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SPONTANEOUSLY ARISING DISEASE: REVIEW ARTICLE

Some Challenges in Forensic Veterinary Pathology:

A Review
R. Munro*,† and H. M. C. Munro†
* Royal Veterinary College, London and † Royal (Dick) School of Veterinary Studies, Edinburgh, UK

Summary
Forensic veterinary pathology is a diverse discipline that is in an early phase of its development. Common chal-
lenges include estimation of the age of skin wounds and bruises, the diagnosis of drowning and estimation of the
time since death. However, many details of the pathological findings related to these various aspects await val-
idation. The ‘multispecies’ nature of veterinary pathology, combined with the preponderance of published ob-
servations originating from animal experimentation, rather than casework, poses two challenges. Firstly,
extrapolation of results between species may jeopardize the reliability (and credibility) of the forensic opinion.
Secondly, experimental studies may not truly reflect the spectrum of changes seen in actual cases (e.g. extent of
injuries, infection, age and health of victim). With regard to drowning, diagnosis based on post-mortem find-
ings remains problematical. Methods for estimation of the time since death (also known as the post-mortem
interval) continue to be a major focus of study, with fresh avenues such as post-mortem diagnostic imaging of-
fering interesting possibilities.
Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: bruising; drowning; forensic veterinary pathology; time since death

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
What is the Meaning of ‘Forensic’? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Brief History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Estimation of the Age of Skin Wounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Estimation of the Age of Bruises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Gross Examination of Bruises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Microscopical Examination of Bruises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Other Tests of Bruises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Bruising in Poultry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Drowning: A Difficult Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Experiments on Drowning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Lung Weights of Drowned Dogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Microscopical Examination of the Lungs from Drowned Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
The Diatom Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Estimation of Time since Death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Methods for Estimating Time since Death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Temperature-Based Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Post-mortem Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Histopathology and Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Post-mortem Radiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

Correspondence to: R. Munro (e-mail: Ranald.Munro@ed.ac.uk).

0021-9975/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jcpa.2012.10.001
58
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Conflict of Interest Statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Introduction
The work of the forensic medical pathologist and the
forensic veterinary pathologist is essentially similar,
but there is a major difference. The work of the former
focuses on one species only, while that of the latter en-
compasses multiple species of great variety, ranging as
it does through cases involving companion animals
(including exotic species), farmed livestock and wild-
life. The complexity of multispecies forensic pathol-
ogy renders a review of all aspects, in all species,
unwieldy and confusing. For this reason, the present
review concentrates on four selected aspects of foren-
sic veterinary pathology that, in the present state of
knowledge, are sources of difficulty. These areas are
(1) estimation of the age of skin wounds, (2) estima-
tion of the age of bruising, (3) the diagnosis of drown-
ing and (4) estimation of the time since death.
Veterinary pathologists are often encouraged to
state when a skin wound was inflicted or to give opin-
ions on the age of bruises. Similarly, it is often of pri-
mary interest to investigators to establish the time of
death as accurately as possible and there is an expec-
tation that the pathologist can provide the answer.
Also included in the challenges facing the veterinary
pathologist is the troublesome subject of drowning,
which has been the focus of extensive research on
both human corpses and experimental drowning of
various other species. Courts rely on veterinary pa-
thologists to unravel the necropsy evidence with
a view to establishing whether an animal, recovered
from water, was alive at the time of immersion. Addi-
tionally, the veterinary pathologist will from time to
time be required to examine a cadaver where there
is concern that the animal may have been drowned
in a bath or other receptacle. Such cases may be fur-
ther complicated if the body has been moved to an-
other location.
What is the Meaning of ‘Forensic’?
Definitions are important in the avoidance of misun-
derstandings and this is particularly relevant when
words or phrases are used both in ‘everyday language’
and in medical terminology. ‘Forensic’ is just such an
example, since its original meaning of ‘relating to the
law’ has broadened over recent years to implying ‘a
detailed investigation and collection of evidence re-
gardless of whether or not there is a specific legal
case or enquiry pending’ (Cooper and Cooper,
R. Munro and H.M.C. Munro
2007b). For the purpose of this review, the word ‘fo-
rensic’ is used in the context of ‘relating to the law’.
Further confusion can arise through differing inter-
pretations of the terms ‘forensic medicine’, ‘forensic
pathology’ and ‘forensic science’.
‘Forensic medicine’ (sometimes also called ‘legal
medicine’) refers to the application of medical and
veterinary knowledge to the elucidation of evidence
for the courts. To some, forensic medicine and forensic
pathology are recognized as being separate, but
closely allied to one another; hence the departmental
title ‘Pathology and Forensic Medicine’ that is found
in many veterinary institutes worldwide. ‘Forensic
science’, however, is generally understood to encom-
pass factors such as scenes of crime examination,
ballistics tests, DNA analysis, toxicology, etc. Never-
theless, it should be recognized that some workers
may include pathology under the term ‘forensic sci-
ence’.
Reports from forensic veterinary pathologists may
be requested in both criminal and civil cases. Civil
cases are concerned with the settlement of private dif-
ferences between members of a community and are
distinct from cases where a criminal charge is in-
volved. The standard of proof necessary in civil cases
is often less exacting than that required in criminal
cases, with the decision depending on the ‘balance
of probabilities’ rather than a need ‘to satisfy the court
beyond reasonable doubt’ that a crime has been com-
mitted (Ross and Chalmers, 2009). Nevertheless, the
task of the forensic pathologist remains the same: to
provide meticulous records, care and attention to de-
tail, clear reporting and recognition that the report is
produced to aid the court to arrive at a just decision
(Munro and Munro, 2008b). Given the nature of civil
proceedings, which frequently are settled out of court
and therefore may not come to the attention of the
general public, very little has been published, in En-
glish language peer-reviewed journals, on the details
of veterinary evidence in civil cases. Thus, this review
is biased towards criminal offences.
Brief History
In the medical field, the development of forensic med-
icine (using the term in its broadest sense) has been
well documented (Fisher and Platt, 1993; Wecht,
2005). At the forefront was Italy, and by the second
half of the 16th century medical legal autopsies were
being conducted in a number of European countries.
 Challenges in Forensic Veterinary Pathology
The first formal academic lectures in forensic pathol-
ogy were held by Johann Michaelis and Johannes
Bohn at the University of Leipzig in the mid to late
17th century (Smith, 1954 cited by Finkbeiner et al.,
2009). Towards the end of the 18th century, three
Chairs in forensic medicine were created in Paris,
Montpellier and Strasbourg, but it was not until
1807 that the first Chair of Legal Medicine in the En-
glish speaking world was established in Edinburgh,
Scotland. Shortly after, the first Professor of Medical
Jurisprudence was created by the College of Physi-
cians and Surgeons of New York City in 1813
(Camps, 1976).
