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Implications of the Investigative Animal Model
Abstract
Although domestic pigs (Sus scrofa domesticus Erxleben) are generally accepted as appropriate human taphonomic proxies, taphonomic studies have used a wide variety of mammalian analogues. These include, among others, cattle (Bos taurus Linnaeus), sheep (Ovis aries Linnaeus), mice (Mus musculus Linnaeus) and brown rats (Rattus norvegicus Berkenhout), and their respective laboratory-reared subspecies. Furthermore, organs/tissue types from different species have been used, both with and without molecular analysis, notably in forensic entomology studies. The relevance, applicability and limitations of common, disparate and novel approaches must, therefore, be deliberated within the multi-disciplinary forensic ecogenomics and related forensic sub-disciplines. The implications of differences in animal model species, their organs/tissues and related parameters are explored and assessed to inform protocol standardization and knowledge transferability to often restricted cadaver-based analyses. The ultimate goal is potential validation and adoption of forensic ecogenomics in real crime scene toolkits in the determinations of postmortem, postmortem submersion, and postburial intervals.
... Also, there is similarity between swine and human cadavers in terms of decomposition where putrefaction progresses at approximately the same rate to that of human bodies of comparable weight (Campobasso et al., 2001) although their body parts surface areas to volume ratios differ (Connor et al., 2018). Without access to donated human bodies and relevant ethical guidelines, animal proxies remain the best alternative to study decomposition and develop forensic investigative tools (Ralebitso-Senior and Pyle, 2018;Williams et al., 2019). ...
Introducing animal carbon-source to soil initiates biochemical and microbial processes that lead to its decomposition and recycling, which subsequently cause successional shifts in soil microbial community. To investigate the use of soil microbial community to inform criminal investigation, this study was designed to mimic clandestine graves. It compared the decomposition of stillborn piglets (Sus scrofa domesticus), as human analogues, to oak (Quercus robur) leaf litter and soil-only controls outdoors for 720 days. Environmental and edaphic parameters were monitored and showed soil microbial community alignment with temperature seasonality, which highlighted the importance of this abiotic factor. Denaturing gradient gel electrophoresis (DGGE) data were used to calculate Hill numbers and diversity indices of the bacterial 16S rRNA community did not distinguish mammalian- from plant-based decomposition consistently during the first or second year of the study. In contrast, the fungal 18S rRNA community allowed clear differentiation between different treatments (beta diversity) throughout the 720-day experiment and suggested the moment of the decomposing mammalian skin rupture. 16S rRNA-based NGS facilitated the identification of e.g., Pirellulaceae, Acidobacteria ii1-15_order and Candidatus xiphinematobacter as Year 2 bacterial markers of gravesoil at family, order and species taxonomic levels, respectively, and confirmed the similarity of the calculated Hill diversity metrics with those derived from DGGE profiling. Parallel soil elemental composition was measured by portable X-ray Fluorescence where calcium profiles for the piglet-associated soils were distinct from those without carrion. Also, soil calcium content and PMI correlated positively during the first year then negatively during the second. This study is one of the first to apply a multidisciplinary approach based on molecular and physicochemical analytical techniques to assess decomposition. It highlights the recognised potential of using soil microbial community in forensic investigations and provides a proof-of-concept for the application of a combined molecular and elemental approach to further understand the dynamics of decomposition. In addition, it sets the scene for further research in different conditions based on Hill numbers metrics instead of the classic ecological indices for soil necrobiome richness, diversity and evenness.
http://repositorio.uchile.cl/handle/2250/178592.
Las discusiones sobre la intensificación en el uso de recursos marítimos por las comunidades arcaicas que habitaron las costas arréicas del desierto de Atacama en la zona de Taltal, descansan mayoritariamente en interpretaciones del registro ictiológico y malacológico, ante la escasa presencia relativa de restos de mamíferos y aves en los sitios estudiados. Aún a pesar de que la evidencia general tiende a confirmar esta hipótesis de intensificación, poca atención se ha puesto en intentar explicar estas diferencias de representación de clases animales en los conjuntos zooarqueológicos. Considerando que estos sitios se insertan en uno de los ecosistemas más activos y dinámicos en términos de reciclaje biológico, mediada por numerosos agentes que intervienen, seleccionan y acumulan restos óseos. Ante esta incertidumbre de potencial sesgo en el registro de tetrápodos para los sitios en Taltal, se elaboró un estudio tafonómico regional a partir de un conjunto actualístico, el cual comparó múltiples variables del registro óseo depositado hoy en las costas desérticas con el proveniente de sitios arqueológicos desde los 5000 cal. A.P. en adelante en la misma zona, información que procesada por medio de un modelo clasificador bayesiano permitió constatar sesgos producto de alteración tafonómica y determinar con precisión aquellos sitios arqueológicos mas proclives a problemas interpretativos producto de dichos sesgos.
Megyesi et al.'s (J Forensic Sci, 2005, 50, 618) paper was important to forensic anthropology as it introduced a quantitative framework for estimating time since death in human cadavers, based upon physical appearance by way of scoring on a novel scale. However, errors concerning rounding, temperature scale, and incorrect use of a statistical regression model render their predictive formula unusable. Based upon only their more reliable data, a more appropriate regression model to predict accumulated degree days (ADD) from total body score (TBS) is presented. The new model is also a superior fit (r(2) = 0.91) and produces markedly narrower confidence intervals than the original, which also allowed impossible, negative ADD values. Explanations of the shortcomings in the original analysis and calculations are presented, which it is hoped will help forensic scientists avoid making similar mistakes.
