The effect of composting on the survival of Escherichia coli O157:H7 in bovine manure.
Animal manure use is well accepted among farmers and demand for manure is projected to increase as organically grown vegetables and fruits gain in popularity. In most countries, however, animal manure is often viewed as a waste management problem (Eghball and Power, 1994) due to the availability of inexpensive man made commercial fertilizer and the prohibitive handling cost of manure from the confinement to the agricultural fields. This results in manure disposal at excessive rates on agricultural field nearby the feed lot or disposed on crop fields without consideration to nutrient values. Lack of storage space is also one of the waste management problems encountered by farmers. In addition, raw cattle manure may not only led to serious pollution problems by fouling water ways and disrupting the flora and fauna in the soil but may play an important role in the persistence and dissemination of pathogens on agricultural environment. Vegetable plants can become contaminated with pathogens before harvest when grown in fields fertilized with fresh or aged manure (Islam et al., 2004). This in no doubt has spelt out clearly the need for intervention strategies such as composting of raw manure to prevent contamination of food and water supplies. Composting is the process of decomposing organic matter, whether manure, crop residue or municipal wastes, by mixed microbial population in a warm, moist aerobic environment. Composted manure is not only a potential source of nutrient needed for organically grown plants, it is free of pathogenic organisms.
Escherichia coli is a normal number of the gastrointestinal microflora of humans and animals, however E. coli serotype O157:H7 can severe hemorrhagic disease, and in some cases lead to serious complications, even death (Buchanan and Doyle, 1997). Escherichia coli O517 H7 has been a public health concern to food processors and consumers since its first recognition in 1982, with incidents of food borne associated with this pathogen occurring in most countries (CDC, 2006). Healthy cattle sporadically harbour E. coli O157:H7 in their gastrointestinal tract and shed the pathogen asymptomatically in their feces (Kudva et al., 1998). Previous studies have revealed long term survival of E. coli O157 H7 in manure held under a variety of conditions (Kudva et al., 1998; Grewal et al., 2006; Franz et al., 2007). In addition, E. coli O157 H7 can survive, replicate, and move within soil, and the presence of manure increase the pathogen's survival time (Gagliardi et al., 2000).
In most developed countries, raw manure must be composted if it to be applied to land used for a crop intended for human consumption. However, in Plateau States, Nigeria--the experimental area, organic manure farmers rarely apply composted manure to their farms. They use aged manure that has been kept for about one month under fluctuating environmental conditions. The application of raw manure or aged manure on agricultural fields has been found to increase the risk of pathogen contamination of farmland and agricultural produce (Armstrong et al., 1996). Research work done on composting of animal in is very limited. Therefore, there is dearth of information on the effect of composting on the destruction pathogens in animal manure. The objective of this research is to evaluate the effect of composting on the survival of E. coli O157:H7 in bovine manure.
MATERIALS AND METHODS
* Composting of bovine manure inoculated with E. coli O157:H7 was carried out in windrows and plastic bins. Manure samples kept under fluctuating environmental conditions to serve as control experiments. The major materials used in this experiment included the following: bovine slurry (BS), bovine feces (BF) and saw dust (S). The composting process was carried out between the months of October 2009 to April 2010.
* Collection of Compost Materials. Manure and sawdust were collected from abattoir and timber market respectively in Jos Plateau State, Nigeria. These materials were loaded in big sacks and transported to the Botanical Garden of the Department of Plant Science and Technology University of Jos, the experimental sites. The manures were sundried to reduce microbial load and moisture content and then re-bagged prior to use.
* Construction of Windrow. Ten freestanding windrows were constructed at the Botanical Garden of the Department of Plant Science and Technology University of Jos. The size of the windrow was adopted from Larney et al.
* Preparation of inocula. E. coli O157:H7 isolated from bovine feces was subcultured on three successive days to reactivate the cells after storage in refrigeration (Sutherland et al., 1995). E. coli O157:H7 was inoculated into portions of the manure with pathogen concentration of [10.sup.5] cfu/g of manure to serve as inocula.
* Experimental Protocol. Saw dust was added to different manure types (BS and BF) to obtain carbon to nitrogen ratio (C: N) of 30 according to the method prescribed by Alberta Government Agriculture and Food Agency (AGAFA) (2005). The (C:N) of manure is a very important factor that affects the whole composting process because microbes need 25 to 30 times more carbon than nitrogen. The manure-sawdust mixture was inoculated with E. coli positive manure (inoculum) and composted in the windrows and plastic bin in replicates of five for each manure types. The amount of manure used in each window and plastic bin was 20kg. The manure saw dust mixture was watered to obtain the recommended moisture content of 60% needed for active composting. Bovine manure types without saw dust comprised of bovine feces and bovine slurry (BF and BS) was each inoculated with E. coli O157:H7 positive manure and kept outside the Botany Garden under fluctuating environmental conditions and used as control experiments as earlier stated. The different types of compost manure were replicated five times and samples were collected from them on weekly basis and taken to the laboratory for analysis. The E. coli O157:H7 counts were determined for each manure types weekly by culturing on selective agar medium--Sorbitol MacConkey agar (March and Ratnam, 1986) and then confirmed both biochemically and serologically as described by Cheesbrough (1991) and Nataro and Kaper (1998) respectively. The temperature in the different manure types were recorded weekly using a thermometer which was inserted inside the manure piles. The pH of the manure samples was determined during each sampling period by homogenizing 5g of the manure with 45ml distilled water. The pH of the resulting homogenate was measured with pH-meter (Model 320). Moisture content was determined by drying 5g portions to constant weight in a hot air oven at 80OC.
