Comparison of selective procedures for isolation and enumeration of Legionella species from hot water systems

E. LEONI AND P.P. LEGNANI. 2001 . Various sample pre-treatment techniques and different growth media for the isolation of Legionellae from hot water supplies in public buildings were compared. A total of 102 hot water samples from taps and showers was examined. The highest recovery frequency was obtained with the heat pre-treatment method and using the selective medium GVPC. However, the results differed according to the concentration of legionellas. In the case of low plate counts (≤5000 cfu l⁻¹), the heat pre-treatment technique gave a significantly higher percentage of positive samples compared with other techniques ( P  < 0·05). With increasing concentration, the differences between the procedures decreased until they became statistically not significant for concentrations above 50 000 cfu l⁻¹. The direct inoculum method allowed a significantly higher detection of concentrations ( P  < 0·001) compared with heat and acid decontamination methods, which brought about a 67–68% reduction in detectable Legionellae. Heat decontamination techniques show greater sensitivity and specificity. However, they underestimate the number of legionellas. In environmental surveillance programmes, this underestimate must be taken into consideration when assessing the health risk.     

Isolation of Legionella from water samples using various culture methods

The efficacy of a non-selective medium and two selective media were compared for the isolation of legionellas from water samples. The effect of acid wash treatment for decontamination of the water samples on the isolation frequency of legionellas was also studied. The 236 samples were taken from cooling, humidifying and drinking water systems; 21% were legionella-positive when inoculated directly on modified Wadowsky-Yee (MWY) medium and 26% were positive when concentrated (x 200) before cultivation on MWY or CCVC media. Inoculation on MWY medium after concentration followed by decontamination by the acid-wash technique gave the highest isolation frequency (31%). The lowest frequency (8%) was found with the non-selective BCYEα medium. An isolation frequency of 28% was achieved with the BCYEα medium after concentration and acid-wash treatment of the samples. Forty per cent of the samples were positive for legionellas when the results from all the culture methods were combined. Not all the legionella-positive samples were identified by a single culture method. Ninety-three of the 95 positive samples were detected with the two best combinations of three culture methods. The best culture method for detecting legionellas depended on the source of the water sample. Some water quality characteristics, like temperature and organic matter content, affected the isolation frequency of Legionella spp.     

A note on legionellas in whirlpools

Water samples from 52 whirlpools (jacuzzi), water temperature 35-40 degrees C, and from 50 swimming pools, water temperature 8-30 degrees C, were investigated for the presence of Legionella pneumophila. This was isolated from 11 of 28 whirlpools with free available chlorine less than 0.3 mg/l. No legionellas were detected in 23 whirlpools with free available chlorine over 0.3 mg/l. Legionella pneumophila was found in two swimming pools. The results indicate that 0.3 mg/l of free available chlorine is sufficient to eliminate legionellas from whirlpools.     

Comparison of polymerase chain reaction and conventional culture for the detection of legionellas in hospital water samples

A detection system for Legionella spp. based on the polymerase chain reaction (PCR) was used to assess the diagnostic value of PCR for the surveillance of contamination of man-made water systems by legionellas. A previously-published primer system was chosen to amplify a fragment of the 5S-ribosomal gene of Legionella spp. A total of 78 water samples from various sources were examined by PCR and culture on MWY Legionella selective agar. Fifty-seven of 78 water samples were positive by both test systems (73%), nine were positive by PCR only (11.5%), another nine were positive by culture but negative by PCR (11.5%), and three were negative by both techniques (3.8%). The PCR was inhibited when large amounts of rust were present in the samples. Culture failed to detect legionellas in samples that contained large numbers of other bacteria capable of overgrowing the legionellas. These results show that PCR is a rapid and sensitive technique for the detection of legionella contamination in water samples and that PCR and culture complement each other in monitoring of environmental water samples.     

A note on legionellas in whirlpools

Water samples from 52 whirlpools (jacuzzi), water temperature 35–40°C, and from 50 swimming pools, water temperature 8–30°C, were investigated for the presence of Legionella pneumophila. This was isolated from 11 of 28 whirlpools with free available chlorine less than 0.3 mg/1. No legionellas were detected in 23 whirlpools with free available chlorine over 0.3 mg/l. Legionella pneumophila was found in two swimming pools. The results indicate that 0.3 mg/l of free available chlorine is sufficient to eliminate legionellas from whirlpools.     

