- Email: [email protected]
Thermogenesis in decomposing carcasses
Forensic Science International 231 (2013) 271–277
Contents lists available at SciVerse ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Thermogenesis in decomposing carcasses Aidan P. Johnson *, Katarina M. Mikac, James F. Wallman Institute for Conservation Biology and Environmental Management, School of Biological Sciences, University of Wollongong, New South Wales 2522, Australia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 December 2012 Received in revised form 25 May 2013 Accepted 28 May 2013 Available online
It is of fundamental importance in forensic entomology that the factors controlling carcass temperatures during decomposition are thoroughly understood. The thermal environment to which fly larvae are exposed is the primary influence on their growth rate, and hence affects any estimate of minimum time since death using such specimens in homicide investigations. To date, much of the entomological research on maggot masses has focused on their elevation of carcass temperatures, with very little focus on the bacteria associated with larval activity. The aim of this study was to investigate the heat associated with decay and the types of bacteria present during the decomposition of a carcass, both in the presence and in the absence of maggots. Three treatments were imposed: fresh, frozen and maggotinfested, each consisting of five replicate pig carcasses. Temperature measurements and bacterial swabs were taken from the gastro-intestinal region of each pig and temperatures and bacterial communities compared between treatments. All carcasses reached average maximum temperatures above 32 8C in a temperature controlled room set at 23 8C. Treatment had no statistically significant effect on the temperatures recorded in each carcass but did significantly affect the community structure of the bacteria. However, bacterial community structure varied across time. This study suggests that bacterial metabolism plays a significant role in carcass thermogenesis, and that maggot masses, while contributing to localised heating within the carcass, may have less of a role in elevating carcass temperatures than previously assumed. ß 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Bacteria Decomposition Heating Maggot mass
1. Introduction The heat generated during decomposition is an important factor in forensic casework, since temperature determines the speed of many of the processes on which minimum post-mortem interval estimations are based, both in pathology and in entomology [1]. Forensic entomology practitioners are focusing increasingly on maggot mass heating in corpses in order to better understand this phenomenon and provide more accurate measures of the minimum time since death [2–6]. However, bacteria associated with the decay of corpses are rarely discussed, despite some authors (e.g. [6–9]) acknowledging that such bacteria could play a role in the heating of carcasses. There is a well-known link between the decomposition of vegetative material and heating due to bacterial metabolism [10–12]; however, this phenomenon has not been specifically examined in reference to the decomposition of carcasses and its forensic application. Such information is needed to predict when, and the degree to which, the temperatures in a carcass will be elevated above ambient.
* Corresponding author. Tel.: +61 2 4221 4117; fax: +61 2 4221 4135. E-mail address: [email protected] (A.P. Johnson). 0379-0738/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.forsciint.2013.05.031
When microorganisms break down organic matter, they produce carbon dioxide, water and heat [10]. During the initial stages of decomposition, mesophilic bacteria are the dominant decomposers and remain active up to temperatures of about 40 8C, at which point the thermophilic bacteria replace them [11,12]. Thermophilic bacteria can survive at temperatures up to about 60 8C and many have the ability to form endospores that are able to lie dormant through heat, cold, and lack of food and water, becoming active when conditions are favourable [10]. Interestingly, even though bacteria play a prominent role in carcass decomposition [6], previous studies that have featured carcasses uninfested with fly larvae have suggested that the temperatures of such carcasses do not differ significantly from ambient [7,9,13]. Bacteria multiply vigorously in decomposing meat, and are thought to be in greater abundance in the presence of maggot masses [6,8]. Whether bacteria play any role in carcass heating in conjunction with such masses, or simply utilise heat generated by the maggots, is unknown [8]. It is also unknown whether the high temperatures recorded among developing maggots might be due to bacterial metabolism arising from mesophilic bacteria or the activation of thermophilic endospores. Therefore, the aim of the current study was to investigate the heat associated with the decay of carrion in the presence and absence of
272
A.P. Johnson et al. / Forensic Science International 231 (2013) 271–277
maggots and to document the types of bacteria associated with this process. 2. Methods 2.1. General experimental setup and sampling protocol To investigate the types of bacteria present during the decomposition of a carcass, 15 pigs, weighing between 6 and 12 kg, were used. Three treatments, fresh, frozen and maggot-infested, were investigated, with five replicate pigs in each. All pigs died of natural causes and, upon death, were immediately stored in airtight barrels and kept cool to prevent premature decay and infestation by flies. All experiments were commenced within three days of death and pigs were randomly allocated to one of the three treatments. Five of the pigs were frozen and then defrosted for the frozen treatment, while the other 10 were fresh (unfrozen). The maggot-infested treatment was established using eggs of the hairy maggot blow fly, Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae). Approximately 1000 eggs were added daily to the mouth of five of the fresh pigs, for the first three days of the experiment, to ensure that a sizeable maggot mass developed within each carcass. The remaining five fresh carcasses provided an uninfested treatment as a control for the other two treatment groups. This allowed for a comparison of the bacteria present in carcasses both infested and uninfested with maggots, as well as the effects of freezing on the bacteria. In addition to the types of bacteria present in these three treatments, any heat generated during the decomposition of the carcasses in these treatments was also recorded. Each pig was positioned in a 970 mm 500 mm 300 mm cardboard box lined with plastic sheeting, on a layer of wheaten chaff 100 mm deep. The sheeting and chaff helped contain the decomposition fluids emanating from the pigs during their decay. A 600 mm length of aluminium tube, with a diameter of 12 mm, was inserted approximately 450 mm into the gastro-intestinal region of each pig via the anus and left in position during decomposition. The tube provided access to the interior of the pig for the purpose of taking bacterial swabs and recording core body temperature. All pig carcasses were maintained at 23 8C in a constant temperature room, with a separate room for each treatment to prevent any cross contamination, particularly from stray maggots. To collect bacterial samples, a sterilised swab (Epicentre, USA) was inserted into each pig via the aluminium tube. A sterile temperature probe was also inserted into the aluminium tube to record the internal temperature at the time of bacterial collection. The swabs were then cultured in liquid Luria broth (LB) and incubated at 37 8C for 24 h using standard culturing practices [14]. Sampling was conducted daily for 14 d in the maggot-infested pigs, by which time the flies’ life cycle had been completed; and every second day for 28 d for the non-infested pigs. An additional swab was taken from each of the maggot-infested pigs. This swab was taken within the most prominent maggot mass to identify the bacteria specifically associated with the larvae, rather than those that may only have been external to the mass in the carcass. This sampling was done after seven days, once the mass in each carcass was well established and starting to invade the torso. 2.2. Temperature analysis and phase selection To determine the key bacterial communities to target for further analysis, the temperatures from the main experiment were graphed over time and four days were selected to represent key thermal phases. These phases were: (1) initial: the start of the experimental period; (2) pre-heating: the day before any additional heating was seen within a pig; (3) peak-heating: the day on which the highest temperatures were reached; and 4) final: the day at the end of the experimental period, when heating had ceased. A parametric F-test was used to test for a difference between the mean curves of temperature against time for each treatment (fresh, frozen and maggot-infested).
2.3. Infrared monitoring of a maggot-infested pig An additional maggot-infested pig was set up as above, with a temperature probe inserted into the gastro-intestinal region via the anus and left permanently in place to record temperature every 15 min. The whole pig was also photographed using an infrared camera (S65 ThermaCAM, FLIR, Australia) every 30 min. This arrangement was used to provide a supplementary comparison of core carcass temperatures (as recorded above) with temperatures of maggot masses over time. 2.4. Bacterial diagnostics The DNA of the bacteria from the LB liquid culture was extracted using the QuickExtractTM Bacterial DNA Extraction Kit (Epicentre, USA) and the One-Tube Bacterial Genomic DNA Extraction Kit (Bio Basic Inc, Canada), following the manufacturer’s specifications. The second DNA extraction kit was used because the first kit became unavailable part way through our research. The 16S rRNA region was targeted for bacterial identification, as this is the most readily sequenced region of bacteria for which an extensive and accessible database of sequences exists that can be searched and compared [15].
