Occurrence of Rhodococcus coprophilus and associated actinomycetes in feces, sewage, and freshwater.

Freshwater, sewage, and fecal samples from various sources were examined for Rhodococcus coprophilus, associated actinomycetes, Escherichia coli, and fecal streptococci. Rhodococcus coprophilus was isolated consistently from feces of farm animals, poultry reared in proximity to farm animals, freshwater, and wastewater polluted with animal fecal wastes. It was not isolated from samples of human feces. The ratio of R. coprophilus total actinomycetes was higher in feces from cattle, sheep, ducks, and geese than in specimens from pigs, horses, and fowl. In samples from two freshwater streams polluted by fecal material from farm animals, the ratios of R. copropilus to total actinomycetes were similar to those found in fecal specimens from cattle and sheep. Ratios of fecal coliform to fecal streptococci could not distinguish between fresh human and animal fecal samples and, furthermore, were not reflected in the stream waters polluted by animal fecal material. R. coprophilus has potential in water and dairy bacteriology as a specific indicator organism of fecal pollution due to farm animal wastes.     

Effect of sunlight on survival of indicator bacteria in seawater.

The stability of the natural populations of fecal coliforms and fecal streptococci in raw sewage diluted 1:1,000 in seawater or phosphate-buffered water at 24 +/- 2 degrees C was markedly affected by the absence or presence of sunlight. In the absence of sunlight, these bacteria survived for days, whereas in the presence of sunlight 90% of the fecal coliforms and fecal streptococci were inactivated within 30 to 90 min and 60 to 180 min, respectively. The bactericidal effect of sunlight was shown to penetrate glass, translucent polyethylene, and at least 3.3 m of clear seawater, suggesting that the visible rather than the ultraviolet light spectrum of sunlight was primarily responsible for the observed bactericidal effect. However, these same sewage-borne bacteria were relatively resistant to the bactericidal effect of sunlight when diluted in fresh mountain stream waters. These results indicate that the presence of sunlight is a major factor controlling the survival of fecal coliforms and fecal streptococci in seawater.     

Enteric Bacterial Growth Rates in River Water

Enteric bacteria, including stocked strains of pathogenic species and organisms naturally present in the stream, were capable of growth in a chemostat with autoclaved river water taken 750 m below a sewage outfall. Maximal specific growth rates for all organisms occurred at 30 C, whereas culture generation times ranged between 33.3 and 116 hr. Of the six laboratory strains of enteric species used, Escherichia coli and Enterobacter aerogenes grew at generation times of 34.5 and 33.3 hr, respectively, while the remaining Proteus, Arizona, Salmonella, and Shigella spp. reproduced at a rate two to three times slower than the coliforms. Little or no growth occurred in the water at incubation temperatures of 20 and 5 C, and death was observed for Salmonella senftenberg at 20 and 5 C and for E. aerogenes and Proteus rettgeri at 5 C. When enteric bacteria naturally present in the river water were employed in similar experiments, coliform bacteria demonstrated a generation time of approximately 116 hr, whereas fecal coliforms failed to grow. Growth of the bacteria from the river demonstrated a periodicity of approximately 100 hr, which suggests that much of the growth of these organisms in the chemostat may be on the glass surfaces. This phenomenon, however, was not observed with any of the stocked enteric species. Neither the stock cultures nor the aquatic strains were capable of growth in autoclaved river water taken above the sewage outfall at the three temperatures tested.     

Survival of the mite Acarophenax lacunatus (Cross & Krantz) (Prostigmata: Acarophenacidae) under starvation

The ability of a natural enemy to tolerate starvation increases its chances to survive in the absence of food, what is an important factor for its success in storage grain environment. The objective of the present work was to assess the survival of Acarophenax lacunatus (Cross & Krantz) in the absence of food. The experiment used individualized physogastric females of A. lacunatus placed in petri dishes (5 cm diameter) and maintained at 20, 25, 28, 30 and 32 degrees C, 50+/-5 % R.H. and 24h scotophase. The number of live mites was recorded every 6h thus assessing the progeny survival without food at different temperatures. The mites died within 60h at the temperatures 30 degrees C and 32 degrees C, while they survived for up to 108h at 20, 25 and 28 degrees C. The mean lethal time for death was 58.6h for the lowest temperatures and 39.3h for the highest temperatures. Thus, A. lacunatus subjected to starvation lived longer under lower temperatures, what is probably due to its lower metabolism. In contrast, the mites survived for about 90h at 28 degrees C, temperature commonly observed in tropical and subtropical climates, what may favor their use as control agents of stored product insects in these regions.     

