Forensic Science International 310 (2020) 110254

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Forensic Science International

journal homepage: www.elsevier.com/locate/forsciint

Dog-bite-related attacks: A new forensic approach

Fabrizio Iarussia,1, Luigi Cipollonib,1, Giuseppe Bertozzib , Luigi Sassob , Michela Ferrarab ,
Monica Salernoc, Giuseppe Tommaso Roberto Rubinoa , Francesca Magliettab ,
Armida Dinisib , Davide Albanoc , Valerio Iarussid , Cristoforo Pomarac,2 ,
Francesco Sessab,* ,2
a
Department of Emergency and Organ Transplants, Veterinary Section, University of Bari, Valenzano, Bari, Italy
b
Department of Clinical and Experimental Medicine, Section of Legal Medicine, University of Foggia, Foggia, Italy
c
Department of Medical, Surgical and Advanced Technologies "G.F. Ingrassia", University of Catania, Catania, Italy
d
Fundamentalist Veterinary Surgery, Small Animal Diseases Specialist, Foggia, Italy

A R T I C L E I N F O A B S T R A C T

Article history: Dog attacks today represent a health hazard considering that prevention strategies have not always been
Received 24 July 2019 successful. The identification of the dog that attacked the victim is necessary, considering the civil or
Received in revised form 4 March 2020 criminal consequences for the animal’s owner. An accurate scene analysis must be performed collecting a
Accepted 11 March 2020
series of important information.
Available online 14 March 2020
Forensic investigations in dog attacks involve different methods, such as the evaluating of the canine
Short Tandem Repeat (STR) typing in saliva traces on wounds or bite mark analysis, however, these
Keywords:
techniques cannot always be applied. The effort to find new methods to identify the dog that attacked the
Forensic science
Forensic pathology
victim represents a very interesting field for the forensic community.
Dog attacks This study aims to propose an innovative approach, based on the identification of the victim's profile in
Cattle genotyping the dog's mouth, using a buccal swab on the suspected aggressor dog, to find the victim’s genetic profile.
Dog identification In addition, a further goal of this study is to determine the persistence time of hexogen DNA in the dog’s
Short tandem repeat mouth to define a timeframe for performing this particular technique.
TGLA53 For this purpose, ten different dogs were used to aggressively bite a bovine sample (reference sample)
TGLA122 to simulate the victim. For each dog two buccal swabs were taken at different time intervals: 300 , 450 , 600 ,
900 , 1200 , 1500 , 1800 and 2400 . The typing of the swabs provided an interpretable profile after 450 while
traces of bovine profile were found until 1500 after the dog attack simulation.
These results could be improved using the human identification kit, which is more sensitive. In the light
of this experimental study, the forensic community should consider using this approach in real casework
studies with the aim of collecting new data, validating this technique for forensic use.
© 2020 Elsevier B.V. All rights reserved.

1. Introduction court to exclude criminal causes and to identify the animal

Dog attacks today represent a health hazard considering that
prevention strategies have not always been successful [1,2]. In
recent years, there has been an increasing number of hospital
admissions due to dog attacks, many of which involving children,
probably because they cannot understand a dog’s behavior thus
causing aggressive reactions [3]. Experts could be called by the
* Corresponding author at: Department of Clinical and Experimental Medicine,
University of Foggia Viale Pinto, 71122, Foggia, Italy.
E-mail address: francesco.sessa@unifg.it (F. Sessa).
1
These authors contributed equally to this work.
2
These authors shared last authorship.
involved in the aggression. In many countries, the law states that
the dog’s owner is responsible for the consequences of the lesions
caused by his/her animal. In dog attacks, a multidisciplinary
approach, including a detailed crime scene investigation, as well as
the study of the victim and the animal, is essential [4,5].
An accurate scene analysis must be performed, collecting
important information such as the age of the victim, the breed type
of the dog and finding possible witnesses. A careful inspection of
where the dog aggression took place is mandatory to collect all
evidence that can be potentially useful to associate the victim to
the animal and vice-versa [6–9].
The classical pattern of a wound produced by a bite is called “a
hole-and-a-tear” [1,10]: the hole is produced by the canine of the
upper or lower dental arch of the dog, that the animal uses to anchor

