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Using phytohaemagglutinin to determine immune
responsiveness in saltwater crocodiles (Crocodylus
porosus)
John W. Finger Jr
A,B,F
, Amanda L. Adams
D
, Peter C. Thomson
B
, Cathy M. Shilton
D
,
Greg P. Brown
E
, Christopher Moran
B
, Lee G. Miles
B
, Travis C. Glenn
A
and Sally R. Isberg
B,C
A
Department of Environmental Health Science, University of Georgia, 150 Green Street, Athens, GA 30602, USA.
B
Faculty of Veterinary Science, University of Sydney, Sydney, NSW 2006, Australia.
C
Centre for Crocodile Research, PO Box 329, Noonamah, NT 0837, Australia.
D
Berrimah Veterinary Laboratories, GPO Box 3000, Darwin, NT 0801, Australia.
E
School of Biological Sciences, University of Sydney, Sydney, NSW 2006, Australia.
F
Corresponding author. Email: finger20@uga.edu
Abstract. Immune responsiveness, the ability of an organism to effectively respond immunologically following antigenic
exposure, is an essential component of life history, as organisms require effective immune functionality in order to grow,
survive and reproduce. However, immune status is also associated with concomitant trade-offs in these physiological
functions. Herein we demonstrate the validation of phytohaemagglutinin (PHA) injection in saltwater crocodiles,
Crocodylus porosus, to assess cellular immune responsiveness. Following injection of 2 mg mL
–1
PHA into the hind toe
webbing, we observed a peak swelling response 12 h after injection, with PHA inducing increased thickness compared with
webs injected with phosphate-buffered saline (PBS) (F
5,518
= 145.13, P < 0.001). Subsequent injections increased
responsiveness relative to the primary injection response (F
5,290
= 2.92, P = 0.029), suggesting that PHA exposure induced
immunological memory, a tenet of acquired immunity. Histological examination revealed that PHA-injected toe webs
displayed increased numbers of leukocytes (granulocytes, macrophages, and lymphocytes) relative to PBS-injected webs,
with peak leukocytic infiltrate observed 12 h after injection. We suggest the use of PHA injection in crocodilians as a
measure of cellular immune responsiveness in agricultural (production and animal welfare), ecological, and toxicological
contexts.
Additional keywords: agriculture, crocodilian, ecology, toxicology.
Received 17 May 2013, accepted 5 August 2013, published online 23 August 2013
Introduction
Of
paramount importance in the life history of an organism is
the capacity to mount an efficacious immune response following
antigenic exposure (Zuk and Stoehr 2002; Vinkler and Albrecht
2011). This capacity, known as immune responsiveness (Vinkler
and Albrecht 2011), is often associated with concomitant
tradeoffs in other physiological parameters, with augmented
immunity negatively affecting growth, reproduction, and
survival (Zuk and Johnsen 1998; Martin et al. 2006, 2008;
McCallum and Trauth 2007; Bascuñán-García et al. 2010; Ruiz
et al. 2010; Gutierrez et al. 2013). Due to these interdependent
effects, determination of immune status is important in several
broad disciplines. An understanding of innate and acquired
immune functions is necessary in immunoecological studies to
determine how immune status may affect fecundity and other
physiological parameters (Smits et al. 1999; Duffy and Ball 2002;
Brzek and Konarzewski 2007; Tschirren et al. 2007; Brown et al.
2011; Demas et al. 2011). In toxicological studies, determination
of immune status following contaminant exposure is required
to understand how exposure may detrimentally impact immune
function (Smits and Williams 1999; Smits et al. 1999; Grasman
2002; Keller et al. 2006; Markman et al. 2011), possibly
culminating in increased disease susceptibility or exaggerated
responsiveness (hypersensitivity) (Youssef et al. 1996;
Fairbrother et al.
2004).
Similarly, in animal production systems,
the
understanding of factors affecting immunity such as microbial
infection or other stressors (Shini et al. 2008), is imperative as
such costs restrict resource allocation for increased growth (Fair
et al. 1999; Chen et al. 2007; Bonato et al. 2009) and reproduction
(Boughton et al. 2007). Indeed, immune responsiveness may
also provide information to develop animal welfare guidelines
(Hanlon et al. 1994; Caipang et al. 2008; Segner et al. 2012).
Thus, techniques to ascertain immune responsiveness are
necessary amongst a variety of scientific disciplines.