* Statistical Analysis. The statistical analyses employed in this study were one way analysis of variance and correlation coefficient analysis.
RESULTS AND DISCUSSION
The results of the effect of composting on the percentage recovery of E. coli O157:H7 from various compost manures are depicted in Fig.1. As the composting process progressed, all the manure began to show a steady decrease in the percentage recovery of E. coli O157:H7 with the windrow compost manure having a significantly higher (p<0.05) death rate of the organism than those of the plastic bin and the controls. E. coli O157:H7 positive manure-saw dust mixtures composted in windrows (BFSw and BSSw) and those composted in plastic bins (BFSp and BSSp) remained culture positive for up to 4 and 6 months respectively. Manure samples without saw dust which served as the controls (BF and BS) and kept at fluctuating environmental conditions remained culture positive throughout the eight months of the composting process. There was no significant difference (p>0.05) was observed in E. coli O157:H7 survival based on the manure types alone, but significant differences (p<0.05) existed in the survival of the organism with regard to the composting methods.
[FIGURE 1 OMITTED]
BF = Bovine Faeces, BS = Bovine Slurry, BFSw = Bovine Faeces-Saw Dust in Windrow, BSSw = Bovine Slurry-Saw Dust in Windrow, BFSp = Bovine Faeces-Saw Dust in Plastic Bin and BSSp = Bovine Slurry-Saw Dust in Plastic Bin.
The results of changes in the composting parameters such as temperature, pH and moisture content of the various compost manures which are likely to affect the percentage recovery of E.coli O157:H7 from the various manures during the eight months of composting process are presented in Figures 2-4. The range between the maximum and the minimum temperatures of the bovine manure composted in windrows (61[degrees]C and 26[degrees]C respectively for BFSw and 60OC and 25[degrees]C respectively for BSSw) was higher than those composted in plastic bins (56[degrees]C and 32[degrees]C respectively for BFSp and 55.5[degrees]C and 32[degrees]C respectively for BSSp). The results also revealed that the range between the maximum and the minimum temperatures of the manure samples that served as controls was the lowest (30[degrees]C and 25[degrees]C respectively for BF and 30[degrees]C and 26[degrees]C respectively for BS).
[FIGURE 2 OMITTED]
Fig.3 shows that the various compost manures rapidly decreased in pH at the initial stage of the composting. The windrow manures (BFSw and BSSw) developed ultimate low pH (5.4 and5.5 respectively) within 3 months of composting process. Beyond this time a rise in pH was recorded for each of the windrow composting manures with the pH of BFSw rising to 7.6, while that of BSSw rose to 7.2 at the end of the composting. Meanwhile, the compost manures of the plastic bins and those of the control experiments continued to maintain low pH throughout the composting period. A slight rise in the controls (BF and BS) was observed as a result of dilutions caused by early rains of April and May.
[FIGURE 3 OMITTED]
Fig.4 reveals that the manure kept at the various environments decreased in moisture content as the compost process progressed. However, the plastic bin manure retained a significantly higher (P<0.05) moisture content than those of the windrows and the controls.
[FIGURE 4 OMITTED]
The correlation coefficients between E. coli O157:H7 population recovered from the compost manure types and the composting variables (temperature, pH and moisture content) within eight months indicate that among the periods of composting, the 3rd month (December) had the highest significant correlation coefficients in the various compost manure and was chosen to further explain the relationship between the organism and the composting parameters (Table 1). High temperatures and low pH values recorded during the 3r month of composting period in windrow and plastic bin manure were positively associated with decline in the percentage recovery of E. coli O157:H7. During this period, low moisture contents of the windrow manure piles were also positively correlated with low percentage recovery of the organism. The moderate moisture contents of the plastic bin compost manures correlated negatively with decline in the percentage recovery of the pathogen. The results of the correlation analysis showed that there was no significant correlation (p>0.05) between the percentage recovery of E. coli O157 H7 from the manure and the composting independent variables in the control experiments.
