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An Afflflnltm Adm/Equal Opportunity Inflation USE OF COMPOSTING AS AN ALTERNATIVE METHOD OF DEAD SWINE DISPOSAL By Josep Garcia Sirera A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Science 1995 ABSTRACT USE OF COMPOSTING AS AN ALTERNATIVE METHOD OF DEAD SWINE DISPOSAL BY Josep Garcia Sirera Three different trials were conducted to evaluate the efficacy, sanitation and safety of using composting as a method for dead swine disposal. In trial one, composting was monitored in a commercial swine operation. Over a one year period, a total of 6000 kgs of porcine tissue were composted. Compost piles reached a temperature of at least 65°C. Winter weather did not limit the composting process. Survival of Pseudorabies herpesvirus (PRV) and Actinobacillus pleuropneumoniae (App)in compost piles was monitored in a second trial. Pigs were infected, euthanized and carcasses composted for 35 d. Temperature of the piles ranged from 270C to 51°C. Samples from d 7 and 14 tested negative for PRV and App. The third trial involved Salmonella cholerasuis (Sc). Pigs were infected, euthanized and carcasses composted for 10 d. Sc was recovered in samples taken on d 0, l and 3 but not on d 7 and 10. Results confirm that composting can be used to dispose of swine mortality on comercial swine_farms. To my parents and to Alicia. iii ACKNOWLEDGMENTS My gratitude to my advisor, Dr Dale Rozeboom who made this thesis possible. Dale supported me in each and every aspect of this project, without his help this thesis would not have been finished. I would also thank the members of my committee, Dr Loudon and Dr Person for their comments and suggestions. The on-farm part of the project was possible thanks to Dick Cheney and his family. Dr Barb Straw contributed as a committee member and she was also a very important part of the Salmonella trial. Dr Thacker, Dr Paula Cray and Dr Larry Granger were very helpful in different aspects of the microorganism survival trials. Fellow graduate students and members of the MSU swine farm also contributed to this work. Thank you very much to everyone. iv TABLE OF CONTENTS List of Tables vii List of Figures viii Introduction 1 Biological Activity 3 On-farm mortality 4 Environmental safety 6 Disease safety 6 Future questions 8 Objectives 8 On-farm composting of dead swine 10 Abstract 10 Introduction 12 Materials and Methods 12 Results and Discussion 16 Cost of Composting 21 Implications 24 Survival of Actinobacillus pleuropneumoniae and Pseudorabies herpesvirus in swine mortality during composting 25 Abstract 25 Introduction 26 Materials and Methods 27 Results 32 Implications 34 Survival of Salmonella cholerasuis in swine mortality during composting 35 Abstract 35 Introduction 36 Materials and Methods 36 Results and Discussion 38 Implications 40 Conclusions 41 List of References 45 Appendices Appendix Appendix Appendix Appendix Appendix Appendix Pseudorabies virus isolation Protocol for analysis Actinobacillus pleuropneumoniae Protocol for analysis ELISA IDEXX test for Pseudorabies virus. Protocol for analysis Pseudorabies Latex Agglutination test. Protocol for analysis Immunoflouresence test for PRV. Protocol for analysis Salmonella cholerasuis. Protocol for inoculation preparation and bacteriologic analysis vi 47 47 49 50 55 59 61 Table Table Table Table LIST OF TABLES Dead swine composting microbial analysis Dead swine composting nutrient analysis Dead swine composting cost comparison Qualitative recovery of S.cholerasuis vii 19 20 23 39 Figure Figure Figure Figure Figure LIST OF FIGURES Compost facilities floor plan Layout of compost pile Compost pile temperature and tissue additions Compost pile temperature, PRV and App trial Compost pile temperature, Salmonella trial viii 13 14 18 33 38 Introduction In recent years swine producers have been challenged with the increasingly difficult task of disposing of dead pigs and after-birth on the farm. Burning, burial, and rendering continue to be the only options available. Incineration eliminates diseases, but has economical and environmental drawbacks. Burial is cheap, but not always convenient in colder climate where the ground is frozen during winter. Also, burial sites can be unearthed by predators, increasing the chance of spreading disease. Where water tables are high, carcasses decay less rapidly and ground water is more likely to become polluted. Incineration raises questions of air pollution. Rendering recovers several animal by-product commodities and effectively controls transmissible diseases, however, not all producers have access to this method of disposal. This inaccessibility may be due to logistics or to the size of the swine farm. Frequently swine operations of 100-200 sows do not produce enough dead animals to justify service by a rendering plant. Also, pre-weaning mortality and afterbirth do not have any value for rendering companies, therefore they are not collected for disposal. 2 Use of composting became important during the 1970's, largely because the Environmental Protection Agency (EPA) sponsored the development of specific composting techniques by the U.S. Department of Agriculture. The main use for composting was the treatment of municipal waste water sludge. Composting was also applicable to municipal solid- waste management. Presently, use of composting as a method of disposing yard-waste is encouraged in several municipalities. Little attention has been directed towards use of composting as a method of dead animal disposal. Composting is currently being used in the U.S. on poultry farms and by only a few swine producers. In most states composting is not an approved method of disposal. Experimental evidence supporting its usefulness as a means of dead pig disposal is lacking and therefore regulations governing dead pig and after-birth composting are unestablished. Composting could be a biologically-safer, more environmentally-friendly alternative disposal method compared to incineration or burial. With composting, there is no risk of ground water contamination or air pollution. In addition, it could be more available than rendering for small to medium sized producers. Research evaluating the efficacy and safety of composting dead pigs and after-birth must be conducted if dead animal disposal laws are to be amended to include composting for swine. E' J . J E . . Composting is a biological process in which organic matter is broken down by aerobic and thermophilic bacteria into a humus-type end product. The process reduces the mass and volume of organic material. The primary factors that affect aerobic composting bacteria are: carbon to nitrogen (C:N) ratio, moisture content, and oxygen content (Henry and White, 1990). Other important factors would be particle size of animal tissue and consistency of bulking agent. If the conditions are correct, composting bacteria will grow and as a result temperature will rise. Temperature may reach 70°C. The ultimate peak temperature and the time needed to achieve it, will depend on conditions for bacterial growth. Peak temperature indicates a loss of conditions to support optimal bacterial growth. Usually a lack of oxygen. This is the reason why compost piles are turned after the internal pile temperature lowers. With the turning process, more oxygen becomes available for bacterial growth. Optimum moisture for composting ranges from 40-60%, depending on particle size of animal tissue. Low moisture retards microbial growth. Moisture contents greater than 60% can be used if proper aeration is maintained. A C:N ratio ranging from 20:1 to 30:1 is best for bacterial growth. C:N ratios are low in animal carcass. Bulking agents are 4 required as a carbon source to raise C:N ratios when animal tissue is composted. Wilts: The majority of studies conducted to-date evaluating composting of dead animals have involved poultry. The use of batch composting for dead bird disposal was reported by Murphy and Handwerker (1988). Their system was available for use year-round on farms of various sizes and it proved to be economically feasible, biologically secure and conforming to water, air and soil pollution standards. Whole dead birds were composted with manure and straw as bulking agents. With poultry, a C:N ratio of 23:1 and a moisture content of around 55% is recommended (Donald and Blake, 1990). Recently, a series of trials involving composting of poultry, were conducted in the laboratory and on-farm by researchers at Michigan State University (Flegal et al., 1993). Results showed: (1) that the decomposition of ground carcasses was faster than using whole birds probably due to the fact that a ground carcass provided composting bacteria quick access to the animal protein tissue, and (2) that the Hemolytic Enteritis virus was destroyed in a three week composting period. The Michigan State University trials confirmed the efficacy and safety of composting as an alternative dead bird disposal method. There are very few reports evaluating the feasibility composting of dead