Effect of water temperature on the development, release and survival of the triactinomyxon stage of Myxobolus cerebralis in its oligochaete host.

The development of the triactinomyxon stage of Myxobolus cerebralis and release of mature spores from Tubifex tubifex were shown to be temperature dependent. In the present work, the effect of temperature over a range of 5-30 degrees C on the development and release of the triactinomyxon stages of M. cerebralis was studied. Infected T. tubifex stopped releasing triactinomyxon spores 4 days after transfer from 15 degrees C to 25 degrees C or 30 degrees C. Transmission electron microscopic examinations of the tubificids held at 25 degrees C and 30 degrees C for 3 days showed that all developmental stages degenerated and transformed to electron-dense clusters between the gut epithelial cells of T. tubifex. In contrast, tubificid worms held at 5 degrees C and 10 degrees C examined at the same time were heavily infected with many early developmental stages of triactinomyxon. At 15 degrees C, the optimal temperature for development, maturing and mature stages of the parasite were evident. Infected T. tubifex transferred from 15 degrees C to 20 degrees C stopped producing triactinomyxon spores after 15 days. However, 15 days at 20 degrees C was not sufficient to destroy all developmental stages of the parasite. When the tubificid worms were returned to 15 degrees C, the one-cell stages and the binucleate-cell stages resumed normal growth. It was also demonstrated that T. tubifex cured of infection by holding at 30 degrees C for 3 weeks and shifted to 15 degrees C could be re-infected with M. cerebralis spores. The waterborne triactinomyxon spores of M. cerebralis did not appear to be as short-lived as previously reported. More than 60% of experimentally produced waterborne triactinomyxon spores survived and maintained their infectivity for rainbow trout for 15 days at water temperatures up to 15 degrees C. In natural aquatic systems, the triactinomyxon spores may survive and keep their infectivity for periods even longer than 15 days.     

Stability of pathogenic bacteria from laboratory animals in various transport media

The stability of pathogenic bacteria from laboratory animals was investigated in various transport media at different temperatures. Bordetella bronchiseptica and Salmonella typhimurium survived for 8 days in phosphate-buffered saline (PBS, pH 7.0) at 37, 24, 4 and -20 degrees C; Brucella canis at 24, 4 and -20 degrees C; Corynebacterium kutscheri at 4 and -20 degrees C; and Pseudomonas aeruginosa at all but -20 degrees C. A marked decrease in numbers of Pasteurella multocida and Past. pneumotropica was observed in PBS at all temperatures. Skimmed milk in PBS improved the survival of Pasteurella spp. and Ps. aeruginosa at -20 degrees C. Neither glycerin, ascorbic acid nor sodium thioglycollate improved survival. The numbers of viable B. canis, Ps. aeruginosa and S. typhimurium were maintained in blood or faecal specimens held for 8 days at 4 degrees C. These results indicated that transport in PBS at 4 degrees C was the only method satisfactory for all species of pathogenic organisms tested, but Pasteurella spp. were the most difficult to maintain.     

Survival and virulence of salmonellae in water

Survival and virulence of salmonellae in drinking and surface waters were tested in a series of model experiments using suspensions of fresh strains of Salmonella enteritidis as a tester strain. Environmental conditions in surface water, modeled by the addition of increasing amounts of municipal sewage, were simulated to have the organic pollution load equivalent COD to 5.3-9.7-15.2 mg O2/l and the coliform counts ranging from 2 X 10 to 2 X 10(3) per ml water. The experiments were carried out at 4 degrees C and 20 degrees C, i.e. at temperatures simulating the two crucial points of the year-round thermal characteristics of water in the external environment. Suspensions of S. enteritidis in water had the initial density ranging between 1 X 10(2) and 1 X 10(4) (per ml, tests for virulence were carried out in the guinea pig eye (conjunctivitis reaction). Time of S. enteritidis survival in the drinking water free of organic pollutants was directly affected by the initial density of strain and indirectly by water temperature, in surface water the most significant variable turned out to be the degree of organic pollution: the time of survival clearly tended to shorten as the complex of organic pollutants in water increased. At the highest degree of organic pollution (COD concentration 15.2 mg per ml) S. enteritidis survival was restricted to less than 24 h whereas in drinking water it could reach up to 30 days. The survival time was always identical with the time of virulence persistence.     

