Veterinary Forensic Pathology Research Articles

Pathological alterations and COHb evaluations as tools for investigating fire-related deaths in veterinary forensic pathology.

Fire-related deaths are usually a consequence of carbon monoxide (CO) poisoning or shock from thermal injuries. In humans, high levels of carboxyhemoglobin (COHb) concentrations in the blood can support a diagnosis of CO poisoning. In veterinary medicine, few studies investigated the pathological changes and blood COHb% in fire victims, and no data are available on post-mortem changes in blood gas composition due to fire. This study aims to investigate the pathological changes and COHb levels in both animal victims of fire and cadavers experimentally exposed to fire. For this purpose, dogs were selected and subdivided into three groups. Group A comprised 9 adult dogs, and Group B comprised 7 puppies that died under fire-related conditions. Group C was represented by 4 dog cadavers experimentally exposed to heat and smoke. A complete macroscopic, histological, and COHb evaluation were performed on each animal. Animals in Groups A and B showed cherry-red discoloration, thermal-injuries and soot deposits along the respiratory tract. Animals in Group C showed thermal injuries and soot deposits limited to the upper respiratory tract. The mean COHb% values in cadavers in Group C were lower than those observed in the other groups but higher compared to the values detected before the heat and smoke treatment. These findings suggest that both pathological changes and COHb analysis are valid tools for investigating fire-related deaths in dogs. However, the increase of COHb levels in cadavers exposed post-mortem to heat and smoke highlights how the COHb analysis should always be evaluated together with macroscopical and microscopical findings to avoid significant misjudgments in investigating fire-related fatalities in veterinary forensic practice.

Agonal Thrombus at Necropsy-A Third Category of Blood Coagulation in Domestic Carnivores.

Agonal thrombus is infrequently discussed in veterinary forensic pathology, being misdiagnosed as postmortem coagulation. The main purpose of the present study is to confirm that agonal thrombosis is an important tool in ruling out sudden death and to characterize it by gross, histological, and immunohistochemical approaches. The investigations have been conducted on 56 domestic carnivores. Fibrin was observed as rosette-like arrangements around platelet aggregates, loose network, wave-like pattern or short fibers and the additional tendency of lines of Zahn being noted inconsistently. All agonal thrombi had positive reactions for anti-CD61 for platelets, disposed in variable-sized clumps or in a linear pattern close to endothelial cells of endocardium. The same positive reaction has been noted to anti-fibrinogen and anti-fibrin antibodies. CD45, CD68 and von Willebrand factor had a very low to absent expression. Cardiac lesions were found in 22 cases (39.29%) suggesting predisposition to agonal thrombosis in animals with cardiovascular diseases. The results prove that agonal thrombus is a third category of blood coagulation that forms strictly during agonal death. The microscopical findings describe the agonal thrombus similar to the morphology of recent thrombus vera. Given the results, the agonal thrombus is a useful tool that confirms the agonal suffering prior to death.

Morphometrical and Immunohistochemical Evaluation of Kidney as an Indirect Parameter to Estimate Age in Puppies in Veterinary Forensic Pathology.

Estimation of age represents a central focus in the veterinary forensic pathology field. Currently, the visual examination of the dentition and the skeletal age are the main methods to estimate the age of puppies. Nevertheless, these methods are affected by a broad range of variables. In contrast, the kidney is characterized by a specific postnatal development. In human glomerulogenesis, fetal mesangial cells change their immunohistochemical phenotypes with maturation. Therefore, we hypothesized that histological and immunohistochemical examinations of the kidney can be used together as an indirect parameter for age determination in puppies' cadavers. Forty-five puppies' cadavers were divided into five groups defined by age (Group A= 0-15 days, Group B = 16-45 days, Group C = 46-85 days, Group D = 86-105 days, Group E= 105-365 days). For each case, kidney samples were collected and processed for histopathological (for morphometrical study of the glomerulus) and immunohistochemical (for the immunolocalization of the α-SMA protein) studies. Morphometrical study allowed us to observe statistical differences in the mean glomerulus numbers per field among assessed groups. Similarly, immunohistochemical examination showed differences in SMA expression among groups. Our findings suggest a potential use of kidney morphometrical and immunohistochemical examinations together as an indirect parameter to assess the age of illegally imported puppies.

