OCAMM Seminar Series
Pathogens

Click on title to view summary.  Presentation date in parenthesis.

Pathogen issues and livestock manure: Environmental impacts of Cryptosporidium
Dr. Lucy Ward, Ohio State University (2000)
Characterization of the microbial populations of swine feces and waste storage pits
Dr. Terry Whitehead, USDA-NCAUAR (2000)

The origin of feces: Factors affecting the concentration, prevalence and persistence of pathogens in livestock manure
Dr. Jeffrey LeJeune, Ohio State University (2003)

Survival of selected animal pathogens and indicator bacteria in on-farm compost and mortality composting systems
Jean Bonhotal, Cornell University (2004)
Public health impact of antimicrobial use on swine farms
Julie Funk, DVM,MS, PhD, Ohio State University (2004)
Using genetic markers to identify bacterial populations in dairy wastewater
Jeff McGarvey, USDA-ARS-FCR (2004)
Composting and anaerobic digestion of manure from antibiotic treated calves
Dr. Osman Atilla Arikan, USDA-ARS (2004)
Comparison of pathogen persistence in composted and liquid stored manures
Dr. Fred Michel, Ohio State University (2007)
Microbial Source Tracking: A tool box for environmental monitoring
Dr. Jorge Santo Domingo, U.S. Environmental Protection Agency (2007)
Fate of foodborne pathogens in manure and soil and its implication for food safety
Dr. Eelco Franz, Wageningen University (2008)

 

 

Pathogen issues and livestock manure: Environmental impacts of Cryptosporidium
Dr. Lucy Ward, Ohio State University

Cryptosporidium is a single-celled protozoan parasite that can cause severe diarrhea in mammals with competent immune systems and death in those with compromised immune systems.  The parasite is shed in the infected animal’s feces and can be food- or water-borne.  The risk from Cryptosporidiumis is increased because billions of oocysts are shed from the host in an infective stage, only a single dose is needed to infect and the parasite can auto-infect the host without being re-ingested.  While there are various species, Cryptosporidium parvum is one species that seems to infect most mammalian hosts.

Traditional methods of reducing the incidence of Cryptosporidium have had minimal impact.  Drug therapy is hampered by the unique niche occupied in the intestine by the organism.  While cattle vaccines have reduced the number of organisms shed by an infected host, the numbers are still too high.  Disinfectants have not been successful because of the thick wall of the oocyst and filtration is difficult because of the oocyst’s small size.  Current research focuses on preventing infection or shedding of the Cryptosporidium oocyst and treating the feces to inactivate the oocyst.  Preliminary studies at OARDC suggest that intestinal microflora may play an important role in determining susceptibility.  Research is planned to evaluate the effectiveness of composting to deactivate the oocyst.

Discussion:

Lynn Willett (OSU) asked at what temperature the oocyst can survive outside the animal.  Ward responded the 120 degrees Fahrenheit for 20 minutes will destroy it.  That is why composting may be effective. However, the temperature must be maintained throughout the compost for the needed time.

Harry Hoitink (OSU) questioned whether the Cryptosporidium can be cultured.  According to Ward, there is no way to culture them other than multiple passages through calves.  Hoitink speculated that if researchers could find a way to develop a titrate to inoculate the soil system, there may be soil factors that will lead to suppression. If so, rotating calf hutches in a specific pattern may reduce the severity.  Ward noted that this may not have much impact since most calves pick up Cryptosporidium at birth and begin shedding within 10 to 21 days.

Mo Saif (OSU) asked if there have been any cases of ground water contamination.  Ward reported that there have been three outbreaks:  1) a break in a pipeline which probably provided the pathway, 2) a shallow ground water aquifer overlain by soils, and 3) an intentional contamination.  Saif questioned how far the oocysts can travel.  Ward noted that it is not well known but that spring runoff probably poses the greatest risk.

Hoitink asked that, if the oocysts are able to survive in the soil, what activates them.  Ward said they are dormant in the soil but are already activated and ready to infect.  Hoitink suggested that if the oocyst passes through a snail or other soil organism, it may cause “germination” of the oocyst and limit its ability to infect a mammalian host.  Ward reported that Cryptosporidium has been shown to be transmitted through an earthworm.

Maurice Watson (OSU) noted that Cryptosporidium was not discovered until 1910 and it must have been a problem 150 years ago.  He questioned why there is so much concern recently.  Ward noted that it was not recognized as a cause of poultry disease until the 1950’s.  The emergence of AIDS has brought it to the forefront for humans at the same time it has received recognition in calves.   Ward commented that research on Cryptosporidium is difficult since reproduction by cloning and in vitro are not possible, resulting in various strains.  While animals have strains that affect humans, humans have strains that do not affect animals. Also diagnostics for infection are relatively recent.

Fred Michel (OSU) asked what is the lowest dose that is infectious.  Ward responded that it can be as little as 10 oocysts in humans and 1 oocyst in animals.  Michel questioned whether a single oocysts can be identified in soil or other areas as that is all that is required for infection.  Ward noted that it is theoretically possible in soil and can be done in feces.

Hoitink questioned whether it was possible to study the effectiveness of a system that could suppress the disease by exposing calves to an ED 50 level dose of Cryptosporidium (i.e., a dose that would cause infection in 50% of the calves?) then subjecting them to a suppressive or non-suppressive system.  Ward explained that because every strain is different, its ED 50 level is different and no single strain is available to study.  There are significant differences among strains because the organism undergoes both sexual and asexual reproduction.

