Slov Vet Res 2009; 46 (3): 105-13 UDC 579.62:631.8:579.871:628.355 Original Research Paper INACTIVATION OF MYCOBACTERIUM AVIUM PARATUBERCULOSIS IN SHEEP MANuRE Ivan Gobec1*, Matjaž Ocepek2, Milan Pogačnik3, Martin Dobeic1 11nstitute of Environmental and Animal Hygiene with Ethology, institute of Microbiology and Parasitology, 3Institute of Pathology, Forensic and Administrative Veterinary Medicine,Veterinary Faculty, Gerbičeva 60, 1000 Ljubljana, Slovenia 'Corresponding author, E-mail: ivan.gobec@vf.uni-lj.si Summary: Livestock manures and composts can be contaminated by Mycobacterium avium subsp. paratuberculosis (MAP) which can cause paratuberculosis - Johne's disease, an intestinal infection of domestic and wild ruminants. Therefore the inactivation of inoculated MAP ATCC 43015 in the compost and in the manure in storage was carried out. MAP (2.0 x 106/g) was inoculated into the compost in three actively ventilated and isolated vessels and in a conventional manure storage pile. Herrold's Egg Yolk medium with supplements for MAP growth, followed by IS900 PCR for isolate identification was used. Direct PCR assessment of the persistence of sequence IS900 in the compost and manure material in parallel was implemented. Moisture, ash and ammonia content in the compost and manure specimens were determined and pH was measured. Salmonella enterica serovar Senftenberg (S. Senftenberg) was introduced into the compost and manure materials to demonstrate the hygienisation process. The presence of MAP on the culture media was confirmed in the samples 16 and 24 hours after exposure in compost and manure, respectively. No MAP was isolated on the medium after 24 hours of exposure. However, using the PCR assay of compost specimens, persistence of MAP was proved in the compost samples until day 7 and in the manure in storage even after 21 days of exposure. S. Senftenberg S-73/98 was not present 24 hours after exposure either in compost or manure storage. Key words: Mycobacterium avium subsp. paratuberculosis; Salmonella enterica serovar Senftenberg; microbial inactivation; bacterial DNA - analysis; composting; sheep manure Introduction MAP infections can cause paratuberculosis and intestinal infections of domestic and wild ruminants and result in considerable economical losses in the livestock production (1). Feces, milk and semen are the main dissemination sources of MAP, and present an important epizootic risk (2, 3) depending on animal management, especially owing to overstocking or to group sizing of young animals (4, 5). The disease manifested in sub clinical forms even increases epidemiological risks, since MAP can be disseminated by infected animals for a long period without any specific clinical signs (6). Received: 20 May 2009 Accepted for publication: 28 August 2009 MAP is highly resistant to physical and chemical factors owing to its special cell wall structure containing mycoside C, mycolic acid, peptidoglycans and lipopolysaccharides (7, 8). Most of the studies regarding the inactivation of MAP in complex environments, were conducted before 1985 (9). However, composting is the one of a most widely applied treatment methods used for bio waste biodegradation and hygienisation influenced by different factors which has an important role on microbiological inactivation (10, 11, 12, 13) (Watanabe et al. 1997; Böhm R 1998; Watanabe et al. 2002; Vinneras et al. 2003. Temperature, oxygen content, microorganisms competition and antagonisms, degradation of organic material, the increase of ammonium concentrations, pH and the composting time, were often selected to control and monitor the composting processes which are responsible for the effect of 106 I. Gobec, M. Ocepek, M. Pogačnik, M. Dobeic proper decomposition and hygienisation processes (14, 15). The objective of this study was to compare the persistence of inoculated MAP during two different manure treatment systems using commonly applied manure storage and composting of sheep manure. Composting is one of the most convenient and optimal manure hygienisation methods, and the application of in-vessel method enables good control of the method (16). Composting of livestock manure can be a method of choice in preventing MAP dissemination into the environment. Materials and methods Design of the study Three ventilated composting vessels, each with the volume of 1m3 (1m x 1m x 1m), and one experimental manure storage pile containing approximately 3 