Peer reviewed Original research Studies on survival of pseudorabies virus, Actinobacillus pleuropneumoniae, and Salmonella serovar Choleraesuis in composted swine carcasses Josep Garcia-Siera, DVM, MS; Dale W. Rozeboom, MS, PhD; Barbara E. Straw, DVM, PhD; Brad J. Thacker, DVM, PhD; Larry M. Granger, DVM; Paula J. Fedorka-Cray, MS, MAS, PhD; Jeffrey T. Gray, MS, PhD.

Summary tored daily, and samples were obtained culture negative for PRV and App. In Ex- Objective: To monitor survival of pseudo- from the carcasses for microbiologic evalu- periment Two, Sc was recovered from rabies virus (PRV), Actinobacillus ation at intervals throughout the samples collected on Composting Days 0, pleuropneumoniae (App), and Salmonella composting period. 1, and 3, but not from samples collected serovar Choleraesuis (Sc) in composted on Days 7 or 10. Results: Temperature of the composting swine carcasses. piles ranged from 27 to 51°C in Experi- Implications: Under the conditions of Methods: In Experiment One, pigs were ment One, and 27 to 62°C in Experiment these experiments, composting can be used infected with PRV, infected 2 days later Two. Composted carcasses degraded rap- to dispose of swine carcasses containing with App, and euthanized 15 to 16 hours idly. After 7 days, only bones, teeth, large PRV, App, and Sc. after App infection. Carcasses were then muscles, and portions of the hide were Keywords: swine, composting, pseudora- composted for 35 days. In Experiment physically recognizable. Muscle and bone bies, Actinobacillus pleuropneumoniae, Sal- Two, pigs were infected with Sc and were discolored, and bones were more eas- monella serovar Choleraesuis euthanized 3 days later, and carcasses were ily crushed or broken. The hide was less composted for 10 days. Compost piles collagenous and tore into several pieces were constructed inside buildings with when carcasses were extracted from piles. Received: December 8, 2000 concrete floors. In both experiments, tem- In Experiment One, tissue samples col- Accepted: March 11, 2001 perature of the composting piles was moni- lected on Composting Days 7 and 14 were

n recent years, swine producers have not produce enough dead animals to justify composting is most widely used to dispose been challenged with the increasingly service by a rendering plant. Also, un- of yard waste. Recently, however, interest I difficult task of disposing of dead pigs weaned pigs and placentas have no value has been directed towards composting as a and placentas on the farm. Incineration, for rendering companies and are not col- method of dead animal disposal. burial, and rendering are frequently the lected for disposal. In addition, rendering In the US, composting of dead animals has only options. Incineration destroys patho- vehicles may serve as potential vectors of been confined primarily to poultry farms, gens, but has economical, environmental, disease as they travel from herd to herd. and aesthetic drawbacks. Burial is cheap, with fewer swine farms making use of this but not always convenient in cold climates Composting initially gained popularity practice. In several states, composting is where the ground is frozen during winter. during the 1970’s, largely because the Envi- not an approved method of carcass dis- Rendering recovers several animal by-prod- ronmental Protection Agency sponsored posal. Experimental evidence supporting its ucts and effectively controls transmissible development of specific composting tech- usefulness and safety as a means of pig car- diseases; however, not all producers have niques by the US Department of Agricul- cass disposal is lacking; therefore, regula- access to this method of disposal because of ture, primarily to treat municipal waste tions and laws governing pig carcass and logistics or the size of the farm. Frequently, water sludge or for solid waste manage- placenta composting have not been swine operations of 100 to 200 sows do ment (sewage sludge). Presently, established. Few reports exist on survival profiles of JGS: Indukern Corp, Miami, Florida 33178; DWR: Department of Animal Science, 2209 Anthony microorganisms in composting piles. Flegal Hall, Michigan State University, East Lansing, Michigan 48824-1225; BES: Department of Large 1 Animal Clinical Sciences, Michigan State University; East Lansing, Michigan 48824-1225; BJT: et al reported destruction of hemolytic Department of Veterinary Diagnostic and Production Animal Medicine, Iowa State University, enteritis virus in poultry composting piles. Ames, Iowa 50011; LMG: Animal Health Division, Michigan Department of Agriculture, Lansing, Morrow et al2 reported partial destruction Michigan; PJFC, JTG: USDA-ARS-SAA, Richard Russell Research Center, Athens, Georgia. of salmonellae and total destruction of Ery- This article is available online at http://www.aasv.org/shap.html. sipelothrix rhusiopathiae and pseudorabies Garcia-Siera J, Rozeboom DW, Straw BE, et al. Studies on survival of pseudorabies virus, virus (PRV) by the heat produced in swine Actinobacillus pleuropneumoniae, and