Willett questioned how long Johnes disease (discussed at last seminar) remains in the soil and whether soil and manure are considered vectors for it.  Ward replied that the length of time was not certain but soil and manure are vectors.

Michel asked how studies of microbial communities in the gut are assessed and oocysts measured.  Ward responded that in a study of mice, one group was colonized with a given microflora and the other group was not.  The results showed a marked reduction in the number and days of shedding for the mice colonized with certain strains of bacteria.  Preliminary data indicated that 10E6 (10 to the sixth) oocysts will kill a gnotobiotic (germ-free) pig while a conventional pig infected with 10E12 oocysts will be ill for two days and recover.  This suggests that the microflora in the gut play a role.

Ken McClure (OSU) asked whether an animal is infective once it is past the symptomatic stage.  Ward noted that Cryptosporidium does not remain dormant after the symptomatic stage.  Because it undergoes sexual and asexual reproduction in the host, it auto-infects the host without re-ingestion.  For immune competent individuals, the immune response will eventually clear the organism after shedding begins.  However, this does not prevent re-infection although more organisms are required each time.

Saif questioned the effectiveness of reducing shedding and allowing the immune system to clear the gut.  Ward noted that reduction in shedding must be significant as only 1 oocyst can infect.  Saif noted that the question becomes: What reduction in shedding is significant to make an impact?

Saif asked if it is possible to completely eliminate Cryptosporidium from the environment.  Ward commented that a young calf cannot be vaccinated in time to prevent infection and shedding.  Because, in older calves, shedding is reduced anyway, a vaccine may have little impact.

Watson asked it Cryptosporidium can be air-borne.  Ward stated that it is possible as the oocyst will survive drying.  Las Vegas, which uses surface water as its primary supply, has the greatest yearly outbreaks.

McClure asked about the implication for manure spreading and whether all manure should be composted.  Ward noted that in Ohio, 80 to 90% of all herds are infected.  Cornell studies have shown that, in a watershed, survivability over a 3 to 4 month period is only 2%.  There are still some viable organisms after 6 months. 

Harold Keener (OSU) suggested that we live with organisms like this all the time.  If we can’t eliminate it, what is the sane approach?  Should we just learn to live with it?  Ward responded that it is a significant public health issue.  It is her opinion that over time humans and animals have increased in number resulting in increased contamination of the environment and that we need to start reducing this contamination so there are fewer threats.  Keener suggested that animal numbers in Ohio have not increased.  He questioned whether we are worse off now (in regards to Cryptosporidium infections) than in the past.  Ward noted that nationwide, animals have increased and that there are no historical measure of infection for comparison.
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Willett asked why people who work in livestock do not appear to be affected by Cryptosporidium while an outbreak in the Milwaukee water supply caused 400,000 to become ill.  Ward remarked that exposure is the key.  Studies indicate that farmers and large animal veterinarians are most resistant.  In cities, where there is little exposure, the population is least resistant.

McClure suggested that a vaccine for those populations not exposed may be the answer.  Ward noted that the current push is not a vaccine for people.  The focus is to protect water supplies and decrease shedding in animals.

Keener noted that it is a losing battle.  Ward responded that although it cannot be eliminated, there is potential for controlling it.  Saif observed that there is always a compromise in living with any disease and combined approaches are often needed.  While elimination is not possible, control is.


Characterization of the microbial populations of swine feces and waste storage pits
Dr. Terry Whitehead, USDA

The decomposition of manure by anaerobic bacteria produces components that create odor. Over 150 different volatile organic compounds (VOCs) associated with swine manure have been identified, including ammonia, organic acids and alcohols and sulfides. To improve methods for reducing odor associated with
swine manure, researchers at the USDA’s National Center for Agricultural Utilization Research have focused on identifying major bacterial species in the swine intestinal tract and swine manure storage pits using molecular and conventional mirobiological analyses. Total DNA was extracted from fecal and manure samples and 16S rDNA genes were amplified using PCR and universal primers. The PCR products were cloned and the 16S rDNA genes sequenced. The sequences were compared to 16S rDNA sequences from known organisms and grouped according to 16S rDNA phylogeny. The majority of sequences obtained were different than, but similar to, those found in known microorganisms. Samples were also diluted, plated onto complex anaerobic media, and colonies from the highest dilutions were isolated for 16S rDNA analysis.  Similarity analyses of the 16S rDNA sequences from total DNA and pure cultures indicated the presence of primarily low G&C, Gram-positive bacteria such as Clostridium sp. and Streptococcus sp.  Additional 16S rDNA sequence analyses of swine waste handling pits using archae-specific primers indicated that the primary archaebacteria present are methanogens. This approach could be used to identify microorganisms that are important in the production of odorous compounds. Probes or PCR primers can also be designed to monitor specific groups of interest (e.g, sulfate reducers or specific pathogens), including the effects of management strategies on odor production. Molecular analyses were also used to identify the presence of erythromycin- and tetracycline- antibiotic resistance genes in bacteria. 