The findings of this study revealed that in all the compost manure in windrow, plastic bin and the control experiments, the numbers of E. coli O157.H7 cells decreased during composting. This may be attributed to the production of toxic metabolites from other microorganisms in the various types of manure, which may be deleterious to E. coli O157.H7 (Martin et al., 1986). There was no significant difference in the percentage recovery of E. coli O157:H7 based on the manure types alone (i.e. BFS and BSS). This may be due to the fact that the C:N ratios of both manure types were balanced before composting thereby making equal nutrient available to the decomposing microorganisms for metabolic activities (Eghball and Power,1994). Proper blending of carbon and nitrogen also helped to ensure that composting temperatures were high enough for the process to work efficiently. This also explains why bovine manure without saw dust did not generate sufficient heat needed to destroy E. coli O157:H7. It was observed from this study that significant differences existed in the rate of death of E. coli O157:H7 cells in the manure based on the method of composting. Hence, the windrow composting method had a more rapid death rate of the pathogen than that of the plastic bin and the control experiments. This report agrees with the work of Kudva et al. (1998). They reported that pathogenic bacteria survived in shorter period of time in manure composted in windrow and periodically aerated by mixing than in manure kept in anaerobic environment; similar to the plastic bins used in the present study. The reason for the high death rate of E. coli O157:H7 observed in the windrow compost manure piles could be due to the presence of indigenous soil microorganism found in the windrow which may be antagonistic to E. coli O157: H7, which is of intestinal origin (Pelczar et al., 2003). Another reason for faster decline of E. coli 0157:H2 recorded in the windrow than in the plastic bin is that E. coli O157:H7 is an anaerobic bacterium and it has the ability to survive longer in plastic bins without aeration. In windrows with sufficient supply of oxygen, the aerobic bacteria are likely to dominate the anaerobes (Martin, 1986).
The results of this study revealed that the manure composted in windrows and plastic bins became E. coli O157:H7 negative before the end of the composting process, while that kept under fluctuating environmental conditions (controls) remained E. coli O157:H7 positive throughout the composting process. This report is in agreement with the findings of Davis and Kendall (2007). The authors reported that raw manure (not composted) deposited on farms and garden soil may serve as a long-term reservoir of E. coli O157:H7 on such environments and may also be a source of infection for humans when it contaminates produce. The long-term survival of E. coli O157:H7 in the manure kept at fluctuating environmental conditions in this study is also supported by the reports of Kudva et al. (1998) and AGAFA (2005). This explains the reason why animal manure should be properly composted in order eliminate or reduce to a minimal level the numbers of pathogenic microorganisms resident in such manure before applying them on the farm land or garden soil.
The association between E. coli O157:H7 population in composting manure and composting parameters (independent variables) such as temperature, pH and moisture content was also determined in this study. The high variances of the composting parameters recorded from windrow and plastic bin compost piles seem to indicate that the pathogen is strongly influenced by these environments. This report agrees with that of Hussein (2000) who reported that the survival and growth of E. coli O157:H7 in any environment, like all bacteria are dependent on the interactions of various factors such as temperature, pH and water availability. The findings of the present investigation showed that high temperature recorded in bovine manure-saw dust mixture composted in windrow and plastic bin correlated positively with decline in the population density of E. coli O157:H7. The population of E. coli O157: H7 cells in the various compost piles at any given time are directly proportional to the moisture contents of the environments. This is because the microbial population in any environment is directly related to the level of moisture content (Pelczar et al., 2003). Water is the major component of bacterial protoplasm and an adequate supply must be available for vegetative development. The findings of this study showed that in all cases low pH exerted negative effect on E. coli O157:H7 cells by reducing the numbers of the organism. This finding agrees with the report of Buchanan and Klawitter (1992) who reported that the growth rate of E. coli O157:H7 declined at low pH level. This study has shown that of the three composting variables, high temperature had the highest correlation coefficient thus suggesting that it may be the major composting variable that inhibited the growth of E. coli O157: H7. This was in agreement with the work of Buchanan and Doyle (1997) who revealed that E. coli: O157: H7 is quite tolerant of acid and dry conditions but can easily be destroyed by high temperature.
The present finding emphasizes the importance of balancing the C:N ratio of the manure using saw dust and other carbon rich materials before composting. This is to facilitate the composting process which consequently helps to destroy the pathogens. The long-term survival of E. coli O157:H7 in manure samples used as the control experiments emphasizes the need for appropriate farm waste management to curtail environmental spread of this bacterium. This study showed that among the composting methods evaluated, the windrow method was found to be the best, as the pathogen was eliminated at a shorter period of time in windrow manure piles. Surprisingly, the best temperatures needed to destroy the pathogen at a very short time were attained in the windrow environment. The present experiment can be extrapolated to farm environment in which poor farmers in Nigeria, who cannot afford inorganic fertilizer, can fall back on windrow method to compost organic manure before using it on their farms.
We thank the Department of Plant Science and Technology University of Jos, Nigeria for providing the materials we used for carrying out this research.
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* Itelima Janet and Agina Samuel
University of Jos, Jos, Nigeria
Table 1: Correlation Coefficients between E. coli O157:H7 Population and Composting Parameters at the 3rd Month Period of Composting in various types of Manure. Parameter BF BS BFSw BSSw BFSp BSSp Temperature -0.31 -0.32 0.97 ** 0.98 ** 0.87 * 0.85 * [degrees]C pH 0.50 0.63 0.88* 0.85 * 0.83 * 0.80 * Moisture 0.55 0.60 0.63 0.67 -0.81 * -0.84 * Content % * Significant at 0.05, ** Significant at 0.01