In contrast, the development of forensic veterinary
medicine has not been formally documented. How-
ever, in recent years the publication of a number of
English language textbooks and journal articles high-
lights the increasing interest in this area (Stroud,
1995; Cooper, 1998; PAW, 2005; Sinclair et al.,
2006a; Cooper and Cooper, 2007a, 2008; Merck,
2007a; Munro and Munro, 2008a, 2011; Newbery
and Munro, 2011). Wildlife forensic investigation
continues to generate attention throughout the world
and two recent textbooks covering this complex area
(Huffman and Wallace, 2012; Cooper and Cooper,
2012) are a welcome addition to the literature.
Estimation of the Age of Skin Wounds
Ohshima (2000) was of the view that in forensic pa-
thology ‘wound examination is the most important
matter and that it requires much experience of foren-
sic practice’. Other pathologists may place the em-
phasis elsewhere, but in many cases it can be of
considerable evidential interest to determine whether
a wound occurred before or after death. This is not al-
ways an easy or simple distinction since the lack of re-
action around the wound margin does not necessarily
indicate that a wound occurred post mortem. How-
ever, a well-developed response around the wound
margin is a clear guide to ante-mortem injury. Even
so, care should be taken when assessing microscopical
changes. For example, it has been recognized for
many years that small numbers of leucocytes at
wound margins do not necessarily signify ante-
mortem injury (Raekallio, 1980). Indeed, Saukko
and Knight (2004d) caution that leucocytic infiltra-
tion can occur in wounds in people ‘even several hours
after death’.
If the wound is adjudged to be ante-mortem in
character, the period that has elapsed from the time
of injury to death of the animal (or presentation for
veterinary examination) may be of significance. Ide-
ally, forensic veterinary pathologists should have
a thorough understanding of the pathophysiology of
59
wound healing in the species of animals under inves-
tigation. However, there may be no such current
knowledge!
Wound healing is complex, but detailed knowledge
of the process has expanded greatly since the 1980s
(Swaim and Krahwinkel, 2006). Much of the under-
standing of the stages of wound healing, based on mi-
croscopy, has been gleaned from experimental studies
in a range of animals including mice (Hiss et al., 1988;
Birch et al., 2005), pigs (Sullivan et al., 2001) and
guinea pigs (Cox et al., 1989). Therein lies a problem,
because experimental studies may not truly reflect the
spectrum of changes related to the size, type and an-
atomical location of the wound, or take account of
the extent of infection in the wound, or the age, spe-
cies or health of the victim. The complications affect-
ing wound healing are considered by Demetriou and
Stein (2011).
Animal models are frequently used in researching
human conditions. The results of these experiments
are then extrapolated despite substantial differences
existing between the reactions of the ‘model’ and hu-
man beings. For this reason, Saukko and Knight
(2004c) consider the use of results from animal exper-
iments as an unreliable basis for opinions in the con-
text of human forensic pathology. An example
concerns the dating of human wounds using rats as
the experimental model. Conclusions based on such
extrapolated data should be viewed with some cau-
tion since the major process involved in wound closure
in rats is contraction, whereas epithelialization is the
primary mechanism in people (Gottrup et al., 2000).
The obverse of that must also be true, where the dat-
ing of wound closure in rats based on the published
rate of epithelialization of human wounds would be
unsound. This has wide implications for forensic vet-
erinary pathology, for it casts doubt on the reliance
paid, in many veterinary publications, on extrapola-
tion of results from studies on people to a range of an-
imal species. As noted above, and by other researchers
(Chvapil et al., 1979), direct inter-species comparisons
suffer from the same uncertainties.
Hosgood (2006) provides a detailed review of the
stages of wound healing. The healing process is tradi-
tionally divided into three phases: inflammatory, pro-
liferative and maturation (Ohshima, 2000).
However, these phases are not defined precisely in
time and all overlap to some extent (Baum and
Arpey, 2005). Early blood clotting and the develop-
ment of provisional extracellular matrix are
followed by scab formation, inflammation and de-
bridement (including migration of leucocytes into
the injury and a shift from neutrophil predomination
in the early inflammatory period to macrophages
in the older lesions), angiogenesis, fibroplasia,
60 R. Munro and H.M.C. Munro

epithelialization, contraction and remodelling/matu- histopathological observations at various stages are

ration. These observations provide the pathologist shown in Table 1.
with guidelines that may facilitate the development The plethora of papers covering numerous aspects
of evidence-based estimates of the time that has of wound healing in people and laboratory rodents
elapsed since injury. Investigators interested in the may tempt the well-meaning, but unwary, to use
detection of vasoactive compounds or mediators of this information during the compilation of forensic
wound healing may find the original papers cited by veterinary reports. However, it would be wise to
Hosgood (2006) helpful. Additionally, the excellent bear in mind the words of Lucius Accius (170e86
overview by Kondo (2007) of collagens, cytokines BC) ‘vigilandum est semper; multae insidiae sunt
and growth factors, as markers for wound viability bonis’ (‘always be on your guard; there are many
and age, points the direction for future research. snares for the good’).
The old veterinary saying ‘cats are not small dogs’
is particularly apposite in forensic work. Wound heal-
ing in cats generally occurs more slowly than in dogs. Estimation of the Age of Bruises
Granulation tissue, for example, appears at approxi-
mately the same time in both species, but Bohling Vanezis (2001) defined a bruise as ‘.a collection of
et al. (2004) found that the subsequent rate of forma- blood, visible to the naked eye as an area of discolou-
tion was more rapid in dogs, resulting in the median ration, which has extravasated into the surrounding
filling time of wounds in dogs to be 8 days compared tissues after vascular disruption, principally as a re-
with 20 days in cats. Epithelialization, contraction sult of trauma or occasionally spontaneously, as a re-
and time to total healing also showed specific differ- sult of a disease process’. A broader definition by
ences, with each of these processes being slower in May and Hamdy (1966) that bruising is ‘tissue in-
cats compared with dogs (Bohling and Henderson jury, without laceration whereby cells and blood
2006). vessels are crushed with a resulting release of cellular
Similarly, ‘ponies are not small horses’ when it fluids and blood into the injured area’ supports the
comes to wound healing. In ponies, the early inflam- view that bruising may involve degeneration and
matory response is more intense than in the horse,
leading to uncomplicated healing. Studies by
Wilmink et al. (1999a) and Wilmink and van Table 1
Weeren (2005) showed second intention healing in Healing of wounds in cattle and buffalo ears following
application of ear tags
ponies to be significantly quicker compared with
horses, with the rate of wound contraction being bet- Time Observations
ter. These authors point out that granulation tissue in
1e3 days Haemorrhage, oedema,
horses remains irregular and purulent for longer. At
necrosis, neutrophilic
the histological level, neutrophil populations in sec- infiltration
ond intention in ponies are high during the first 7 days Appearance of healing in
3 weeks then decrease rapidly. In horses, they are the form of vascular
slower to rise, but persist for longer. Additionally, connective tissue
2 weeks Regeneration of
there is strict organization of myofibroblasts in ponies
epithelium begins
compared with horses, particularly in metatarsal 3 weeks Healing of auricular
wounds (Wilmink et al., 1999b). It is also worthy of cartilage observed in
note that metatarsal wounds in horses are unrepresen- both cattle and buffalo
tative of other cutaneous wounds and may increase in and epithelialization
in cattle complete
size during the first 2 weeks post injury (Wilmink et al.,
4 weeks Complete healing of the
1999a). dermis and epidermis
Responses to cutaneous wounding differ in horses in cattle but
and cattle, with horses showing a more rapid develop- epithelialization in
ment of granulation tissue, while growth and differen- buffalo incomplete
6 weeks Epithelialization
tiation of connective tissue in cattle 10 days post
complete in buffalo
injury is more pronounced than in horses (Dinev 8e26 weeks Scar tissue avascular and
and Dzhurov, 1987). Shehata et al. (1992) conducted contracted
an interesting investigation into the healing of the accompanied by
wounds in the pinnae of cattle and buffalo ears follow- absence of hair follicles
and both sweat and
ing the application of plastic ear tags. Species differ-
sebaceous glands
ences in the rate of healing were highlighted. The
 Challenges in Forensic Veterinary Pathology
inflammation of muscle and adipose tissue
(Thornton and Jolly, 1986).