Discussion:
Fred Michel, OSU, asked whether the resistant genes correspond to antibiotics fed to the pigs.  Whitehead responded that tetracycline-resistant bacteria were found even though tetracycline was not included in the feed.  Additional research is needed to determine if tetracycline in feed will influence the number or distribution of resistant species.

Harry Hoitink, OSU, asked whether this approach could be extended to analysis of compost samples.  Whitehead said that it is appropriate for a variety of samples. 

Kevin Elder (?), ODNR, questioned the number of buildings and samples used in the study.  Whitehead noted that only 1 building (which is a criticism) but this was used to establish a baseline of which organisms are present.  The research needs to be expanded to see if the relationship of organisms stands with different samples.

Hoitink asked whether 18S RNA analysis had been used.  Whitehead responded that using the 18S RNA was not successful in the pits, perhaps due to the presence of fungi.

Michel questioned using the clone/sequencing approach in subsequent studies.  Whitehead noted that a checkerboard hybridization approach is favored initially as it allows for more rapid analysis for comparison with other barns.

Elder asked if the approach has been used on septic tanks.  Whitehead said not the he knows but it has been used for anaerobic digesters.

Dave Elwell (OSU), siting recent paper out of Minnesota on odor production, asked if identifying odor producers at the species level will improve odor control.  Whitehead commented that most data so far has been identifying microbial populations at the genus level.  There has been little correlation at the species level using known gene sequences.  This suggests many unknown populations of bacteria.

Hoitink noted that work by his group indicates that analysis at the genus level gives the information needed for the study of odor control.  Whitehead observed that it depends on the question being asked, specifically what the organism is accomplishing.  Hoitink responded that if interested in stability/odor control, genus shifts will give the needed information.  Whitehead noted that two different species may generate different odors but a shift in the population of the two may not be detected at the genus level.

Mark Morrison, OSU, asked if data back up an increase in fecal populations on enumerated (?) plates when using ruminal fluid while a swine slurry additive shows no growth of organisms.  Using a 16S rDNA approach, do fecal samples and slurry samples at different depths still break out into different genera or only at the species level?  Whitehead that Lactobaccillus sp. was not found in the pit and that  Clostridium sp. was found in the feces and pit.  A pure culture from the pit that matches a pure culture from the feces at the species level was not found, indicating a shift in the temperature, nutrient and other variables in the ecosystem.

Michel asked how many clones were analyzed for each sample.  Whithead said 135-140 for each.  This provides a snapshot of the bacteria present.  Extensive analysis is limited by cost, time and effort.

Burk Dehority, OSU, asked whether a source of inoculation in the pits could be from environmental sources rather than feces.  Whitehead responded that some environmental inoculation is possible but the amount of feces added daily lead to speculation that it is the primary source.  Dehority noted that it would be interesting to start with an empty, sterile pit.

Jody Tischmack, Purdue University, questioned the effect of an increase in the pH of the sludge.  Whitehead noted that it would depend on the situation.  While there would be an ecological shift, the specifics of the bacteria present is not known.

Tishmack asked about cost comparison of the classical techniques for analyzing bacteria present to the molecular techniques used in the study.  Whitehead responded that because non-monetary factors are important, it is difficult to assign a cost.  For example, more space is needed for classical analyses and PCR is faster, but the gene sequencing is costly.  However, using agar has costs and may increase the chance of missing an organism.

Tishmack asked if this technique is available commercially so that anyone could send an example to a lab for analysis.  Whitehead said yes but noted that the 16S rDNA is fast but only provides a snapshot of the primary components.  There is a risk of missing other components of the system.

Elder commented that most swine manure odors are the end products of fermentation of the substrate that exists in the pit.  How would you cause a change in the substrate or microbial population to reduce odors?  Whitehead noted that they are not trying to stop the fermentation process.  Companies are looking at additives that can inhibit odor production.  These analyses can be used to evaluate the impact of these additives on the microbial population and its effect on odor generation.

Elder noted that the breakdown of amino acids generate odors and questioned whether they should be removed from diets.  Whitehead noted that changes in diet from bulk protein to specific amino acids is being studied to determine the impact on odor.  The correlation of the change in microbial population with the change in odor production is important.

Lynn Willet, OSU, commented that quicklime was used in outhouses to reduce odors.  Tischmack noted that this practice can increase ammonia odors due to the quick increase in pH.  Hoitink commented that lime stabilization is a problem because few farmers can use the end product.

Whitehead confirmed Elder’s assumption that the samples were from unagitated pits.  Elder asked if the lowest samples in the pit were solid.  Whitehead said that the whole pit was about 90% liquid but that there was more particulate near the bottom.  Population shifts at this depth may result from analysis of dead bacteria that have migrated downward as the technique does not differentiate between living and dead.

A participant in Lima asked for an estimate of the bacteria that would remain viable in an aerobic environemnt such as during land application.  Whitehead observed that in general, most bacteria will not survive unless tolerant to air.  Some work is being done to compare bacterial survival to the movement of genes.

Elder noted that a next step in this research would be to evaluate population shifts in a waste treatment pit designed for odor control. Whitehead noted that such a pit had not been tested at this time since Illinois and other states have banned the use lagoons for new facilities.  However, the technique is the same.