Thermal and water source effects upon the stability of enteroviruses in surface freshwaters

The long-term survival of three human enterovirus serotypes, Coxsackievirus B3, echovirus 7, and poliovirus 1 was examined in samples of surface freshwater collected from five sites of physically different character. These were an artificial lake created by damming a creek, a small groundwater outlet pond, both a large- and a medium-sized river, and a small suburban creek. Survival was studied at temperatures of -20, 1, and 22 degrees C. The average amount of viral inactivation was 6.5-7.0 log10 units over 8 weeks at 22 degrees C, 4-5 log10 units over 12 weeks at 1 degree C, and 0.4-0.8 log10 units over 12 weeks at -20 degrees C. The effect of incubation temperature upon viral inactivation rate was statistically significant (p less than 0.00001). As determined by pairing tests, survival was also significantly related to both viral serotype and water source at each of the three incubation temperatures (p less than or equal to 0.05). Efforts were made to determine whether the rate of viral inactivation observed at the different incubation temperatures was related to characteristics inherent to the water that was collected from the different locations. The characteristics examined included physical and chemical parameters, indigenous bacterial counts, and the amount of bacterial growth that the waters would support (measured as the maximum number of generations which seeded bacteria could undergo after being placed into either pasteurized or sterile-filtered water samples). Analysis of viral inactivation rate versus these characteristics revealed three apparent effectors of viral persistence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Application of polymerase chain reaction (PCR) and TaqMan PCR techniques to the detection and identification of Rhodococcus coprophilus in faecal samples.

Rhodococcus coprophilus, a natural inhabitant of herbivore faeces, has been suggested as a good indicator of animal (as opposed to human) faecal contamination of aquatic environments. However, conventional detection methods limit its use for this as they require up to 21 days to obtain a result. In this paper an optimised method for extracting R. coprophilus DNA from faecal samples is described. PCR and 5'-nuclease (TaqMan) PCR methods were developed to allow the detection and enumeration of R. coprophilus in faecal samples within 2-3 days. Both PCR methods targeted the 16S rRNA gene, producing an amplicon of 443 bp which was specific for R. coprophilus. Sixty cells were required to produce an amplification product by conventional PCR, while as little as one cell was required for the TaqMan PCR method. The latter approach gave a linear quantitative response over at least four log units with both bacterial cells and DNA. Successful amplification by PCR was achieved using DNA extracted from cow, sheep, horse and deer faeces but was negative for samples from humans, pig, possum, duck and rabbit. These PCR methods enhance the feasibility of using R. coprophilus to distinguish faecal pollution of farmed herbivores from human pollution.     

Temperature affects physiological stress responses to acute confinement in sunshine bass (Morone chrysops x Morone saxatilis)

Sunshine bass (Morone chrysopsxMorone saxatilis) were subjected to a 15-min low-water confinement stressor at temperatures ranging from 5 to 30 degrees C. Physiological responses were evaluated by measuring hematocrit, and plasma chloride, glucose and cortisol. Fish acclimated to 30 degrees C had initial glucose concentrations of 3.13 mM (564 mg/L) which were significantly lower than in fish acclimated to 5 and 10 degrees C (4.32 and 4.82 mM or 779 and 868 mg/l, respectively). Fish survived the conditions imposed at every temperature except 30 degrees C, where 15 out of 42 fish died during the stress and recovery protocol. The general pattern was an initial increase in hematocrit, followed by a delayed decrease in hematocrit and chloride, and an increase in plasma glucose and cortisol. In general, fish stressed at temperatures below 20 degrees C had lower and more delayed changes in plasma glucose and cortisol than fish tested at 20, 25 and 30 degrees C. Initial cortisol concentrations were 65 ng/ml and increased to above 200 ng/ml in fish held at 20 degrees C and above. At the higher temperatures, glucose concentrations were twice the initial concentration after stress and cortisol changes were four to five times the initial concentration after the stress. Quantitative responses for glucose and cortisol were moderate and recovery rapid in fish stressed at 10 and 15 degrees C; therefore, this range of water temperature is recommended when handling sunshine bass.     