Trauma is often the primary focus in a forensic in-
vestigation. More specifically, assessment of when
trauma occurred vis-a-vis the time of death can be ev-
identially crucial. The pressure to define the time that
has elapsed since injury has generated considerable
research into methods for dating of bruises in a range
of species including guinea pigs, rats, rabbits,
poultry, calves, adult cattle and lambs (Hamdy
et al., 1957; May and Hamdy, 1966; McCausland
and Dougherty, 1978; Thornton and Jolly, 1986).
There are, however, no objective reports on the devel-
opment and healing of bruises in, for example, dogs,
cats or horses.
Inter-species variability in the detail of the timing
of the appearance of markers such as haemosiderin
can be considerable (Vanezis, 2001). Consequently,
despite the confidence expressed by Thornton and
Jolly (1986) that ‘experiments using animals will re-
main the most practicable means of developing and
refining wound ageing techniques for eventual foren-
sic application’, considerable caution should be exer-
cised in the extrapolation of data from one species to
another.
Economic loss related to bruising of livestock going
to slaughter is recognized as a worldwide problem
(Anon, 1954; McManus and Grieve, 1964;
Marshall, 1977; McNally and Warriss, 1996; Gallo
et al., 2005; Andrade et al., 2009; Huertas, 2009;
Alende, 2010). The potential financial benefit of elim-
inating these unnecessary costs to the industry has
driven efforts to identify the causes of bruising and
the times when it occurs (McCausland and Millar,
1982). Central to these investigations is the develop-
ment of reliable ways of dating bruises (Hamdy
et al., 1957).
Various approaches have been attempted to deter-
mine the length of time that has elapsed between the
event causing the injury and the post-mortem exam-
ination of the bruise. These investigations have
considerable relevance to forensic pathology when
improper use of sticks or poor livestock handling are
the focus of welfare prosecutions.
Gross Examination of Bruises
Gross examination of bruises may permit estimation
of an approximate age based on the criteria set out
by Gracey et al. (1999). These authors consider that
at 0e10 h post injury, bruises are red and haemor-
rhagic. By 24 h the colour has darkened and the bruise
becomes watery in consistency between 24 and 38 h.
Bruises over 3 days old appear rustyeorange and
have a ‘soapy’ feel.
61
Earlier experimental work by McCausland and
Dougherty (1978) found that lesions inflicted at
slaughter showed reddening of subcutaneous and
muscle tissue. Examination of the injuries 8 and
24 h after they were induced showed no significant
difference in the appearance of the lesions at these
two times. However, compared with earlier lesions,
the reddened area was wider and deeper, with a small
quantity of clear fluid detectable in the muscle fascia
and subcutaneous tissues of both calves and lambs.
‘Pockets of un-clotted blood’ affected the reddened
areas in some lambs. By 48 h, some differences were
noted between lambs and calves. Generally, the le-
sions in lambs were similar to those seen during the
8e24 h post-injury period, although the fluid collec-
tions tended to be yellow or green. In calves, at
48 h, the affected muscle was yellowered and the le-
sions were drier and smaller.
Tramline bruising caused by sticks or poles is often
referred to as ‘stick marks’ (McNally and Warriss,
1996) and are described by Wilson (2005) as ‘two
straight red weals with a clear area in between’.
Such lesions are commonly noted in pigs, but can
also affect cattle (McNally and Warriss, 1996). Simi-
lar lesions are well described in medical forensic texts
(Saukko and Knight, 2004b). The parallel bruises re-
sult from stretching and rupture of small blood vessels
at the margins of the line of compression correspond-
ing to the strike of the stick.
Microscopical Examination of Bruises
McCausland and Dougherty (1978) studied the histo-
logical changes in bruises induced experimentally in
calves and lambs. Based on the extent of haemor-
rhage, fibrin formation, relative numbers of neutro-
phils and macrophages and muscle fibre damage,
they proposed a scheme for ageing bruises up to
2 days old. Observations in this study were made at
0, 8, 24 and 48 h and reference should be made to
the original paper for the details of the changes de-
scribed. Little difference was found between the reac-
tions in these two species and the authors further
believe that the results are equally applicable to ma-
ture animals.
To overcome some of the inherent variability asso-
ciated with observer error and interpretation of histo-
pathological changes, Thornton and Jolly (1986)
investigated ageing of ovine bruises over 0e72 h by
application of a Bayesian probability model. The con-
fidence values suggested that the Bayesian probability
model could be applied successfully to age bruises into
two broad categories only: 1e20 h and 24e72 h (c.f.
the more optimistic view of McCausland and
Dougherty, 1978). However, Thornton and Jolly
62 R. Munro and H.M.C. Munro

(1986) do point out that accuracy was increased when Johnston (1996) consider these signs are suitable for
more than one tissue sample was examined and that use in ‘fresh carcasses’ (although these authors do
for forensic purposes multiple sampling would, pre- not define a ‘fresh carcasse’), but they agree that the
sumably, be routine. yellow bruising fades as the carcasse ages.
The influence of environmental temperature on the
Other Tests of Bruises colour of bruises in chickens is a consequence of hae-
moglobin being degraded to biliverdin in birds kept
Many attempts have been made to estimate the age of at 30
C and to bilirubin in poultry reared at 21
C
bruises using biochemical and histochemical methods or below.
