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Aims: This study was carried out to determine the survival time of Escherichia coli, Salmonella choleraesuis, Aujeszky’s Disease virus and Blue Eye Disease virus in ensilages based on the solid fraction of pig faeces.
Methods and Results: The four micro-organisms were inoculated into microsilos based on the solid fraction of pig faeces, sorghum and molasses. They were left for 0, 7, 14, 28 and 56 days, after which the state of each microsilo was evaluated, and isolation of the inoculated agents was attempted. The four inoculated agents were isolated only on day 0 of ensilage. The viral agents were identified through the cytopathic effect and fluorescence.
Conclusions, Significance and Impact of the Study: It is concluded that ensilages based on the solid fraction of pig faeces appear to reduce the risk of the transmission of the agents inoculated in this study and help to reduce the environmental impact by using the solid in animal feed.
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Pig farming is a potentially highly polluting activity. Four causes have been identified for the impact of this activity on the environment: (i) the high concentration of animals; (ii) the lack of a relationship with agricultural activities, as a result of which pig faeces are deposited on non-agricultural lands or discharged into rivers or lagoons without treatment; (iii) the high-nutrient feeding systems which are not completely assimilated by the pigs and from which originates a great volume of faeces; and (iv) the use of hydraulic systems to remove faeces (Pérez 1997).
Pig faeces have a high content of organic matter which is 55% biodegradable, as well as numerous polluting components among which are pathogenic micro-organisms and minerals such as copper, zinc and arsenic (Taiganides 1994). The amount of faeces produced by pigs depends on several factors, but a 100 kg pig excretes approximately 6·17 kg of faeces and urine per day (Taiganides 1994). Extrapolating this value to the inventory of pigs in Mexico, it represents an important source of pollution which is not subjected to a treatment system (Pérez 1997).
The dilution of faeces in water, its storage in tanks or lagoons and its later disposal or treatment, is the method most used in pig farms in Mexico (Taiganides 1994; Moser 1997). Once the dilution has been accomplished, the solids are allowed to settle and mechanical separation takes place. The liquids are sent to anaerobic lagoons for storage and later used as fertilizer (Andreadakis 1992), or they are deposited in aerobic lagoons which allow the recycling of the water. The nutrient-rich solids may be used as fertilizers, compost and ingredients for ruminant and pig feed (Molina 1997).
The recycling of pig faeces for animal feed helps reduce the environmental pollution caused by the solids and makes it possible to take advantage of the high nutrient content, decreasing feeding costs (Iñigo et al. 1991). However, some authors have observed that the use of faeces without treatment increases risks through the recycling of pathogenic micro-organisms and other pollutants (Strauch and Ballarini 1994; Hernández 1997; Molina 1997).
Strauch and Ballarini (1994) and Olson (1995) have stated that a variety of pathogenic micro-organisms may survive and multiply in pig faeces during storage. Among these are Salmonella spp., Escherichia coli, Erisipelothrix rhusiopathiae, Yersinia enterocolitica, Staphylococcus aureus, Clostridium perfringens, Bacillus anthracis, Brucella spp., Leptospira spp. and Brachyspira hyodysenteriae.
This is in agreement with other authors (Iñigo et al. 1991; Mateu et al. 1992) who detected Enterobacteria in pig faeces stored in stabilization lagoons and aerobic fermentation systems for up to 18 days. Plym and Ekesbo (1993) found that Salmonellasenftenberg and Salm. typhimurium survive 7 days and Salm. derby survives 14 days in sow faeces.
The survival of some viruses in pig faeces has also been reported. Among these are Enterovirus, with a titre of 106 infecting doses per cellular culture (CCID50) (Strauch and Ballarini 1994), Aujeszky’s Disease virus for three to 15 weeks, the African Swine Fever virus for 60 days, the Foot and Mouth Disease virus for 21–103 days (Strauch and Ballarini 1994) and the Pig Parvovirus for up to 14 weeks (Mengeling 1999). Ming and Cliver (1995) have detected the persistence of Type I Poliovirus and Hepatitis A virus in the sediment of mixtures of human and pig faeces for up to four and five weeks, respectively.