GoTaq Master Mix (Promega, USA) was used at a final concentration of 1 with 16S rRNA primers (F63-D4: 50 -CAGGCCTAACACATGCAAGTC-30 ; R1389: 50 AGCGGCGGTGTGTACAAG-30 ). Primers were used at 0.4 mM of each final concentration. PCR amplifications were based on the conditions outlined by Smith et al. [16], consisting of: an initial denaturation step of 94 8C for 2 min; followed by 27 cycles at 94 8C for 45 s, 55 8C for 1 min and 74 8C for 2 min; and 1 cycle at 74 8C for 7 min. Quality control was practised as follows: (1) all water was nano-purified and autoclaved to minimise contamination with non-target DNA that could produce artefacts during PCR; (2) PCR assays were carried out using filter tips to eliminate the possibility of aerosol contamination from carryover products; and (3) negative controls were included in all PCR assays. PCR products were visualised using 1 TBE (Tris/Borate/EDTA) and 1% agarose gels (stained with 3 GelRed nucleic acid stain (Biotium, USA)) and electrophoresed for 60 min at 80 V. PCR fragment size was inferred by comparison with a 100 base pair (bp) size standard (New England Biolabs, UK). To minimise user-selected bias during culturing, bacteria were indiscriminately amplified by PCR. Multiple PCR bands or fragments, possibly representing multiple bacterial strains, were excised from 1 TAE (Tris/Acetic/EDTA) and 1% agarose gels (also stained with 3 GelRed nucleic acid stain (Biotium, USA)) that were electrophoresed for 60 min at 80 V. Excised fragments were cleaned using the Spin Column DNA Gel Extraction Kit (Bio Basic Inc, Canada) following the manufacturer’s protocol. PCR products were not cloned prior to sequencing. Rather, each discrete PCR fragment (visible on the TAE agarose gels) was excised from the gel (as above), purified and sequenced. Purified PCR products were then commercially sequenced by Macrogen Inc. (Korea). Macrogen Inc. conduct standard capillary sequencing using a high throughput Applied Biosystems 3730XL DNA analyser and the Applied Biosystems BigDye1 Terminator. Bacterial sequences were subjected to BLAST searches (using the blastn algorithm) of the 16S ribosomal RNA sequences (Bacteria and Archaea) database in GenBank, using sequences > 550 bp in length and with sequence matches (maximum identity) > 80%. Using the aforementioned criteria, bacteria were identified to genus by retaining the highest match for each sequence. 2.5. Analysis of bacterial communities Non-parametric multivariate techniques were used to determine differences among pig carcasses based on the presence or absence of bacteria according to treatments and time. The multivariate analyses of PERMANOVA (permutational multivariate ANOVA) and SIMPER (similarity percentage) were conducted using PRIMER V6.0 [17]. To examine multivariate differences in treatment and time a PERMANOVA was used. A PERMANOVA design was created with three factors: ‘treatment’ and ‘thermal phase’, which were fixed effects, and ‘pig’, which was a random factor nested within ‘treatment’. The PERMANOVA was run initially with the default program and parameters. The PERMANOVA was then run again using the ‘pairwise test’ to determine where significant differences occurred. A two-way crossed SIMPER [18] analysis, examining the factors ‘treatment’ and ‘thermal phase’, was run with a 90% cut-off imposed that was based on the Bray– Curtis similarity matrices (presence or absence of bacteria). The SIMPER was used to identify the bacteria that were most important in contributing to treatment and time differences.
3. Results 3.1. Temperature analysis and phase selection The F-test revealed no significant difference between the three mean temperature curves for each treatment (F21,169 = 0.101, P = 1.00). Average initial carcass temperatures were approximately the same as room temperature (23 8C), with temperatures gradually increasing through the pre-heating phase to an average peak heating of 32 8C, before returning to just above ambient (24 8C) during the final thermal phase (Fig. 1). Maximum temperatures above 40 8C were achieved in some pigs in both the fresh treatment and the maggot-infested treatment, while one pig from each of the fresh and frozen treatments failed to heat at all. Based on this thermal profile, the four thermal phases were defined as Day 0 (initial), Day 8 (preheating), Day 12 (peak-heating), and Day 20 (final). It should be noted that there was no final thermal phase for the maggot-infested pigs, as, by day 14, maggots had finished feeding, pupated and the resultant flies had eclosed. Within the maggot-infested pigs, the Ch. rufifacies maggots had moved from the head into the torso by Day 6. However, many maggots preferred to mass around the edges of each carcass rather than directly within it, so it is unlikely that these core temperature
A.P. Johnson et al. / Forensic Science International 231 (2013) 271–277
273
45
40
35
Temperature (ºC)
30
25
20 Fresh
15
Frozen Maggot
10
Average fresh Average frozen
5
Average maggot 0
0
5
10
15
20
25
Day Fig. 1. Daily temperature recorded in each pig throughout the experimental period. Lines represent the mean daily temperature for each treatment.
recordings reflected the maximum temperatures among the maggots. 3.2. Infrared monitoring of a maggot-infested pig The internal probe placed in the single pig studied with the infrared camera recorded similar heating patterns to those noted above, with temperatures peaking at 42 8C after Day 7, slightly earlier than the average peak seen in the main experiment. The infrared images revealed maximum carcass temperatures of 42 8C after only 3 days, which declined steadily, aligning with the internal temperatures after Day 8. This initial peak temperature was seen in the region of the mouth, where the maggot mass was created. Infrared images revealed that the position of the maximum carcass temperatures shifted gradually into the main body cavity of the pig, coinciding with the movement of the maggot mass into this region. 3.3. Bacterial diagnostics Based on the criteria used for diagnosis, this study identified ten genera of bacteria within the three treatments of decomposing pigs: Bacteroides, Budvicia, Escherichia, Ignatzschineria, Morganella, Proteus, Providencia, Serratia, Shigella and Wohlfahrtiimonas. No genera were unique to any one treatment and none of these genera were found in all 15 pigs. However, Budvicia, Escherichia, Morganella, Proteus, Providencia and Shigella were found in at least two of the pigs in all three treatments. The percentage of pigs in which each genus was found varied across the four thermal phases (Fig. 2). Ignatzschineria and Wohlfahrtiimonas were identified in the maggot mass in each of the five maggot-infested pigs. Serratia was also identified in one of these masses. 3.4. Analysis of bacterial communities The PERMANOVA analysis revealed that the effect of treatment on the presence of bacterial genera did not differ significantly between thermal phases (Pseudo-F5,32 = 1.37, P = 0.149). There were significant differences between replicate pigs in the types of
bacteria found within all treatments (Pseudo-F12,32 = 1.55, P = 0.020). There was also a significant difference in the types of bacteria found between treatments (Pseudo-F2,32 = 2.42, P = 0.038). The pairwise test revealed that the fresh, non maggot-infested treatment was not significantly different from the frozen (t = 1.10, P = 0.29) or maggot-infested (t = 1.33, P = 0.13) treatments. The frozen and maggot-infested treatments were, however, significantly different (t = 0.26, P = 0.001). SIMPER revealed that treatments had average similarities of 16%, 39% and 44% within fresh, frozen and maggot-infested pigs, respectively (Table 1). Significant differences in bacterial genera were found between thermal phases (Pseudo-F3,32 = 5.04, P = 0.001). The pairwise test revealed all thermal phases to be significantly different from each other, except the peak-heating and final phases (t = 1.20, P = 0.26). SIMPER average similarity values of 53%, 27%, 25% and 20% were calculated for the initial, pre-heating, peak heating and final thermal phases, respectively (Table 2). The bacterial genus Proteus was a major contributor to the similarity within the first two phases, while Providencia dominated the final two phases. Morganella was prominent during pre-heating and remained prominent until the final phase. Escherichia and Shigella were both prominent in the initial and peak-heating phases, but were much less dominant during the pre-heating and final phases. Budvicia was another contributor to the peak-heating phase (Fig. 2).
4. Discussion This is the first study to show experimentally that exposed carcasses are capable of heating without any thermal contribution from maggot masses, solar radiation or any other external heat source. This suggests that bacterial metabolism during decomposition causes the bulk of such heat, and that maggot masses, while contributing to localised heating within the mass, may have less of a role in carcass thermogensis than previously assumed. Given this finding, it is imperative to understand the nature of the bacteria involved. As such, our study also documented the bacterial genera
A.P. Johnson et al. / Forensic Science International 231 (2013) 271–277
274
Fig. 2. Percentage of pig carcasses with specified bacteria present across the four thermal phases: (1) initial; (2) pre-heating; (3) peak-heating; and (4) final.
present at key points along the thermal profile throughout the decomposition process. 4.1. Carcass temperatures The thermal profiles for all treatments were not significantly different, with average peak temperatures above 30 8C across all treatments. It is worth noting that temperatures above 40 8C
occurred not only in the maggot-infested pigs, but also in some of the fresh (uninfested) pigs. However, it cannot be discounted that maggots add additional heat to the system, as seen in the supplementary pig monitored using infrared imaging. Mass temperatures as high as 42 8C (19 8C above ambient) were generated within three days in the mass created in the mouth of this pig, compared to the core temperature, which took seven days to reach temperatures of equivalent elevation.
