Bioresource Technolo~, 41 (1992) 97-100
Effect of the Earthworm
(Eisenia foetida) on
Salmonella enteritidis in Horse Manure Acie C. Murry, Jr" & Lynn S. Hinckley:' "Animal Science Department, :'Pathobiology Department, University of Connecticut Storrs, Connecticut 06269, USA (Received 18 March 1991 ; revised version received and accepted 15 July 1991 )
Abstract Salmonella enteritidis, an enteropathogen of human,s, is a common isolated serotype of infected domestic animals and may be excreted in the feces Of these attimals. Because the manure from some infected animals may be applied to agricultural land as fertilizer, Salmonella may be introduced into the environment, possibly causing heavy conrumination and posing a health hazard to human beings and other susceptible animals'. Earthworm biotechnology has been suggested as a method for managing organic waste on soil. The present study was conducted to determine the effect of earthworms!Eisenia foetida) on Salmonella enteritidis inoculated to cultural dishes containing horse manure. Results indicated that 48 h after inoculation, the per cent decrease in the concentration of Salmonella was different (P < 0"05) in the cultures with earthworms compared to cultures without earthworms (8% versus 2%). In addition, normal bacterial flora was reduced (3%) in the presence of earthworms, and increased (2%) in their absence. These .ndings suggest that earthworm biotechHology may be beneficial in the management of animal manure by decreasing pathogen persistence.
Key words: Earthworm (Eisenia foetida), horse manure, Salmonella enteritidis, pathogen, soil biodegradation, waste management.
INTRODUCTION Salmonella enteritidis is a c o m m o n isolated pathogenic serotype found in infected horses (Smith et al., 1979; Gibbons 1980; Ikeda et al., 1986). It has been reported in horses in the United States
(Carter et al., 1986), Australia (Begg et al., 1988) and Japan (Sato et al., 1988). Pathological findings indicate that many horses appear to harbor the Salmonella in the intestinal tract. Salmonella in horse manure may enter the soil through clinical states in which the horses have acute colitis with severe diarrhea. In the diseased horse, large volumes of fluid feces may be passed for several days (Smith et al., 1979). Furthermore, because a common disposal method for horse manure is land spreading, Salmonella may be continuously introduced into the environment. These passageways of Salmonella into the soil may cause heavy environmental contamination, thus posing a hazard not only to other horses, but also to other animals and humans (Hird e: al., 1984: Carter et al., 1986). The earthworm (Eisenia foetida)is a common inhabitant of composts and manures and has a marked influence on their biological and physical characteristics (Edwards & Lofty, 1972). Earthworms require microorganisms, cellulose and soil as essential ingesta (Fiack & Hartenstein, 1984). Although microorganisms are indigenous to manures and sewage, they serve as primary consumers and decomposers of the labile organic component. However, earthworms are secondary consumers of organic components, and in their presence the rate of oxidation of organic matter is accelerated via the ongoing consumption of the indigenous microorganisms (Hartenstein & Neuhauser, 1985). The earthworm (Eisenia foetida) can enhance the efficiency of managing biological wastes on land (Watanable & Tsukamoto, 1976; Hartenstein, 1978; Loehr etal 1984; Hassett etal., 1988; Hartenstein & Bisesi, 1989). Bisesi (1990) conducted an experiment to determine the effect of
97 Bioresource Technology 0960-8524/92/S05.00 © 1992 Elsevier Science Publishers Ltd, England. Printed in Great Britain
Acie C Murry, Jr &Lynn & Hinckley
variable temperatures and seasonal changes on vermial and microbial activities. T h e results suggest that vermial growth and activity is accelerated under ambient conditions of temperature and seasonal changes. Brown and Mitchell ( 1981 ) conducted a series of experiments to test the effect of earthworm feeding on the survival of Salmonella enteritidis ser. typhimurium in wastewater effluent and sewage sludge. Their findings indicated that the earthworms caused a maximum decrease in Salmonella of 29% per day in contrast to a m a x i m u m decrease of 14% in their absence. Recently, Hassett et al. (1988) and Amaravadi et al. (1990) observed a reduction in the infectivity of both cowpea mosaic and tobacco mosaic virus fed to earthworms and concluded that E. foetida may possess a virucidal enzyme system and may contribute to the inactivation of pathogenic viruses potentially associated with land application of sewage sludges and livestock manures. Currently, the majority of research on earthworms and waste management is focused on wastewater effluent and sewage sludge. However, limited evidence exists on issues related to the utilization of earthworms for the reduction of Salmonella enteritidis in manure of domestic animals. In this study, laboratory experiments were conducted to determine the effect of the earthworms (E. foetida) on Salmonella enteritidis inoculated to horse manure.
METHODS Experimental design E. foetida obtained from laboratory cultures were placed on filter paper saturated with distilled water and allowed to gut-void for 4 h prior to entering the sterile growing media. Horse manure was collected from the University of Connecticut horse research facility and saturated to approximately 20% solids with distilled water. T h e manure was autoclaved under 15 lb pressure (1.035 x 105 Pa), at 121°C for 70 min. Ten gram aliquot cultures of sterile worm growing horse media were placed into 100 x 15 m m Petri dishes. A population of 10 E. foetida (0.25-0-35 g wet wt) was added into each of three replicated Petri dishes and incubated at 25°C for 48 h. T h e dishes (21) contained the following cultural types: horse manure with no worms; horse manure with worms; horse manure and Salmonella with no worms; horse m a n u r e and Salmonella with worms.