http://dx.doi.org/10.1016/j.forsciint.2020.110254
0379-0738/© 2020 Elsevier B.V. All rights reserved.
2 F. Iarussi et al. / Forensic Science International 310 (2020) 110254
the tissues, while the tear is due to the shaking of the dog’s head. This
typical lesion is associated with various types of lesions, including
punctiform “V”-shaped holes, abrasions, lacerations of the skin and
soft tissues penetrating wounds and bone fractures [11].
Fatal dog attacks are rare, and usually involve innocent people,
such as children or the elderly and death is secondary to
decapitation, massive bleeding, asphyxia, or air embolism [12–14].
To date, the most used methods to identify an offending dog are
odontological analysis of bite marks and DNA analysis.
Odontological analysis can exclude various breed types of dogs
and recognize the breed type of the offending dog, as the shape of
the bite mark characterizes each breed type. Inter-canine distances
are variable depending on the breed type of the dog, thus their
measurement can be useful to identify the suspected animal
[15,16]. For this purpose an approach using 3D processing/
modelling software has been described that demonstrates the
correspondence between the dog's dental arches and the wound
[17]. It is essential to note that this kind of analysis can be used
effectively when two dogs are very different under this criterion.
Contrariwise, other evidence must be collected.
The DNA method is based on the search for the canine profile,
typing the so-called microsatellites or Short Tandem Repeats (STRs)
at the wound site [18,19]. Indeed, the most used DNA technique
consists in the extraction of STRs from saliva traces of the offender
dog on the victim’s wound [20]. This practice has proved to be a valid
support in the identification of attacking dogs, but has several
limitations. When a dog attack occurs, the time between the accident
and sample collection is very variable, passing from several tens of
minutes to hours. Moreover, the first thing to do is the rescue
operation: during this phase, the use of several medications on the
victim’s tissue could erase the dog’s biological traces. Another
method that could be used to identify the dog is the genetic analysis
of dog hairs: it has been described that a direct amplification of the
hair can be used for this purpose [21].
This study proposes an innovative approach, based on the
identification of the victim's profile in the dog's mouth, using a
buccal swab on the suspected aggressor dog, to find the victim’s
genetic profile. In addition, a further goal of this study is to
determine the persistence time of hexogen DNA in the dog’s mouth
to define a timeframe for performing this particular technique.
2. Materials and methods
2.1. Experimental model
Ten different dog types were enrolled for this study: two German
Shepherds, two Italian Mastiffs, two Bull Terriers, two Rottweilers,
and two mongrels. In agreement with the owners, the dogs were
selected for their aptitude to bite deeply into rubber toys. As a
Fig. 1. The experimental model: ten dogs bit a bovine meat sample for 5 min; after the established period of time, two swabs were obtained from two different anatomical
regions of the mouth.
replacement for a victim, a single large piece of bovine meat was
used, dividing it into several samples of about two hundred grams.
Written informed consent forms were obtained from all dog owners.
All experimental procedures were performed in strict accordance
with the international regulation on animal welfare (EU Directive
2010/63/EU for animal experiments) and were approved by the
Scientific Committee of the University of Foggia.
Before the experiment, bovine DNA was extracted from two
samples (designated E1 and E2) in order to collect the reference
profiles of the control. The complete genotyping is reported in the
supplementary file (Figs. S1–S3).
To simulate the aggression, each dog bit into the beef sample for
five minutes, to ensure a tightening action with the canines and
tearing with the molars as would happen in a real attack.
At the end of each "attack", two upper and lower dento-gingival
swabs were performed, both on the buccal and mesial part, with
particular attention to the region of canines and incisors. For each
dog, the samplings were repeated at different time intervals: 300 ,
450 , 600 , 900 , 1200 , 1500 , 1800 and 2400 . During the time between the
simulation of the aggression and the application of the swab, the
animal was monitored to record any behavior that could have
compromised the test. Particularly, the dog was maintained in
water and food deprivation.
At the end of each test time, a total of 10 samples for the upper
jaw (u.j.) and 10 samples for the lower jaw (l.j.) were obtained. Each
one was identified by a specific code and frozen at 20  C
immediately after collection. At the end of the experiment 160