Salmonella serovar Choleraesuis in composted swine carcasses. composting piles (internal temperatures in J Swine Health Prod. 2001;9(5):225-231. excess of 60°C). In that study, salmonellae Journal of Swine Health and Production — Volume 9, Number 5 225 and Erysipelas organisms were grown in lethargy, inappetence). Lung and brain tis- from bottom to top was St-Sd-C-Sd-C-Sd- culture tubes that were sealed and buried in sue from three carcasses (controls) were C-Sd, with each layer approximately 15 cm compost piles. Nine of 15 salmonellae cul- collected immediately after euthanasia deep. Strata in Pile Two were the same as in ture tubes were culture negative when re- (Composting Day 0) to determine the Pile One, but there were only two layers of trieved on day 127 of composting, and on presence of PRV and App. The remaining carcasses. In each pile, a layer of carcasses day 177, 11 of 14 tubes were culture nega- 15 whole carcasses were placed in compost contained three whole pigs. Total carcass tive. All 18 Erysipelas tubes were culture piles and tested for viral and bacterial sur- weight was 92 kg for Pile One and 63 kg negative when withdrawn from the com- vival after composting for 7, 14, or 35 for Pile Two. The internal temperature of post pile on days 245 and 351 (9 tubes on days. the compost piles was recorded with a 90- each day). In the same study, tonsils from cm, probe-type thermometer. Temperature PRV-infected pigs, in sterile wide mouth Challenge inocula and procedures measurements were taken every 1 to 3 days glass bottles, and carcasses of four PRV- The strain of PRV used in this study was by randomly inserting the probe approxi- infected pigs, securely wrapped in plastic prepared as previously described.3 A 0.5- mately 30 to 45 cm into several areas of the biohazard bags, were placed in compost mL dose of challenge inoculum, containing pile. Early in the composting process, car- piles. After extraction from the compost 106 Tissue Culture Infective Doses (TCID) casses were struck with the probe and pro- pile on days 29 and 53, tonsillar tissue of PRV per mL, was instilled into each nos- hibited complete insertion. The thermom- samples from bottles and bagged carcasses tril over a 30-second period.3 Infectivity of eter was then inserted adjacent to the were placed in a common scintillation vial, the inoculum was confirmed by titration carcasses. After approximately 7 to 10 days stored frozen for an unspecified length of using the Crandall-Reese Feline Kidney of decomposition, the probe passed time, and used to prepare an inoculum (CRFK) cell line. through carcasses. Piles were intentionally which was injected into PRV-free sentinel aerated on Day 21, after pile temperatures An App serotype 1 strain (Shope) was pre- pigs. At the end of a 28-day observation had peaked. Contents were manually pared for intratracheal challenge as previ- period, serum samples collected from the turned and mixed using a garden shovel ously described4 and contained 10 colony pigs were negative for PRV antibodies. and fork. As a consequence, each pile was forming units (CFU) of App in 10 mL of moved from its original location to a spot While results from Morrow et al2 suggest normal saline. Pigs were anesthetized by IV immediately adjacent. Prior to this, piles that pathogenic organisms may be inacti- administration of a mixture of ketamine were aerated to an unknown but lesser ex- vated by composting, the conditions of the hydrochloride (Ketaset; Fort Dodge Labo- tent by the random temporary removal of trial did not reflect an “on-farm” situation. ratories, Ft Dodge, Iowa), 4.4 mg per kg carcasses from their respective piles when In their experiment, the pathogenic micro- BW, and xylazine hydrochloride (Rompun; samples were obtained. organisms were not directly in contact with Miles Inc, Shawnee, Kansas), 1.65 mg per the compost material, and conditions in- kg BW. The inoculum was placed in the Microbiologic techniques side the culture tubes may not have ad- trachea via percutaneous injection using an Animal tissue or compost material was col- equately reflected those surrounding an 18-gauge spinal needle. The inoculating lected on Composting Days 0, 7, 14, and infected carcass in a compost pile. The dose was confirmed by back titration on 35 to test for presence of infective microor- study reported here investigated the effect brain-heart infusion agar (BHI; Difco ganisms. Carcasses were randomly desig- of composting the carcasses of experimen- Laboratories, Detroit, Michigan ) contain- nated for sampling on Days 7 and 14. On tally infected pigs under simulated swine ing V factor (nicotinamide adenine di- Day 7, three whole pigs or their remains farm conditions in late spring to summer, nucleotide; Sigma, St Louis, Missouri) as were extracted from each pile. One 15-g in a structure with a roof and impermeable previously described.4 tissue sample was collected from the brain floor. of each pig for PRV isolation, and another Compost pile construction, 15-g sample was collected from the lungs temperature, and aeration