Boar hunt or Manhunt? fatality during a wild boar hunting expedition. Need for a multidisciplinary forensic approach.

Hunting is commonly regarded as a prevalent leisure activity in many Western countries. Moreover, hunting-related shooting injuries and fatalities are an important issue in the forensic world. However, there is limited research investigating the necessity of a multidisciplinary approach to provide a thorough analysis of these deaths. Being able to distinguish between homicide and accidental death is crucial in hunting-related incidents. In cases of hunting incidents, it also becomes essential to identify the shooter among the participants of the hunting expedition. The authors report a case of death occurred during a wild boar hunting expedition. The autopsy revealed a penetrating gunshot wound that tore the internal iliac artery and caused massive blood loss. A careful and detailed examination of the events leading up to the hunter's death revealed that the bullet first hit a wild boar and then, due to the deflection of the bullet on the animal's humerus, the victim. These deductions represent the culmination of an exhaustive forensic investigation led by experts in multiple scientific and forensic disciplines.

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.

Threats to the Reintroduction Program of the Northern Bald Ibis (Geronticus eremita) in Italy: A Forensic Investigation.

The Northern Bald Ibis Geronticus eremita is an endangered species, and recently it was reintroduced in Europe by the Waldrappteam. The reintroduction program has been strongly threatened by several sudden deaths, mainly in Italy. The present study used a forensic approach to analyze all of the 27 Northern Bald Ibises found dead in Italy between 2016 and 2022, which were suspected to have been victims of poaching, and it followed the veterinary forensic guidelines. Human-related causes accounted for 60% of the deaths, including illegal hunting (30%), blunt force trauma (26%), and electrocution (4%). Natural causes, starvation (15%), predation (11%) and disease (7%), accounted for 33% of the deaths. 7% of the causes of deaths remained undetermined. This study uses a forensic approach to analyze, for the first time, the main causes of death in Geronticus eremita and highlights the relevance of detecting illegal actions related to endangered species and stopping the phenomenon of poaching.

The first evidence for Late Pleistocene dogs in Italy.

The identification of the earliest dogs is challenging because of the absence and/or mosaic pattern of morphological diagnostic features in the initial phases of the domestication process. Furthermore, the natural occurrence of some of these characters in Late Pleistocene wolf populations and the time it took from the onset of traits related to domestication to their prevalence remain indefinite. For these reasons, the spatiotemporal context of the early domestication of dogs is hotly debated. Our combined molecular and morphological analyses of fossil canid remains from the sites of Grotta Paglicci and Grotta Romanelli, in southern Italy, attest of the presence of dogs at least 14,000 calibrated years before present. This unambiguously documents one of the earliest occurrence of domesticates in the Upper Palaeolithic of Europe and in the Mediterranean. The genetic affinity between the Palaeolithic dogs from southern Italy and contemporaneous ones found in Germany also suggest that these animals were an important common adjunct during the Late Glacial, when strong cultural diversification occurred between the Mediterranean world and European areas north of the Alps. Additionally, aDNA analyses indicate that this Upper Palaeolithic dog lineage from Italy may have contributed to the genetic diversity of living dogs.

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.

A combined morphological and molecular approach for hair identification to comply with the European ban on dog and cat fur trade.