Over the last decades, studies on juvenile development of forensically important Diptera were performed by using non-human tissues (e.g., beef liver) as a nutrition medium. Such developmental data are used as a reference in determining the age of juvenile necrophagous insects sampled from a human body and thereby to estimate the minimum postmortem interval (PMImin). Despite the acceptance of these studies in the forensic community, some might ask whether such data appropriately reflect the growth of blow flies on human tissue. We, therefore, studied larval growth rates and development times of Calliphora vicina (Diptera: Calliphoridae) on human muscle tissue as well as on pork loin, pork liver, and minced pork at 25 °C. Larval growth rates were significantly (p < 0.001) slower on pork loin and pork liver compared to human muscle tissue. Nonetheless, the time at which the examined developmental landmarks "post-feeding larvae," "pupae," and "adult fly" were reached was similar for all tissues, with significant delays in first fly eclosion only for specimens reared on pork loin (p = 0.027) and pork liver (p = 0.036). Our results highlight the fact that not all porcine tissues are similarly suitable for producing sound growth data for necrophagous Diptera. At present, we recommend the use of minced pork as a non-human nutrition medium, since our results show no developmental differences on this diet compared to human tissue.
The thanatomicrobiome (thanatos, Greek for death) is a relatively new term and is the study of the microbes colonizing the internal organs and orifices after death. Recent scientific breakthroughs in an initial study of the thanatomicrobiome have revealed that a majority of the microbes within the human body are the obligate anaerobes, Clostridium spp., in the internal postmortem microbial communities. We hypothesized that time-dependent changes in the thanatomicrobiome within internal organs can estimate the time of death as a human body decays. Here we report a cross-sectional study of the sampling of 27 human corpses from criminal cases with postmortem intervals between 3.5–240 hours. The impetus for examining microbial communities in different internal organs is to address the paucity of empirical data on thanatomicrobiomic succession caused by the limited access to these organs prior to death and a dearth of knowledge regarding the movement of microbes within remains. Our sequencing results of 16S rRNA gene amplicons of 27 postmortem samples from cadavers demonstrated statistically significant time-, organ-, and sex-dependent changes. These results suggest that comprehensive knowledge of the number and abundance of each organ's signature microorganisms could be useful to forensic microbiologists as a new source of data for estimating postmortem interval.
The scuttle fly, Megaselia scalaris (Loew) (Diptera: Phoridae), is of medical, veterinary, and forensic importance. In the case of the latter, M. scalaris is commonly associated with indoor death or neglect cases of humans or household animals, and its larvae are useful in determining time of colonization (TOC). This study is the first to examine the effects of different temperatures and tissues from two vertebrate species on the growth rate and larval length of M. scalaris . A preliminary validation of these data was also conducted. Immatures of M. scalaris were reared on either bovine or porcine biceps femoris at 24 °C, 28 °C, and 32 °C. Temperature significantly impacted immature development time, including egg eclosion, eclosion to pupation, and pupation to adult emergence, to favor faster development at higher temperatures. From ovipostion to eclosion, development rate was 32.1% faster from 24 °C to 28 °C, 13.9% faster from 28 °C to 32 °C, and 45.5% faster from 24 °C to 32 °C. Development from eclosion to pupation displayed similar results with differences of 30.3% between 24 °C and 28 °C, 15.4% between 28 °C and 32 °C, and 45.2% between 24 °C and 32 °C. Development from pupation to adult emergence, likewise, displayed a 44.4% difference from 24 °C and 28 °C, 7.3% from 28 °C to 32 °C, and 51.2% from 24 °C to 32 °C. From oviposition to adult emergence, M. scalaris needed ∼32.7% more hours to complete development when reared at 24 °C than 28 °C, 8.5% when reared at 28 °C rather than 32 °C, and 38.4% more time when reared at 24 °C over 32 °C. Tissue type did not significantly impact development.
A preliminary validation study was conducted in four indoor environments (two attics, a closet, and a bathroom) spanning two different buildings. Utilizing minimum and mean lengths, time of colonization estimates were underestimated in all instances. The predicted range encompassed the actual TOC for two of the four environments. On average, when using minimum length, time of colonization was underestimated by 45%, but overestimated by only 2% when using maximum development range. Data generated from this research could be useful when estimating a TOC of decomposing vertebrate remains. Future research will need to examine development for each stadium in order to increase precision of such estimates.
Following a mass disaster, it is important that victims are rapidly located as the chances of survival decrease greatly after approximately 48 h. Urban search and rescue (USAR) teams may use a range of tools to assist their efforts but detector dogs still remain one of the most effective search tools to locate victims of mass disasters. USAR teams can choose to deploy human scent dogs (trained to locate living victims) or human remains detection (HRD) dogs (trained to locate deceased victims). However, little is known about the variation between live human scent and postmortem human remains scent and the timeframe during which one type of scent transitions to the other. The aim of the current study was to measure the change in the scent profile of human decomposition analogues during the first 72 h postmortem by measuring the volatile organic compounds (VOCs) that comprise the odour. Three pig carcasses (Sus scrofa domesticus L.) were placed on a soil surface and allowed to decompose under natural conditions. Decomposition odour was sampled frequently up to 75 h postmortem and analysed using comprehensive two-dimensional gas chromatography – time-of-flight mass spectrometry (GC×GC-TOFMS). A total of 105 postmortem VOCs were identified during the early postmortem period. The VOC profile during the early postmortem period was highly dynamic, changing both hourly and daily. A transition period was observed after 43 h postmortem, where the VOC profile appeared to shift from a distinct antemortem odour to a more generalised postmortem odour. These findings are important in informing USAR teams and their use of detector dogs for disaster victim recovery.
Forensic entomology involves the use of insects and other arthropods to estimate the minimum time elapsed since death, referred to as minimum postmortem interval (min PMI). This is based on the assemblage of insects found in association with remains, and most often, the time required for development of the first colonizing insects to develop to their size/ life stage at time of collection. This process involves the accumulation of appropriate data for the development of the species of insect at a variety of relevant temperatures and consideration of the other biotic and abiotic factors that may affect developmental rate. This review considers the approaches to the estimation of min PMI, focusing largely on the age estimation of specimens collected from remains and the limitations that accompany entomology-based PMI estimations. Recent advances and newly developed techniques in the field are reviewed in regard to future potential.