The Origin of Feces:  Factors Affecting the Concentration, Prevalence
and Persistence of Pathogens in Livestock Manure

Dr. Jeffrey LeJeune, Ohio State University

Cattle manure can harbor bacteria, viruses and parasites that can cause illness in humans.  Since the first outbreak of E coli 0157 in 1982, this bacteria infects 70,000 people in the US each year with approximately 10% of those infected experiencing renal failure.   With only a very small infectious dose (~100 CFUs) needed to affect humans, outbreaks have been associated with undercooked beef, non-pasteurized milk and cider, fruits and vegetables, drinking water, lake swimming, and both animal and human contact.  Although cattle are not the reservoir for E coli 0157 and are asymptomatic, they can amplify the organism shedding up to 1 million human  infectious doses per day in fresh manure.  While only a small percentage shed at this rate, they contribute to 80% of the total.   Analysis of four herds indicates point sources, such as watering troughs or feed, for infection and greater prevalence from July to September which is also reflected in human trends.  E coli 0157 can remain infectious for up to 250 days in non-sterile water, more than 6 months on surfaces such as concrete and straw and more than 1 year in manure piles.  Composting has the potential to destroy the organism if temperatures of 55 degrees Celsius are maintained for at least 24 hours.  Although E coli 0157 will never be eradicated, additional research is needed to understand the ecology of the organism in the farm environment and gastrointestinal tract of cattle and to develop handling procedures that minimize the risk to humans.  Areas of investigation include minimizing infection from feed, field testing probiotics to inhibit growth and developing vaccines to protect against gastrointestinal colonization.

Discussion:

Steve Loerch (OSU) asked if the specific source of the corn and barley associated with contamination and been checked prior to feeding it to cattle.  LeJeune noted that it was not feasible to test before feeding due to the large number of shipments.

Brian McSpadden-Gardner (OSU) questioned whether the seasonal fluctuation of the organism could be attributed to the susceptibility of the host or to survival in the environment.  LeJeune responded that, while there may be variations in the animal’s susceptibility with the seasons, none has been identified.  Analysis of soil and other environments such as sawdust bedding do show higher counts during the summer.

Ted Short (OSU) asked about the treatment for humans infected with E coli 0157.  LeJeune responded that for those with symptoms (bloody diarrhea), increased fluids are recommended.  Antibiotics are contraindicated and may increase the amount of toxin produced by the bacteria.  Children and the elderly are most susceptible to contamination and development of renal failure.

Loerch questioned whether the sterile environment created by human’s sanitation efforts impacts contamination.  That is, is there more immunity among farm families that have a greater potential for contact with the pathogen?  LeJeune noted that research is just beginning to explore the relationship between immunity and exposure.

Short asked if swine or poultry are susceptible to E coli 0157.  LeJeune observed that pigs get a different disease from a different E coli.  The distribution of receptors affect whether the animal becomes sick or not.  E coli 0157 has only recently been detected in swine but is very rare.  Neither pork nor sausage has ever been implicated in an outbreak.  E coli 0157 is not found in poultry.

Loerch asked if there is the potential to test for E coli 0157 as a screening procedure at the slaughterhouse.  LeJeune responded that it is not feasible as such testing would result in a large number being culled, negatively impacting marketability.

Jim Skeeles (OSU Extension) asked if county fair boards should adopt policies to minimize the risk of contamination.  LeJeune identified two safety precautions:  1) wash hands and 2) only consuming food outside animal barns. Hand washing can reduce risk of contamination by 50% and those consuming food in barns increase their risk by about 20 times.


Select Pathogens and Bacteria in Compost
Jean Bonhotal. Cornell University

The Cornell Waste Management Institute (CWMI) collaborated with thirty farms, primarily dairy, to evaluate composted manure for a variety of parameters.  Because the definition of finished compost was not standardized, the material analyzed varied widely between farms.  For the dairy farms, fecal coliform and weed seeds were low and pH, chloride and maturity levels were acceptable.  Some dairies that used hoof dip had high levels of copper.  For the seven poultry composts tested, some were high in pH and chlorides and low in maturity but all had low fecal coliform and weed seeds.  A windrow of dairy manure inoculated with Johne’s disease showed no contamination after six days of composting and similar results were found for finished compost from four dairy farms with high incidence of the disease.  One farm that used an anaerobic digester prior to composting achieved good reduction of both fecal coliform and Johne’s.  CWMI also assessed the impact of composting dead animals and butcher waste on Clostridium, E. coli, and Listeria and found that results vary with the composting method.  The preferred practice is to use large woodchips to allow passive aeration and absorb moisture and to apply the compost to forests and non-root crops.

Discussion:

David Elwell (OSU) asked if attempts had been made to increase the rate of composting by mixing butcher waste with wood chips to increase the contact between nitrogen and carbon sources.  Bonhotal replied that the method used is to layer 18-24 inches of woodchips, then 6-12 inches of waste, then 12 inches woodchips, etc.  The material is not turned as with manure composting.  For blood waste, a trench can be formed at the top of the windrow that contains other feedstock and the blood incorporated from there.  The same method has been successfully used for liquid manure as well as waste materials from Ben & Jerry’s ice cream.

Fred Michel (OSU) questioned the composting regulations in New York State.  Bonhotal noted that NY regulations are less stringent than Ohio’s.  Universities and property owners (e.g., custom butchers) are exempted from permits unless there is a problem. 