Materials and Methods for App. On Day 14, carcasses were se- Two compost piles were constructed in an Experiment One, Part A: verely decomposed and individual soft tis- isolated, environmentally controlled build- Composting and microbiological sues were no longer recognizable. Samples ing at the Michigan State University Veteri- testing of carcasses infected with were collected near the intended anatomic nary Research Farm. Air temperature inside PRV and App location, which was determined by finding the building was maintained at 23 to 27°C. pieces of skin, hair, and large bones. Experimental design One pile was placed in each of two rooms Samples were thus composed of a mixture The sequence of events followed in Experi- (3.1 × 4.3 m) with concrete block walls of animal tissue and compost material. Be- ment One is shown in Figure 1. Eighteen and concrete floors. cause of further decomposition and the pigs, weighing approximately 10 to 15 kg, Pile One consisted of three layers of car- aerating process, Day 35 samples were also were infected intranasally with PRV. Two casses (C), one layer of straw (St), and four a mixture of unrecognizable animal tissue days later, the pigs were inoculated layers of fresh sawdust (Sd) free of foreign and sawdust and could not be attributed to intratracheally with App. All pigs were material and received directly from a saw specific carcasses in either pile. Pile One euthanized by intravenous injection of so- mill that cut dried hard- and softwood samples may have contained animal tissue dium pentobarbital 15 to 16 hours post lumber. Straw was used as the bottom layer from both previously sampled carcasses and challenge with App, when severe signs of to provide porosity and maintain aerobic the three carcasses not sampled previously. App and (or) PRV had developed (fever, conditions longer. The layout of the pile Day 35 samples from Pile Two contained 226 Journal of Swine Health and Production — September and October, 2001 Figure 1: Timeline of events for inoculation of pigs, composting and microbiological testing of carcasses, and infection and testing of sentinel pigs in Experiments One and Two. PRV: pseudorabies virus; App: Actinobacillus pleuropneumoniae; Sc: Salmonella serovar Cholerasuis var kunzendorfχ3246

Experiment One

Part A: Infection Part A: Composting Part B: Sentinel animals 18 pigs, 10-15 kg 15 pigs 12 pigs

Carcasses removed from piles for Weaned Serology Pigs euthanized and Inoculations microbiologic examination (one or two at 7 -10 days confirming pigs necropsied for from each pile each sampling) of age are PRV and App microbiologic negative examination PRV App

Day of study 0 1 2 3 4 11 18 25 32 39 46 74 88 95 Composting Day 0 7 14 21 28 35

Selection All pigs euthanized, three pigs Both Compost material Compost necropsied for microbiologic piles frozen for future material fed evaluation, 15 pigs composted aerated use as feed and and used as (nine in Pile one and six in Pile two) bedding bedding

Experiment Two

Inoculum preparation: Pigs inoculated Three carcasses Three carcasses Three carcasses Three carcasses 12-wk old pig intranasally with removed and removed and removed and removed and inoculated with strain 3246 necropsied for necropsied for necropsied for necropsied for Sc microbiologic microbiologic microbiologic microbiologic evaluation evaluation evaluation evaluation

Day of study -7 to -5 012345678910111213 Composting Day 0 12345678910

All pigs euthanized, four pigs necropsied for microbiologic evaluation, 12 pigs composted (two piles, six in each) tissues from the six previously sampled car- and then centrifuged for 20 minutes at prior to inoculation in triplicate onto 5% casses. After collection, all samples were 1300g (Model TJ6; Beckman, Palo Alto, blood agar plates with Staphylococcus immediately placed in an ice-filled con- California). The supernatant was collected epidermidis feeder colonies. One plate was tainer and transported to the laboratory for and 0.2 mL was inoculated onto Maden- incubated aerobically, one plate in an at- processing. Darby Bovine Kidney (MDBK) cell culture mosphere containing 5% CO2, and one monolayers in 24-well microtiter plates. plate anaerobically. Plates were incubated Pseudorabies virus in brain tissue and com- Cell cultures were observed for cytopathic for 18 to 24 hours at 37°C. In addition, a post samples was detected by virus isolation effect for 7 days. Negative cultures were second culture was attempted after over- at the Iowa State University Veterinary Di- subcultured on MDBK cells for 7 more night enrichment of 2.5 g of each sample agnostic Laboratory, Ames, Iowa, using a days. in 25 mL of Pleuropneumonia-Like Or- procedure previously described by Cook et ganism broth (PPLO; Difco), at 37°C in al5 with the following modifications. For Lung tissue collected on Day 0 for App an atmosphere containing 5% CO . sample preparation, approximately 5 g of isolation was processed as previously de- 2 4 brain or compost sample was mixed with scribed. For lung tissue collected from Experiment