Human Versus Animal: Contrasting Decomposition Dynamics of Mammalian Analogues in Experimental Taphonomy

Taphonomic studies regularly employ animal analogues for human decomposition due to ethical restrictions relating to the use of human tissue. However, the validity of using animal analogues in soil decomposition studies is still questioned. This study compared the decomposition of skeletal muscle tissues (SMTs) from human (Homo sapiens), pork (Sus scrofa), beef (Bos taurus), and lamb (Ovis aries) interred in soil microcosms. Fixed interval samples were collected from the SMT for microbial activity and mass tissue loss determination; samples were also taken from the underlying soil for pH, electrical conductivity, and nutrient (potassium, phosphate, ammonium, and nitrate) analysis. The overall patterns of nutrient fluxes and chemical changes in nonhuman SMT and the underlying soil followed that of human SMT. Ovine tissue was the most similar to human tissue in many of the measured parameters. Although no single analogue was a precise predictor of human decomposition in soil, all models offered close approximations in decomposition dynamics.     

Odor analysis of decomposing buried human remains

This study, conducted at the University of Tennessee's Anthropological Research Facility (ARF), lists and ranks the primary chemical constituents which define the odor of decomposition of human remains as detected at the soil surface of shallow burial sites. Triple sorbent traps were used to collect air samples in the field and revealed eight major classes of chemicals which now contain 478 specific volatile compounds associated with burial decomposition. Samples were analyzed using gas chromatography-mass spectrometry (GC-MS) and were collected below and above the body, and at the soil surface of 1.5-3.5 ft. (0.46-1.07 m) deep burial sites of four individuals over a 4-year time span. New data were incorporated into the previously established Decompositional Odor Analysis (DOA) Database providing identification, chemical trends, and semi-quantitation of chemicals for evaluation. This research identifies the "odor signatures" unique to the decomposition of buried human remains with projected ramifications on human remains detection canine training procedures and in the development of field portable analytical instruments which can be used to locate human remains in shallow burial sites.     

Potential Use of Bacterial Community Succession in Decaying Human Bone for Estimating Postmortem Interval,,

Bacteria are taphonomic agents of human decomposition, potentially useful for estimating postmortem interval (PMI) in late‐stage decomposition. Bone samples from 12 individuals and three soil samples were analyzed to assess the effects of decomposition and advancing time on bacterial communities. Results indicated that partially skeletonized remains maintained a presence of bacteria associated with the human gut, whereas bacterial composition of dry skeletal remains maintained a community profile similar to soil communities. Variation in the UniFrac distances was significantly greater between groups than within groups (p < 0.001) for the unweighted metric and not the weighted metric. The members of the bacterial communities were more similar within than between decomposition stages. The oligotrophic environment of bone relative to soft tissue and the physical protection of organic substrates may preclude bacterial blooms during the first years of skeletonization. Therefore, community membership (unweighted) may be better for estimating PMI from skeletonized remains than community structure (weighted).     

Characterization of human DNA in environmental samples

Environmental samples from indoor surfaces can be confounded by dust, which is composed largely of human skin cells and has been documented to contain roughly tens of micrograms of total DNA per gram of dust. This study complements previous published work by providing estimates of the quantity of amplifiable human DNA found in environmental samples from a typical indoor environment, categorized by the intensity of human traffic and visible quantity of dust. Dust was collected by surface swabbing standard 576 cm² areas in eight locations, and evaluated for total DNA quantity, presence of human DNA (mitochondrial and nuclear loci using conventional PCR), quantity of human nuclear DNA using quantitative PCR, and STR analysis. The total DNA content of 36 dust samples ranged from 9 to 28 ng/cm², and contained 0.2–1.1 pg/cm² of human DNA. Overall, human DNA was detected in 97% of 36 dust samples and 61% of samples yielded allele distributions of varying degrees of complexity when subjected to STR analysis. The implications of this study are twofold. First, the presence of dust in evidence can be a significant contamination source in forensic investigations because the human DNA component is of sufficient quality and quantity to produce allele calls in STR analysis. This can be effectively managed by implementing stringent protocols for collection and analysis of potential biological samples. A second implication is the use of dust as a source of evidence for identification of inhabitants within a defined location. In the latter case, a number of additional studies would be necessary to identify relevant pretreatments for environmental dust samples and to develop the necessary deconvolution techniques to separate the composite genotypes obtained.     

