The effect of summer and winter seasons on the survival of Salmonella typhimurium and indicator micro-organisms during the storage of solid fraction of pig slurry

Abstract

Aims: Investigations were carried out to observe the influence of winter/spring and summer periods on the survival of Salmonella typhimurium and indicator bacteria (psychrophilic, mesophilic, coliform and faecal coliform bacteria and faecal streptococci) in the solid fraction of pig slurry from agricultural wastewater treatment plant.

Methods and Results: Leather squares and PVC bottles with openings served as test carriers. They were inoculated with broth culture of Salm. typhimurium and introduced directly into the solid fraction. During the experiment, quantitative and qualitative examinations were carried out to determine the presence of Salm. typhimurium and observe the dynamics of indicator bacteria in the solid fraction.

Conclusions: Salmonella typhimurium survived for 26 d in summer and for 85 d in winter/spring. The T₉₀ values of indicator bacteria in summer ranged from 35·44 d (coliform bacteria) up to 100·29 d (mesophilic bacteria). The winter T₉₀ values of indicator bacteria ranged from 74·58 d (faecal coliform bacteria) to 233·07 d (coliform bacteria).

Significance and Impact of the Study: The present study demonstrated that it is necessary to pay increased attention to the manipulation of slurry solid fraction.

INTRODUCTION

The operation of large-capacity farms housing high number of animals in one location creates problems with regard to possible spreading of various diseases, some of them transmissible also to humans, either directly through excrement or via animal products. Diseases that can be transmitted include helminthoses, fungal diseases (e.g. trichophytosis), salmonellosis, leptospirosis, tularaemia and colibacillosis.

Farms with litterless technology, producing liquid excrements, pose a serious risk. This technology of housing fails to create conditions for biothermal processes in the manure which, together with other factors, encourage destruction of pathogens.

One way to solve these problems is the operation of wastewater treatment plants (WTP) on livestock farms. Wastewaters from large-capacity farms are processed by technologies which separate the solid and liquid fractions on vibrating screens or filter belts. The solid fraction is disposed to field manure storage or to landfills which could be a reservoir of many pathogens. On the other hand, it is necessary to remember that excrements of farm animals are an important source of organic substances which should be returned to the soil. Therefore, all methods of manure treatment should concentrate on the improvement of its properties and a maximal utilization of its manuring value (Tofant et al. 2000).

Venglovskýet al. (1994), Novák (1994) and colleagues concluded that the most suitable way of processing this material was a biothermic composting in the thermophilic temperature region which can ensure destruction of pathogens. Despite recommendations, insufficient attention has been paid to a practical manipulation of slurry solid fraction in practice. From this point of view we investigated survival of Salmonella typhimurium and a series of indicator micro-organisms in solid fraction of slurry. Salmonella typhimurium was chosen as a model strain since it can cause serious zoonoses disseminated via faecal contamination.

One of the most important factors affecting the survival of micro-organisms in the environment is temperature. Therefore we investigated the influence of winter and summer conditions on survival of the above-mentioned model strain and of indicator micro-organisms.

This study is intended to provide more information on pathogen survival in solid fraction of pig slurry. Currently, similar information is only available for slurry, sewage sludge and manures.

MATERIALS AND METHODS

Solid fraction of slurry

The solid fraction of slurry used in our experiments was obtained by slurry mechanical separation by vibrating screens. This is the first stage of the treatment process of pig slurry from a farm in Košická Polianka, Slovak Republic with capacity of 23 000 fattening pigs. Slurry was treated by an aerobic process in a WTP with mechanical, chemical and biological treatment stages.

Storage

The solid fraction was placed into a 50-l container. This container was located outdoors in a fenced space and covered with a netting to prevent contamination with insects. Experiments were carried out in the summer period in the months of June to August and in winter/spring from January to June. The temperature of the environment and in the solid fraction was recorded during the experiments in 1-h intervals by means of a programmable registration thermometer Commeter (COMET System, Roz✓nov p. R., Czech Republic).

Test carriers

Two test carriers were used. These were sterile leather squares (4 × 4 cm) and sterile PVC bottles (100 ml, with side openings to ensure the contact with the environment). Sterile 20 cm long glass ampoules, 1 cm in diameter, were used for controls.