Simple and sensitive method for quantification of low tobramycin concentrations in human plasma using HPLC-MS/MS

After oral administration of tobramycin, as part of selective decontamination of the digestive tract (SDD) in critically ill patients, absorption of tobramycin from the gut into the blood may take place. To quantify low concentrations of tobramycin in human plasma, we developed and validated a simple (sample pre-treatment consisting of protein precipitation with acetonitrile using 200microl plasma), rapid (runtime 3min using a Pathfinder MR reversed-phase column) and sensitive (concentration range of 0.05-1.0mg/l using MS/MS detection) method.     

Automatic fermentation control based on a real-time in situ SIRE biosensor regulated glucose feed

Monitoring and regulation of fermentations is of a paramount industrial and academic importance in order to keep conditions optimal during the entire process. Established techniques employed today include HPLC and spectrophotometry, which both have the disadvantage that broth samples have to be drawn from the fermentor and that they often require sample pre-treatment. The objectives of this study was to design and evaluate a software controlled automatic real-time SIRE biosensor connected to a glucose feed solution pump for in situ based monitoring and regulation of the glucose concentration during a yeast fermentation process. The maximal frequency for the measuring-regulation cycles was 30/h. A 10 mM mean glucose concentration level was successfully maintained within +/-0.013 mM during 60 min fermentations at various concentrations of yeast (10, 20, 40 and 80g/l). The on/off-regulator used caused some expected fluctuations (oscillations) of the glucose concentration around the mean value (+/-0.12 mM at 10 g/l, +/-0.26 mM at 20 g/l, +/-0.51 mM at 40 g/l, and +/-0.99 mM at 80 g/l). A 7-h fermentation process (10 mM glucose and 20 g/l yeast) was successfully monitored and regulated. The obtained measuring data were found to be 8.5-22.9% lower than data obtained with a commercially available spectrophotometric kit. The difference increased linearly (-0.26 mM/h), during the fermentation process and indicated that some clogging of the in situ positioned probe occurred. The speed and the automatisation adaptability of the presented device suggest advantages compared to established techniques.     

Survival of Listeria monocytogenes in commercial food-processing equipment cleaning solutions and subsequent sensitivity to sanitizers and heat

AIMS: To determine the ability of Listeria monocytogenes to survive exposure to commercial food-processing equipment cleaning solutions and subsequent treatment with sanitizers or heat. METHODS AND RESULTS: Cells of five strains of L. monocytogenes were suspended in 1% solutions of eight commercial cleaners (pH 7.1-12.5) or in water (control) and incubated at 4 degrees C for 30 min or 48 h before populations were determined by plating on tryptose phosphate agar. After exposure of cells to cleaning solutions for 30 min, populations of the most resistant strain of L. monocytogenes were reduced by < or = 1.63 log10 cfu ml(-1). In only three highly alkaline cleaning solutions (pH 11.6-12.4) were populations reduced significantly (P < or = 0.05) compared with reductions in water. After 48 h, populations were significantly higher in one cleaning solution (pH 10.4) than in water, while populations in six of the other seven cleaning solutions were reduced by > or = 4.72 log10 cfu ml(-1). Cells exposed to cleaning solutions for 30 min became sensitive to 4.0 or 6.0 mg l(-1) free chlorine and to 50 or 100 mg l(-1) benzalkonium chloride and cetylpyridinium chloride, common components of quaternary ammonium sanitizers. Cells exposed to four of the five test cleaners had D56 degrees C values less than or equal to those of the control cells. CONCLUSIONS: Listeria monocytogenes tolerates exposure to a high concentration of alkaline cleaning solutions but consequently becomes sensitized to sanitizers. SIGNIFICANCE AND IMPACT OF THE STUDY: The elimination of L. monocytogenes surviving exposure to alkaline cleaning solutions widely used for food-processing equipment is essential and the appropriate use of sanitizers for subsequent application to equipment is important in achieving this goal.     

Risk factors for contamination of domestic hot water systems by legionellae.

