Fate of Pathogens Present in Livestock Wastes Spread onto Fescue Plots

Fecal wastes from a variety of farmed livestock were inoculated with livestock isolates of Escherichia coli O157, Listeria monocytogenes, Salmonella, Campylobacter jejuni, and Cryptosporidium parvum oocysts at levels representative of the levels found in naturally contaminated wastes. The wastes were subsequently spread onto a grass pasture, and the decline of each of the zoonotic agents was monitored over time. There were no significant differences among the decimal reduction times for the bacterial pathogens. The mean bacterial decimal reduction time was 1.94 days. A range of times between 8 and 31 days for a 1-log reduction in C. parvum levels was obtained, demonstrating that the protozoans were significantly more hardy than the bacteria. Oocyst recovery was more efficient from wastes with lower dry matter contents. The levels of most of the zoonotic agents had declined to below detectable levels by 64 days. However, for some waste types, 128 days was required for the complete decline of L. monocytogenes levels. We were unable to find significant differences between the rates of pathogen decline in liquid (slurry) and solid (farmyard manure) wastes, although concerns have been raised that increased slurry generation as a consequence of more intensive farming practices could lead to increased survival of zoonotic agents in the environment.     

Declines of zoonotic agents in liquid livestock wastes stored in batches on-farm

AIM: To measure the decline rates of zoonotic agents introduced into liquid livestock wastes in on-farm storage tanks. METHODS AND RESULTS: Salmonella spp., Escherichia coli O157, Campylobacter jejuni, Listeria monocytogenes and Cryptosporidium parvum, propagated in laboratory-controlled conditions, were inoculated into 35,000-l volumes of fresh livestock wastes (pig slurries, cattle slurries and dirty waters). D-values for bacteria were six to 44 days, and for C. parvum were 133 to 345 days. Campylobacter jejuni declined significantly more rapidly than the other bacterial pathogens, while E. coli O157 declined significantly more slowly. On average, bacterial declines were not affected by the season of waste deposition and storage or by the dry matter content of the wastes, but were more rapid in dirty waters than in pig slurries. The physiciochemical composition of wastes in each category varied significantly. CONCLUSIONS: Zoonotic agents can survive for several months during storage of liquid livestock wastes. Livestock wastes should be batch-stored and not subjected to continuous additions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that batches of liquid livestock waste, if contaminated with bacterial pathogens, should be stored for 6 months to reduce contamination levels. Alternative strategies for reducing C. parvum levels in liquid livestock wastes should be explored.     

Decline of zoonotic agents in livestock waste and bedding heaps

AIMS: To measure the rates of decline of zoonotic agents introduced into heaps of spent bedding and faecal wastes generated by commercially farmed livestock and managed in a similar way to that of a working farm. METHODS AND RESULTS: Livestock isolates of Salmonella, pathogenic Listeria, Campylobacter and Escherichia coli O157 were laboratory cultured and used to inoculate 5 m3 heaps of cattle, sheep or pig wastes mixed with bedding materials. Decline of each of the infectious agents was monitored with time as was the temperature inside each heap. Temperatures of >50 degrees C were typically achieved at the core of the heaps. Pathogen decline was rapid, typically <3 days for a 1-log reduction in levels. The longest time that zoonotic agents were isolated from the heaps was 93 days. CONCLUSIONS: Movement of heaps of livestock bedding waste from animal pens to a secondary store, and storing them under conditions conducive for increased temperature is a simple and cost-effective treatment for rapidly lowering levels of zoonotic agents in solid farm wastes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates a simple and cheap treatment that can be used to help prevent the spread of zoonotic agents through agricultural environments.     