The biochemical alteration of soil beneath a decomposing carcass

The processes associated with cadaver decomposition in outdoor settings, particularly those that occur during the extended postmortem interval (>30 days) are poorly understood. Thus, few methods are currently available to accurately estimate the extended postmortem interval (PMI). Of these methods, a soils-based approach has the potential to address the postmortem interval between which entomology and anthropology are the most valuable. Although the validity of soil-based methods has previously been established, little work has been conducted to explain the processes that have been designated for forensic application. As a consequence, we investigated the dynamics of carbon, nitrogen, and phosphorus-based compounds in soil beneath pig (Sus scrofa) cadavers (gravesoil) that were placed on the soil surface over a period of 100 days. Decomposition was assessed through the physical characteristics of the cadaver, soil pH, soil moisture content, and the concentration of total carbon, total nitrogen, soil-extractable phosphorus, and lipid-phosphorus in soil. Cadaver decomposition did not result in a significant difference in soil carbon and moisture content. However, significant (P < 0.05) increases were observed in the concentration of soil pH, total nitrogen, soil-extractable phosphorus, and lipid-phosphorus. Based on the current results, a significant increase in the concentration of gravesoil nutrients represented a maximum PMI of 43 days (lipid-P), 72 days (total nitrogen), or 100 days (soil-extractable phosphorus). This work provides further evidence that a soil-based method has the potential to act as a tool for the estimation of extended PMI.     

Modelling the buried human body environment in upland climes using three contrasting field sites

Despite an increasing literature on the decomposition of human remains, whether buried or exposed, it is important to recognise the role of specific microenvironments which can either trigger or delay the rate of decomposition. Recent casework in Northern England involving buried and partially buried human remains has demonstrated a need for a more detailed understanding of the effect of contrasting site conditions on cadaver decomposition and on the microenvironment created within the grave itself. Pigs (Sus scrofa) were used as body analogues in three inter-related taphonomy experiments to examine differential decomposition of buried human remains. They were buried at three contrasting field sites (pasture, moorland, and deciduous woodland) within a 15 km radius of the University of Bradford, West Yorkshire, UK. Changes to the buried body and the effect of these changes on hair and associated death-scene textile materials were monitored as was the microenvironment of the grave. At recovery, 6, 12 and 24 months post-burial, the extent of soft tissue decomposition was recorded and samples of fat and soil were collected for gas chromatography mass spectrometry (GCMS) analysis. The results of these studies demonstrated that (1) soil conditions at these three burial sites has a marked effect on the condition of the buried body but even within a single site variation can occur; (2) the process of soft tissue decomposition modifies the localised burial microenvironment in terms of microbiological load, pH, moisture and changes in redox status. These observations have widespread application for the investigation of clandestine burial and time since deposition, and in understanding changes within the burial microenvironment that may impact on biomaterials such as hair and other associated death scene materials.     

Mitochondrial DNA Extraction from Burial Soil Samples at Incremental Distances: A Preliminary Study

Preservation variance of soil DNA is neglected in the literature, and exceptional cases exaggerate amplification capabilities. This study sought to amplify a short mitochondrial fragment (212 bp) specific to Sus scrofa domesticus from the soil surrounding decomposing pig remains from an open‐air locale. Samples collected above the body at incremental distances after 145 days of initial placement yielded pig DNA. A secondary sampling was collected in 2017, approximately 768 days after burial. Inhibition tests corroborated that pig DNA was no longer present in the soil resulting in a loss of original DNA between 145 and 768 days. The results provide evidence that genetic material leaches out radially from the source and DNA fragments longer than 200 bp do not persist in soil for a relatively short timeframe in western Montana. The conclusions support the collection of soil in crime scene investigation procedures within the first few months of decomposition.     

Decomposition chemistry of human remains: a new methodology for determining the postmortem interval

This study was conducted to characterize the chemistry associated with the decomposition of human remains with the objective of identifying time-dependent biomarkers of decomposition. The purpose of this work was to develop an accurate and precise method for measuring the postmortem interval (PMI) of human remains. Eighteen subjects were placed within a decay research facility throughout a four-year time period and allowed to decompose naturally. Field autopsies were performed and tissue samples were regularly collected until the tissues decomposed to the point where they were no longer recognizable (encompassing a cumulative degree hour (CDH) range of approximately 1000 (approximately 3 weeks)). Analysis of the biomarkers (amino acids, neurotransmitters, and decompositional by-products) in various organs (liver, kidney, heart, brain, muscle) revealed distinct patterns useful for determining the PMI when based on CDHs. Proper use of the methods described herein allow for PMIs so accurate that the estimate is limited by the ability to obtain correct temperature data at a crime scene rather than sample variability.     