samples were obtained (20  8 tested time).
The experimental model is illustrated in Fig. 1.
2.2. DNA extraction and quantitative analysis of DNA
DNA extraction from the two kinds of samples (portion of beef
tissue for control samples and canine buccal swabs) was
performed using the QIAamp1 DNA Mini Kit (Qiagen, Hilden,
Germany), following protocols for the extraction of DNA from
tissue and from buccal swabs. DNA was then eluted into 30 mL of
AE buffer. To monitor reagent contamination a negative control
was also tested. Obtained samples were frozen and stored at 20 
C before being processed for the subsequent phases.
QubitTM ds DNA HS assay kit (InvitrogenTM, Thermo Scientific
Inc., USA) and QubitTM 3.0 fluorometer were used to quantify DNA
isolated from the samples following the manufacturer’s recom-
mended protocol.
2.3. Bovine meat typing
To obtain the genetic profile, the co-amplification Stock-
MarksTM for Cattle Genotyping Kit (Catalog number: 4307480,
 F. Iarussi et al. / Forensic Science International 310 (2020) 110254
Applied Biosystems, Europe BV) was used. The amplification
primer mix for the StockMarks1 for Cattle contains 11 primer sets:
all investigated loci are dinucleotide repeats. Dinucleotide repeats
give specific stutter patterns: for this reason, in order to define the
reference profile, the criteria of the consensus method were
applied [22]; moreover, the electropherogram interpretation was
obtained following the kit’s user guide suggestions reported in the
section "Interpretation of results".
The control DNA provided by the kit, the reference sample
(bovine meat) and all swabs obtained in this experimental work,
were amplified. In addition, following the manufacturer’s recom-
mended protocol, all samples were standardized to a concentra-
tion of 5 ng/mL (samples need to be at a concentration between
110 ng/mL). For each PCR run, a negative control was amplified, in
order to verify the absence of exogenous contamination.
The PCR reaction master mix was used in an Eppendorf1
Mastercycler1 gradient thermal cycler (Eppendorf, Hamburg,
Germany), according to the manufacturer’s guidelines.
The amplified product was analyzed in a 10 mL reaction volume
that consisted of 1 mL amplified product plus 9 mL formamide/
GeneScan TM 500 LIZTM dye size standard (Applied Biosystem)
mixture, using capillary electrophoresis on an ABI Prism1 3130
genetic analyzer (Applied Biosystems Inc., Foster City, CA, USA) - an
automated capillary electrophoresis system based on fluorescence
- and data were analyzed using the GeneMapper Analysis Software
version 4.0. The DNA profiles obtained were analyzed using an
analytical threshold of 50 RFU. At the end of the analysis, 160 swab
profiles, 2 reference profiles obtained from bovine meat, 16 blank
extraction profiles, 16 negative PCR profiles, and 16 reference
profiles were obtained and analyzed. Considering the negative
results obtained from all swabs performed after 2400 , they were
used as negative controls.
2.4. Statistical analysis
Descriptive analyses were performed using frequency percen-
tages (total number of alleles observed/total number of alleles
expected). Data were analyzed with the software package SPSS 22.0
for Windows. The one-way variance analysis (ANOVA) was used to
determine any statistically significant differences among the groups.
3. Results
The first step of the proposed experimental model was the
typing of the bovine meat used as the reference sample,
Fig. 2. The box plot analysis on the DNA concentrations obtained at the established times showing that the DNA concentrations diminished over time.
3
considering that in the bovine kit there are no reference standards
(allelic ladder) unlike the human kit used in forensic investigations
where reference standards are provided. The differentiation
between the various STRs is based on their molecular weight
and the respective dye Label. The DNA profile of the reference
sample (bovine meat) used in this experiment is reported in the
supplementary file, indicating the molecular weights obtained for
each marker. A univocal complete profile was obtained from both
samples analyzed (E1 and E2), and was used as a reference sample.
Subsequently, DNA extraction was performed on the buccal
swabs. The box plot analysis was applied to present the data about
DNA concentrations. The values of DNA concentrations are
summarized in the supplementary file (Fig. S4), under the criteria
of the sampled anatomical region. No statistical differences were
reported in the box plot analysis, comparing the data obtained
sampling the u.j. and l.j. (one-way ANOVA [F = 2.48, p = 0.11]).
The complete genotyping is reported in the supplementary file
(Figs. S1– S3).
The concentration data are compared and summarized in Fig. 2,
evaluating interval time between the bite on the meat and
