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Journal of Veterinary Diagnostic Investigation
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© 2016 The Author(s)
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DOI: 10.1177/1040638716642268
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Full Scientific Report
Introduction
Crocodile farming is a growing industry in Australia, pro-
ducing high-quality skins for the luxury leather market. Aus-
tralian farmed crocodiles are almost exclusively saltwater
crocodiles (Crocodylus porosus) obtained from either cap-
tive breeding or regulated sustainable egg harvesting from
the endemic wild population. Crocodile farming is intensive
as most farms contain thousands of crocodiles, incubating
eggs and raising crocodiles from hatching to harvest at 2–4
years of age (Foster M. Emerging animal and plant indus-
tries: their value to Australia. Barton, ACT: Australian Gov-
ernment Rural Industries Research and Development
Corporation Publication No. 14/069, 2014).
Three new disease syndromes have emerged in recent years
among farmed crocodiles in the Northern Territory of Austra-
lia. The first syndrome to emerge was conjunctivitis and/or
pharyngitis (CP). Chlamydiaceae bacteria have been noted in
the literature as being associated with conjunctivitis in various
Crocodylus spp.,15–17,32 and CP in saltwater crocodiles was
initially attributed to this agent. However, the role of the bacte-
rium became unclear when a survey on the prevalence of Chla-
mydiaceae in live saltwater crocodiles revealed a poor
association between the syndrome and presence of the bacteria
(Jerrett I, et al. Chlamydial infection in farmed crocodiles [final
report to funding body]. Barton, ACT: Australian Government
642268VDIXXX10.1177/1040638716642268Herpesvirus infection in farmed crocodiles in AustraliaShilton et al.
research-article2016
Berrimah Veterinary Laboratories, Northern Territory Government,
Darwin, Northern Territory, Australia (Shilton, Davis, Walsh, Benedict,
Melville); AgriBio Centre, Victorian Government, Bundoora, Victoria,
Australia (Jerrett); The University of Sydney, Sydney, New South Wales,
Australia (Isberg, Phalen, Brown); Centre for Crocodile Research,
Noonamah, Northern Territory, Australia (Isberg); Wildlife Management
International Pty Limited, Berrimah, Northern Territory, Australia (Webb,
Manolis); Research Institute for the Environment and Livelihoods, Charles
Darwin University, Darwin, Northern Territory, Australia (Webb); and
School of Veterinary and Life Sciences, Murdoch University, Murdoch,
Western Australia, Australia (Hyndman).
1Corresponding Author: Catherine M. Shilton, Berrimah Veterinary
Laboratories, Northern Territory Government, GPO Box 3000, Darwin,
Northern Territory, 0801, Australia. cathy.shilton@nt.gov.au
Diagnostic investigation of new disease
syndromes in farmed Australian saltwater
crocodiles (Crocodylus porosus) reveals
associations with herpesvirus infection
Catherine M. Shilton,1 Ian V. Jerrett, Steven Davis, Susan Walsh,
Suresh Benedict, Sally R. Isberg, Grahame J. W. Webb, Charlie Manolis,
Timothy H. Hyndman, David Phalen, Gregory P. Brown, Lorna Melville
Abstract. Since 2006, 3 new disease syndromes have emerged in farmed saltwater crocodiles (Crocodylus porosus) in the
Northern Territory of Australia. We describe the syndromes through a retrospective study of laboratory findings from 187
diagnostic cases submitted to Berrimah Veterinary Laboratories between 2005 and 2014. The first syndrome was characterized
by conjunctivitis and/or pharyngitis (CP), primarily in hatchlings. Herpesviruses were isolated in primary crocodile cell
culture, or were detected by polymerase chain reaction (PCR) directly from conjunctiva or pharyngeal tissue, in 21 of 39 cases
of CP (54%), compared with 9 of 64 crocodiles without the syndrome (14%, p < 0.0001). Chlamydiaceae were detected by
PCR in conjunctiva or pharyngeal tissue of 55% of 29 CP cases tested, and of these, 81% also contained herpesvirus. The
second syndrome occurred in juveniles and growers exhibiting poor growth, and was characterized histologically by systemic
lymphoid proliferation and nonsuppurative encephalitis (SLPE). Herpesviruses were isolated or detected by PCR from at least
1 internal organ in 31 of 33 SLPE cases (94%) compared with 5 of 95 crocodiles without the syndrome (5%, p < 0.0001). The
third syndrome, characterized by multifocal lymphohistiocytic infiltration of the dermis (LNS), occurred in 6 harvest-sized
crocodiles. Herpesviruses were isolated from at least 1 skin lesion in 4 of these 6 cases. Although our study revealed strong
associations between herpesvirus and the CP and SLPE syndromes, the precise nature of the role of herpesvirus, along with
the pathogenesis and epidemiology of the syndromes, requires further investigation.
Key words: Australia; crocodiles; conjunctivitis; herpesvirus; lymphoproliferation; pathology.
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Shilton et al.
2
Rural Industries Research and Development Corporation Pub-
lication No. 08/188, 2008). The second syndrome to emerge
was systemic lymphoid proliferation with nonsuppurative
encephalitis (SLPE), and the third was lymphonodular skin
lesions (LNS). Two novel herpesviruses, crocodyline herpesvi-
rus 1 and crocodyline herpesvirus 2 (CrHV-1 and -2, respec-
tively), were identified in 8 saltwater crocodiles affected with
these 3 syndromes. Bayesian phylogenetic analysis of the
amino acid sequences of a segment of the polymerase gene
revealed the 2 viruses to be distinct and clustered within the
subfamily Alphaherpesvirinae. CrHV-1 was isolated from con-
junctiva of 2 crocodiles with CP syndrome, whereas CrHV-2
was isolated from conjunctiva of 1 crocodile with CP syn-
drome, internal organs from 3 crocodiles with SLPE syndrome,
skin from 1 crocodile with LNS syndrome, and skin from 1
crocodile with ulcerative skin disease.18
Herpesviruses are enveloped, large DNA viruses with
intranuclear replication and generally very high host fidelity.28
Transmission typically occurs following close contact, par-
ticularly mucosal, with an infected individual but it can also
be via aerosol or vertical. Herpesviruses exhibit latency,
commonly persisting in lymphocytes and neurons. Infection
is considered to be life-long and is often subclinical. Clinical
disease is typically associated with very young or otherwise
immunocompromised individuals, concurrent disease, infec-
tion in an alternate host, or stressful conditions.23
Diseases caused by herpesviruses in mammals and birds
are often characterized by mucosal inflammation and ulcer-
ation (e.g., infectious rhinotracheitis and pustular vulvovagi-
nitis caused by Bovine herpesvirus 1,6,23 rhinotracheitis and
conjunctivitis caused by Felid herpesvirus 1,10 and laryngo-
tracheitis caused by Gallid herpesvirus 112). Herpesvirus
infection may also be associated with lymphoproliferative
disease (e.g., malignant catarrhal fever caused by alcephaline
herpesvirus 1 or Ovine herpesvirus 239 and Marek’s disease
caused by Gallid herpesvirus 233).