Journal compilation CSIRO 2013 www.publish.csiro.au/journals/ajz
CSIRO PUBLISHING
Australian Journal of Zoology
http://dx.doi.org/10.1071/ZO13041
Techniques to determine immune functionality generally
fall under two general approaches: immunomonitoring or
immunochallenge, with the former often highly criticised for
lack of standardisation and the latter allowing for treatment
standardisation among individuals and direct quantification of
response following administration (Norris and Evans 2000).
One such immunochallenge technique involves intradermal
injection of phytohaemagglutinin (PHA), a non-pathogenic,
antigenic-lectin derived from the red kidney bean (Phaseolus
vulgaris), with repeated measurements of the subsequent
swelling response at the injection site enabling evaluation of
the organism’s immune response to antigenic stimulation
(Vinkler and Albrecht 2011; Gutierrez et al. 2013). PHA was first
utilised as a measure of cell-mediated immunity, with in vitro
exposure inducing augmented T-lymphocyte mitogenesis in
human leukocytes (Nowell 1960; Morgan et al. 1976 ). Following
in vivo injection, PHA acts as a polyclonal antigen inducing
enhanced endothelial permeability, triggering oedema and
leukocyte migration out of blood vessels into tissues (Turmelle
et al. 2010; Brown et al. 2011). Initial injections trigger a
primary (innate, non-specific) immune response, with infiltration
of a whole host of innate cells including granulocytes and
macrophages (Martin et al. 2006; Kennedy and Nager 2006),
along with lymphocytes, the mediators of acquired (adaptive,
specific) immunity. During a normal microbial infection, these
innate cells activate lymphocytes, which differentiate into
effector and memory cells, the former of which aid in elimination
of antigen and the latter of which are important in repeated
exposures (Abbas et al. 2010). Compared with primary
injections, subsequent injections of PHA exhibit an increased
responsiveness (more swelling), showing that PHA can induce
immunological memory (Tella et al. 2008; Brown et al. 2011),
a tenet of acquired immunity. Thus, PHA stimulation may
elucidate both the primary immune response (primary injection)
and acquired immune response (repeated injections) in an
individual (Demas et al. 2011).
The impetus for this study on saltwater crocodiles (Crocodylus
porosus) was two-fold. First, as with other crocodilians,
saltwater crocodiles occupy the top trophic status in their
semiaquatic environment. Therefore, crocodilians are important
environmental indicators for events such as toxicant exposure
(Milnes and Guillette 2008). Second, saltwater crocodile skin is
highly sought by the international skin trade, and consequently
farming crocodiles for their skin has become a sustainable
economic enterprise in Northern Australia with proven
conservation benefits (references within Fukuda
et al. 2011).
In
this respect, an understanding of both the innate and adaptive
immune system would be beneficial to alleviate the on-farm risk
of disease, increase survival (Isberg et al. 2006, 2009), enhance
growth (Isberg et al . 2005) and, furthermore, develop animal
welfare guidelines. Finally, crocodilian immune function is
very poorly characterised (Finger and Isberg 2012), so the
development of inexpensive and easy techniques will provide
a foundation for further research.
Before PHA can be used to assess cellular immune
responsiveness in a species, the appropriate concentration and
volume for injection must be determined, the time points for
repeated measurements defined and qualified according to the
infiltration of different immunological cell types, and an
immunological memory demonstrated following reinjection.
Herein we achieve these objectives on juvenile saltwater
crocodiles, using an experimental design similar to that described
by Brown et al.(2011) for cane toads (Rhinella marina).
Methods and materials
Study species
Three-month-old saltwater crocodiles from Darwin Crocodile
Farm, Noonamah, Northern Territory, Australia were used in
this study. The animals were housed in pens (35 in each; pen
dimensions: 116.5 cm wide and 209.5 cm long) with the water
temperature maintained at 32
C(1
C). The animals were fed
in excess five days per week in the evenings and the pens were
cleaned the following morning with a chlorine-based detergent.
For sampling, crocodiles were randomly selected and injected
as described below, then placed in individual containers and
placed within a dark humidified (90– 100%) incubator at a
constant temperature of 32
C(0.5
C). As our study coincided
with normal non-feeding periods, crocodiles were not provided
with food during the sampling period. Upon completion,
animals were returned to their respective pens.