sampling: the trend suggests a lower level of DNA for longer times
between bite and sampling.
In the supplementary files, the electropherograms obtained for
two swabs collected after bite with two different interval times:
450 and 1200 are summarized. After the amplification and runs, the
electropherograms obtained were compared with the reference
profile evaluating the ratio between detected and expected alleles
(Fig. 3). The ratio calculation is made dividing the number of
detected alleles by the number of expected for each swab: the
number of expected alleles is always the same (all alleles of the
reference sample, 20 alleles), while the detected amount is related
to the time between the bite and sampling.
As shown in Fig. 3, the complete profile of the reference sample
was obtained after 30 min (98.5% for u.j.; 99.5% for l.j.). The ratio
percentage decreased in an inversely proportional manner,
comparing the interval of time between the bite and sampling:
the more time passed, the lower the number of alleles detected.
The data obtained were statistically significant (one-way ANOVA [F
= 115.24, p = 2.94  106]).
After 45 min, 86% of the alleles were detected for samples
obtained from the u.j. and 86.5% for the l.j. After 60 min, the
percentage was 73% for the u.j. and 74.5% for the l.j. Moreover, the
percentage went down after 900 (54% for the u.j. and 58% for the l.j.)
and 1200 (44% for the u.j. and 33% for the l.j.). In the supplementary
file were reported a profile obtained after 450 and another one
4 F. Iarussi et al. / Forensic Science International 310 (2020) 110254
Fig. 3. This graph shows the ratio percentage of detected/expected alleles with error bar; the data were divided under the criteria of the anatomical region swabbed.
obtained after 1200 (Figs. S4–S6) are reported. After 150 min, only
the alleles related to the TGLA53 and TGLA122 loci were detected,
obtaining 20% for the u.j. and 18% for the l.j. No peaks were detected
for all loci in all samples collected in the time intervals after 150
min. No statistical differences were reported comparing the data
obtained sampling the u.j. and the l.j. (one-way ANOVA [F = 0.002, p
= 0.96]).
4. Discussion
Dog attacks and dog-pack attacks represent a hazard for public
health both because of the severe injuries caused and, in some
cases, because of fatalities [2,8]. Moreover, even if every nation
tries to reduce the phenomenon by introducing new laws, they do
not always produce a reduction in dog attacks and dog-bite–
related fatalities [8]. It has been described that dog attacks are
frequently related to the patterns of animal behavior [1,10]. The
defense of territory, the role of hunger, environmental stimuli, and
social interaction, represent different important aspects involved
in dog attacks. Moreover, the high-pitched cries, rapid movements,
and strategy of defense of the victim can be considered as usual
factors involved in the animal attacks [9,13]. Furthermore, the
gravity of damages generated by dog attacks is related to the
vulnerability of the victim: it is clear that when an attack affects
the elderly, young children and disabled persons, it could be fatal
for the victim [9,23,24].
Animal DNA represents a new type of evidence and the
potential applications are very wide-ranging. Animal DNA has
been used to match canine saliva recovered from a murder victim
to an attack dog (individual DNA profiling). Canine DNA analysis of
STR markers has become an effective tool in forensic investigations
allowing the guilty dog identification [25–27].
Furthermore, Eichmann et al. suggested looking to canine
STRs, not just on a victim’s wound, but also on gauzes used to
wrap the injuries [28]. They describe that of fifty-two cases of
fatal attacks, the complete profile of the dog was obtained only in
fifteen, regardless of the sampling method used (wounds or
gauze). The limitation of this approach is related to the difficulty
to obtain a complete dog profile in all cases, thus it is not always
possible to demonstrate the involvement of the animal in the
aggression.
It is important to note that biological traces of domestic animals
on human skin are frequently found, as interactions with dogs are
frequent in everyday life, especially if the victim is a dog owner.
This situation could be very complex to analyze for the forensic
team in the reconstruction of the crime scene.
For these reasons, the efforts to find new methods to identify
aggressor dogs represents a very interesting field for the forensic
community. The research of canine STRs on wounds in dog attacks
does not always represent a successful procedure, because of
different factors. After a dog attack, first aid workers operate before
the intervention of the forensic team, treating the wound with
antiseptics, which alter dog residues.
In this experimental study, meat was used to mimic the object
