Legionella pneumophila-induced suppression of early blastogenic responsiveness of murine spleen cells to specific and nonspecific stimulation in vitro

Legionella pneumophila whole cells, including viable organisms or a killed vaccine, early after injection into mice suppressed the blastogenic responses of mouse spleen cells to both specific (i.e.,Legionella) and nonspecific (i.e., plant mitogen andEschericia coli lipopolysaccharide) stimulators. Mice given injections of sublethal numbers of viableLegionella or of a killed vaccine evidenced 3–4 weeks thereafter a marked increase in blastogenic sensitivity of their spleen cells to theLegionella antigen, either whole cells or soluble antigen, but no increase in responsiveness to nonspecific mitogens (i.e., concanavalin A, phytohemagglutinin, andE. coli lipopolysaccharide) was evident. In contrast, during the first week or so after injection of mice with either viable or killedLegionella, marked suppression of blastogenic responsiveness of spleen cells toLegionella antigens was evident. Concomitant suppression also occurred to concanavalin A and phytohemagglutinin, as well as toE. coli lipopolysaccharide. However, by the second week after injection of the animals with live or killedLegionella, such suppression disappeared. The importance of such early specific suppression of a cellular immune response early after exposure toLegionella antigen, in contrast with the early and sustained rise in specific antibody formation is being further investigated.     

牛种布鲁氏菌2308不同途径小鼠感染模型的建立与评估

[背景] 布鲁氏菌可经口、皮肤、黏膜和呼吸道感染人和动物。小鼠是布鲁氏菌研究中最常用的模型动物。[目的] 建立牛种布鲁氏菌2308不同途径和剂量感染BALB/c小鼠的模型,为布鲁氏菌小鼠感染试验提供参考。[方法] 用10¹-10⁵ CFU这5个不同感染剂量,分别经注射、口服和点眼方式感染BALB/c小鼠。在感染后不同时间点采集小鼠血清,检测IgG、IgM、IgA抗体含量、脾脏重量及脾脏含菌量,评价布鲁氏菌经不同途径感染BALB/c小鼠的效果。[结果] 10 CFU是注射感染BALB/c小鼠的最小感染剂量;10⁵ CFU是口服感染BALB/c小鼠的最小感染剂量。10¹-10⁵ CFU这5个不同感染剂量经点眼途径均未能成功感染BALB/c小鼠。在10⁵ CFU感染剂量下,口服与注射感染组小鼠每克脾脏平均含菌量分别为10^5.673 CFU/g和10^5.009 CFU/g,无显著差异（P>0.05）,但口服感染组小鼠脾脏平均重量为0.310 g,显著高于注射感染组0.165 g （P<0.01）。在试验期内,注射感染组和口服感染组小鼠体内IgG抗体的滴度均随感染时间延长而持续升高;整体上,口服感染组IgG抗体峰值显著高于注射感染组;2组IgM抗体变化趋势一致;口服感染组有2只小鼠在感染28 d后产生IgA抗体,注射感染组均未检测到IgA抗体。[结论] 建立了牛种布鲁氏菌2308通过不同途径感染BALB/c小鼠的模型。     

Inhibitory effects of silver ions on Legionella pneumophila grown on agar, intracellular in Acanthamoeba castellanii and in artificial biofilms

Aims: We undertook a series of experiments to investigate the susceptibility of Legionella pneumophila grown under extracellular and intracellular conditions and other water‐related bacteria to silver ions. Methods and Results: In this study, the antimicrobial effect of silver ions to intra‐ and extra‐cellular grown Legionella bacteria was investigated. The minimal inhibitory concentration (MIC) after 24 h exposure, leading to a 5 log reduction, was c. 64 μg l^?1 AgNO₃ for extracellular grown Legionella and other tested Gram‐positive and Gram‐negative bacteria. In contrast, the MIC for intracellularly grown Legionella was up to 4096 μg l^?1 AgNO₃ after 24 h. Furthermore, the heterotrophic bacteria grown within a biofilm model were killed at a concentration of 4–16 μg l^?1 AgNO₃. In contrast, biofilm‐associated Legionella were less sensitive (MIC 128–512 μg l^?1 AgNO₃). Conclusion: Intracellularly and biofilm‐grown legionellae are less sensitive against silver compared with agar‐grown bacteria. Significance and Impact of the Study: The reduced sensitivity of Legionella grown in amoebae might explain why the effect of silver decontamination requires an extended exposure in field trials.     