Michel asked if there was any difference for those selling the compost off-site.  Bohnotal said that food waste and manure composters do not need a permit.  Biosolids composting facilities must be permitted and yardwaste facilities must register

Srinand Sreevatsan (OSU) asked if containment is used to compost dead animals to protect wildlife from BSE or similar wasting diseases.  Bohnotal replied that regulations do not require containment.  If the pile is well constructed, there should not be runoff.

Sreevatsan questioned whether pH levels were very high during mortality composting.  Bohnotal responded that it is generally not the case.

Elwell asked if there were any data to indicate that the prions that cause the mass wasting diseases breakdown when applied to fields after composting.  Bohnotal responded that to date, the only way to disable the prion is by alkaline digestion.  Sreevatsan noted a National Institute of Health study that found no deterioration of the prion when infected brains were buried (no composting).  Elwell suggested that something, perhaps a bacterium, seems to destroy prions over time otherwise there would be more incidences of infection.

Warren Dick (OSU) noted that as a soil scientist he has never found a protein that will not degrade in soil.  While a prion (a protein) may be more resistant it would appear that they do degrade otherwise greater accumulation in the soil would be expected.  Sreevatsan suggested that some may not agree with that conclusion.

Dick asked for the source of the copper found in some of the composted dairy manure.  Bonhotal said that copper sulfate is found in some hoof dips which are used to control problems with hoof diseases that tend to occur in the high moisture environment associated with liquid manure systems.

Michel asked whether the higher concentration of fecal coliform in the edge of the windrow compared to the middle could be the result of temperature differences.  Bohnotal noted that the difference was found in storage piles that had been turned.  The fact that the middle of the pile had lower concentrations than the edges was unexpected as the middle is wetter.

Sreevatsan asked whether it is acceptable to spread composted manure after 6 days of composting since the data indicate that Johne’s disease had been eliminated by then.  Bohnotal responded that veterinarians have given the ok but for animals on pasture, more mature compost is more palatable for the animals.  Also, although Johne’s was not found after day 6 (days 2-5 were not tested), there is a possibility that it still exists as it may be found in isolated areas in the compost.


Using Genetic Markers to Identify Bacterial Populations in Dairy Wastewater
Jeffrey McGarvey, USDA/ARS/FCR

Research on a California dairy used molecular methods to determine the type of bacteria that inhabit a wastewater treatment system, how they travel through the system and whether pathogenic organisms are a risk. Total DNA was extracted from samples from three major components of the system, manure from the free stall barn, separator pit water and circulated holding lagoon water, and 16S rDNA genes were amplified using PCR and universal primers. The PCR products were cloned, the 16S rDNA genes sequenced then compared to known 16S rDNA sequences. The analysis indicated a decrease in the bacterial density and diversity as the manure moved through the system. Few pathogenic bacteria were detected and none were identified with a specific PCR. A second study compared the bacterial communities between a stagnant and a circulated lagoon from two dairies located within a 1-mile radius both using a balanced feed. Although gross plate counts showed similar microbial and nutrient content, rarefraction analysis indicated that the stagnant lagoon contained a more diverse population. Additional analysis revealed that green non-sulfur bacteria were more prevalent in the stagnant lagoon whereas purple sulfur bacteria were more common in the aerated lagoon. The observance that the aerated lagoon had minimal odors as compare to the stagnant lagoon may be due to the purple sulfur bacteria which have been shown to degrade volatile organic carbons. Additional research is planned to evaluate volatile organics on a dairy farm that has the option of diverting wastewater to either a stagnant or an aerated lagoon.

Discussion:

Srinand Sreevatsan (OSU) asked if the bacterial population fluctuates with the season. McGarvey noted that the amount of photosynthetic purple sulfur bacteria decreases with decreasing sunlight but that others go up.  The weather plays a role as does the fact that the animals are on pasture more often during the summer.

Sreevatsan asked whether the farm has a Johnes testing program.  McGarvey responded that according to the producer, Johnes is not a problem.  While McGarvey’s research tested for but did not detect Johnes, it did not enrich the sample for a stronger analysis.


Public Health Impact of Anitmicrobial Use on Swine Farms
Julie Funk, DVM, MS, PhD, Ohio State University

Although the estimated $0.15 – 3 billion dollar public health cost from antimicrobial resistance is primarily attributed to human use of antibiotics, antimcrobials used for livestock may also have an impact.  Currently, antibiotics in agriculture do not require veterinary prescriptions and are used to 1) treat individuals for disease (therapeutic); 2) treat a herd to stop the spread of disase (metaphylactic); 3) prevent an outbreak (prophylactic); and 4) promote growth.  As the percentage of healthy animals treated increases, so does public concern* that the antibiotics will be less effective in treating human diseases.  To assess the risk of antibacterial use in animals on human microbial resistance, the probabilities of the presence of resistant bacteria in the animal, human ingestion of it and adverse health consequences must be evaluated.  Some studies seems to indicate that there is an impact on humans but more data on in vivo transfer is needed.  For example, while fluoroquinolones were approved in 1986 to treat campylobacter infections in humans, resistant isolates did not appear until two years after they were approved for use in chickens (1995).  However, the data may be influenced by the increased efforts to identify resistance in the past few years.  In addition, the extent of human resistance attributed to antibacterial use in animals is difficult to quantify especially as there is a large variablity in estimating the proportions of human, therarpeutic animal and non-therapeutic animal anitmicrobial use.  Analysis of resistant isolates in European countries where some antibiotics have been banned for growth promotion, indicate that resistant eteroccoci have decreased but there has been no impact on human health.  There have been no changes in resistant salmonella and campylobacter, but pigs have shown increased diarrhea and decreased growth in the nursery while there has been no effect on the growth performance of finishers.  Current research at OSU includes evaluating the effect of sub-therapeutic chlortetracycline on the reservoir of antimicrobial resistance in swine.   Additional research is underway to compare the source, diversity and resistance of three foodborne bacteria for swine reared in both conventional and antibiotic-free environments.