Effect of length of time before incorporation on survival of pathogenic bacteria present in livestock wastes applied to agricultural soil

In response to reports that the contamination of food can occur during the on-farm primary phase of food production, we report data that describes a possible cost-effective intervention measure. The effect of time before soil incorporation of livestock wastes spread to land on the rate of decline of zoonotic agents present in the waste was investigated. Fresh livestock wastes were inoculated with laboratory-cultured Salmonella, Listeria, and Campylobacter spp. and Escherichia coli O157 before they were spread onto soil. Incorporation of the spread wastes was either immediate, delayed for 1 week, or did not occur at all. Bacterial decline was monitored over time and found to be significantly more rapid for all waste types when they were left on the soil surface. There were no significant differences in initial bacterial decline rates when wastes were spread in summer or winter. Our results indicate that not incorporating contaminated livestock wastes into soil is a potential intervention measure that may help to limit the spread of zoonotic agents further up the food chain. The implications of these findings are discussed in relation to current advice for livestock waste disposal.     

Effect of Length of Time before Incorporation on Survival of Pathogenic Bacteria Present in Livestock Wastes Applied to Agricultural Soil

In response to reports that the contamination of food can occur during the on-farm primary phase of food production, we report data that describes a possible cost-effective intervention measure. The effect of time before soil incorporation of livestock wastes spread to land on the rate of decline of zoonotic agents present in the waste was investigated. Fresh livestock wastes were inoculated with laboratory-cultured Salmonella, Listeria, and Campylobacter spp. and Escherichia coli O157 before they were spead onto soil. Incorporation of the spread wastes was either immediate, delayed for 1 week, or did not occur at all. Bacterial decline was monitored over time and found to be significantly more rapid for all waste types when they were left on the soil surface. There were no significant differences in initial bacterial decline rates when wastes were spread in summer or winter. Our results indicate that not incorporating contaminated livestock wastes into soil is a potential intervention measure that may help to limit the spread of zoonotic agents further up the food chain. The implications of these findings are discussed in relation to current advice for livestock waste disposal.     

Analyses of livestock production, waste storage, and pathogen levels and prevalences in farm manures

Survey results describing the levels and prevalences of zoonotic agents in 1,549 livestock waste samples were analyzed for significance with livestock husbandry and farm waste management practices. Statistical analyses of survey data showed that livestock groups containing calves of <3 months of age, piglets, or lambs had higher prevalences and levels of Campylobacter spp. and Escherichia coli O157 in their wastes. Younger calves that were still receiving milk, however, had significantly lower levels and prevalence of E. coli O157. Furthermore, when wastes contained any form of bedding, they had lowered prevalences and levels of both pathogenic Listeria spp. and Campylobacter spp. Livestock wastes generated by stock consuming a diet composed principally of grass were less likely to harbor E. coli O157 or Salmonella spp. Stocking density did not appear to influence either the levels or prevalences of bacterial pathogens. Significant seasonal differences in prevalences were detected in cattle wastes; Listeria spp. were more likely to be isolated in March to June, and E. coli O157 was more likely to be found in May and June. Factors such as livestock diet and age also had significant influence on the levels and prevalences of some zoonotic agents in livestock wastes. A number of the correlations identified could be used as the basis of a best-practice disposal document for farmers, thereby lowering the microbiological risks associated with applying manures of contaminated livestock to land.     

The air-borne distribution of zoonotic agents from livestock waste spreading and microbiological risk to fresh produce from contaminated irrigation sources