To assess risk factors associated with the contamination of the domestic environment by legionellae, 211 houses in the Quebec City area were randomly selected and water samples were collected from the hot water tank, the shower heads, and the most frequently used faucet. After centrifugation, concentrated samples were seeded in triplicate on BCYE and GPV media. Data on the characteristics of the hot water system and plumbing in the house and on the personal habits of the occupants were collected for each house. Among these 211 houses, hot water was provided by either an oil or gas heater in 33 and by an electric heater in 178. Legionellae were isolated from none of the samples from houses with oil or gas heaters and from 39% (69 of 178) of those with electric water heaters (P less than 0.0001). This association remained highly significant after control for water temperature and other variables in a stratified analysis. In the 178 houses with an electric heater, 12% of the faucets, 15% of the shower heads, and 37% of the water heaters were contaminated. Legionella pneumophila serogroups 2 and 4 were the most frequently isolated strains. Logistic regression showed that factors associated with electric water heater contamination were (i) location of the house in older districts of the city (P less than 0.0001), (ii) old age of the water heater (P = 0.003), and (iii) low water temperature (P = 0.05). Contamination of the water heater was the only factor significantly associated with the contamination of peripheral outlets (P less than 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Risk factors for contamination of domestic hot water systems by legionellae.

To assess risk factors associated with the contamination of the domestic environment by legionellae, 211 houses in the Quebec City area were randomly selected and water samples were collected from the hot water tank, the shower heads, and the most frequently used faucet. After centrifugation, concentrated samples were seeded in triplicate on BCYE and GPV media. Data on the characteristics of the hot water system and plumbing in the house and on the personal habits of the occupants were collected for each house. Among these 211 houses, hot water was provided by either an oil or gas heater in 33 and by an electric heater in 178. Legionellae were isolated from none of the samples from houses with oil or gas heaters and from 39% (69 of 178) of those with electric water heaters (P less than 0.0001). This association remained highly significant after control for water temperature and other variables in a stratified analysis. In the 178 houses with an electric heater, 12% of the faucets, 15% of the shower heads, and 37% of the water heaters were contaminated. Legionella pneumophila serogroups 2 and 4 were the most frequently isolated strains. Logistic regression showed that factors associated with electric water heater contamination were (i) location of the house in older districts of the city (P less than 0.0001), (ii) old age of the water heater (P = 0.003), and (iii) low water temperature (P = 0.05). Contamination of the water heater was the only factor significantly associated with the contamination of peripheral outlets (P less than 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence of Legionella pneumophila in some arthropods and related natural aquatic habitats

Abstract Using direct fluorescent antibody (DFA) staining technique, Legionella pneumophila SG 1, 3 and 5 was evident in water samples collected from different localities of central Italian regions, Marche and Abruzzi; L. pneumophila SG 1 and 3 was also detected in aquatic stages of arthropods living in the Legionella -positive waters. Diptera, Coleoptera, Collembola and Isopoda were found to be positive for legionellas by DFA. Diptera, the most common in the waters surveyed, were represented by Chironomidae and Culicidae families, the latter being larval and pupal stages of genus Anopheles and Culex . Mosquito adults, emerged in laboratory from pupae collected in one sample of positive water, were also positive. The findings that aquatic arthropods harbor legionellas and whether they could be involved in the maintenance and dissemination of legionellas in nature are discussed.     

Exposure to non-acid fresh meat decontamination washing fluids sensitizes Escherichia coli O157:H7 to organic acids

AIMS: To investigate whether Escherichia coli O157:H7 maintains acid tolerance in water meat decontamination washing fluids. METHODS AND RESULTS: A rifampicin-resistant derivative of E. coli O157:H7 strain ATCC 43895 was inoculated (10(5) cfu ml(-1)) in spray-washings from meat sprayed with cold (10 degrees C) or hot (85 degrees C) water, stored at 10 degrees C for up to 14 days, and its acid tolerance was assessed at 2 and 8 days by exposure to broth or new washings adjusted to pH 3.5 or 3.7 with lactic or acetic acid. The pathogen survived in the water washings, but it was outgrown by the natural, Pseudomonas-like flora, and it was sensitized to acid. CONCLUSIONS: The acid tolerance of E. coli O157:H7 decreases following exposure to non-acid, but otherwise stressful, conditions prevailing in water meat washings at 10 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings suggest that the more intense use of water-based technologies should be included in meat decontamination strategies because they may contribute to enhanced meat safety by inducing acid sensitization in E. coli O157:H7.     