Genomic studies have estimated there are approximately 10(3)-10(6) bacterial species per gram of soil. The microbial species found in soil associated with decomposing human remains (gravesoil) have been investigated and recognized as potential molecular determinants for estimates of time since death. The nascent era of high-throughput amplicon sequencing of the conserved 16S ribosomal RNA (rRNA) gene region of gravesoil microbes is allowing research to expand beyond more subjective empirical methods used in forensic microbiology. The goal of the present study was to evaluate microbial communities and identify taxonomic signatures associated with the gravesoil human cadavers. Using 16S rRNA gene amplicon-based sequencing, soil microbial communities were surveyed from 18 cadavers placed on the surface or buried that were allowed to decompose over a range of decomposition time periods (3-303 days). Surface soil microbial communities showed a decreasing trend in taxon richness, diversity, and evenness over decomposition, while buried cadaver-soil microbial communities demonstrated increasing taxon richness, consistent diversity, and decreasing evenness. The results show that ubiquitous Proteobacteria was confirmed as the most abundant phylum in all gravesoil samples. Surface cadaver-soil communities demonstrated a decrease in Acidobacteria and an increase in Firmicutes relative abundance over decomposition, while buried soil communities were consistent in their community composition throughout decomposition. Better understanding of microbial community structure and its shifts over time may be important for advancing general knowledge of decomposition soil ecology and its potential use during forensic investigations.
The anatomical features distinctive to each of the very large array of species used in today's biomedical research must be born in mind when considering the correct choice of animal model(s), particularly when translational research is concerned. In this paper we take into consideration and discuss the most important anatomical and histological features of the commonest species of laboratory rodents (rat, mouse, guinea pig, hamster, and gerbil), rabbit, and pig related to their importance for applied research.
In a forensic context, microbial-mediated cadaver decomposition and nutrient recycling cannot be overlooked. As a result, forensic ecogenomics research has intensified to gain a better understanding of cadaver/soil ecology interactions as a powerful potential tool for forensic practitioners. For this study, domestic pig (Sus scrofa domesticus) (4 g) and grass (Agrostis/Festuca spp) cuttings (4 g) were buried (July 2013 to July 2014) in sandy clay loam (80 g) triplicates in sealed microcosms (127 ml; 50 × 70 cm) with parallel soil only controls. The effects of the two carbon sources were determined by monitoring key environmental factors and changes in soil bacterial (16S rRNA gene) and fungal (18S rRNA gene) biodiversity. Soil pH changes showed statistically significant differences (p < 0.05) between the treatments. The measured ecological diversity indices (Shannon–Wiener, HꞋ; Simpson, D; and richness, S) of the 16S rRNA and 18S rRNA gene profiles also revealed differences between the treatments, with bacterial and fungal community dominance recorded in the presence of S. scrofa domesticus and grass trimming decomposition, respectively. In contrast, no statistically significant difference in evenness (p > 0.05) was observed between the treatments.
Human decomposition is a complex biological process driven by an array of variables which are not clearly understood. The medico-legal community have long been searching for a reliable method to establish the post-mortem interval (PMI) for those whose deaths have either been hidden, or gone un-noticed. To date, attempts to develop a PMI estimation method based on the state of the body either at the scene or at autopsy have been unsuccessful. One recent study has proposed that two simple formulae, based on the level of decomposition humidity and temperature, could be used to accurately calculate the PMI for bodies outside, on or under the surface worldwide. This study attempted to validate 'Formula I' [1] (for bodies on the surface) using 42 Canadian cases with known PMIs. The results indicated that bodies exposed to warm temperatures consistently overestimated the known PMI by a large and inconsistent margin for Formula I estimations. And for bodies exposed to cold and freezing temperatures (less than 4°C), then the PMI was dramatically under estimated. The ability of 'Formulae II' to estimate the PMI for buried bodies was also examined using a set of 22 known Canadian burial cases. As these cases used in this study are retrospective, some of the data needed for Formula II was not available. The 4.6 value used in Formula II to represent the standard ratio of time that burial decelerates the rate of decomposition was examined. The average time taken to achieve each stage of decomposition both on, and under the surface was compared for the 118 known cases. It was found that the rate of decomposition was not consistent throughout all stages of decomposition. The rates of autolysis above and below the ground were equivalent with the buried cases staying in a state of putrefaction for a prolonged period of time. It is suggested that differences in temperature extremes and humidity levels between geographic regions may make it impractical to apply formulas developed in one region to any other region. These results also suggest that there are other variables, apart from temperature and humidity that may impact the rate of human decomposition. These variables, or complex of variables, are considered regionally specific. Neither of the Universal Formulae performed well, and our results do not support the proposition of Universality for PMI estimation.
Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
A strategy to understand the microbial components of the human genetic and metabolic landscape and how they contribute to normal physiology and predisposition to disease.
Experiments were conducted to determine whether a specific larval substrate impacted immature development rates. Protophormia terraenovae was raised on beef organs and compared with whole carcasses (rat), to determine if discrepancies in development times were observed. The minimum development time on beef liver was the most consistent with the rat carcass but a significant difference between all substrates was found after the third instar. These differences can be explained by the differences found between heart and muscle and the other substrates. Often length of the larvae is used to estimate insect age and so Day 2 measuring of weight, length, and width of the pupae was undertaken. Significant differences were found for all parameters measured on each of the substrates. As a result, the use of these measurements should not be done for P. terraenovae due to conflicting rearing substrates. Significant differences within substrates were only found for weight of insects developing on heart and length of insects developing on muscle. There was no significant difference in survival from first instar to the adult stage on any of the substrates but personal observation suggested that mortality was higher in insects developing on the brain tissue.