Temperature effects on survival and excystment of cercariae of Halipegus occidualis (Trematoda)

Survival of the cystophorous cercariae of Halipegus occidualis (Trematoda: Hemiuridae) from naturally infected Helisoma anceps (Gastropoda: Planorbidae) was determined following constant temperature storage at 4, 16, 22, 26 and 30 degrees C. At 4 degrees C, 90% of cercariae survived for 14 weeks and 50% survived for 30 weeks. Survival varied inversely with temperature, and at 30 degrees, 90% survived for 1.5 weeks and 50% survived for 2.5 weeks. At the time of release from snails, cercariae excysted within 0.5 s of stimulation. Excystment times ultimately increased to 1-3 s at all temperatures as length of storage increased, but no connection was detected between excystment time and the activity of the cercaria. Excystment times began increasing while survival was still above 90%. Temperature specific mortality rates of cercariae were estimated, and used to simulate the survival of cercariae released naturally at various times of the year. The results of these simulations suggest that cercariae of H. occidualis are present in the environment at all times of the year, but that cercariae present in late winter (January to mid-March) may have an impaired excystment mechanism that reduces their infectivity.     

A comparison of different culture media for the membrane filter quantification of Aeromonas in water

A comparative assessment of culture media for the membrane filter enumeration of Aeromonas spp. in water was performed, testing the effects of different incubation conditions (aerobic and anaerobic), temperatures (30 and 37 degrees C) and times (24 and 48 h). Different water samples seeded with test suspensions of Aeromonas spp., fecal material or raw sewage were examined. Results indicate clearly that plates should be incubated aerobically at 30 degrees C for 24 h. If the bacterial contamination is likely to be low, the use of most sensitive culture media, such as SAA, mA, ADA or PADE Agar, is recommended. By contrast, samples with an expected high level of background microbial flora should be analysed through more selective media, such as MIX Agar. However, the low selectivity of all media tested and the high likelihood of false negatives based upon the macroscopic examination of colonies means that further research directed to the development of more efficient media is needed.     

Influence of temperature on Ranavirus infection in larval salamanders Ambystoma tigrinum

Temperature strongly influenced percent mortality and time to death of salamanders exposed to the Ambystoma tigrinum virus (iridovirus) (ATV). Most salamanders survived when exposed at 26 degrees C, whereas all died at 18 degrees C and nearly all died at 10 degrees C. Some asymptomatic salamanders that survived 60 d at 10 or 26 degrees C were found to be carrying virus. Polymerase chain reaction (PCR) confirmed the presence of virus in ATV-exposed salamanders but was found to be less sensitive than cell culture in detecting ATV at low concentrations. PCR products were 100% identical to ATV in the major capsid protein sequence. Virus titer was higher in salamanders held at 10 degrees C than at 18 degrees C but little virus, if any, was present in the small number of salamanders that died at 26 degrees C. These results may help explain periodic viral epizootics in field populations of A. tigrinum where water temperatures fluctuate widely.     

Sensitivity of Encephalitozoon cuniculi to various temperatures, disinfectants and drugs.

Spores of Encephalitozoon cuniculi were exposed to various temperature or to disinfectants, and their infectivity was then tested on monolayer cultures of canine kidney cells. The maximum survival time for spores suspended in medium 199 was 1 day at -20 degrees C, 98 days at 4 degrees C, 6 days at 22 degrees C, and 2 days at 37 degrees C. Only 2.5% survived 30 min at 56 degrees C. Boiling for 5 min or autoclaving at 120 degrees C for 10 min killed all spores. Dry spores survived less than a week at 4 degrees C but at least 4 weeks at 22 degrees C. Exposure for 30 min to recommended working concentrations of 9 of the 11 disinfectants tested killed all spores. The growth-inhibition effect of 7 antibiotics and chemotherapeutics was studied on canine kidney cell culture inoculated with E. cuniculi. None could completely inhibit growth. The most effective was chloroquine phosphate which, at a concentration of 12.5 mg per 1000 ml culture medium and during a test period of 8 weeks, reduced the harvest of E. cuniculi to 31% of that from inoculated, untreated cultures.     