(see McCausland and Dougherty, 1978; Vanezis, Previous work by Kaiser and Smith (1958) showed
2001). Cattle and rabbits were the subjects of experi- that breed and bodily condition affected the appear-
ments to assess the usefulness of chemical means in the ance and detection of bruises. Hamdy et al. (1961b)
determination of the age of bruises (Hamdy et al., noted that bruises in young birds (4e6 weeks of
1957). Bilirubin, formed during the degradation age) tend to heal faster than in older birds.
of haemoglobin, was not detectable (using a Table 3 shows the ages of poultry bruises, based on
10e20 min exposure to Fouch
e’s reagent at room the colour reactions using Fouch
e’s reagent (Hamdy
temperature) until 50e60 h post injury. Bruises et al., 1961a). These experiments adopted the method-
60e72 h old developed a very light blue colour at ology developed by Hamdy et al. (1957) for bruises in
60 h and were blue by 72 h. Three to 5-day-old bruises cattle and rabbits.
were distinguished by a diffuse dark green at the pe-
riphery with a brown centre. Old bruises (5e8
Drowning: A Difficult Diagnosis
days) showed little or no blue colour.
Electrical conductivity tests on bruised tissues were Anecdotal reports indicate that accidental drowning
also undertaken by Hamdy et al. (1957), but appear to occurs in all classes of domestic animals. These inci-
be of limited practical value. In addition to the re- dents may be more common in companion animals
quirement to have un-bruised control samples from than other groups, but the frequency of drowning/
symmetrical areas, it was found that the size of the near drowning, including in the dog and cat, is un-
bruise and the quantity of fat present in the tissue af- clear.
fected the conductivity. Heffner et al. (2008) examined a series of 28 cases
(25 dogs and three cats) in which, on separate occa-
Bruising in Poultry sions, nine dogs and all three cats were found sub-
merged in water with no indication of how this
Bruising in chickens may be visible within seconds fol-
lowing trauma (Hamdy et al., 1961b). Table 2 sets out
Table 3
(as modified by May and Hamdy, 1966) the gross Estimation of age of bruises in poultry using Fouch

e’s
morphological colour changes observed over 120 h reagent
post injury following experimental bruising of
chickens (Hamdy et al., 1961a). Age of bruise Tissue colour after 20 min in
Fouch
e’s reagent
It is important to note that these descriptions apply
to live birds, but after death all bruises revert to dark Normal tissue No colour
redepurple and lose the yellow and green pigments 0e13 h Pink, within minutes
(Hamdy et al., 1961b). Nevertheless, Bremner and turning brown
14e24 h Diffused light blue along
with pink and brown
Table 2 24e36 h Diffuse light green,
Gross morphological colour changes in poultry bruises especially at the
periphery, with
Age of External appearance in External appearance in
a brown colour in the
bruise 21
C environment 30
C environment
centre of the bruise
Control Normal Normal 2e3 days Diffuse dark green along
2 min Red Red with brown centre
12 h Diffused dark redepurple Diffused redepurple 3e4 days Dark green spots or
24 h Diffused light greenepurple Diffused light greenepurple crystals embedded in
36 h Yellowegreenepurple Diffused greenepurple the bruised area, with
48 h Yellowegreen (orange) Dark green no brown colouration
72 h Yelloweorange Almost normal 5 days Sometimes slight blue
96 h Slight yellow Normal colour
120 h Normal Normal
(Adapted from Hamdy et al., 1961a)
 Challenges in Forensic Veterinary Pathology
happened. The remaining 16 dogs had various histo-
ries including falls into water, thin ice, swimming ac-
cidents, seizure near water and two cases of
‘intentional’ drowning. Munro and Thrusfield
(2001), in their study of ‘battered pets’, recorded
three cases of attempted drowning of adult cats. Nev-
ertheless, Munro and Munro (2008c) stress that even
in instances which appear, at first sight, to be clear
cases of drowning, it is essential to eliminate other pos-
sible causes of death. Careful necropsy examination
and objective interpretation of the findings are crucial
if the frequently asked question ‘was the animal dead
before being immersed in water?’ is to be considered.
Experiments on Drowning
Numerous experiments involving the drowning of
cats, dogs, guinea pigs, mice, rabbits and rats have
been conducted. The purpose of these experiments,
based on the assumption that the process of drowning
in ‘animal models’ in fresh and salt water would be
the same as that in people, was to unravel the patho-
physiology of drowning in people (Swann et al., 1947).
Early experiments (Brouardel and Vibert, 1880;
Yamakami, 1922, 1923; Martin, 1932; Karpovich,
1933; Banting et al., 1938; Lougheed et al., 1939;
Jetter and Moritz, 1943; Kylstra, 1962) were undeni-
ably inhumane and unethical by modern standards,
involving, as they did, conscious animals. In addition,
the scientific methodologies were seriously flawed by
(1) asphyxiation of control animals by clamping the
trachea or by strangulation and (2) clamping the oe-
sophagus to prevent swallowing of water. These pro-
cedures rendered the results invalid in the context of
‘natural drowning’.
In 1963, Fuller reviewed freshwater drowning in
people and pointed out that the course of human
drowning does not follow the course predicted by
Swann and colleagues’ experiments (1947) on dogs.
Nevertheless, this did not prevent further experimen-
tal studies using lightly anaesthetized dogs in the com-
ing years (Farthmann and Davidson, 1965; Modell
et al., 1966; Giammona and Modell, 1967). Eventu-
ally, in 2005, Lunetta and Modell stated ‘It is gener-
ally agreed that although pathophysiological
differences between drowning in freshwater or saltwa-
ter may be observed in experimental models, these
have little or no clinical significance for human
drowning’. These authors also noted the recurring ex-
perimental design fault, whereby the quantities of wa-
ter inhaled during experimental drowning of dogs are
greatly in excess of those that have been calculated to
be aspirated in 85% of human drownings.
This background raises serious moral and ethical
questions regarding the use of data generated by cruel
63
or scientifically flawed studies. In this regard, the ex-
cellent appraisals of the ethics of citing results of Nazi
medical experiments (Moe, 1984; Talia, 2002) pro-
vide balanced guidance to the dilemma. From
a human drowning perspective, the results of the ex-
perimental drowning studies in animals lack validity
and applicability and as such there seems no scientific
or moral justification for their citation. As far as foren-
sic veterinary pathology is concerned, and without
condoning these distasteful experiments, which
make for distressing reading, it may be possible to sal-
vage some good from them. As a consequence of im-
proving ethics on the use of experimental animals,
these experiments will not be repeated, but they re-
main a source of pathology data on drowning, partic-
ularly in dogs. Table 4 summarizes a number of gross
observations recorded in the various reports (cited
above) on experimental drowning.
Lung Weights of Drowned Dogs
Hyde et al. (1989) reported that, using computed to-
mography (CT) and re-breathable gases, the lungs
of healthy mongrel dogs weighed 19  5 g/kg body
weight (BW). In comparison, Giammona and
Modell (1967) found the lung weights of their lightly
anaesthetized drowned mongrel dogs to be
34.9  5.3 g/kg BW, 35.4  3.6 g/kg BW and
38.7  4.7 g/kg BW, depending on whether the dogs
were drowned with distilled water, seawater or chlo-
rinated distilled water, respectively. Despite the in-
creased weights of lungs from drowned dogs, it
should be noted that Lougheed et al. (1939) and
Swann et al. (1947) found that most lungs from exper-
imentally drowned dogs floated in water.