In reptiles, disease associated with herpesvirus infection
includes chronic proliferative stomatitis in lizards42,43; acute
and necrotizing, or chronic and proliferative, stomatitis and
rhinitis in tortoises27; conjunctivitis, necrotizing, or prolifer-
ative pharyngitis and tracheitis and proliferative broncho-
pneumonia in sea turtles20; and papular to ulcerative skin
lesions in sea turtles.30,36 Reports of herpesviral disease in
crocodilians are limited. Mild skin disease associated with
herpesvirus-like particles was reported in a 6-month-old salt-
water crocodile that had been farm-hatched in the Northern
Territory of Australia, and transported to a facility in Victo-
ria, Australia for stress research.25 There is also a report asso-
ciating cloacal lymphoid nodules in juvenile farmed
American alligators (Alligator mississippiensis) with herpes-
virus infection.11 However, the herpesvirus was subsequently
determined to have likely been a contaminant (GenBank
accession AY913769.1).
We provide a retrospective study analyzing the results of
diagnostic laboratory testing in saltwater crocodiles with CP,
SLPE, and LNS syndromes, including detailed descriptions
of the pathology. We also establish the strength of associa-
tion between herpesviruses and the syndromes by comparing
virus isolation and generic herpesvirus polymerase chain
reaction (PCR) results from crocodiles with each syndrome
to crocodiles not affected with any of the syndromes.
Materials and methods
Crocodile farms and animals
Saltwater crocodiles submitted for the study originated from
4 farms within 60 km of Darwin, in the Northern Territory of
Australia. Crocodiles were reared in pens containing rela-
tively small numbers of animals (<20) or hundreds of ani-
mals. Grading of pens occurred every few months to ensure
each pen continued to contain similarly sized animals. Pens
contained water maintained at ~32°C for hatchlings and
juveniles or ambient temperatures for older animals, and at
sufficient depth for crocodiles to submerge, with dry areas to
allow crocodiles to leave the water. Pens were in sheds that
were either only partly enclosed, or had windows that could
be opened to provide ventilation. Crocodiles were fed 3–7
times per week. Hatchlings were fed finely minced red meat
(horse or buffalo) supplemented with a vitamin and mineral
mix, and larger crocodiles were fed whole or minced chicken
heads, without any supplementation. The water in pens was
emptied and replaced with fresh water either from a dam or
bore on the day following feeding.
Case material for the study consisted of 187 crocodiles that
were submitted to Berrimah Veterinary Laboratories for post-
mortem examination between 2005 and 2014. Crocodiles
were either submitted dead, after being found dead at a farm,
or submitted alive if they were moribund, weak, exhibited
poor growth, had unusual lesions, or had findings suggestive
of the syndromes described herein. Crocodiles submitted live
were euthanized with 80 mg/kg of intravenous pentobarbital.a
Total length (snout to tail tip) was measured for each case.
Exact ages were not known, but animals were categorized as
hatchling (1–6 months of age, mean total length 39.5 cm,
standard error [SE] 0.9), juvenile (6–12 months of age, mean
total length 52.3 cm, SE 2.0), grower (12–24 months of age,
mean total length 93.6 cm, SE 8.6), or harvest-size (24–48
months of age, mean total length 150.3 cm, SE 12.8).
Pathology, histology, and syndrome
classification
A full gross postmortem examination was performed on each
crocodile in the study, with the exception of 2 crocodiles with
LNS in which only the skin was examined. Histologic exami-
nation of selected tissues (based on those that were deemed
necessary for a case diagnosis) was also performed in most
cases of CP and all cases of SLPE and LNS. Tissues col-
lected for histologic examination were fixed in 10% neu-
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Herpesvirus infection in farmed crocodiles in Australia 3
tral buffered formalin, processed using standard techniques,
and stained with hematoxylin and eosin. To enhance visual-
ization of gram-negative and -positive bacteria, acid-fast bac-
teria, and fungal elements, Gram–Twort, Ziehl–Neelsen, and
periodic acid–Schiff stains,4 respectively, were used to exam-
ine lesions in several cases from each of the syndromes
Based on gross and/or histopathological findings, croco-
diles were categorized as being affected with 1 of the 3 syn-
dromes or as being a nonsyndrome control animal. Crocodiles
demonstrating grossly reddened and swollen conjunctivae
with or without fibrinonecrotic exudate and/or pharyngeal
fibrinonecrotic exudate were diagnosed as having CP syn-
drome (39 cases). SLPE syndrome was diagnosed based on
the histological findings of systemic lymphoid proliferation
and nonsuppurative encephalitis (33 cases). If a case met the
inclusion criteria for both SLPE and CP, it was placed in the
SLPE category. LNS syndrome was characterized by multiple
grossly evident dermal nodules underlying intact epidermis,
confirmed histologically as being comprised of primarily
lymphocytes and macrophages (6 cases). The remaining 109
cases in the study did not meet the criteria for any of the syn-
dromes, and in most cases were diagnosed with another dis-
ease. Consequently, these animals served as nonsyndrome
control animals.
Bacteriology and PCR screening for
Chlamydiaceae
Based on gross and/or histopathological findings, ancillary
testing was performed to diagnose the cause of the morbidity or
mortality. Aerobic bacterial culture of liver, spleen, kidney,
and/or brain was used to screen for bacterial septicemia or
meningitis, which are commonly associated with morbidity or
mortality in farmed crocodiles.5,21 Aerobic bacterial culture and
speciation were performed using standard veterinary bacteriol-
ogy phenotypic and biochemical techniques. Briefly, samples
were homogenized and plated onto sheep blood agarb and Mac-
Conkey agarb and incubated at 35°C for 48 h. The bacterial
isolates were initially characterized by gram staining, colony
morphology, and oxidase and catalase tests. The appropriate
commercial kits were then used to speciate the isolates.c,d Aero-
bic bacterial culture of organs was performed in 13 CP cases,
25 SLPE cases, and 78 nonsyndrome control cases. Aerobic
bacterial culture was performed on conjunctival swabs in 8 CP
cases and 9 samples of affected skin from 4 LNS cases. Testing
for Chlamydiaceae was performed on conjunctiva and/or pha-
ryngeal tissue or swabs in 29 CP cases using PCR targeting the
16S gene of Chlamydiaceae as previously described.8
Virus isolation and electron microscopy
of isolates
Isolation of cytopathic viruses was attempted from conjunc-
tiva and/or pharyngeal swabs or tissue, at least 1 internal tissue
(liver, brain, spleen, kidney, lung, or thymus), or skin. For
virus isolation, primary cell lines were derived from saltwater
crocodile hatchlings less than 24 h old. For each sample, virus
isolation was attempted on 2 cell lines: 1 of epithelial mor-
phology (derived from kidney) and 1 of fibroblast morphology
(derived from liver, subcutaneous connective tissue, heart, or
trachea).18 Cultures showing cytopathic effects, seen as loss of
confluence of the cell monolayer, rounding up of cells, and, in
some cases, syncytia formation, were categorized as virus iso-
lation positive (VI+) and were stored at –70°C for later analy-
ses. If no cytopathic effect was observed at any time during 2
passages, further testing was not pursued and the culture was
considered negative for cytopathic viruses (VI–).