Evaluation of fire-related death in veterinary forensic pathology

Evaluation of fire-related death in veterinary forensic pathology

Evaluation of Muscle Proteins for Estimating the Post-Mortem Interval in Veterinary Forensic Pathology.

Postmortem cadaveric changes are commonly used to estimate the postmortem interval (PMI) in humans and animals. However, these modifications have been poorly investigated in animals of interest to veterinary forensic pathology. The aim of this study was to investigate the potential use of muscle proteins (desmin and dystrophin) as biomarkers for estimating the PMI in dogs. For this study, 10 dead adult dogs were evaluated for 4 days in a temperature-controlled room at 19 ± 1 °C. For each animal, at 3, 24, 48, 72, and 96 h after death, a 1 × 1 × 1 cm cube of muscle tissue was removed from the vastus lateralis and triceps brachii. Protein expression levels were analyzed by immunohistochemical examination and immunoblot analysis. The obtained results showed rapid dystrophin degradation, with complete disappearance at 72 h after death. In contrast, desmin-positive fibers and desmin protein bands detected by immunoblot were observed on all 4 days of observation. Our findings suggest the potential use of muscle proteins as biomarkers for estimating the PMI in dogs.

Use of the international classification of diseases (ICD)-11 method applied to veterinary forensic pathology for coding the cause and manner of death in wildlife.

The growth of human population has led, in recent years, to increasingly frequent contacts with the wild animals with which we share the territory, sometimes leading to negative interactions with them. The purpose of the study is to apply the codes contained in the 11th Revision of the International Classification of Diseases (ICD-11) method to investigate the cause and the manner of death, also to entrust the veterinarian with the task of recognizing and describing a suspected animal abuse as a sentinel indicator of violence toward humans and non-humans, thus expanding the concept of “One Health” from a forensic investigation perspective. The subjects recruited are wild mammals submitted for autopsy to the Pathology Unit of the Department of Veterinary Science, University of Parma, Italy, from 2015 to 2018. The manner and the cause of death of 167 wild animals of 16 different species have been investigated. When possible, an on-site inspection where the corpse was found was performed. Injuries were classified according to the on-line 11th Revision of the International Classification of Diseases method. Section 22 (Injury, poisoning or certain other consequences of external causes) was used to record the “immediate cause of death” (cause of death) and Section 23 (External causes of morbidity or mortality) was used to record the “underlying cause of death” (manner of death) for each animal. In most cases, death occurred as a result of road trauma but in some cases, abuse and voluntary killing were investigated. The recognition of non-accidental injuries is particularly important for both the defense in court of animals and for the connection between crimes committed against animals and against humans, known as “The Link”. The use of the ICD-11 method, as a sort of summary of the autopsy report, was confirmed to be of great value for the clarity and simplicity of processing the data collected also by veterinary pathologists. The veterinary pathologists can use this evidence-based method with the aim of creating a national register and therefore, to understand the real extent of the human impact on wildlife and document it in a scientific and statistically usable way.

Evaluation of Muscle Proteins and DNA Degradation to Estimate the Post-Mortem Interval in Veterinary Forensic Pathology

Evaluation of Muscle Proteins and DNA Degradation to Estimate the Post-Mortem Interval in Veterinary Forensic Pathology

Morphometrical and Immunohistochemical Examination of Kidney as An Indirect Parameter to Assess Age in Puppies in Veterinary Forensic Pathology

Morphometrical and Immunohistochemical Examination of Kidney as An Indirect Parameter to Assess Age in Puppies in Veterinary Forensic Pathology

The use of Corticotropin-releasing factor (CRF) as a marker of agonic stress in abused dogs: Preliminary results

In Veterinary Forensic Medicine, determination of the degree of animal suffering is an essential element for the prosecution of perpetrators of animal abuse. The purpose of this study is to find a suitable immunohistochemical marker for the assessment of suffering to be routinely used in Veterinary Forensic Pathology, by analyzing the expression of Corticotropin-releasing factor (CRF) in formalin-fixed brains of dogs as a measurement of the agonic stress. CRF, a key peptide element in exogenous and endogenous stressors adaptation, can regulate endocrine-behavioral responses to stress stimulating pituitary ACTH release and consequent adrenal secretion of glucocorticoids. Since CRF acts in days or weeks, this study investigates its role as a potential distinctive marker between sudden death and death associated with a longer agonic period. The study used immunohistochemistry (IHC) to evaluate the CRF expression in the brain of dogs that suffered sudden death, as compared to dogs that died after long-term agonic stress. IHC labelling analysis was performed with machine-learning-based software and the results were statistically evaluated. Our results demonstrate for the first time that CRF is a promising marker of stress in abused patients also in Veterinary Medicine.