A.P. Johnson et al. / Forensic Science International 231 (2013) 271–277 Table 1 The average similarities within each treatment (fresh, frozen and maggot-infested), and the main contributing genera of bacteria as determined by SIMPER. Treatment
Genus
Fresh
Shigella Escherichia Proteus Providencia
Average similarity 7.50 3.75 2.92 1.67
Contribution % 47.37 23.68 18.42 10.53
Frozen
Escherichia Proteus Shigella Morganella
12.17 9.38 8.42 7.96
30.83 23.74 21.33 20.17
Maggot-infested
Morganella Proteus Providencia
19.33 15.00 5.22
44.39 34.44 11.99
Another study that revealed heating during decomposition in the absence of maggots is that of Rodriguez and Bass [19], who measured the temperatures of buried human bodies. These workers found that bodies buried 1.2 and 0.6 m deep showed no signs of insect activity, but began heating after 4 and 2 weeks, respectively. These corpses heated approximately 10 and 20 8C above the surrounding soil temperatures. These temperature elevations were similar to those noted in the current study, although the time until peak heating was greatly increased. Rodriguez and Bass [19] stated that lower environmental temperatures and fluctuating pH and oxygen levels should decrease bacterial metabolic heating, which may explain the delay in heating observed in their study. While these authors highlighted the need for greater understanding of temperatures in buried cadavers, there is clearly an even more pressing need to understand bacteria-induced heating in bodies above ground. The role of insulation in maintaining temperatures throughout the heating process could be the key to scenarios in which heating is observed in decomposing carrion. In the case of the increased temperature noted in the abovementioned buried bodies [19], the surrounding soil would have insulated the corpse and thus aided in heat retention during bacterial metabolism. In insect-infested remains it is also likely that maggot masses act as insulators, trapping heat within the system and allowing the build up of metabolic heat. In addition to providing insulation from the surrounding environment, the constant movement of maggots through the carcass could aid in aerating the decaying material to ensure that adequate oxygen levels are maintained and aerobic decay continues [20]. When obligate or facultative anaerobes are present, anaerobic decay can continue in the absence of oxygen. However, anaerobic decay lacks the high temperatures generated
Table 2 The average similarities within each temperature phase, and the main contributing genera of bacteria as determined by SIMPER. Thermal phase
Genus
Average similarity
Contribution %
Initial
Shigella Proteus Escherichia
19.44 18.89 14.44
36.84 35.79 27.37
Pre-heating
Morganella Proteus
15.00 11.67
56.25 43.75
Peak-heating
Escherichia Morganella Providencia Shigella Budvicia
8.12 5.29 5.22 3.12 2.29
32.65 21.26 21.00 12.54 9.19
Final
Morganella Providencia
14.50 3.33
74.36 17.09
275
during aerobic decay and thus the aeration of carcasses is essential for heating to occur [20,21]. This has important implications not only for our understanding of how maggot mass heat is generated, but also the factors that might influence the temperatures achieved within these masses, such as mass volume, maggot density and maggot movement, as well as the size and position of the carcass. It is important to note that, during the current study, one pig carcass in each of the fresh and frozen treatments failed to heat. This lack of carcass heating in the absence of maggots has also been noted in the few studies in which such conditions have been met [7,9,13], but the underlying reason for this is unknown. Deonier [9] suggested that an uninfested goat carcass in winter did not heat due to bacterial metabolism being suppressed at low ambient temperatures. Wallman [7] also failed to note any appreciable heat in an uninfested pig carcass studied in autumn, but in this case ambient temperatures fluctuated within the relatively mild range of 10–25 8C. Low temperatures also do not explain the lack of heating in the two pigs in our study, as all pigs were exposed to a constant temperature of 23 8C. Since maggots were not present in either of the non-heating carcasses, internal decay may have been predominantly anerobic rather than aerobic due to the lack of aeration of the carcass by the fly larvae. 4.2. Bacterial communities All of the bacteria found in these carcasses are mesophilic, with some known to be associated with the decay of vegetative material [11,12]. Mesophilic bacteria generally dominate during the initial part of the decay process and remain active, and hence continue producing heat, up to temperatures of around 40 8C. At temperatures above 40 8C thermophiles replace mesophiles and continue the heating process [10–12]. No thermophilic bacteria were identified within any of the carcasses, but since temperatures in the pigs reached, but did not exceed 40 8C, it seems that temperature did not limit the activity of the mesophiles. The two bacterial genera associated with the maggot masses, Ignatzschineria and Wohlfahrtiimonas, are of the family Xanthomonadaceae. They have both been recorded in association with the larvae of the flesh fly Wohlfahrtia magnifica, particularly in cases of myiasis of both humans and other animals [22–24]. While this genus of flesh fly is not found in Australia, these bacterial taxa may have a broader association with other carrion fly larvae. The bacterial genera identified as diagnostic of the thermal phases (Fig. 2) were Budvicia, Escherichia, Morganella, Proteus, Providencia and Shigella. These are all gram-negative bacteria of the family Enterobacteriaceae. This is unsurprising, given that enterobacteria are facultative anaerobes generally associated with the intestines of vertebrates [25]. While Shigella and Escherichia have been separated historically into different genera, there are few biochemical properties that can be used to distinguish them. Many bacteriologists suggest that they should be considered a single genus due to their high degree of nucleotide sequence similarity [26,27]. Their presence throughout the four thermal phases of the current study reflects this close link, with both also being strong contributors to the similarity between the initial and peak heating phases. The genera Proteus, Morganella and Providencia are all quite closely related and are considered to be opportunistic pathogens [25]. Despite this close relationship, the three genera were found to have unique thermal phase preferences. Proteus was a major contributor to the initial and pre-heating phases, while Providencia was a major contributor to the peak-heating and final phases and not diagnostic of the initial and pre-heating phase. Morganella appeared in sequence between these two genera, being a major contributor to the preand peak heating phases, as well as the final phase.
276
A.P. Johnson et al. / Forensic Science International 231 (2013) 271–277
This study utilised 16S rRNA gene sequences for identifying the bacteria present because this is the most common genetic marker used in bacterial taxonomy and phylogenetics [28]. 16S rRNA gene sequencing has successfully identified genera in over 90% of studies reviewed by Janda and Abbot [28], but is less discriminating at the level of species (for 65–83% of these studies). Some of the difficulties in reliably identifying the species of a given bacterium are the occurrence of undescribed taxa or putative species sharing similar or identical 16S rRNA sequences [28]. Additional difficulties arise from the use of databases such as the GenBank 16S ribosomal RNA database (Bacteria and Archaea) [29], the EzTaxon-e server [30] or the Ribosomal database project [31], in which sequences may be incomplete or erroneous. It is due to these recognised issues in bacterial identification that our study did not examine the taxonomy of bacterial taxa below the level of genus. There was no significant difference between the bacterial genera present in the fresh, non maggot-infested pigs compared to the maggot-infested pigs. This means that, while maggot infestation increases decomposition, it does not appear to affect the bacterial community within the carcass. However, flies are known vectors of many pathogenic bacteria, including Escherichia and Proteus [32]. These bacteria are consumed during development by maggots, survive the pupation process and are present in the emergent adults, which then act as dispersers of the bacteria [33]. In this study the adult flies did not lay the eggs directly on the carcasses, and so they would not have been able to spread bacteria and act as vectors. Since Ch. rufifacies eggs were transferred onto the carcasses and maggots had not yet been exposed to bacteria from other sources, the introduction of bacteria into the system would have been minimal. The results also revealed no significant differences in bacterial composition between the fresh and frozen carcasses, although the frozen and maggot-infested carcasses did differ. The former finding is at odds with Micozzi [34], who found frozen-thawed carcasses to be more susceptible to invasion by both insects and microorganisms than fresh carcasses. Caution should therefore continue to be exercised by both forensic entomologists and anthropologists when using frozen carcasses for experimental work. With future advances in the taxonomic resolution of bacteria, it is possible that bacterial species could have utility as diagnostic tools for identifying minimum time since death. Some preliminary work using bacteria for estimating death times has already been carried out, examining particular species rather than focusing on the overall community structure [35]. There is also need for research that examines carcasses that are free of bacteria, in order to strengthen the link between bacteria and heating. This could be done by gamma irradiation of the carcasses and their subsequent maintenance in a bacteria-free room (or laminar flow hood). Furthermore, irradiated carcasses could be selectively contaminated with specific bacteria, either aerobic or anerobic, to examine their role in the heating and decay process.
5. Conclusions This study is the first to show a significant increase in carcass temperature in the absence of maggot masses or solar radiation. It has highlighted the need to better understand the contribution of bacteria to carcass heating and define the thermogenic contribution of maggot masses. This study has also provided a preliminary assessment of the bacterial genera involved in carcass decay. However, quantification of the density of these bacteria, and more specific identification of them, should help determine not only how bacteria affect carcass heating but also improve our understanding of the role of maggot masses in promoting or suppressing bacterial activity.