One, Part B: Sentinel 20 mL of Earles balanced salt solution con- composted carcasses and compost material, animal testing taining a fungistat and antibiotic (amphot- App isolation was performed by standard ericin B and gentamicin sulfate, respec- methods at the Iowa State University Vet- Sentinel animals tively; Grand Island Biologics Co, Grand erinary Diagnostic Laboratory, Ames, Iowa, Twelve pigs were weaned at 7 to 10 days of Island, New York). The mixture was placed with the following modifications. Approxi- age. Segregated early weaning (SEW) was in a stomacher 80 bag (Tekmar, Cincin- mately 3 g of each sample was soaked in 30 used to avoid dam-to-offspring transmis- nati, Ohio), blended for 20 to 25 seconds, mL of phosphate buffered saline solution sion of PRV, App, or other pathogens that Journal of Swine Health and Production — Volume 9, Number 5 227 might interfere with the study. The herd of At the end of the feeding period (8 weeks collected for bacteriologic culture included origin was tested monthly for PRV, had no of age), pigs were euthanized by IV injec- mandibular (mandib-LN), bronchial history of PRV or App, was seronegative tion of sodium pentobarbital. Brain tissue (bronch-LN), ileocolic (ICLN), and co- for PRV by ELISA (S:P<0.7) and latex ag- was collected for assay of PRV, and lung lonic (CLN) lymph nodes. Also collected glutination tests, and was seronegative for tissue for assay of App. Assays for PRV in- for bacteriologic culture were 6- to 8-g App by complement fixation test (ti- cluded immunofluoresence testing and vi- samples of tonsil, lung, spleen, liver, ter<1:8). Blood samples obtained from the rus isolation (MSU Animal Health Diag- middle ileum (ileum-mid), ileocolic junc- 12 sentinel pigs at 5 weeks of age were nostic Lab, East Lansing, Michigan) as tion (ICJ), cecum, and colon. Lastly, 25 g negative for antibodies to PRV and App. previously described.3 Isolation of App of cecal contents (CC) was collected and Latex agglutination and ELISA tests were from lung tissue was performed at the Iowa cultured. used to test for antibodies to PRV (Michi- State University Veterinary Diagnostic The remaining 12 pigs were placed in two gan Department of Agriculture Diagnostic Laboratory as described. compost piles as described below. Necrop- Laboratory, East Lansing, Michigan) and sies were performed on three different pigs an indirect ELISA was used to detect anti- Experiment Two: Composting of on each of Composting Days 1, 3, 7, and bodies to App (Oxford Labs, Worthington, carcasses infected with Sc 10. Tissues were recognizable on Days 1 Minnesota; S:P>0.4 considered positive). Bacterial strain, challenge culture, and and 3, allowing for the collection of the preparation of inoculum desired 13 samples. All but two specific Housing and feeding Wild type Salmonella serovar Choleraesuis samples were obtained on Day 7. However, Pigs were housed in an environmentally var kunzendorf χ32466 was kindly pro- by Day 10, organs had severely decom- controlled isolation facility, in a single pen vided by the laboratory of Roy Curtiss III, posed, and unrecognizable tissue, lying in a (2.54 × 3.55 m) with solid concrete Washington University, St. Louis, Medical similar anatomic location to the desired flooring, two nipple waterers, and a three- Officer of Health to the National Animal tissue or material, was collected. space, fence line feeder. Throughout this Disease Center at Ames, Iowa. A 12-week- experiment, pigs were provided with ad old pig was inoculated intranasally, and the Compost pile construction libitum access to non-medicated feeds and isolate was recovered from the ileocolic Two compost piles were constructed, con- water. From weaning to 4 weeks of age, lymph node (ICLN), stored at -70°C in sisting of one layer of straw (bottom), one pigs were fed two commercially available, glycerol, and used as the challenge strain layer of spelt hulls, one layer of carcasses pelleted diets for early-weaned pigs (EW (3246pp).7 Strain 3246pp was streaked (six pigs), and one layer of spelt hulls (top). and HE; United Feeds, Sheridan, Indiana) onto a trypticase soy agar (TSA) plate Each layer was approximately 20 cm deep. according to manufacture’s feeding direc- (Difco) which was sealed with parafilm and After the piles had been constructed, water tions. After 4 weeks of age, the pigs were shipped to Michigan State University for was added to increase the moisture content fed ground corn-soybean meal-based diets preparation of challenge cultures. Sterile to about 60%, based on the proximate manufactured at the Michigan State Uni- cotton-tipped swabs were used to prepare analysis of the saw dust samples (ranging versity Feed Mill. The phase three diet lawns of strain 3246pp on 20 TSA plates. from 8 to 12% moisture)8 and the reported (1.25% lysine, containing dried whey and After incubation of plates overnight at moisture content (about 45%) of young fish meal) was fed from 4 to 6 weeks of age, 37°C, growth was harvested with a cotton- pigs.9 Each of the two sampling areas in and the phase