pigs and after-birth. Morris et al. (1994) has tested two different management schedules and two 5 different bulking agents. One schedule consisted of a 2 month loading, 2 month primary composting, and 2 month secondary composting period. The second schedule consisted of a 3 month loading and primary composting period and 3 month secondary composting. Piles contained either straw or sawdust. In each pile, six swine carcasses, each weighing approximately 100 kg, plus one sow carcass weighing approximately 200 kg were composted. Composting continued year round. The authors reported that if the composting is underway in the winter months, the results are satisfactory, but composting piles should not be started in severe cold weather. Piles containing straw showed less carcass degradation than piles containing sawdust. Results of a few small, field trials including composting of dead swine have been reported in popular press. In one such a trial, carried out by the University of Missouri and also reported in an extension publication (Fulhage, 1994), composting proved effective in disposing of animal remains. The composting bins consisted of bales of hay placed end to end to form walls for three-sided enclosures. The animal carcasses were added and covered with sawdust (bulking agent). Before turning into the secondary composting bin, the primary bins were allowed to compost for _3 months after the last carcass was added. Secondary composting was also allowed for three months or longer. WW One concern regarding composting as a disposal method is possible contamination of the environment. One possible cause of contamination would be runoff of leachate from the compost pile. Morris et a1. (1994), studied this possibility. Leachate from different compost piles which were uncovered, was collected and analyzed. The results showed an average N content of 55 ppm and an average P content of 111 ppm. In order to make composting a safe method, the leachate problem should be addressed. A simple solution would be to build composting facilities with a roof and concrete base to avoid excessive water runoff due to rainfall. WW As discussed earlier temperatures may reach (60 — 70°C) in compost piles, due to bacterial activity and growth. The destruction of pathogen microorganisms in infected animal tissue is possible. To date, few reports on microorganism survival on composting piles are available. As mentioned before, Flegal et al. (1993) reported the destruction on Hemolytic Enteritis virus in poultry composting piles. In an unpublished manuscript, Morrow et a1. (1995) reported the partial destruction on Salmonella and the total destruction of Erypsipelthrix rhusiopathiae and Pseudorabies herpesvirus (PRV) in swine composting piles. In this experiment, Salmonella and E.rushiopathiae in the composting 7 piles were isolated in culture tubes. Salmonella tubes were retrieved from the composting piles on day 127, when the piles were turned, and again on day 177. E.rushiopathiae tubes were retrieved from another pile on day 245 (first turning) and on day 351. For the PRV test, tonsils from PRV infected pigs were placed into sterile widemouth glass bottles along with aseptically collected muscle tissue to mimic possible thermal insulating properties of the skull, and four PRV infected pigs were wrapped in plastic biohazard bags. Two bottles and two bags were retrieved from the compost pile on day 29 (first pile turning) and on day 53. After extraction from the compost pile, tonsilar tissue samples were obtained from the carcasses placed into the biohazard bags. All tonsilar tissue samples, from the glass bottle and from the bags, were used to inoculate PRV free pigs. The pigs were observed for 28 days, and serum samples were collected on day 28 for serologic evaluation. The results from this trial showed that all samples for PRV and Erypselas were negative. Indicating that temperature achieved in the compost piles were adequate to destroy the virus and bacteria. Nine of 15 Salmonella culture tubes retrieved at day 127 were negative. On day 177, ll of 14 tubes were negative, indicating that the temperatures achieved were adequate to kill some, but not all, of the Salmonella. Temperatures in all piles exceeded 60°C. While this experiment gives an indication of the ability of composting to destroy pathogen organisms, the 8 conditions of the trial do not reflect an "on-farm" situation. In that experiment, the pathogen microorganisms were not directly in contact with the compost material, therefore the conditions inside the culture tubes were different than those of the compost pile. W In order to further test if composting pile conditions are capable of destroying common swine pathogens experimental methods and conditions need to be similar to those found in a commercial swine production setting. Due to the high biological risk involved in the survivability experiments, the experiments have to be conducted with caution and strict control. Other questions that need to be addressed are use of different bulking agents and the addition of afterbirth to compost piles. Objectives The objectives of this research were: (1) to monitor the effectiveness of composting on a commercial Michigan swine operation under Michigan weather conditions, and (2) to determine the safety of composting dead pigs as a method of disposal by monitoring viral and bacterial survival over time, in a controlled experimental setting resembling the 9 management and conditions likely to be used in composting swine mortality on farm. In order to achieve these objectives, the project was divided into two parts: 1. On-farm trial with cost analysis comparing burial, incineration rendering and composting. 2. On-campus experiments to determine safety of composting regarding pathogens microorganisms survivals over time in composting piles. Microorganisms to include, Actinobacillus pleuropneumoniae (App), Pseudorabies herpesvirus (PRV) and Salmonella cholerasuis (Sc). App was selected because of the high incidence of pneumonia in swine production. PRV was selected because of the importance to eradicate it as stated by the national eradication program in progress. Sc was selected because of viability in extreme environments and because it is a meat safety concern to meat quality safety. On-farm.composting of dead swine Abstract Composting was used to dispose of swine mortality and afterbirth in a commercial 250 sow, farrow-to-finish operation to demonstrate the efficacy of composting in a standard swine production with Michigan weather conditions. A cost analysis was included to show the economic advantage of composting when compared with burial, rendering and incineration. Four 2.25 x 4.5 m bins were constructed to serve as primary and secondary composting bins. In each primary bin, straw was added as the first layer, directly on top of concrete. The straw layer provided space for oxygen and helped absorb leakage. Layers of bulking agent, animal tissue, bulking agent, animal tissue, and so on, were added respectively until a height of 1.5 m was obtained. Fresh bulking agent was always added as the final layer in order to minimize odor and control flies. Animal tissue was placed no closer than 15 cm from the sides of the compost bin. Spelt hulls were used as bulking agent. Temperature was monitored with a 90 cm probe-type thermometer. Turning from primary to secondary bins was planned after 1.5 to 2 months. Temperature change over time in the pile, space availability and the appearance of animal tissue were considered as factors when deciding turning dates. A close observation of 10 11 the first piles was made to establish a practical effective turning schedule. Microbial analysis of final compost material was performed to identify populations surviving during the composting procedure. Nutrient analysis (N, P, K) of final compost material was also performed to establish the value of compost as a soil amendment for crop production. 1 Over a one year period, a total of 6000 kgs of porcine tissue were composted. Seven primary compost batches were produced, averaging 857 kg of animal tissue per batch. After recording the turning time (3 months) of initial primary composting piles and observing the degree of degradation of the animal tissue, a turning pattern was established. The average turning point of the primary batches was 1.8 months. Piles were turned 2 or 3 wk after the final addition to a given primary pile. All compost piles reached a temperature of at least 65°C . An all-year peak temperature (70°C) was recorded during winter time and lasted several weeks. The results from the trial clearly indicated the feasibility of using composting as a method of dead swine disposal in Michigan. Winter weather was not an obstacle for a correct composting process of the piles. Management of the piles did not require any special skills or excessive amount of time. The cost analysis showed composting to be close in cost to burial and approximately half the cost of that of rendering and incineration. 