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.     

Efficacy against lung metastasis with a tumor-targeting mutant of Salmonella typhimurium in immunocompetent mice

Salmonella typhimurium double leu-arg auxotrophs have been shown to be highly effective as antitumor agents in nude mouse models of human metastatic cancer. In order to proceed to clinical development of the S. typhimurium double auxotroph, termed A1-R, it is necessary to evaluate antitumor efficacy in immunocompetent mice. In the present study, we have observed the efficacy of A1-R on the Lewis lung (LLC) carcinoma in vitro as well as in C57BL/6 (C57) immunocompetent mice. In vitro, A1-R treatment of LLC began to induce cell death within one hour. Various doses and schedules of A1-R were administered to C57 mice implanted with LLC, including bolus single intravenous injection; medium dose with weekly intravenous administration and metronomic treatment with small intravenous doses twice a week. Bolus treatment was toxic to the immunocompetent host, in contrast to nude mice. Lower-dose weekly doses and metronomic doses were well tolerated by the immunocompetent host. Weekly intravenous injection with 2 x 10⁷ bacteria and twice a week intravenous injection with 10⁷ bacteria significantly inhibited metastasis formation, while bolus injection was toxic. Intra-thoracic administration was carried out with 10⁸ bacteria A1-R injected into Lewis lung-bearing C57 mice weekly for three weeks. Lung metastasis was significantly inhibited by intrathoracic bacterial administration, without toxicity. The results in this report, demonstrating the anti-metastatic efficacy of S. typhimurium A1-R in immunocompetent mice, indicate the clinical potential of bacterial therapy of cancer.     

THE CONVULSANT ACTION OF METHYLDITHIOCARBAZINATE: A COMPARISON WITH OTHER SULPHUR-CONTAINING HYDRAZIDES

—The convulsant action of methyldithiocarbazinate (MDTC), thiocarbohydrazide (TCH) and thiosemicarbazide (TSC) has been studied in mice. The relationship between dose and time to convulsions indicated that MDTC has a dual action and is more potent than TSC. Pretreatment of mice with pyridoxal phosphate (0.25 mmol/kg) protected against convulsions and death produced by low doses of MDTC or TCH, and low or high doses of TSC. Pretreatment with pyridoxine hydrochloride (0.25 mmol/kg) protected mice against TSC but not against TCH. It protected against low doses of MDTC (0.12 mmol/kg), but shortened the latency to convulsions after intermediate doses of MDTC (0.37 mmol/kg). Glutamate decarboxylase activity (GAD, EC 4.1.1.15) in whole brain homogenates from mice killed at the onset of seizures, was significantly reduced by all 3 drugs at all doses. This inhibition did not exceed 30% after any dose of TSC or TCH, but was 64% in mice killed 4 min after the injection of MDTC (0.98 mmol/kg). The addition of pyridoxal phosphate to brain homogenates abolished GAD inhibition after MDTC but not after TCH. In vitro brain GAD was 50% inhibited by 10⁻⁴m -MDTC, 18% by 10⁻⁴m -TSC and 8% by 10 ⁻⁴m -TCH. Kinetic studies suggested that at low concentrations MDTC inhibits by competing with pyridoxal phosphate. At the onset of convulsions the cerebral content of pyridoxal phosphate was reduced after low or high doses of TSC (0.27 and 2.2 mmol/kg) and after high doses of MDTC (0.98 mmol/kg). All three drugs (at 10⁻⁵−10⁻⁴m ) inhibited pyridoxal phosphokinase (EC 2.7.1.35) in vitro. Short latency convulsions after MDTC (0.37–0.98 mmol/kg) very probably arise from inhibition of cerebral GAD, due to competition for coenzymic sites and/or unavailability of coenzyme. Long-latency convulsions after MDTC (0.12–0.37 mmol/kg) are comparable to those seen after TSC (0.27–2.2 mmol/kg) and may depend on a mechanism additional to inhibition of GAD.     