Fecal indicator bacteria persistence under natural conditions in an ice-covered river.

Total coliform (TC), fecal coliform (FC), and fecal streptococcus (FS) survival characteristics, under natural conditions at 0 degrees C in an ice-covered river, were examined during February and March 1975. The membrane filter (MF) technique was used throughout the study, and the multiple-tube (MPN) method was used in parallel on three preselected days for comparative recovery of these bacteria. Survival was studied at seven sample stations downstream from all domestic pollution sources in a 317-km reach of the river having 7.1 days mean flow time (range of 6.0 to 9.1 days). The mean indicator bacteria densities decreased continuously at successive stations in this reach and, after adjustment for dilution, the most rapid die-off was found to occur during the first 1.9 days, followed by a slower decrease. After 7.1 days, the relative survival was TC less than FC less than FS, with 8.4%, 15.7%, and 32.8% of the initial populations remaining viable, respectively. These rates are higher than previously reported and suggest that the highest survival rates for these bacteria in receiving streams can be expected at 0 degree C under ice cover. Additionally, the FC-FS ratio was greater than 5 at all stations, indicating that this ratio may be useable for determining the source of fecal pollution in receiving streams for greater than 7 days flow time at low water temperatures. The MPN and MF methods gave comparable results for the TC and FS at all seven sample stations, with both the direct and verified MF counts within the 95% confidence limits of the respective MPNs in most samples, but generally lower than the MPN index. Although FC recovery on membrane filters was comparable results at stations near the pollution source. However, the results became more comparable with increasing flow time. The results of this study indicate that heat shock is a major factor in suppression of the FC counts on the membrane filters at 44.5 degree C. Heat shock may be minimized by extended incubation at 35 degrees C before exposure to the higher temperature.     

Survival of F-specific RNA coliphage,feline calicivirus,and Escherichia coli in water: a comparative study

The relationship between the survival of enteric viral pathogens and their indicators (coliform bacteria and coliphages) is not well understood. We compared the survival rates of feline calicivirus (FCV), Escherichia coli, and a male-specific RNA coliphage MS2 at 4, 25, and 37 degrees C for up to 28 days in dechlorinated water. The survival rates of E. coli and FCV, a surrogate of noroviruses (NV), had a high degree of correlation at 4 and 25 degrees C, while MS2 phage survived significantly longer (P < 0.05) at these two temperatures. At 37 degrees C, the survival rates for all three organisms were highly correlated. Decimal reduction values indicating the number of days needed for 90% reduction in titer (D values) decreased for all three organisms as storage temperatures increased. FCV had the shortest D value among all three organisms at all temperatures investigated. These findings indicate that F-specific RNA phages may be useful indicators of NV in the environment.     

Effects of high temperatures on functional responses of haemocytes in the clam Chamelea gallina

The effects of high temperatures on the clam, Chamelea gallina, generally recognised as a low tolerant bivalve species, were studied by evaluating some functional responses of the haemocytes. The animals were kept for 7days at 20, 25 and 30 degrees C and total haemocyte count (THC), phagocytosis, lysozyme activity (in both haemocyte lysate and cell-free haemolymph), activity and expression of the antioxidant enzyme superoxide dismutase (SOD) (in both haemocyte lysate and cell-free haemolymph) were chosen as biomarkers of exposure to high temperatures. The survival-in-air test was also performed. During the experiment, the clams showed differing burrowing behaviour: the animals kept at 20 and 25 degrees C burrowed completely, whereas at 30 degrees C the clams progressively emerged from the sediment and then remained on the surface. The highest temperature significantly increased THC, whereas it decreased the phagocytic activity of haemocytes. The haemocyte size frequency distribution in clams kept at 30 degrees C showed that the cell population of about 8-10microm was markedly reduced compared to clams kept at 20 and 25 degrees C. In clams maintained at 25 degrees C, lysozyme activity was significantly increased in haemocyte lysate, whereas it was markedly decreased in cell-free haemolymph. Total SOD activity significantly decreased in haemocytes from clams held at 30 degrees C whereas it increased in cell-free haemolymph from clams held at 25 degrees C and 30 degrees C. A significant decrease in haemocyte Mn-SOD and Cu/Zn-SOD activities was found with increasing temperature. In cell-free haemolymph, the highest Mn-SOD activity was recorded at 30 degrees C, whereas the Cu/Zn-SOD activity showed no significant changes in clams maintained at different temperatures. SOD isoform expression exhibited different patterns in haemocyte lysate and cell-free haemolymph. The resistance to air exposure of clams kept at 30 degrees C was shown to decrease significantly, LT(50) values fell from 6days in clams kept at 20 degrees C and 25 degrees C to 4days in those kept at 30 degrees C.     