Test bacterium and inoculation of test carriers

A lyophilized strain of Salm. typhimurium SK 14/39 (SZÚ Prague, Czech Republic) was used in our experiments.

Aliquots of 0·2 ml of 24-h broth cultures (Nutrient Broth No. 2, Imuna, Šarišské Michal’any, Slovakia) of Salm. typhimurium were used to inoculate leather squares. Dried leather pieces were transferred to a thermostat controlled at 37°C and incubated for 24 h to achieve good adhesion of salmonellae to the carriers.

A 20-g sample of slurry solid fraction was transferred to each PVC bottle, to which 0·5 ml aliquots of broth culture of Salm. typhimurium were applied directly into the solid fraction. Additionally, 1 ml aliquots of broth culture of Salm. typhimurium were transferred to control glass ampoules which were sealed.

Leather pieces, PVC bottles and control glass ampoules were inserted directly into the solid fraction placed in the 50-l container.

Quantitative and qualitative examinations of Salm. typhimurium

Both carriers were examined by the method of Müller (1973). Each leather carrier was transferred to 50 ml of sterile 0·9% saline solution under sterile conditions and 20 g of the material from the PVC bottle carrier was transferred to 180 ml of sterile 0·9% saline solution, and both shaken for 30 min by mechanical shaker. After 30 min sedimentation, each carrier was examined quantitatively and qualitatively for presence of salmonellae. In quantitative examination we prepared series of decimal dilutions up to 10^–10 from both carriers and the control ampoule and inoculated 0·1 ml on to Xylose Lysine Deoxycholate Agar (XLD, Imuna) and on to Salmonella Shigella Agar (SS, Imuna). Both plates were incubated at 37°C for 24 and 48 h.

In qualitative examination we used for the purpose of nonselective cultivation, Buffered Peptone Water (BPW, Becton Dickinson, Cockeysville, MD, USA) as a pre-enrichment medium which was incubated at 37°C for 24 h. The selective cultivation was then carried out in two enrichment media: Selenite medium (Imuna) at 37°C for 48 h, and in a medium according to Rappaport and Vassiliadis (Merck, Darmstadt, Germany) at 43°C for 48 h. A loopful from each of the selective broth was plated on XLD and SS agars (Imuna) and both plates were incubated at 37°C for 24 and 48 h.

The presumptive salmonella colonies were examined biochemically using a system for identification of bacteria from the family of Enterobacteriaceae (BBL Enterotube, Becton Dickinson). Serological examinations were carried out at the State Veterinary Institute in Košice (Slovakia), Department of Microbiology.

The carriers were examined in the time intervals presented in Table 1 (summer) and Table 4 (winter/spring). The values obtained by quantitative examination, presented in the tables, are arithmetic means of three parallel examinations. Qualitative tests were considered negative in those cases in which all three parallel examinations for the presence of Salm. typhimurium were negative.

Table 1.   Detection time of Salmonella typhimurium in solid fraction – summer

Table 4.   Detection time of Salmonella typhimurium in solid fraction – winter/spring

Presence of Salmonella spp. in the solid fraction of slurry

At the beginning of our experiments we examined the original solid fraction for the presence of Salmonella spp. using the method of Philipp et al. (1990). For the nonselective cultivation we added 5 g of the solid fraction to 45 ml of BPW (Becton Dickinson) and incubated the mixture at 37°C for 24 h. Selective cultivation was carried out in a Selenite medium (Imuna) and Rappaport-Vassiliadis medium (Merck). Aliquots from both selective media were inoculated in parallel on XLD agar (Imuna) and Rambach agar (Merck). The procedure used was the same as that described above for the qualitative examination of carriers.

Indicator bacteria – media and bacteriological procedures

Samples of the solid fraction for observation of dynamics of indicator micro-organisms were examined in time intervals specified in Table 2 (summer) and Table 5 (winter/spring). Twenty grams of the sampled material were transferred into a 500-ml bottle, 180 ml of 0·9% saline solution were added and the mixture was shaken for 5 min. Then, after 1 min of sedimentation, the supernatant was examined to determine the number of indicator micro-organisms. The samples were diluted with 0·9% saline solution down to the concentration of 10^–6.