Initial investigations involved electron microscopy of
VI+ cultures. After the media were removed, cells were fixed
in 2.5% glutaraldehyde in phosphate-buffered saline and
then processed. Ultrathin sections were double-stained with
uranyl acetate and lead citrate, as previously described,
except Sorenson phosphate buffer was used (300 mOsm/kg,
pH 7.2).41 VI+ cell cultures were analyzed from 11 cases:
conjunctiva and/or pharynx from 6 CP cases; liver or kidney
from 3 SLPE cases; skin from 1 LNS case; and pharynx from
1 nonsyndrome control case.
Herpesvirus PCR
Based on the preliminary findings of electron microscopy,
either tissue homogenates or frozen-thawed cell culture
homogenates from VI+ cultures were tested for herpesvirus
using a pan-Herpesviridae nested PCR.40 We determined
that this PCR was suitable for screening samples for croco-
dyline herpesviruses by sequencing the PCR product from
8 isolates (representing 3 cases of CP, 3 cases of SLPE, 1
case of LNS, and 1 case of ulcerative skin disease) and con-
firming that every pan-Herpesviridae PCR product that we
sequenced was a crocodyline herpesvirus.18 DNA was
extracted from a 200-µL aliquot of each cell culture or tis-
sue homogenate using a commercial kit according to the
manufacturer’s instructions.e Bovine herpesvirus 2 was
used as a positive control, and tissue culture medium from
uninfected crocodile cell culture was used as a negative
control. Virus isolation with subsequent detection of her-
pesvirus by PCR in VI+ cultures was used to determine if a
sample was herpesvirus positive or negative in 91 conjunc-
tiva and/or pharyngeal samples, 140 internal organ sam-
ples, and 18 skin samples. PCR performed directly on tissue
homogenates was used to detect herpesvirus in 30 conjunc-
tiva and/or pharyngeal samples, 9 internal organ samples,
and 4 skin samples. Uninfected cell lines were tested by
PCR to ensure the cell lines were not contaminated with
herpesvirus and were negative.
Statistical analyses
If either the conjunctiva or pharyngeal swab, or any internal
organ, was positive for herpesvirus, the animal was considered
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Shilton et al.
4
to be infected with herpesvirus. Chi-square tests were used to
test for statistical differences in herpesvirus detection rates
between cases with the disease syndromes and nonsyndrome
control animals. For the internal organ testing, the numbers
of internal organs tested varied between syndrome animals
and the nonsyndrome control animals. To remove any bias
this may have caused, the number of organs tested in a case
was introduced as a covariate in a multiple logistic regres-
sion analysis. To remove the potential bias of some internal
organs being more likely than others to be herpesvirus posi-
tive, statistics were also compiled for liver samples only, as
this was the most commonly tested internal organ. If the chi-
square test revealed a significant difference between groups,
the odds ratio (OR, also known as the cross-products ratio)
was calculated to compare the likelihood of a crocodile hav-
ing a syndrome if it was herpesvirus positive versus herpes-
virus negative, using nonsyndrome cases as the case control
group.24 If one of the values used for calculating the OR was
0, 0.5 was added to each of the 4 values in the calculation. In
order to refine the comparisons, and control for any effect of
age category on herpesvirus-positive rate, statistics as
described above, were also calculated for the age groups that
were most commonly affected by a syndrome: hatchlings for
CP and juveniles and growers for SLPE. A chi-square test
and OR were used to compare herpesvirus and Chlamydia-
ceae detection rates in samples that were tested for both.
Results
Animal information
CP syndrome was first recognized by crocodile farmers in
mid-June 2006. On the most extensively affected farm,
nearly 50% of more than 2,000 hatchlings were noted to have
some degree of conjunctivitis and/or pharyngitis. Over the
next 6 weeks, the farm lost an estimated 70% of their hatch-
lings, presumably mainly a result of starvation, because they
were unable to see the food, or suffocation because of occlu-
sion of the larynx with pharyngeal exudate. At the time of
writing (2015), outbreaks of CP were still occurring in hatch-
lings approximately every 2–4 years. The 39 CP cases in this
study were submitted from 2006 to 2013, and 27 of them
were hatchlings (Table 1).
The first cases of SLPE were submitted for examination
in October 2009. The syndrome was recognized only on 1
farm, mainly in association with outbreaks in 2 pens. The
first outbreak was in a large pen composed of several smaller
interconnected pens, in all containing ~1,400 juvenile croco-
diles. Crocodiles in these pens had ill thrift and poor growth,
and farmers reported a “moderately increased” mortality rate
compared to similarly aged, unaffected crocodiles. Eighteen
of the 33 cases of SLPE described in this study (Table 1)
were crocodiles that were either from this pen or had been in
contact with crocodiles from this pen. Of these 18 SLPE
cases, 13 were submitted live in 2 groups for diagnostic post-
mortem examinations: 8 crocodiles in March 2010 and 5
crocodiles in August 2010. The 5 crocodiles in the latter
group were judged by farmers to be clinically normal apart
from exhibiting poor growth. Nine of the 33 SLPE cases
were from a second pen outbreak that occurred in November
2013, involving a pen of 48 crocodiles. None of these ani-
mals died spontaneously, as they were in good body condi-
tion but exhibited poor growth. The majority had mildly
reddened conjunctivae. One live crocodile with reddened
conjunctivae from this pen was initially submitted for diag-
nostic postmortem examination, after which 8 more were
randomly selected from the pen for the same purpose.
For the LNS syndrome, 6 harvest-sized crocodiles were
submitted to the laboratory for assessment: 4 in 2009 and 2
in 2010 (Table 1). Crocodiles with this syndrome were gen-
erally in good body condition, and lesions were detected
only by close inspection of the skin prior to harvest.