Experimental protocol
PHA (PHA-P #L8754; Sigma-Aldrich, St Louis, MO, USA) was
aseptically dissolved in sterile phosphate-buffered saline (PBS)
and 0.02 mL was injected into the toe web between the first
and second hind digits using a 0.3-mL syringe with a 29-gauge
needle. Sterile PBS was used as the control and 0.02 mL was
injected into the opposing toe web of each animal. PBS was
autoclaved and subsequently aliquoted into 1.5-mL tubes and
stored at 4
C before and after administration. The volume to inject
was standardised at 0.02 mL as this amount was readily observed
subcutaneously in the toe web upon successful injection, the
whole amount could be injected without any fluid leaking out
the point of injection, and the fluid quickly dissipated within a
short time after injection (Brown et al. 2011). One person (JWF)
performed all the injections to standardise the procedure. Prior
to administration, toe webs were swabbed with alcohol in an
attempt to prevent pathogenic entry upon injection.
Toe web thickness was measured before (0 h) and after (6, 12,
24, 48 and 72 h) injection using a dial thickness gauge (Peacock
G-1A; Ozaki Manufacturing Ltd, Japan). Three measurements
were taken in quick succession at each time point, allowing an
initial contact of less than 2 s to determine swelling thickness.
Gauge contact pushed fluid out of the immediate area, thus quick
measurements were of paramount importance to ascertain true
thickness (Brown et al. 2011). Measurements were performed
by one person (JWF) to standardise time of contact with swelled
skin. The three measurements were averaged to determine the
average swelling of each individual at each respective time
point. Head length of each individual was measured using digital
calipers (to the nearest 0.01 mm) from snout tip to the median
posterior of the cranial platform as a measure of overall length
(Webb and Messel 1978; Isberg et al. 2005).
Dose effects
An appropriate concentration was determined by injecting
0.02 mL of 1, 2, or 5 mg mL
–1
of PHA and examining the effects
B Australian Journal of Zoology J. W. Finger Jr et al.
of swelling. In total, 24 animals were injected, with eight
animals randomly allocated into each dosage group. Following
statistical analysis, 2 mg mL
–1
was selected as the concentration
of PHA used in all subsequent experiments.
Temporal effects and histological verification
Whilst acknowledging that the swelling response is a
combination of innate and cell-mediated immune activation that
needs to be further qualified at various time points (see below), the
next objective was to determine the temporal response after
injection. As such, 60 animals (together with 8 injected with
2mgmL
–1
from the previous experiment, for a total of 68) were
injected with 0.02 mL PHA and PBS (control) in the left and right
toe webs, respectively. The toe web thickness was measured
before injection (0 h) and then at 6, 12, 24, 48 and 72 h after
injection. Of these 60 animals, 30 were also used in the
histological examination of cell-type (five at each time point) so
the swelling response of 60, 55, 50, 45, 40 and 35 animals were
available for analysis at each time point respectively.
To qualify the cellular basis of the swelling response, toe
webs were histologically examined as previous studies have
shown that PHA increases T lymphocyte migration, a
component of acquired immunity (Tella et al. 2008; Brown et al.
2011), but other cell types are also attracted to the site of
injection, including innate cells such as macrophages and
granulocytes (Martin et al. 2006). Biopsies (3-mm diameter)
from both the PHA and PBS (control) toe webs, juxtaposed to
the site of injection, were taken from five animals at each time
point (0, 6, 12, 24, 48, and 72 h) and stored in 10% buffered
formalin. Biopsies were processed for histological examination
by embedding in paraffin, sectioning at 4 mm and staining
with haematoxylin and eosin. As histological examination is
subjective, to minimise bias, biopsies were examined
independently by two individuals (JWF and ALA) who were
unaware of the time point at which the biopsy was taken.
Macrophages, granulocytes, and lymphocytes were quantified
using a light microscope at 400 magnification following
standard staining characteristics attributed to each cell type
(Glassman et al. 1981; Can field 1985; Zayas et al. 2011).
Biopsies were divided into adjacent fields (Brown et al. 2011),
with cell types enumerated in each field and subsequently
summed, providing a total count of all leukocytes in the biopsy.
Total counts by JWF and ALA at respective time points were
averaged. Lymphocyte type cannot be distinguished with
haematoxylin and eosin under light microscopy, so the counts
reflect total number of lymphocytes and are not restricted to
T-cells (Brown et al. 2011). Similarly, granulocytes with
eosinophilic granules were not differentiated but instead reflect
the total sum of heterophils and eosinophils.
Temporal replication
As well as determining the temporal response, one of the tenets
of cell-mediated immunity is immunological memory. That is,
subsequent injections of PHA should induce an increased
swelling response when compared with the initial injection
(Tella et al. 2008; Brown et al. 2011). To determine the role of
memory upon subsequent injection with PHA, the 30 animals
not used in the histological examination were reinjected. The
second injection occurred 21 days after the initial injection
when web thickness was assumed to have returned to
preinjection thickness. The second PHA injection was
administered into the opposite toe web of the initial injection
(i.e.