* The US Food and Drug Adminiatration maintains a website with links to guidance documents and other information regarding antimicrobial resistance and the use of antibiotics for livestock, click here .

Discussion:

Fred Michel (OSU) asked why more resistant isolates have been found in humans than in animals.  If transfer is from animals to humans, wouldn’t you expect more in the animals?  Funk agreed but noted that a Danish study indicated that there may be more than one source of the isolates.  Also, animals are surveyed at slaughter rather than when infection is apparent.  However, isolates are detected in humans when diagnosing illness.

A participant in Columbus questioned data on resistance to tetracycline.  Funk noted that in the literature, swine seem to harbor a higher percentage of fecal e. coli resistant to tetracycline but it is not clear why.

Mo Saif (OSU) noted that tetracycline is used in poultry but rarely for growth.  Funk noted that it also is not used for growth promotion in swine.  The research underway used tetracycline because the grant funded research for assessing the impact of antimicrobials for growth promotion on foodborne pathogens.  However, most antimicrobial used for growth are active against gram positive bacteria and most foodborne pathogens are gram negative.  Thus, tetracycline was chosen and administered at 50 grams per ton because at that concentration there are no labeled claims for therapeutic use.

Linda Saif (OSU) asked whether the studies in Denmark and Sweden found an increase in other diseases when specific antimicrobials were eliminated.  Funk responded that there have been some increases but they are primary viral.  The increased incidence of diarrhea in nursery swine was treated with copper and zinc but those before environmental concerns were prevalent.  Claims have been made that bedding nursery swine on deep straw is effective in reducing diarrhea; however, US studies indicate other problems with deep straw.

L. Saif asked about other studies on whether antibiotics used for growth promotion are effective.  Funk noted that there are not many and none are observational.  One in Thailand evaluated the DNA of bacterial communities in feces.  It looked at shifts in population in response to antibiotics.  Current thought of how antibiotics promote growth is that they shift the bacterial population in the gastrointestinal tract which increases the feed efficiency.  There is some evidence to support this.

L. Saif asked about the use of antibiotics for parasitic control (e.g., cryptosporidia).  Funk replied that they are still used for such control but data of the effectiveness is not clear.


Composting and Anaerobic Digestion of Manure from Antibiotic Treated Calves
Osman Atilla Arikan, USDA-ARS

The use of antibiotics for therapeutic purposes and growth promotion in livestock production results in the excretion of the antibioitic and/or its metabolites in manure and the potential for surface and groundwater contamination when the manure is applied to land.  Oxytetracycline (OTC), a broad-spectrum antibiotic, has been shown to reside in sediments for several months negatively impacting algae and crustaceans and increasing the potential to contribute to antimicrobial resistance to tetracyclines.  The objective of this study was to determine whether anaerobic digestion and composting would degrade OTC and its metabolites excreted in cattle manure and to assess the effects of OTC on anaerobic digestion and composting processes.  Manure from five beef cattle fed rations with OTC at standard concentrations was evaluated.  For the composting process, 99.8% of the OTC was removed compared to 27.4% for the anaerobic digestion.  In addition, the OTC residuals in the manure caused significant inhibition of biogas production during anaerobic digestion.

Discussion:
Referring to a graph depicting the rate of biogas production from OTC contaminated manure decreasing after five days in a digester, a Columbus participant suggested one might expect the opposite to  occur as microbes become more resistant to the antibiotic.  Arikan replied that during the first few days, the most biodegradable portion of the mixture is broken down thus masking the effect of the OTC and its metabolites.

Lynn Willett (OSU) asked if the composition of the biogas was analyzed.  Arikan noted that one sample was analyzed as having 50-60% methane.  Future studies will use gas chromatography to analyze the biogas, including fatty acids and hydrogen, to better understand the process.

Srinand Sreevatsan (OSU) asked how much antimicrobial activity is retained by the metabolites.  Arikan responded that he did not have the data for comparison with OTC.  Future studies will address the issue.

Harry Hoitink (OSU) noted that the strong adsorption of the OTC to the solid matrix in the composting studies may be affected by the maturity of the compost.

Fred Michel (OSU) asked if the photosynthetic bacteria are phototrophic and heterotrophic and whether they can use different substrates.  What effect on the bacterial populations would lagoon covers have?  McGarvey suggested that the photosynthetic bacterial populations would be expected to decrease.  Lagoon covers can be effective in trapping odors but can cause other problems.

A Columbus participant asked about the differences in bacteria between the manure, separator pit and lagoon in the first study.  McGarvey noted that the most common bacterial groups identified, unknown and firmicutes, were the most common from all three locations so are fit for each environment.  However, this is not true for all the bacteria as the third most common bacteria in the manure and separator pit, actinobacter, differed from that in the holding lagoon, proteobacter.