Comparison of Detection Methods for Legionella Species in Environmental Water by Colony Isolation,Fluorescent Antibody Staining,and Polymerase Chain Reaction

Three detection methods for Legionella species in water samples from cooling towers and a river were examined. Direct counting of bacteria stained with fluorescent antibody (FA) for L. pneumophila (serogroups 1 to 6) could detect the cell of 10⁴ to 10⁶ cell/100 ml in all 14 samples, while colony counting method detected 10 to 10³ CFU/100 ml only in 8 samples from cooling towers. Polymerase chain reaction (PCR) assay with primers to amplify 16S ribosomal DNA sequence of most Legionella species (LEG primer) detected legionellae in 13 samples, while species-specific primers for L. pneumophila detected the DNAs from 3 samples. In laboratory examination, LEG primers could amplify DNAs of 29 species of genus Legionella with high sensitivity, even from 1 cell of L. pneumophila GIFU 9134. The PCR assay with LEG primers was specific and sensitive methods to be satisfied the survey of legionellae. Thus, PCR assay is a suitable method to detect and monitor Legionella species in an environment.     

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.     

The use of copper and silver in carbon point-of-use filters for the suppression of Legionella throughput in domestic water systems

Aims: To evaluate throughput of seeded Legionella pneumophila bacteria in domestic point‐of‐use filters. Methods and Results: The filters were challenged with tap water seeded with Leg. pneumophila. After multiple challenge events (4·25 × 10¹¹ CFU per filter), the levels of Legionella were lower in the effluent from the filter containing both copper and silver (mean 4·48 × 10³ CFU ml^?1) than in the effluent from the filter containing copper only (1·26 × 10⁴ CFU ml^?1; P < 0·001). After a single challenge event of approx. 5 × 10⁹ CFU L. pneumophila per filter, there was no significant difference between the levels of Legionella in the effluents from a carbon filter containing copper and a carbon filter with no metals (mean 6·87 × 10² and 6·89 × 10² CFU ml^?1, respectively; P = 0·985). Conclusions: Legionella was detected in filter effluent up to 6 weeks after being challenged, indicating that while filters may reduce the levels during an initial contamination event, the exposure is extended as the accumulated bacteria slough off over time. Significance and Impact of the Study: This study has provided an understanding of the response of Legionella to the use of silver and copper in domestic point‐of‐use carbon filters.     

Incidence of Legionella and heterotrophic bacteria in household rainwater tanks in Azumino,Nagano prefecture,Japan

Many administrative agencies in Japan are encouraging installation of household rainwater‐storage tanks for more effective use of natural rainwater. Water samples were collected periodically from 43 rainwater tanks from 40 households and tested for the presence of Legionella species and the extent of heterotrophic bacteria in Azumino city, Nagano prefecture, Japan. PCR assays indicated the presence of Legionella spp. in 12 (30%) of the 43 tank water samples. Attempts were made to identify correlations between PCR positive samples, topography, pH, chemical oxygen demand (COD), atmospheric temperature and the numbers of heterotrophic bacteria. Between June and October, 2012, the numbers of heterotrophic bacteria in rainwater tanks and the values of COD positively correlated with the presence of Legionella species. In most of the Legionella‐positive cases, heterotrophic bacterial cell counts were >10⁴ CFU/mL. Moreover, Legionella species were less frequently detected when the COD value was >5 mg KMnO₄/L. Therefore, at least in Azumino, Japan between June and October 2012, both heterotrophic bacterial counts and COD values may be considered index parameters for the presence of Legionella cells in rainwater tanks. Much more accumulation of such data is needed to verify the accuracy of these findings.     

Detection of culturable and nonculturable Legionella species from hot water systems of public buildings in Japan