Aims:  To assess the risks of zoonotic agents in dissemination of livestock wastes into the environment by airborne distribution. To subsequently assess the survival time of zoonotic agents, introduced in irrigation water, on the phylloplane of produce.
Methods and Results:  An Escherichia coli marker was introduced into pig slurry which was spread using a rain gun sprayer. Air sampling was undertaken to determine the distance reached by the marker. No recoveries were observed at a distance of 250 m. Borehole water, contaminated with zoonotic agents, was used to irrigate field plots sown with lettuce and spinach. Decline in bacterial numbers on the phylloplane was observed with time. After initial rapid decreases, we were unable to detect any pathogen from the phylloplane, 1 month after contamination.
Conclusions:  These preliminary results suggest that the risks to public health from the aerosolized spread of bacteria during slurry spreading by rain gun are low. Although, zoonotic agents on crop phylloplanes perish quickly, the risks of overhead irrigation of fresh produce 3 weeks before harvest should still be considered.
Significance and Impact of the Study:  These preliminary results improve our understanding on the fate of zoonotic agents in the environment. Spreading liquid livestock wastes by an airborne mechanism may not pose a significant public health risk. Detection of zoonotic agents 3 weeks after contamination of lettuce and spinach means that consideration should be given by the farmers until the time of harvest, when irrigating fresh produce with water that may have been directly or indirectly contaminated by livestock wastes.     

Analyses of Livestock Production, Waste Storage, and Pathogen Levels and Prevalences in Farm Manures

Survey results describing the levels and prevalences of zoonotic agents in 1,549 livestock waste samples were analyzed for significance with livestock husbandry and farm waste management practices. Statistical analyses of survey data showed that livestock groups containing calves of <3 months of age, piglets, or lambs had higher prevalences and levels of Campylobacter spp. and Escherichia coli O157 in their wastes. Younger calves that were still receiving milk, however, had significantly lower levels and prevalence of E. coli O157. Furthermore, when wastes contained any form of bedding, they had lowered prevalences and levels of both pathogenic Listeria spp. and Campylobacter spp. Livestock wastes generated by stock consuming a diet composed principally of grass were less likely to harbor E. coli O157 or Salmonella spp. Stocking density did not appear to influence either the levels or prevalences of bacterial pathogens. Significant seasonal differences in prevalences were detected in cattle wastes; Listeria spp. were more likely to be isolated in March to June, and E. coli O157 was more likely to be found in May and June. Factors such as livestock diet and age also had significant influence on the levels and prevalences of some zoonotic agents in livestock wastes. A number of the correlations identified could be used as the basis of a best-practice disposal document for farmers, thereby lowering the microbiological risks associated with applying manures of contaminated livestock to land.     

Survival of Escherichia coli O157:H7 and Salmonella typhimurium in cow manure and cow manure slurry

An exponential linear destruction was observed for Escherichia coli O157:H7 and Salmonella typhimurium in cattle manure and manure slurry stored at 4, 20 or 37 degrees C. The resulting decimal reduction times ranged from 6 days to 3 weeks in manure and from 2 days to 5 weeks in manure slurry. The main effects of time as well as temperature were pronounced with the most rapid destruction at 37 degrees C. The ammonia concentration in manure increased slightly during storage but did not exceed 0.1%. pH values in the deeper layers of manure remained constant except at 37 degrees C when the pH increased by 1 unit in 60 days. In the surface layers of manure, pH increased by 1.5-2 units, the oxidation-reduction potential of the manure declined rapidly to values below -200 mV. These changes do not seem to be reflected in changing rates of bacterial destruction. The observed order of destruction makes it possible to predict storage conditions (temperature and time) that will lead to a predetermined level of reduction of the two pathogens.     

Single-chain variable fragment antibodies selected by phage display against the sporozoite surface antigen P23 Of Cryptosporidium parvum

Cryptosporidium parvum, an apicomplexan parasite transmitted via animal fecal wastes, is the causative agent of cryptosporidiosis. Clones were selected from 2 synthetic na?ve human single-chain variable fragment (scFv) phagemid libraries that bound to the recombinant P23 protein of C. parvum. Panning the Tomlinson I and J phagemid libraries resulted in 6 distinct clones. Two clones had full-length scFv sequences, while the remaining clones were either truncated or missing a section of the heavy chain. Despite these differences, all clones were able to detect both native C. parvum proteins and recombinant P23. None of the selected clones cross-reacted with Escherichia coli, Streptococcus pyogenes, Listeria monocytogenes, Bacillus cereus, Giardia lamblia (cysts or trophozoites), or with S16, another dominant surface antigen on C. parvum sporozoites. Clones expressed as the scFv-gIIIp fusion construct in soluble form detected C. parvum. Panning from na?ve libraries is a useful method for isolation and identification of recombinant antibodies that have the potential for use in pathogen detection and immunotherapy.     