four diet (1.15% lysine) was tipped swab and resuspended in 20 mL of each pile contained three carcasses. fed from 6 to 8 weeks of age. All diets met phosphate buffered saline (PBS; 0.02M, or exceeded NRC (1998) nutrient recom- Compost piles were contained in separate pH 7.2). Final concentration of the inocu- mendations for pigs of like maturities. bays in an open front, naturally ventilated, lum was determined by plate count. multiple–bay pole barn, which had a roof Experimental design and a concrete floor. Bays were approxi- Experimental design The sequence of events followed in this mately 3 × 5 m and had three solid Figure 1 shows the sequence of events in experiment is shown in Figure 1. Potential wooden walls. The fourth side of each bay Experiment Two. Sixteen pigs were housed was closed with square straw bales so that exposure of pigs to PRV and App was ac- in isolation facilities and challenged at 8 compost piles measured approximately 1.5 complished by mixing material obtained weeks of age (Study Day 0). The strain × 3 m. This experiment was conducted in from compost piles (Experiment One, Part 3246pp inoculum, containing 2×1010 the spring, with daily low air temperatures A) into the feed, and using the same com- CFU per mL in PBS, was administered ranging from -2 to 14°C (Capitol City Air- post material as bedding. Both practices intranasally, 0.5 mL in each nostril port, Lansing, Michigan; average, 7°C) and began when pigs were 7 weeks of age and dropwise on inspiration. Clinical signs of daily high air temperatures ranging from continued for 7 days. Compost material lethargy, anorexia, and dyspnea were moni- 12 to 27°C (Capitol City Airport, Lansing, had been stored frozen (-20°C) for 7 weeks tored by observation on Days 1 through 3 Michigan; average, 19°C). before it was blended by hand into the while feeding the pigs. On Day 3 post phase four diet (compost-to-feed ratio, challenge, all pigs showed various degrees Bacteriologic techniques 1:17) or used as bedding. Throughout the of lethargy, anorexia, and dyspnea and were Tissues were processed according to the exposure period, pigs were monitored daily euthanized with sodium pentobarbital method described by Gray et al.7 Briefly, for signs of PRV and App infection, such as (IV). Necropsies were immediately per- tissues collected at necropsy were minced increased respiration rate, labored respira- formed on four pigs and 13 samples were using a sterile scalpel, then homogenized in tion, lethargy, and inappetence. collected per pig. Entire lymph nodes (LN) 228 Journal of Swine Health and Production — September and October, 2001 a stomacher 80 laboratory blender Figure 2: Internal temperature changes in two compost piles containing (Tekmar, Cincinnati, Ohio). All tissues carcasses of pigs infected with pseudorabies virus and Actinobacillus were incubated aerobically for 18 to 24 pleuropneumoniae. Pile One contained nine carcasses, and Pile Two contained hours at 37°C in GN-Hajna broth with six carcasses. Both piles were fully aerated on Day 21 and were disturbed to streptomycin sulfate (200 µg per mL; GN- take culture samples on Days 7 and 10. S; Difco), then 1 loopful was streaked on brilliant green agar with sulfadiazine (1 g 55 per L) and streptomycin sulfate (200 µg Pile Two (six carcasses) per mL; BGS-S; Difco). Additionally, at 18 50 to 24 hours, 100 µl of GN-S suspension C) was transferred to Rappaport-Vassiliadis ° 45 (RV) medium,10 incubated aerobically at 37°C for 18 hours, then streaked on BGS- 40 S. All BGS-S plates were incubated aerobi- cally for 24 hours at 37°C. Colonies having 35 the appearance of Salmonella serovars were picked and inoculated into triple sugar iron and lysine iron agar slants. Isolates having 30 Pile One (nine carcasses) biochemical reactions typical of Salmonella Internal temperature ( serovars were confirmed as group C by ag- 25 glutination with Salmonella serovar antise- Sampling (Days 7,10) Aeration (Day 21) rum group C1O (Difco). Representative 20 isolates were serotyped at the National Vet- 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 erinary Services Laboratories (NVSL; Composting Day Ames, Iowa). Bacterial counts were conducted on the small amount of brain tissue in liquid form microorganisms. ICJ samples using the five-tube most prob- was obtained from the skull cavity of one able number method (Wood and Rose, pig in Pile One. All other samples collected Experiment Two 1992)11 with GN-S, BGS-S, and RV me- on Day 7 were mixtures of animal tissue dia as described above and reported as the Bacterial strains and challenge cultures and sawdust. Carcasses appeared to be de- mean value. Bacterial counts were also con- Salmonella serovar Choleraesuis 3246pp composed except for pieces of bone and ducted on the ICLN samples by direct was isolated from 44 of the 52 samples col- hide. Collections on Days 14 and 35 con- plate count using limiting dilution. lected on Composting Day 0. In all four sisted of a mixture of animal tissue, saw- pigs, Sc was isolated from the ICLN, ICJ, dust, and