12 Introduction Many swine producers face the problem of dead pig disposal with few options. Rendering, the best option for large producers, recovers several animal by-product commodities and controls transmissible diseases. But rendering is usually not available to small and mid-size producers because they do not produce enough dead animals to justify service by a rendering plant. Burial and incineration are the most common options, but both methods have environmental drawbacks such smoke pollution with incineration, and ground water contamination with burial. Composting is currently being used in the US by a few producers (Fulhage, 1994). However, its feasibility under colder weather conditions remains to be proven. The objective of the trial was to demonstrate the feasibility of composting in a commercial swine production unit under Michigan weather conditions. Materials and Methods Based on guidelines approved for composting of poultry (Flegal et al.,1993), composting facilities were built on a standard swine production operation near Mason, Michigan. 13 Facilities included primary and secondary bins, covered with a roof (to prevent seepage), and constructed with sufficient sidewalls (treated lumber) to minimize infiltrating precipitation, and the windblow movement of compost. One side of the facility had a reinforced vertical wall to confine material during mechanical turning and handling. Floors were reinforced concrete. Four 2.25 x 4.5 m bins were constructed in an existing machinery shed. Approximately .12 UP of bin space per sow was calculated as necessary, based on estimations by Fulhage (1992). Thus, the facilities were oversized according to the initial estimates (2.25 m x 4.5 m x 4 bins : 250 sows = .16 US per sow). The extra space was included to compensate for the slope of the pile which will prevent use of total bin space. 9.30 m A V ‘ 2.25 m , 4.5 m a: 7.5 cm separation between walls b: 5 cm on bottom of wall for cleaning perforated walls for air flow c: reinforced endwall Figure l. Compost facilities floor plan 14 The layout of the pile is shown in Figure 2. The first layer, directly on top of concrete was straw, to provide air (02) and to absorb leakage. The following layers were bulking agent, animal tissue, bulking agent, animal tissue, and so on, until a height of 1.5 m was obtained. The final layer was fresh bulking agent to minimize odor and flies. Animal tissue was placed no closer than 15 cm from the sides of the compost bin. Spelt hulls from a nearby milling company were used as bulking agent (spelt is a German wheat variety). Sawdua Dead pigs Sawdust Dead pigs Sawdust Straw Concrete Figure 2. Layout of compost pile 15 Temperature was monitored with a 90 cm probe-type thermometer. Measurements were taken twice a week from the front and the back of the pile. Turning from primary to secondary bins was planned after 1.5 to 2 months, time sufficient for initial compost activity (Fulhage,1992). Temperature change over time in the pile and space availability were considered deciding factors for turning dates. Because of little previous experience, a close observation of the appearance of animal tissue in the first piles was made to establish a practical effective turning schedule. Microbial analysis (standard bacteriology assay) of final compost material was performed to identify microbial populations surviving during the composting procedure. Nutrient analysis (N, P, K) of final compost material was also performed to establish the value of compost as soil amendment for crop production. A cost comparison between composting, burial, incineration and rendering was made considering labor and cost of facilities. 16 Results and Discussion Based on recommendations of Fulhage (1994), the first primary pile was turned after 3 months of composting (time after the first addition of animal tissue). At that time, it was observed that the degradation of carcasses was almost complete. Only small pieces of bone remained visually recognizable. For this reason, a turning time was established of 2 mo after the initiation of the pile, or 3 wk after the final addition to the pile. Another 2 mo of secondary composting (after turning) followed, for a finish degradation of animal tissue Over a one year period, a total of approximately 6000 kg of animal tissue was composted, averaging 175 kg of animal tissue per wk. Seven primary compost batches were produced, averaging 857 kg of animal tissue per batch. The average turning point of the Primary batches was 7 weeks.. The composting facilities proved to be larger than necessary for the dead pig production of the test farm. Only three bins were consistently used as composting bins. One bin was used for the primary pile. The other two bins were used as secondary compost areas. The last of the original four bins was used for storage of fresh bulking agent. After operating a year, an average of .12 HQ of composting surface area was established as necessary per sow in the farrow-to-finish herd. 17 Once the initial two batches were finished as primary piles and secondary, the composted material was used as bulking agent in establishing new piles on a 50:50 % basis with fresh bulking agent. Usage of compost material as bulking agent is recommended to inoculate the pile with the desired composting bacteria. This practice is only possible when the compost material being used is not completely composted, that is, there is still some bacterial activity, but the material does not contain any remains of visually recognizable animal tissue. In this on-farm demonstration, after 4 mo of composting (2 mo primary, 2 mo secondary), the resulting product still contained active bacteria, as indicated by the temperature of the pile being higher than ambient temperature. The use of final compost as bulking agent for subsequent primary piles also minimized the need for fresh bulking agent. The top layer though was always fresh bulking agent in order to avoid odor and flies. During the duration of the demonstration, no strong odor or flies were observed. Temperature and animal tissue additions of a standard pile (batch #3) are described on Figure 3. Temperature from the front and back of the pile are shown. Temperature increased rapidly and reached a peak near d 30. The back area of the pile showed a constant increase in temperature, with little oscillation. This area of the composting pile remained undisturbed after the initial additions, thus the bacterial growth was not affected by constant tissue additions. The front of the 18 pile, while increasing in temperature overall, showed different peaks and drops in temperature. This was due to the constant additions of new animal tissue until the bin was full. Bacterial growth was affected by these additions, with the composting process continuously being reinitiated. This fact would explain the peaks and drops in temperature in this area of the pile. T rature 0p 70 FL 65 Water added (lite 22 60 ....... E--- W...” .. Ti). 95 .. Days - - Front — Back ] Figure 3. Compost pile temperature and tissue additions The highest temperature in all batches was obtained during the months of January and February (batch #6), with a peak of 69.4°C. Several factors could have been involved. The differences in temperature between composting pile and environment could create a convection effect which would result in drafts, thus increasing oxygen available for bacterial growth. Increase moisture content due to partial 19 snow coverage of the pile and use of final compost as bulking agent could have been another reason for reaching a peak temperature during the winter time. The high temperature in winter was contrary to our expectations. Moisture appeared to be limiting in the pile during the summer, especially with the initial primary pile in which only fresh bulking agent was used. Some water was added to elevate moisture in the compost(Figure 3). The low moisture content of piles was less of a problem when previously composted material was used as bulking agent. Microbial analysis of final compost material is presented on Table 1. All species found in the compost sample are non-pathogen. Nutrient content of final compost material is described on Table 2. Table 1. Dead swine composting microbial analysis Staphylococcus > 100 cfu Coliforms > 100 cfu Proteus < 50 cfu Bacillus < 50 cfu Corinebacterium > 100 cfu Mycoa < 5 cfu Streptomices < 50 cfu 20 Table 2. Dead swine composting nutrient analysis Sample Compost Spelts hulls 121%” NE; 236. Kit 89 2.43 .47 .51 88 .78 .11 .28 21 Cost of Composting A cost comparison between composting, burial, incineration and rendering is shown in Table 3. Cost in all four cases is based on a 250 sows farrow-to-finish operation with an estimated dead pig production of 12200 kg of animal tissue per year including afterbirth, preweaning mortality, postweaning mortality and sow/boar losses. The final mass of animal tissue was based on mortality estimates of Smith and Aitken (1980) estimation on dead pig production. For cost comparison purposes, 20 pigs weaned per sow per year was assumed. Another assumption made in determining the cost of composting was that the final compost material consisting of animal tissue and bulking agent would be 1/3 the initial animal tissue weight. This assumption was based on our experience in the on-farm experiment. This fact was considered when calculating the return for land application of compost as fertilizer. A $3.75 per tm value was estimated for composting material when used as fertilizer based on N, P and K content. Cost of burial included all mortality produced. Cost of incineration and rendering did not include preweaning mortality and afterbirth. A common practice is to use burial for preweaning mortality and a manure disposal system for disposal of afterbirth when using incineration or rendering as the methods of dead swine disposal in the swine 22 production unit. The incinerator considered in this estimate was a Burn-EasyTM model 36. It measures 180 x 75 cm and holds up to 180 kg. It can burn up to 2.4 kg of animal tissue per liter of fuel (for calculation purposes the price of fuel was estimated at $0.26 per liter). The manufacturer claims that meets or exceeds EPA guidelines for pollution and does not produce unpleasant odors or smoke during burning process. Rendering companies considered for this estimate are Darling International Inc.