PHA in right toe web first and into left toe web second).
Following primary injections, crocodiles were fitted with
individual tags (National Band and Tag Co., USA) on their first
single-row scute (Isberg et al. 2004) to enable easy identification
for the subsequent injections.
Statistical analysis
Analyses were conducted using restricted maximum likelihood
(REML) in G
ENSTAT (ver. 14, VSN International) with web
thickness at their respective time points serving as the response
variable. In all analyses, Animal was included as a random effect
to account for paired observations between PHA- and PBS-
injected toe webs. Animal size, as indicated by head length (HL;
covariate), time, treatment and a time treatment interaction
were included as fixed effects in all models. The term Replication
was also included for the temporal replication analysis along
with all possible interactions. A serial correlation structure was
accounted for within the model structure for each analysis.
Following histological examination, enumerated leukocytes
(granulocyte, macrophage, or lymphocyte) were analysed using
a generalised linear mixed model (GLMM) with a Poisson
distribution with cell type count used as the response variate
and time, treatment and their interaction used as fixed effects.
Animal was again included as a random effect to account for
paired observations. Reported estimates are presented as back-
transformed means.
Results
Effects of body size on web thickness
Prior to injection, the average thickness of left and right toe
webs were similar (F
1,82
= 0.29, P = 0.593). However, there was
a significant effect of crocodile size (HL) on initial toe webbing
thickness (1.11 10
2
mm mm
–1
; s.e. = 9.76 10
4
; F
1,81
=
129.27, P < 0.001) (Fig. 1), although one animal was removed
from the analysis as its head length (HL) was an extreme outlier.
Consequently, HL was used as a covariate in subsequent analyses
whenever significant.
Dose effects
There was a significant time by dose interaction (F
15,22
= 7.12,
P < 0.001). Using the least significant difference (5% l.s.d.), the
1mgmL
–1
dose was significantly different from the control
(PBS) at 24 and 48 h only, whereas the 2 and 5 mg mL
–1
were
significantly different at all time points with the exception of
2mgmL
–1
at 72 h (Fig. 2). As a result, we chose to conduct all
further examinations using the lower dose of 2 mg mL
–1
(Smits
and Williams 1999). Animal size also had a significant effect
on swelling (F
1,20
= 11.03, P = 0.003).
Temporal profile of PHA-induced swelling
To clarify the time effect on the swelling response, 60 additional
crocodiles were injected, allowing for 68 crocodiles to be
examined when combined with the original eight from the dose
effect trial. However, of these additional 60 crocodiles, 30 (five
Validation of PHA in crocodiles Australian Journal of Zoology C
at each time point) were randomly chosen for biopsies and could
not be remeasured after the biopsy was taken. Therefore, there
were totals of 68, 63, 58, 53, 48 and 43 crocodiles (i.e. the decrease
reflecting animals unavailable for measurement due to biopsy)
available for measurement of toe web thickness at each time
point. PHA-injected toe webs exhibited amplified swelling
compared with PBS-injected webs although this varied over
time (time treatment interaction F
5,55
= 75.44, P < 0.001), with
PHA-induced swelling significantly increased (5% l.s.d.) at 6, 12,
24, 48, and 72 h (Fig. 3). Peak swelling response was observed
12 h after injection for both PHA- and PBS-injected toe webs,
with an increase in thickness of 66.7% (0.45 to 0.75 mm) and
15.6% (0.45 to 0.52 mm), respectively (Fig. 3). As expected,
larger crocodiles showed a greater response (HL: F
1,36
= 90.75,
P < 0.001).
Temporal replication
As PHA is purported to stimulate the cell-mediated response
of the acquired immune system, we would expect increasing
responsiveness upon subsequent stimulation with the same
antigen. Secondary administration had a significant effect on
0.55
0.50
0.45
0.40
0.35
0.30
0.25
54 56 58 60 62 64 66
Head length (mm)
Web thickness (mm)
68 70 72 74
0.60
Fig. 1. Relationship between crocodile size, as indicated by head length (HL, mm) and pretreatment toe web
thickness (mm). Closed and open circles indicate the left and right toe web thicknesses, respectively.
