First evidence of Kunjin strain of West Nile virus associated with saltwater crocodile ( Crocodylus porosus ) skin lesions

Abstract

Recently, the Kunjin strain of West Nile virus (WNVKUN) has been detected using qRT‐PCR in belly skin lesions of farmed juvenile saltwater crocodiles. This follows an established association between similar lesions and West Nile virus in American alligators. The lesions present as cutaneous lymphohistiocytic aggregates in the dermal layers of both species. While these lesion do not create an obvious defect on the live crocodile, upon tanning the lesion area collapses and does not uptake the dye evenly, thus reducing its aesthetic appeal. As a result, skins are being rejected jeopardising the economic viability of the Australian crocodile industry. Over 50 skin lesions have since been confirmed as WNVKUN‐positive and preliminary evidence of lesion restructuring is presented. Horizontal transmission of WNVKUN by mosquitoes is well‐established but other transmission routes, such as ingestion and cloacal shedding, need further evaluation. An infection trial is currently underway to ensure WNVKUN is the causative agent of these skin lesions.

Full text

SHORT CONTRIBUTION
First evidence of Kunjin strain of West Nile virus associated with
saltwater crocodile (Crocodylus porosus) skin lesions
SR Isberg,
a
* JL Moran,
a
R De Araujo,
b
N Elliott,
b
SS Davis
b
and L Melville
b
Recently, the Kunjin strain of West Nile virus (WNV
KUN
) has been
detected using qRT-PCR in belly skin lesions of farmed juvenile
saltwater crocodiles. This follows an established association
between similar lesions and West Nile virus in American alligators.
The lesions present as cutaneous lymphohistiocytic aggregates in
the dermal layers of both species. While these lesion do not cre-
ate an obvious defect on the live crocodile, upon tanning the
lesion area collapses and does not uptake the dye evenly, thus
reducing its aesthetic appeal. As a result, skins are being rejected
jeopardising the economic viability of the Australian crocodile
industry. Over 50 skin lesions have since been confirmed as
WNV
KUN
-positive and preliminary evidence of lesion restructuring
is presented. Horizontal transmission of WNV
KUN
by mosquitoes is
well-established but other transmission routes, such as ingestion
and cloacal shedding, need further evaluation. An infection trial is
currently underway to ensure WNV
KUN
is the causative agent of
these skin lesions.
Keywords Kunjin strain; West Nile virus; flavivirus; saltwater
crocodile; Crocodylus porosus; skin lesions
Abbreviation BVL, Berrimah Veterinary Laboratories, Berrimah,
Northern Territory; PCR, Polymerase chain reaction; qRT-PCR,
Quantitative real-time polymerase chain reaction; TL, Total length;
VNT, Virus neutralisation test; WNV, West Nile virus; WNV
KUN
,
Kunjin strain of WNV
Aust Vet J 2019 doi: 10.1111/avj.12862
“Pix”, as it is commonly referred to in the crocodilian skin
tanning industry, was first reported by Dickson et al.
1
in
2002 as small, 1 mm round depressions in the tanned
skins of American alligators (Alligator mississippiensis). The lesions
were so named as they resembled “small pits in the [tanned] hide
made with an ice pick”.
1
These detract from the overall aesthetic
appeal of the hide and downgrade its value with many hides being
completely rejected.
1
Histology of the lesions revealed dermal
lymphohistiocytic aggregates that have been firmly associated with
WNV infection by qRT-PCR.
2–4
Saltwater crocodile (Crocodylus porosus) belly skins are produced in
Australia for the luxury fashion market. “Pix”lesions first began to
cause C. porosus skin downgrading and rejection in 2009. The lesions
were not visible to the naked eye on harvest size C. porosus but were
clearly evident as translucent foci on the light-table (light shone
through from underneath) during grading (Figure 1A). Some of
these lesions were followed through the tanning process and were
consistently shown to become depressions with uneven dye uptake
(Figure 1B). Since the Kunjin strain of WNV (WNV
KUN
)
5
is
endemic to Australia, it was proposed as the causative agent. Previ-
ously, farmed harvest-size crocodiles had reported positive WNV
KUN
serology
6
but histology had not revealed any dermal
lymphohistiocytic aggregates as reported in alligators.
4
This could be
due to the different harvest sizes between the two species whereby
C. porosus are produced for larger fashion items such as handbags
and shoes. This requires the crocodiles to be harvested at a total
length between 1.5–2.2 m, corresponding to a belly width of
35–50 cm (2–5 years of age). Although some A. mississippiensis skins
are produced for this market, the majority are produced for the
watchstrap and smaller leather product market.
7
These alligators are
harvested as yearling between 91–122 cm TL (12–15 months of age)
7
and it was at this size class that Nevarez et al.
4
identified the cutane-
ous lymphohistiocytic aggregates.
As such, during some skin inspections on yearling (80–150 cm TL)
crocodiles on one Northern Territory farm in August 2016, nine
dark grey-red 1–2 mm focal lesions were identified. Similar to early
active poxvirus lesions
8
, these lesions did not have any contour
(depression or protrusion) compared to the surrounding skin.
*Corresponding author.
a
Centre for Crocodile Research, PO Box 329, Noonamah, Northern Territory, Australia
0837; sally@crocresearch.com.au
b
Berrimah Veterinary Laboratories, Northern Territory Government, Darwin, Northern
Territory, Australia
Figure 1. A“pix”lesion (arrows) on A. raw and B. tanned C. porosus
skin. Black arrowheads are integumentary sensory organs (ISO). A. On
the raw skin, the “pix”lesion has no contour or abnormal keratin but is
lucent on the light-table (light shone through from underneath). B. On
the tanned hide, the “pix”lesion has become depressed and has a