- Saginaw (company A), and Darling International Inc.- Coldwater (company B). Both companies serve the Mid-Michigan area. Prices for each company were based on present collection practices in the Mid-Michigan area. Company A indicated that their price was based on a per-animal basis. Company B charges on a per-trip bases with an average load of 12 animals per trip. Refrigerating facilities are needed to keep dead animals until time of delivery in case of rendering. Cost of such facilities was not included in this comparison. The cost should be included in the setup cost. It is clear from the table that burial and composting are the cheapest alternatives, with composting being approximately 50% of the cost of incineration and rendering. In addition to cost, composting and burial where methods in which disposal of all mortality was included. 23 Table 3. Dead swine composting cost comparison SEMMEQSJDJKii Setup cost: facilities (.12 m2/sow; $110/m2) = $3300 Operating cost: facility depreciation (10 years) ’ = $330/year tractor/loader/spreader (1 hr / month) - $20/hr x 12 = $240/year Return for land application of compost - 4078 kg(1/3 initial wt)x $3.75/tm = +$7.65/year Total = $562.35/year anzuuui: Operating cost: tractor, back-hoe (1.75 hr / month) x $20/hr x 12 = $420/year JJKZUNEBAJflEnli Setup cost: incinerator = $1256 Operating cost: incinerator depreciation (8 years) = $157.00/year fuel- 7740 kg x 1 l/2.4 kg x 26 ct/l $838.50/year Total $995.50/year JBENDEEUDflii 7740 kg / 114 kg/animal = 68 animals (approx.) Company A: 68 x $15/animal = $1020/year Company B: 68 / 12 animal/trip x $160/trip (Lansing) = $800/year 24 Implications The results from this trial clearly indicate the feasibility of using composting as a method of dead swine disposal in Michigan. All mortality on the farm can be successfully composted. Winter weather, in northern climates should not hinder the composting process. Management of the piles will not require any special skills or excessive amount of time, with little problems with odor, flies or predators disturbing. Composting will be economical. Survival of Actinobacillus Pleuropneumoniae and Pseudorabies herpesvirus in swine mortality during composting Abstract Survival of PRV and App in compost piles containing infected swine mortality was determined in order to evaluate the safety of composting as a method of carcass disposal. Twenty one pigs from 7 to 15 kg were infected intranasaly with 106 TCIB of PRV and intratrachealy with 108 cfu of App. All pigs were euthanized approximately 16 h after infection and whole carcasses were then composted in two piles which contained 92 kg and 63 kg of animal tissue, respectively. Each compost pile consisted of a single layer (15 cm) of straw on the bottom, covered by alternating layers (15 to 20 cm) of uncontaminated sawdust and animal tissue. Pile depth was 1 to 1.25 m. Piles temperatures were monitored with a 90 cm probe-type thermometer and ranged from 27°C to 51°C. Piles were turned once, 21 d after composting was initiated. Composting was terminated after 35 d. All resulting compost material was then used as feed and/or bedding in a live-pig assay. Eleven pigs were segregated at weaning (8 to 9 d of age) to prevent infection with App and PRV. Laboratory assay of serum samples taken at about 6 wk of age confirmed that these pigs had not been infected with either disease organism. Compost material was then fed (15% of ration) and 25 26 used as bedding for these animals. During the feeding and bedding period, pigs were monitored for symptoms of disease. No signs of disease were observed. After a 7 d feeding and bedding period, tissue (lung, brain and tonsil) samples were obtained and analyzed for the presence of App and PRV. Assay results for both microorganisms were negative suggesting that both organisms did not survive in infected swine mortality composted for a period of 5 wk. Results of this study further establish the safety and effectiveness of composting swine mortality as a mean of dead animal disposal. Introduction Composting has the potential to be an economical and effective method of dead swine disposal. Little research has been conducted examining the survival of virus and bacteria in compost piles. The safety of composting is not known. The survival of PRV outside the living host is dependent on pH and temperature. Davies and Beran (1981) have shown that PRV is inactivated at 0.6 log10 per day at 37‘C at a pH 6-8. Morgan et a1. (1995) reported the destruction of PRV in compost piles after 29 d. In this experiment tonsil tissue samples from infected swine were placed inside composting piles together with muscle tissue and inside glass bottles, resembling thermal conditions in the skull, and as part of 27 whole carcass infected pigs inside plastic bags. After composting for 29 d, tonsil samples from tubes were used to inoculate PRV free pigs (sentinell pigs). None of the sentinell pigs develop any symptoms of disease or tested serum positive for PRV after 28 days. This experiment did not expose animal tissue to the conditions in composting piles, where infected dead animals would be totally exposed to composting bacteria and other environmental elements. In order to determine the biological safety of composting, more experiments need to be conducted using infected carcasses in piles. App is a common pathogen in swine operations, and pneumonia has a high incidence in swine production. PRV is currently the target for a national erradication program because of its economic importance in the swine industry. Survival of PRV and App in compost piles containing infected swine mortality was determined in order to evaluate the safety of composting as a method of carcass disposal. Materials and Methods Two experiments (Experiment 1 and 2) were dessigned to test for the survival of App and PRV in composted swine carcasses. In Experiment 1, the survival of pseudorabies virus (PRV) and Actinobacillus pleuropneumoniae (App) in 28 composted swine mortality was evaluated by laboratory assay. The validity of results obtained in experiment 1 were questioned. Experiment 2 was planned, to determine the survival of PRV and App in composted infected swine mortality by exposing non-infected live pigs to material obtained from experiment 1 containing mortality infected with these organisms. Validity of experiment 1 results was in question because bacteria important for decomposition may interfere (false negative) with laboratory assays for PRV and App. ExperimenLl Animal Tissue: Twenty-one pigs, approximately 10 to 15 kg BW, were infected intranasaly with 106 TCIB of PRV. Two days later, the pigs were infected intratracheally with 5 x 107 cfu of App. Pigs were anesthetized before intra-tracheal inoculation using Ketamine C>(4.5 mg/kg BW) and Rompun @> (165 mg/kg BW). All pigs were euthanized 15 to 16 hours post App infection with invtravenous injection of Fatal Plus G) (.22 cc/kg BW). Lung and brain samples from three carcasses (control) were cultured after euthanasia (d0) to determine presence of PRV and App. The remaining 18 pigs were used as animal tissue to test for virus and bacterial survival in the composting piles. Pile Construction: Two piles were constructed. Pile One consisted of 3 layers of animal tissue (At), 1 layer of 29 straw (St), and 4 layers of uncontaminated saw dust (Sd). Each layer was 15 cm deep. The layout of the pile was, St- Sd-At-Sd-At-Sd-At-Sd. Pile Two had the same strata as Pile One but it contained only two layers of animal tissue. Each layer of animal tissue in both piles contained 3 whole pigs. Total animal tissue weight was 92 kg for Pile One and 63 kg for Pile Two. Pile Temperature and Turning: The