Thermal Resistance of Salmonellae and Staphylococci in Foods

The heat-resistant Salmonella senftenberg 775W and two strains of Staphylococcus aureus were tested at temperatures up to 68.3 C (71.1 C for S. senftenberg) in four different media. From the survival data, decimal reduction times (D values) were calculated for each set of conditions, and decimal reduction time curves were constructed for each bacterial strain in each medium. Slopes of decimal reduction time curves (Z(D)) ranged from 4.52 to 6.38 C with a single exception. There was no statistical heterogeneity among the remaining values. Results were in close agreement with published results of similar studies conducted at somewhat lower temperatures and support the practice of using a slope value (Z(D)) of 5.56 C for establishing time-temperature relationships for food processing. It is recommended that such a decimal reduction time curve not be extrapolated to temperatures more than 5.56 C higher than those actually tested.     

Metabolic activity of pathogenic bacteria during semicontinuous anaerobic digestion.

In natural environments such as anaerobic digesters, bacteria are frequently subjected to the stress of nutrient fluxes because of the continual changes in the flow of nutrients, and to survive, they must be capable of adapting readily to nutrient changes. In this study, the metabolic activities of Escherichia coli, Salmonella typhimurium, Yersinia enterocolitica, Listeria monocytogenes, and Campylobacter jejuni were studied within culture bags (Versapor-200 filters, 0.22-microns pore size) in laboratory anaerobic digesters. The metabolic activity of these bacteria was indicated by their adenylate energy charge (EC) ratios and their ability to incorporate [3H]thymidine, which was related to the respective changes in viable numbers within the culture bags during anaerobic digestion. Fluctuations in the adenylate EC ratios, the uptake of [3H]thymidine, and the viable numbers of E. coli, S. typhimurium, Y. enterocolitica, and L. monocytogenes cells were probably due to constant changes in the amount of available nutrients within the anaerobic digesters. The viability of S. typhimurium increased quickly after a fresh supply of nutrients was added to the system as indicated by the uptake of [3H]thymidine and an increase in the adenylate EC ratios. The viable numbers of E. coli, S. typhimurium, Y. enterocolitica, and L. monocytogenes organisms declined rapidly from 10(7) to 10(8) CFU/ml to 10(3) to 10(4) CFU/ml and remained at this level for an indefinite period. The decimal reduction time calculated during the period of exponential decline ranged from 0.8 to 1.2 days for these bacteria. C. jejuni had the greatest mean decimal reduction time value (3.6 days).(ABSTRACT TRUNCATED AT 250 WORDS)     

Zoonotic cryptosporidiosis

The widespread usages of molecular epidemiological tools have improved the understanding of cryptosporidiosis transmission. Much attention on zoonotic cryptosporidiosis is centered on Cryptosporidium parvum. Results of genotype surveys indicate that calves are the only major reservoir for C. parvum infections in humans. The widespread presence of human-adapted C. parvum, especially in developing countries, is revealed by recent subtyping and multilocus typing studies, which have also demonstrated the anthroponotic transmission of C. parvum subtypes shared by humans and cattle. Developing and industrialized countries differ significantly in disease burdens caused by zoonotic species and in the source of these parasites, with the former having far fewer human infections caused by C. parvum and little zoonotic transmission of this species. Exclusive anthroponotic transmission of seemingly zoonotic C. parvum subtypes was seen in Mid-Eastern countries. Other zoonotic Cryptosporidium spp. are also responsible for substantial numbers of human infections in developing countries, many of which are probably transmitted by anthroponotic pathways. The lower pathogenicity of some zoonotic species in some populations supports the occurrence of different clinical spectra of Cryptosporidium spp. in humans. The use of a new generation of molecular diagnostic tools is likely to produce a more complete picture of zoonotic cryptosporidiosis.     

