Dr P.J. Quinn, Department of Veterinary Microbiology and Parasitology, Faculty of Veterinary Medicine, University College Dublin, Ballsbridge, Dublin 4, Ireland (e-mail: vetmp@ucd.ie).
Abstract
This research was undertaken to evaluate volatile chemicals which retained mycobactericidal activity in cattle slurry. Mycobacterium bovis, suspended in sterilized cattle slurry, was treated with different concentrations of five volatile chemicals with mycobactericidal activity. Following treatment of the slurry for specified time intervals, the reaction mixture was lyophilized to remove the volatile chemicals, and samples of the reconstituted slurry were used to inoculate flasks of Lowenstein–Jensen medium to determine survival or inactivation of the mycobacteria. Acetone, at a concentration of 22·5%, inactivated M. bovis in less than 24 h. Ammonium hydroxide, at a concentration of 1%, was mycobactericidal after 36 h. Chloroform at a concentration of 0·5%, ethyl alcohol at a concentration of 17·5% and xylene at a concentration of 3% inactivated the mycobacteria within 48 h. Some of the volatile chemicals with mycobactericidal activity are potentially useful at farm level.
Mycobacterium bovis, the causative agent of tuberculosis in cattle, causes chronic disease characterized by the progressive development of tubercles in many tissues, especially in the lungs and lymph nodes. This pathogen may also infect other domestic animals and wildlife species such as badgers, deer and possums, which may serve as reservoir hosts for M. bovis with possible dissemination to cattle. Bovine tuberculosis is a zoonotic disease which can be transferred to humans through consumption of unpasteurized milk, dairy products and meat. The disease is of economic importance, as cattle giving positive reactions to the tuberculin test are removed from herds, and carcasses with tuberculous lesions detected during meat inspection are condemned. Susceptible animals usually acquire infection with M. bovis by the respiratory route. Occasionally, infection may be acquired orally. When generalized tuberculosis occurs in cattle, M. bovis may be shed in mucus, milk, urine and faeces. The faecal shedding of M. bovis by tuberculous cattle was first reported by Schroeder and Cotton (1907) and by Reynolds and Beebe (1907). Other workers have also confirmed the presence of this pathogen in the faeces of reactor cattle (Williams and Hoy 1927; Maddock 1936; Reuss 1955; Neill et al. 1988).
Mycobacterium bovis, shed in the faeces of tuberculous cattle or in other excretions which enter slurry tanks, may be capable of surviving for long periods both in stored cattle slurry and in the environment after land spreading. Prolonged survival of pathogenic bacteria increases the risk of animals acquiring infection from environmental sources. Organic matter of animal origin, which may prolong the survival of pathogens in the environment, also interferes with the biocidal activity of many chemical disinfectants. Acid-fast bacteria such as M. bovis are well known for their ability to survive dehydration, fluctuations in temperature, moderate pH changes and exposure to sunlight. In addition, the mycobacterial cell wall has a high lipid and wax content which confers hydrophobicity, rendering these bacterial cells less susceptible to many chemical disinfectants than other conventional bacteria (Russell 1996).
The presence of pathogenic bacteria in slurry which is spread on pasture is a potential source of infection for susceptible grazing animals (Jones 1980). Health risks to humans and animals arise from the disposal of slurry from herds with reactor animals. Vigorous mechanical agitation of contaminated cattle slurry prior to removal from holding tanks, and during land application, creates aerosols. The production of aerosols containing M. bovis is a potential health risk for humans and animals if these infectious particles are inhaled. Methods used for land application of slurry, and prevailing climatic conditions during spreading, can greatly influence the dispersal of aerosols. If slurry containing M. bovis is spread on land during windy weather, dissemination of these potentially infective aerosols can occur, resulting in contamination of adjoining land, waterways and the environment. Such contamination, together with the long survival of M. bovis, may be a potential source of infection for susceptible livestock and wildlife.