internal temperature of the compost piles was recorded with a 90 cm probe-type thermometer. The thermometer measurements were taken once a day by inserting it in areas of the pile originally containing the carcasses. Piles after pile temperature had peaked (d 21). Microorganism survival: Animal tissue for microorganism survival test was collected on days 7, 14 and 35. Each collection time, three whole pigs, or their remainings were extracted from each pile and tissue samples were collected. When animals and tissues were no longer recognizable, samples of compost material closest to the original location of the pig carcasses were collected. Tests to determine presence of PRV and App were carried out as follows: PRV presence was tested in brain tissue and compost samples on d 0, 7 and 14, using standard virus isolation procedures (Iowa State University Veterinary Diagnostic Laboratory, Ames, Iowa, Appendix A). App isolation in lung tissue (d0) 30 followed a standard culture procedure of the Michigan State University Animal Health Diagonostic Laboratory. App testing in compost samples on d 7, 14 and 35 was performed at the Iowa State University Veterinary Diagnostic Laboratory, Ames, Iowa (Appendix B). ExperimenLZ Animals: Twelve pigs were weaned early at 7-10 d of age and taken from MSU Swine Farm to isolation in the MSU Veterinary Research Center. The segregated early weaning (SEW) method was used to avoid dam-to-offspring infection with PRV and App. Blood samples were obtained at about 5 wk of age by vena puncture to ensure that all pigs were free of PRV and App. Assays for App and PRV are described in Microorganisms Survival. Housing: Pigs were housed in isolation (room 9, Barn G, Veterinary Research Center). Temperature was kept at 30- 320C from wk 1 to week 2, 24 to 260C from wk 3 to 6, gradually decreasing to 18 to 21°C for wk 7 through 9 (experimental period). Treatments: During the first 7 wk of age, pigs were provided ad libitum free access to a commercially available SEW dietary program and water. At 7 wk of age, the diet was changed to the experimental diet, consisting of a ground diet mixed with compost material obtained from compost piles 31 used in experiment 1. Animals were reared to 7 wk of age to obtain 1.5 to 2 kg feed intake of the experimental diet consisting of approximately 15% compost material. .Microorganisms survival: ELISA IDEXX and Latex aglutination tests were used for blood tests of PRV (Michigan Department of Agriculture Diagonostic Lab, East Lansing MI, protocols in Appendix C and Appendix D) and Indirect ELISA was performed for blood tests of App (Oxford Labs©,Worthington, MN, sesitivity 100%, specificity 100%) . During the feeding period, pigs were monitored for symptoms of PRV and App diseases such coughing, sneezing, depressive behavior and heavy breathing. At the end of the feeding period, pigs were euthanized via IV infection of sodium pentobarbital. Brain and lung tissues were collected for assay of PRV and App. PRV assays included immunofluoresence test (Appendix E) and standard virus isolation (MSU Animal Health Diagnostic Lab, E.Lansing, MI). App isolation in lung tissue followed a standard culture procedure of the Michigan State University Animal Health Diagonostic Laboratory. 32 Results and Discussion Experimeml Brain and lung samples from all three control carcasses, tested positive for PRV and App on d 0. Bacteriology culture from all three pigs contained more than 1000 cfu of App. PRV was also isolated from the brain samples obtained from all control pigs. Positive results for both App and PRV proved that the infection procedures for both microorganisms were successfull. Temperature in compost piles ranged from 26.7 °C (room temperature) to 51.7 °C (Figure 4). The highest temperature was reached at about d 7 of composting and remained about 50 °C until about d 14. After d 14, temperature in both piles started to drop, more rapidly in Pile One than Pile Two. At d 21, piles were turned to aerate the compost material. About 2 d after turning, the temperatures peaked again at 50 °C , likely a growth response by the microbial to added Oz. When the trial was terminated at d 35, both piles had higher internal temperature than room temperature, suggesting that biological activity of the composting process was not completely finished. 33 Temperature 0c: 60 55 50 7¥flFS§§::A‘ g 45 '1 \\ l V \I ”m\l l] \ 7<\ 35 % V \‘ 25 207 'l'l'l'l‘l'l’l'T—fT'l'l'l'T’ T'l‘l'l O 2 4 6 81012141618202224262830323436 turning [— Pile One Pile Twoj Figure 4. Compost pile temperature, PRV and App trial. By day 7, or the first day of tissue collection, carcasses were physically unrecognizable. A small amount of brain tissue in a liquid form was obtained from the skull cavity of one pig in Pile One. All other samples were mixtures of animal tissue and sawdust. Pigs appeared to be degraded except for pieces of bone and hide. Collections on d 14 and d 35 consisted of mixed bulking agent and animal tissue. By d 14, dead pigs were totally unrecognizable, except for 15 cm wide pieces of hide. Piles were dark brown in color with small bone pieces vissually detectable. Some of the skulls were still recognizable on d 14. By d 35, no major bones were recognizable. 34 Microorganisms Survival: Live virus were not isolated in compost samples taken on d 7 and d 14. Therefore d 35 samples were not analyzed. There were no App colonies recovered from the compost samples on d 7, d 14 or d 35. W Results of blood analysis at 5 wk of age were negative for all pigs, proving that the early-weaning procedure was succesfull in avoiding contamination by PRV and App. Pigs did not develope any symptoms related to a possible outbreak of App or PRV. All analyses of tissues at the end of the experiment tested negative for both microorganism in all samples. Implications Results from both experiments suggest that conditions in the compost piles were sufficient to eliminate PRV and App from contaminated dead pigs. Doubts of false negatives in experiment 1 on d 35 were ruled out with results from experiment 2. Survival of Salmonella cholerasuis in swine mortality during composting Abstract Survival of Salmonella cholerasuis in compost piles containing infected swine mortality was monitored in order to determine the safety of composting as a method of carcass disposal. Sixteen pigs, 10-15 kg BW were challenged intranasaly with 2 x 1010 cfu of Salmonella cholerasuis (Sc). All pigs were euthanized after infection and whole carcasses were then composted in two piles containing six pigs each. Each compost pile consisted of a single layer (15 cm) of straw on the bottom, covered by layers (15 to 20 cm) of spelt hulls, animal tissue and spelt hulls, alternatively. Pile temperatures were monitored with a 90 cm probe-type thermometer and ranged from 27°C to 62°C. Animal tissue was collected on d 0, l, 3, 7 and 10. Each collection was involved three carcasses. Tissue collected for analysis include samples of tonsil, mandibular lymph nodes, lung, bronchial lymph nodes, liver, spleen, cecum, cecal contents, ileocolic junction, ileocolic lymph nodes, middle ileum, colon and colonic lymph nodes. By d 7 tissue differentiation was impossible because of carcass decomposition. Sc was recovered in samples from d 0, l and 3. Samples from d 7 and 10 did not contain Sc. These results prove that conditions of the compost piles were effective in destroying Sc by d 7. 35 36 Introduction Salmonellosis is a common cause of septicemia and diarrhea in weaned pigs. Salmonella multiply at 7-45°C, survive freezing and remains viable in certain organic substrates for weeks. Salmonellae have been reported to last in manure oxidation ditches for 47 days (Will et al. 1973). The microorganism is not capable of forming spores, and it is inactivated by sunlight and heat. Salmonella cholerasuis is usually considered the most frequent serotype causing disease in swine (Wilcock et al. 1976). Because of its high resistance to environmental conditions, Sc was considered a good example of an ubiquitous swine pathogen to be tested for survival in composting piles. Materials and Methods Animal Tissue: Sixteen pigs, approximately 10-15 kg BW were infected with Sc (2x101° cfu; source described in Appendix F). All pigs were euthanized 2 d after infection (Pentobarbital, lcc / 4.5 kg BW, IV). Pigs were infected and euthanized in room 9, G Barn, Veterinary Research Center to allow for the necessary isolation of infected animals. Following euthanasia, three pigs were necropsied and tissue samples analyzed to determine if pigs were indeed infected. The remaining 13 pigs were used as animal tissue in compost 37 piles to test for bacterial survival. The composting piles were constructed at the MSU Swine Barn composting unit (Endocrine site). Pile construction: Two piles were constructed, consisting of one layer of straw at bottom, one layer of spelt hulls, one layer of animal tissue, and one layer of spelt hulls on top. The animal tissue layer contained six pigs in each pile. After constructing the compost piles, water was added to increase the moisture content of the pile. Each pile had two sampling areas containing three pigs each. Tissue collection and measurements: Animal tissue was collected on d 0, 1, 3, 7 and 10. Each collection was taken from three carcasses contained in one sampling area. The remaining sampling areas remained undisturbed until time of collection. Tissues collected for analysis include samples of tonsil, mandibular lymph nodes, lung, bronchial lymph nodes, liver, spleen, cecum, cecal contents, ileocolic junction, ileocolic lymph nodes, middle ileum, colon and colonic lymph nodes. Tissue samples from each collection were sent for bacteriological analysis the same day to National Animal Disease Center, Ames IA (protocol included in Appendix F). Temperature was monitored two times a day in all sampling areas, during the whole experimental period. Analysis S.cholerasuis infection was were added to animal tissue, 38 Results and Discussion of tissue samples on d 0 yielded an average concentration of'lffi cfu, proving that accomplished. Dead pigs from d O collection for extra the piles (one pig in each pile) and as a way of disposal. These carcasses were not sampled again. Temperature °C 70 50 fissue 40 ; smumMngég \. 