of the dog’s aggression. Indeed, for obvious reasons, it was not
possible to use human subjects to simulate a victim. Two buccal
swabs were taken from each animal in order to define the latency
time of bovine DNA (simulating the victim) in the dog's mouth
(simulating the aggressor). Moreover, in this experimental model
we investigated if the anatomical region of the dog’s mouth could
be related with the successful rate of detection of exogenous DNA.
As previously discussed, the DNA concentration obtained
decreased over time: indeed, the largest quantity of DNA was
obtained in the samples collected at 300 and 450 after the bites,
while only traces of cattle DNA, unsuitable for identification, were
found after 1200 and 1500 ’. No data were obtained at 1800 and 2400 .
These data were confirmed analyzing the ratio of the detected/
expected alleles: our results demonstrate an inverse correlation
between the time interval after the bite and DNA sampling.
Moreover, in this study the DNA concentration was measured
through a no species-specific test: therefore, the concentration
goes down because at high interval times the bovine DNA was not
detected, reducing the DNA concentration values. These results
show that this novel approach can represent an evolution of
previous ones, because it overcomes the previous bias, despite the
study limitations.
 F. Iarussi et al. / Forensic Science International 310 (2020) 110254
The major limitation of the discussed experimental model is
related to the use of beef samples for the test. Firstly, the research
in the analysis of bovine microsatellites is not as advanced as that
for forensic human identification [29]. If this choice represents a
limit for the experimental work, it could, on the other hand, be
considered an advantage in real casework. The results of the
present study could be improved using the human identification
kit, obtaining a complete profile at more than 450 . In our results, the
meat DNA traces were detected until 1500 , even if only the shorter
loci were amplified. In consideration of the limitation of the kit
used to obtain the profile, it could be thought that in real casework
the use of the human identification kit could obtain genetic profiles
from minimal DNA traces, increasing the time between the attack
and sampling to obtain a positive result.
The timing of the intervention of forensic experts at a crime
scene is variable, depending on various situations. For this reason,
establishing the latency time of human traces in an aggressor dog’s
mouth represents a starting point to try and draw up a new
successful protocol in cases of canine aggression. Furthermore, the
swab samples can be stored for several years, in order to be used as
evidence in court.
In the light of this experimental study, the forensic community
should use this approach in real casework with the aim of
collecting new data on human cases, thus validating this technique
for forensic use.
Compliance with ethical standards
The present paper is in compliance with the ethical standards of
the Journal.
Funding
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors
Informed consent
Neither patients nor research participants were involved.
Ethical approval
All experimental procedures were performed in strict accor-
dance with international regulations on animal welfare and were
approved by the Scientific Committee of the University of Foggia.
CRediT authorship contribution statement
Fabrizio Iarussi: Conceptualization, Methodology, Validation,
Writing - original draft. Luigi Cipolloni: Conceptualization,
Methodology, Validation, Writing - review & editing. Giuseppe
Bertozzi: Formal analysis, Investigation, Writing - review &
editing. Luigi Sasso: Formal analysis, Investigation, Writing -
review & editing. Michela Ferrara: Formal analysis, Investigation,
Writing - review & editing. Monica Salerno: Formal analysis,
Investigation, Writing - review & editing. Giuseppe Tommaso
Roberto Rubino: Formal analysis, Investigation, Writing - review &
editing. Francesca Maglietta: Formal analysis, Investigation,
Writing - review & editing. Armida Dinisi: Formal analysis,
Investigation, Writing - review & editing. Davide Albano: Formal
analysis, Investigation, Writing - review & editing. Valerio Iarussi:
Formal analysis, Investigation, Writing - review & editing.
Cristoforo Pomara: Conceptualization, Methodology, Validation,
Writing - review & editing. Francesco Sessa: Conceptualization,
Methodology, Validation, Writing - review & editing.
5
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgements
We wish to thank the Scientific Bureau of the University of
Catania for language support.
Appendix A. Supplementary data
Supplementary material related to this article can be found,
in the online version, at https://doi.org/10.1016/j.for-
sciint.2020.110254.
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