Manual Strangulation of a stray cat

Veterinary forensic pathology plays a crucial role in animal death investigations. Recent years have seen increased attention given to this field in Taiwan. Here we report a grave crime that aroused public indignation in Taiwan, when in December 2015, a missing free-roaming cat was allegedly strangled by a suspect, according to witnesses. Surveillance video footage of the cruel incident surfaced that showed the cat, without visible signs of struggle, strangled by both hands of the suspect and kicked in the abdomen’s left side. The body was stored in a travel bag when found in the suspect’s motorcycle trunk three days after the cat had been missing. The radiographs revealed a suspected luxation of cervical vertebrae 6 and 7 (C6–C7). At forensic necropsy and histopathology, neck compression with subcutaneous hemorrhage at the left submandibular area, bruising of the neck, tears in the wall of the left external jugular vein, and pulmonary lesions were identified, all consistent with asphyxiation. Hemorrhages of the epidural space of the C7 vertebra, the spinal nerve at the level of C7, and surrounding soft tissues were noted. The pathological findings were in line with the suspect’s confession, the witnesses’ statements, and the video footage. The process and outcome of this case reflected society’s growing awareness of animal welfare and the increased attention that the authorities are giving crimes against animals in Taiwan, and the link between pathologic findings and the crime.

Diatom Test and Intrapulmonary Aquaporins Expression as New Tools for the Diagnosis of Drowning in Veterinary Forensic Pathology

Diatom Test and Intrapulmonary Aquaporins Expression as New Tools for the Diagnosis of Drowning in Veterinary Forensic Pathology

Causes and Manners of Death of 100 Alleged Animal Cruelty Cases During 2011–2016: The Development of Veterinary Forensic Pathology in Taiwan

Causes and Manners of Death of 100 Alleged Animal Cruelty Cases During 2011–2016: The Development of Veterinary Forensic Pathology in Taiwan

Diagnosis of Drowning and the Value of the Diatom Test in Veterinary Forensic Pathology.

The detection of diatoms into the organs is considered an important “biological marker” for the diagnosis of drowning in human pathology, but it still has a high possibility for false positive results. The aims of this study were: (1) to evaluate the contribution of pathological examination in drowning cases and (2) to investigate the differences in the number and location of diatoms between animals who died in drowning and non-drowning conditions. For these purposes, 30 dead adult dogs were selected for the study and subdivided into five groups. The group A comprised six cadavers dead for drowning; the group B comprised six control animals; the groups C, D, and E comprised six animals dead for causes other than drowning and subsequently immersed in water for 24, 48, and 72 h, respectively. On each animal, a complete macroscopic and histological examination and diatom test were performed. Diatoms test and quantification were also performed on drowning mediums. Pathological findings of the animals in the group A showed pulmonary congestion, oedema, and hemorrages in the lung. However, similar injuries were also observed in control and experimentally submerged cadavers. In contrast, we observed a statistically differences between drowning animals and all experimentally submerged groups and control animals regarding diatom numbers recovered from organ tissue samples (p < 0.05). Therefore, these findings suggest that the number of diatoms may be used as a valid tool to differentiate animals who died in drowning and non-drowning conditions, even if the latter were found in an aquatic environment.

Assessment of Google Glass for Photographic Documentation in Veterinary Forensic Pathology: Usability Study.