small pieces of straw. By Day 14, Results liver, mid-IL, colon, and CC; in three of carcasses were totally unrecognizable, ex- the four pigs, Sc was isolated from the Experiment One: Part A cept for a few 15-cm pieces of hide. Piles mandib-LN, bronch-LN, CLN, tonsil, On Composting Day 0, PRV was isolated were dark brown with small bone segments lung, and cecum; and in two of the four from three of three brain samples, and App visually detectable. Some skulls were still pigs, Sc was isolated from the spleen. On (>1000 CFU) was recovered from three of recognizable on Day 14. By Day 35, few Composting Day 1, Sc was not isolated three lung samples, collected from the con- major bones were recognizable. from the ICJ or CC of one pig, but was trol pigs immediately after euthanasia. isolated from the other 37 of the 39 sites Microorganism survival Temperature in compost piles ranged from sampled. On Day 3, Sc was isolated from Viruses were not detected in compost all 39 sites sampled. However, on Days 7 26.7°C (near ambient temperature in the samples taken on Days 7 and 14. There- and 10, Sc was not isolated from any building) to 52.2°C (Figure 2). The highest fore, virus isolation was not performed on sample. temperature in Pile One (51.7°C) was samples collected on Day 35. No App colo- reached on Day 8 of composting, and the The mean number of Sc recovered from nies were recovered from compost samples highest temperature in Pile Two (52.2°C) tissue samples was 3.6 × 105 CFU per g of on Days 7, 14, or 35. was reached on Day 9 of composting. Tem- ICJ and 1.0 × 103 CFU per g of ICLN. peratures in the piles remained above 45°C Experiment One: Part B All isolates submitted to NVSL were until Day 14, then started to drop, more During the final week of the experiment, confirmed by serotyping to be Salmonella rapidly in Pile One than Pile Two. On Day sentinel pigs consumed approximately 1.5 serovar Choleraesuis var kunzendorf. 23, 2 days after the piles were aerated, the kg of feed and compost material per pig temperatures peaked again at 50°C. per day. The pigs did not develop signs re- Discussion By Day 7, the first day of tissue collection, lated to infection with App or PRV, and all Isolation of App and PRV from all three specific anatomic structures within car- tissues collected at the end of the experi- control pigs in Experiment One and Sc casses were physically unrecognizable. A ment tested negative for both from all four control pigs in Experiment

Journal of Swine Health and Production — Volume 9, Number 5 229 Two demonstrates that the infection proce- logical activity of the composting process killed by freezing (–18°C) for 35 days. dures were successful. Therefore, the patho- was not complete. Actinobacillus pleuropneumoniae was killed gen content in the carcasses of these experi- These trials were completed in the spring within 7 days in piles that became active mentally infected pigs probably closely when ambient temperatures were between quickly, as indicated by the rapid rise in resembled that found in clinical cases of -2 and 25°C. The composting process temperature. This outcome was expected, these diseases. Usually, young pigs, similar would have been different if the new piles as Nicolet15 reported that App survives in age to those used in this study, and not had been constructed outdoors during win- outside the body for only a few days, even mature swine, die from active infections ter, as we have observed in on-farm when protected by mucus and organic mat- with PRV, App, and Sc. Therefore, the pigs composting demonstrations in Michigan ter. Results in the sentinel pigs could be in these experiments are representative of (unpublished data). During the winter, seen as confirmation of pathogen destruc- animals that might be composted under temperatures in new piles are slower to rise, tion by Day 35 or earlier. However, draw- clinical and commercial conditions. but gradually reach peak temperatures ing a conclusion about the survival in the Multiple small compost piles were used in comparable to those found in this study, compost material is questionable for App, these studies, both to facilitate sample re- provided that carcasses are not frozen. Fro- as it was for PRV, because App was frozen trieval without greatly disturbing the piles zen carcasses compost poorly during cold and stored for 7 weeks before the sentinel and to better represent conditions that weather, especially if the pile is being con- pigs were exposed to it. would be experienced on the outside layers structed with new material and the car- Isolation of Sc from Day 0 tissue samples of larger piles. Microbial activity and de- casses are 10 kg or greater in size. If car- confirmed that all pigs were infected prior struction of potential pathogens harbored casses are cold but not frozen when added to composting. The pattern of tissue colo- in carcasses are slower in the outer layers of to an already active compost pile in winter, nization and the mean numbers of Sc re- piles, possibly because of the greater poros- the onset