Animal furs are encountering more and more the detriment of public opinion, that is increasingly sensitive to animals, their welfare and protection. The feeling of outrage against animal suffering is particularly intense when cats and dogs are involved, since these are the most popular pets in Western countries. However, in some Asian countries breeding of dogs and cats for the fur industry is a common practice. These furs and their finished garments are often mislabelled in order to be imported and sold to unaware consumers in Western countries. The European Union has issued the Regulation 1523/2007, which bans the use and trade of dog and cat furs. The main purposes of the Regulation were to normalise the internal market and to address the concerns of European consumers about the risk of inadvertently buying products containing these species. The Regulation states that several analytical methods (microscopy, DNA testing and mass spectrometry) can be used to exclude dogs and cats as source species, but an official analytical protocol was not provided. In this paper, we report on the development of a reliable and affordable method for species identification in furs, based on a combined morphological and molecular approach. Our protocol provides an initial morphological analysis as a time and cost effective screening test. Only samples that are morphologically not excluded as canid/felid furs, based on few selected microscopic features, are then submitted to DNA testing. The application of this protocol on seized furs reached 92% identification of species. Our approach assists in identifying frauds and reinforcing the ban on dog and cat fur trade, allowing (1) rapid inexpensive recognition of fake furs, (2) exclusion of non-canid/non-felid furs through fast microscopic morphological screening, (3) overall cost reduction with lower number of samples to be submitted to DNA analysis, (4) analytical protocol to stand in court in case criminal sanctions are to be applied.

Systemic tuberculosis by MYCOBACTERIUM BOVIS in a free-ranging MARSICAN brown bear (URSUS ARCTOS MARSICANUS): a Case report.

BACKGROUND: Mycobacterium bovis is known to have a wide host range and has been isolated from numerous free-ranging wildlife species, carnivores included. In bears, M. bovis has been previously reported only from a culture of pooled lymph nodes of a black bear (Ursus americanus) in the absence of lesions. The aims of this study were to describe gross and microscopic pathological findings of M. bovis tuberculosis in a deceased Marsican brown bear (Ursus arctos marsicanus). CASE PRESENTATION: In March 2014, an adult female Marsican brown bear was found in the Abruzzo, Lazio and Molise National Park (Italy) showing severe non-specific clinical signs. The animal died soon after its discovery and the carcass was submitted to post-mortem examination to identify the cause of death. The bear was diagnosed with a severe Mycobacterium bovis infection, with both pathological and microbiological aspects suggesting ongoing generalization. A presumptive diagnosis of mycobacterial infection was initially made based on gross findings. Histopathology showed the presence of acid-fast bacilli in all sampled tissues along with poorly organized granulomatous lesions. Slow-growing Mycobacterium sp. was isolated from multiple organs (intestine, mesenteric lymph nodes, liver, spleen, lung and kidneys). The PCR and sequencing algorithm identified the Mycobacterium sp. isolate as M. bovis. Spoligotyping demonstrated that the M. bovis isolate belonged to spoligotype SB0120. CONCLUSIONS: This is the first report of lethal M. bovis tuberculosis infection in a free-ranging brown bear. This pathogen could have serious adverse effects in an endangered relic population such as the Marsican brown bear. Stricter application of health regulations in force, surveillance of M. bovis infections in wild ungulates and carnivore scavengers, along with dismissal of supplementary feeding points intended for cattle or wildlife, are warranted to control the presence of bovine tuberculosis in wild and domestic animals in protected areas.

Urinary capillariosis in a free-ranging Marsican brown bear (Ursus arctos marsicanus).

Extraintestinal nematodes have been seldom investigated in the brown bear (Ursus arctos). In this study, a case of urinary capillariosis and bladder associated lesions is reported in a deceased free-ranging Marsican brown bear (Ursus arctos marsicanus) from Central Italy. Gross lesions in the urinary bladder consisted of scattered foci of mucosal hyperemia, while at histological examination mild cystitis was observed. At microscopic examination of urine and bladder lavage fluid, capillariid adult female nematodes and eggs were found, suspected of belonging to the genus Pearsonema based on their location in the urinary bladder. This is the first report of Pearsonema infection and associated bladder lesions in a brown bear.

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

BACKGROUND: Google 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. OBJECTIVE: The objective of our study was to determine whether Google Glass is fit for veterinary forensic pathology purposes. METHODS: A 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. RESULTS: The 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. CONCLUSIONS: These 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.

Cardiac laceration following non-penetrating chest trauma in dog and cat.