In recent years, there has been an increased awareness of the risks arising from the spreading of slurry containing viable pathogenic micro-organisms on agricultural land. Consequently, statutory legislation and voluntary guidelines have been introduced in many countries to regulate the management of animal waste in order to minimize environmental contamination and to limit the spread of infectious diseases. When bovine tuberculosis has been confirmed in herds, statutory regulations may require the treatment of animal waste with approved disinfectants. Regulations may also stipulate a minimum storage period for contaminated animal waste, and may specify how land application of manures and slurry should be carried out. In order to minimize the risk of spreading disease by contaminated animal waste, methods aimed at reducing the numbers of pathogens, or eliminating them completely from slurry, have been proposed. Two methods are feasible and appropriate for use at farm level: long-term storage to ensure that the numbers of pathogens have declined to acceptable levels, and chemical disinfection to inactivate the pathogens present.
Long-term storage of slurry is an inexpensive but slow method for allowing many infectious agents to decline to low numbers. Pathogenic micro-organisms can survive for longer periods in slurry than in solid composted manure (Strauch 1981). Dokoupil (1964) reported that M. bovis survived for 176 days in liquid manure stored at 5 °C. This indicates that storage for up to 6 months may be necessary before all M. bovis organisms in contaminated slurry are naturally inactivated. Frequently, however, the slurry-holding capacity on many farms may be inadequate for the volumes produced. In these circumstances, long-term storage to allow for the natural decline in M. bovis in cattle slurry may not be feasible.
Chemical treatment of slurry containing pathogenic micro-organisms offers a suitable alternative when long-term storage is not possible. A fundamental requirement for a chemical selected for the treatment of slurry contaminated with M. bovis is retention of mycobactericidal activity in the presence of high concentrations of organic matter. The evaluation of chemicals or disinfectants for mycobactericidal activity is a prerequisite for the selection of suitable chemical compounds for this purpose.
In the evaluation of chemicals or disinfectants for biocidal activity, a fundamental step in the procedure involves the inactivation or elimination of residual disinfectant activity at the end of the reaction period. This may be achieved by neutralization, dilution or physical methods. Apart from dilution, dialysis, centrifugation, filtration, chromatography and electrophoretic procedures may be used to physically remove compounds from reaction mixtures (Quinn and Carter 1999). When using cattle slurry as a suspending medium for M. bovis, many of these procedures are impractical because of the semi-solid nature and high organic matter content of the material. To overcome these technical difficulties, a method has been developed, based on freeze-drying, which is suitable for the elimination of volatile chemicals from high concentrations of particulate organic matter (Quinn and Scanlon, 2000). Using this method, five volatile chemicals have been investigated for their mycobactericidal activity. Mycobacterium bovis suspended in sterilized cattle slurry was used as test organism. Sterilization of the cattle slurry was necessary to facilitate the recovery of surviving M. bovis from the slurry by culture following treatment with chemicals.
Materials and methods
Cattle slurry
Cattle slurry, which had been well agitated, was collected from a slurry tank in a slatted unit. The dry matter content of the slurry used was 8·5% and the pH value was 7·2.
Sterilized cattle slurry
When isolating Mycobacterium bovis from clinical specimens, it is first necessary to decontaminate the material to eliminate contaminating organisms prior to culturing. This can be achieved by treating the specimen with chemicals such as 4% sodium hydroxide. This procedure inactivates contaminating bacteria and allows the recovery of M. bovis. In cattle slurry, however, high numbers of bacterial endospores are present which are resistant to chemical decontamination and can survive treatment which inactivates M. bovis. Therefore, chemical decontamination of the cattle slurry was not practical and it proved necessary to autoclave the slurry at 1·75 kgf/cm2 (132 °C) for 70 min to render it sterile. The slurry was autoclaved in 600 ml volumes in 2·5 litre capacity bottles. After autoclaving, the pH of the sterilized slurry, which was 9·15, was adjusted to 7·28 by the addition of a small volume of glacial acetic acid (50% v/v concentration).
Preparation of Lowenstein–Jensen medium containing sodium pyruvate
Lowenstein-Jensen medium was prepared using dehydrated Bacto Lowenstein base (Difco) and sterile egg homogenate, aseptically prepared from fresh eggs, free from antimicrobial substances. Sodium pyruvate (Sigma) was added at a concentration of 0·4% to enhance the growth of M. bovis. The medium was dispensed in 50 ml volumes into 75 cm2 tissue culture flasks (Costar) and inspissated at 85 °C, in flowing steam, for 1 h.