In all of this, it should be borne in mind that the ra-
tio of normal lung weight/body weight will vary sub-
stantially according to breed, age and bodily
condition of the dog (e.g. a young, fit greyhound or
collie, vis-a-vis a fat, middle-aged Labrador).
Microscopical Examination of the Lungs from Drowned
Animals
Microscopical changes in the lung after drowning
may include alveolar overdistension, attenuation of
alveolar septae, narrowing of alveolar capillaries, al-
veolar rupture and intra-alveolar haemorrhage and
flooding by pale eosinophilic fluid (Munro and
Munro, 2008c). However, Reidbord (1980) cau-
tioned that in most cases of human drowning ‘the pa-
thologist is faced with a variety of non-specific
histologic changes that preclude a definitive diagno-
sis’. As a consequence, no single histopathological
change, or combination of changes, is considered
64 R. Munro and H.M.C. Munro
Table 4
Gross changes in experimental drowning
 Large quantities of froth and fluid flow from the
nostrils.
 Clear or pink frothy foam is present in the trachea
and major airways in 80% of dogs drowned in
freshwater. More froth is noted in seawater
drowning than in freshwater drowning.
 The entire bronchial system is usually filled with
a frothy fluid.
 The dependent parts of lung lobes appear atelec-
tatic and haemorrhagic.
 Congested or haemorrhagic areas may be scattered
elsewhere on the lung surfaces.
 Crepitus can occur.
 Various authors (Lougheed et al., 1939; Swann
et al., 1947; Modell et al., 1966) agree that the gross
appearance of the lungs in freshwater and seawater
drowning is similar.
 The right side of the heart and the great veins are
usually engorged, while the chambers of the left
heart may be virtually empty.
 Large quantities of water may be present in the
stomach when artificial respiration or vomiting
have not already affected its removal.
pathognomonic for drowning in people. This may
also be the case for other species.
The ultrastructural changes described in rat lung
following experimental intratracheal perfusion of ei-
ther fresh or salt water showed clear differences. In
freshwater drowning there was severe disruption of
all cellular organelles with marked endothelial vesic-
ulation. Salt water caused relatively little ultrastruc-
tural alteration, resembling the changes seen in
acute hypoxia (Reidbord and Spilz, 1966;
Reidbord, 1966, 1967).
The Diatom Test
Diatoms are unicellular algae which are found in al-
most all aquatic and damp terrestrial habitats
(Horton et al. 2006). Although literally hundreds of
peer-reviewed papers have been published on the po-
tential of diatom testing for the diagnosis of drowning,
its reliability remains controversial (Bortolotti et al.,
2004; Lunetta and Modell, 2005; Horton et al.,
2006). Problems confounding the reliability of this
test include (1) contamination of the samples at the
time of necropsy examination or during processing
of tissues, (2) passive penetration into bodies following
immersion, (3) absence of diatoms in some cases of
human drowning and (4) the occurrence of
falsepositives, whereby diatoms are identified in bod-
ies that have had no contact with water.
Nevertheless, Merck (2007b), citing literature re-
ferring entirely to human cases, considers that suit-
ably controlled diatom testing is a valid means of
confirming drowning as the cause of death in veteri-
nary cases.
The rather sketchy account of an investigation by
Giri and Tripathi (1994) on immersion of anaesthe-
tized and dead dogs in water, suggests that examina-
tion of liver, spleen, brain and bone marrow for
diatoms might be helpful in distinguishing between
submersion before or after death. However, the lack
of information on the time interval between immer-
sion and necropsy examination rather undermines
the application of their findings. This report also con-
firms that low numbers of diatoms can be recovered
from lungs, heart and kidney in dogs that died from
causes other than drowning and which had no contact
with water.
Recent investigations involving immersion, in
freshwater, of piglets that had died from natural
causes (Giancamillo et al., 2010) confirmed the exis-
tence of ante-mortem contamination by diatoms
and that post-mortem contamination also occurs. In
contrast, Bortolotti et al. (2011) found no diatoms in
the lungs or the bone marrow of the sternum of people
who had died from causes other than drowning.
Because many species of diatoms are
habitatspecific, considerable efforts have been made
to use diatoms to identify the location where a person
may have drowned. This research led to the develop-
ment of the ‘diatom-based quantitative reconstruc-
tion technique’ (Horton et al., 2006) but, to the best
of the authors’ knowledge, this methodology has not
been employed in veterinary forensic pathology.
At the present time, it seems prudent to proceed on
the basis that ‘diatom testing cannot be accepted, in
a court of law, as a definitive method for establishing
death by drowning’ (Lunetta and Modell, 2005).
Estimation of Time since Death
Establishment of the time since death is a daily task in
human forensic casework (Henssge and Madea,
2007). Routine murder enquiries usually attempt to
discover whether particular individuals were in the
 Challenges in Forensic Veterinary Pathology
area at the time and had the opportunity to commit
the offence, or if they can provide an alibi. Estimation
of the post-mortem interval (i.e. the period between
death and medical examination) may assist investiga-
tors in narrowing this ‘window of opportunity’,
thereby eliminating specific events and suspects. Be-
cause of the importance of this task, much time and
energy have been invested in researching an almost
bewildering variety of methods for determining the
time since death.
Great caution must be taken when providing an es-
timate of the post-mortem interval. Knight (1988)
states ‘The margin of error remains large and unpre-
dictable, even in controlled research conditions,
let alone for the more variable environment of an ac-
tual scene of death’. The lack of accuracy and reliabil-
ity of most methods was also of concern to Henssge
(1988). Over the last 30 years some improvements
have been made, but Swift (2010) concludes that ‘It
remains debatable whether there is any single, reli-
able and accurate means of estimating the time since
death during the early post-mortem interval’. In a vet-
erinary context, the picture is particularly complex
since requests from prosecutors, police or defence
agents for time of death estimates are driven by di-
verse objectives depending on the category and spe-
cies of animal involved.
In companion animals, for example, investigations
of suspected non-accidental injury (i.e. deliberate in-
jury) are in many ways similar to human assault cases,
where placing the accused in the location at the esti-
mated time of the crime can be of importance. How-
ever, in contrast to human forensic casework, it may
(depending on the jurisdiction involved) be a legal re-
quirement, central to a prosecution, to determine
whether suffering occurred in the period between as-
sault and death. It follows that in such cases establish-
ment of the time of death is important.
Wildlife crime investigations are less concerned
with issues of suffering because they are usually fo-
cused on regulatory matters (e.g. ‘out of season’ shoot-
ing of game animals, poaching and breaches of
statutory time limits). Many countries have enacted
laws to provide protection of wildlife through limiting
hunting to specific periods of the year and by requir-
ing trappers to visit their traps and snares at least once
in any 12 or 24 h period. Estimation of the post-
mortem interval can be helpful during investigation
of breaches of these regulations. Establishment of
the approximate time of death of protected species
(e.g. badgers) can serve the same purpose of deter-
mining alibi and opportunity, just as for human and
companion animal deaths.