Cardiac laceration with non-penetrating chest trauma is reported as a common cause of death in human following rapid deceleration in high-speed vehicular accident. In contrast, in veterinary medicine, traumatic rupture of heart and great-vessel structures appears to be an uncommon cause of death. Here we report three cases of cardiac laceration following non-penetrating chest trauma in a one cat and two dogs. In two of these cases, necropsy revealed a rupture of the heart associated with fractures of the ribs and lung contusion; only one case did not exhibit any external chest injury but revealed pericardial tear associated with hemothorax following rupture of the right auricle of the heart. However, in all three presented cases, the thoracic location of the injuries allowed to conclude that the cause of the cardiac rupture was due to a direct impact of the chest wall with a high speed object and consequent transmission of the kinetic force and compression of the heart between left and right thorax. These case reports underline the importance of a systematic and complete macroscopic evaluation of the heart in all cases of death following non-penetrating chest trauma in dog and cat such as in human. They also highlight how, in clinical and forensic practice, the cardiac injury following blunt chest trauma should be ruled out even in the cases of absence of external chest injury.

Veterinary forensic sciences to solve a fatal case of predation on flamingos (Phoenicopterus roseus).

The present case study concerns a case of predation of 4 individuals of captive pink flamingo in Emilia Romagna Region, Northeastern Italy. The pink flamingo (Phoenicopterus roseus) is a species included in the Red List of Threatened Species established by the International Union for Conservation of Nature (IUCN) which lists species in danger of extinction. During the Winter of 2013, 4 flamingos (2 in the Comacchio area, and 2 from Argenta and Codigoro oases - Ferrara province) were found dead some of them headless, with their bodies severely bitten. At first, a fox (Vulpes vulpes) was suspected to be the predator responsible for the killing and the birds were taken to the laboratory for further investigations. The investigations included: field observations, study of the predator behaviour, necropsy examinations, assessment of the intercanine distance, and genetic analysis on the predator's traces. The intercanine distance indicated that the predator could not have been a fox. The analysis of salivary DNA samples enabled us to establish that the predator was in fact a dog. This case highlights the importance of co-operation among the various branches of forensic sciences and the great usefulness of the roles filled by other veterinary forensic experts involved in solving crime.

Hindering the illegal trade in dog and cat furs through a DNA-based protocol for species identification.

In Western countries dogs and cats are the most popular pets, and people are increasingly opposed to their rearing for the fur industry. In 2007, a Regulation of the European Union (EU) banned the use and trade of dog and cat furs, but an official analytical protocol to identify them as source species was not provided, and violations of law are still frequent in all Member States. In this paper we report on the development and validation of a simple and affordable DNA method for species detection in furs to use as an effective tool to combat illegal trade in fur products. A set of mitochondrial primers was designed for amplification of partial cytochrome b, control region and ND1 gene in highly degraded samples, like furs and pelts. Our amplification workflow involved the use of a non-specific primer pair to perform a first test to identify the species through sequencing, then the application of species-specific primer pairs to use in singleplex end-point PCRs as confirmation tests. The advantage of this two-step procedure is twofold: on the one hand it minimises the possibility of negative test results from degraded samples, since failure of amplification with a first set of primers can be offset by successful amplification of the second, and on the other it adds confidence and reliability to final authentication of species. All designed primers were validated on a reference collection of tissue samples, obtaining solid results in terms of specificity, sensitivity, repeatability and reproducibility. Application of the protocol on real caseworks from seized furs yielded successful results also from old and dyed furs, suggesting that age and chemical staining do not necessarily affect positive amplifications. Major pros of this approach are: (1) sensitive and informative primer sets for detection of species; (2) short PCR amplicons for the analysis of poor quality DNA; (3) binding primers that avoid contamination from human DNA; (4) user-friendly protocol for any laboratory equipped for analysis of low-copy-number DNA. Our molecular procedure proved to be a good starting point for enforcing the EU Regulation against dog and cat fur trade in forensic contexts where source attribution is essential to the assignment of responsibilities.