Removal of pathogen and indicator microorganisms from liquid swine manure in multi-step biological and chemical treatment

Concern has greatly increased about the potential for contamination of water, food, and air by pathogens present in manure. We evaluated pathogen reduction in liquid swine manure in a multi-stage treatment system where first the solids and liquid are separated with polymer, followed by biological nitrogen (N) removal using nitrification and denitrification, and then phosphorus (P) extraction through lime precipitation. Each step of the treatment system was analyzed for Salmonella and microbial indicators of fecal contamination (total coliforms, fecal coliforms, and enterococci). Before treatment, mean concentrations of Salmonella, total coliforms, fecal coliforms, and enterococci were 3.89, 6.79, 6.23 and 5.73 log(10) colony forming units (cfu)/ml, respectively. The flushed manure contained 10,590 mg/l TSS, 8270 mg/l COD, 688 mg/l TKN and 480 mg/l TP, which were reduced >98% by the treatment system. Results showed a consistent trend in reduction of pathogens and microbial indicators as a result of each step in the treatment system. Solid-liquid separation decreased their concentrations by 0.5-1 log(10). Additional biological N removal treatment with alternating anoxic and oxic conditions achieved a higher reduction with average removals of 2.4 log(10) for Salmonella and 4.1-4.5 log(10) for indicator microbes. Subsequent P treatment decreased concentration of Salmonella and pathogen indicators to undetectable level (<0.3 log(10) cfu/ml) due to elevated process pH (10.3). Our results indicate that nitrification/denitrification treatment after solids separation is very effective in reducing pathogens in liquid swine manure and that the phosphorus removal step via alkaline calcium precipitation produces a sanitized effluent which may be important for biosecurity reasons.     

The susceptibility of the giant freshwater prawn Macrobrachium rosenbergii to Lactococcus garvieae and its resistance under copper sulfate stress.

Addition of copper sulfate (0.1 to 0.4 mg l(-1)) to tryptic soy broth (TSB) had no effect on growth rate of the bacterial pathogen Lactococcus garvieae. Giant freshwater prawns Macrobrachium rosenbergii were injected with L. garvieae (4 x 10(6) colony-forming units [cfu] prawn(-1)) grown in TSB or TSB containing copper sulfate at 0.1, 0.2, 0.3 or 0.4 mg l(-1). After 48 h, the cumulative mortality was significantly (p < 0.05) higher for prawns exposed to L. garvieae grown in 0.4 mg l(-1) copper sulfate than at the lower concentrations examined. In other experiments, prawns were injected with TSB-grown L. garvieae (4 x 10(6) and 2 x 10(5) cfu prawn(-1)), then held in water containing copper sulfate. After 8 h the mortality of L. garvieae-exposed prawns held in water containing 0.4 mg l(-1) copper sulfate was significantly higher than prawns held in water containing 0.2 and 0.3 mg l(-1) copper sulfate. At the lower L. garvieae density, cumulative mortality of prawns increased directly with ambient copper sulfate concentrations in the range of 0.2 to 0.4 mg l(-1). All prawns survived a 168 h exposure to 0.1 mg l(-1) copper sulfate. Prawns exposed to different concentrations of copper sulfate were examined for hemocyte density, phenoloxidase activity and respiratory burst. No significant differences in hemocyte density were observed among treatments. In prawns following a 48 h exposure to 0.1 mg l(-1) copper sulfate, phenoloxidase activity was decreased, but respiratory burst was increased. In conclusion, copper sulfate increased the virulence of L. garvieae to M. rosenbergii and modulated its immune system. Copper sulfate at 0.1 mg l(-1) decreased susceptibility of M. rosenbergii to L garvieae infection, whereas at 0.2 mg l(-1) the susceptibility was increased. The generation of superoxide anion by M. rosenbergii exposed to copper sulfate at a concentration higher than 0.2 mg l(-1) was considered to be cytoxic.     