of microbial decomposition covered from tissue samples were similar to ity.2 Use of small piles also made it easier to would be delayed only slightly, by 3 to 7 those described by Gray and others.7 identify the location of composted car- days depending on ambient temperature, casses. Piles were constructed inside a to- until the heat of the compost mass warms Salmonella serovar Choleraesuis was de- tally enclosed building in Experiment One the carcasses. An active pile would already stroyed in infected swine carcasses by and in an open-front structure in Experi- be warm, with substantial microbial de- composting for 3 to 7 days. This conclu- ment Two. Some states, including Michi- composition already occurring. Small fro- sion is consistent with the report of gan, Illinois, and Minnesota, presently re- zen pigs would probably thaw when added Forshell and Ekesbo,16 which states that quire that on-farm composting be to an active compost pile during winter Salmonella serovar Senftenberg and Salmo- conducted in a facility with a minimum of months, and microbial decomposition nella serovar Typhimurium do not survive concrete floor, bin walls, and roof. would probably begin within 7 to 14 days. more than 7 days in composted swine ma- These observations are in agreement with nure. In contrast, the results of other stud- Temperatures in our piles attained 40 to those of other researchers.12 ies suggest that some salmonella may sur- 50°C for prolonged periods, indicating vive the composting process much longer. that conditions (porosity, moisture con- In this study, the conditions achieved in Droffner and Brinton17 observed that Sal- tent, and carbon-to-nitrogen ratio) were the composting process killed PRV and the monella serovar Typhimurium Q survived conducive to microbial population growth. two species of bacteria in the carcasses. Our 59 days during the composting of indus- Different composting materials, also results are in agreement with those of Mor- trial sludge, even though temperatures of known as bulking agents or carbon sources, row et al,2 who reported that composting 60°C were attained. Morrow and cowork- were intentionally used in Experiment One temperatures effectively and quickly de- ers2 reported that salmonellae in culture (sawdust) and Two (spelt hulls), with no stroyed PRV. Similarly, Davies and Beran13 tubes survived after more than 6 months in apparent impact on the composting pro- have shown that at 37°C and pH 6 to 8, compost piles containing swine carcasses. cess. Both provided adequate amounts of PRV outside the living host is inactivated In their experiment, salmonellae were carbon. The decision to use spelt hulls in at a rate of 0.6 log 10 per day. Failure to grown in trypticase soy broth in culture Experiment Two was based on cost (none), isolate PRV from tissue samples collected tubes which were then sealed, creating availability, and producer interest in using from composting carcasses indicates that nearly anaerobic conditions. When re- this milling industry by-product. PRV was destroyed by Day 7. moved from the pile 127 days after the Previously composted material may be used Failure to infect sentinel pigs with PRV by composting process began, 40% of the to inoculate compost piles to speed the exposing them to composted material in tubes contained live salmonellae, and 21% composting process.12 In these experi- feed or bedding appears to be a of the tubes still contained live salmonellae ments, temperatures in the piles rose confirmation that the PRV was destroyed by Day 177. In Morrow’s study, salmonel- quickly, suggesting that sufficient numbers prior to Day 35 of composting. This con- lae were exposed to heat alone. These au- of composting microbes must have been clusion is debatable, however, as the com- thors explained that by isolating the bacte- naturally present on the fresh bulking post material was stored frozen at –20°C ria in culture tubes, they “underestimated agents and the carcasses. When the trial for 7 weeks from the time it was taken the bactericidal effect of composting.” was terminated on Day 35, both piles had from the pile until its use in feed and bed- Droffner and Brinton17 similarly con- internal temperatures higher than the am- ding. Previous research14 has shown that cluded that the destruction of salmonellae bient temperature, suggesting that the bio- PRV in pig muscle and bone marrow is “during aerobic composting is complex and