Bacterial cultures Two concentrations of Salmonella enteritidis inocula were prepared by making spread plates of nutrient agar (Buck & Cleverdon, 1960). These plates were incubated for 24 h at 37°C then flooded with 10 ml of distilled water. T h e bacterial suspension was added to 1000 ml of distilled water; 25 ml of the suspension was combined with 25 ml of distilled water to yield 50 ml of inoculum at 400 000 cells/ml for concentration 1. Concentration 2 was prepared by adding 12.5 ml of the Salmonella suspension to 37.5 ml of distilled water resulting in 50 ml of inoculum at 200 000 cells/ml. Bacterial counts Standard serial 10-fold dilution procedures were followed using 0"5 g inoculum (Richardson, 1985) to determine the initial and final bacteria count (N =3). After the dilutions were prepared, the samples were cultured on duplicate plates of the appropriate media. Dilutions at 10- 2 and 10-4 were needed to yield countable plates having 3 0 - 3 0 0 colonies. T h e 10-2 dilutions were plated on brilliant green sulfa (BGS) and xylose lysine sodium desoxycholate (XLD) media, while the 10 -4 were plated on to XLD and blood agar. These plates were incubated at 37°C for 24 h then counted. T h e BGS and XLD plates provided counts for Salmonella and lactose positive colonies. T h e blood agar plates provided counts for all environmental bacteria. A n electronic manostat colony counter was used to determine all bacterial counts. Statistical methods A completely randomized design was used for this study. Differences between means, due to treatment, were determined by the Duncan's Multiple Range Test.
R E S U L T S AND D I S C U S S I O N Results from this study indicated that the per cent change in bacteria populations was not influenced by the concentrations of Salmonella (Table lt). Means of bacteria populations were statistically
tNote: Similarities in standard errors (Tables 1-3). The best estimate of within variation is the experiment within group error. This value was used in calculating the standard error of individual means. Therefore, all means based on the same number of observations will have the same standard error.
Effect o f earthworm on Salmonella in m a n u r e
Table 1. Effect of Sahnonella concentration on bacteria
Table 2. Effect of earthworms on normal bacterial flora"
counts" in horse manure in relation to time
present in horse manure in relation to time
~ 4,~ Mean difference
5"556 +_0'054 5"243 _+0"042 + 0"313/,
5'276 + 0"054 5"048 + 0-042 + 0"228/,
(I 48 Mean difference
5'058 _+0"043 5'155 _+0"057 - 0.097/'
5" 175 _+0"043 5-017 _+0"057 + 0.158'
"kogH. bacterial density (N= 3). /'Means with the same superscript are not different (P> 0.3).
"Loglo bacterial density (normal bacterial flora)/0-5 g (N= 3/. l,.,. Means with different superscripts are different (/' < 0.05 ).
different at 0 versus 48 h, however, in the presence of earthworms, b a s e d on the D u n c a n ' s Multiple Range Test. Table 2 presents a s u m m a r y of the normal bacterial flora ( N B F ) of horse manure in the presence and absence of earthworms. T h e n u m b e r of N B F was r e d u c e d by approximately 3% ( P < 0 " 0 5 ) in the presence of e a r t h w o r m s 48 h after inoculation. T h e r e was a 2% increase, however, in N B F in cultural dishes without earthworms. Forty-eight hours after inoculation with S a l m o n e l l a enteritidis, the p o p u lation of S a l m o n e l l a was lower in cultures with e a r t h w o r m s when c o m p a r e d to cultures without earthworms. S a l m o n e l l a population was r e d u c e d approximately 8% ( P < 0 . 0 5 ) in the presence of e a r t h w o r m s but r e d u c e d by only approximately 2% in their absence (Table 3). Similar findings have been r e p o r t e d by B r o w n and Mitchell (1981) w h o inoculated S a l m o n e l l a enteritidis ser. ~ p h i r n u r i u m to cultures of wastewater effluent and sludge, and c o m p a r e d survival rates from 4 to 28 days after inoculation of the bacterium with and without earthworms. T h e y suggested that the reduction in S a l m o n e l l a p o p u lations induced by the earthworms may have b e e n due to antibiosis of, or competition with, bacteria that inhabit the intestines of the earthworms. Brown and Mitchell (1981) also suggested that earthworms may indirectly cause a reduction of S a l m o n e l l a by exposing the organism to light and oxygen and desiccation from the mixing of organic substrate, all of which cause S a h n o n e l l a mortality ( M o r r i s o n & Martin, 1977). In addition, as the e a r t h w o r m s c o n s u m e the microorganisms, they may concomitantly contribute to accelerated decimation of pathogenic microbial agents (Biscsi, l 9 90 ). T h e results of the current experiment indicated that pathogenic persistence in horse m a n u r e can be r e d u c e d by earthworms. In addition, these data provide additional information to justify the use of e a r t h w o r m s for biological degradation of animal manure that may be applied to agricultural land as
Table 3. Effect of earthworms on Salmonella enwritidis" in
horse manure in relation to time Time (h)
0 48 Mean difference
5"458 _+0"054 5"335 _+0"042 - 0.123/'
5"373 _+0"054 4"956 + 0"042 - 0.417'
"Ix)gLo bacterial density (Sahnonella enteritidis )/0"5 g ( N = 3).
/'-'Means with different superscripts are different (I' < 0"05).
a fertilizer. Given the practical p r o b l e m s of the utilization of manure p r o d u c e d by various farm animal species, and the impact of S a l m o n e l l a on human and animal health, continued research into elimination or determining m a x i m u m reduction of S a l m o n e l l a enteritidis in manure of domestic farm animals would be beneficial.
This work is a contribution from the Center fl~r Environmental Health, D e p a r t m e n t of Animal Science; a p p r o v e d by the Director. T h e authors express special thanks to Dr John Post, Mr Seth Wilner, and Mr T i m o t h y G o r t o n , Pathobiology Department, for assistance with the microbiological analysis. T h e authors also express thanks to Dr John Riesen, Animal Science Department, for assistance with the statistical analysis.
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