Aims: To investigate the prevalence of culturable and nonculturable Legionella species in hot water systems of public buildings in Japan and assess the risk factors associated with Legionella contamination in hot water systems. Methods and Results: Legionella species were detected by conventional culture and molecular methods in 130 water samples collected from 40 buildings. A total of 26 (20·0%) water samples from 17 (42·5%) buildings were positive by culture, qualitative PCR or both methods: Legionella pneumophila and Leg. anisa were detected in four samples by a culture method, whereas 23 samples were positive by qualitative PCR, with the presence of various Legionella species confirmed by sequencing. Of these 23 samples, bacterial counts were quantifiable in 21 by real‐time PCR (from 1·7 × 10⁵ to 2·6 × 10¹¹ cells per litre). Phylogenetic analysis of amplified partial 16S rRNA gene showed close relations to various species of Legionella, including Leg. anisa and Leg. micdadei, all of which have been associated with respiratory diseases or increased antibody titres in human sera. Assessment of risk factors showed that turbidity, free chlorine concentration, iron concentration and heterotrophic plate count (HPC) were significantly associated with Legionella contamination (P < 0·05). Conclusions: Contamination of hot water systems of public buildings with culturable and nonculturable Legionella species may be a potential risk factor for Legionella infection in Japan. Adequate levels of chlorine, low levels of iron and HPC are important maintenance measures in the reduction of Legionella contamination in hot water systems. Significance and Impact of the Study: More than 40% of hot water systems in the Japanese public buildings examined were contaminated by not only culturable Leg. pneumophila and Leg. anisa but also by nonculturable pathogenic species. To our knowledge, this is the first report of both culturable and nonculturable Legionella contamination in hot water systems of public buildings in Japan.     

Systemic candidiasis after thermal injury

The effect of thermal injury on the host response to systemic infections withCandida albicans was studied by use of a 20% total body surface, full-thickness scald burn in inbred BALB/c mice. The lethal dose of intravenously injectedC. albicans required to kill 50% of the population (LD₅₀) of control, sham-burned mice was 4.7×10⁵ organisms, whereas the LD₅₀ of burned mice was 300 organisms (p<0.001). Mice injected intraperitoneally with 2×10⁷ C. albicans were assayed at two, four, seven, and ten days after burn for the presence of yeasts in the kidneys, skin, or burn wounds. At each time period examined, fewer yeasts were recovered from the kidneys of sham-burned mice than from burned mice. In addition, only one (5%) of 20 sham-burned mice had organisms in the skin at the time of sacrifice, whereas seven (37%) of 19 burned mice had organisms isolated from the burn wound (p<0.05). Wound histology demonstrated that the organisms were localized in the subeschar area and not colonizing the wound surface. This study describes the enhanced susceptibility of burned mice to systemic candidiasis and shows that a systemic infection withCandida can lead to organisms contaminating the wound.     

Usefulness of real-time PCR as a complementary tool to the monitoring of Legionella spp. and Legionella pneumophila by culture in industrial cooling systems

Aims: This study was designed to evaluate the usefulness of quantification by real‐time PCR as a management tool to monitor concentrations of Legionella spp. and Legionella pneumophila in industrial cooling systems and its ability to anticipate culture trends by the French standard method (AFNOR T90‐431). Methods and Results: Quantifications of Legionella bacteria were achieved by both methods on samples from nine cooling systems with different water qualities. Proportion of positive samples for L. pneumophila quantified by PCR was clearly lower in deionized or river waters submitted to a biocide treatment than in raw river waters, while positive samples for Legionella spp. were quantified for almost all the samples. For some samples containing PCR inhibitors, high quantification limits (up to 4·80 × 10⁵ GU l^?1) did not allow us to quantify L. pneumophila, when they were quantified by culture. Finally, the monitoring of concentrations of L. pneumophila by both methods showed similar trends for 57–100% of the samples. Conclusions: These results suggest that, if some methodological steps designed to reduce inhibitory problems and thus decrease the quantification limits, could be developed to quantify Legionella in complex waters, the real‐time PCR could be a valuable complementary tool to monitor the evolution of L. pneumophila concentrations. Significance and Impact of the Study: This study shows the possibility of using real‐time PCR to monitor L. pneumophila proliferations in cooling systems and the importance to adapt nucleic acid extraction and purification protocols to raw waters.     