There were 109 other cases from the farms in this study
that were determined, by gross and histologic examination,
to not be affected with any of the syndromes. These croco-
diles were categorized as nonsyndrome control cases
(Table 1). The majority of these crocodiles were hatchlings
or juveniles and were routine diagnostic submissions to the
laboratory during the same time interval as the crocodiles
affected by the 3 syndromes described in our report. Virus
isolation and/or herpesvirus PCR was performed on these
individuals for survey and comparative purposes. Forty-two
of these 109 cases were diagnosed with some form of bacte-
rial infection involving (in descending order of frequency)
septicemia, meningitis, wound infection, or yolk sacculitis.
Of the remaining animals, a variety of diagnoses were made,
including runts (18 animals; described previously),34 cause
of death not apparent (16), hepatic chlamydiosis (5), spinal
trauma (4), ulcerative or erosive skin disease (4), and single
cases of other conditions: systemic coccidiosis, drowning,
irritant eye lesions, metabolic bone disease, and nephrosis.
The remaining 15 of these 109 animals were in good health
and had been euthanized for unrelated research projects from
which stored samples were scavenged for this investigation.
Regarding the ulcerative or erosive skin disease nonsyndrome
control cases noted above, 2 of the crocodiles were growers
and 2 were harvest-sized. The skin lesions in the 4 cases did
not fit the inclusion criteria for LNS syndrome and were suf-
ficiently grossly and histologically distinct from each other
that they could not be combined into a separate syndrome.
Gross pathology and histopathology
CP syndrome. Gross autopsy findings in CP syndrome were
usually limited to the eyes, pharynx, and larynx. Eye lesions
were typically bilateral. In relatively mild cases, there
was thickening, edema, and reddening of the nictitating
membrane and palpebral conjunctiva associated with scant
mucopurulent exudate (Fig. 1A). In more severe cases, the
exudate was copious, fibrinocaseous, and often adherent to
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Herpesvirus infection in farmed crocodiles in Australia 5
the conjunctiva (Fig. 1B). In most cases, there was also con-
current mild to marked opacity of the cornea, and in the most
severe cases, rupture of the cornea and copious, fibrinocase-
ous exudate filling the ocular chambers. Multifocal to region-
ally extensive mucosal erosion or ulceration with associated
scant to abundant lightly adherent fibrinocaseous exudate
were present in the pharynx and/or larynx. The pharynx at
the junction of the base of the tongue and the gular flap (Fig.
1A), and surrounding the larynx (Fig. 1C), were frequently
the most severely affected regions.
Histologically, eye lesions varied in appearance depend-
ing on severity, and presumably chronicity. In relatively
acute lesions, there was moderate to severe edema and necro-
sis of the conjunctival epithelium resulting in erosion or
ulceration with associated mild to marked epithelial and sub-
mucosal heterophil and macrophage infiltration and overly-
ing coagula composed of fibrin, heterophils, and necrotic
cell debris. In more chronic lesions, or often intermingled
with lesions containing the above described acute features,
there were segments of moderate to marked conjunctival epi-
thelial hypertrophy and hyperplasia, with mild to marked
epithelial and submucosal lymphoplasmacytic and macro-
phage infiltration. The cornea varied from normal to edema-
tous, with epithelial erosion or ulceration and epithelial and
stromal heterophil and/or lymphocyte infiltration. In many
affected eyes, there was mild to marked heterophil, macro-
phage, and/or lymphocyte infiltration of the iris (Fig. 1D).
The pharyngeal and laryngeal mucosal epithelium exhibited
similar lesions as the conjunctiva (Fig. 1E). In the mucosal
epithelia of both the conjunctiva and pharynx, where the epi-
thelium had not yet ulcerated, superficial epithelial cells
sometimes appeared to have condensed, dark nuclei, or
enlarged nuclei undergoing karyorrhexis (Fig. 1D, inset).
However, convincing intranuclear bodies suggestive of viral
inclusions were not observed. In severe conjunctival and
pharyngeal lesions, there were frequently mixed gram-nega-
tive and -positive bacterial rods and cocci and occasionally
fungal hyphae visible histologically in the necrotic superfi-
cial exudate, although none appeared to be infiltrating viable
tissue.
SLPE syndrome. Gross postmortem findings were minimal
and limited to affected crocodiles being in poor body condi-
tion with variable splenomegaly and/or pulmonary edema.
Six crocodiles with this syndrome (3 juveniles and 3 grow-
ers) had mild gross lesions of conjunctivitis and/or pharyngi-
tis as described above for the CP syndrome.
Histologically, SLPE was characterized by lymphohistio-
cytic infiltration of a wide variety of tissues, including the
pulmonary septae (Fig. 2A), hepatic periportal regions, pan-
creatic interstitium (Fig. 2B), gastrointestinal submucosa,
pericardium, epicardium, and in severe cases, the wall of
large blood vessels (Fig. 2C). There was abundant protein-
aceous fluid in pulmonary air spaces (Fig. 2A). In the spleen,
perivascular lymphoid cuffs were large and confluent, and
the red pulp was expanded by abundant macrophages, many
having vacuolated cytoplasm containing variegated fine pale
eosinophilic material (presumably phagocytized cell debris).
The tonsils were frequently markedly active, with oblitera-
tion of the tonsillar epithelium by lymphocytes and solid
dense sheets of lymphocytes and macrophages expanding
the tonsillar folds. In the brain, there was diffuse mild to
Table 1. Summary of herpesvirus testing results by disease syndrome and for nonsyndrome control crocodiles.
Syndrome Hatchlings Juveniles Growers
Harvest
size Total cases
% cases herpes+
conjunctiva or
pharynx by any
test (+/total)*†
% cases herpes+
in an internal
tissue by any test
(+/total)*†
% cases
herpes+ liver
by VI (+/
total)†‡
Conjunctivitis/
pharyngitis (CP)
27 3 5 4 39 54% (21/39) 0% (0/5) 0% (0/3)
p < 0.0001 p = 0.3 p = 0.7
χ2 = 18.5,
OR = 7
χ2 = 1.0 χ2 = 0.2
Systemic lymphoid
proliferation with
nonsuppurative
encephalitis (SLPE)
0 17 16 0 33 58% (11/19) 94% (31/33) 61% (19/31)
p = 0.0002 p < 0.0001 p < 0.0001
χ2 = 13.8,
OR = 8
χ2 = 69.8,
OR = 358
χ2 = 45.3,
OR = 57
Lymphonodular skin
(LNS)
0 0 0 6 6 33% (1/3) 0% (0/4) 0% (0/3)
p = 0.4 p = 0.6 p = 0.7
χ2 = 0.7 χ2 = 0.3 χ2 = 0.2
Nonsyndrome controls 64 29 14 2 109 14% (9/64) 5% (5/95) 3% (2/74)
* Tests included virus isolation (VI) and subsequent confirmation of isolate by either herpesvirus polymerase chain reaction (PCR) or electron microscopy,
or herpesvirus PCR directly on tissue. Virus isolates were identified by cytopathic effect in primary crocodile cell lines. + = positive.