Effects of several factors on the heat-shock-induced thermotolerance of Listeria monocytogenes.

The influence of the temperature at which Listeria monocytogenes had been grown (4 or 37 degrees C) on the response to heat shocks of different durations at different temperatures was investigated. For cells grown at 4 degrees C, the effect of storage, prior to and after heat shock, on the induced thermotolerance was also studied. Death kinetics of heat-shocked cells is also discussed. For L. monocytogenes grown at 37 degrees C, the greatest response to heat shock was a fourfold increase in thermotolerance. For L. monocytogenes grown at 4 degrees C, the greatest response to heat shock was a sevenfold increase in thermotolerance. The only survival curves of cells to have shoulders were those for cells that had been heat shocked. A 3% concentration of sodium chloride added to the recovery medium made these shoulders disappear and decreased decimal reduction times. The percentage of cells for which thermotolerance increased after a heat shock was smaller the milder the heat shock and the longer the prior storage.     

Non-specific induction of increased resistance in mice to Trypanosoma congolense and Trypanosoma brucei by immunostimulants.

Administration of the immunostimulants Corynebacterium parvum, Bacillus Calmette-Guérin (BCG) or Bordetella pertussis prior to, or at the same time as, challenge with Trypanosoma congolense significantly increased survival times in mice, both of trypano-susceptible (A/J) and trypano-resistant (C57Bl) strains. The increased survival time was associated with significant alterations in parasitaemia, which included lengthening of the pre-patent period, a delay in the time taken to reach the first peak of parasitaemia and a reduction in the level of parasitaemia. Similar results were obtained when these strains of mice were challenged with Trypanosoma brucei following pre-treatment with C. parvum. Thus, by the use of immunostimulants it was possible to reduce the susceptibility of mice to trypanosomiasis and the hope is that this can also be achieved with domestic livestock.     

The zoonotic transmission of Giardia and Cryptosporidium

The molecular characterisation of Giardia and Cryptosporidium has given rise to a more epidemiological meaningful and robust taxonomy. Importantly, molecular tools are now available for 'typing' isolates of the parasites directly from clinical and environmental samples. As a consequence, information on zoonotic potential has been obtained although the frequency of zoonotic transmission is still poorly understood. Analysis of outbreaks and case-control studies, especially when coupled with genotyping data, is slowly providing information on the public health significance of zoonotic transmission. Such studies support the hypothesis that Cryptosporidium hominis is spread only between humans but that the major reservoir for Cryptosporidium parvum is domestic livestock, predominantly cattle, and that direct contact with infected cattle is a major transmission pathway along with indirect transmission through drinking water. The situation is less clearcut for Giardia duodenalis but the evidence does not, in general, support zoonotic transmission as a major risk for human infections. However, for both parasites there is a need for molecular epidemiological studies to be undertaken in well-defined foci of transmission in order to fully determine the frequency and importance of zoonotic transmission.     

The effect of slurry storage and anaerobic digestion on survival of pathogenic bacteria

The decline in viable numbers of Salmonella typhimurium, Yersinia enterocolitica and Listeria monocytogenes in beef cattle slurry is temperature-dependent; they decline more rapidly at 17°C than at 4°C. Mesophilic anaerobic digestion caused an initial rapid decline in the viable numbers of Escherichia coli, Salm. typhimurium, Y. enterocolitica and L. monocytogenes. This was followed by a period in which the viable numbers were not reduced by 90%. The T₉₀ values of E. coli, Salm. typhimurium and Y. enterocolitica ranged from 0.7 to 0.9 d during batch digestion and 1.1 to 2.5 d during semi-continuous digestion. Listeria monocytogenes had a significantly higher mean T₉₀ value during semi-continuous digestion (35.7 d) than batch digestion (12.3 d). Anaerobic digestion had little effect in reducing the viable numbers of Campylobacter jejuni.     