A pathological Late Pleistocene canid from San Sidero (Italy): implications for social- and feeding-behaviour.

Evidence of diseases on vertebrate fossil bones can provide detailed information on many aspects of extinct animals. This study focused on pathological craniodental remains (left maxilla and dentary) referred to the canid Cuon alpinus unearthed from a Late Pleistocene karst filling deposit at San Sidero (Apulia, southern Italy). These fossils show clear evidence of a chronic periodontitis that caused the animal's death. Clinical diagnosis of the disease and the timing of its development have been defined on the basis of a veterinary odontostomatology approach, in addition to radiographic and tomographic techniques. From the initiation of the infection until death, a time span of at least 6 months occurred, and three main steps have been defined: (1) the bacterial infections of the buccal cavity turning into severe periodontitis, (2) the fracture of the lower carnassial and (3) the loss of teeth due to the worsening infection that deformed and/or eroded maxillary and mandibular bones and enlarged alveoli. The analysis of the palaeopathology also provides information about the biomechanics of the bite, on the feeding behaviour and on the relationships of injured members in a pack of Late Pleistocene canids.

First notification in Italy of cardiopulmonary filariosis (heartworm disease) in a wolf (Canis lupus).

The authors report on the first notification of filariosis (heartworm disease) caused by Dirofilaria immitis in a wolf (Canis lupus) in Italy. On account of this exceptional finding, the parasite was typed not only using traditional methods, such as stereomicroscopic examination, but also using highly innovative diagnostic methods, such as scanning electron microscope and molecular identification with the application of various recently developed methods (polymerase chain reaction and sequencing). Certain aspects regarding the epidemiology of the disease are discussed in the light of this first case in Italy that occurred in an area in which cardiopulmonary filiariasis had not previously been reported in wild or domestic carnivores.

Mechanisms of exchange modulation in trimethylenemethane-type biradicals: the roles of conformation and spin density.

The molecular structures and magnetic properties of six dinitroxide biradicals are described. Five of the dinitroxides are trimethylenemethane-type (TMM-type) biradicals; that is, the intramolecular exchange parameter, J, is modulated by a carbon-carbon double bond. However, the efficacy of the carbon-carbon double bond as an exchange coupler is determined by the molecular conformation. Our results show that the exchange parameters correlate with phenyl-ring torsion angles (phi) via a simple Karplus-Conroy-type relation: J = 44 cos(2) phi - 17. Comparison of these results to those obtained for our isostructural series of bis(semiquinone) biradicals shows that both the magnitude of J and the resistance of ferromagnetic J to bond torsions is proportional to the spin density adjacent to the exchange coupler.

Trends in exchange coupling for trimethylenemethane-type Bis(semiquinone) biradicals and correlation of magnetic exchange with mixed valency for cross-conjugated systems.

A magnetostructural correlation (conformational electron spin exchange modulation) within an isostructural series of biradical complexes is presented. X-ray crystal structures, variable-temperature electron paramagnetic resonance spectroscopy, zero-field splitting parameters, and variable-temperature magnetic susceptibility measurements were used to evaluate molecular conformation and electron spin exchange coupling in this series of molecules. Our combined results indicate that the ferromagnetic portion of the exchange couplings occurs via the cross-conjugated pi-systems, while the antiferromagnetic portion occurs through space and is equivalent to incipient bond formation. Thus, molecular conformation controls the relative amounts of ferro- and antiferromagnetic contributions to exchange coupling. In fact, the exchange parameter correlates with average semiquinone ring torsion angles via a Karplus-Conroy-type relation. Because of the natural connection between electron spin exchange coupling and electronic coupling related to electron transfer, we also correlate the exchange parameters in the biradical complexes to mixed valency in the corresponding quinone-semiquinone radical anions. Our results suggest that delocalization in the cross-conjugated, mixed-valent radical anions is proportional to the ferromagnetic contribution to the exchange coupling in the biradical oxidation states.