Culture of Mycobacterium bovis
A pathogenic strain of mycobacteria, Mycobacterium bovis ATCC 19210, was used as test organism in this study. This bacterium was grown aerobically on Lowenstein–Jensen medium containing sodium pyruvate at 37 °C for 6 weeks. The culture was then stored at 4 °C until use.
Procedure
Volumes of sterilized cattle slurry were added to sterile, screw-capped, 200 ml capacity bottles containing sufficient glass balls (8 mm diameter) to cover the bottom. The volume of sterilized cattle slurry used varied in accordance with the other constituents added. Volumes (1 ml) of well mixed suspensions of M. bovis containing 0·1 mg ml−1 (estimated to yield approximately 2 × 106 cfu ml−1) were added to each bottle of sterilized slurry and the contents were mixed. The chemicals being evaluated were added to yield the required concentrations in slurry and the contents were mixed thoroughly. In each instance, the final volume was 100 ml per bottle. Controls consisting of 1 ml suspensions of M. bovis in sterilized slurry without added chemicals were included in each experiment.
Samples of 10 ml volumes were removed from the reaction mixtures at specified time intervals and transferred to sterile, pre-weighed, 200 ml capacity Erlyenmeyer flasks which were then capped with sterile cotton wool enclosed in sterile gauze. The flasks were re-weighed and the weights of the added samples were calculated. The flasks and contents were frozen at – 80 °C for 1 h and were then transferred to the drying chamber of an Edwards Modulyo freeze-drying apparatus (Edwards High Vacuum International, Crawley, West Sussex, UK). Freeze-drying continued for 15–20 h, or until the contents of the flasks were dry. The flasks were then removed and placed in an incubator at 37 °C for 1 h to minimize the absorption of water vapour from the atmosphere prior to weighing.
The flasks containing the dried material were re-weighed and the dried material was reconstituted to the original weight of the slurry samples with sterile phosphate-buffered saline. The reconstituted material was mixed thoroughly and held at room temperature for 1 h. Volumes of 0·33 ml of the reconstituted material from each flask were inoculated onto Lowenstein–Jensen medium in triplicate. Each inoculum was spread evenly over the surface of the medium with a sterile glass spreader, and each inoculated flask was incubated aerobically at 37 °C for up to 8 weeks. At the end of this period, colonies of M. bovis were counted and recorded. The experimental procedures followed are illustrated in Fig. 1.
Procedure used to determine the activity of volatile chemicals against Mycobacterium bovis suspended in sterilized cattle slurry
Strict biosafety precautions, which included the use of a Class 1 microbiological safety cabinet, were observed for all hazardous operations involving M. bovis.
Results
Small numbers of M. bovis survived in slurry for 48 h after treatment with 20% acetone. When the concentration of acetone was increased to 25%, few viable organisms were recovered at 12 h and none were recovered at 24 h (Table 1). At a concentration of 22·5%, acetone had mycobactericidal activity at 24 h (Table 2).
Table 1. The effect of selected concentrations of five volatile chemicals on the survival of Mycobacterium bovis, suspended in sterilized cattle slurry
Chemical/concentration
cfu ml−1 of slurry at
12 h
24 h
48 h
*
Results are expressed as the mean number of M. bovis colonies growing on three flasks each of which was inoculated with 0·33 ml of treated slurry.
Table 2. Determination of the time required to inactivate Mycobacterium bovis suspended in sterilized cattle slurry by 22·5% acetone
Acetone concentration
cfu ml−1 of slurry at
6 h
12 h
24 h
30 h
22·5%
79
5
0
0
Control
739
not sampled
608
not sampled
Mycobacterium bovis in sterilized cattle slurry survived for more than 12 h when treated with 2% ammonium hydroxide. When treated with 1% ammonium hydroxide, survival continued for more than 24 h, but no viable M. bovis were recovered at 48 h (Table 1). When the slurry treated with 1% ammonium hydroxide was sampled at other specified intervals, few surviving mycobacteria were recovered at 24 and 30 h, and no viable M. bovis were recovered at 36 h (Table 3).