230 Journal of Swine Health and Production — September and October, 2001 not simply the result of thermal physical unopened, it may take longer for the 8. AOAC Official Methods of Analysis. 16th ed. Gaithersburg, Maryland: Association of Official environment.” In our experiment, Sc was composting process to destroy pathogens Analytical Chemists International; 1995. exposed to other potentially inactivating such as PRV, App, or Sc. However, once 9. Shields RG Jr, Mahan DC, Graham PL. Changes factors, such as water, air, and other mi- exposed to microbes, the rate of decompo- in swine body composition from birth to 145 kg. J crobes and the by-products of their activity, sition of muscle, internal organs, and other Anim Sci. 1983;57:43–54. explaining the negative culture results for tissues would probably be similar, regard- 10. Vassiliadis P. The Rappaport-Vassiliadis (Rv) enrichment medium for the isolation of salmonellas: Sc in all samples on Day 7. less of animal age. an overview. J Appl Bacteriol. 1983;48:167–174. The number of swine pathogens examined 11. Wood RL, Rose R. Populations of Salmonella in this study was intentionally limited to Implications typhimurium in internal organs of experimentally • Conditions in composting piles are infected carrier swine. Am J Vet Res. 1992;53:653– three. We know of no other studies where adequate to kill pseudorabies virus, 658. the carcasses of infected swine have been Actinobacillus pleuropneumoniae, and 13. Davies EB, Beran GW. Influence of environ- composted to evaluate the survival of mental factors upon the survival of PRV. Res Vet Sci. Salmonella serovar Cholerasuis in the pathogens. There is much concern world- 1981;31:32–36. carcasses of pigs weighing 10 to 30 kg. wide about the spread of prions associated 14. Durham PJK, Gow A, Poole WSH. Survival of • Composting is a safe method of Aujeszky’s disease virus in frozen pig meat. Res Vet with transmissible spongiform encephalo- disposal of swine carcasses of this size. Sci. 1980;28:256–258. pathies and of the foot-and-mouth disease 15. Nicolet J. Actinobacillus pleuropneumoniae. In: Leman AD, Straw BE, Mengeling WL, D’Allaire S, (FMD) virus. There are no published re- th ports of swine being infected by prions, Acknowledgements Taylor DJ, eds. Diseases of Swine. 7 ed. Ames, The Michigan Pork Producers Association, Iowa: Iowa State University Press; 1992:401–408. possibly because the short life span of The Sycamore Creek Watershed Project, 16. Forshell LP, Ekesbo I. Survival of Salmonellas in swine limits infectivity. The agents of trans- and The Michigan Department of Agricul- composted and not composted solid animal ma- missible spongiform encephalopathies are nures. J Vet Med. 1993;40:654–658. ture provided funding for this research. very difficult to destroy18 and would prob- 17. Droffner ML, Brinton WF. Survival of E. coli ably survive the composting process. Foot- and Salmonella populations in aerobic thermophilic References — refereed composts as measured with DNA gene probes. Zbl and-mouth disease is caused by an 2. Morrow WE, O’Quinn P, Barker J, Erickson G, Hyg. 1995;197:387–397. aphthovirus which is easily destroyed at pH Post K, McCaw M. Composting swine as a suitable 18. Brown P, Rau EH, Johnson BK, Bacote AE, above 9 and below 6.19 The pH of com- technique for managing swine mortalities. Swine Gibbs Jr, CJ, Gajdusek DC. New studies on the post piles containing swine carcasses may Health Prod. 1995;6:236–243. heat resistance of hamster-adapted scrapie agent: 3. Vilnis A, Sussman MD, Thacker BJ, Senn M, Threshold survival after ashing at 600°C suggests an range from 5.5 to 7.2, depending on car- Maes RK. Vaccine genotype and route of adminis- inorganic template of replication. Proc Natl Acad Sci bon source.2 Whether compost pH and tration affect pseudorabies field virus latency load USA. 2000;97:3418–3421. other conditions within the pile would in- after challenge. Vet Microbiol. 1998;62:81–96. activate the FMD virus is not known. 4. Jolie RAV, Mulks MH, Thacker BJ. Cross-protec- References — non refereed tion experiments in pigs vaccinated with Actinobacil- 1. Flegal CJ, Gerrish J, Schwartz LD, Maes R, Mature animals were not used in the lus pleuropneumoniae subtypes 1A and 1B. Vet Endres F. Animal Protein Composting - Final Report. Microbiol. 1995;45:383–391. Michigan State University, 1993. Poultry Research present study primarily because of their Report. 5. Cook DR, Hill HT, Kinker DR. Efficacy of a inconvenient size. Because of the greater killed gpX deleted pseudorabies virus vaccine. Can J 12. Fulhage C. Composting dead swine. University of collagen content of the hide and the greater Vet Res. 1990;54:438–445. Missouri; 1994. Extension Publication WQ225. mineralization of large bones in older 6. Kelly, SM, Bosecker BA, Curtiss R, III. Charac- 19. AVIS. Consortium consisting of the Institute for terization and protective properties of attenuated Animal Health, UK; The Food and Agriculture swine, microbial decomposition is slower Organisation, Rome; L’Office International des 12 mutants of Salmonella choleraesuis. Infect Immun. to initiate and longer to complete. The 1992;60:4881–4890. Epizooties, Paris and Telos; ALEFF Ltd., UK. Avail- rate of decomposition depends on pile able at: http://www.aleffgroupl.com/avisfmd/A010- 7. Gray JT, Fedorka-Cray PJ, Stabel TJ, Kramer TT. fmd/mod0/0123-ph.html. Accessed June 28, 2001. management, environment, and whether Natural transmission of Salmonella choleraesuis in or not the carcasses are cut open prior to swine. Appl Environ Microbiol. 1996;62:141–146. composting. If carcasses are left intact or

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