With regard to farm livestock, the desire to estab-
lish the time of death may be related to questions on
65
(1) duration of neglect, (2) failure to dispose of car-
casses within statutory time limits, (3) uncertainties
over death during transport, (4) suspected fraudulent
insurance claims and (5) time of onset of serious hus-
bandry problems (e.g. interruption of water supply to
poultry houses).
Methods for Estimating Time since Death
There are two basic approaches to estimation of time
of death: (1) measurement of change that takes place
at a known rate (e.g. rigor mortis, cooling of the body
and putrefaction), and (2) comparison of the occur-
rence of events known to have taken place at a specific
time with the time of death (e.g. extent of digestion of
last meal).
The sources of evidence that are relied on when at-
tempting to estimate the time of death in people in-
clude those present in the body (corporeal), those
present in the vicinity of the body (environmental
and associated evidence) and anamnestic evidence
based on the deceased’s ordinary habits, movements
and day-to-day activities (Pounder, 1995). The first
two categories are important in animal cases. How-
ever, it could be postulated that evidence based on
‘day-to-day activities’ could also be helpful in certain
instances of a forensic veterinary investigation e for
example a case of suspected illegal shooting of ducks
that have a regular pattern of flights to and from par-
ticular ponds or feeding sites.
Techniques and procedures used to estimate the
post-mortem interval in mammals and birds have
been outlined by Edge (1984), Erlandsson (2003),
Cooper and Cooper (2007c), Merck (2007c),
Munro and Munro (2008d), Okene (2010) and
Sinclair et al. (2006b). The specific issues related to de-
termination of the post-mortem interval in reptiles
and amphibians are highlighted and discussed by
Cooper (2012). In human forensic casework,
Henssge and Madea (2007) believe that ‘most
methods for estimation of the time since death are of
only academic interest’. These authors are of the opin-
ion that all methods of estimation of time since death
must demonstrate quantitative measurement and in-
clude a mathematical description. They also suggest
that any proposed method should be accompanied
by proof of precision of the method on independent
material, and that quantification of those factors
that influence the method needs to be provided. In
general, field studies are necessary to demonstrate
the practicality of any method. Similar strictures ap-
ply to methods adopted in veterinary forensic investi-
gations.
Techniques for estimation of time of death are listed
in Table 5. However, many of these are, currently, of
66 R. Munro and H.M.C. Munro
Table 5
Methods for the estimation of the post-mortem interval
Temperature-based methods
Post-mortem chemistry
Electrical stimulation of muscle and nerves
Gross appearance of body:
Rigor mortis
Eye shape, colour and luminosity, etc
Decomposition
Histopathology and electron microscopy
Radiology
DNA and RNA analyses
Entomology
Environmental and associated evidence
limited practical value in legal cases because lack of
validation of the methods renders the results open to
challenge. For example, the use of electrical excitabil-
ity of muscle to determine the time of death in the
early post-mortem period may be questioned on the
basis that the reaction of the muscle is affected by
the manner of death (Madea and Henssge, 1990;
Elmas et al., 2002). Similarly, although studies on
post-mortem degeneration of DNA extracted from
porcine muscle suggest that this technique may be
useful during the first 72 h after death (Watson,
2010), validation of this method awaits detailed inves-
tigation of the impact of environmental variables such
as ultraviolet radiation, heat, high humidity and fun-
gal/bacterial contamination.
There is an accepted body of knowledge and expe-
rience on rigor mortis and decomposition that allows
experienced veterinary pathologists to estimate the
post-mortem period within approximate blocks of
time such as <24 h, 1e3 days, 3e7 days, 7e21
days, weeks, months or years (Munro and Munro,
2008d). These changes are well described in standard
veterinary pathology texts (e.g. Maxie, 2007) and are
not considered further in this review. Nevertheless,
experimental studies (Turner and Wiltshire, 1999;
Wilson et al., 2007; Schotsmans et al., 2012) designed
to simulate ‘clandestine burials’ revealed interesting
insights into the effects of soil type, hydrated lime
and quicklime on the process of decay of pig cadavers.
These findings might have relevance to some veteri-
nary investigations.
It is also worth noting that in the field of human fo-
rensic pathology, experienced pathologists frequently
underestimate the time since death (James and
Knight, 1965) and it is probable that similar errors
are commonplace in veterinary pathology. This is
a source of concern when a Court seeks veterinary
guidance in relation to the time of death.
Forensic entomology can be of considerable value
in veterinary cases (Anderson and Huitson, 2004). Al-
though identification of the types and stages of mag-
gots and beetles is outside the competence of most
veterinary pathologists, knowledge of the correct pro-
cedures for the collection of entomological evidence is
a necessary skill. Byrd et al. (2010) provide up-to-date
guidance on appropriate methods.
Some research conducted on temperature-based
methods, post-mortem chemistry, histopathology,
electron microscopical changes and post-mortem ra-
diology is, however, of relevance to forensic veterinary
pathology. These methods may provide the means of
refining the rather crude estimates based on evidence
of decomposition or open fresh avenues for further in-
vestigation and independent validation.
Temperature-Based Methods. A carefully controlled
study of beagle dogs weighing between 8.5 and
15 kg demonstrated the potential usefulness of rectal
probes for estimation of time of death over the initial
10 h after death (Erlandsson and Munro, 2007). The
data collected in this study suggested that the post-
mortem interval can be estimated in 2 h bands or pe-
riods. For example, the rectal temperature at 60 min
post mortem was clearly distinguishable from that
taken 3 h post mortem. Similarly, the body tempera-
ture 5 h post mortem was distinct from that recorded
7 h post mortem etc. After 10 h the rate of drop of rec-
tal temperatures of individual dogs showed greater
variability, resulting in overlap and less clear separa-
tion. Nevertheless, under the conditions prevailing in
this study (ambient temperature of 10.9e16.8
C), by
17e17.5 h post mortem the rectal temperature of each
dog was <19
C. Body temperatures approximated
ambient temperature 24e48 h post mortem. Con-
trary to the findings for human cadavers (Knight,
1988; Henssge, 2002), Erlandsson and Munro
(2007) found no evidence of a plateau in the rectal
temperature curve in the early part of the post-
mortem period.
Two studies from Malaysia provide data on the
cooling of dogs at ambient temperatures of
24e36
C. Abdulazeez and Noordin (2010) inserted
stirring thermometers into liver and rectum while
Okene (2010) recorded external ear canal, rectal
and hepatic cooling using probes and thermocouples.
These authors are of the opinion that the exponential
and linear equations developed during their studies
may prove useful for the estimation of the time of
death in the initial 7 h post mortem. Although the ex-
ponential model may provide a slightly more accurate
estimate, the linear model appeared mathematically
easier to use in the field. Abdulazeez and Noordin
(2010) found cooling under tropical conditions was
less consistent than in temperate climates. When
body temperature was close to ambient temperature,
the cooling curve showed peaks and short plateaux
 Challenges in Forensic Veterinary Pathology
that lasted for an average of 70 min. Under these trop-
ical conditions, body temperature reached ambient
temperature in 26  8 h.