We investigated diverse genomic selections using high-density SNP data of five distinct cattle breeds. Based on allele frequency differences, we detected hundreds of candidate regions under positive selection across Holstein, Angus, Charolais, Brahman, and N'Dama. In addition to well-known genes such as KIT, MC1R, ASIP, GHR, LCORL, NCAPG, WIF1 and ABCA12, we found evidence for a variety of novel and less-known genes under selection in cattle, such as LAP3, SAR1B, LRIG3, FGF5, and NUDCD3. Selective sweeps near LAP3 were then validated by next generation sequencing. Genome wide association analysis involving 26,362 Holsteins confirmed that LAP3 and SAR1B were related to milk production traits, suggesting that our candidate regions were likely functional. In addition, haplotype network analyses further revealed distinct selective pressures and evolution patterns across these five cattle breeds. Our results provided a glimpse into diverse genomic selection during cattle domestication, breed formation, and recent genetic improvement. These findings will facilitate genome-assisted breeding to improve animal production and health.
Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution 2014. This work is written by US Government employees and is in the public domain in the US.
Since their domestication in the Neolithic, cattle have belonged to our cultural heritage. The reconstruction of their history is an active field of research1 that contributes to our understanding of human history. Archeological data are now supplemented by analyses of modern and ancient samples of cattle with DNA markers of maternal, paternal, or autosomal inheritance. The most recent genetic data suggest that maternal lineages of taurine cattle originated in the Fertile Crescent with a possible contribution of South-European wild cattle populations, while zebu cattle originate from the Indus Valley. Subsequently, cattle accompanied human migrations, which led to the dispersal of domestic cattle of taurine, indicine, or mixed origin over Asia, Africa, Europe, and the New World. This has resulted in their adaptation to different environments and considerable variation in appearance and performance. More recently, rational management of breeding led to international movements of sires, which again changed the global patterns of genetic diversity.
Blowflies have major medical and sanitary importance because they can be vectors of viruses, bacteria, and helminths and are also causative agents of myiasis. Also, these flies, especially those belonging to the genus Chrysomya, are among the first insects to arrive at carcasses and are therefore valuable in providing data for the estimation of the minimum postmortem interval (PMImin). The PMImin can be calculated by assessing the weight, length, or development stage of blowfly larvae. Lack of information on the variables that might affect these parameters in different fly species can generate inaccuracies in estimating the PMImin. This study evaluated the effects of different types of bovine tissues (the liver, muscle, tongue, and stomach) and chicken heart on the development rates of larvae of Chrysomya albiceps Wiedemann, Chrysomya megacephala Fabricius, and Chrysomya putoria Wiedemann (Diptera: Calliphoridae). The efficiency of each rearing substrate was assessed by maggot weight gain (mg), larval development time (h), larval and pupal survival (%), and emergence interval (h). The development rates of larvae of all blowfly species studied here were directly influenced by the type of food substrate. Tissues that have high contents of protein and fat (muscle and heart) allowed the highest larval weight gain. For bovine liver, all Chrysomya species showed slower growth, by as much as 48 h, compared to the other tissues. Different rates of development are probably associated with specific energy requirements of calliphorids and the nutritional composition of each type of food.
Many authors produced carrion insect development data for predicting the age of an insect from a corpse. Under some circumstances, this age value is a minimum postmortem interval. There are no standard protocols for such experiments, and the literature includes a variety of sampling methods. To our knowledge, there has been no investigation of how the choice of sampling method can be expected to influence the performance of the resulting predictive model. We calculated 95 % inverse prediction confidence limits for growth curves of the forensically important carrion flies Chrysomya megacephala and Sarconesia chlorogaster (Calliphoridae) at a constant temperature. Confidence limits constructed on data for entire age cohorts were considered to be the most realistic and were used to judge the effect of various subsampling schemes from the literature. Random subsamples yielded predictive models very similar to those of the complete data. Because taking genuinely random subsamples would require a great deal of effort, we imagine that it would be worthwhile only if the larval measurement technique were especially slow and/or expensive. However, although some authors claimed to use random samples, their published methods suggest otherwise. Subsampling the largest larvae produced a predictive model that performed poorly, with confidence intervals about an estimate of age being unjustifiably narrow and unlikely to contain the true age. We believe these results indicate that most forensic insect development studies should involve the measurement of entire age cohorts rather than subsamples of one or more cohorts.
Vertebrate scavengers and decomposers compete for animal carcasses in all temperate and tropical ecosystems. We examined the influence of carcass size, forest type, and air temperature on the fate of rodent carcasses at the Savannah River Site, South Carolina, USA. Three hundred rodent carcasses were placed at random locations in forested habitats and scavengers were identified using remote photography. Seventeen species of vertebrates removed 104 of 300 (35%) rodent carcasses over a year. Raccoons (Procyon lotor (Linnaeus, 1758)) and Virginia opossums (Didelphis virginiana Kerr, 1792) scavenged most frequently. For scavenged carcasses, the mean time to carcass removal was 2.58 days after placement. Carcass acquisition by scavengers and decomposers was influenced moderately by forest type and carcass size, although ambient air temperature considerably influenced the fate of carcasses. Vertebrates removed fewer carcasses as temperatures increased: only 28 of 144 (19%) carcasses were scavenged when temperatures exceeded 17°C. The temporal pattern of carcass removal by vertebrates, however, did not vary with temperature. Consistent rates of carcass removal by vertebrates across the year and increased activity by insects during warm weather led to elevated levels of decomposition during summer months. This study confirms the complexity and dynamic nature of competitive relationships among scavengers and decomposers.