Long-term operation of slurry bioreactor for decomposition of food wastes

A pilot scale slurry bioreactor was used for the treatment of food wastes. Food wastes were continuously added (750 g wet weight per day) into the reactor and successfully decomposed to inorganic carbon without intermittent removal of suspended solids. During operation for 90 days, 91% reduction of food wastes was achieved. Microorganisms actively grew during the initial 20 days of operation, and reached a stationary phase with a cell concentration of around 5 x 10(10) cells ml(-1), which indicated that food waste was utilized as a respiratory substrate during this phase. Using data for time variation of dissolved oxygen, the oxygen requirement for decomposition of food wastes was estimated to be 5.0 g O2 g(-1) dry weight of food wastes.     

Persistence of inoculated hepatitis A virus in mixed human and animal wastes.

The persistence of hepatitis A virus (HAV) was determined both in mixtures of septic tank effluent (STE) with dairy cattle manure slurry (DCMS) and in mixtures of STE with swine manure slurry (SMS). HAV was consistently inactivated more rapidly in the two types of mixed wastes than in STE alone or in the control Dulbecco's phosphate-buffered saline (PBS). At 5 degrees C, the D values (time, in days, for a 90% reduction of virus titer) were 34.6 for the mixed STE and DCMS, 48.5 for the mixed STE and SMS, 58.5 for STE, and 217.4 for the Dulbecco's PBS control. At 22 degrees C, the D values were 23.0, 17.1, 35.1, and 90.1 for the four suspension media, respectively. A comparison of HAV inactivation in mixed wastes subjected to different treatments at the same pH and temperatures showed that the virus inactivation in the mixed wastes was related, at least in part, to microbial activity. In mixed STE and DCMS, the D values at 25 degrees C were 8.3 for raw mixed wastes, 15.1 for autoclaved mixed wastes, and 9.6 for bacterium-free filtrate of raw mixed wastes; D values at 37 degrees C were 6.8, 10.1, and 7.0 for these three suspension media, respectively. In mixed STE and SMS, the D values at 25 degrees C were 8.1 for raw mixed wastes, 14.3 for autoclaved mixed wastes, and 9.1 for bacterium-free filtrate of raw mixed wastes; the D values at 37 degrees C were 6.8, 9.4, and 6.9 for the three suspensions, respectively.     

Morphologic, host specificity, and molecular characterization of a Hungarian Cryptosporidium meleagridis isolate

This study was undertaken in order to characterize Cryptosporidium meleagridis isolated from a turkey in Hungary and to compare the morphologies, host specificities, organ locations, and small-subunit RNA (SSU rRNA) gene sequences of this organism and other Cryptosporidium species. The phenotypic differences between C. meleagridis and Cryptosporidium parvum Hungarian calf isolate (zoonotic genotype) oocysts were small, although they were statistically significant. Oocysts of C. meleagridis were successfully passaged in turkeys and were transmitted from turkeys to immunosuppressed mice and from mice to chickens. The location of C. meleagridis was the small intestine, like the location of C. parvum. A comparison of sequence data for the variable region of the SSU rRNA gene of C. meleagridis isolated from turkeys with other Cryptosporidium sequence data in the GenBank database revealed that the Hungarian C. meleagridis sequence is identical to a C. meleagridis sequence recently described for a North Carolina isolate. Thus, C. meleagridis is a distinct species that occurs worldwide and has a broad host range, like the C. parvum zoonotic strain (also called the calf or bovine strain) and Cryptosporidium felis. Because birds are susceptible to C. meleagridis and to some zoonotic strains of C. parvum, these animals may play an active role in contamination of surface waters not only with Cryptosporidium baileyi but also with C. parvum-like parasites.