Table 2.   Dynamics of indicator micro-organisms in solid fraction – summer

Table 5.   Dynamics of indicator micro-organisms in solid fraction – winter/spring

Psychrophilic and mesophilic bacteria.

Plate counts of psychrophilic and mesophilic bacteria were determined using Nutrient agar No. 2 (Imuna). The psychrophilic bacteria were incubated at 20°C for 72 h and the mesophilic bacteria at 37°C for 24 h.

Faecal coliforms and coliform bacteria.

Faecal coliforms and coliform bacteria were determined using Endo Agar (Imuna), incubated at 43°C for 48 h and at 37°C for 24 h, respectively.

Faecal streptococci.

Plate counts of faecal streptococci were determined using Selective agar for isolation of faecal streptococci (Imuna), incubated at 37°C for 24 h.

Physico-chemical parameters

The physico-chemical parameters – pH, dry matter content, ammonia nitrogen and total nitrogen – were determined according to Placháet al. (1997) and total phosphorus according to the Standard Methods for Examination of Water and Wastewater (APHA 1985). The samples for physico-chemical examinations were taken in time intervals specified in Table 3 (summer) and Table 6 (winter/spring).

Table 3.   Physico-chemical parameters in solid fraction – summer

Table 6.   Physico-chemical parameters in solid fraction −winter/spring

Statistical analyses

The influence of physico-chemical parameters on the survival of Salm. typhimurium and on the dynamics of indicator bacteria was evaluated by means of correlation analysis of logarithmically transformed data. The time of survival of the observed micro-organisms was expressed as T₉₀ values. The decimation time (T₉₀), as defined by Schlundt (1984), is the time taken for viable counts of a population to decrease by one logarithmic unit (log₁₀) which is equivalent to a 90% reduction. The decimation time T₉₀ was calculated according to the formula:

where α is a decimation constant corresponding to the line slope compared to the axis x. The T₉₀ values were determined using the method of minimum squares. In addition to the values of T₉₀, the tables also contain the gradient values of regression lines fitted through the given points.

RESULTS

Summer period

Survival of Salm. typhimurium.

During the summer period, Salm. typhimurium survived the longest period of 26 d in the PVC bottle carrier (Table 1). During this time a change in the pH value from alkaline to acidic was recorded (from the original value of 8·08–6·50 – Table 3). The counts of Salm. typhimurium bacteria in the control glass ampoules decreased only by one order of magnitude at the end of the experiment. The values of T₉₀ for Salm. typhimurium bacteria were 2·35 d on the leather carrier, 2·33 d in the PVC bottle and 66·63 d in the control ampoule (Table 1).

Indicator bacteria.

The numbers of indicator and other bacteria in the solid fraction, ranged from 10³ to 10⁷ cfu ml^–1 at the beginning of the experiment (Table 2). The most pronounced decrease (by two orders of magnitude) was observed in psychrophilic bacteria and faecal streptococci, and a lesser decrease (by one order) was observed in coliforms and mesophilic bacteria. Faecal coliforms persisted on the same level during the entire experiment with the exception of d 26, when an increase by one order was recorded followed by a subsequent decrease to the original value on d 48. The decimation time values ranged from 35·44 d (coliform bacteria) to 100·29 d (mesophilic bacteria, Table 2).

Physico-chemical parameters.

Of the physicochemical parameters observed, the most marked decrease was detected in the values of pH, which declined from the starting value of 8·08 to the final value of 5·91 at the end of the experiment. The values of NH₄⁺-N showed a decreasing tendency with the exception of d 5 of the experiment when a slight increase with regard to the starting value was observed. Other values showed slightly decreasing tendency (Table 3).

The temperature of the outer environment ranged from 10°C to 33°C and the temperature in the solid fraction varied between 17°C and 26°C.

Winter/spring period

Survival of Salm. typhimurium.