Chemical, physical and mineralogical characterization of soils from the Curitiba Metropolitan Region for forensic purpose

The objective of this paper is to propose a soil characterization methodology for forensic use, based on physical (textural and spectroscopic analyses), chemical (extractions with hydrofluoric acid, ammonium oxalate, sodium dithionite-citrate-bicarbonate and NaOH solution) and mineralogical (thermal analyses and X-ray diffraction) analyses. The study was carried out in the State of Paraná, Brazil, in three neighborhoods of Curitiba city and in two cities within the Curitiba Metropolitan Region. In order to verify the similarity between samples, four composite samples (repetitions) were prepared in each of the five studied sites. It was obtained a great number of quantitative variables (56) from a reduced amount of soil sample (1g). The variables selected from the chemical extractions (16) were more precise in grouping similar samples (same horizon and sampling site) as well as in separating samples collected in different horizons or sites. Seven distinct groups were formed, each with high intragroup similarity, but the unexpected dispersion of two samples (from a total of 40 samples) reduced the distinction of three other studied groups. The placement of the two samples in a different group and the higher dispersion (24 samples) for samples collected in the city of Curitiba are due to the great pedological homogeneity of this area (physical-chemical horizon characteristics, soil color and parent material). The methodology used in this work (analytical method and data treatment) presents high potential for forensic studies and can be easily validated for other areas.     

A new sensitive short pentaplex (ShoP) PCR for typing of degraded DNA

Analysis of short tandem repeat makers has become the most powerful tool for DNA typing in forensic casework analysis. Unfortunately, typing of DNA extracted from telogen shed hairs, bones buried in the soil or from paraffin-embedded, formalin-fixed tissue often reveals no results due to the degradation of DNA. The reduction in size of the target fragments by development of new primers and their combination in multiplex approaches open a new field of DNA analysis. Here we present a new sensitive short pentaplex PCR including the loci amelogenin, TH01, VWA, D3S1358 and D8S1179. Validation tests of our new method included sensitivity, mixtures, human specificity, artificial degradation of DNA by DNase I and case work analysis on a panel of different forensic samples. The detection limit was 12.5 pg of human DNA, and mixtures of 50 pg in a total of 1000 pg were clearly detectable and revealed complete profiles. Only DNA extracts of human primates displayed a few signals, whereas other animal, fungal or bacterial DNA showed no signals. Our method proved extremely valuable in the analysis of artificially degraded DNA and in forensic cases, where only poorly preserved DNA was available. This approach and other similar methods can aid in the analysis of samples where allelic drop out of larger fragments is observed. It is highly recommended to develop more of these multiplexes to improve poor quality DNA typing.     

Spatial and temporal influences on bacterial profiling of forensic soil samples

Bacterial content may be helpful in differentiating forensic soil samples; however, the effectiveness of bacterial profiling depends on several factors, including uniqueness among different habitat types, the level of heterogeneity within a habitat, and changes in bacterial communities over time. To examine these, soils from five diverse habitats were tested over a 1 year period using terminal restriction fragment length polymorphism (TRFLP) analysis. Soil samples were collected at central locations monthly, and 10 feet in cardinal directions quarterly. Similarity indices were found to be least related among habitats, while the greatest bacterial similarities existed among collection locations within a habitat. Temporally, however, bacterial content varied considerably, and there was substantial overlap in similarity indices among habitats during different parts of the year. Taken together, the results indicate that while bacterial DNA profiling may be useful for forensic soil analysis, certain variables, particularly time, must be considered.     

Time since death determinations of human cadavers using soil solution.

This study was conducted to collect data on specific volatile fatty acids (produced from soft tissue decomposition) and various anions and cations (liberated from soft tissue and bone), deposited in soil solution underneath decomposing human cadavers as an aid in determining the "time since death." Seven nude subjects (two black males, a white female and four white males) were placed within a decay research facility at various times of the year and allowed to decompose naturally. Data were amassed every three days in the spring and summer, and weekly in the fall and winter. Analyses of the data reveal distinct patterns in the soil solution for volatile fatty acids during soft tissue decomposition and for specific anions and cations once skeletonized, when based on accumulated degree days. Decompositional rates were also obtained, providing valuable information for estimating the "maximum time since death." Melanin concentrations observed in soil solution during this study also yields information directed at discerning racial affinities. Application of these data can significantly enhance "time since death" determinations currently in use.     