† Statistical comparison was performed between a disease syndrome category and the nonsyndrome controls category; p = level of statistical significance
(significant comparisons in boldface), χ2 = chi-squared statistic. Odds ratio (OR) was calculated for statistically significant comparisons. For internal tissue
statistical comparisons, number of internal tissues tested by VI for a case was entered as a covariate in multiple logistic regression.
‡ Liver tested by VI and subsequent identification of isolate by herpesvirus PCR.
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Shilton et al.
6
moderate gliosis and lymphohistiocytic infiltration surround-
ing parenchymal vessels and in the meninges and choroid
plexus (Fig. 2D). Histology of the eyes was conducted on 25
crocodiles with SLPE syndrome, and all had a mild to mod-
erate degree of chronic hyperplastic lymphocytic conjuncti-
vitis, with most crocodiles for which the iris was examined
also having mild to marked lymphoid infiltration of the iris.
No gram-positive or -negative or acid-fast bacteria, fungal
elements, or protozoa were appreciable within lymphohistio-
cytic infiltrates in any of the tissues.
LNS syndrome. Grossly, skin lesions in this syndrome were
characterized by multiple pale, soft, raised, well-delineated
4–20 mm foci most commonly involving the lateral abdomi-
nal scales. Lesions occurred either in the interscalar region,
on part or all of a single scale, or a few contiguous scales
(Fig. 3A). Lesions that involved only part of a scale were
often centered on the integumentary sensory organ. Occa-
sionally, large lesions were ulcerated and covered with
caseous exudate. On cut section, lesions appeared as pale
pink soft glistening tissue between the epidermis and deep
dermal collagen (Fig. 3A, inset). In the 4 cases where a full
autopsy was performed, there were similar soft raised foci
involving the tongue or commissures of the mouth, at times
with an ulcerated surface covered in caseous exudate. Tonsils
were diffusely enlarged with a fine multinodular appearance.
Other gross lesions present in 2 cases were discrete soft
white foci present in subepithelial tissue of the conjunctiva,
multinodular swelling of the cloacal mucosa, and a few scat-
tered discrete white soft 1–3 cm foci expanding the paren-
chyma of the myocardium, liver, or kidney.
Histologically, the main feature of all LNS skin lesions
was expansion of the superficial and mid-dermis by large
dense aggregates of lymphocytes of various sizes and macro-
phages, sometimes arranged in clusters and containing varie-
gated fine pale eosinophilic material, which was presumed to
be phagocytized cell debris (Fig. 3B). In many lesions, the
overlying epidermis was normal, whereas in a few lesions it
Figure 1. Typical gross pathology and histology of conjunctivitis and/or pharyngitis (CP) syndrome in hatchling saltwater crocodiles
(Crocodylus porosus). A. Reddening and swelling of the conjunctivae of the eyelids and nictitating membrane. Abundant fibrinocaseous
exudate adherent to the caudal tongue and oropharynx. Bar = 1 cm. B. Partial to complete bilateral closure of eyes as a result of accumulation
of abundant fibrinocaseous conjunctival exudate (anterodorsal view). Bar = 1 cm. Inset: enlarged lateral view of right eye. C. Ventral
aspect of closed jaw with tongue and underlying skin excised from the margins of the mandible and retracted caudally to reveal abundant
fibrinocaseous exudate covering the larynx (arrow). Bar = 1 cm. D. Marked lymphocyte and moderate heterophil infiltration of variably
hyperplastic, eroded, or ulcerated conjunctival epithelium (upper part of image). Moderate heterophil infiltration of cornea (middle of
image). Marked lymphocyte infiltration of iris and scant proteinaceous exudate containing heterophils in anterior chamber (lower part of
image). Hematoxylin and eosin (HE). Bar = 200 µm. Inset: higher magnification of conjunctival epithelium. E. Marked lymphocyte and
heterophil infiltration of variably hyperplastic, eroded, or ulcerated pharyngeal (upper part of image) and laryngeal (middle and lower parts
of image) epithelium. Fibrinocaseous exudate in both pharynx (upper arrow) and larynx (lower arrow). HE. Bar = 200 µm.
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Herpesvirus infection in farmed crocodiles in Australia 7
was mildly infiltrated with lymphocytes. In the lesions in
which the overlying epidermis was eroded or ulcerated, there
was heterophil infiltration and an overlying crust of necrotic
cell debris with superficial mixed bacteria and/or fungi. Oral
lesions were histologically similar to skin lesions. Tonsils
were markedly hyperplastic and contained dense solid sheets
of lymphocytes and intermingled macrophages expanding
the tonsillar folds and infiltrating the epithelium. The dis-
crete white foci in internal organs, noted grossly in 2 cases,
were histologically characterized by dense lymphohistio-
cytic aggregates, similar to those described in the dermis. No
gram-positive or -negative or acid-fast bacteria, fungal ele-
ments, or protozoa were appreciable within lymphohistio-
cytic infiltrates in the skin or other tissues.
Bacteriology
Aerobic bacterial culture of the conjunctival swabs from the
8 CP cases that were tested yielded no significant growth in
2 cases and light to moderate growth of a variety of bacteria
in 6 cases. Predominant organisms were Streptococcus sp.
(3 cases), Aeromonas hydrophila (1 case), Morganella mor-
ganii (1 case), and Enterobacter sp., Staphylococcus sp.,
and Corynebacterium sp. (all from 1 case). For 13 CP cases
in which there was a suspicion of concurrent bacterial septi-
cemia based on history (found dead with relatively mild CP
lesions) and gross and/or histological findings, bacterial cul-
ture was positive in 7 cases from liver, kidney, and/or spleen.
Bacteria isolated were Streptococcus sp. (1 case) and gram-
negative environmental species: Providencia rettgeri (2
cases), Aeromonas sp. (2 cases), Edwardsiella tarda (1
case), and Salmonella sp. (1 case).
Aerobic bacterial culture of internal organs from 25 croc-
odiles with SLPE was negative in all but 3 cases in which P.
rettgeri, Salmonella sp., Aeromonas sp., and M. morganii
were isolated (the latter 2 organisms both from the same
case).
Aerobic bacterial culture that was performed on 9 skin
lesions from 4 of the LNS skin cases showed no growth for 4
lesions and light to moderate mixed bacterial growth in 5
lesions, with predominant isolates being Dermatophilus sp.
