 days after infection. In the majority of cats microfilarial counts remained very steady between 2 and 10 microfilariae per mm³ for long periods. At autopsy 10·7% of the infective larvae injected were recovered as adult worms. The recovery of adult worms was directly related to the number of larvae injected. The microfilarial level did not increase significantly with an increase in the number of adult worms.     

Analysis of Cellular Response and Gamma Interferon Synthesis in Bronchoalveolar Lavage Fluid and Lung Homogenate of Mice Infected with Pneumocystis carinii

The cellular and cytokine responses in the lungs of mice infected with Pneumocystis carinii were examined on both lung homogenates and bronchoalveolar lavage (BAL) fluids. In the lungs of infected mice, the number of P. carinii cysts rapidly decreased by day 7, then started to increase with a peak on day 14, and thereafter decreased gradually. When the presence of P. carinii was examined at the DNA level by dot blot hybridization, a similar clearance curve was obtained, and the organisms were shown to be completely eliminated on day 28. In the late phase of infection, leukocytes, mainly lymphocytes, increased in number when analyzed on lung homogenates, while no significant increase of inflammatory cells was observed in BAL fluids. An accumulation of both CD4⁺ and CD8⁺ T cells and an increase of activated T cells expressing IL-2Rα were observed in lung homogenates of the infected mice. In addition, a considerable amount of IFN-γ was detected in lung homogenates, but not in BAL fluids. These data indicate that lung homogenates are more suitable than BAL fluids for the analysis of cellular and cytokine responses in the lungs of mice infected with P. carinii. To define the involvement of IFN-γ in host defense against P. carinii, the effect of this cytokine on the killing activity of macrophages against P. carinii was examined in vitro. IFN-γ was found to augment this activity by increasing nitric oxide synthesis of the macrophages. Thus, it is suggested that IFN-γ plays an important role in the protection of mice from P. carinii infection.     

Active Immunization with an Octa-Valent Staphylococcus aureus Antigen Mixture in Models of S. aureus Bacteremia and Skin Infection in Mice

Proteomic studies with different Staphylococcus aureus isolates have shown that the cell surface-exposed and secreted proteins IsaA, LytM, Nuc, the propeptide of Atl (pro-Atl) and four phenol-soluble modulins α (PSMα) are invariantly produced by this pathogen. Therefore the present study was aimed at investigating whether these proteins can be used for active immunization against S. aureus infection in mouse models of bacteremia and skin infection. To this end, recombinant His-tagged fusions of IsaA, LytM, Nuc and pro-Atl were isolated from Lactococcus lactis or Escherichia coli, while the PSMα1-4 peptides were chemically synthesized. Importantly, patients colonized by S. aureus showed significant immunoglobulin G (IgG) responses against all eight antigens. BALB/cBYJ mice were immunized subcutaneously with a mixture of the antigens at day one (5 μg each), and boosted twice (25 μg of each antigen) with 28 days interval. This resulted in high IgG responses against all antigens although the response against pro-Atl was around one log lower compared to the other antigens. Compared to placebo-immunized mice, immunization with the octa-valent antigen mixture did not reduce the S. aureus isolate P load in blood, lungs, spleen, liver, and kidneys in a bacteremia model in which the animals were challenged for 14 days with a primary load of 3 × 10⁵ CFU. Discomfort scores and animal survival rates over 14 days did not differ between immunized mice and placebo-immunized mice upon bacteremia with S. aureus USA300 (6 × 10⁵ CFU). In addition, this immunization did not reduce the S. aureus isolate P load in mice with skin infection. These results show that the target antigens are immunogenic in both humans and mice, but in the used animal models do not result in protection against S. aureus infection.     

Prophylactic effect of human lactoferrin against Streptococcus mutans bacteremia in lactoferrin knockout mice

Streptococcus mutans is the primary agent of dental caries, which is often detected in transient bacteremia. Lactoferrin is a multifunctional glycoprotein showing antibacterial activities against several Streptococcus species. We reported here the prophylactic effect of human lactoferrin (hLF) in a lactoferrin knockout mouse (LFKO^−/−) bacteremic model. The hLF treatment significantly cleared S. mutans from the blood and organs of bacteremic mice when compared to the non-hLF treated mice. Further, analysis of serum cytokines, spleen and liver cytokine mRNA levels revealed that hLF prophylaxis modulates their release differently when compared to the non-hLF treated group. C-reactive protein level (P = 0.003) also decreased following hLF prophylaxis in S. mutans induced bacteremic mice. Additional quantitative RT-PCR analysis revealed that hLF prophylaxis significantly decreased the expression level of IFN-γ, TNF-α, IL-1β, IL-6, MPO and iNOS in spleen and liver. These results suggested that the hLF protects the host against S. mutans-induced experimental bacteremia.     