BackgroundGoogle Glass is a head-mounted device designed in the shape of a pair of eyeglasses equipped with a 5.0-megapixel integrated camera and capable of taking pictures with simple voice commands.ObjectiveThe objective of our study was to determine whether Google Glass is fit for veterinary forensic pathology purposes.MethodsA total of 44 forensic necropsies of 2 different species (22 dogs and 22 cats) were performed by 2 pathologists; each pathologist conducted 11 necropsies of each species and, for each photographic acquisition, the images were taken with a Google Glass device and a Nikon D3200 digital single-lens reflex (DSLR) camera. The pictures were collected, divided into 3 groups (based on the external appearance of the animal, organs, and anatomical details), and evaluated by 5 forensic pathologists using a 5-point score system. The parameters assessed were overall color settings, region of interest, sharpness, and brightness. To evaluate the difference in mean duration between necropsies conduced with Google Glass and DSLR camera and to assess the battery consumption of the devices, an additional number of 16 necropsies were performed by the 2 pathologists. In these cases, Google Glass was used for photographic reports in 8 cases (4 dogs and 4 cats) and a Nikon D3200 reflex camera in the other 8 cases. Statistical evaluations were performed to assess the differences in ratings between the quality of the images taken with both devices.ResultsThe images taken with Google Glass received significantly lower ratings than those acquired with reflex camera for all 4 assessed parameters (P<.001). In particular, for the pictures of Groups A and B taken with Google Glass, the sum of frequency of ratings 5 (very good) and 4 (good) was between 50% and 77% for all 4 assessed parameters. The lowest ratings were observed for the pictures of Group C, with a sum of frequency of ratings 5 and 4 of 21.1% (342/1602) for region of interest, 26% (421/1602) for sharpness, 35.5% (575/1602) for overall color settings, and 61.4% (995/1602) for brightness. Furthermore, we found a significant reduction in the mean execution time for necropsy conduced with the Google Glass with respect to the reflex group (P<.001). However, Google Glass drained the battery very quickly.ConclusionsThese findings suggest that Google Glass is usable in veterinary forensic pathology. In particular, the image quality of Groups A and B seemed adequate for forensic photographic documentation purposes, although the quality was lower than that with the reflex camera. However, in this step of development, the high frequency of poor ratings observed for the pictures of Group C suggest that the device is not suitable for taking pictures of small anatomical details or close-ups of the injuries.

The intensity of the inflammatory response in experimental porcine bruises depends on time, anatomical location and sampling site

The assessment of the age of bruises inflicted on livestock is an important component of veterinary forensic pathology investigations. However, the sampling site within a bruise, the anatomical location and the mass and speed of the object inflicting the blunt trauma might influence the intensity of the inflammatory reaction. In the present study, the variation of the inflammatory reaction within and along experimental porcine bruises was evaluated in order to determine the optimal sampling site. Moreover, we evaluated if a combination of histological characteristics and gene expression signatures was able to differentiate bruises according to anatomical location, age of bruises and the speed and mass of the object used to cause the impact.Twelve experimental slaughter pigs were anesthetized, and on each animal four blunt traumas were inflicted on the back using either a plastic tube or an iron bar, respectively. The pigs were euthanized at 2, 5 or 8 h after infliction. Following gross examination, skin and underlying muscle tissue were sampled from the center and both ends of bruises and evaluated histologically. Subcutaneous fat tissue from the center of the bruises was sampled for quantitative real-time polymerase chain reaction to evaluate mRNA expression of 13 selected genes. Uninjured tissue was sampled from the right thigh of all pigs and served as control tissue.The amount of tissue damage and the intensity of the inflammatory reaction in bruises depended on the sampling site within and along a bruise, the anatomical location and the age of the bruise. The optimal site for sampling, i.e. the most pronounced inflammatory reaction, was at the center of the bruises where the plastic tube or iron bar first struck the skin. Moreover, bruises inflicted in areas with a thin layer of subcutaneous fat tissue showed more damage and inflammation in the underlying muscle tissue compared to bruises inflicted in areas with a thicker layer of subcutaneous fat tissue. In addition, hemorrhage in the muscle tissue was more likely present when bruises were inflicted with an iron bar compared to a plastic tube. Combining histology and mRNA expression of the 13 genes showed that the age of bruises could be determined with a precision of ±2.04 h. Moreover, the age of bruises could be determined with a precision of ±1.84 h based solely on mRNA expression of a selection of four genes.

Forensic aspects of gene expression signatures for age determination in bruises as evaluated in an experimental porcine model.

Determining the age of bruises and the force used to inflict the trauma is of crucial importance in both human and veterinary forensic pathology. In the present study, the expression of more than 50 different genes in subcutaneous fat and muscle tissue from experimental bruises in pigs was investigated. The aim was to evaluate if expression signatures of selected genes were capable of determining bruises according to age and the force of impact. Eighteen experimental pigs were anesthetized, and on each animal four blunt traumas were inflicted on the back with a low, moderate or high force. The pigs were euthanized from 1 to 10h after infliction of the trauma and subcutaneous fat and muscle tissues were sampled. As control, subcutaneous fat and muscle tissues were sampled from two un-injured pigs. Quantitative real-time polymerase chain reaction was performed to evaluate mRNA expression of genes involved in inflammation, tissue damage and repair. Expression signatures of thirteen selected genes in subcutaneous fat but not in muscle tissue reflected the age of bruises with a precision of approximately ±2h. Moreover, the gene expression signature in the subcutaneous fat was to some extend able to separate bruises inflicted with different forces. Expression signatures of selected genes in the subcutaneous fat will increase the precision of the age determination of bruises in pigs. Further, due to the similarity of porcine and human skin physiology and immunity, these results might also provide valuable information in human forensic science.