Ethical standards All pig carcasses used in this study died of natural causes. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements This project was funded by ARC Linkage Grant LP0883711. We thank the Australian Federal Police and the NSW Police Force for their financial support, and the Victorian Institute of Forensic Medicine, Victoria Police and Forensic Science SA for their in-kind support. We are also grateful to Windridge Pig Farm, Young, NSW, for providing the pig carcasses, Axton Aguiar, Jacinta Rice and Samuel Wighton for their assistance with transporting the pigs and Stephanie Ivkosic for assisting with the bacterial culturing and DNA extractions. Statistical assistance was provided by Kristine French. References [1] G. MacMaster, Environmental Forensics and its Effects on Investigation, PageFree Publishing Inc, Michigan, 2006. [2] D.H. Slone, S.V. Gruner, Thermoregulation in larval aggregations of carrionfeeding blow flies (Diptera: Calliphoridae), J. Med. Entomol. 44 (2007) 516–523. [3] A.P. Johnson, M.S. Archer, L. Leigh-Shaw, M. Pais, C. O’Donnell, J.F. Wallman, Examination of forensic entomology evidence using computed tomography scanning: case studies and refinement of techniques for estimating maggot mass volumes in bodies, Int. J. Legal Med. 126 (2012) 693–702. [4] A.P. Johnson, M.S. Archer, L. Leigh-Shaw, M. Brown, C. O’Donnell, J.F. Wallman, Non-invasive visualisation and volume estimation of maggot masses using computed tomography scanning, Int. J. Legal Med. 127 (2013) 185–194. [5] T.J. Cianci, J.K. Sheldon, Endothermic generation by blow fly larvae Phormia regina developing in pig carcasses, Bull. Soc. Vector Ecol. 15 (1990) 33–40. [6] M.L. Goff, A Fly for the Prosecution: How Insect Evidence Helps Solve Crimes, Harvard University Press, London, 2000. [7] J.F. Wallman, Systematics and Thermobiology of Carrion-breeding Blowflies (Diptera: Calliphoridae), The University of Adelaide, Adelaide, 1999(PhD Thesis). [8] B. Greenberg, J.C. Kunich, Entomology and the Law, Cambridge University Press, Cambridge, 2002. [9] C.C. Deonier, Carcass temperatures and their relation to winter blowfly populations and activity in the Southwest, J. Econ. Entomol. 33 (1940) 166–172. [10] N. Trautmann, E. Olynciw, Compost Microorganisms, in: Cornell Composting, Cornell Waste Management Institute, Ithaca, 1996, Available at: http://compost.css.cornell.edu/microorg.html (accessed 02.09.12). [11] S. MacGregor, F. Miller, K. Psarianos, M. Finstein, Composting process control based on interaction between microbial heat output and temperature, Appl. Environ. Microb. 41 (1981) 1321–1330. [12] J. Ryckeboer, J. Mergaert, K. Vaes, S. Klammer, D. De Clercq, J. Coosemans, H. Insam, J. Swings, A survey of bacteria and fungi occuring during composting and self-heating processes, Ann. Microbiol. 53 (2003) 349–410. [13] G. Wobeser, E.A. Galmut, Internal temperature of decomposing duck carcasses in relation to botulism, J. Wildlife Dis. 20 (1984) 267–271. [14] M.R. Green, J. Sambrook (Eds.), Molecular Cloning: A Laboratory Manual, fourth ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2012. [15] S. Altschul, T. Madden, A. Scha¨ffer, J. Zhang, Z. Zhang, W. Miller, D. Lipman, Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res. 25 (1997) 3389–3402. [16] C.J. Smith, B.S. Danilowicz, A.K. Clear, F.J. Costello, B. Wilson, W.G. Meijer, T-Align, a web-based tool for comparison of multiple terminal restriction fragment length polymorphism profiles, FEMS Microbiol. Ecol. 54 (2005) 375–380. [17] K.R. Clarke, R.N. Gorley, PRIMER v6: User Manual/Tutorial, PRIMER-E, Plymouth, 2006. [18] K.R. Clarke, Non-parametric multivariate analyses of changes in community structure, Aust. J. Ecol. 18 (1993) 117–143. [19] W.C. Rodriguez, W.M. Bass, Decomposition of buried bodies and methods that may aid in their location, J. Forensic Sci. 30 (1985) 836–852. [20] M. Baker, B. Knoop, S. Quiring, A. Beard, B. Lesikar, J. Sweetan, R. Burns, The Decomposition Process, in: Don’t Bag ItTM – Compost It!!, Texas A&M University System, College Station, 2009, Available at: http://aggie-horticulture.tamu.edu/ earthkind/landscape/dont-bag-it/chapter-1-the-decomposition-process/ (accessed 13.08.12). [21] J. Peirce, R. Weiner, P. Vesilind (Eds.), Environmental Pollution and Control, fourth ed., Butterworth-Heinemann, Oxford, 1998. [22] E. To´th, G. Kava´cs, P. Schumann, A. Kova´cs, U. Steiner, A. Halbritter, K. Ma´rialigeti, Schineria larvae gen. nov., sp. nov., isolated from the 1st and 2nd larval stages of
A.P. Johnson et al. / Forensic Science International 231 (2013) 271–277
[23]
[24] [25] [26]
[27]
[28]
Wohlfahrtia magnifica (Diptera: Sacophagidae), Int. J. Syst. Evol. Microbial. 51 (2001) 401–407. E. To´th, P. Schumann, A. Borsodi, Z. Ke´ki, A. Kova´cs, K. Ma´rialigeti, Wohlfahrtiimonas chitiniclastica gen. nov., sp. nov., a new gammaproteobacterium isolated from Wohlfahrtia magnifica (Diptera: Sarcophagidae), Int. J. Syst. Evol. Microbial. 58 (2008) 976–981. S. Rebaudet, S. Genot, A. Renvoise, P. Fournier, A. Stein, Wohlfartiimonas chitiniclastica bacteremia in homeless woman, Emerg. Infect. Dis. 15 (2009) 985–986. C.M. O’Hara, F.W. Brenner, J.M. Miller, Classification, identification, and clinical significance of Proteus, Providencia, and Morganella, Clin. Microbiol. Rev. 13 (2000) 534–546. M. Dworkin, S. Falkow, E. Rosenberg, K. Schleifer, E. Stackebrandt, Proteobacteria: gamma subclass, in: M. Dworkin (Ed.), third ed., The Prokaryotes: A Handbook on the Biology of Bacteria, vol. 6, Springer, New York, 2006. F. Yang, J. Yang, X. Zhang, L. Chen, Y. Jiang, Y. Yan, X. Tang, J. Wang, Z. Xiong, J. Dong, Y. Xue, Y. Zhu, X. Xu, L. Sun, S. Chen, H. Nie, J. Peng, J. Xu, Y. Wang, Z. Yuan, Y. Wen, Z. Yao, Y. Shen, B. Qiang, Y. Hou, J. Yu, Q. Jin, Genome dynamics and diversity of Shigella species, the etiologic agents of bacillary dysentery, Nucleic Acids Res. 33 (2005) 6445–6458. J. Janda, S. Abbot, Minireview: 16S rRNA gene sequencing for bacterial identification in the diagnostic laboratory: pluses, perils, and pitfalls, J. Clin. Microbiol. 45 (2007) 2761–2764.
277
[29] D.A. Benson, I. Karsch-Mizrachi, D.J. Lipman, J. Ostell, D.L. Wheeler, GenBank, Nucleic Acids Res. 1 (2006) 16–20. [30] O.S. Kim, Y.J. Cho, K. Lee, S.h. Yoon, M. Kim, H. Na, S.C. Park, Y.S. KJeon, J.H. Lee, H. Yi, S. Won, J. Chun, Introducing EzTaxon-e: a prokaryotic 16S rRNA Gene sequence database with phylotypes that represent uncultured species, Int. J. Syst. Evol. Microbial. 62 (2012) 716–721. [31] J.R. Cole, Q. Wang, E. Cardenas, J. Fish, B. Chai, R.J. Farris, A.S. Kulam-SyedMohideen, D.M. McGarrel, T. Marsh, G.M. Garrity, J.M. Tiedje, The ribosomal database project: improved alignments and new tools for rRNA analysis, Nucleic Acids Res. 37 (2009) 141–145. [32] Q. Ma, A. Fonseca, W. Liu, A.T. Fields, M.L. Pimsler, A.F.T. Spindola, A.M. Crippen, T.L.J.K. Tomberlin, T.K. Wood, Proteus mirabilis interkingdom swarming signals attract blow flies, Int. Soc. Microbial Ecol. J. 6 (2012) 1356–1366. [33] A. Ahmad, A. Broce, L. Zurek, Evaluation of significance of bacteria in larval development of Cochliomyia macellaria (Diptera: Calliphoridae), J. Med. Entomol. 43 (2006) 1129–1133. [34] M.S. Micozzi, Experimental study of postmortem change under field conditions: effects of freezing, thawing, and mechanical injury, J. Forensic Sci. 31 (1986) 953–961. [35] I.C.F. Corcobado, La Microbiologia forense se renueva: bioindicadores de data de muerte, Microbiologia 20 (2009) 16–18.