A central concept in forensic entomology is that arthropod succession on carrion is predictable and can be used to estimate the postmortem interval (PMI) of human remains. However, most studies have reported significant variation in successional patterns, particularly among replicate carcasses, which has complicated estimates of PMIs. Several forensic entomology researchers have proposed that further integration of ecological and evolutionary theory in forensic entomology could help advance the application of succession data for producing PMI estimates. The purpose of this essay is to draw attention to the role of spatial aggregation of arthropods among carrion resources as a potentially important aspect to consider for understanding and predicting the assembly of arthropods on carrion over time. We review ecological literature related to spatial aggregation of arthropods among patchy and ephemeral resources, such as carrion, and when possible integrate these results with published forensic literature. We show that spatial aggregation of arthropods across resources is commonly reported and has been used to provide fundamental insight for understanding regional and local patterns of arthropod diversity and coexistence. Moreover, two suggestions are made for conducting future research. First, because intraspecific aggregation affects species frequency distributions across carcasses, data from replicate carcasses should not be combined, but rather statistically quantified to generate occurrence probabilities. Second, we identify a need for studies that tease apart the degree to which community assembly on carrion is spatially versus temporally structured, which will aid in developing mechanistic hypotheses on the ecological factors shaping community assembly on carcasses.
Carcass mass and carcass clothing are factors of potential high forensic importance. In casework, corpses differ in mass and kind or extent of clothing; hence, a question arises whether methods for post-mortem interval estimation should take these differences into account. Unfortunately, effects of carcass mass and clothing on specific processes in decomposition and related entomological phenomena are unclear. In this article, simultaneous effects of these factors are analysed. The experiment followed a complete factorial block design with four levels of carcass mass (small carcasses 5-15 kg, medium carcasses 15.1-30 kg, medium/large carcasses 35-50 kg, large carcasses 55-70 kg) and two levels of carcass clothing (clothed and unclothed). Pig carcasses (N = 24) were grouped into three blocks, which were separated in time. Generally, carcass mass revealed significant and frequently large effects in almost all analyses, whereas carcass clothing had only minor influence on some phenomena related to the advanced decay. Carcass mass differently affected particular gross processes in decomposition. Putrefaction was more efficient in larger carcasses, which manifested itself through earlier onset and longer duration of bloating. On the other hand, active decay was less efficient in these carcasses, with relatively low average rate, resulting in slower mass loss and later onset of advanced decay. The average rate of active decay showed a significant, logarithmic increase with an increase in carcass mass, but only in these carcasses on which active decay was driven solely by larval blowflies. If a blowfly-driven active decay was followed by active decay driven by larval Necrodes littoralis (Coleoptera: Silphidae), which was regularly found in medium/large and large carcasses, the average rate showed only a slight and insignificant increase with an increase in carcass mass. These results indicate that lower efficiency of active decay in larger carcasses is a consequence of a multi-guild and competition-related pattern of this process. Pattern of mass loss in large and medium/large carcasses was not sigmoidal, but rather exponential. The overall rate of decomposition was strongly, but not linearly, related to carcass mass. In a range of low mass decomposition rate increased with an increase in mass, then at about 30 kg, there was a distinct decrease in rate, and again at about 50 kg, the rate slightly increased. Until about 100 accumulated degree-days larger carcasses gained higher total body scores than smaller carcasses. Afterwards, the pattern was reversed; moreover, differences between classes of carcasses enlarged with the progress of decomposition. In conclusion, current results demonstrate that cadaver mass is a factor of key importance for decomposition, and as such, it should be taken into account by decomposition-related methods for post-mortem interval estimation.
Post mortem or even normal changes during life occurring in major gut bacterial populations are not known. We investigated Bacteroides sp., Bifidobacterium sp., Clostridium leptum, Clostridium coccoides, Streptococcus sp., Lactobacillus sp. and Enterobacteriacaea ratios in 7 fecal samples from healthy volunteers and in 61 autopsies rectum and cecum samples and studied the effect of post mortem time using quantitative real-time PCR. Bacterial ratios in stool samples from volunteers and rectum samples from autopsy cases were similar and did not change significantly up to 5 days post mortem. In cecum, significant post mortem time-dependent differences were observed in ratios of Bacteroides sp. (p = 0.014) and Lactobacillus sp. (p = 0.024). Our results showed that ratios of Bacteroides sp., Bifidobacterium sp., Clostridium leptum, Clostridium coccoides, Streptococcus sp., Lactobacillus sp. and Enterobacteriacaea can be investigated in autopsy rectum samples up to 5 days after death.
Human decomposition is a mosaic system with an intimate association between biotic and abiotic factors. Despite the integral role of bacteria in the decomposition process, few studies have catalogued bacterial biodiversity for terrestrial scenarios. To explore the microbiome of decomposition, two cadavers were placed at the Southeast Texas Applied Forensic Science facility and allowed to decompose under natural conditions. The bloat stage of decomposition, a stage easily identified in taphonomy and readily attributed to microbial physiology, was targeted. Each cadaver was sampled at two time points, at the onset and end of the bloat stage, from various body sites including internal locations. Bacterial samples were analyzed by pyrosequencing of the 16S rRNA gene. Our data show a shift from aerobic bacteria to anaerobic bacteria in all body sites sampled and demonstrate variation in community structure between bodies, between sample sites within a body, and between initial and end points of the bloat stage within a sample site. These data are best not viewed as points of comparison but rather additive data sets. While some species recovered are the same as those observed in culture-based studies, many are novel. Our results are preliminary and add to a larger emerging data set; a more comprehensive study is needed to further dissect the role of bacteria in human decomposition.