m3 of material (2m x 2m x 1.5 m) were used for the study. Composting material was the mixture of sheep deep litter manure and pine bark with addition of water, so that the moisture content reaches approximately 65%. The composting mixture had been prepared fresh before experimental composting started. The compost temperature was controlled and limited up to 68 oC using radial fans which were also used to achieve aerobic composting process. (Fig. 1) The temperature of compost was controlled by PT 100 probes and by the "Visi DaQ"® computer program (Advantech, USA). The probes were placed in three vertical levels (16 cm, 50 cm and 66 cm above the vessel bottom) and inserted 30 cm deep into the material. The experimental manure storage pile, consisted of sheep deep litter manure only. The temperature was also measured by PT 100 probes at three vertical levels (25 cm, 75 cm and 125 cm above the ground), inserted 30 cm deep into the manure. (Fig. 1) The temperatures were measured every minute and recorded by data loggers (Testo 175 T3, Germany). Physical-chemical analyses of compost and sheep manure Samples were analysed in two parallels. Moisture content was determined in the specimens as a loss upon drying at 105 oC - 110 oC after 24 h by weighing with the accuracy of 0.001 g (Exacta, Tehtnica Železniki, Slovenia). The pH was determined in the liquid extract after the specimens had been oven dried (105 oC - 110 oC) (5 g). mixed with distilled water (25 g) and stabilized (10 minutes) using a calibrated pH meter (Iskra, Slovenia). Ammonia was determined by titration, with 0.1 M NaOH, of 150 ml of distillate (acquired out of 10 g of the specimen, 250 ml H2O and 3 g MgO) blended with 50 ml of 0.1 M H2SO4 and metal red dye. The ash content was determined as solid residua after the samples had been incubated at 550 oC for 30 minutes and weighed with the accuracy of 0.0001 g. Preparation of the samples for exposure and sampling To apply the bacteria in compost and manure, we used diaphyses of bovine long bones as carriers. The carriers were filled up with approximately 5 grams of composting mixture or manure respectively and inoculated with the tested bacterial suspension (Fig. 2). Bone holes stand open, and therefore the Figure 1: Design of the study, placing of the clusters of samples and temperature probes Inactivation of Mycobacterium avium paratuberculosis in sheep manure 107 material in carriers had direct contact with the surrounding material. The filled and inoculated carriers were placed in metal mesh baskets. Two holders with MAP inoculum and one carrier with S. Senften-berg inoculum were placed in each basket. The rest of the baskets' volume was filled up with composting material or manure respectively, and was also used for chemical analyses. Completely filled up baskets were placed in horizontal clusters (sample positions), each containing 9 mesh baskets at three different altitudes and at least 20 cm from the walls (Fig. 1). One filled up mesh basket was removed from the material at each sampling time according to the sampling scheme (Tab 1) covering the period of 21 days. So each sampling (No) comprised six samples of MAP from the composting vessels (54 samples in 21 days) and two samples of MAP from the manure in storage (18 samples in 21 days). S. Senftenberg was sampled simultaneously, and comprised three samples from compost (27 samples in 21 days) and one sample from manure in storage (9 samples in 21 days). During transport to the laboratory, the mesh baskets were sealed in plastic bags and cooled below 6 oC. Single aliquots (200 |l) inoculated into carriers contained 2.0 x 106 of live MAP ATCC 43015 in suspension. The number of MAP was ascertained after 6 weeks of incubation at 37 oC by counting the colonies on Middlebrook 7H10 medium. Serial dilutions of suspension from 10-1 to 10-7 were used for inoculating (100 |l) the medium for enumeration. The used suspension of S. Senftenberg (S-73/98) contained 11.7 x 109/ml of live bacteria. The suspension was inoculated into the compost/manure at the ratio of 1:10. The number of live S. Senften-berg was determined by bacteriological method in three different media (blood agar, Rambach, xylose lysine deoxycholate agar (XLD)), and in two parallels each. Serial dilutions of the suspension from 10-1 to 10-7 were used for inoculating on the media. The colonies were counted after 24 hours of incubation. Bacteriological analyses of