30,- .............................. .M— “H”... .. .. 20 a"" “ 4’ ' 0 l l i l l l l l f l 1 2 3 4 5 6 7 8 9 10 Days -- Ambient — Site1 -- Site 2 - - Site 3 - Site 4 Figure 5. Temperature Necropsy on d recognizable. "cooked meat". unrecognizable. Compost pile temperature, Salmonella trial. ranged from 27°C to 62°C (Figure 5). 1 and 3 was performed with all tissues being On d 3 the apperance of the carcass was of On d 7 and 10, pigs carcasses were Only small bone pieces with some meat 39 attached could be obtained. Any attempt to recognize any of the intended tissues for analysis was futile. The compost material looked like spelt with a darker color. Qualitative recovery of Sc from each necropsy is shown in Table 4. Sc was recovered from pigs on d 1 and 3, not on d 7 and 10 . The mean number of Sc in the ileocolon junction and ileocolon lymph nodes was 3.6 x 105 cfu/g and 1.0 x 103 cfu/g of tissue, respectively. Table 4. Qualitative recovery of S.cholerasuis Tissue 3pca lacb 3ac 7acc 10acd (na4) (n-3) (n-3) (n-3) (ns3) Tonsil 3 3 3 0 0 Mandib-LN 3 3 3 0 0 Lung 3 3 3 0 O Bronch-LN 3 3 3 0 0 Liver 4 3 3 O 0 Spleen 2 3 3 0 0 Cecum 3 3 3 0 O Cecal Cont. 4 2 3 0 0 Ileocolic Jc. 4 2 3 0 0 Ileocolic LN 4 3 3 0 O Mid-Ileum 4 3 3 0 0 Colon 4 3 3 0 0 Colonic-LN 3 3 3 0 0 a 3 :Eys post-challenge, b ac = after composting, C 13 tissue parts from 2 pigs and 11 tissue parts from 1 pig were cultured. Parts were not recognized as individual tissues d 14 samples from 1 pig taken. Parts were not recognized as individual tissues 4O Implications Ccomposting is capable of destroying the environment- resistant microorganism Sc in composted swine mortality. The elimination of Sc in infected carcasses in compost piles confirms the safety of composting as a method of dead swine disposal. Conclusions This master's research was designed in order to provide scientific evidence for the feasibility of composting as a dead swine disposal method. Before the start of the project, it was already clear that "composting works". Small research experiments and on-farm projects had been made, but none had provided scientific data. The intention of this thesis was then to develop a series of trials that would test, in a scientific manner, the feasibility of composting The thesis was divided into two main categories: (1) on-farm experiment and (2) biological safety experiments. During the completion of this thesis, it was learned that other researchers were evaluating the feasibility of composting (Morris et al. 1994, Morgan et a1. 1995). Although some similarities exist among these studies and the preSent study, there were significant differences as well. Most notable were the use of facilities with both concrete floor and full roof, and the use of whole carcasses known to have been infected by selected pathogens. On—farm composting: The objective of the on—farm experiment was to demonstrate the practicability of composting in a standard swine production unit under Michigan weather conditions. One of the questions before the experiments was whether composting would be possible under colder winter temperatures. After a full year of composting, it was clear 41 42 that pile temperatures, management and labor requirements of the piles in winter, were not concerns. It was during winter that composting pile temperatures achieve an all-year high which lasted several weeks. A cost-comparison between composting and the other methods of dead swine disposal (burial, rendering and incineration) was also included in the thesis. The analysis was based on a 250 sows farrow-to-finish operation, since it is for this size of operation (small to medium size) that composting is an attractive disposal alternative. The cost-comparison demonstrated the economic feasibility of composting. The calculated cost was close to that of burial and approximately half of incineration and rendering. The comparison was made based on an operation in the Mid-Michigan area (Lansing). It is difficult to generalize in terms of labor, construction material cost, rendering charges and other factors included in the economic analysis, but the wide margin of error (composting was calculated to be 1/2 cheaper than other methods), leaves no doubts about composting economic advantages. Biological-safety experiments: Two different experiments were designed to test the feasibility of composting from a biological safety aspect. The main objective of the trials were to demonstrate if composting pile conditions were sufficient to destroy common swine pathogens. 43 .A.pleuropneumoniae (App), Pseudorabies herpesvirus (PRV) and S.cholerasuis (Sc) were chosen as the pathogens to be tested because of their importance in the swine industry as health- risks and their resistance to adverse environment conditions. The first experiment included App and PRV as swine pathogens. The second experiment included Sc. Both trials had basically the same design. A group of pigs was infected with the pathogens. After euthanasia, the carcasses were composted. Samples of the carcasses and compost material, when the tissues were no longer recognizable, were taken overtime to test for survival of the microorganisms. Both experiments demonstrated the total absence of pathogen microorganisms by d 7 of composting. Results from the biological-safety experiments proved the safety of composting as a method of disposal. It is clear that other microorganisms could be tested, but it is the authors' opinion, that the ones chosen represent three which are of serious concern. The trials were conducted resembling on-farm conditions, with composting of pigs infected with the pathogen microorganisms. This approach allows to conclude than composting would be capable of destroying infected swine mortality in an on-farm setting. Results from all three trials in this thesis indicate both, the feasibility of composting in a commercial swine operation, and the safety of composting as a method of dead 44 swine disposal. Hopefully this thesis can be used as a scientific reference when examining the possibilities of composting for the swine industry. Ideas for future research in this area should include use of different bulking agents, destruction of swine pathogens that are important in some geographical areas, possibility of disease transmission from compost material to crops when used as fertilizer, and alternative designs of composting facilities. Answers to these questions are important to progress in the advantages of composting as a method of dead swine disposal. LIST OF REFERENCES LIST OF REFERENCES Davies, E.B. and G.W. Beran. 1981. Influence of environmental factors upon the survival of Aujeszky's disease virus. Research in Veterinary Science 31:32-36. Donald, J.O. and J.P. Blake. 1990. Dead poultry composter construction. In: Proceedings of National Poultry Waste Management Symposium, Oct. 3-4, 1990, Raleigh, NC. Auburn University Printing Service, Auburn University, Auburn, Ala. pp. 38-45. Flegal, C.J., J. Gerrish, L.D. Schwartz, R. Maes and F. Endres. 1993. Animal Protein Composting -Final Report. Poultry Research Report. Michigan State University. Fulhage, C. 1994. Composting Dead Swine. University of Missouri, University Extension Publication, Sheet WQ 225 Water Quality Series. Gray, J. T., P.J. Fedorka-Cray, and T.J. Stabel. 1995. Influence of inoculation route on the carrier state of Salmonella cholerasuis in swine. Vet. Microbiol. In press. Henry, S.T. and R.K. White. 1990. Composting broiler litter - effects of two management system. In: Agricultural and Food Processing Waste. ASAE, St. Joseph, MI. pp. 1-9. Morrow, W.E., P. O'Quinn, J. Barker, G. Erickson, K. Post and M. McCaw. 1995. Composting as a suitable technique for managing swine mortalities. Submitted for publication. Morris, J., T. O'Connor and F. Kains. 