reduced dye uptake compared to the surrounding area. Bar = 5 mm.
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However, in contrast, they had normal overlying keratin and were
distinctly darker in colour (Figure 2). Lesion material was collected
after wiping the overlying skin area with 70% ethanol. A sterile
pipette tip was used to extrude the lesion and stored in a sterile
1.5 mL microcentrifuge tube at −20C until processing. DNA/RNA
was extracted using the QIAamp DNA Mini Kit (Qiagen, Australia)
as per the manufacturer’s instructions for viral DNA.
These lesions were all confirmed negative for poxvirus using the
PCR protocol described by Moore et al.
8
but confirmed to be
WNV
KUN
using qRT-PCR using the protocol described by Pyke
et al.
9
Further sampling has confirmed WNV
KUN
in over 50 addi-
tional skin lesions. To provide further confirmation of causation, one
crocodile with qRT-PCR confirmed WNV
KUN
lesions was also
harvested for histology and confirmed the dermal lymphohistiocytic
aggregates in multiple lesions as reported by Nevarez et al.
4
(Figure 3). Some lesions have been collected for virus isolation but
have not been successful to date.
The farm decided to track another crocodile, identified in April 2017
with 47 lesions, to observe how long it would take for these lesions
to heal. Photos of each skin lesion were taken (e.g. Figure 4A, E) and
two lesions were confirmed to be WNV
KUN
by qRT-PCR. A blood
sample submitted to BVL returned a VNT titre of 72 confirming
WNV
KUN
exposure. This crocodile was harvested six months later as
Figure 2. Three WNV
KUN
lesions (arrows) on the belly scales of a farmed
yearling C. porosus. The arrow heads are integumentary sensory organs
(ISO). Bar = 5 mm.
Figure 3. Lesions on the live crocodile (A & D) and on the light-table (B & E) indicated by arrows. The arrow heads are integumentary sensory
organs (ISO). Bar = 5 mm. Histology (C & F) shows lymphohistiocytic aggregates
4
HE. Bar = 500 μm. Insets show lymphohistocytic cells within
aggregate. Bar = 50 μm.
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Figure 4. Lesions (black arrows) as first identified (A, E) and again six months later (B, F) on the belly scales of a farmed C. porosus. C and G shows
the same scales on the light-table with the red arrow indicates another lesion that was not noticeable on the live crocodile. Bars = 5 mm. Histology
(D & H) shows reduced lymphoid aggregation with the influx of ground substance. HE. Bar = 500 μm.
© 2019 The Authors and Northern Territory Government Australian Veterinary Journal 3
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part of routine farm procedures and the lesions were re-
photographed (Figure 4B, F). Grossly, the lesions appeared less obvi-
ous after six months but were clearly visible on the light-table
(Figure 4C, G). From histology (Figure 4D,H), it is postulated that
the degree of collagen displacement from the lymphohistiocytic
aggregates will be restructured with less-structured, finer collagen
fibres and relatively more ground substance compared to normal
dermis.
10
Thus, the lesions will appear to “fade”but will be lucent
when inspected on a light table leading to rejection. Since tanning
removes the epidermal layers, basement membrane and the ground
substance between the collagen,
10
as well as any lymphocytic mate-
rial, leaving the lesions’finer restructured collagen to collapse creat-
ing the characteristic “small pits in the hide”.
1
WNV causes encephalitis in humans, horses and birds but in 2011,
WNV
KUN
caused an unprecedented outbreak of encephalitis in
horses in Australia.
5
So far, WNV
KUN
does not appear to cause mor-
bidity or mortality in C. porosus. In contrast, WNV has been
reported to cause up to 60% mortality of A. mississippiensis after
showing neurological symptoms.
11,12
Histology and immunohisto-
chemistry reveal lymphocytic-histiocytic infiltrates in multiple
organs and tissues
11,13
as well as the skin.
2–4,12
Similar lymphocytic-
histiocytic infiltrates have not been found in moribund or dead
C. porosus, with the exception of the recent skin lesions described
herein.
WNV strains are flaviviruses transmitted by mosquitoes
5,14
but other
forms of transmission are also possible. For instance, feeding juvenile
A. mississippiensis WNV-positive horsemeat was suggested to be the
primary route of infection in a 2002 outbreak in south Georgia,
USA.
13
Klenk et al.
14
confirmed that ingestion is a likely transmission
path by feeding alligators WNV-infected mice that produced the
same magnitude of viremia as alligators injected subcutaneously with
WNV, although the viremia was offset by one day. In Australia, red
meat is also fed including horse, buffalo, camel and kangaroo.
Testing will begin to validate if ingestion could be a possible source
of infection on Australian farms.
In addition, Klenk et al.
14
showed vertical transmission. Viral shed-
ding from the cloaca of infected alligators was detected within three
days of viremia onset followed by non-infected conspecifics becom-
ing viremic 10–14 days post-infection. Despite this evidence, Klenk
et al.
14
did not detect WNV in the water. So far we have detected
WNV
KUN
in three water samples from the farm (unpublished data).
WNV
KUN
obviously poses a large threat to the Australian crocodile
industry’s ability to produce the skin quality demanded by the fash-
ion industry. Research is underway to fill the knowledge gaps as
outlined above, including an infection trial, to develop prevention
strategies.
Conflicts of interest and sources of funding
The authors declare no conflicts of interest. Funding was partially
provided by AgriFutures, Australia (PRJ-010453) and protocols were
approved by the Charles Darwin University Animal Ethics Commit-
tee (A16005).
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(Accepted for publication 20 June 2019)
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