In view of the potential risk for the transmission of pathogenic agents from pig faeces to animal feed, it is imperative that the faeces be treated before being used as raw material in animal feed.
Among the treatments recommended for recycling faeces, the one that is preferred is ensilage. This decreases the smell of the faeces, preserves most of the nutrients and favours the anaerobic fermentation of the soluble carbohydrates, which in turn decreases the pH, increases the temperature within the silos and changes the osmotic pressure, all of which inhibit the growth of bacteria, viruses and other pathogens (Iñiguez 1991; Russell and Diez-González 1998). It is important to determine whether any of the pathogens that infect pigs remain viable in the pig faeces ensilages.
MATERIAL AND METHODS
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- MATERIAL AND METHODS
This research was carried out in the Centro de Enseñanza, Investigación y Extensión en Producción Porcina (CEIEPP) and in the diagnosis laboratory of the Departamento de Producción Animal: Cerdos, of the Facultad de Medicina Veterinaria y Zootecnia, of the Universidad Nacional Autónoma de México.
In order to gather information on the sanitary state of the farm from which faeces were obtained to prepare the ensilages, samples were collected from 30 pigs of different ages following the methods of DiGiacomo and Koepsell (1986) and Carpenter and Gardner (1996), and the presence of antibodies for Aujeszky’s Disease virus and Blue Eye Disease virus was determined.
Based on the method of Morilla’s (1997), the 30 animals included 10 reproductive females, five gilts, five weaned pigs, five pigs between three and four months of age, and five pigs between five and six months of age.
In order to determine the presence of Escherichia coli, Salmonella choleraesuis, Aujeszky’s Disease virus and Blue Eye Disease virus in the faeces, 100 samples were collected of which five were obtained from each of the following areas of the farm: the liquids collected from the solids separator, the solids, the sedimentation pit (at the surface and at a depth of 1 m), the storage pit for separated liquids, lactating sows from each maternity area (three areas, with a total of 15 samples), each weaning area (five buildings, with a total of 25 samples), each fattening area (four buildings, with a total of 20 samples), each gestation area (two buildings, with a total of 10 samples), and from replacement gilts.
In the case of the liquids from the solids separator, five 10 ml samples were taken, one every 5 min, in sterile jars. For the sampling at the sedimentation pit, 10 ml were taken from five different points at the surface and at a depth of 1 m. The five samples (50 ml) were mixed and from this, 15 ml represented the final sample.
In the case of the solids, 10 g were collected from five points within the storage tank in new 0.5 kg plastic bags. The samples were mixed as for the liquids.
A 10 g faecal sample was collected from the rectum of each of five randomly chosen animals, each one from a different pen within each type of area throughout the farm. In order to obtain the samples directly from the animals, defecation was stimulated at the anus and the faeces were collected in sterile plastic bags. Using one disposable plastic spoon per animal, 10 g faeces were taken from each plastic bag and deposited in a glass jar of the same type as those used for the collection of liquid matter from the pits. One glass jar was used for the faeces of the five animals of each area, the faeces were mixed, and 15 g of this mixture represented the sample for a particular area.
Isolation, identification and quantification of Salmonella spp., E. coli, Aujeszky’s Disease virus and Blue Eye Disease virus were carried out for each sample in order to determine the levels in the faeces used for the preparation of the microsilos.
Serological tests, bacterial isolation and viral isolation were carried out at the end of this phase.
Preparation and inoculation of the microsilos
Microsilos were prepared with sorghum, molasses and the solid fraction of pig faeces obtained from the solids separator, in 0·5 kg (ground grain), 13 cm high, 7 cm diameter plastic jars with screw tops (Envases de Plástico, México D.F.).
The ensilage contained 10% ground sorghum (7 kg), 82% solids (57·4 kg) and 8% molasses (5·6 kg). The components were mixed by hand to produce 70 kg of the mixture (Hernández 1997). The flasks were filled with this mixture by adding 3 cm thick layers and compacting them with a flat-bottomed flask until they were full and compacted up to the rim. These were weighed, and the difference between the heavier and the lighter flask was 0·012 kg (0·596 kg vs 0·584 kg). The pH of the mixture determined before silage was 6·0.