compost/manure samples MAP Two grams of the MAP inoculum was removed from each bone holder, mixed with water (50 ml), homogenized (for 30 sec.) in a stomacher (IUL, Spain), shaken for 30 minutes, and left at room temperature for 30 minutes to settle. Afterwards, 5 ml of supernatant was mixed with 25 ml of 0.9% hexadecylpy-rimidum chloride (Sigma, USA) while 1 ml of supernatant was shaken again for 30 minutes and left at room temperature for 18 hours. The samples were centrifuged for 20 minutes at 1400 g (Heraeus 1.0 R, Germany). Meanwhile the supernatant was discarded, the sediment was diluted by 1500 |l of H2O. 200 |l of diluted sediment was inoculated on the Herrold's Egg Yolk medium with supplements: 1. without any mycobactin; 2. with mycobactin, "3. with mycobactin and egg yolk, malachite green, penicillin and amfotericin, and on Bectom Dickinson medium. The inoculated media were incubated in 25 ml tubes in horizontal position (at 37 oC for two weeks) and vertical position (for 3 months). S. Senftenberg The number of S. Senftenberg was determined on Rambach and XLD (Xylose lysine deoxycholate agar) medium in three parallels in up to 24 hours of exposure. Portions of 1 g each of inoculated material from the carriers was mixed, i.e. diluted with 9 ml of sterile physiological solution, and the specimen was further diluted to up to 10-5. 0.1 ml of each dilution was inoculated on the medium. After 24 hours of incubation at 37 oC, the colonies of bacteria were counted. Results with 15 - 300 colonies Bacterial holders Inoculation of bacteria to Placing of the mash bas- Order of sampling holders ket with holders Figure 2: Application of bacteria to the compost, placing of filled-up baskets and sampling order 108 I. Gobec, M. Ocepek, M. Pogačnik, M. Dobeic per plate were considered as adequate for counting analysis. The presence of S. Senftenberg in specimens was determined on the basis of pre- enrichment incubation of 1 g of specimens in buffered peptone medium - BPW at 37 oC for 16 - 20 hours. After pre-enrich-ment, aliquot of 0.1 ml of medium with colonies was transferred to selective enrichment media - Rappa-port Vassiliadis Broth (RVS) and Muller-Kauffmann Tetrathionate-Novobiocin Broth (MKTTn). The specimens were incubated on RVS at 41.5 oC for up to 27 hours and on MKTTn at 37 oC also for 21 - 27 hours. After incubation, S. Senftenberg was determined on Rambach and XLD media by counting typical colonies after incubation at 37 oC for 21 to 27 hours. DNA extraction and direct molecular determination of MAP in compost material After extraction by QIAamp DNA Stool Mini Kit (Qiagen, Germany), DNA was amplified for IS900 using P90 (5' GTT CGG GGC CGT CGC TTA GG 3') and P91 (5' GAG GTC GAT CGC CCA CGT GA 3') primers, respectively. After the amplification, 400-bp PCR-products were separated using electrophoresis (Power Pac 300, BioRad) on 2% agar gel stained by ethidium bromide and analyzed by scanning and a visualization system (Gel Doc 1000, BioRad, USA). Results Physico-chemical analysis During the first 16 hours, average hourly temperatures exceeded 50 oC in the upper and lower positions, and in the next 8 hours reached the limited maximum of 68 oC in the upper positions of composts (Fig. 3). Mean hourly temperatures (43.5 oC ± 5.2 as the 95% confidence interval of the difference, S.D. 6.8) in composts were higher than the average temperatures (23.1°C±14.2, S.D. 5.7) of the sheep manure with no correlation to outdoor temperatures in the first 24 hours (Fig. 3). In the upper positions of the composts in vessels, the temperatures over 60 oC lasted from day 2 to day 7 (148 hours), while they persisted in the range of 50 to 60 oC from day 2 to day 6 on the upper (135 hours), middle (108 hours) and lower (62 hours) positions, respectively (Fig. 4). Sheep manure was heated to more than 60 oC in the first 3 to 9 days of the test (120 hours), but after this period, the temperatures did not exceed 50oC (Fig. 2). Mean daily temperatures (47.4oC) were significantly (P=0.00) higher in sheep manure than in the composts at the middle (38.8oC±4.27, S.D. 9,3) and lower (33.2oC±4,8, S.D. 10,5) positions; however, the temperatures were lower (44.1oC±7.39, S.D. 16.3) at the upper position of compost as well (Fig. 4). Mean hourly and daily temperatures of the composts and in the sheep manure are presented in Fig. 3 and Fig. 4. \ 9 3 n t e i s « io n «13 u mi v in il zo ii aa —mvatnstan ----Mlttf S pWUen--Li»«r msm