In the winter period, Salm. typhimurium was detected qualitatively until d 85 on both carriers (Table 4). Quantitative determinations provided positive results until d 4 on the leather carrier and until d 54 in the PVC bottle. The values of decimation times T₉₀ reached 0·96 d on the leather carrier and 12·91 d in the PVC bottles (Table 4).

Indicator bacteria.

The counts of indicator bacteria in the solid fraction during winter ranged from 10² to 10⁸ cfu ml^–1 of the sample (Table 5). During the storage of the solid fraction the numbers of psychrophilic and mesophilic bacteria decreased by one order of magnitude. The numbers of faecal streptococci and faecal coliforms decreased by two orders and the numbers of coliforms decreased by one order by d 54 of the experiment. However, on d 120 an increase to original numbers was observed. The values of T₉₀ ranged from 74·58 d (faecal coliforms) to 233·07 d (coliform microorganisms) (Table 5).

Physico-chemical parameters.

The physicochemical examination showed considerable decrease in ammonia nitrogen, which declined to 38% of the initial concentration (Table 6). The values of total nitrogen and total phosphorus showed a slightly increasing tendency. The value of pH decreased from the starting 8·52 to 7·51 and the values of volatile solids also decreased. Dry matter content was decreasing till d 40 of the experiment and then it increased to the starting value (Table 3).

The ambient temperatures fluctuated in the range between −11°C and +34°C and the temperature in the solid fraction ranged from −1°C to 30°C.

Statistical evaluation

In the summer period a statistically significant correlation was observed between survival of Salm. typhimurium and pH on both leather and PVC carrier (r=0·904, r=0·907, respectively). Significantly positive correlations were also observed between survival of salmonellae and the level of N-NH₃ (r=0·959), N-total (r=0·972), volatile solids (r=0·973), P-total (r=0·999), and a negative correlation between temperature and survival of salmonellae on PVC carrier (r=0·936). Temperature and the survival of salmonellae on the leather carrier exhibited a tendency to negative correlation (r=0·869) while dry matter and survival of the tested salmonellae correlated positively (r=0·842). The influence of other physico-chemical parameters on the survival of salmonellae was not significant. In this period a significantly positive correlation was observed between survival of coliform bacteria and P-total (r=0·908). Positive relationships were detected between faecal streptococci and volatile solids and dry matter (r=0·865, r=0·859, respectively). A tendency to positive correlations was also observed between coliform and psychrophilic bacteria and dry matter (r=0·891, r=0·837, respectively). No significant relationship was observed between other investigated parameters.

In the winter/spring period, comparison of survival of Salm. typhimurium and physico-chemical parameters showed significant correlations between survival and temperature on PVC and leather carriers (r=0·997, r=0·995, respectively). Salmonellae on the leather carrier were affected positively by dry matter and volatile solids (r=0·822, r=0·886, respectively) and negatively by N-total (r=0·888). A tendency to positive correlations between survival of salmonellae and pH and N-NH₃ was observed on the PVC carrier (r=0·862, r=0·847, respectively). A tendency to a positive correlation was also recorded between pH and faecal streptococci (r=0·802) and of negative correlations between temperature and faecal coliform bacteria and faecal streptococci (r=0·800, r=0·876, respectively).

DISCUSSION

The results indicate that the survival of Salm. typhimurium and indicator bacteria was considerably affected by temperature. Our results point to the differences in the survival of salmonellae during summer (26 d) and winter/spring (85 d) and therefore confirm the findings of a number of authors (Dean and Lund 1981; Strauch 1991; Ahmed and Sorensen 1995; Cabadaj et al. 1995). This observation is also supported by Müller (1973) who stated that different strains of salmonellae survived in slurry for 4–97 d in summer and up to 87 d in winter/spring.

In our study we refer to a winter/spring period because the experiment began in January and terminated in May. The tested strain was recovered qualitatively up to March but to terminate the experiment three subsequent negative analyses were needed. Shorter time of survival of Salm. typhimurium in the summer period in comparison with winter/spring period may be explained, besides other physicochemical factors, also by higher temperature in the summer. The increase in temperature in the spring was most likely one of the important factors contributing to devitalization of salmonellae in the winter/spring period. This is supported also by our statistical analyses (significant correlation between survival and temperature on PVC and leather carriers r=0·997, r=0·995, respectively).