The flasks were randomly distributed for inoculation; 15 flasks were inoculated with 2 ml of 1 × 107 cfu ml–1Salm. choleraesuis from a 6 h culture (growth phase), and another 15 flasks were inoculated with 2 ml of a 1 × 107 cfu ml–1E. coli 6 h culture (growth phase). A 5 ml syringe and a 5 cm 20 calibre needle were used in both cases to place the inoculum into the middle of the flasks. The surface of the mixture was compacted again before each flask was closed and sealed.
The Salm. choleraesuis strain was a donation of the Centro de Investigación de Estudios Avanzados of the Instituto Politécnico Nacional in Mexico, and the haemolytic E. coli serotype F4, O149, K91, H10 culture was donated by the Universidad Autónoma de Yucatán from a strain derived from the reference centre at Pennsylvania State University, USA.
Following the same procedure, 15 flasks were inoculated with 5 ml of a 1 × 107 cfu ml–1 Aujeszky’s Disease virus culture, and another 15 flasks were inoculated with 5 ml containing 1 × 107 infecting doses per cellular culture (CCID-50 ml) of the Blue Eye Disease virus using Karber’s method for virus concentration determination (Snyder et al. 1981). Each inoculation was carried out in a different flask. These viral agents were obtained from the strains collection of the Departamento de Producción Animal: Cerdos, of the Universidad Nacional Autónoma de México.
Once inoculated, the plastic flasks were closed with screw tops to which a rubber stopper was fixed in the middle to be used as a valve. The flasks were sealed around the top edge and around the rubber stopper with silicon, and the air inside was vacuumed out through the rubber valve using a 2.5 cm 22 calibre disposable needle connected to a rubber hose and then to a vacuum pump (Curtis Mathison Scientific, Michigan, USA) with a vacuum capacity of 400 mm mercury 30 s–1.
All the flasks were incubated at room temperature in the laboratory (15–20°C) for 0, 7, 14, 28 and 56 days, with three duplicates for each inoculated agent (Table 1). The silos were labelled according to the inoculated infectious agent as follows: microsilos inoculated with E. coli (EC), microsilos inoculated with Salm. choleraesuis (SC), microsilos inoculated with Aujeszky’s Disease virus (AD) and microsilos inoculated with Blue Eye Disease virus (BED). Additionally, six silos that were not inoculated were used for each ensilage time as negative controls, three for bacterial inocula (BC) and three for viral inocula (VC).
Table 1. Number of microsilos per inoculated agent and time of fermentation
Positive controls were prepared in another 15 flasks that were filled with the same mixture and inoculated with the same agents and doses, but were not subjected to ensilage (NS). Isolation and titration of the different inoculated aetiological agents were carried out from these flasks after 28 days.
After fermentation had taken place, each microsilo was opened and the colour, smell and presence of fungi on the surface of the silo were recorded. For pH evaluation, a 2 g sample from each flask was placed in 25 ml buffer solution for acid pH determination using a pH meter (Hanna Instruments, USA). The first 5 cm of the ensiled material was then discarded, and a 20 g sample was taken with a sterile spatula for isolation and bacteriological and viral quantification of the inoculated micro-organisms. Only the aetiological agent initially inoculated in each microsilo was identified and quantified.
Bacteriological isolation and quantification were carried out in accordance with the following procedure.
For the isolation of Salm. choleraesuis, a 15 g sample was taken from each microsilo and mixed with 45 ml Tetrathionate broth (Broth Base Tetrathionate, Merck) (Pradal-Roa 1994). This suspension was incubated aerobically for 72 h at 36°C in a bacteriological incubator (Incubadora Blue, Revco Scientific, Ashville, USA). A subculture was carried out every 24 h in Salmonella-Shigella (SS) agar (Difco) and Brilliant Green agar (BG) (Merck), and was incubated again for 72 h at the same temperature. The colonies suggestive of being non-lactose fermenters were subjected to biochemical tests, and culture under aerobic conditions was carried out in Triple Sugar Iron Agar (TSI) (Merck), SIM Medium (Merck), Simmons Citrate Agar (Merck), Urea (Becton-Dickinson) and the sugars Arabinose and Trehalose, for 24 h; the data obtained were recorded in a bacterial identification format. The colonies whose morphology corresponded to Salm. choleraesuis were subjected to a serological test (Bacto Salmonella O Antiserum Group C1, Factors 6, 7. Difco) to confirm their identification.