The literature on estimation of time of death of peo-
ple makes frequent reference to a lag phase, or pla-
teau, in the cooling curve derived from rectal
temperature measurement in the early period after
death. However, the studies by Baccino et al. (1996)
demonstrated no lag phase if the temperature mea-
surements were taken from the external ear canal.
Erlandsson (2003), Abdulazeez and Noordin (2010)
and Okene (2010) failed to demonstrate a plateau
in dogs, irrespective of the organ where the tempera-
ture readings were taken. This is of some importance
as it highlights the caution and attention to detail nec-
essary when extrapolating between species. Further
consideration needs to be given to potential differ-
ences arising through the use of differing methodolo-
gies and equipment.
Merck (2007c) notes the ‘rule of thumb’ cooling
rate of 1.5
F/h that is sometimes used in estimating
the time of death of people and that it is recommen-
ded to add 1e2 h to the post-mortem interval in cases
where a plateau may have occurred. She comments
that ‘It makes sense to apply this rule to animal cases
as well’. Sinclair et al. (2006b), citing Rauch (2003),
also comment on the temperature plateau and the
1.5
F loss per hour in rectal temperature. However,
the studies by Erlandsson (2003), Abdulazeez and
Noordin (2010) and Okene (2010) suggest there is
no scientific evidence to support the extrapolation,
to canine cadavers, of guidelines formulated for hu-
man cadavers. The same caveat may well apply to
other species.
Establishment of reliable evidence to support pros-
ecutions for out-of-season killing of deer was the pri-
mary aim of a number of studies conducted in
North America. The widespread practice of ‘field
dressing’ culled deer, whereby the abdominal cavity
is opened and the gastrointestinal tract removed
soon after the deer has been shot, renders rectal tem-
perature measurement impossible or meaningless in
these animals. Consequently, thigh and/or nasopha-
ryngeal temperatures in culled North American
deer were recorded by Neubrech (1960), Gill and
O’Meara (1965), Pex et al. (1983), Woolf et al.
(1983), Kienzler et al. (1984), Cox et al. (1994) and
Hadley et al. (1999). In addition to the thigh and na-
sopharyngeal temperatures, these investigators re-
corded ambient temperature, deer weight and the
approximate time that elapsed between shooting
and sampling. These studies allowed the construction
of tables, temperature charts (Kienzler et al., 1984)
and a field manual (Kienzler and Fuller, 1983) to as-
sist rangers and law enforcement agencies to estimate
67
time since death, taking into account BW and prevail-
ing ambient temperature. Although affected by un-
certainties (e.g. variations in body temperature at
time of death), this approach was endorsed 20 years
ago as being of practical value (Oates, 1992). The de-
velopment of computer software programmes by
Kienzler (1985) and Cox et al. (1994) made this task
easier.
Experiments on the cooling rates of pig cadavers
were conducted by Kaliszan et al. (2005). These stud-
ies aimed to assess the practical value of the ‘two-ex-
ponential’ model for time of death estimation, in
comparison with the single exponential model. Al-
though these experiments were undertaken to assist
in human forensic investigations, they provide useful
insights into the estimation of the time since death
in pigs.
Pin probes (connected to two channel thermome-
ters) inserted into the eyeball, orbital tissues, ‘rump’
[sic] muscle and rectum measured the decrease in
body temperature, beginning 75 min after death.
The single exponential model applied to eyeball cool-
ing allowed very precise estimation of the time of
death up to 13 h post mortem. Thereafter, better
time-of-death estimations were obtained from muscle
or rectal probes. Limited numbers of pigs were used in
these experiments. Consequently, it is desirable that
other research groups validate this method.
Post-mortem Chemistry. The search for reliable chemi-
cal markers that correlate strongly with time since
death continues to be a popular area of research in hu-
man forensic medicine. In the past many of these stud-
ies focused on vitreous humour (Madea et al., 1989)
because it is generally less affected by autolysis and
is isolated from the influences of the large organs in
the chest and abdomen (Saukko and Knight, 2004a).
Following the experiences in human studies, at-
tempts were made to correlate potassium levels in vit-
reous humour and glucose in aqueous humour with
the time since death in deer. Pex et al. (1983) demon-
strated the fall in glucose in aqueous humour in black-
tailed deer to be a useful marker during the first 8 h
post mortem. Rising potassium levels in vitreous hu-
mour of mule deer were considered to have a logarith-
mic, rather than a linear (arithmetical), relationship
with the lengthening post-mortem interval (Johnson
et al., 1980). However, Woolf and Gremillion-Smith
(1983) advised caution because this technique is
based on intraocular post-mortem autolysis, which
can vary widely.
A variable rate of autolysis is not the sole drawback
of this type of marker. Lack of knowledge of the ‘nor-
mal’ ante-mortem levels of chemicals in the vitreous
or aqueous humour undermines confidence in the
68 R. Munro and H.M.C. Munro
relevance of the findings. Similarly, the effects of dis-
ease or nutritional status on the distribution and con-
centration of these chemicals remains to be studied.
Consequently, in a veterinary context, these uncer-
tainties result in chemical markers in vitreous and
aqueous humour being viewed as providing support-
ing evidence, rather than the primary means, of esti-
mating time since death for deer.
The situation in dogs is slightly further advanced
with regard to vitreous humour. Schoning and
Strafuss (1980) examined 60 adult mongrel dogs
and helped to establish baseline values for ante-
mortem and post-mortem levels of sodium, chloride,
potassium, urea nitrogen, glucose and creatinine.
However, these tests do not appear to have been
adopted as standard methods of estimation of the
post-mortem interval in domestic dogs.
Research has also been conducted into the use of
protein markers for estimation of the time since death.
Analysis for cardiac troponin 1 in cattle (Sabucedo
and Furton, 2003), although semi-quantitative in na-
ture and temperature dependent up to 37
C, shows
potential, but awaits validation. Nagaraj et al.
(2005) studied changes in skeletal muscle in goat car-
casses stored at 5
C for 3e20 days, with the primary
purpose of understanding the processes involved in
the tenderization of meat. Proteolytic breakdown
products appeared after 6 days storage and the detec-
tion of a 55 kDa polypeptide was a consistent feature
of muscle samples from 12-day-old carcasses. Again,
these findings are interesting, but validation will,
however, be necessary if they are to form the basis of
alternative methods for estimation of the time since
death, for legal proceedings.