The domestic pig originates from the Eurasian wild boar (Sus scrofa). We have sequenced mitochondrial DNA and nuclear genes from wild and domestic pigs from Asia and Europe. Clear evidence was obtained for domestication to have occurred independently from wild boar subspecies in Europe and Asia. The time since divergence of the ancestral forms was estimated at approximately 500,000 years, well before domestication approximately 9,000 years ago. Historical records indicate that Asian pigs were introduced into Europe during the 18th and early 19th centuries. We found molecular evidence for this introgression and the data indicated a hybrid origin of some major "European" pig breeds. The study is an advance in pig genetics and has important implications for the maintenance and utilization of genetic diversity in this livestock species.
Decomposition is a complex process with inputs from several interacting biotic and abiotic components. A full understanding of how these factors influence the ecology of decomposition is crucial for helping law enforcement and forensic personnel. Although it has long been known that microbes play a role in decomposition, they have historically remained understudied, mainly due to their abundance and difficulties in culturing such diversity in the laboratory. Due to the power of next-generation sequencing for profiling microbial communities and the exponentially decreasing cost of such sequencing efforts, a number of investigators have recently begun focusing on the role of microorganisms in decomposition. Here we review the current knowledge of the role of microorganisms—bacteria, fungi, and other eukaryotes—in decomposition, providing an extensive overview of microbial communities associated with gravesoils; leaf litters; fish and whales (aquatic decomposition); and mice, rats, and swine (terrestrial decompositions) and the pioneering work on human corpses. Finally, the potential for this knowledge to be applied to postmortem interval estimates is also discussed.
Recent advances in microbiology have facilitated an increased interest in forensic microbiology research. Some of this research has focused on the decomposition microbial ecology of human remains, with some studies using nonhuman animal surrogates as models and others using donated human cadavers. There are pros and cons of both approaches to decomposition research and potential relevance to forensics. In addition, the experimental context and design of decomposition microbiology studies will continue to be an invaluable consideration for basic research translation into forensic practice. This chapter provides a framework for researchers considering forensic microbiology research that is focused on the decomposition of remains, outlining the challenges with this kind of research and offering guidance on how to approach such studies from a rigorous scientific perspective.
1. Ecological processes are maintained in different environments by different species performing similar functional roles. Yet, little is known about the role of the environment in shaping insect biodiversity associated with a process that is ephemeral and patchy.
2. In this study, the mass loss of carrion in response to contrasting habitat types (grassland or tree) was quantified experimentally, as well as the presence, diversity and composition of insect assemblages. Differences in insect assemblages between these two habitats were also examined.
3. It was found that the presence of insects led to a doubling in mass loss, but that grassland or tree habitat type had no effect on this process. By contrast, habitat type had a significant effect on the composition of generalist ant and beetle assemblages, but not on specialist fly assemblages. Given the colonisation of insects, carrion mass loss was negatively associated with increasing evenness of fly assemblages and increasing ant abundance. Variation in fly assemblage composition was also found to correlate with variation in carrion mass loss.
4. This study highlights the major role of habitat type in shaping the composition of generalist insects at carrion, but the minor role in affecting specialist and highly vagile insects. This complements the authors' findings that insect colonisation of carrion was critical to accelerated mass loss, and that fly assemblages were responsible for variation in this process, regardless of habitat. The present study sheds new light on the contribution of insect biodiversity to decomposition in variable environments, with consequences for carrion food webs and nutrient cycling.
Previous postmortem microbiome studies have focused on characterizing taxa turnover during an undisturbed decomposition process. How coexisting conditions (e.g., frozen, buried, burned) affect the human microbiome at the time of discovery is less well understood. Microbiome data were collected from two pediatric cases at the Wayne County Medical Examiner in Michigan. The bodies were found frozen, hidden in a freezer for an extended time. Microbial communities were sampled from six external anatomic locations at three time points during the thawing process, prior to autopsy. The 16S rRNA V4 gene amplicon region was sequenced using high-throughput sequencing (Illumina MiSeq). Microbial diversity increased, and there was a distinct shift in microbial community structure and abundance throughout the thawing process. Overall, these data demonstrate that the postmortem human microbiome changes during the thawing process, and have important forensic implications when bodies have been substantially altered, modified, and concealed after death.
The popular assumption persists that wounded or injured carcasses and by association human bodies, provide enhanced attraction and opportunity for the oviposition of forensically important flies and therefore lead to accelerated putrefaction. Recent studies, however, contradict this assumption. In this study piglet carcasses were exposed in early spring and late summer to ensure seasonal comparability of decay. To assess possible influencing factors injuries of varying depths were inflicted postmortem (e.g. abrasions, superficial and deeper lacerations of the skin) and additional blood was applied onto wounds. It could be confirmed for small piglet carcasses that injuries and trauma might have an influence on the pattern of decomposition but not on the rate of decomposition. The data gained might be valuable in estimating postmortem intervals for babies or small children.
Necrophagous species of insects provide useful complementary data to estimate the postmortem interval in forensic
cases. Here, for the first time, we report on insect specimens collected from human corpses in Riyadh,
Kingdom of Saudi Arabia. During the study, 14 beetle larvae were collected from the outdoor corpse (case report
one) and five flies and seven beetles were collected from the indoor corpse (case report two). Sequencing
was performed to study the mitochondrial DNA (mtDNA) as the prospective basis of an identification technique.
The sequencing focused on a section of the cytochrome oxidase I encoding region of mtDNA. Two beetle species, Dermestes frischii (Kugelann) and Dermestes maculatus (De Geer) (Coleoptera: Dermestidae), and one fly species, Chrysomya albiceps (Wiedemann) (Diptera: Calliphoridae), were identified. These results will be instrumental in the implementation of a Saudi database of forensically relevant insects.