Table 3. Determination of the time required to inactivate Mycobacterium bovis suspended in sterilized cattle slurry by 1% ammonium hydroxide
Ammonium hydroxide concentration
cfu ml−1 of slurry at
6 h
12 h
24 h
30 h
36 h
1%
162
71
1
1
0
Control
1651
not sampled
876
not
not sampled
Chloroform, at a 1% concentration, was mycobactericidal within 12 h. When used at 0·5% concentration, no viable mycobacteria were recovered at 48 h (Table 1). These results were confirmed when samples were collected at more closely spaced intervals (Table 4).
Table 4. Determination of the time required to inactivate Mycobacterium bovis suspended in sterilized cattle slurry by 0·5% chloroform
Chloroform concentration
cfu ml−1 of slurry at
6 h
12 h
24 h
36 h
48 h
0·5%
159
100
38
2
0
Control
1338
not sampled
1015
not sampled
899
Mycobacterium bovis survived for more than 48 h in slurry treated with 15% ethyl alcohol. When the concentration of ethyl alcohol was increased to 20%, no viable mycobacteria were recovered at 12 h (Table 1). There was a steady decline in the number of surviving mycobacteria following treatment with 17·5% ethyl alcohol. By 48 h, no viable organisms were detected (Table 5).
Table 5. Determination of the time required to inactivate Mycobacterium bovis suspended in sterilized cattle slurry by 17·5% ethyl alcohol
Ethyl alcohol concentration
cfu ml−1 of slurry at
12 h
24 h
36 h
48 h
17·5%
87
27
1
0
Control
988
794
915
696
Xylene, at a 2% concentration, was not mycobactericidal after 48 h (Table 1). When used at a 3% concentration, however, few surviving mycobacteria were detected at 30 and 36 h and no viable M. bovis was recovered at 48 h (Table 6).
Table 6. Determination of the time required to inactivate Mycobacterium bovis suspended in sterilized cattle slurry by 3% xylene
Xylene concentration
cfu ml−1 of slurry at
6 h
12 h
24 h
30 h
36 h
48 h
3%
641
318
23
3
1
0
Control
1643
not
927
not
not
727
sampled
sampled
sampled
Discussion
Chemical disinfection of cattle slurry contaminated with M. bovis presents many difficulties, some relating to the large volumes of slurry requiring treatment and others to the selection and evaluation of effective chemicals. It is recognized that mycobacteria are more resistant to disinfectants than most other non-spore-forming bacteria (Croshaw 1971). Chemical compounds with activity against mycobacteria include alcohols (ethyl and isopropyl), aldehydes (formaldehyde, glutaraldehyde, glyoxal and succinaldehyde), halogens (chlorine-releasing and iodine-releasing agents), some peroxygen compounds, some phenolic compounds (particularly o-phenylphenol) and the sterilizing agents ethylene oxide and β-propiolactone (Russell 1996).
Specific information on the mycobactericidal properties of many disinfectants is not available. Discrepancies in reports relating to the mycobactericidal activity of different compounds may be due to lack of uniformity in evaluation procedures, the use of pathogenic and non-pathogenic mycobacteria as test organisms, the nature of the organic matter included in the test procedures and other variables relating to the test methods employed.