Contents lists available at SciVerse ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Thermogenesis in decomposing carcasses Aidan P. Johnson *, Katarina M. Mikac, James F. Wallman Institute for Conservation Biology and Environmental Management, School of Biological Sciences, University of Wollongong, New South Wales 2522, Australia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 December 2012 Received in revised form 25 May 2013 Accepted 28 May 2013 Available online
It is of fundamental importance in forensic entomology that the factors controlling carcass temperatures during decomposition are thoroughly understood. The thermal environment to which fly larvae are exposed is the primary influence on their growth rate, and hence affects any estimate of minimum time since death using such specimens in homicide investigations. To date, much of the entomological research on maggot masses has focused on their elevation of carcass temperatures, with very little focus on the bacteria associated with larval activity. The aim of this study was to investigate the heat associated with decay and the types of bacteria present during the decomposition of a carcass, both in the presence and in the absence of maggots. Three treatments were imposed: fresh, frozen and maggotinfested, each consisting of five replicate pig carcasses. Temperature measurements and bacterial swabs were taken from the gastro-intestinal region of each pig and temperatures and bacterial communities compared between treatments. All carcasses reached average maximum temperatures above 32 8C in a temperature controlled room set at 23 8C. Treatment had no statistically significant effect on the temperatures recorded in each carcass but did significantly affect the community structure of the bacteria. However, bacterial community structure varied across time. This study suggests that bacterial metabolism plays a significant role in carcass thermogenesis, and that maggot masses, while contributing to localised heating within the carcass, may have less of a role in elevating carcass temperatures than previously assumed. ß 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Bacteria Decomposition Heating Maggot mass
1. Introduction The heat generated during decomposition is an important factor in forensic casework, since temperature determines the speed of many of the processes on which minimum post-mortem interval estimations are based, both in pathology and in entomology [1]. Forensic entomology practitioners are focusing increasingly on maggot mass heating in corpses in order to better understand this phenomenon and provide more accurate measures of the minimum time since death [2–6]. However, bacteria associated with the decay of corpses are rarely discussed, despite some authors (e.g. [6–9]) acknowledging that such bacteria could play a role in the heating of carcasses. There is a well-known link between the decomposition of vegetative material and heating due to bacterial metabolism [10–12]; however, this phenomenon has not been specifically examined in reference to the decomposition of carcasses and its forensic application. Such information is needed to predict when, and the degree to which, the temperatures in a carcass will be elevated above ambient.
* Corresponding author. Tel.: +61 2 4221 4117; fax: +61 2 4221 4135. E-mail address: [email protected] (A.P. Johnson). 0379-0738/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.forsciint.2013.05.031
When microorganisms break down organic matter, they produce carbon dioxide, water and heat [10]. During the initial stages of decomposition, mesophilic bacteria are the dominant decomposers and remain active up to temperatures of about 40 8C, at which point the thermophilic bacteria replace them [11,12]. Thermophilic bacteria can survive at temperatures up to about 60 8C and many have the ability to form endospores that are able to lie dormant through heat, cold, and lack of food and water, becoming active when conditions are favourable [10]. Interestingly, even though bacteria play a prominent role in carcass decomposition [6], previous studies that have featured carcasses uninfested with fly larvae have suggested that the temperatures of such carcasses do not differ significantly from ambient [7,9,13]. Bacteria multiply vigorously in decomposing meat, and are thought to be in greater abundance in the presence of maggot masses [6,8]. Whether bacteria play any role in carcass heating in conjunction with such masses, or simply utilise heat generated by the maggots, is unknown [8]. It is also unknown whether the high temperatures recorded among developing maggots might be due to bacterial metabolism arising from mesophilic bacteria or the activation of thermophilic endospores. Therefore, the aim of the current study was to investigate the heat associated with the decay of carrion in the presence and absence of
272
A.P. Johnson et al. / Forensic Science International 231 (2013) 271–277
maggots and to document the types of bacteria associated with this process. 2. Methods 2.1. General experimental setup and sampling protocol To investigate the types of bacteria present during the decomposition of a carcass, 15 pigs, weighing between 6 and 12 kg, were used. Three treatments, fresh, frozen and maggot-infested, were investigated, with five replicate pigs in each. All pigs died of natural causes and, upon death, were immediately stored in airtight barrels and kept cool to prevent premature decay and infestation by flies. All experiments were commenced within three days of death and pigs were randomly allocated to one of the three treatments. Five of the pigs were frozen and then defrosted for the frozen treatment, while the other 10 were fresh (unfrozen). The maggot-infested treatment was established using eggs of the hairy maggot blow fly, Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae). Approximately 1000 eggs were added daily to the mouth of five of the fresh pigs, for the first three days of the experiment, to ensure that a sizeable maggot mass developed within each carcass. The remaining five fresh carcasses provided an uninfested treatment as a control for the other two treatment groups. This allowed for a comparison of the bacteria present in carcasses both infested and uninfested with maggots, as well as the effects of freezing on the bacteria. In addition to the types of bacteria present in these three treatments, any heat generated during the decomposition of the carcasses in these treatments was also recorded. Each pig was positioned in a 970 mm 500 mm 300 mm cardboard box lined with plastic sheeting, on a layer of wheaten chaff 100 mm deep. The sheeting and chaff helped contain the decomposition fluids emanating from the pigs during their decay. A 600 mm length of aluminium tube, with a diameter of 12 mm, was inserted approximately 450 mm into the gastro-intestinal region of each pig via the anus and left in position during decomposition. The tube provided access to the interior of the pig for the purpose of taking bacterial swabs and recording core body temperature. All pig carcasses were maintained at 23 8C in a constant temperature room, with a separate room for each treatment to prevent any cross contamination, particularly from stray maggots. To collect bacterial samples, a sterilised swab (Epicentre, USA) was inserted into each pig via the aluminium tube. A sterile temperature probe was also inserted into the aluminium tube to record the internal temperature at the time of bacterial collection. The swabs were then cultured in liquid Luria broth (LB) and incubated at 37 8C for 24 h using standard culturing practices [14]. Sampling was conducted daily for 14 d in the maggot-infested pigs, by which time the flies’ life cycle had been completed; and every second day for 28 d for the non-infested pigs. An additional swab was taken from each of the maggot-infested pigs. This swab was taken within the most prominent maggot mass to identify the bacteria specifically associated with the larvae, rather than those that may only have been external to the mass in the carcass. This sampling was done after seven days, once the mass in each carcass was well established and starting to invade the torso. 2.2. Temperature analysis and phase selection To determine the key bacterial communities to target for further analysis, the temperatures from the main experiment were graphed over time and four days were selected to represent key thermal phases. These phases were: (1) initial: the start of the experimental period; (2) pre-heating: the day before any additional heating was seen within a pig; (3) peak-heating: the day on which the highest temperatures were reached; and 4) final: the day at the end of the experimental period, when heating had ceased. A parametric F-test was used to test for a difference between the mean curves of temperature against time for each treatment (fresh, frozen and maggot-infested).
2.3. Infrared monitoring of a maggot-infested pig An additional maggot-infested pig was set up as above, with a temperature probe inserted into the gastro-intestinal region via the anus and left permanently in place to record temperature every 15 min. The whole pig was also photographed using an infrared camera (S65 ThermaCAM, FLIR, Australia) every 30 min. This arrangement was used to provide a supplementary comparison of core carcass temperatures (as recorded above) with temperatures of maggot masses over time. 2.4. Bacterial diagnostics The DNA of the bacteria from the LB liquid culture was extracted using the QuickExtractTM Bacterial DNA Extraction Kit (Epicentre, USA) and the One-Tube Bacterial Genomic DNA Extraction Kit (Bio Basic Inc, Canada), following the manufacturer’s specifications. The second DNA extraction kit was used because the first kit became unavailable part way through our research. The 16S rRNA region was targeted for bacterial identification, as this is the most readily sequenced region of bacteria for which an extensive and accessible database of sequences exists that can be searched and compared [15].