In addition to the temperature effect, the survival of salmonellae is also affected significantly by the dry matter content. Mitscherlich and Marth (1984) compared the survival of Salm. typhimurium in three types of manure differing by dry matter content and concluded that Salm. typhimurium micro-organisms survived for 84 d in the manure with dry matter content of 17·3%, comparable with dry matter percentage in the solid fraction used in our experiments (Table 6), at a temperature of 10°C. This result is comparable with our results, which showed that Salm. typhimurium micro-organisms survived for 85 d in the solid fraction during the winter period (Table 4).

The longer survival of salmonellae at lower temperatures is also indicated by decimation time values, T₉₀, determined in our experiment. The T₉₀ values for the tested strain Salm. typhimurium on leather carriers and in the PVC bottles were 0·96 d and 12·91 d, respectively, in winter and 2·35 d and 2·33 d, respectively, in summer.

The influence of temperature on the survival of indicator micro-organisms is evident also in our experiments. The counts of indicator bacteria during summer and winter are comparable with the counts obtained by Ondrašovic✓ováet al. (1994) during the storage of slurry, with the difference that the results show considerable decrease in the number of coliform and faecal coliform micro-organisms during 6-week storage of slurry while in our experiment only psychrophilic micro-organisms in summer and faecal streptococci in winter exhibited pronounced decreases.

Our results are also comparable with the findings of Jepsen et al. (1997) from the storage of sludges in which a significant reduction of salmonellae was observed in summer (average temperature 20°C) and a slower reduction in winter (average temperature below 10°C). These authors did not observe systematic reduction in faecal streptococci during the storage and did not recommend to use them as indicator micro-organisms in the process of improving the hygienic quality of sludges by storage.

During our experiments we detected an increase of total nitrogen. Nitrogen is the major nutrient required by micro-organisms in the assimilation of the carbon substrates in organic wastes. Golueke (1977) postulated that 2/3 of the carbon in the material consumed is given off as CO₂ and the other 1/3 is combined with nitrogen in the living cell leading to an increase in total N.

Our results proved that the decrease of the pH value has a devitalization effect on micro-organisms. This was also confirmed by statistical evaluation which showed that the more apparent pH change from d 85 in winter and from d 48 in summer resulted in reduction in the counts of indicator micro-organisms. Our statistical evaluations point to the fact that the pH changes affect essentially the vitality of micro-organisms. The observations by Strauch (1987), according to whom 90% of salmonellae reduction is connected with pH decrease in the substrate, could be confirmed by our results. According to Strauch (1987) the decrease of the pH value during storage is influenced by the natural bacterial flora producing fatty acids which have toxic effects upon salmonellae. The latter, in contrast to natural bacterial flora, are not able to secure nutrients and this probably causes their die-off.

Numbers of indicator bacteria in the solid fraction detected by Placháet al. (1997) were higher than in an influent to a WTP found by Venglovskýet al. (1994), Fig. 1.

Figure 1.  Indicator bacteria in an influent (□) and in a solid fraction (▮)

Our results, well as those of other authors (Venglovskýet al. 1994; Pac✓ajová and Venglovský 1997), indicate that the solid fraction of pig slurry is highly contaminated with pathogenic micro-organisms and therefore its treatment deserves increased attention.

Niewolak (1994) stated that micro-organisms (Escherichia, Salmonella) that reach the soil by means of application of contaminated pig slurry can penetrate to the depth of 160–180 cm. Henry et al. (1995) isolated salmonellae from pasture 2 months, and from soil 8 months, after the application of contaminated pig slurry. This is the reason why it is necessary to ensure proper sanitation of contaminated slurry. The most suitable way of processing of the solid fraction of slurry is composting. Several authors, including Niewolak and Szelagiewicz (1997), Novák (1994) and Plachý (1995), have reported that composting results in significant decreases in pathogenic bacteria, fungi and helminth eggs, and resulting high quality organic manure with a considerable portion of humic substances.

The present study demonstrated that it is necessary to pay increased attention to the manipulation of slurry solid fraction.