Effects of cysteamine onPseudomonas infections in mice

A non-lethal dose of cysteamine — a radioprotective agent — when administered intraperitoneally was demonstrated to increase the susceptibility of mice to infection byPseudomonas aeruginosa. At numbers of cells (about 10⁶) which killed 30–40 per cent of the untreated mice, the intraperitoneal treatment with 5 mg per day of cysteamine raised the mortality to 60 per cent. In contrast, the presence of 10 mg/ml of cysteamine in drinking water appeared to give some protection against the same number ofPseudomonas cells for the first three days after infection, but increased the susceptibility when the oral treatment was prolonged beyond three days. Such effects could have some significance in the study of sulfhydryl compounds as radioprotective agents.     

A murine pneumonia model to study pathogenesis of <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis>

Early attempts to develop an animal model of infection appeared to support the hypothesis that Stenotrophomonas maltophilia does not cause serious sepsis when bacteria are intravenously administered to mice. This species has also been implicated in an increasing number of infections such as, bacteremia, endocarditis, ophthalmological syndromes, skin lesions, urinary, respiratory tract and gastrointestinal infections. Despite this clinical importance, the mechanisms involved in the pathogenesis of S. maltophilia infections have not been elucidated and the virulence factors of importance in the pathogenesis of S. maltophilia associated pulmonary infection remain to be characterized. The purpose of this study was to establish an infection model using 5 clinical isolates of S. maltophilia in a mouse pneumonia model. All strains were able to establish themselves in respiratory tract with peak of infection occurring at 24 h post infection. The strains were able to cause neutrophil influx, were taken up and intracellularly killed by alveolar macrophages except Sm2 that persisted for a slightly longer time in the macrophages. All strains were resistant to lytic action of serum and survived in blood confirming their ability to cause bacteremia. The strains were cleared from spleen and liver by 7th and 4th day but caused tissue damage that was measured in terms of lipid peroxidation, lactate dehydrogenase activity and histopathological examination of lung tissue homogenate. All strains caused interstitial pneumonitis in lungs of mice.     

Potential use of probiotic <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> SS2 isolated from a fermented plant beverage: safety assessment and persistence in the murine gastrointestinal tract

A strain of Lactobacillus plantarum SS2 isolated from a fermented star fruit (Averrhoa carambola) beverage product in Thailand has been reported to have some probiotic properties, in vitro. In the present study, its in vivo safety and gastrointestinal survival following oral administration to mice was investigated. The acute toxicity test on ICR mice by force-feeding of a single dose of 10⁹ and 10¹² cells per mouse over 14 days after ingestion showed no adverse effects related to the normal behavior of the laboratory mouse. Although the weight gains of the dosed mice were lower than the control they were still within normal values. There were no significant differences in liver weight ratio or spleen weight index among tested mice and control mice and no evidence of bacteremia. Live SS2 cells labeled with a fluorescent dye (cFDA-SE) and administered orally at 10⁹ cells per mouse were shown to persist in the gastrointestinal tract for 7 days. Colonies similar to the SS2 were detected in fecal samples from the test mice even though the fecal lactic acid bacterial count showed no significant difference in any mice. The strain SS2 is therefore considered to be a possible alternative choice for an inoculum to produce fermented plant beverages.     

Activation of trehalase by heat shock in yeast ascospores: Correlation with total cellular cyclic-AMP content

An animal model resembling the human disease caused byCampylobacter jejuni has been developed. Characteristic illness followed intragastric challenge of neonatal mice with strains ofC. jejuni enhanced for virulence by serial intraperitoneal passage in weanling mice of organisms suspended in either mucin or iron dextran. Such passage lowered the LD₅₀ in weanlings from 2×10¹¹ colony-forming units (CFU) to 2×10⁵ CFU per mouse. Neonatal mice chellenged by intragastric intubation with 2×10⁹ CFU of the virulence-enhanced organisms suspended in mucin or iron dextran showed signs of infection by day 5, including severe diarrhea, increased musus discharge occasionally with blood, and reduced weight gain. Diarrhea contionued for eight days, after which most animals recovered. This mouse infection model provides a means for assessing the determinants of virulence among strains ofC. jejuni.     