GoTaq Master Mix (Promega, USA) was used at a final concentration of 1 with 16S rRNA primers (F63-D4: 50 -CAGGCCTAACACATGCAAGTC-30 ; R1389: 50 AGCGGCGGTGTGTACAAG-30 ). Primers were used at 0.4 mM of each final concentration. PCR amplifications were based on the conditions outlined by Smith et al. [16], consisting of: an initial denaturation step of 94 8C for 2 min; followed by 27 cycles at 94 8C for 45 s, 55 8C for 1 min and 74 8C for 2 min; and 1 cycle at 74 8C for 7 min. Quality control was practised as follows: (1) all water was nano-purified and autoclaved to minimise contamination with non-target DNA that could produce artefacts during PCR; (2) PCR assays were carried out using filter tips to eliminate the possibility of aerosol contamination from carryover products; and (3) negative controls were included in all PCR assays. PCR products were visualised using 1 TBE (Tris/Borate/EDTA) and 1% agarose gels (stained with 3 GelRed nucleic acid stain (Biotium, USA)) and electrophoresed for 60 min at 80 V. PCR fragment size was inferred by comparison with a 100 base pair (bp) size standard (New England Biolabs, UK). To minimise user-selected bias during culturing, bacteria were indiscriminately amplified by PCR. Multiple PCR bands or fragments, possibly representing multiple bacterial strains, were excised from 1 TAE (Tris/Acetic/EDTA) and 1% agarose gels (also stained with 3 GelRed nucleic acid stain (Biotium, USA)) that were electrophoresed for 60 min at 80 V. Excised fragments were cleaned using the Spin Column DNA Gel Extraction Kit (Bio Basic Inc, Canada) following the manufacturer’s protocol. PCR products were not cloned prior to sequencing. Rather, each discrete PCR fragment (visible on the TAE agarose gels) was excised from the gel (as above), purified and sequenced. Purified PCR products were then commercially sequenced by Macrogen Inc. (Korea). Macrogen Inc. conduct standard capillary sequencing using a high throughput Applied Biosystems 3730XL DNA analyser and the Applied Biosystems BigDye1 Terminator. Bacterial sequences were subjected to BLAST searches (using the blastn algorithm) of the 16S ribosomal RNA sequences (Bacteria and Archaea) database in GenBank, using sequences > 550 bp in length and with sequence matches (maximum identity) > 80%. Using the aforementioned criteria, bacteria were identified to genus by retaining the highest match for each sequence. 2.5. Analysis of bacterial communities Non-parametric multivariate techniques were used to determine differences among pig carcasses based on the presence or absence of bacteria according to treatments and time. The multivariate analyses of PERMANOVA (permutational multivariate ANOVA) and SIMPER (similarity percentage) were conducted using PRIMER V6.0 [17]. To examine multivariate differences in treatment and time a PERMANOVA was used. A PERMANOVA design was created with three factors: ‘treatment’ and ‘thermal phase’, which were fixed effects, and ‘pig’, which was a random factor nested within ‘treatment’. The PERMANOVA was run initially with the default program and parameters. The PERMANOVA was then run again using the ‘pairwise test’ to determine where significant differences occurred. A two-way crossed SIMPER [18] analysis, examining the factors ‘treatment’ and ‘thermal phase’, was run with a 90% cut-off imposed that was based on the Bray– Curtis similarity matrices (presence or absence of bacteria). The SIMPER was used to identify the bacteria that were most important in contributing to treatment and time differences.
3. Results 3.1. Temperature analysis and phase selection The F-test revealed no significant difference between the three mean temperature curves for each treatment (F21,169 = 0.101, P = 1.00). Average initial carcass temperatures were approximately the same as room temperature (23 8C), with temperatures gradually increasing through the pre-heating phase to an average peak heating of 32 8C, before returning to just above ambient (24 8C) during the final thermal phase (Fig. 1). Maximum temperatures above 40 8C were achieved in some pigs in both the fresh treatment and the maggot-infested treatment, while one pig from each of the fresh and frozen treatments failed to heat at all. Based on this thermal profile, the four thermal phases were defined as Day 0 (initial), Day 8 (preheating), Day 12 (peak-heating), and Day 20 (final). It should be noted that there was no final thermal phase for the maggot-infested pigs, as, by day 14, maggots had finished feeding, pupated and the resultant flies had eclosed. Within the maggot-infested pigs, the Ch. rufifacies maggots had moved from the head into the torso by Day 6. However, many maggots preferred to mass around the edges of each carcass rather than directly within it, so it is unlikely that these core temperature
A.P. Johnson et al. / Forensic Science International 231 (2013) 271–277
273
45
40
35
Temperature (ºC)
30
25
20 Fresh
15
Frozen Maggot
10
Average fresh Average frozen
5
Average maggot 0
0
5
10
15
20
25
Day Fig. 1. Daily temperature recorded in each pig throughout the experimental period. Lines represent the mean daily temperature for each treatment.
recordings reflected the maximum temperatures among the maggots. 3.2. Infrared monitoring of a maggot-infested pig The internal probe placed in the single pig studied with the infrared camera recorded similar heating patterns to those noted above, with temperatures peaking at 42 8C after Day 7, slightly earlier than the average peak seen in the main experiment. The infrared images revealed maximum carcass temperatures of 42 8C after only 3 days, which declined steadily, aligning with the internal temperatures after Day 8. This initial peak temperature was seen in the region of the mouth, where the maggot mass was created. Infrared images revealed that the position of the maximum carcass temperatures shifted gradually into the main body cavity of the pig, coinciding with the movement of the maggot mass into this region. 3.3. Bacterial diagnostics Based on the criteria used for diagnosis, this study identified ten genera of bacteria within the three treatments of decomposing pigs: Bacteroides, Budvicia, Escherichia, Ignatzschineria, Morganella, Proteus, Providencia, Serratia, Shigella and Wohlfahrtiimonas. No genera were unique to any one treatment and none of these genera were found in all 15 pigs. However, Budvicia, Escherichia, Morganella, Proteus, Providencia and Shigella were found in at least two of the pigs in all three treatments. The percentage of pigs in which each genus was found varied across the four thermal phases (Fig. 2). Ignatzschineria and Wohlfahrtiimonas were identified in the maggot mass in each of the five maggot-infested pigs. Serratia was also identified in one of these masses. 3.4. Analysis of bacterial communities The PERMANOVA analysis revealed that the effect of treatment on the presence of bacterial genera did not differ significantly between thermal phases (Pseudo-F5,32 = 1.37, P = 0.149). There were significant differences between replicate pigs in the types of
bacteria found within all treatments (Pseudo-F12,32 = 1.55, P = 0.020). There was also a significant difference in the types of bacteria found between treatments (Pseudo-F2,32 = 2.42, P = 0.038). The pairwise test revealed that the fresh, non maggot-infested treatment was not significantly different from the frozen (t = 1.10, P = 0.29) or maggot-infested (t = 1.33, P = 0.13) treatments. The frozen and maggot-infested treatments were, however, significantly different (t = 0.26, P = 0.001). SIMPER revealed that treatments had average similarities of 16%, 39% and 44% within fresh, frozen and maggot-infested pigs, respectively (Table 1). Significant differences in bacterial genera were found between thermal phases (Pseudo-F3,32 = 5.04, P = 0.001). The pairwise test revealed all thermal phases to be significantly different from each other, except the peak-heating and final phases (t = 1.20, P = 0.26). SIMPER average similarity values of 53%, 27%, 25% and 20% were calculated for the initial, pre-heating, peak heating and final thermal phases, respectively (Table 2). The bacterial genus Proteus was a major contributor to the similarity within the first two phases, while Providencia dominated the final two phases. Morganella was prominent during pre-heating and remained prominent until the final phase. Escherichia and Shigella were both prominent in the initial and peak-heating phases, but were much less dominant during the pre-heating and final phases. Budvicia was another contributor to the peak-heating phase (Fig. 2).
4. Discussion This is the first study to show experimentally that exposed carcasses are capable of heating without any thermal contribution from maggot masses, solar radiation or any other external heat source. This suggests that bacterial metabolism during decomposition causes the bulk of such heat, and that maggot masses, while contributing to localised heating within the mass, may have less of a role in carcass thermogensis than previously assumed. Given this finding, it is imperative to understand the nature of the bacteria involved. As such, our study also documented the bacterial genera
A.P. Johnson et al. / Forensic Science International 231 (2013) 271–277
274
Fig. 2. Percentage of pig carcasses with specified bacteria present across the four thermal phases: (1) initial; (2) pre-heating; (3) peak-heating; and (4) final.
present at key points along the thermal profile throughout the decomposition process. 4.1. Carcass temperatures The thermal profiles for all treatments were not significantly different, with average peak temperatures above 30 8C across all treatments. It is worth noting that temperatures above 40 8C
occurred not only in the maggot-infested pigs, but also in some of the fresh (uninfested) pigs. However, it cannot be discounted that maggots add additional heat to the system, as seen in the supplementary pig monitored using infrared imaging. Mass temperatures as high as 42 8C (19 8C above ambient) were generated within three days in the mass created in the mouth of this pig, compared to the core temperature, which took seven days to reach temperatures of equivalent elevation.