In order to identify E. coli, a loopful from a 1 g sample of each microsilo was plated using a bacteriological wire loop onto a MacConkey agar plate (Difco) and incubated aerobically at 36°C for 24 h. The identity of the bacteria from the lactosa MacConkey agar plates was confirmed by colony morphology and Gram staining. To confirm the identity of the bacteria isolated, the API 20E System (API 20E, Biomeriux, France) was used on the characteristic colonies. A plate agglutination test was carried out for the identification of the serotype using antisera donated by Bayer de México, S.A.
In order to enumerate the lactose fermenter Enterobacteria present in the sample, 10-fold serial dilutions were made by placing 1 g of the sample in 9 ml sterile physiological saline solution (10% sodium chloride, J.T. Baker) at pH 7. The final 1 : 10 dilutions were homogenized using a rotamixer (Vortex Genie 2 Scientific Industries, USA), after which 1 ml was transferred to another tube to prepare the next dilution, and so on. Then, 0·05 ml was taken from each dilution; a culture was made on MacConkey agar plates with lactose (Difco) and incubated at 37°C for 24 h, after which the number of colony-forming units per g (cfu g–1) was recorded in accordance with the plate counting method described by Miles et al. (Henry et al. 1983).
For the isolation of viruses, 3 ml cell culture medium (MEM) (Sigma Aldrich) were added to 2 g sample, after which the supernatant fluid was placed into a 24 multi-well plate (In Vitro, México) containing cellular monolayer specific for each viral agent; monolayers of kidney cells from African green monkeys (VERO) were used for the Blue Eye Disease virus and bovine kidney cells (MDBK) were used for Aujeszky’s Disease virus. A positive control, a negative control and a cell control were placed into each well of the 24 multi-well plate.
The inoculated monolayers were incubated for 90 min at 37°C (Nabco Controlled Enviroment Incubator, Portland, USA), after which they were frozen at –70°C for 24 h, thawed, and centrifuged for 10 min at 2000 g. The supernatant fluids were filtered through 0·22 μm diameter pore size membranes (Millipore); after filtration they were again inoculated into a 24 multi-well plate, with the previously mentioned monoestrate with MEM and 2% fetal cattle serum. These were incubated for 90 min, the medium was decanted and they were placed in a maintenance medium (99% MEM and 1% fetal cattle serum). The monoestrates were checked every 24 h for 3 days. Then, 72 h post-inoculation, the supernatant fluids were removed and the cells fixed with anhydrous alcohol (J.T. Baker) in order to evaluate the cytopathic effect in the MBDK cells.
An indirect inmunofluorescence test was carried out on the monoestrates of VERO cells previously inoculated with the material from the microsilos, for the identification of both Aujeszky’s Disease virus and the Blue Eye Disease virus. To each plate was added 0·05 ml bovine serum albumin; this was washed three times with BSS and the first antibody was added, in this case pig antibodies against both viruses. The plate was incubated for 45 min at room temperature and washed again with BSS. The conjugate was then added (AntiPig-FITC Conjugated, Sigma Diagnostics), incubated for another 45 min and washed with distilled water. Next, 0·02 ml Evans Blue (Sigma Diagnostic) was added and the preparation observed with an inverted fluorescence microscope (Olimpus IX70, Tokyo) (Buerleson et al. 1992).
As a support technique for the viral identification, a 1 g sample previously inoculated with the same virus was taken from each jar at days 0, 7 and 14 after inoculation, in order to identify the viruses inoculated using electronic transmission microscopy. This was carried out in the Laboratorio de Microscopía Electrónica of the Instituto de Fisiología Celular, Universidad Nacional Autónoma de México. Additionally, a sample of each viral inoculum was taken as a reference for observation under the electron microscope.