1994. A Method for the Bio-Degradation of Dead Pigs. Conference proceedings of the 1994 Waste Management Conference. Chicago Ill. 373-382 45 46 Smith, P. and G. Aitken. 1980. Dealing with dead pigs. Pig Farming, vol. 28 No.9 Murphy, D.W. and T.S. Handwerker, 1988. Preliminary Investigations of Composting as a Method of Dead Bird Disposal. Proceedings of the National Poultry Waste Management Symposium, Columbus, Ohio, April 18-19, 1988. pp. 65-72. Vassiliadis, P. 1983. The Rappaport-Vassiliadis (RV) enrichment medium for the isolation of salmonellas: An Overview. J. Appl. Bacteriol. 48:167-174. Wilcock, B. P.; Armstrong, C. H.; and Olander, H. J. 1976. The significance of the serotype in the clinical and pathologic features of naturally occurring porcine salmonellosis. Can J. Comp Med 40:80-88. Will, L. A.; Diesch, S. L.; and Pomeroy, B. X. 1973. Survival of Salmonella typhimurium in animal manure disposed in a model oxidation ditch. Am J. Public Health 63:322-336. APPENDICES APPENDIX A APPENDIX A Pseudorabies virus isolation Protocol for analysis Iowa State University Veterinary Diagnostic Laboratory Ames, IA A representative sample was selected from tissue and compost samples submitted for analysis and placed in a StomacherC>tmg'for processing. To each was added approximately 20 ml of chilled EBSS (Earles balanced salt solution) containing amphotericin B (FungizoneCn Apothecon) and gentamicin sulfate (GentocinCL Shering-Plough). Each bag containing the compost sample was placed in a StomacherC>400 blender (TekmarCU and mixed for 20-25 seconds. The resulting homogenate had the consistency of a milkshake. This homogenate was transferred by pouring into a 50 ml centrifuge tube (SarstedtCU and processed using a refrigerated tabletop centrifuge at 2,500 rpm for 20 minutes. After centrifugation, the supernatant was decanted into a sterile snap-cap tube (Falcon) for use in the tissue culture challenge. The pellet that remained in the centrifuge was held in storage at -70°C until the isolation experiment was completed. 47 48 The MDBK (Madden-Darby bovine kidney) cell line was selected for use. Twenty-four well tissue culture cluster plates (Costar) were seeded and incubated at 37°C and 4% C02. After 24 hours, the confluent monolayer was challenged with a portion of the supernatant. Four wells of the cluster plate were dedicated for each sample; 0.2 m1 of the sample were used per well. The plates were placed in an incubator (37°C, 5% C02); after 1 hour, the fluid containing the sample inoculum was removed from the plates and replaced with MEM maintenance media (Minimal Essential Medium, Grand Island Biologics), supplemented with 2% fetal calf serum (Hyclone Laboratories). The plates were placed back into the incubator and were observed daily for viral CPE (cytopathic effects). At the end of seven days, the plates containing samples not exhibiting CPE characteristic of a herpesvirus were placed into a freezer. The plates were quickly thawed in a 37°C waterbath, and 0.2 ml from each negative well was transferred to a plate containing a fresh monolayer of MDBK cells. These plates were observed daily for an additional seven days, and closed as negative samples if no CPE was observed during the time period described. Samples exhibiting CPE characteristics for a herpesvirus were confirmed as positive by fluorescent antibody staining of the cell monolayer using a direct conjugate specific for PRV (NVSL). APPENDIX B APPENDIX B Actinobacillus pleuropneumoniae Protocol for analysis Iowa State University Veterinary Diagnostic Laboratory Ames, IA Compost samples were soaked in phosphate buffered saline (1:10 W/V) and then plated and streaked on the following media: three 5% blood agar plates (#1 Plate - aerobic incubation with S.epidermidis feeder colony, #2 Plate - 5% CO: incubation with S.epidermidis feeder colony, #3 Plate - anaerobic with no feeder colony), Tergitol-7, MacConkey, Hectoen-Enteric and brilliant green. After overnight incubation, plates were evaluated and colonies identified by conventional techniques. In addition, the samples were subjected to overnight incubation in a PPLO enrichment broth. Following the 18 hour incubation, all samples were plated according to the aforementioned protocol. 49 k. :1 files; N '1'.’ 13"". APPENDIX C APPENDIX C ELISA IDEXX test for Pseudorabies Virus Protocol for analysis Michigan Department of Agriculture Animal Industry Division Lansing, MI Reagents and Materials: - PRV coated plates - Anti-porcine: Horseradish Peroxidase (HRPO) Conjugate in Tris Buffer with protein stabilizers. Preserved with Gentamicin. - Strong Positive Control. Porcine Anti-PRV in Phosphate Buffer with protein stabilizers. Preserved with Sodium Azide. - Weak Positive Control. Porcine Anti-PRV in Phosphate Buffer with protein stabilizers. Preserved with Sodium Azide. - Negative Control. Porcine serum non-reactive to PRV in Phosphate Buffer with protein stabilizers. Preserved in Sodium Azide. 50 51 - Sample diluent. Phosphate Buffer with protein stabilizers. - ABTS Concentrate. Preserved with Gentamicin. — ABTS Diluent. Citrate-Phosphate Buffer containing Hydrogen Peroxide. Preserved with Gentamicin. - Wash solution. 10x concentrate. Phosphate buffer. Preserved with Gentamicin. - 0.12% Hydrofluoric Acid (HF) - Precision Pipettes: suitable for delivering 25, 50, 100ul and 475ul or multiple delivering pipette devices. - Disposable pipette tips. - Graduated cylinders - 96 well plate reader - Uncoated plate for blanking. Procedure: 1. Dilute test samples twenty fold. 2. Samples should be well mixed prior to dispensing into the PRV coated plates. 3. The ABTS concentrate must be added to the ABTS Diluent before use (0.1 ml ABTS concentrate per 10.9 ml of ABTS Diluent). The ABTS substrate Solution must be stored at room temperature and used within 60 minutes after preparation. The ABTS diluent should not be exposed to any metal during the preparation. 4. The wash concentrate should be brought to room temperature and mixed to assure dissolution of any 52 precipitated salts. The wash concentrate must be diluted 1 to 10 with distilled/deionized water before use. 5. Obtain antigen coated plate(s). 6. Dispense 100 pl of undiluted Negative Control into wells A1, A2 and A3. 7. Dispense 100 pl of undiluted Weak Positive Control into wells A4, A5 and A6. 8. Dispense 100 pl of undiluted Strong Positive Control into wells A7 and A8. 9. Dispense 100 pl of diluted sample into appropriate wells. 10. Incubate for 30 minutes at room temperature. 11. Aspirate liquid contents of all wells. 12. Wash each well with appropriate 300 pl of phosphate buffered wash solution four times. Aspirate liquid contents of all wells after each wash. Avoid plate drying between plate washing and prior to the addition of conjugate. Following the final wash, aspiration, gently but firmly tap residual wash fluid from each plate onto absorbent material. 13. Dispense 50 pl of anti-porcine: HRPO Substrate Solution into each well. 14. Incubate for 30 minutes at room temperature. 15. ABTS substrate solution should be prepared at this point. 16. Repeat steps 11 and 12. 17. Dispense 100 pl of ABTS substrate solution into each test plate well. In addition, dispense 100 pl of Substrate solution into 8 wells in an uncoated plate for blanking. 53 18. Incubate for 20 minutes at room temperature. 19. Dispense 50 pl of HF in each well of the test plate to stop the reaction and into the wells of the blanking plate containing substrate. 20. Blank reader by using the uncoated plate and measuring the absorvance values at 410 nm, A(410), of each of the eight wells. Blank the instrument on the well with the lowest absorbance reading. 21. Measure and record the A(410) for samples and controls. 22. Calculate results. Calculate the negative control mean by adding the absorbances reading of wells A1, A2 and A3 together and dividing this number by 3. Calculate the weak positive control mean by adding the absorbance value of wells A4, A5 and A6 together and dividing this number by 3. Calculate the strong positive control by adding the absorbance value of wells A7 and A8 and dividing this number by 2. Calculate the sample to weak positive ratio (S/P) by subtracting the absorbance value of the negative control mean form the sample absorbance value and dividing this number by the absorvance value of the weak control mean minus the absorbance value of the mean of the negative control. 