Forensic aspects of incised wounds and bruises in pigs established post-mortem

Recognizing post-mortem (PM) changes is of crucial importance in veterinary forensic pathology. In porcine wounds established PM contradicting observations regarding infiltration of leukocytes have been described. In the present study, skin, subcutis and muscle tissue sampled from experimental pigs with PM incised wounds (n=8), PM bruises (n=8) and no lesions, i.e. controls (n=4), were examined for signs of vitality over time. All tissue samples were subjected to gross and histopathological evaluation.Hemorrhages were present along the edges of PM incised wounds but deposits of fibrin were never observed. PM bruise led to hemorrhage in the subcutis visible on cross section of the skin in 3 out of 8 pigs. Histologically, hemorrhages in the subcutaneous tissue and disrupted muscle fibers were observed in PM bruises and could not be differentiated from similar lesions in ante-mortem (AM) bruises. Vital reactions, i.e. infiltrating leukocytes, hyper-leukocytosis and pavement of leukocytes, were absent in all incised wounds and bruises regardless of the time of sampling after traumatization.In conclusion, a vital reaction was not present in PM incised wounds, regardless of the time of sampling. Moreover, it was found that AM bruises free of leukocyte infiltration cannot be distinguished from PM bruises, an observation which is of crucial importance when timing bruises in forensic cases.

Cerebellar Histomorphometry for Age Determination of Puppies in Veterinary Forensic Pathology

Cerebellar Histomorphometry for Age Determination of Puppies in Veterinary Forensic Pathology

A Survey of Attitudes of Board-Certified Veterinary Pathologists to Forensic Veterinary Pathology.

An electronic survey was conducted to determine the attitudes of veterinary pathologists toward forensic pathology and the adequacy of their training in the discipline. The survey was sent to 1933 diplomates of the American College of Veterinary Pathologists and 311 completed responses were analyzed. Of respondents, 80% report receiving at least 1 type of medicolegal case, with cases from law enforcement received most frequently. Most (74%) of the respondents indicated that their previous training did not prepare them adequately to handle forensic cases and almost half of the respondents (48%) indicated that they needed more training on serving as an expert witness. Relative risk ratios (RRR) and odds ratios (OR) were generated to determine the strength of a statistically significant association. Responses from a free-text entry question determining additional training needs could be grouped into 3 main categories: (1) veterinary forensic pathology science and procedures, (2) documentation, evidence collection and handling, and (3) knowledge of the medicolegal system. Last, a field for additional comments or suggestions regarding veterinary forensic pathology was completed by 107 respondents and many reinforced the need for training in the categories previously described. The survey highlights that a significant proportion of diplomates of the American College of Veterinary Pathologists are currently engaged in veterinary forensic pathology but feel their training has not adequately prepared them for these cases. Hopefully, the survey results will inform the college and residency training coordinators as they address the training requirements for an important emerging discipline.

Veterinary Forensic Pathology: Drowning and Bodies Recovered From Water.

Determining the cause of death in animals recovered from bodies of water, swimming pools, or other water-containing vessels is challenging. Animals recovered from water may or may not have drowned. The diagnosis of drowning is usually one of exclusion, requiring information from the crime scene, recovery scene, the medical history or reliable witness accounts. While there are characteristic macroscopic and microscopic lesions of drowning, none are specific and are dependent on the volume and tonicity of the drowning medium. Beyond interpreting the postmortem findings, the court may ask pathologists to comment on the behavioral and welfare implications of drowning. This requires an understanding of the drowning process, which is a complex series of sequential, concurrent, and overlapping cardiorespiratory reflexes, electrolyte and blood gas abnormalities, aspiration, physical exhaustion, and breathlessness eventually culminating in death. This review addresses the mechanisms, lesions, and diagnostic issues associated with drowning in nonaquatic companion animals.