Developing a new scoring method to evaluate human decomposition in a humid,continental (Dfb) climate in Quebec

The published literature shows a lack of methods to evaluate the patterns and extent of decomposition of human remains and to estimate the post-mortem interval (PMI) in humid, continental (Dfb) climates such as Quebec. The aim of this study was to address this gap in the current knowledge base by providing the first observations from human corpses studied under controlled conditions in Quebec. A 12-month study was conducted at the site for Research in Experimental and Social Thanatology; the first human taphonomy facility in Canada. Six human donors with known time of death were deposited across spring (n = 1), summer (n = 3), and autumn (n = 2) 2021. The lack of suitability of the total body score method to evaluate the extent of decomposition at the facility prompted the development of a new scoring system based on the macromorphoscopic changes observed. The scoring system was applied to the donors to evaluate decomposition throughout seasons. All donors followed comparable decomposition trajectories, regardless of the season of deposition. Eighty-five percent of taphonomic patterns appeared in the first 25 experimental days or 5000 Kelvin accumulated degree days (350 ADD). Extensive desiccation of tissues was observed at a median of 21 experimental days across donors, resulting in a plateau within decomposition with no extensive skeletonization. To the authors' knowledge, this is the first published report of experimentally observed desiccation in such a form in a Dfb climate. This study provides new data on the types of decomposition patterns to expect in forensic investigations in southern Quebec and comparable climates.     

Identifying background microbiomes in an evidence recovery laboratory: A preliminary study

16S rRNA profiling of bacterial communities may have forensic utility in the identification or association of individuals involved with criminal activities. Microbial profiling of evidence may, in the future, be performed within environments currently utilised for human DNA recovery, such as a forensic biology laboratory. It would be important to establish the background microbiome of such an environment to determine the potential presence of human or environmental microbial signatures to assist forensic scientists in the appropriate interpretation of target microbial communities. This study sampled various surfaces of an Evidence Recovery Laboratory (ERL) on three occasions including (a) before a monthly deep-clean, (b) immediately following the deep-clean, and (c) immediately after the laboratory’s use by a single participant for the purposes of routine item examinations. Microbial profiles were also generated for the involved participant and researcher for comparison purposes. Additionally, human nuclear DNA was profiled for each of the samples collected, using standard forensic profiling techniques, to provide a prospective link to the presence or absence of a background microbial signature within the ERL after its use. Taxonomic distributions across ERL samples revealed no consistent signature of any of the items sampled over time, however, major phyla noted within all ERL samples across the three timepoints were consistent with those found in human skin microbiomes. PCoA plots based on the Unweighted Unifrac metric revealed some clustering between participant microbial reference samples and surfaces of the ERL after use, suggesting that despite a lack of direct contact, and adherence to standard operating procedures (SOPs) suitable for human DNA recovery, microbiomes may be deposited into a forensic setting over time. The reference samples collected from the involved participant and researcher generated full STR profiles. Human DNA was observed to varying degrees in samples taken from the ERL across each of the sampling timepoints. There was no correlation observed between samples that contained or did not contain detectable quantities of human nuclear DNA and microbial profile outputs.     

Seasonal Variation of Carcass Decomposition and Gravesoil Chemistry in a Cold (Dfa) Climate

It is well known that temperature significantly affects corpse decomposition. Yet relatively few taphonomy studies investigate the effects of seasonality on decomposition. Here, we propose the use of the Köppen‐Geiger climate classification system and describe the decomposition of swine (Sus scrofa domesticus) carcasses during the summer and winter near Lincoln, Nebraska, USA. Decomposition was scored, and gravesoil chemistry (total carbon, total nitrogen, ninhydrin‐reactive nitrogen, ammonium, nitrate, and soil pH) was assessed. Gross carcass decomposition in summer was three to seven times greater than in winter. Initial significant changes in gravesoil chemistry occurred following approximately 320 accumulated degree days, regardless of season. Furthermore, significant (p < 0.05) correlations were observed between ammonium and pH (positive correlation) and between nitrate and pH (negative correlation). We hope that future decomposition studies employ the Köppen‐Geiger climate classification system to understand the seasonality of corpse decomposition, to validate taphonomic methods, and to facilitate cross‐climate comparisons of carcass decomposition.     