Examination of microbial contaminants of emergency showers and eyewash stations

To evaluate the effects of regular flushing, water from fifty emergency eyewash and shower stations was cultured for the presence of potentially pathogenic protozoa, heterotrophic bacteria, and Legionella species. This study also provided the opportunity to evaluate a commercially available molecular assay for the direct detection of Legionella sp in environmental samples. The Perkin Elmer Legionella EnviroAmp polymerase chain reaction (PCR) kit and culture on buffered charcoal yeast extract agar were used to detect Legionella species in water samples. Chemical and physical parameters of station water measured included: pH, hardness, alkalinity, turbidity, conductivity, total chlorine and assimilable organic carbon. Protozoal isolates were identified by classical identification methods, and isolates from the stations were identified as Hartmannella sp, Vexillifera sp, Vahlkampfia sp, Acanthamoeba sp, and Vanella sp. Heterotrophic plate counts ranged from 10² to 10⁶ CFU ml⁻¹ and acridine orange total counts ranged from 10³ to 10⁶ cells ml⁻¹ after regular flushing. PCR and gene probe analysis showed that 89% of the stations (eyewash and shower) were positive for Legionella species by PCR, while 6% of the samples were culture positive. These results indicate that routine flushing alone is not sufficient to control microbial contamination and disinfection must also be included in a routine maintenance program. In addition, regular maintenance, disinfection, and monitoring of emergency eyewash and shower stations is important in preventing potential secondary microbial infections by either direct inoculation or aerosol transmission. Received 02 September 1997/ Accepted in revised form 29 November 1997     

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.     

Legionella in aquatic habitats in the Mount Saint Helens blast zone

Illnesses of undiagnosed etiology among researchers exposed to lakes and streams in the Mt. St. Helens blast zone after the 18 May 1980 eruption prompted us to determine the occurrence and potential virulence ofLegionella (Legionnaries' disease bacteria) in aquatic habitats near Mt. St. Helens during the summers of 1981 and 1982. Concentrations ofL. pneumophila, L. micdadei, L. gormanii, L. dumoffii, andL. bozemanii, determined by microscopic counts using direct immunofluorescent staining, ranged from <10⁴ to 10⁵ cells/l in lakes and rivers outside the Mt. St. Helens blast zone while the numbers ofLegionella in aquatic habitats inside the blast zone were from 10⁵ to 10⁷ cells/l.Legionella numbers were consistently highest in North Coldwater and Spirit lakes, which received water from hydrothermal seeps.Legionella pneumophila serogroups 4 and 6 were isolated from North Coldwater Lake in 1981 and from South Coldwater Creek in 1982, indicating that potentially virulent strains ofLegionella persist in aquatic habitats in the blast zone of Mt. St. Helens.Technical paper no. 6923, Oregon Agricultural Experiment Station.     

A rapid detection method using flow cytometry to monitor the risk of Legionella in bath water

Legionella species are the causative agents of human legionellosis, and bathing facilities have been identified as the sources of infection in several outbreaks in Japan. Researchers in Japan have recently reported evidence of significant associations between bacterial counts and the occurrence of Legionella in bathing facilities and in a hot tub model. A convenient and quantitative bacterial enumeration method is therefore required as an indicator of Legionella contamination or disinfection to replace existing methods such as time-consuming Legionella culture and expensive Legionella-DNA amplification. In this study, we developed a rapid detection method (RDM) to monitor the risk of Legionella using an automated microbial analyzing device based on flow cytometry techniques to measure the total number of bacteria in water samples within two minutes, by detecting typical patterns of scattered light and fluorescence. We first compared the results of our RDM with plate counting results for five filtered hot spring water samples spiked with three species of bacteria, including Legionella. Inactivation of these samples by chlorine was also assessed by the RDM, a live/dead bacterial fluorescence assay and plate counting. Using the RDM, the lower limit of quantitative bacterial counts in the spiked samples was determined as 3.0 × 10³ (3.48 log) counts mL^− 1. We then used a laboratory model of a hot tub and found that the RDM could monitor the growth curve of naturally occurring heterotrophic bacteria with 1 and 2 days' delayed growth of amoeba and Legionella, respectively, and could also determine the killing curve of these bacteria by chlorination. Finally, samples with ≥ 3.48 or < 3.48 log total bacterial counts mL^− 1 were tested using the RDM from 149 different hot tubs, and were found to be significantly associated with the positive or negative detection of Legionella with 95% sensitivity and 84% specificity. These findings indicated that the RDM can be used for Legionella control at bathing facilities, especially those where the effectiveness of chlorine is reduced by the presence of Fe²⁺, Mn²⁺, NH₄⁺, skin debris, and/or biofilms in the water.     