A laboratory hot tub model for disinfectant efficacy evaluation

This paper describes a novel laboratory hot tub (LHT) apparatus and associated standard operating procedure (SOP) designed to reproduce the key biological, chemical, and engineering parameters associated with recreational and therapeutic hot tubs. Efficacy, as measured quantitatively by log reduction values, was determined against both biofilm and planktonic bacteria. When the LHT was run according to the SOP, with no antimicrobial treatment, a consistent level of bacterial contamination occurred. The means of log10 viable cell densities (+/- the repeatability standard deviation of log densities) were 7.2 (+/-0.31) for the bulk water (density in units of cfu ml-1), 5.3 (+/-0.56) for the coupons (density in units of cfu cm-2), and 6.6 (+/-0.50) for the filters (density in units of cfu cm-2). When control and chlorine treated LHTs were run in parallel, the log reduction increased significantly with chlorine concentration for samples of planktonic bacteria in the bulk water (p=0.016), biofilm bacteria on the coupons (p=0.09) and biofilm bacteria on the filter (p=0.005), indicating that the method was sensitive to chlorine concentration. The method also displayed sensitivity by differentiating between chlorine and bromine treatments; in every case, chlorine produced a greater log reduction than did the same concentration of bromine. The model and SOP were shown to be rugged with respect to slight changes in fluid mixing intensity, water chemistry (saturation index), inoculum size, and organic loading. The LHT and associated SOP provide a reliable second tier in a three-tiered testing process, in which the first tier is a suspension test and the final tier is a field test.     

Effect of chemical decontamination and refrigerated storage on the isolation of Mycobacterium avium subsp. paratuberculosis from heat-treated milk

AIMS: To assess the impact of chemical decontamination and refrigerated storage before culture on the recovery of Mycobacterium avium subsp. paratuberculosis from heat-treated milk. METHODS AND RESULTS: Five-millilitre samples of ultra heat-treated (UHT) milk spiked with Myco. paratuberculosis NCTC 8578, B4 or 806R (ca 10(6) CFU ml(-1)) were heated at 63 degrees C for 20 or 30 min by submersion in a water bath. Heat-treated milk (0.5 ml) was cultured immediately into BACTEC 12B medium or refrigerated at 4 degrees C for 48 h before culture. Milk samples that received a 20-min heat treatment were also subjected to decontamination with 0.75% cetylpyridinium chloride (CPC) for 5 h at room temperature before inoculation into BACTEC 12B medium when tested immediately and after 48 h at 4 degrees C. BACTEC vials were monitored for evidence of growth over an 18-week incubation period at 37 degrees C. CPC decontamination resulted in a significant reduction in the number of culture-positive milk samples recovered immediately after heating (P < 0.05) and after refrigerated storage for 48 h (P < 0.01). Refrigerated storage for 48 h before testing did not have any significant effect, beneficial or detrimental, on Myco. paratuberculosis recovery rates. CONCLUSIONS: CPC decontamination applied to milk immediately or 48 h after heating will adversely affect the recovery of viable Myco. paratuberculosis, possibly leading to nonrecovery of the organism although viable cells are present in the original milk sample. SIGNIFICANCE AND IMPACT OF THE STUDY: Published pasteurization studies in which milk samples were decontaminated before culture will have underestimated the survival capability of Myco. paratuberculosis after high-temperature, short-time pasteurization. CPC decontamination should not be applied to pasteurized milk in future studies.     

Effect of potassium permanganate stress on immune resistance and susceptibility to Lactococcus garvieae in the giant freshwater prawn Macrobrachium rosenbergii

This work is part of a continuing series of investigations on the effect of commonly used aquaculture chemicals on the immune resistance and susceptibility of the giant freshwater prawn Macrobrachium rosenbergii to Lactococcus garvieae. The methodology has been described in earlier publications of the series. Potassium permanganate at 1.0 mg l(-1) in tryptic soy broth (TSB) had no effect on the growth rate of L. garvieae. The mortality of M. rosenbergii challenged with 4 x 10(6) colony-forming units (cfu) prawn(-1) of TSB-grown L. garvieae was significantly greater than that of challenged controls. Addition of potassium permanganate at 1.0 mg l(-1) in TSB significantly increased the virulence of L. garvieae to M. rosenbergii. Exposure of M. rosenbergii to potassium permanganate prior to challenge with TSB-grown L. garvieae at 4 x 10(6) and 3 x 10(6) cfu prawn(-1) revealed that 96 h mortality was significantly lower for prawns held in water containing 0.3 mg l(-1) of the chemical than for prawns in water containing 1.0 mg l(-1) or no chemical. Potassium permanganate caused no significant changes in total hemocyte counts and differential hemocyte counts, compared to the control treatments. However, a concentration of 1.0 mg l(-1) or more for 96 h resulted in decreased phenoloxidase activity, phagocytic activity and clearance efficiency. Respiratory burst increased with exposure to 0.3 mg l(-1). In conclusion, treatment with potassium permanganate at 0.3 mg 1(-1) was effective in reducing M. rosenbergii mortality from L. garvieae infection, but higher concentrations had a negative effect, probably due to reduced prawn defenses.     