Extraintestinal infection of Helicobacter cinaedi induced by oral administration to Balb/c mice

Although Helicobacter cinaedi was initially considered an opportunistic pathogen in immunocompromised patients, it was later shown to also infect immunocompetent and healthy individuals. Sporadic bacteremia due to H. cinaedi has frequently been reported; however, whether the bacterium can be translocated after passage through the intestinal mucosa remains unclear. In the present study, a preclinical small animal model that faithfully reproduces H. cinaedi infection in humans was developed. Balb/c male mice were orally inoculated with a single dose of 6.8 × 10⁷ CFU of a human clinical H. cinaedi strain. The organism persistently colonized the intestinal tract of the mice, particularly the cecum and colon, for at least 56 days, and the bacteria were excreted in the feces. Although inoculated bacteria were recovered from the spleen, liver, kidney, lung, bladder and mesenteric lymph nodes during the first 2 weeks of bacteremia, the organism was not isolated from these organs after 4 weeks, suggesting that complement‐ and antibody‐mediated serum sensitivity account for the relatively low frequency of systemic infection. However, H. cinaedi was isolated from the biceps femoris, triceps branchii, latissimus dorsi, and trapezius muscles beyond 2 weeks after infection and after production of specific anti‐H. cinaedi IgM and IgG antibodies. The present findings suggest that experimental infection of Balb/c mice with H. cinaedi may be a useful model for further studies of H. cinaedi pathogenesis, prophylaxis or therapeutic interventions in vivo.     

Pathogenicity of Hansenula anomala in a model of immunocompromised mice

Systemic infections caused by opportunistic fungi have shown an increased frequency in the past 10 years, particularly in immunocompromised patients. Hansenula anomala is an ascosporogenous yeast of the Ascomycetes class found in the skin, throat, and digestive tract transient normal flora. This study was conducted to compare the pathogenicity of H. anomala and Candida albicans in a model of immunocompromised mice. Thirty-eight Swiss mice were divided into two groups as follows: 30 animals received an intraperitoneal (i.p.) injection of cyclophosphamide (200 mg/kg) four days before the induction of infection with H. anomala (1 × 10⁶ yeasts/mL), and 8 animals received 100 mg/kg of cyclophosphamide at 3-day intervals during 3 weeks before inoculation of 1 × 10⁷ yeasts/mL. All animals were treated with amoxicillin/clavulanic acid (40 mg/kg) four days before induction of infection. A group of mice inoculatd with C. albicans (ATCC 64548) served as control. Tissue samples from the lung, spleen, liver, and kidney for histological and mycologic studies were obtained at necropsy. In each animal, the number of viable yeasts per gram of kidney was determined. The organs most frequently infected by H. anomala were the kidneys and the liver (20%), and the lung (10%). However, in conditions of sustained immunosuppression, H. anomala was found in 65.5% of the organs examined. It is concluded that in an experimental model of immunocompromised mice, the pathogenicity of H. anomala was low. This revised version was published online in August 2006 with corrections to the Cover Date.     

Infectivity of hepatic strain Klebsiella pneumoniae in diabetic mice

Besides urinary tract infection (UTI) and pneumonia, increased severe liver abscesses caused by Klebsiella pneumoniae (KP), especially in diabetic patients, have been observed in infections acquired in hospitals. This indicates that different KP strains with higher virulence have emerged in recent years. Our goal was to investigate the infectivity of KP isolates in mice from liver abscess or UTI patients. Mice were injected with streptozotocin to induce diabetes. Male ICR mice were infected with KpU1 (UTI strain CG3 for survival experiment only) and KpL1 (liver abscess strain CG5) by tail-vein injection of 5 x 10(4) colony-forming units (CFU) bacterial suspension. The mice survival rates, cytokine level by enzyme-linked immunosorbent assay (ELISA), and bacterial presence in liver tissue by Giemsa stain were examined. The survival rates for the KpL1-infected animals were 28% and 0% in normal and diabetic groups, respectively, whereas, for the KpU1-infected mice, the rates were 100% and 75% during a 30-day observation. Nonsurviving KpL1-infected mice showed > 10(5) bacteria/ml blood and the bacteria appeared in the liver sinus area and inside liver cells. The KpL1-infected mice showed a tendency to increase the blood interleukin 1beta (IL-1beta) level in both nondiabetic and diabetic groups, whereas the tumor necrosis factor-alpha (TNF-alpha) level was significantly decreased in the KpL1-infected diabetic mice (P = 0.002). In conclusion, the KP strain from liver abscess showed a greater virulence in mice than the KP from UTI and was more virulent in diabetic than in nondiabetic mice. The infection with KP from liver abscess significantly decreased the blood TNF-alpha level in diabetes mellitus (DM) mice and the blood IL-1beta level tended to increase in both infected nondiabetic and diabetic groups. High blood bacterial count and appearance of bacteria in liver sinus and cells usually contribute to death of the animals.     