The survival of the lactose fermenter Enterobacteria inoculated in the microsilos at different times during ensilage was analysed using descriptive statistics, as lack of data for the growth of the Enterobacteria at any time during the ensilage made it impossible to carry out any analysis.
A completely randomized design was used for the statistical analysis of the pH variations. The differences between the averages were analysed by analysis of variance and a Tukey’s test with the SAS statistical package (Statistical Analysis System 1988).
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The number of lactose fermenter Enterobacteria observed in the samples of the different areas of the farm is within the range reported by Pradal-Roa (1994), who recorded between 102 and 107 cfu g–1 in 10–12 kg pigs. It is important to point out that the average recorded here of 7·6 × 105 includes the samples taken from the solids and liquids and from the sedimentation and storage pits, all of which had a lower content of lactose fermenter Enterobacteria.
The genera of bacteria found in the different faecal samples, both from the animals and from the storage and processing areas, coincide with the flora that is frequently isolated from pig faeces and does not cause important clinical signs, with the exception of E. coli and Salm. choleraesuis (Iñigo et al. 1991; Pradal-Roa 1994; Hernández 1997).
The isolation of Salm. choleraesuis from fattening pigs confirms other reports (Chandler and Creven 1981; Plym and Ekesbo 1993; Strauch and Ballarini 1994) with respect to the elimination of this agent through the faeces, and confirms its presence in the sampled population. The lack of Salm. choleraesuis isolation in other areas may have resulted from a low occurrence as, considering that this is a serious health problem in a pig herd, whenever affected animals are detected they are treated with high doses of antimicrobials, after which the amount of the micro-organism in the pig population and environment decreases.
The isolation of Salm. choleraesuis from the liquids collected from the separator confirms its presence in the farm. This is in agreement with Chandler and Creven (1981) who isolated Salm. havana from pig effluents in a farm with no reports of disease in 10 years, and Plym and Ekesbo (1993) who found Salm. senftemberg and Salm. typhimurium in a 7 day pig faeces compost, as well as Salm. dublin in a 14 day pig faeces compost.
Henry et al. (1995) also isolated 11 serovarieties of Salmonella spp. from a storage pit of anaerobic, aerobic and facultative faeces, as well as from the liquid effluent of three different farms. These authors stated that some of the processes used in these farms do not establish the ideal pH conditions for the elimination of Salmonella spp.
In the case of the viral agents, the facts that Aujeszky’s Disease virus and the Blue Eye Disease virus were not isolated, either from the animals or from the deposits of solid and liquid faeces, that no antibodies against these diseases were detected, that these diseases have not been recorded in this farm since its beginning in 1992, and that no external genetic material has been added to the population, indicate that these agents are absent in this population. Additionally, it is well documented that adverse conditions of temperature, ultraviolet light, acidity, heat, other micro-organisms and residues of disinfectants inactivate several viruses (Ward et al. 1986; Mateu et al. 1992; Ming and Cliver 1995).
The organoleptic characteristics of the ensilages proved that fermentation was adequate, with the elimination of the smell from the pig faeces and an improvement in the texture. These coincided with those reported by Hernández (1997) and Cabrera (1998), who recorded a brown colour, an acid smell and no growth of fungi on the surface of microsilos with pig faeces prepared in 2·5 kg plastic flasks throughout 60 days of ensilage.
The presence of fungi on day 28 of ensilage in two flasks and on day 56 in six flasks may have resulted from air entering through some points along the edge of the jars. This may be explained by the strong production of gas that occurred during the first 72 h. As the flasks were compacted to full capacity, leaving no space for gas, the gas pressure broke through some points along the edge of several flasks, allowing the entrance of air and the growth of fungi. This occurs frequently in bigger ensilages and does not interfere with the fermentation process in the centre of the silo (Hernández 1997; Cabrera 1998).