A.P. Johnson et al. / Forensic Science International 231 (2013) 271–277 Table 1 The average similarities within each treatment (fresh, frozen and maggot-infested), and the main contributing genera of bacteria as determined by SIMPER. Treatment
Genus
Fresh
Shigella Escherichia Proteus Providencia
Average similarity 7.50 3.75 2.92 1.67
Contribution % 47.37 23.68 18.42 10.53
Frozen
Escherichia Proteus Shigella Morganella
12.17 9.38 8.42 7.96
30.83 23.74 21.33 20.17
Maggot-infested
Morganella Proteus Providencia
19.33 15.00 5.22
44.39 34.44 11.99
Another study that revealed heating during decomposition in the absence of maggots is that of Rodriguez and Bass [19], who measured the temperatures of buried human bodies. These workers found that bodies buried 1.2 and 0.6 m deep showed no signs of insect activity, but began heating after 4 and 2 weeks, respectively. These corpses heated approximately 10 and 20 8C above the surrounding soil temperatures. These temperature elevations were similar to those noted in the current study, although the time until peak heating was greatly increased. Rodriguez and Bass [19] stated that lower environmental temperatures and fluctuating pH and oxygen levels should decrease bacterial metabolic heating, which may explain the delay in heating observed in their study. While these authors highlighted the need for greater understanding of temperatures in buried cadavers, there is clearly an even more pressing need to understand bacteria-induced heating in bodies above ground. The role of insulation in maintaining temperatures throughout the heating process could be the key to scenarios in which heating is observed in decomposing carrion. In the case of the increased temperature noted in the abovementioned buried bodies [19], the surrounding soil would have insulated the corpse and thus aided in heat retention during bacterial metabolism. In insect-infested remains it is also likely that maggot masses act as insulators, trapping heat within the system and allowing the build up of metabolic heat. In addition to providing insulation from the surrounding environment, the constant movement of maggots through the carcass could aid in aerating the decaying material to ensure that adequate oxygen levels are maintained and aerobic decay continues [20]. When obligate or facultative anaerobes are present, anaerobic decay can continue in the absence of oxygen. However, anaerobic decay lacks the high temperatures generated
Table 2 The average similarities within each temperature phase, and the main contributing genera of bacteria as determined by SIMPER. Thermal phase
Genus
Average similarity
Contribution %
Initial
Shigella Proteus Escherichia
19.44 18.89 14.44
36.84 35.79 27.37
Pre-heating
Morganella Proteus
15.00 11.67
56.25 43.75
Peak-heating
Escherichia Morganella Providencia Shigella Budvicia
8.12 5.29 5.22 3.12 2.29
32.65 21.26 21.00 12.54 9.19
Final
Morganella Providencia
14.50 3.33
74.36 17.09
275
during aerobic decay and thus the aeration of carcasses is essential for heating to occur [20,21]. This has important implications not only for our understanding of how maggot mass heat is generated, but also the factors that might influence the temperatures achieved within these masses, such as mass volume, maggot density and maggot movement, as well as the size and position of the carcass. It is important to note that, during the current study, one pig carcass in each of the fresh and frozen treatments failed to heat. This lack of carcass heating in the absence of maggots has also been noted in the few studies in which such conditions have been met [7,9,13], but the underlying reason for this is unknown. Deonier [9] suggested that an uninfested goat carcass in winter did not heat due to bacterial metabolism being suppressed at low ambient temperatures. Wallman [7] also failed to note any appreciable heat in an uninfested pig carcass studied in autumn, but in this case ambient temperatures fluctuated within the relatively mild range of 10–25 8C. Low temperatures also do not explain the lack of heating in the two pigs in our study, as all pigs were exposed to a constant temperature of 23 8C. Since maggots were not present in either of the non-heating carcasses, internal decay may have been predominantly anerobic rather than aerobic due to the lack of aeration of the carcass by the fly larvae. 4.2. Bacterial communities All of the bacteria found in these carcasses are mesophilic, with some known to be associated with the decay of vegetative material [11,12]. Mesophilic bacteria generally dominate during the initial part of the decay process and remain active, and hence continue producing heat, up to temperatures of around 40 8C. At temperatures above 40 8C thermophiles replace mesophiles and continue the heating process [10–12]. No thermophilic bacteria were identified within any of the carcasses, but since temperatures in the pigs reached, but did not exceed 40 8C, it seems that temperature did not limit the activity of the mesophiles. The two bacterial genera associated with the maggot masses, Ignatzschineria and Wohlfahrtiimonas, are of the family Xanthomonadaceae. They have both been recorded in association with the larvae of the flesh fly Wohlfahrtia magnifica, particularly in cases of myiasis of both humans and other animals [22–24]. While this genus of flesh fly is not found in Australia, these bacterial taxa may have a broader association with other carrion fly larvae. The bacterial genera identified as diagnostic of the thermal phases (Fig. 2) were Budvicia, Escherichia, Morganella, Proteus, Providencia and Shigella. These are all gram-negative bacteria of the family Enterobacteriaceae. This is unsurprising, given that enterobacteria are facultative anaerobes generally associated with the intestines of vertebrates [25]. While Shigella and Escherichia have been separated historically into different genera, there are few biochemical properties that can be used to distinguish them. Many bacteriologists suggest that they should be considered a single genus due to their high degree of nucleotide sequence similarity [26,27]. Their presence throughout the four thermal phases of the current study reflects this close link, with both also being strong contributors to the similarity between the initial and peak heating phases. The genera Proteus, Morganella and Providencia are all quite closely related and are considered to be opportunistic pathogens [25]. Despite this close relationship, the three genera were found to have unique thermal phase preferences. Proteus was a major contributor to the initial and pre-heating phases, while Providencia was a major contributor to the peak-heating and final phases and not diagnostic of the initial and pre-heating phase. Morganella appeared in sequence between these two genera, being a major contributor to the preand peak heating phases, as well as the final phase.
276
A.P. Johnson et al. / Forensic Science International 231 (2013) 271–277
This study utilised 16S rRNA gene sequences for identifying the bacteria present because this is the most common genetic marker used in bacterial taxonomy and phylogenetics [28]. 16S rRNA gene sequencing has successfully identified genera in over 90% of studies reviewed by Janda and Abbot [28], but is less discriminating at the level of species (for 65–83% of these studies). Some of the difficulties in reliably identifying the species of a given bacterium are the occurrence of undescribed taxa or putative species sharing similar or identical 16S rRNA sequences [28]. Additional difficulties arise from the use of databases such as the GenBank 16S ribosomal RNA database (Bacteria and Archaea) [29], the EzTaxon-e server [30] or the Ribosomal database project [31], in which sequences may be incomplete or erroneous. It is due to these recognised issues in bacterial identification that our study did not examine the taxonomy of bacterial taxa below the level of genus. There was no significant difference between the bacterial genera present in the fresh, non maggot-infested pigs compared to the maggot-infested pigs. This means that, while maggot infestation increases decomposition, it does not appear to affect the bacterial community within the carcass. However, flies are known vectors of many pathogenic bacteria, including Escherichia and Proteus [32]. These bacteria are consumed during development by maggots, survive the pupation process and are present in the emergent adults, which then act as dispersers of the bacteria [33]. In this study the adult flies did not lay the eggs directly on the carcasses, and so they would not have been able to spread bacteria and act as vectors. Since Ch. rufifacies eggs were transferred onto the carcasses and maggots had not yet been exposed to bacteria from other sources, the introduction of bacteria into the system would have been minimal. The results also revealed no significant differences in bacterial composition between the fresh and frozen carcasses, although the frozen and maggot-infested carcasses did differ. The former finding is at odds with Micozzi [34], who found frozen-thawed carcasses to be more susceptible to invasion by both insects and microorganisms than fresh carcasses. Caution should therefore continue to be exercised by both forensic entomologists and anthropologists when using frozen carcasses for experimental work. With future advances in the taxonomic resolution of bacteria, it is possible that bacterial species could have utility as diagnostic tools for identifying minimum time since death. Some preliminary work using bacteria for estimating death times has already been carried out, examining particular species rather than focusing on the overall community structure [35]. There is also need for research that examines carcasses that are free of bacteria, in order to strengthen the link between bacteria and heating. This could be done by gamma irradiation of the carcasses and their subsequent maintenance in a bacteria-free room (or laminar flow hood). Furthermore, irradiated carcasses could be selectively contaminated with specific bacteria, either aerobic or anerobic, to examine their role in the heating and decay process.
5. Conclusions This study is the first to show a significant increase in carcass temperature in the absence of maggot masses or solar radiation. It has highlighted the need to better understand the contribution of bacteria to carcass heating and define the thermogenic contribution of maggot masses. This study has also provided a preliminary assessment of the bacterial genera involved in carcass decay. However, quantification of the density of these bacteria, and more specific identification of them, should help determine not only how bacteria affect carcass heating but also improve our understanding of the role of maggot masses in promoting or suppressing bacterial activity.