The Influence of Preburial Insect Access on the Decomposition Rate

Abstract: This study compared total body score (TBS) in buried remains (35 cm depth) with and without insect access prior to burial. Sixty rabbit carcasses were exhumed at 50 accumulated degree day (ADD) intervals. Weight loss, TBS, intra‐abdominal decomposition, carcass/soil interface temperature, and below‐carcass soil pH were recorded and analyzed. Results showed significant differences (p << 0.001) in decomposition rates between carcasses with and without insect access prior to burial. An approximately 30% enhanced decomposition rate with insects was observed. TBS was the most valid tool in postmortem interval (PMI) estimation. All other variables showed only weak relationships to decomposition stages, adding little value to PMI estimation. Although progress in estimating the PMI for surface remains has been made, no previous studies have accomplished this for buried remains. This study builds a framework to which further comparable studies can contribute, to produce predictive models for PMI estimation in buried human remains.     

The use of crossover immunoelectrophoresis to detect human blood protein in soil from an ambush scene in Kosovo

This study examines the survivability of human blood proteins in soils from a year and a half old ambush scene in Kosovo. A total of 72 soil samples were collected, a number of which were directly associated with bone fragments or bullet projectiles. The samples were examined using crossover immunoelectrophoresis (CIEP) to determine the presence of blood protein and species affiliation. Human blood proteins were identified in 44 of the 72 samples (61%) with the majority of the positive observations (29 of 44) found 0.0-4.5 cm below ground surface (65%). Chi-squared and two-sample difference of proportions tests confirmed significant differences between samples with and without associated physical evidence and the presence and depth of human blood proteins. While DNA has largely replaced immunological analysis in forensic analyses, our results suggest that in particular situations, CIEP may still be a valuable tool in criminology.     

Assessing the potential of bacterial DNA profiling for forensic soil comparisons

A pilot study was undertaken to evaluate DNA profiling of the bacterial community in soil as an alternative to geological methods for forensic soil comparisons. Soil samples from three different ecosystems were compared, and the variation within and between ecologically different sites was determined by using terminal restriction fragment (TRF) analysis of 16S ribosomal DNA. Comparison of TRF profiles revealed that samples from within a specific ecosystem (e.g., a field) showed a significantly higher similarity to each other than to those from another ecosystem (e.g., a forest). In addition, some profile features were unique to specific ecosystems. These features may allow the determination of characteristic profiles that will facilitate identification of ecologically different sites, so that a given sample collected from a suspect could be identified as originating from, for example, a field, rather than a forest. The implications of these preliminary findings for forensic investigations are discussed.     

Microsatellite stability in human post-mortem tissues

Human identification and forensic criminal casework may involve DNA profiling of decomposed material. Somatic microsatellite (STR) instability may lead to false exclusions and theoretically to false inclusions, both in criminal cases and in human identification. Hence, the somatic and postmortal stability of the actual sequences is crucial to the reliability of such analyses. Somatic STR stability in human tissues has been documented in small series only and the effect of postmortal tissue decomposition on microsatellite stability remains to be elucidated. On this basis, we have systematically searched for somatic STR mutations in 26 deceased humans without signs of decomposition at autopsy and 25 autopsy cases with obvious signs of postmortal decomposition. A blood sample and six tissue samples were collected from each case.Seven STRs were chosen for study, the tetranucleotides HUMVWA31/A, HUMTH01, HUMF13A1, and HUMFES/FPS, and the hyperpolymorphic markers HUMAPOAI1, D11S554 and HUMACTBP2. Denaturing gel electrophoresis was performed on an ABD Prism 377 gene sequencer with Genescan 672 software (Applied Biosystems, Inc.).The bone DNA profile of each case was chosen as the standard DNA profile. All cases gave profiles from additional tissues. By intraindividual comparison of DNA profiles in the cases without signs of degradation we find that the short repetitive sequences under study are stable, that is without evidence of somatic mutations. The cases with varying degree of decomposition display postmortal microsatellite stability, we detect no somatic mutations or other possible postmortal changes that could lead to between-organ non-matches.In conclusion, PCR-based STR analyses are suitable in human identification and forensic casework dealing with different tissues, even when the substrate is heavily decomposed.