Sensitivity and Specificity of the Indirect Fluorescent Antibody Test in the Study of Four Murine Coccidia1

The sensitivity and specificity of the indirect fluorescent antibody (IFA) test for the detection of serum antibodies were examined in mice that were infected with Eimeria falciformis, E. ferrisi, E. papillata, or E. vermiformis. For the study of each species, five groups of mice were given graded inoculation doses of 10, 10², 10³, 10⁴, or 10⁵ sporulated oocysts in a primary infection. The sixth group was infected with three sequential doses of 1.5 times 10³, 1.5 times 10⁴, and 1.5 times 10⁵ sporulated oocysts per mouse at two- to three-week intervals. All groups of infected mice developed serum antibodies. Sera were titrated by the IFA test with purified sporozoites. Strong fluorescence and high IFA titers were observed with homologous reactions mainly with the sera from mice infected with the higher inoculation dose levels in primary infections and from those given three sequential inoculation doses. Immunological cross reaction among the four species of Eimeria occurred at dilutions of 1:10 to 1:160. Very weak or no fluorescence of free sporozoites was observed with sera from noninfected mice, and there was no fluorescence of sporozoites contained in intact sporocysts.     

LIGHT AND ELECTRON MICROSCOPY OF GRAZING BY POTERIOOCHROMONAS MALHAMENSIS (CHRYSOPHYCEAE) ON A RANGE OF PHYTOPLANKTON TAXA1

Grazing of fluorescent latex beads, bacteria, and various species of phytoplankton by Poterioochromonas malhamensis (Pringsheim) Peterfi (about 8.0 μm in diameter) was surveyed. The alga ingested fluorescent beads and various live or killed and nomnotile or motile organisms including bacteria, blue-green algae, green algae, diatoms, and chrysomonads. The size range of grazed prey was from 0.1 to 6.0 μm for latex beads and from 1.0 μm (bacteria) to about 21 μm (Carteria inverse) for organisms. As many as 17 latex beads (2.0 μm) or more than 10 Microcystis cells (5–6 μm) were ingested by a single P. malhamensis cell. Following such grazing, the cell increased in volume by up to about 30-fold. The range of cell volume of ingested prey was from 0.52 μm³ (bacteria) to about 3178 μm³(Carteria inversa). This study demonstrates for the first time that P. malhamensis is capable of grazing algae 2–3 times larger in diameter than its own cell and of grazing intact motile algae. Poterioochromonas malhamensis is an omnivorous grazer. Food vacuole formation and digestion processes were examined. The membrane that was derived from the plasma membrane and surrounded the prey disappeared sometime after ingestion. The food vacuole was then formed by successive fusion of numerous homogeneous vesicles accumulated around the prey. The prey was enclosed in a single membrane-bound food vacuole and then digested.     

The Influence of Immunization with Live or Killed Staphylococcus aureus Vaccines on the Early Development of Opsonizing and Bactericidal Factors in Lymph and Blood of Sheep

In order to obtain sensitive measurements on the synthesis of opsonins following immunization with live or killed S. aureus vaccines, lymph was collected from the efferent popliteal lymphatic duct of sheep during the early phase of the immune response. Lymph and blood serum were assayed for opsonizing capacity using ³H-labeled S. aureus. Within 1 hr after vaccination there was a rapid, transitory decrease in uptake by neutrophils of bacteria opsonized with lymph from sheep given the killed vaccine (Group 2). These results were in contrast to the relatively constant uptake rates of bacteria opsonized with lymph from sheep given the live vaccine (Group 1) and non-vaccinated controls (Group 3) at this time. At 72, 96, and 120 hr post-injection mean uptake values for bacteria opsonized with lymph from either vaccinated group were significantly greater than comparable values for controls. Mean uptakes for organisms opsonized with blood serum from Group 1 at 72 and 96 hr post-injection were significantly greater than comparable values for the control group. The percentage of viable neutrophil-associated bacteria decreased when lymph collected from animals in Group 2 in the first hour post-injection was used to opsonize the organisms. Percentages of viable, neutrophil-associated S. aureus for assays in which blood serum was used to opsonize remained relatively constant at around 45% for Groups 2 and 3. In contrast, however, values of viable neutrophil-associated bacteria for Group 1 decreased during the 120 hr after immunization.