(2 cases), Nocardia sp. (2 cases), Streptomyces sp. (1 case),
and Streptococcus sp. (1 case).
Figure 2. Typical histology of systemic lymphoid proliferation and encephalitis syndrome (SLPE) in juvenile saltwater crocodiles
(Crocodylus porosus). A. Proteinaceous fluid in small pulmonary airways and moderate diffuse interstitial lymphocyte infiltration (arrows).
Hematoxylin and eosin (HE). Bar = 100 µm. B. Marked pancreatic interstitial mononuclear cell infiltration. HE. Bar = 100 µm. Inset: higher
magnification of pancreatic infiltrate composed of mixed lymphocytes and macrophages. HE. C. Moderate lymphocyte infiltration of the
tunica intima and tunica adventitia (arrow) of a large artery at the base of the heart. HE. Bar = 50 µm. D. Marked primarily perivascular
mononuclear cell infiltration of the white and gray matter of the brain. HE. Bar = 100 µm. Inset: higher magnification of perivascular mixed
lymphocyte and macrophage brain infiltrate. HE.
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Shilton et al.
8
Of the 78 nonsyndrome control cases in which aerobic
bacterial culture of internal organs was performed, bacteria
were isolated in 47 (60%). Providencia rettgeri was the most
common bacteria cultured (19 cases), with less common iso-
lates (<10 cases each), including Salmonella sp., M. morga-
nii, Streptococcus agalactiae, Aeromonas sp., Providencia
stuartii, and E. tarda.
Virus isolation, electron microscopy, and PCR
for herpesvirus and Chlamydiaceae
Electron microscopy of VI+ cultures revealed herpesvirus-
like virus in 10 of the 11 cultures tested. There were no virus
structures observed in 1 VI+ culture from the liver of an
SLPE case. Seven of these VI+ cultures examined by elec-
tron microscopy were also tested by herpesvirus PCR and all
were positive, including the VI+ culture in which no virions
were observed.
Herpesvirus was detected in the conjunctiva and/or phar-
ynx in significantly more CP cases (54%) than nonsyndrome
control cases (14%; Table 1). The OR between these 2 groups
indicated that the rate of CP syndrome in crocodiles with
herpesvirus-positive conjunctiva and/or pharynx was 7 times
greater than the rate of CP syndrome in crocodiles with her-
pesvirus-negative conjunctiva and/or pharynx. The strength
of this relationship increased substantially if the age group-
ing was made more comparable by just including hatchlings,
as 59% of 27 hatchlings with CP were positive for herpesvi-
rus in the conjunctiva and/or pharynx, whereas none of 32
nonsyndrome control hatchlings were positive (p < 0.0001,
χ2 = 24.8, OR = 93). Of the 29 CP cases in which the con-
junctiva and/or pharynx were tested for both Chlamydiaceae
and herpesvirus, 18 (62%) were positive for herpesvirus.
Thirteen of these 18, plus 3 other cases that were negative for
herpesvirus, were positive for Chlamydiaceae (55%). The
odds of detecting herpesvirus in a crocodile with Chlamydi-
aceae were 6.9 times greater than the odds of detecting it in
one without (p < 0.05).
For the SLPE syndrome, results for herpesvirus detection
in any internal organ revealed that the odds of having the
syndrome were over 350 times greater for crocodiles with
detectable herpesvirus infection than for crocodiles with no
detected herpesvirus (Table 1). The relationship between
SLPE and herpesvirus remained highly significant when
potential bias resulting from which organ(s) were tested was
controlled for by calculating statistics for virus isolation and
subsequent PCR confirmation of VI+ cultures from only
liver samples (Table 1). These 2 comparisons (any internal
organ and liver only) between SLPE cases and nonsyndrome
control cases remained significant when the comparisons
were made more age comparable by including only juveniles
and growers from the nonsyndrome controls (all SLPE cases
were juveniles and growers). For herpesvirus detection in
any internal organ, 11% of 38 nonsyndrome control juvenile
and/or grower cases were positive (significantly less than
SLPE cases, p < 0.0001, χ2 = 42.0, OR = 125), whereas for
herpesvirus detection in liver, 8% of 25 nonsyndrome control
juvenile and/or grower cases were positive (significantly less
than SLPE cases, p < 0.0001, χ2 = 18.8, OR = 18). Of the 21
crocodiles with SLPE from the 2 pen outbreaks that were
submitted live for euthanasia and diagnostic autopsy, all
were positive for herpesvirus from at least 1 internal organ.
There was also a significant relationship between the
SLPE syndrome and detection of herpesvirus in the con-
junctiva and/or pharynx (Table 1). If the comparison of her-
pesvirus-positive rates of conjunctiva and/or pharynx
between SLPE cases and nonsyndrome control cases was
made more age comparable by just including juveniles and
Figure 3. Typical gross pathology and histology of lymphonodular skin syndrome (LNS) in a harvest-sized saltwater crocodile
(Crocodylus porosus). A. Crocodile in dorsal recumbency. Lateral abdominal scales either completely (3 white arrows at left) or partially (2
white arrows at right) pale, soft, and raised. Bar = 1 cm. Inset: cut section of an affected scale showing pale pink glistening tissue between the
epidermis and deep dermal collagen (black arrow). B. Marked expansion and displacement of collagen of the superficial and mid-dermis by
intense multinodular mononuclear cell infiltrate. Note normal overlying epidermis (upper left part of image). Hematoxylin and eosin (HE).
Bar = 200 µm. Inset: higher magnification of mixed lymphocytes and macrophages composing the dermal infiltrate. Some macrophages
have vacuolated cytoplasm containing variegated fine pale eosinophilic material and are occasionally arranged in small clusters (arrow). HE.
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Herpesvirus infection in farmed crocodiles in Australia 9
growers, the relationship was weaker but still significant
(26% of 31 nonsyndrome control cases were positive, p =
0.02, χ2 = 5.1, OR = 4.0).
In the LNS syndrome, virus isolation was attempted from
all 6 cases on a total of 13 skin lesions. Four out of the 6 LNS
cases had at least 1 herpesvirus-positive skin lesion. Across
all skin lesions, there were 5 herpesvirus-positive skin sam-
ples and 8 herpesvirus-negative samples. Therefore, any
relationship between LNS skin lesions and herpesvirus infec-
tion is equivocal. In the 4 ulcerative or erosive skin cases in
the nonsyndrome control cases, herpesvirus was detected in
skin samples from all 4 cases.