The number of lactose-fermenting Enterobacteria recorded 60 min after inoculation was in accordance with the inoculum used, and there was practically no difference between the quantity inoculated and that measured 1 h after inoculation. A decrease from 4 × 106 to 7·6 × 105 was recorded in the control microsilos 60 min after inoculation; this was the same quantity of Enterobacteria as was found in the solids in the initial samples, and coincided with the results of Hernández (1997) of 1·6 × 106 recorded 60 min after ensilage. The number of Enterobacteria recorded on day 0 was slightly lower than that found by Hernández (1997) of 2·4 × 107 on the same day.
The lack of growth of Enterobacteria from day 7 onwards coincides with the observation of Iñiguez et al. (1990) that all coliform bacteria were destroyed after 7 days of fermentation, and with the data of Hernández (1997) of no Enterobacteria after 30 days of ensilage. McCaskey et al. (1996) reported that E. coli and Salm. typhimurium inoculated into ensilages based on ruminal contents, maize, alfalfa hay, cotton seed husk and poultry manure, were absent after 5 days.
The destruction of the Enterobacteria in this study seems to be closely related to the moment when the lowest pH level was reached within the microsilos, and this is in accordance with several authors who have pointed out that decrease in pH, absence of oxygen, presence of volatile fatty acids and the temperature during the ensilage process are factors that destroy bacteria (Iñiguez 1991; Henry et al. 1995; Russell and Diez-González 1998).
The effect of the pH on inoculated Enterobacteria is supported by the findings of several researchers. Hernández (1997) found that E. coli does not survive a pH of 4·4, Salm. paratyphi a pH of 4·5, Salm. schottmuelleri a pH of 4·5 and Salm. typhi a pH of 4·0. Henry et al. (1995) found that Salm. typhimurium does not survive a pH of 4·0 after 24 h, and at a pH of 5·0 only three of four samples recuperated after 48 h. Strauch and Ballarini (1994) found that 90% of E. coli, Salmonella spp., Y. enterocolitica, Staph. aureus and Brachyspira hyodysenteriae populations are destroyed after a week at a pH lower than 6·5.
The lack of lactose-fermenting Enterobacteria in 28 day flasks with a mixture that was not subjected to ensilage may have resulted from the presence of oxygen and a pH of 5·0. As mentioned above, E. coli does not resist a pH close to 5·0, as was found in the material in these flasks, and Salmonella spp. resists pH 5·0 for only 48 h (Henry et al. 1983).
Although the effect of ensilage on the survival of viruses has not been well established, it is known that few viruses resist the acidity found in the microsilos of this study. For example, it is known that Aujeszky’s Disease virus becomes inactive at a pH of 4·3 and a temperature of 39°C between one and seven days (Kluge et al. 1999). The Blue Eye Disease virus becomes inactive at 56°C after 30 min, but at 37°C it conserves its haemoagglutinant and infective properties in cellular culture for 110 days (Stephano 1999). There is no information on its susceptibility to an acid pH.
Both the temperature and the pH are important factors in the inactivation of viruses. Ward et al. (1986) reported a 99% elimination of echovirus and rotavirus in liquids fermented at a pH of 6·5 for 24 h. Ming and Cliver (1995) recorded viral inactivation with high temperatures and changes in the pH, as these denature viral proteins, and observed that enteric viruses are both more stable at a neutral pH than at an acid pH and more susceptible to an alkaline pH than to an acid pH.
With respect to the results obtained from the different tests that were carried out for viral identification, the observation of a cytopathic effect and fluorescence in some samples may be explained by the low number of viruses present in these samples. This explains why the cytopathic effect was moderate and detectable in only some areas of the affected pits, whereas it was extensive in the positive controls. Fluorescence was observed only in one point of some cells, whereas it was observed over the whole culture surface in the positive controls.
These results coincide with those of Bolin et al. (1985) of a greater number of positive samples with the viral isolation test than with the inmunofluorescence test in the study of Aujeszky’s Disease virus using brain, lung, liver and tonsil tissues. In the case of the Blue Eye Disease virus, McNeilly et al. (1997) found that the viral isolation test was more sensitive for the isolation of the virus than the inmunofluorescence test. It can be concluded that ensilages based on the solid fraction of pig faeces reduce the risk of transmission of the bacterial and viral agents used in this study.