Comparison of Pathogen Persistence in Composted and Liquid Stored Manures
Fred Michel, The Ohio State University

The effect of common manure management practices on the persistence of pathogens were compared.  Dairy manure was inoculated with M. paratuberculosis and other naturally occurring pathogens such as Salmonella, E.Coli and Listria monocytogenes then treated under conditions simulating three systems:  thermophilic composting at 55º C, bedded pack composting at 25º C and liquid lagoon storage.  Each treatment was sampled on days 0, 3, 7, 14, 28, and 56 for the detection of pathogens.  The high temperature (thermophilic) composting system was most effective in reducing the pathogens.  It is recommended that the thermophilic composting be used for treatment of manure that will be used in pathogen-sensitive environments such as vegetable production, residential gardening or application to rapidly draining fields.

A second study evaluated the persistence of antibiotic resistant (AR) genes in swine manure.  The study involved developing quantitative methods to analyze the microbial ecology of AR genes and to determine the prevalence and concentration in four full scale treatment systems: a conventional liquid storage lagoon, a High Rise Hog facility (composting system), and ambient temperature anaerobic digester and an aerobic Ekokan biofiltration system (similar to wastewater treatment).  It was found that real time PCR assays can be used to adequately quantify the reservoirs of several classes of AR genes.  Also, the effect of treatment varied with a conventional storage lagoon having the least ability to reduce AR genes and anaerobic digestion and composting having the greatest.

Additional Resources:

Grewal SK, S Rajeev, S Sreevatsan, FC Michel Jr. 2006. Persistence of Mycobacterium avium subsp paratuberculosis and other zoonotic pathogens during simulated composting, manure packing, and liquid storage of dairy manure. Applied and Environmental Microbiology 72: 565-574. Click here.

Jing Chen, Zhongtang Yu, FC Michel, Jr., Thomas Wittum, and Mark Morrison. 2008. Development and application of real-time PCR assays for quantification of erm genes conferring resistance to macrolide-lincosamides-streptogramin B in livestock manure and manure management systems. Applied and Environmental Microbiology 02799-06v1 (on ASM website).

Grewal, S, S. Sreevatsan, FC Michel Jr. 2007. Persistence of Listeria monocytogenes and Salmonella enterica serovar typhimurium during simulated composting, pack storage, and aerated and unaerated liquid storage of swine manure. Compost Science and Utilization 15(1):53-62.

Discussion:
Columbus participant:  What happens to solids in the Ekokan system (aerobic filtration)?
Michel: Initially, the plan was to separate the solids for composting or land application, so they were not evaluated.

Hoitink: In the anaerobic digestion system, are virus and antibiotic resistant (AR) gene concentrations reduced during nitrification?
Michel: Reduction appears to be in the digester.  The viruses and AR genes are more prevalent in the effluent entering the lagoon but are significantly reduced in the effluent leaving the lagoon.

Hoitink:  Is there concern that antibiotic resistant gene markers are not destroyed and may be passed into the food chain?

Michel:  Those working with the marker genes are concerned because they are becoming ubiquitous in the environment.


Microbial Source Tracking: A tool box for environmental monitoring
Dr. Jorge Santo Domingo, U.S. Environmental Protection Agency

The 2000 National Water Quality Inventory reported that 13% of the impaired surface waters in the U.S. were impaired due to the presence of fecal indicator bacteria primarily from non-point sources, including septic systems, domesticated animals and wildlife.  While traditional monitoring assesses the presence of harmful bacteria and the associated health risk, microbial source tracking (MST) methods attempt to identify the source of contamination, allowing for improved risk analysis and better risk management.  MST is based on the hypothesis that conditions in the gut, including temperature, pH and diet, will select for specific microbial populations which can be identified using a variety of techniques. Several MST methods, such as host-specific PCR, do not require the development of large culture-based fingerprint databases, allowing for faster and more economic means of fecal source detection.  Since detection limits and host specificity vary for different assays, as a result there is a need for methods that can provide information about multiple host-specific genes to help identify the source of contamination.

Additional Resources:
Microbial Source Tracking Guide Document, a U.S. EPA reference guide, click here.

Microbial Source Tracking. 2007. M. J. Sadowsky and J. W. Santo Domingo (ed.), ASM Press, Washington. DC

Santo Domingo, J.W., D.G. Bambic, T.A. Edge, and S. Wuertz. 2007. Quo vadis source tracking? Towards a strategic framework for environmental monitoring of fecal pollution. Water Res. 41:3539-3552.

Simpson, J.M., J.W. Santo Domingo, and D.J. Reasoner.  2002. Microbial source tracking: state of the science.  Environ. Sci. Technol. 36:5279-5288.

Discussion:
Jim Hoorman (OSU):  What is the cost of testing samples in a watershed? 
Santo Domingo:  It depends on the goal of the study.  If you are trying to differentiate between cattle and humans it can be relatively inexpensive.  However, if you want to quantify the amount of contamination from different sources, it will be more expensive.

Fred Michel (OSU):  Can MST be used to identify a specific farm? 
Santo Domingo: It is very difficult with the current technology.  Metagenomics has the potential but a large sample is needed.  As techniques become less expensive and more accurate it may be more likely, particularly if we want to discriminate between dairy and beef cattle. But it still depends on the detecting differences in the genetic sequence in the fecal microbes on each farm, a task that is more difficult than if you want to discriminate between animal types.