Discussion
Diseases are having a significant impact on the number of
crocodiles that can be produced in commercial operations in
the Northern Territory in Australia. The purpose of our study
was to retrospectively examine the results of diagnostic labo-
ratory investigations related to 3 emerging disease syn-
dromes in farmed saltwater crocodiles and to determine the
strength of association of these syndromes with the presence
of herpesviruses.
The association between the CP syndrome and herpesvi-
rus was not strong, as the virus was not detected in conjunc-
tival and/or pharyngeal samples from 46% of crocodiles with
the syndrome. However, given that there was a highly sig-
nificant difference in the detection rate between crocodiles
with and without CP, it seems likely that herpesvirus at least
plays some role in the syndrome, or is more likely to infect
animals with the CP syndrome, and is not simply present
incidentally in the conjunctiva or pharynx.
The conjunctiva and pharynx lesions in crocodiles with
CP were characterized by acute erosive or ulcerative necro-
tizing lesions with fibrinocaseous exudate. These lesions
were consistent with herpesvirus infection, being similar to
conjunctivitis, stomatitis, or pharyngitis associated with her-
pesvirus infection in a wide variety of other species, includ-
ing sea turtles,20 tortoises,27 lizards,42,43 chickens (infectious
laryngotracheitis),12 and cats.2,45
The lack of histologically visible inclusion bodies in the
crocodile CP lesions was unexpected, given that inclusion
bodies are frequently seen in herpesvirus mucosal infections
in some species of reptiles.19,27,42 However, in mucosal her-
pesvirus infections in other species (e.g., monitor lizards,43
chickens,12 bovids,6 and cats45), typical herpesvirus inclu-
sions have either not been appreciable, or are transient,
appearing only in the early stages of infection. Further evi-
dence for rapid loss of herpesvirus from lesions comes from
molecular studies of Gallid herpesvirus 1 and Felid herpes-
virus 1 infections in which, even when using highly sensitive
PCR assays, herpesvirus was frequently undetectable within
30 days of infection.38,47 Therefore, the absence of herpesvi-
rus inclusions in CP-affected crocodiles does not rule out a
causal association of the virus infection and disease, but
could represent either herpesvirus infection without inclu-
sion formation, or transient presence of herpesvirus.
In addition to transience, the incomplete association
between CP syndrome and herpesvirus could arise from mul-
tifactorial causation. For example, in infectious bovine kera-
toconjunctivitis, Moraxella bovis is considered the primary
etiological agent, but there is frequently concurrent infection
with other agents, including Bovine herpesvirus 1 and Myco-
plasma sp. as well as potential exacerbation by environmen-
tal factors (dust, ultraviolet radiation) that may contribute to
lesion severity.45 Another example of multifactorial conjunc-
tivitis is in cats, in which the condition may be caused by
Felid herpesvirus 1 or Chlamydophila felis, with a question-
able minor role for concurrent infection with Mycoplasma
sp.29,31,45 In the investigation of the earliest cases of CP that
occurred during 2006, pan-Mycoplasma PCR was performed
on 6 conjunctiva and 4 pharyngeal samples, with only a sin-
gle conjunctiva sample returning a positive result (Jerrett
et al. 2008, as referenced in their introduction), suggesting
that any Mycoplasma spp. involvement in CP cases in salt-
water crocodiles is perhaps sporadic or transient. However,
the possible involvement of Mycoplasma spp. in CP syn-
drome warrants further investigation.
There remains uncertainty in the role that Chlamydiaceae
plays in the pathogenesis of CP syndrome. In our study, cases
tested for both Chlamydiaceae and herpesvirus failed to
determine which organism had a stronger association with
the presence of lesions. Similar percentages of the cases
were positive for each agent (62% for herpesvirus and 55%
for Chlamydiaceae), and 45% of them were positive for
both, with herpesvirus being significantly (6.9 times) more
likely to be detected in crocodile conjunctiva and/or pharynx
if Chlamydiaceae was also present. The significance of the
combined presence of herpesvirus and Chlamydiaceae is
unknown, but a similar situation has been noted in a herpes-
virus investigation of Australian marsupials in which the
presence of Chlamydia pecorum was a significant predictor
of herpesvirus presence in 68 koalas (OR = 60).37 In the lit-
erature, there is allusion to conjunctivitis being caused by
Chlamydiaceae infection in Crocodylus niloticus in
Africa,15,16 C. porosus in Papua New Guinea,17 and recently,
C. siamensis in Thailand.32 However, in all of these investi-
gations, the diagnosis appears to be based primarily on iden-
tification of Chlamydiaceae in internal organs, most
commonly the liver, because unlike the CP cases detailed in
our study, the infections also involved internal organs. In a
previous epidemiological investigation sampling live croco-
diles from the same farms as the present study, there was a
very poor association between PCR presence of Chlamydia-
ceae in combined conjunctival and pharyngeal swabs and
presence of CP syndrome (as defined by a crocodile having
eyelid swelling or discharge and/or pharyngeal fibrin; Jerrett
I, et al., 2008). In that study, in 224 hatchlings up to 4 months
of age, 26% were positive for Chlamydiaceae but only 4%
had CP syndrome. In 476 crocodiles 1–3 years of age, 18%
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Shilton et al.
10
were positive for Chlamydiaceae, whereas 8% had CP syn-
drome. Within this group of 476 crocodiles, there were 120
crocodiles from one farm that were all positive for Chlamyd-
iaceae despite none of the tested crocodiles having CP syn-
drome. Conversely, on another farm, there were 120
crocodiles sampled in which none were positive for Chla-
mydiaceae but 29 had CP syndrome.
In our study, the preliminary results of electron micros-
copy focused our screening for herpesviruses. The possible
involvement of other viruses was not explored. Examples of
other viruses associated with conjunctivitis and/or pharyn-
gitis are iridovirus in chelonians,35,44 paramyxoviruses in
birds (e.g., Newcastle disease),1 morbillivirus in mammals
(e.g., canine distemper),6 calicivirus in cats,22 and adenovi-
ruses in humans.3 Future work should include efforts to
examine the role that other viruses may play in CP syn-
drome. Unbiased molecular techniques, such as next-gener-
ation sequencing that are capable of detecting a number of
infectious agents, including agents with fastidious culture
requirements, should be considered.
We found a strong association between SLPE and the
prevalence of herpesvirus in internal tissues, with SLPE-
affected crocodiles having a substantially higher prevalence
of infection (94%) compared to nonsyndrome control croco-
diles (5%). The 100% prevalence of herpesvirus in SLPE-
affected crocodiles that were euthanized for diagnostic
postmortem examination from the 2 pen outbreaks suggests
that chronic infection or latency may be common in some
circumstances. Histologic examination of crocodiles with
SLPE did not reveal lesions suggestive of a known disease,
and aerobic bacterial culture of internal organs provided
equivocal results.