Many conventional disinfectants are inappropriate for treating slurry due to the high content of organic matter and the inherent cost of treating large volumes of the material. Consequently, decontamination of cattle slurry containing M. bovis requires moderately-priced chemicals which are mycobactericidal at low concentrations. Chemicals selected for the inactivation of M. bovis in cattle slurry should be mycobactericidal, relatively non-toxic, cheap, biodegradable and non-polluting. Chemicals which have been used for the treatment of animal waste and sewage sludge include ammonia, urea, peracetic acid, ammonium hydroxide, sodium hydroxide, chlorine, formalin, xylene and lime products. Chemical treatment of animal waste contaminated with pathogenic mycobacteria has been reported. Grishaev et al. (1988) described the disinfection of liquid manure with a solution of ammonia on farms in the former Soviet Union where bovine tuberculosis had been confirmed. Tickhonov (1976) reported the disinfection of liquid pig and poultry manure contaminated with mycobacteria by the addition of 3% ammonium hydroxide solution. A 5% solution of formalin inactivated M. avium, M. tuberculosis and M. bovis in a mixture of blood, urine and manure (Genov 1965). Mycobacterium paratuberculosis was inactivated in cattle slurry 2 weeks after treatment with 1·5% formalin (Genov 1965). The use of lime products for the inactivation of pathogenic mycobacteria has also been reported. Genov (1965) reported that 5% chloride of lime inactivated M. avium, M. tuberculosis and M. bovis in a mixture of blood, urine and manure in a laboratory experiment. Mycobacterium paratuberculosis in cattle slurry was inactivated within 4 weeks by a 2% concentration of calcium cyanamide (Ley 1992). A lime product termed ‘thick lime milk’ consisting of a mixture of calcium hydroxide and water is sometimes used to treat slurry and sewage sludge. In Germany, Strauch (1983) stated that it was mandatory to treat slurry with ‘thick lime milk’ when certain notifiable diseases, including bovine tuberculosis, had been confirmed.
In this study, five volatile chemicals were assessed for their potential mycobactericidal activity. These selected chemicals did not appear to form complexes with the organic matter in the slurry and were effectively removed by freeze-drying. The lipid solvent properties of some of these chemicals may account for their mycobactericidal activity. Acetone, at a concentration of 22·5% in slurry, inactivated M. bovis within 24 h, but this concentration was at the highest level of the five chemicals evaluated. This highly flammable chemical can produce explosive mixtures with air. Toxic effects and health symptoms in humans associated with exposure to acetone include central nervous system depression, headache, fatigue and irritation of the eyes and mucous membranes (Satoh et al. 1996). Acetone is biodegradable in the environment and its fast rate of evaporation may prevent soil and water contamination. However, its cost, flammability, toxicity and irritancy are undesirable properties which may limit its usefulness for the treatment of slurry.
A strong aqueous solution of ammonia gas (27–30%) is generally referred to as ammonium hydroxide. In the experiments reported here, a relatively low concentration (1%) was effective for inactivation of M. bovis in slurry (Table 3). Strong solutions of ammonium hydroxide emit pungent fumes which have a suffocating effect on humans and animals. Concentrated solutions of ammonium hydroxide cause severe skin irritation due to the caustic nature and high pH value of the chemical (Payne 1991). When added to the slurry to yield a final concentration of 1%, the pH of the reaction mixture was raised to a value above 10. It has been reported that in general, viability of micro-organisms is adversely affected by high pH values (Strauch 1981). It is probable that in this instance, the raised pH value in the slurry led to the inactivation of M. bovis. The use of ammonium hydroxide and ammonia for the treatment of animal manure has been reported by other workers. Tickhonov (1976) reported the disinfection of liquid pig and poultry manure, contaminated with mycobacteria, using ammonium hydroxide at a final concentration of 3% in the treated material. Grishaev et al. (1988) used a solution of ammonia, at an application rate of 30 kg m−2, to disinfect slurry on farms where bovine tuberculosis had been confirmed. The relatively low concentration of ammonium hydroxide required to treat slurry offers a realistic alternative to other mycobactericidal chemicals. Because of its caustic properties, workers using ammonium hydroxide should adopt appropriate safety precautions. Protective clothing, gloves, face and eye protection should be worn, and exposure to ammonia released from the chemical should be kept to a minimum. Ammonium hydroxide is a volatile compound which releases ammonia to the atmosphere. Residual ammonium hydroxide in treated slurry is biodegraded after land application, and presents a negligible risk of pollution in the environment.
Chloroform was effective for inactivating M. bovis within 48 h at a concentration of 0·5% (Table 4). This chemical was mycobactericidal at a concentration lower than any of the other chemicals evaluated in this study. The most consistently observed toxic effect of chloroform is liver damage, but the amount which is normally acquired by inhalation from the atmosphere is not sufficient to produce these toxic effects. Chloroform is highly volatile and preferentially transfers from water to air (Wolf and Butterworth 1997). Cattle slurry contains approximately 92% water and, as chloroform is not a chemically-reactive compound, it does not form a stable chemical complex with the organic matter in the slurry. Consequently, the use of low concentrations of chloroform for the treatment of slurry poses a minimal health risk to persons using the chemical, and is unlikely to cause pollution of the environment after land application of the chemically-treated slurry.