Measurement of interstitial albumin in human skeletal muscle and adipose tissue by open-flow microperfusion

The absolute concentration of albumin was measured in the interstitial fluid of subcutaneous adipose tissue and skeletal muscle in six healthy volunteers by combining the method of open-flow microperfusion and the no-net-flux calibration technique. By use of open-flow microperfusion, four macroscopically perforated double lumen catheters were inserted into the tissue regions of interest and constantly perfused. Across the macroscopic perforations of the catheters interstitial fluid was partially recovered in the perfusion fluid. Catheters were perfused with five solutions, each containing different concentrations of albumin. Absolute interstitial albumin concentrations were calculated by applying linear regression analysis to perfusate vs. sampled albumin concentration (no-net-flux calibration technique). Interstitial albumin concentrations were significantly lower (P < 0.0001) in adipose tissue (7.36 g/l; r = 0.99, P < 0.0003; range: 4.3-10.7 g/l) and in skeletal muscle (13.25 g/l; r = 0.99, P < 0.0012; range: 9.7 to 15.7 g/l) compared with the serum concentration (48.9 +/- 0.7 g/l, mean +/- SE, n = 6; range: 46.4-50.4 g/l). Furthermore, interstitial albumin concentrations were significantly higher in skeletal muscle compared with adipose tissue (P < 0.01). The study indicates that open-flow microperfusion allows stable sampling of macromolecules from the interstitial space of peripheral tissue compartments. Moreover, the present data report for the first time in healthy humans in vivo the true albumin concentrations of interstitial fluid of adipose tissue and skeletal muscle.     

Detection of Legionella pneumophila at thermal spas

Water samples were collected at three therapeutic thermal spas in the area of Brescia, between February and October 2000: 34.8% of the samples contained Legionella pneumophila; the predominant isolates (30%) belonged to Legionella pneumophila serogroup 1. The microorganism was present in the spa water at high concentrations, generally higher than 10000 cfu/l. The large number of positive Legionella pneumophila samples indicates a potential risk of infection to patients, especially those undergoing inhalation treatment with thermal water, or those using a whirlpool or taking a shower even if, during the study, no clinical cases of Legionnaires' disease were observed. In some inhalators in use we detected Legionella pneumophila: after a treatment to eradicate the microorganism, no sanitary fittings currently show contamination. Thus, in our opinion, they are not sources of infection when they are mantained and serviced properly. Thermal disinfection and service checks at regular intervals are suggested for contaminated systems.     

HSP72 gene expression progressively increases in human skeletal muscle during prolonged, exhaustive exercise.

To examine the effect of exercise on heat shock protein (HSP) 72 mRNA expression in skeletal muscle, five healthy humans (20 +/- 1 yr; 64 +/- 3 kg; peak O(2) uptake of 2.55 +/- 0.2 l/min) cycled until exhaustion at a workload corresponding to 63% peak O(2) uptake. Muscle was sampled from the vastus lateralis, and muscle temperature was measured at rest (R), 10 min of exercise (Min10), approximately 40 min before fatigue (F-40 = 144 +/- 7 min), and fatigue (F = 186 +/- 15 min). Muscle samples were analyzed for HSP72 mRNA expression, as well as glycogen and lactate concentration. Muscle temperature increased (P < 0.05) during the first 10 min of exercise but then remained constant for the duration of the exercise. Similarly, lactate concentration increased (P < 0.05) when Min10 was compared with R but decreased (P < 0.05) thereafter, such that concentrations at F-40 and F were not different from those at R. In contrast, muscle glycogen concentration fell progressively throughout exercise (486 +/- 74 vs. 25 +/- 7 mmol/kg dry weight for R and F, respectively; P < 0.05). HSP72 mRNA was detected at R but did not increase by Min10. However, HSP72 mRNA increased (P < 0.05) 2.2 +/- 0.5- and 2.6 +/- 0.9-fold, respectively, when F-40 and F were compared with R. These data demonstrate that HSP72 mRNA increases progressively during acute cycling, suggesting that processes that take place throughout concentric exercise are capable of initiating a stress response.