Michel:  If organisms are specific to the gut, how will the environment of the surface water affect their survival or cause changes over time?
Santo Domingo: This is an important issue that has not received a lot of attention by the source tracking community, and consequently we have very little data to predict how host-specific populations survive in environmental waters. Lets say that only one organism/marker was being evaluated, then the chances of detecting them in the environment, and therefore correctly identify the source of pollution, depend on the survival skills outside of the gut of only one population. That is why if we target multiple genetic markers of multiple populations we increase the chances of picking up a source tracking signal, as any given host-specific population must survive long enough to allow identification of a specific location.

Mark Fritz (SWCD):  Could samples from a specific pollution event be used to identify the species causing the impairment?
Santo Domingo:  Yes, but environmental samples need to be collected very shortly after a runoff event.  The ability to identify contamination from a species will depend on the level of the microbes in the sample, although it may be possible to enrich for them. Potential hotspots should be identified during dry weather.

Jeff LeJuene (OSU):  For commercial companies providing MST services, how can the validity of their results be assessed?
Santo Domingo:  Limitations of the methodology need to be understood.  A good sampling scheme is important and the data should be validated with fecal samples.


Fate of Foodborne Pathogens in Manure and Soil and its Implication for Food Safety
Dr. Eelco Franz, RIKILT-Institute of Food Safety, Wageningen University

Increases in outbreaks of illness associated with ready-to-eat-food (RTEV) have been observed internationally, with E coli O157:H7 and Salmonella contamination of lettuce and other salad foods most frequently implicated.  Sources of contamination include irrigation water, manure amended soils, and proximity to livestock and wildlife.  Reducing contamination cannot focus only on post-harvest sanitation; it requires a systems approach that includes reducing the prevalence of pathogens in livestock and reducing pathogen survival in the plant, manure and soil.  A study using lettuce plants demonstrated that E coli can be found on the root surface and in intercellular spaces of vegetables grown in contaminated soil and appears to impact plant growth.  Research on dairy farms in the Netherlands indicate that manure storage systems indicate that lower temperatures increase the survival rate of the pathogens but greater fluctuations in temperature decrease it.  Anaerobic conditions also decrease the survival rate.  A study of 25 dairy farms concluded that native microflora play a significant role in reducing the survival rate of the pathogens, which may reflect the importance of biodiversity in competing for nutrients or the presence of microorganisms harmful to the pathogens.  Research on the role of nutrients in pathogen survival in manure indicated that survival decreased as pH and fiber content increased, indicating the role of nutrient availability in survival. The level of easily available carbon sources per unit microbial biomass was the most important factor in determining E. coli O157 survival in manure-amended soils (negative relation). Manure handling practices that increase the aging of the manure and the interval between application and planting can also help reduce contamination of RTEV’s.

Discussion:
Warren Dick (OSU): In reference to the finding that the animal pathogens can reside in the plant, what is the risk that it could re-inoculate the soil?  Franz:  The survival of the pathogen in the plant was relatively early in the production; there was no follow through to harvest. As long as we do not know the fate of pathogens residing on or into crops up to harvest, we cannot answer this question. It might however be relevant for green manuring.

Fred Michel (OSU):  How was the biomass carbon (C) measured in the study that found that an increase in the dissolved organic C per unit of biomass C correlated with an increase in contamination?  Franz: The biomass was fumigated then the carbon extracted.

Michel:  Were E coli O157:H7 present in the natural flora?  If so, how do you differentiate between it and the inoculated bacteria?  Franz: Some manure were naturally contaminated with E. coli O157. We related the manure characteristics to this. In order to study risk factors for survival, we added an GFP E. coli O157.

Harold Keener (OSU):  The pathogen survival rate showed little difference for sandy soils compared to loamy ones.  At what temperature was the study conducted?  Would you expect any change with different temperature regimes?  Franz;  The study was conducted at 15o C.  Different results would be expected for outside conditions with variations in temperature.

Keener:  The higher survival rate of the pathogens at the soil surface compared to below surface was unexpected as it is usually assumed that the UV light at the surface is effective in killing pathogens.  Franz:   Yes, that was the original thought but there is actually very little published to document that effect.  It might be that pathogens below surface are more prone to the microbial activity and competition of the soil.

Question:  Is the decreased survival correlated to increased microbial diversity the result of competition or another mechanism?  Franz:  Both.  Competition for nutrients does negatively impact the pathogens. Generally, oligotrophic conditions lead to higher microbial diversity. Subsequently, there is more chance oon the presence of specific antagonists or competitors.

Mo Saif (OSU):  What is the mechanism for contamination of the vegetables: surface injury or uptake via root system?  Franz:  That has not yet been determined.

Question:  What is the location of the internalized E coli, i.e, is it in the roots or edible portion?  Franz: There is high variability in the research results. Some only found E. coli in the below ground parts, others also in the edible part.

Saif:  Does this research imply that human pathogens are becoming plant pathogens?  Franz:  No. While the pathogen does negatively impact plant growth, there is no mechanism for the organism to enter the plant as a plant pathogen would.  The E coli probably enters passively when there is an opportunity.