The SLPE lesions are similar to those seen in other spe-
cies with herpesvirus infections that have the ability to trans-
form lymphocytes. Histologically, SLPE bears a strong
similarity to Marek’s disease in chickens, which is caused by
the cell-associated, lymphotropic alphaherpesvirus Gallid
herpesvirus 2. Marek’s disease is characterized by lymphop-
roliferative disorders ranging from lymphoid infiltrations in
various tissues, including the iris and other tissues of the eye,
to lymphomatous masses in various internal organs.33
Another herpesviral disease with some pathological similari-
ties to SLPE is malignant catarrhal fever, caused by several
ruminant gammaherpesviruses. In this lymphoproliferative
disease, lymphohistiocytic infiltrates are widespread, includ-
ing in the brain and blood vessels, and the virus causes pan-
ophthalmitis.23,39
Crocodiles with SLPE frequently had a mild degree of CP,
and herpesvirus was detected in 58% of these tissues, sug-
gesting that SLPE and CP syndromes may be related. Her-
pesvirus infections can manifest differently depending on
various factors, including host age, immune competence,
exposure circumstances, and latency status.10,13,23,33 Most of
the CP cases were hatchlings, and all of the SLPE cases were
in juveniles and growers. It is conceivable that CP and SLPE
represent manifestations of herpesvirus infection at different
ages or levels of immune competency. This supposition
would benefit from more information regarding the specific
identities of herpesviruses involved in the syndromes, a limi-
tation of this study being that we did not go beyond generic
PCR for herpesvirus identification. CrHV-1 was found in 2
of 3 cases of CP tested, and CrHV-2 was found in the third
CP case tested and 3 of 3 SLPE cases tested.18 Sequencing of
greater numbers of herpesviruses from CP and SLPE cases is
warranted to clarify the respective roles of these 2 crocodyl-
ine herpesviruses.
There are only 6 cases of LNS described in this study, and
herpesvirus presence in affected skin was inconsistent. No
alternate etiology for these lymphohistiocytic dermal nod-
ules was evident histologically, and aerobic bacterial culture
of intact and ulcerated lesions yielded several genera of bac-
teria, with none being convincing primary etiological agents.
Herpesviruses have been associated with lymphocytic skin
infiltrates in malignant catarrhal fever,39 and in lymphocytic
nodules associated with feather follicles in Marek’s disease.33
However, given that the association of herpesvirus infection
with LNS was weak, further investigation is required, includ-
ing consideration of other viral etiologies. For example, ret-
roviruses in birds and mammals cause lymphoproliferative
syndromes.9,23 Endogenous retroviruses have been found in
C. porosus in the Northern Territory of Australia7; however,
disease associations with retroviruses in C. porosus have yet
to be investigated. In American alligators, proliferative lym-
phohistiocytic cutaneous lesions have been associated with
West Nile virus infection.26
In addition to the LNS cases, there were 4 cases in the
nonsyndrome control cases that had erosive and/or ulcerative
skin lesions. Although a detailed description of these cases
was beyond the scope of this study, they bear mention
because herpesvirus was detected in skin from all cases. Her-
pesvirus infection is capable of causing ulcerative or necro-
tizing skin lesions in several species, including turtles,30
cats,14 and wallabies.46 Notably, the only published finding
of a herpesvirus in a crocodilian relates to relatively mild,
erosive skin lesions in a young crocodile from the Northern
Territory of Australia that was involved in a stress experi-
ment and in which herpesvirus-like particles were detected in
superficial keratinocytes using electron microscopy.25
Our study provides evidence of widespread herpesvirus
infection in farmed populations of a crocodilian species and
presents data correlating the infection with emerging disease
syndromes on the farms. The associations at this stage are far
from being established as causal. Further research should
attempt to clarify the temporal and spatial association of viral
presence with lesions using immunohistochemistry or in situ
hybridization, and to experimentally reproduce the disease
syndromes with transmission studies. If a causal relationship
can be established between the virus and 1 or more of the
syndromes, further epidemiological research to establish
prevalence and latency should be pursued to determine how
by guest on April 14, 2016vdi.sagepub.comDownloaded from
Herpesvirus infection in farmed crocodiles in Australia 11
common these viruses may be in farmed saltwater crocodiles
in Australia, to detect any insidious effects they may have on
health (and therefore growth rates and skin quality), and to
clarify any relationships among the syndromes. Most impor-
tant to the farming industry, preventative measures would
need to be established, including how to prevent exposure
and transmission or any triggers that may exist for clinical
expression of disease. Finally, the risk of these viruses to
wild populations should be established.
Acknowledgments
We thank all the staff at the crocodile farms and Berrimah Veteri-
nary Laboratories for assistance with this study. The electron
microscopy was performed by Dr. Alex Hyatt, at the Australian
Animal Health Laboratories, Geelong, Victoria, Australia. We
thank the reviewers for their thorough editing and constructive
comments regarding an earlier version of this manuscript.
Authors’ contributions
CM Shilton contributed to conception and design of the study; con-
tributed to acquisition, analysis, and interpretation of data; and
drafted the manuscript. IV Jerrett contributed to conception and
design of the study; contributed to acquisition of data; and critically
revised the manuscript. S Davis contributed to conception and
design of the study; contributed to acquisition and interpretation of
data; and critically revised the manuscript. S Walsh and S Benedict
contributed to acquisition and interpretation of data, and critically
revised the manuscript. SR Isberg, GJW Webb, and C Manolis con-
tributed to acquisition of data, and critically revised the manuscript.
TH Hyndman and D Phalen contributed to interpretation of data,
and critically revised the manuscript. GP Brown contributed to
analysis of data, and critically revised the manuscript. L Melville
contributed to conception and design of the study, and critically
revised the manuscript. All authors gave final approval, and agreed
to be accountable for all aspects of the work in ensuring that ques-
tions relating to the accuracy or integrity of any part of the work are
appropriately investigated and resolved.
Sources and manufacturers
a. Lethabarb euthanasia injection (325 mg/mL), Virbac Animal
Health, Milperra, New South Wales, Australia.
b. Oxoid Australia, Thebarton, South Australia, Australia.
c. Api 20 Strep and api Coryne, bioMérieux, Marcy-l’Etoile, France.
d. Microbact gram-negative identification system, Oxoid Ltd.,
Basingstoke, Hants, United Kingdom.
e. MagMAX viral RNA isolation kit, Ambion, Austin, TX.
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect
to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support
for the research, authorship, and/or publication of this article: This
research was funded in part by the Australian Government Rural
Industries Research and Development Corporation project no.
PRJ-002461, and results summarized in a non–peer-reviewed pub-
licly available report for the funding body.
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