Deleterious effects of mild simulated overwintering temperatures on survival and potential fecundity of rose-galling Diplolepis wasps (Hymenoptera: Cynipidae)

Prepupae of the rose galling Diplolepis spinosa from areas with relatively cold winters in southern Canada, and Diplolepis variabilis from a milder locale in western Canada, were used to test the hypothesis that mild winter temperatures are detrimental to the survival and potential fecundity of insects. Prepupae of D. spinosa held within or removed from their galls were exposed to simulated overwintering temperatures (-22, 0, 5, or 10 degrees C) for approximately four months before measuring their survival, body size, and potential fecundity. Similar studies were conducted using prepupae of D. variabilis that were removed from their gall and subjected to 0 degrees C or 10 degrees C treatments. Diplolepis spinosa, with or without their galls, averaged 66% more mortality at 10 degrees C than at 0 degrees C. Female D. spinosa that survived the 10 degrees C treatment had 32% fewer eggs than those held at 0 degrees C. In contrast, there was no difference in survival or numbers of eggs between D. variabilis held at 0 degrees C and 10 degrees C. Body size of adult females and size of eggs did not differ among temperature treatments for either species. We conclude that mild overwintering temperatures may be detrimental for insects by raising their metabolism, and consequently reducing energetic reserves needed for development to the adult stage and subsequent production of eggs the following spring.     

Effects of temperature and humidity on the development of eggs of Toxocara canis under laboratory conditions

The influence of temperature and humidity on the survival and development of Toxocara canis eggs in an in vitro model system was investigated. Two soil samples were inoculated with T. canis eggs and maintained at 3% and 50% humidity and temperatures of 19-24 degrees C. Nine soil samples were inoculated with T. canis eggs of which three samples were kept at 4 degrees C with humidities at 3%, 15%, and 30%; three were maintained at 21 degrees C and three more were incubated at 34 degrees C, and at the same three humidity levels. Samples were monitored every 7 days for a total of 2 months, for the presence and development of eggs. With increasing temperature, the number of eggs undergoing development increased (P<0.01); the number of deformed eggs decreased, the number of infective eggs increased (P<0.01), and egg maturation was accelerated. A decrease in the survival of infective eggs occurred at 34 degrees C. An increase in humidity produced a rise in the number of developed eggs at all three temperatures (P<0.01). This study suggests that elevated temperatures accelerated the development as well as the degradation of eggs of T. canis, whereas the range in humidity was directly correlated with egg development.     

Influence of temperature on survival, growth and fecundity of the freshwater snail Indoplanorbis exustus (Deshayes).

To note the effect of temperature on survival, growth and fecundity, newly hatched (zero day old) snails Indoplanorbis exustus were cultured at 10 degrees, 15 degrees, 20 degrees, 25 degrees, 30 degrees and 35 degrees C constant temperatures and room temperature (17.5 degrees-32.5 degrees C). Individuals exposed to 10 degrees C died within 3 days while those reared at 15 degrees, 20 degrees, 25 degrees, 30 degrees, 35 degrees C and room temperature survived for a period of 6, 27, 18, 16, 12 and 17 weeks respectively. An individual added on an average 0.21 mm and 0.45 mg, 0.35 mm and 7.94 mg, 0.63 mm and 15.5 mg, 0.81 mm and 27.18 mg, 1.07 mm and 41.48 mg and 0.78 mm and 31.2 mg to the shell diameter and body weight respectively at those temperatures per week. The snails cultured at 15 degrees C died prior to attainment of sexual maturity. On an average, an individual produced 31.9 and 582.77, 54.86 and 902.18, 56.01 and 968.45, 49.32 and 798.68 and 62.34 and 1143.97 capsules and eggs respectively at 20 degrees, 25 degrees, 30 degrees, 35 degrees C and room temperature (17.5 degrees-32.5 degrees C).