In this study, ethyl alcohol, at a concentration of 17·5%, inactivated M. bovis in slurry within 48 h (Table 5). Ethyl alcohol has many desirable attributes as a chemical disinfectant and is frequently used at a 70% concentration for skin disinfection. It is recognized that both ethyl alcohol and isopropyl alcohol are mycobactericidal at concentrations of 60–90% (McDonnell and Russell 1999). A relatively high concentration of ethyl alcohol was required to inactivate M. bovis in cattle slurry within the specified time. As alcohols are flammable, strict precautions should be observed when using these chemicals. Ethyl alcohol is volatile, relatively non-toxic, non-corrosive and biodegradable and, consequently, its use for the treatment of cattle slurry contaminated with M. bovis is unlikely to be a source of environmental pollution after land application of the treated slurry.
Xylenes are aromatic hydrocarbon solvents which are obtained as by-products of the petroleum industry. In this study, a commercial grade of mixed xylenes containing the isomers ortho, meta and para-xylene was used. A concentration of 3% xylene in slurry inactivated M. bovis within 48 h (Table 6). The use of xylene for the treatment of cattle slurry has been reported. Plommet (1972) used xylene at a concentration of 0·1% in slurry for inactivation of Brucella abortus. Toxic effects in humans due to exposure to xylene include eye and respiratory tract irritation, central nervous system changes and damage to the liver and kidneys (Fay et al. 1998).
A relatively low concentration of xylene (3%) in slurry inactivated M. bovis within 48 h (Table 6). Because of its volatile nature, release of xylene into the environment is through evaporation. As xylene does not form a stable chemical complex with the organic matter in slurry, low concentrations would be released to water and soil.
The use of M. bovis as a test organism for disinfectant evaluation presents a number of difficulties. This pathogenic organism can infect animals and humans and, consequently, strict biosafety precautions must be observed. Reliable standardization of mycobacterial suspensions is difficult to achieve due to the production of cording factor and other substances which promote aggregation of the bacteria. Accordingly, it is difficult to obtain reproducible results in sequential experiments. Despite attention to detail, variation in the findings reported here can be attributed, in part, to the test organism employed. Another problem arising from the experimental design was the difficulty in measuring samples of slurry accurately due to its semi-solid consistency. Decreases in the numbers of viable mycobacteria recovered in the controls probably relate to some inactivation of the mycobacteria by freeze-drying in the absence of a cryoprotective agent.
Under farm conditions, chemical treatment of cattle slurry requires careful planning and proper supervision. Cost, safety considerations and risks of environmental pollution due to the chemicals selected should be carefully reviewed in advance. Chemicals used for the inactivation of microbial pathogens should be critically evaluated beforehand to determine their effectiveness for a particular application. If used for slurry treatment, chemicals should be added to yield the correct biocidal concentration and thoroughly mixed to achieve a uniform concentration. When chemically treating slurry containing M. bovis, a number of factors may influence the outcome. A limited number of chemicals have mycobactericidal activity and the effectiveness of some of these may be adversely affected by high concentrations of organic matter. The results presented in this paper demonstrate that a number of volatile chemicals are mycobactericidal and that they retain their activity in cattle slurry. Apart from acetone at a concentration of 22·5%, which inactivated M. bovis in less than 24 h, the other volatile chemicals took up to 48 h to exert mycobactericidal activity. It is evident that chemicals which retain their mycobactericidal activity in slurry can also be used for the effective disinfection of contaminated farm buildings, equipment and transport vehicles.
The methods described here for evaluating the mycobactericidal activity of volatile chemicals may be used with other test organisms also. An advantage of this method over many of the other systems described is that it can be used with soluble or particulate organic matter, thereby approximating the conditions obtaining at farm level.
Acknowledgements
The work reported in this paper was funded, in part, by the Eradication of Animal Disease Board (ERAD).
1365-2672/asset/olalertbanner.jpg?v=1&s=a90144ecfab279fb549fe78913f236cb55332bd0)