Histopathology and Electron Microscopy. In addition to
collecting temperature data, Erlandsson (2003) noted
post-mortem gross and microscopical changes in bea-
gle dogs. Subsequently, Erlandsson and Munro
(2007) drew up a table of differential findings at var-
ious time points (<1 day, 3 days, 7 days and 23 days)
after death. They found heart, liver, lungs, pancreas,
tonsils, thyroid and urinary bladder to be the most
useful organs for highlighting histological changes at
different times in these animals. However, the usual
caveats apply to extrapolation of these data to other
breeds of dog or different species. Nevertheless, this
study raises interesting possibilities for the use of histo-
pathology and immunohistochemistry in refining the
estimation of time since death. Similarly, the investi-
gation of time-dependent changes in post-mortem tes-
tis histopathology in the rat (Bryant and Boekelheide,
2007) provides valuable background information on
autolytic changes at specific times. Expansion of this
research to encompass other species and different en-
vironmental circumstances would inevitably establish
a sounder basis for exploring this approach to estima-
tion of the post-mortem interval.
Although somewhat more esoteric, but nevertheless
valuable, is the work of Pallot et al. (1992) who showed
that autolytic changes in the carotid body may be mis-
interpreted if account is not taken of the delay between
death and fixation of the tissue sample. The ‘beyond
reasonable doubt’ principle used in criminal proceed-
ings applies equally to veterinary pathology as it does
to other types of evidence. Therefore, elimination of
doubt over the significance of particular histological
observations could be of crucial importance.
The study by Nagaraj et al. (2005) outlines changes
to Z-disks in goat skeletal muscle observed by transmis-
sion electron microscopy (TEM). Six days after death
there was little alteration of these structures, but degra-
dation of the Z-disks was noted to be ‘considerable’ by
12 days. Munoz et al. (1999) employed TEM to relate
autolytic changes in canine myocardial cells to the
post-mortem period. Morphometrics characterized
(1) decreased numbers of mitochondria, (2) increased
mitochondrial volume and (3) increased surface areas
of both outer and inner mitochondrial membranes, as
the post-mortem interval extended from 0 to 240 min.
Post-mortem Radiology. The concept of the ‘virtual
autopsy’ (also known as ‘virtopsy’) using CT and
magnetic resonance imaging (MRI) is a developing
sub-speciality of post-mortem radiology (O’Donnell
and Woodford, 2008). In human forensic pathology,
CT scanning of the deceased is increasingly being
adopted as a routine procedure before conventional
post-mortem examination. However, significant dif-
ferences between clinical radiology and post-mortem
imaging are potential pitfalls (Flach et al., 2010).
There is a considerable shortage of information on
the use of advanced imaging techniques in veterinary
post-mortem examination. Exceptions include the es-
sentially practical experimental studies on the com-
parative sensitivity of conventional necropsy
examination and CT scanning in detection of bone
fractures in piglets (Cattaneo et al., 2006). More fun-
damental, and less likely to become a mainstream
procedure, is the use of proton magnetic resonance
spectroscopy to detect new metabolites of autolysis
and putrefaction in pig and sheep brains (Cecil
et al., 1998; Ith et al., 2002; Banaschak et al., 2005).
Heng et al. (2008), using conventional radiography,
described post-mortem changes in the abdomen of
41 cats that had been humanely destroyed and kept
at 4
C for up to 12 h before radiography. Of these
cats, 11 (27%) had intravascular gas, with the liver
being the most common site. Intravascular gas was
also detected in the aorta, femoral artery, coeliac
 Challenges in Forensic Veterinary Pathology
and cranial mesenteric arteries and the caudal super-
ficial epigastric artery. Interestingly, only two cats
showed distension of the small intestines, while a soli-
tary cadaver was affected by gas dissecting the tissues
in the wall of the large intestine (pneumatosis coli).
Heng et al. (2009b) subsequently studied the post-
mortem radiographic appearance of the abdominal
organs of dogs. However, these studies were conducted
on canine cadavers kept at ambient temperatures of
22e33
C. Consequently, the results are not directly
comparable to the feline study. The high ambient tem-
peratures resulted in rapid decomposition and pre-
vented the study extending beyond 24 h after death.
Increased gas in the gastrointestinal tract was ob-
served as early as 8 h. By 16 h, gas was observed in
the liver, caudal vena cava and peritoneal cavity in
all cadavers. Abdominal distension was a feature in five
of the six dogs at this time. The authors suggest that, in
ambient temperature of 22e33
C, detection of gas in
the abdominal cavity indicates that death occurred
at least 8e16 h before radiography. Okene (2010),
who also conducted his experiments in ambient tem-
peratures of 22e33
C, found that the intestines, liver
and hepatic vein were the radiological markers of
choice for estimation of time of death. The observa-
tions made in this latter study included radiographs
taken 6 h after death, reducing by 2 h the time selected
by Heng et al. (2009b) as the start point.
Serial thoracic radiographs detected post-mortem
gas accumulation in the pleural cavity in one dog af-
ter 8 h and in all dogs by 16 h (Heng et al., 2009a).
Okene (2010) found that 90% of dogs in his study
showed varying degrees of gas accumulation in the
pleural cavity 12 h after humane destruction. How-
ever, he cautioned that compression of thoracic or-
gans by abdominal distension may limit accurate
interpretation of the development of pneumothorax.
In the study by Heng et al. (2009a), gas in the car-
diovascular system was detected first in the right ven-
tricle, coronary vessels, main pulmonary artery and
caudal vena cava. This contrasts with Okene’s inves-
tigations (2010) where the right atrium and cranial
vena cava had evidence of gas 6 h after death. This
study also recorded a time dependent increase in ac-
cumulation of gas in the cranial vena cava from
12e24 h. There being no trauma or surgical interven-
tions in the dogs before humane destruction, in either
study, it can be concluded that all the gas in the car-
diovascular system was a consequence of putrefaction.
Clearly, more research is needed to determine the
value of post-mortem radiology in relation to estima-
tion of time of death in veterinary cases. However, there
is another aspect to this research. Gas accumulation oc-
curred in the scapulohumeral joints of dogs in the ab-
sence of joint disease or a history of stress to the joints.
69
It appears that this gas results from post-mortem de-
composition and is not a manifestation of local bone is-
chaemia or other degenerative changes (‘vacuum
phenomenon’) (Heng et al., 2009a). This finding rein-
forces the importance of being aware of post-mortem
artefacts that may lead to misinterpretation.
Conclusion
This review focuses on four aspects of forensic veteri-
nary pathology. Although each presents particular di-
agnostic challenges, there are difficulties that are
common to all. These include lack of detail of the tim-
ing of changes, the danger of extrapolation of findings
from one species to another, limitations of experimen-
tal models to reflect the spectrum of changes seen in
actual cases, and the constant requirement for the pa-
thologist to provide a balanced, factual interpretation
of post-mortem findings. Great reliance is placed on
forensic pathologists to guide investigations into al-
leged offences. Investigators wish for clear, unequivo-
cal answers. Currently, however, much remains to be
discovered about the development and resolution of
lesions in different species; therein lie exciting possibil-
ities for the expansion and refinement of the forensic
veterinary pathology knowledge base.
Conflict of Interest Statement
The authors have not declared any conflict of interest
that may arise from being named as an author on the
manuscript.
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½ Received,
Accepted, October 6th, 2012 
July 4th, 2012