The development of a methodology that estimates the postmortem interval (PMI) from stages of decomposition is a goal for which forensic practitioners strive. A proposed equation (Megyesi et al. 2005) that utilizes total body score (TBS) and accumulated degree days (ADD) was tested using longitudinal data collected from human remains donated to the Forensic Anthropology Research Facility (FARF) at Texas State University-San Marcos. Exact binomial tests examined the rate of the equation to successfully predict ADD. Statistically significant differences were found between ADD estimated by the equation and the observed value for decomposition stage. Differences remained significant after carnivore scavenged donations were removed from analysis. Low success rates for the equation to predict ADD from TBS and the wide standard errors demonstrate the need to re-evaluate the use of this equation and methodology for PMI estimation in different environments; rather, multivariate methods and equations should be derived that are environmentally specific.
© 2015 American Academy of Forensic Sciences.
This is the first report on an ongoing study conducted to collect data on the specific insects that are found in association with decaying human cadavers. Four nude unembalmed human cadavers were each placed, at various times of the year, within a decay research facility located in open wooded area. Data were collected daily throughout the entire decay cycle on the various insect populations that frequented each cadaver. Analysis of the data shows that there is a direct correlation between the rate of decay and the succession of insect families and species found in association with a decaying cadaver. Application of this entomological information can contribute to a more accurate estimation of 'time since death' of an individual.
Postmortem succession of human-associated microbial communities ("human microbiome") has been suggested as a possible method for estimating postmortem interval (PMI) for forensic analyses. Here we evaluate human gut bacterial populations to determine quantifiable, time-dependent changes postmortem. Gut microflora were repeatedly sampled from the proximal large intestine of 12 deceased human individuals as they decayed under environmental conditions. Three intestinal bacterial genera were quantified by quantitative PCR (qPCR) using group-specific primers targeting 16S rRNA genes. Bacteroides and Lactobacillus relative abundances declined exponentially with increasing PMI at rates of Nt = 0.977e(-0.0144t) (r(2) = 0.537, p < 0.001) and Nt = 0.019e(-0.0087t) (r(2) = 0.396, p < 0.001), respectively, where Nt is relative abundance at time (t) in cumulative degree hours. Bifidobacterium relative abundances did not change significantly: Nt = 0.003e(-0.002t) (r(2) = 0.033, p = 0.284). Therefore, Bacteroides and Lactobacillus abundances could be used as quantitative indicators of PMI.
© 2015 American Academy of Forensic Sciences.
Little is known about how variables, such as carcass mass, affect the succession pattern of microbes in soils during decomposition. To investigate the effects of carcass mass on the soil microbial community, soils associated with swine (Sus scrofa domesticus) carcasses of four different masses were sampled until the 15th day of decomposition during the month of June in a pasture near Lincoln, Nebraska. Soils underneath swine of 1, 20, 40, and 50 kg masses were investigated in triplicate, as well as control sites not associated with a carcass. Soil microbial communities were characterized by sequencing the archaeal, bacterial (16S), and eukaryotic (18S) rRNA genes in soil samples. We conclude that time of decomposition was a significant influence on the microbial community, but carcass mass was not. The gravesoil associated with 1 kg mass carcasses differs most compared to the gravesoil associated with other carcass masses. We also identify the 15 most abundant bacterial and eukaryotic taxa, and discuss changes in their abundance as carcass decomposition progressed. Finally, we show significant decreases in alpha diversity for carcasses of differing mass in pre-carcass rupture (days 0, 1, 2, 4, 5, and 6 postmortem) versus post-carcass rupture (days 9 and 15 postmortem) microbial communities.
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).
© 2015 American Academy of Forensic Sciences.
Global approach with case studies and contributions from six continents. Most of the research to date has been concentrated in Europe or North America.
This research examined differences in decomposition rate and manner of domestic pig subjects (Sus scrofa) in never frozen (control) and previously frozen (experimental) research conditions. Eight control and experimental subjects were placed in an identical outdoor research environment. Daily quantitative and qualitative measurements were collected: abdominal circumference, total body score (TBS), temperature, photographs, descriptive decomposition stages, and visual observations. Field necropsies were performed at accumulated degree days (ADD) between 50 and 300 (Celsius). Paired samples t-tests of ADD to TBS >3.0, TBS >9.5, and TBS >16.0 indicate the rate of decomposition of experimental subjects was significantly slower than controls at both TBS >3 and >9.5 (p = 0.003 and p = 0.002, respectively). A suite of qualitative indicators of predecomposition freezing is also reported. The differences between experimental and control subjects suggest previously frozen subjects should not be used in taphonomic research, as results do not accurately reflect the "normal" taphonomic condition.
© 2015 American Academy of Forensic Sciences.
The hairy maggot blow fly, Chrysomya rufifacies (Diptera: Calliphoridae), is a forensically important fly often encountered on human and other vertebrate remains in temperate and tropic regions throughout the world including Australia, Asia, Central America and North America. C. rufifacies was reared under controlled laboratory conditions on three muscle types (i.e., porcine, equine and canine) at three temperatures (i.e., 20.8, 24.8 and 28.3 °C). Rate of larval weight gain across time was statistically significant between muscle types (P ≤ 0.0001) and approaching significance across time between temperatures (P = 0.0511). This research represents the first development study for C. rufifacies from central Texas, USA and the first study to examine the impact of tissue type on its development. Furthermore, these data, when compared to those available in the literature, indicate developmental differences that could be due to genetic differences in populations or possibly methods employed during the studies. Caution should be emphasized when applying development data for this species from one region to forensic investigations in other ecoregions as such differences in development based on tissue fed upon by larvae, population genetics, and methodologies used in the studies could represent error in estimating the time of colonization.