54 Results: For the assay to be valid, the difference (P—N) between the weak positive control mean (WPCx), and the negative control mean (NCx) must be greater than or equal to 0.150. In addition, the NCx must be less than or equal to 0.100 and the ratio of the strong positive control mean (SPCx) to the WPCx must be greater or equal to 2.0. Interpretation: The presence or absence of antibody to PRV is determined by sample to weak positive (S/P) ratio for each sample. The weak positive control has been standardized and represents a significant level of antibody to PRV in swine serum. Serum samples with S/P ratios of less than 0.4 are classified as Negative for PRV antibodies. If the S/P ratio is greater than or equal to 0.4, the sample is classified Positive for PRV antibodies. When samples are classified as positive in a screening test, they must be retested using a verification method as either the "Pseudorabies Virus Verification Test Kit" or the "Pseudorabies Latex Agglutination Plate Test" which are both approved as verification tests. APPENDIX D APPENDIX D Pseudorabies Latex Agglutination test Protocol for analysis Michigan Department of Agriculture Animal Industry Division Lansing, MI Significance: This method provides laboratory confirmation of a swine disease that is state and federally regulated. Reagents and Materials: - 1 vial of Pseudorabies virus (PRV) latex reagent - 1 bottle containing 30 ml of serum dilution buffer - 1 vial containing 0.75 ml of undiluted PRV negative control serum - 1 vial containing 0.75 ml of undiluted PRV positive control serum 100 plastic Spreaders 1-21 gauge plastic tip needle glass slides pippettes or syringes capable of delivering 50 pl test tubes 55 56 - Rotator with a humidified cover, capable of rotating at 80 to 120 rpm. Procedure: 1. Label and identify one test tube for each serum sample to be assayed. This tube is for preparation of a 1:4 dilution of the serum sample or control. 2. With a micropipettor, add 150 pl of serum dilution buffer to each labeled test tube. 3. With a micropipettor, add 50 pl of each serum to a test tube containing 150 pl of sample dilution buffer. Mix by drawing up and down with the micropipettor six times. 4. With the same micropipettor, transfer 50 pl of the diluted specimen to a 1.4cm well on the glass slide. 5. When all of the specimens to be tested on one slide have been diluted and transferred to the slide, use a spreader to spread each diluted sample to cover the entire well surface. Use a clan spreader for each sample. 6. Mix the latex reagent by vortexing or shaking for a few seconds. Hold the latex reagent bottle in an inverted position and drop one drop of sensitized latex on each slide well containing a diluted serum sample. 7. Place the slide under a humidified coer on a rotator and rotate for 8 minutes at 80 to 120 rpm in a circumscribed circle of approximately two centimeters in diameter. 57 8. The slide should be read in a wet state immediately. Examine macroscopically under incandescent light or sunlight for the presence of visible aggregates of latex that are similar to those observed with the positive control serum. Gently rotate the slide by hand while reading facilitates the visualization of weak positive reactions. 9. Agglutination of the latex is indicative of the presence of antibody and should be reported as positive. The negative serum produces no agglutination and the latex remains as a smooth dispersed solution. Protocol for Evaluating Sera Positive Latex Agglutination: Occasional false positive results may be obtained with the Latex Agglutination test. Most of these have low titers. Therefore, it is recommended that all samples tested Positive at the initial 1:4 screening dilution be retested according to the following protocol. 1. Screen all sera at 1:4 dilution as specified in the test procedure. 2. Serum samples that are Negative at this solution should be considered Negative. 3. Serum samples that are Positive should be retested at a 1:40 dilution. 58 4. Serum samples that are Positive at both serum dilutions should be considered Positive and should be confirmed by another test. 5. Serum samples that are Positive at the 1:4 dilution but Negative at the 1:40 serum dilution should be Heat Inactivated and retested. 6. Heat inactivate the sample by placing the original undiluted sample in a water bath set at 56 degrees C for 30- 33 minutes. Retest the inactivated serum at a 1:4 dilution. 7. Serum samples converting to Negative at the 1:4 dilution after Heat Inactivation should be considered Negative. 8. Serum remaining Positive after the heat inactivation step may be Positive and should be confirmed by another test such as Serum Neutralization. APPENDIX E “W W APPENDIX E Immunofluoresence Assay for PRV Protocol for analysis Michigan State University Animal Health Diagnostic Laboratory East Lansing, MI 1. Make slide with two tissue sections. Use poly—L-Lysine treated slides. 2. Air dry slides for 30 minutes. 3. Fix slides in acetone for 10 minutes; air dry for 30 minutes. 4. Apply 100 pl FITC conjugate to slide and spread evenly over tissue section with wooden stick. (Spread gently to avoid scraping tissue off of slide). 5. Incubate in a humid chamber for 30 minutes. Cover chamber with paper towel and turn off overhead lights. 6. Rinse with distilled water. 7. Wash in 0.85% NaCl for 10 minutes (Change saline every two days). 8. Rinse with distilled water. 9. Immerse in 0.01% Evan's blue counterstain for 1.5 - 2 minutes. 59 6O 10. Rinse in distilled water for 1 minute or until blue clears. 11. Air dry or place on vent in hood. 12. Mount coverslip with 90% glycerol in PBS (0.01M, pH 7.2). APPme F APPENDIX F Salmonella Cholerasuis Protocol for inoculation preparation and bacteriologic analysis National Animal Disease Center Ames, IA Inoculation Preparation The following material and instructions were used and followed for preparation of the inoculation solution to infect pigs with Salmonella cholerasuis and tissue collection. Material - approximately 31 TSA agar plates - 3 TSA agar plates with S.cholerasuis 3246pp cultures on them. approximately 20, 9 ml PBS dilution tubes - One 50 ml blue top tube with 25 ml of PBS - sterile swabs - Whirl-pack bags for tissues (17 pigs, pre-labeled) - 1 package of 1 ml disposable pipettes - several 1 ml syringes for infecting pigs 61 62 1. Day before infection day gather 17 of the sterile TSA plats, the 3 plates containing pure cultures of S.cholerasuis 32466pp and several packages of sterile swabs. 2. Make laws of S.cholerasuis on the sterile TSA plate by picking up 5 - 10 colonies of S.cholerasuis on a dry sterile swab and streaking the bacteria across the entire plate. Two lawns (2 plates) can be made with each swab of bacteria. 3. Place the 17 lawn plates face down at 37°C and allow to grow overnight 18 - 20 hours. 4. Remove the plates from the incubator. Use a fresh swab for each plate to scrape the bacterial growth off of the plate and place the swab in the blue cap tube containing 25 ml PBS, vigorously rotate the swab then squeeze the excess liquid out of the swab before discarding. (a conservative estimate is that each plate contains approximately 2x10lo CFU of S.cholerasuis, which is the same dose desired to give each pig) 5. After removing the bacteria from all 17 plates and resuspending in PBS, mix thoroughly. Do not plate count on the culture in the blue cap tube. Take 1 ml from the blue top tube into 9 ml (1:10 dilution; 9 ml PBS tubes provided). The first tube is the -1 dilution. Continue this, using a 63 new sterile 1 ml pipette for each dilution and vortexing the tube after adding the bacteria through the -12 dilution. 6. Plate the —8, -9, -11 and —12 dilution tubes by taking 0.1 ml out of the respective tube and plating it on each of 2 fresh TSA plates. Spread the liquid evenly over the agar surface and allow to dry before turning the plates over . 7. Incubate the plates at 37°C overnight. The following day count the colonies on each dilution plate (as long as they are less than 250) and record. 'E'E' E' Using the culture of S. cholerasuis in the blue cap tube. 1. Mix the culture well before starting. It is also important to mix in between each pig. 2. Draw up 1 ml of culture in the 1 ml syringe provided and while holding the pigs nose up instill intranasally dropwise on inspiration 0.5 ml in each nostril, alternating nostrils, 1 drop at a time. 3. Do this for all pigs changing syringes after 3 or 4 pigs. 64 4. It works well if one person is catching and holding pigs, one person is infecting the pigs and a third person is drawing up the culture. Bacteriologic Analysis Tissues are processed according to the method as described by Gray et a1. (1995). Briefly, tissues (collected at necropsy) are minced using a sterile scalpel then homogenized in stomacher 80 lab blender (Tekmar, Cincinnati, OH). All tissues are incubated at 37°C in GN-Hajna broth with 200 pgr/ml streptomycin sulfate (BGS-S, Difco, Detroit, MI). Additionally, at 18 to 24 hours 100 pL of GN-S is transferred to Rappaport-Vassiliadis (RV) medium (Vassiliadis, 1983), incubated at 37°C for 18 hours, then streaked to BGS-S. All BGS-S plates are incubated 24 hours at 37°C. Colonies having the appearance typical of Salmonella are picked and inoculated into triple sugar iron and lysine iron agar slants. Isolates having biochemical reactions typical of Salmonella are confirmed as group C by agglutination with Salmonella antiserum group CHO (Difco, Detroit, MI). Representative isolates are serotyped at the National Veterinary Services Laboratories (NVSL). nICHIan STATE UNIV. LIBRQRIES 111111111111111111111”11111111111in 31293014100493