Luca Botta

502306
Matriculation number: 901879

Contacts

Supervisor

Phd thesis

Scientific background/state of the art
Veterinary forensic medicine provides support in civil and criminal cases, aiding not only in determining the cause of death but also in identifying and analysing the physical evidence associated with it^1,2. Nevertheless, veterinary forensic pathology is a subject of ongoing research and requires the integration of various animal species to explore the inherent discrepancies within comparative studies¹. Most of the studies have been conducted in pigs, often used as proxies in humans, and rodents, which can easily bred under controlled conditions³. Conversely, only few studies are available on companion animals warranting further investigation.

In forensic medicine, it is important to assess not only the cause of death, but also the time elapsed between the death and the autopsy, named Post-Mortem Interval (PMI), to correctly identify when the crime occurred. Methods used to calculate PMI in humans and specific animal species include analysing gross and histological alterations associated with Accumulated Degree Days (ADD)^4,5, as well as methods for DNA, RNA, and proteins detection, as reported in prior research⁶.The ADD method uses the concept of heat-time units as a metric for estimating decomposition rates, achieved by computing the average daily temperature. This method has demonstrated accuracy and reliability in estimating the PMI4,7.

This project focuses on the domestic cat (Felis catus), which offers a standardized model due to its consistent size, weight and coat coverage across breeds. Additionally, cats are frequently involved in legal cases.

Aims
This project aims to investigate the PMI in cats via macroscopic, microscopic and biomolecular analysis and will be carried out on animals recovered within 1 hour from death and held under different postmortem artificial conditions (in the field at various ADD, refrigerated at +4^o C and frozen at -20^o C) at various time intervals.
Macroscopic and microscopic observations will be utilized to develop a standardized score system for postmortem alterations. These evaluations will be correlated with the time elapsed since death and the ADD.

At last, this research aims to identify the most suitable technique to estimate PMI through RNA degradation rate, comparing automated electrophoresis, qPCR and ddPCR analysis. RNA has been favoured over DNA or proteins due to its comparatively lower stability, which facilitates the more linear degradation patterns within each sampled organ across different time intervals and/or ADD.

Materials and methods
The study will be conducted on 78 cats donated after death by their owners, following the signing of an informed consent. The cats will be divided into controls (6 cats necropsied within 1 hour after death) and “cases”. The “cases” will be preserved “in field” (maintained in an “open air” environment, with restricted access limited to insects), “refrigerated” and “frozen” (respectively, in a +4^o or -20^o C cold room) and subjected to a forensic autopsy⁸ after 1, 3, 14 or 28 days (6 animals for each time and condition).

Macroscopic evaluation of the body and of each organ will be carried out on photographs obtained during necropsy. All the sampled organs will be submitted to histologic examination. All the data collected will be organized to develop macro- and micro-scoring systems on indicators of alterations.

The RNA Integrity Number (RIN) will be estimated using automated electrophoresis across different tissues collected during necropsy. The results will be compared with those obtained by qPCR and ddPCR using RPL17 as reference gene.
Descriptive and inferential statistics will be conducted on macroscopic and histological scores and biomolecular results, and a correlation will be applied to compare the different PMI estimation methods.

Expected results
The macroscopic and microscopic evaluations will serve to establish a scoring system for every sampled organ, as well as for the cadaver. The system will allow the classification of alterations related to the preservation conditions and the PMI.

The molecular analysis results will integrate PMI estimation. Comparing the three methods for detecting RNA quality (RIN, qPCR, ddPCR) will help defining the most suitable technique for assessing RNA degradation rates, whether used individually or in combination, and determining the tissue or organ most suitable for PMI estimation.

References
1. R. Munro and H. M. C. Munro, “Some challenges in forensic veterinary pathology: A review,” Journal of Comparative Pathology, vol. 149, no. 1. pp. 57–73, Jul. 2013. doi: 10.1016/j.jcpa.2012.10.001.
2. R. Touroo, J. W. Brooks, R. Lockwood, and R. Reisman, “Medicolegal Investigation,” in Veterinary Forensic Pathology , Springer., vol. 1, J. W. Brooks, Ed., Springer International Publishing, 2018, pp. 1–8.
3. A. Dautartas, M. W. Kenyhercz, G. M. Vidoli, L. Meadows Jantz, A. Mundorff, and D. W. Steadman, “Differential Decomposition Among Pig, Rabbit, and Human Remains,” J Forensic Sci, vol. 63, no. 6, pp. 1673–1683, Nov. 2018, doi: 10.1111/1556-4029.13784.

4. M. S. Megyesi, S. P. Nawrocki, N. H. Haskell, “Using accumulated degree-days to estimate the postmortem interval from decomposed human remains,” Journal of Forensic Science, May 2005, doi: 50(3):618-26. PMID: 15932096.
5. M. Erlandsson and R. Munro, “Estimation of the post-mortem interval in beagle dogs,” Science and Justice, vol. 47, no. 4, pp. 150–154, Dec. 2007, doi: 10.1016/j.scijus.2007.09.005.
6. A. Sangwan, S. P. Singh, P. Singh, O. P. Gupta, A. Manas, and S. Gupta, “Role of molecular techniques in PMI estimation: An update,” Journal of Forensic and Legal Medicine, vol. 83. Churchill Livingstone, Oct. 01, 2021. doi: 10.1016/j.jflm.2021.102251.
7. A. Wahyono and D. P. Alim, “Estimation of Post Mortem Interval using Accumulated Degree Days (ADD) Method,” Malaysian Journal of Medicine and Health Sciences, vol.18, Nov. 2022

8. O. Paciello, R. Fico, E. Antuofermo, C. Cantile, A. M. Cantoni, L. Della Salda, G. Di Francesco, L. R. Gibelli, V. Grieco, G. Lanteri, E. Lepri, G. E. Magi, S. Mazzariol, L. Meomartino, G. Miletti, G. Militerno, F. Mutinelli, G. Passantino, D. Pintus, … S. Zoppi, “Linee guida nazionali per le autopsie a scopo forense in medicina veterinaria,” Associazione Italiana Di Patologia Veterinaria, 2019.