Laboratory rearing of Phormia regina Meigen larvae on pork and venison was conducted as part of a study to determine whether forensic entomology approaches can be used in wildlife poaching investigations. Larvae were reared at 30°C, 75% relative humidity, and a photoperiod of 14:10 (L:D) h on pork or venison diets, and samples were collected every 8 h until >90% of the maggots reached the third-instar wandering or prepupal stage. Significant differences were found in the distribution of lengths of the third instar and combined instars for maggots reared on the two different meat sources. Maggots reared on venison reached the prepupal wandering stage significantly faster (≈6 h) compared with maggots on the pork diet. Mean adult weight and wing length of venison-reared flies were significantly greater than for flies reared on pork. The lower crude fat content of venison appears to make this meat source a more suitable medium than pork for larvae of P. regina. The difference in growth rate could introduce error into PMImin estimations from third-instar maggots in deer poaching cases if estimates are based on data from studies in which maggots were reared on pork.
The breakdown of DNA and RNA in decomposing human tissue represents a major obstacle for postmortem forensic molecular analysis. This study investigated the feasibility of performing PCR-based molecular analysis of blood and muscle tissue from 45 autopsy cases with defined postmortem intervals ranging from one to more than 14 days. It was not possible to collect blood from 38 % of the autopsy cases due to severe coagulation and hemolysis, whereas muscle tissue was available for all cases. PCR-amplifiable DNA could be extracted from 96 % of the frozen muscle specimens and from 93 % of the formalin fixed and paraffin embedded (FFPE) muscle specimens. A quality assessment of muscle-derived DNA showed increased fragmentation with advancing body decomposition and generally more fragmentation in DNA from FFPE tissue than in DNA from frozen tissue. It was possible to amplify 1,000 basepair (bp) DNA fragments from all samples with postmortem intervals below 3 days whereas 400-600 bp long fragments typically could be amplified from the most decomposed muscle specimens. RNA was less stable than DNA in postmortem muscle tissue, yet selected mRNA molecules could be detected by reverse-transcriptase PCR in all samples up to 3 days after death. We conclude that analysis of DNA from bodies with a wide postmortem interval range is usually possible whereas the consistency of RNA analyses decreases considerably 3 days postmortem. We showed that muscle tissue is a highly usable source of DNA and RNA for postmortem forensic molecular analysis as well as for retrospective research projects based on archived FFPE specimens.
Carrion decomposition is an ecologically important natural phenomenon influenced by a complex set of factors including temperature, moisture, and the activity of microorganisms, invertebrates, and scavengers. The role of soil microbes as decomposers in this process is essential, but not well understood and represents a knowledge gap in carrion ecology. To better define the role and sources of microbes in carrion decomposition, lab-reared mice were decomposed on either 1) soil with an intact microbial community or 2) soil that was sterilized. We characterized the microbial community (16S rRNA gene for bacteria and archaea, and the 18S rRNA gene for fungi and microbial eukaryotes) for three body sites along with the underlying soil (i.e. gravesoils) at time intervals coinciding with visible changes in carrion morphology. Our results indicate that mice placed on soil with intact microbial communities reach advanced stages of decomposition 2-3 times faster than those placed on sterile soil. Microbial communities associated with skin and gravesoils of carrion in stages of Active and Advanced Decay were significantly different between soil types (sterile vs. untreated), suggesting that substrates on which carrion decompose may partially determine the microbial decomposer community. However, the source of the decomposer community (soil versus carcass associated microbes) was not clear in our data set, suggesting that greater sequencing depth needs to be employed to identify the origin of the decomposer communities in carrion decomposition. Overall, our data show that soil microbial communities have a significant impact on the rate at which carrion decomposes and has important implications understanding carrion ecology.
Vertebrate scavengers can modify surface deposited human remains which can hinder
forensic investigations. The effects of such scavenging vary between species and regions.
Published research into the effects of the scavenging of human remains is dominated by
work from North America with few studies covering Northwestern Europe. Forensic
investigators in Northwestern Europe are often left questioning on a basic level as to which
scavengers are active and how they might affect human remains. This paper presents the
results of a field study utilizing deer (Cervus nippon; Capreolus capreolus) as surface
deposits observed by motion detection cameras in a British woodland. The most common
avian and rodent scavenger species recorded included the buzzard (buteo buteo), carrion
crow (Corvus corone), wood mouse (Apodemus sylvaticus) and gray squirrel (Sciurus
carolinensis). The scavenging behaviors observed were affected by seasonality, rates of
decomposition and insect activity. Scavenging by buzzards, unlike carrion crows, was most
frequent during fall to winter and prior to insect activity. Overall, avian scavengers modified
and scavenged soft tissue. Rodents scavenged both fresh and skeletonised remains with
gray squirrels only scavenging skeletal remains. Wood mice were most active in winter and
scavenged both soft tissue and bone.
'Organs-on-chips' are microengineered biomimetic systems containing microfluidic channels lined by living human cells, which replicate key functional units of living organs to reconstitute integrated human organ-level pathophysiology in vitro. These microdevices can be used to test efficacy and toxicity of drugs and chemicals, and to create in vitro models of human disease. Thus, they potentially represent low-cost alternatives to conventional animal models for pharmaceutical, chemical and environmental applications. Here we describe a protocol for the fabrication, microengineering and operation of these microfluidic organ-on-chip systems. First, microengineering is used to fabricate a multilayered microfluidic device that contains two parallel elastomeric microchannels separated by a thin porous flexible membrane, along with two full-height, hollow vacuum chambers on either side; this requires ∼3.5 d to complete. To create a 'breathing' lung-on-a-chip that mimics the mechanically active alveolar-capillary interface of the living human lung, human alveolar epithelial cells and microvascular endothelial cells are cultured in the microdevice with physiological flow and cyclic suction applied to the side chambers to reproduce rhythmic breathing movements. We describe how this protocol can be easily adapted to develop other human organ chips, such as a gut-on-a-chip lined by human intestinal epithelial cells that experiences peristalsis-like motions and trickling fluid flow. Also, we discuss experimental techniques that can be used to analyze the cells in these organ-on-chip devices.