Ethical standards All pig carcasses used in this study died of natural causes. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements This project was funded by ARC Linkage Grant LP0883711. We thank the Australian Federal Police and the NSW Police Force for their financial support, and the Victorian Institute of Forensic Medicine, Victoria Police and Forensic Science SA for their in-kind support. We are also grateful to Windridge Pig Farm, Young, NSW, for providing the pig carcasses, Axton Aguiar, Jacinta Rice and Samuel Wighton for their assistance with transporting the pigs and Stephanie Ivkosic for assisting with the bacterial culturing and DNA extractions. Statistical assistance was provided by Kristine French. References [1] G. MacMaster, Environmental Forensics and its Effects on Investigation, PageFree Publishing Inc, Michigan, 2006. [2] D.H. Slone, S.V. Gruner, Thermoregulation in larval aggregations of carrionfeeding blow flies (Diptera: Calliphoridae), J. Med. Entomol. 44 (2007) 516–523. [3] A.P. Johnson, M.S. Archer, L. Leigh-Shaw, M. Pais, C. O’Donnell, J.F. Wallman, Examination of forensic entomology evidence using computed tomography scanning: case studies and refinement of techniques for estimating maggot mass volumes in bodies, Int. J. Legal Med. 126 (2012) 693–702. [4] A.P. Johnson, M.S. Archer, L. Leigh-Shaw, M. Brown, C. O’Donnell, J.F. Wallman, Non-invasive visualisation and volume estimation of maggot masses using computed tomography scanning, Int. J. Legal Med. 127 (2013) 185–194. [5] T.J. Cianci, J.K. Sheldon, Endothermic generation by blow fly larvae Phormia regina developing in pig carcasses, Bull. Soc. Vector Ecol. 15 (1990) 33–40. [6] M.L. Goff, A Fly for the Prosecution: How Insect Evidence Helps Solve Crimes, Harvard University Press, London, 2000. [7] J.F. Wallman, Systematics and Thermobiology of Carrion-breeding Blowflies (Diptera: Calliphoridae), The University of Adelaide, Adelaide, 1999(PhD Thesis). [8] B. Greenberg, J.C. Kunich, Entomology and the Law, Cambridge University Press, Cambridge, 2002. [9] C.C. Deonier, Carcass temperatures and their relation to winter blowfly populations and activity in the Southwest, J. Econ. Entomol. 33 (1940) 166–172. [10] N. Trautmann, E. Olynciw, Compost Microorganisms, in: Cornell Composting, Cornell Waste Management Institute, Ithaca, 1996, Available at: http://compost.css.cornell.edu/microorg.html (accessed 02.09.12). [11] S. MacGregor, F. Miller, K. Psarianos, M. Finstein, Composting process control based on interaction between microbial heat output and temperature, Appl. Environ. Microb. 41 (1981) 1321–1330. [12] J. Ryckeboer, J. Mergaert, K. Vaes, S. Klammer, D. De Clercq, J. Coosemans, H. Insam, J. Swings, A survey of bacteria and fungi occuring during composting and self-heating processes, Ann. Microbiol. 53 (2003) 349–410. [13] G. Wobeser, E.A. Galmut, Internal temperature of decomposing duck carcasses in relation to botulism, J. Wildlife Dis. 20 (1984) 267–271. [14] M.R. Green, J. Sambrook (Eds.), Molecular Cloning: A Laboratory Manual, fourth ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2012. [15] S. Altschul, T. Madden, A. Scha¨ffer, J. Zhang, Z. Zhang, W. Miller, D. Lipman, Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res. 25 (1997) 3389–3402. [16] C.J. Smith, B.S. Danilowicz, A.K. Clear, F.J. Costello, B. Wilson, W.G. Meijer, T-Align, a web-based tool for comparison of multiple terminal restriction fragment length polymorphism profiles, FEMS Microbiol. Ecol. 54 (2005) 375–380. [17] K.R. Clarke, R.N. Gorley, PRIMER v6: User Manual/Tutorial, PRIMER-E, Plymouth, 2006. [18] K.R. Clarke, Non-parametric multivariate analyses of changes in community structure, Aust. J. Ecol. 18 (1993) 117–143. [19] W.C. Rodriguez, W.M. Bass, Decomposition of buried bodies and methods that may aid in their location, J. Forensic Sci. 30 (1985) 836–852. [20] M. Baker, B. Knoop, S. Quiring, A. Beard, B. Lesikar, J. Sweetan, R. Burns, The Decomposition Process, in: Don’t Bag ItTM – Compost It!!, Texas A&M University System, College Station, 2009, Available at: http://aggie-horticulture.tamu.edu/ earthkind/landscape/dont-bag-it/chapter-1-the-decomposition-process/ (accessed 13.08.12). [21] J. Peirce, R. Weiner, P. Vesilind (Eds.), Environmental Pollution and Control, fourth ed., Butterworth-Heinemann, Oxford, 1998. [22] E. To´th, G. Kava´cs, P. Schumann, A. Kova´cs, U. Steiner, A. Halbritter, K. Ma´rialigeti, Schineria larvae gen. nov., sp. nov., isolated from the 1st and 2nd larval stages of
A.P. Johnson et al. / Forensic Science International 231 (2013) 271–277
[23]
[24] [25] [26]
[27]
[28]
Wohlfahrtia magnifica (Diptera: Sacophagidae), Int. J. Syst. Evol. Microbial. 51 (2001) 401–407. E. To´th, P. Schumann, A. Borsodi, Z. Ke´ki, A. Kova´cs, K. Ma´rialigeti, Wohlfahrtiimonas chitiniclastica gen. nov., sp. nov., a new gammaproteobacterium isolated from Wohlfahrtia magnifica (Diptera: Sarcophagidae), Int. J. Syst. Evol. Microbial. 58 (2008) 976–981. S. Rebaudet, S. Genot, A. Renvoise, P. Fournier, A. Stein, Wohlfartiimonas chitiniclastica bacteremia in homeless woman, Emerg. Infect. Dis. 15 (2009) 985–986. C.M. O’Hara, F.W. Brenner, J.M. Miller, Classification, identification, and clinical significance of Proteus, Providencia, and Morganella, Clin. Microbiol. Rev. 13 (2000) 534–546. M. Dworkin, S. Falkow, E. Rosenberg, K. Schleifer, E. Stackebrandt, Proteobacteria: gamma subclass, in: M. Dworkin (Ed.), third ed., The Prokaryotes: A Handbook on the Biology of Bacteria, vol. 6, Springer, New York, 2006. F. Yang, J. Yang, X. Zhang, L. Chen, Y. Jiang, Y. Yan, X. Tang, J. Wang, Z. Xiong, J. Dong, Y. Xue, Y. Zhu, X. Xu, L. Sun, S. Chen, H. Nie, J. Peng, J. Xu, Y. Wang, Z. Yuan, Y. Wen, Z. Yao, Y. Shen, B. Qiang, Y. Hou, J. Yu, Q. Jin, Genome dynamics and diversity of Shigella species, the etiologic agents of bacillary dysentery, Nucleic Acids Res. 33 (2005) 6445–6458. J. Janda, S. Abbot, Minireview: 16S rRNA gene sequencing for bacterial identification in the diagnostic laboratory: pluses, perils, and pitfalls, J. Clin. Microbiol. 45 (2007) 2761–2764.
277
[29] D.A. Benson, I. Karsch-Mizrachi, D.J. Lipman, J. Ostell, D.L. Wheeler, GenBank, Nucleic Acids Res. 1 (2006) 16–20. [30] O.S. Kim, Y.J. Cho, K. Lee, S.h. Yoon, M. Kim, H. Na, S.C. Park, Y.S. KJeon, J.H. Lee, H. Yi, S. Won, J. Chun, Introducing EzTaxon-e: a prokaryotic 16S rRNA Gene sequence database with phylotypes that represent uncultured species, Int. J. Syst. Evol. Microbial. 62 (2012) 716–721. [31] J.R. Cole, Q. Wang, E. Cardenas, J. Fish, B. Chai, R.J. Farris, A.S. Kulam-SyedMohideen, D.M. McGarrel, T. Marsh, G.M. Garrity, J.M. Tiedje, The ribosomal database project: improved alignments and new tools for rRNA analysis, Nucleic Acids Res. 37 (2009) 141–145. [32] Q. Ma, A. Fonseca, W. Liu, A.T. Fields, M.L. Pimsler, A.F.T. Spindola, A.M. Crippen, T.L.J.K. Tomberlin, T.K. Wood, Proteus mirabilis interkingdom swarming signals attract blow flies, Int. Soc. Microbial Ecol. J. 6 (2012) 1356–1366. [33] A. Ahmad, A. Broce, L. Zurek, Evaluation of significance of bacteria in larval development of Cochliomyia macellaria (Diptera: Calliphoridae), J. Med. Entomol. 43 (2006) 1129–1133. [34] M.S. Micozzi, Experimental study of postmortem change under field conditions: effects of freezing, thawing, and mechanical injury, J. Forensic Sci. 31 (1986) 953–961. [35] I.C.F. Corcobado, La Microbiologia forense se renueva: bioindicadores de data de muerte, Microbiologia 20 (2009) 16–18.