Blastogenic responsiveness of spleen cells fromLegionella pneumophila-infected mice immunosuppressed with cyclophosphamide

Although guinea pigs are highly susceptible to experimental infection withLegionella pneumophila, mice are considered resistant. In the present study it was found that, although untreated mice resisted lethal infection with up to 10⁷ L. pneumophila, mice treated with three divided doses of cyclophosphamide became 10–100 times more susceptible. Injection of mice with 150 mg cyclophosphamide/kg body weight 96 and 48h prior to and on the same day as intraperitoneal challenge with graded dose ofL. pneumophila resulted in markedly increased lethality. Approximately half of the mice pretreated with cyclophosphamide succumbed to 10⁶ legionellae within 4–10 days after infection, and all treated animals given 10⁷ bacteria died. Legionellae were readily recovered from spleen, lymph nodes, and liver of surviving mice 4–10 days after infection, but not thereafter. Sensitization of mice with Legionella antigen was evident by the lymphocyte blastogenic test in vitro, by use of spleen cells at various times after infection. Mice given graded doses ofL. pneumophila evinced enhanced responsiveness to either formalin-killed whole cell vaccine, cell-free sonicate, or purified outer membrane antigen when tested in vitro on days 3 and 5. Peak responses generally occurred 20–35 days after infection. Mice given none or one dose of cyclophosphamide and injected with legionellae showed enhanced responses on day 5 of culture in vitro, a time when spleen cells from control nonsensitized animals showed much lower responses. Surviving mice given three doses of cyclophosphamide had lower blastogenic responses, generally as low as that occurring with spleen cells from nonsensitized animals. Thus suppression of immune responses of mice by cyclophosphamide substantially increased susceptibility toL. pneumophila and depressed blastogenic responsiveness.     

Reduction of Experimental Salmonella and Campylobacter Contamination of Chicken Skin by Application of Lytic Bacteriophages

Lytic bacteriophages, applied to chicken skin that had been experimentally contaminated with Salmonella enterica serovar Enteritidis or Campylobacter jejuni at a multiplicity of infection (MOI) of 1, increased in titer and reduced the pathogen numbers by less than 1 log₁₀ unit. Phages applied at a MOI of 100 to 1,000 rapidly reduced the recoverable bacterial numbers by up to 2 log₁₀ units over 48 h. When the level of Salmonella contamination was low (< log₁₀ 2 per unit area of skin) and the MOI was 10⁵, no organisms were recovered. By increasing the number of phage particles applied (i.e., MOI of 10⁷), it was also possible to eliminate other Salmonella strains that showed high levels of resistance because of restriction but to which the phages were able to attach.     

Specific protection of cyclophosphamide-immunosuppressed mice by vaccination withLegionella pneumophila

Mice are highly resistant to infection byLegionella pneumophila compared with guinea pigs. In the present study injection of mice with moderate doses of cyclophosphamide before and at the time of challenge injection with viable legionellae made the animals much more susceptible to these organisms. The doses of cycloplosphamide used for the short time suppressed the mice in terms of their ability to form antibody and develop cellular immunity toLegionella antigens as measured by the splenic blastogenic assay. Vaccination of mice with a sublethal dose ofLegionella (10⁶ organisms) or with 10⁷ killed bacteria in complete Freund's adjuvant protected the cyclophosphamide-treated mice from infection with liveLegionella. It seems apparent that a vaccine prepared from live organisms or killed bacteria in adjuvant may be protective for immunosuppressed mice.     

Staphylococcal Footpad Infection in Mice

Local footpad infection in mouse was investigated with 55 clinically isolated strains of Staphylococcus aureus. When 10⁷ viable cells were inoculated into the footpad, local swelling and bacterial growth resulted after 24 hr. With a dose of 10⁶ cells, moderate swelling was observed after a few hours but the reaction had almost disappeared after 24 hr. About 75% of the staphylococcal strains tested caused footpad edema in mice at doses of 10⁷ cells. A statistical comparison of the virulence of the organisms on intravenous and intraperitoneal injection with that in inducing footpad swelling is also reported.     

Epithelial entry rather than the ensuing systemic immune response determines the pathogenicity of two Salmonella enterica serovar Typhimurium strains in a mouse model

Most studies of Salmonella enterica serovar Typhimurium infection focus only on the pathogenicity of one strain. We investigated whether differences in pathogenicity of two wild-type S. Typhimurium strains; DT120 and SL1344, were related to gut invasion or the resulting immune response.Oral administration of a ten-fold lower number of SL1344 (10⁶ CFU) as compared to DT120 (10⁷ CFU) resulted in higher bacterial counts in liver and lymph nodes, and led to massive neutrophil infiltration of the spleen, while DT120 administration did not. In contrast, administration of the same dose (10³ CFU) of the two strains intravenously resulted in the same levels of bacteria and neutrophils in spleen and bone marrow. Oral administration of SL1344 led to an increase in neutrophil apoptosis in both spleen and the bone marrow and four out of five mice died before Day 8, while in DT120 mice, no increase in neutrophil apoptosis was observed and all mice survived until Day 8. This study reveals that two wild-type S. Typhimurium strains, despite evoking highly comparable immune responses upon intravenous injection, exhibit diverse pathogenicity in mice and thus suggests that differences in their invasiveness and survival during gut passage determines the success of the ensuing immune response.