0.75
0.70
0.65
0.60
0.55
0.50
0.45
0.40
0.35
0612 24 48
1 mg mL
–1
2 mg mL
–1
5 mg mL
–1
PBS
72
Web thickness (mm)
Time (h)
Fig. 2. The effects of PBS (dashed black line, open circles) and 1 (squares), 2 (triangles), and 5 mg mL
–1
(solid
circles) PHA on average (s.e.) swelling response (mm) over time (h).
D Australian Journal of Zoology J. W. Finger Jr et al.
swelling response (replication time treatment F
5,693
= 2.26,
P = 0.047). Secondary injection of PHA significantly induced
more swelling than primary injection at all time points except 6 h
(replication time treatment 5% l.s.d.) (Fig. 4). Interestingly,
secondary injection of PBS also induced more swelling than
primary injection, with significant increases observed at 0, 24,
and 48 h (replication time treatment 5% l.s.d.). The results
observed at 0 h with both PBS and PHA may suggest that the
period of 21 days between injections was not sufficient to reduce
swelling back to preinjection levels, as PHA injection may have
long-lasting effects on the immune system (see Sarv and Horak
2009). However, these differences may also reflect a natural
increase in thickness between the two injection periods, as
farmed saltwater crocodiles experience pronounced growth
during the first few years of life (Isberg et al. 2004, 2005).
Secondary responsiveness was not affected by animal size
(F
1,32
= 3.85, P = 0.058).
Histological examination
Examination revealed a significant infiltration of granulocytes
(F
5,25
= 3.00, P = 0.03), lymphocytes (F
5,26
= 3.34, P = 0.018),
and macrophages (F
5,24
= 5.87, P = 0.001) at all time points after
injection (0 h) (approximate 5% l.s.d. = 2 SED) in PHA-
0.75
0.80
0.70
0.65
0.60
0.55
0.50
0.45
Web thickness (mm)
Time (h)
0 6 12 24 48
PBS
PHA
72
Fig. 3. The effects of injection of PHA (solid circles) and PBS (dashed black line, open circles) on average (s.
e.) swelling response (mm) in juvenile saltwater crocodile toe webs over time (h).
0.75
0.80
0.85
0.90
0.70
0.65
0.60
0.55
0.50
0.45
0.40
Time (h)
12 24 48 72
Web thickness (mm)
PBS1
PBS2
PHA1
PHA2
06
Fig. 4. The effects of first and second injections of PHA (open and solid squares, respectively) and PBS (open
and solid circles) on average (s.e.) swelling response (mm) over time (h).
Validation of PHA in crocodiles Australian Journal of Zoology E
50
PBS
PHA
(a)
(b)
(c)
45
40
35
30
25
20
15
10
5
0
30
25
20
15
10
10
20
30
40
50
60
70
80
5
0
0
Average macrophage countAverage lymphocyte countAverage granulocyte count
0
0612 24 48 72
Time (h)
Fig. 5. Changes in average (s.e.) cell counts following administration with PHA (solid circles) and PBS
(open circles): (a) macrophages, (b) lymphocytes and (c) granulocytes.
F Australian Journal of Zoology J. W. Finger Jr et al.
injected webs compared with control webs. Peak infiltration of
macrophages (Fig. 5a) and lymphocytes (Fig. 5b) occurred at 12 h
after injection (Figs 6 and 7), whereas peak granulocyte
infiltration (Fig. 5c) occurred at 6 h. Animal size had no effect on
cellular infiltration (granulocytes: F
1,14
= 0.01, P = 0.937;
lymphocytes: F
5,25
= 0.60, P = 0.447; macrophages: F
1,19
= 0.83,
P = 0.374).
Discussion
Herein we have validated the use of PHA and suggested its use
to measure immune responsiveness in the saltwater crocodile
(Crocodylus porosus). After injection, the augmented swelling
peaked at 12 h, with larger responses observed in larger
animals. Histological examination revealed that primary injection
initially stimulated innate cellular infiltration by first recruiting
granulocytes (peaking at 6 h), followed by peak numbers of
macrophages and lymphocytes at 12 h; however, it is doubtful
that these lymphocytes embody newly formed progeny of
PHA-stimulated lymphocytes this soon after injection (Brown
et al. 2011). However, subsequent administration revealed an
enhanced swelling response, suggesting that PHA induces an
adaptive immune response in juvenile saltwater crocodiles.
These results correspond with what others have observed in
birds (Tella et al. 2008) and anurans (Brown et al. 2011), with
subsequent injections of PHA increasing swelling response.
Analogous to our results, Zimmerman et al.(2010) observed
a peak swelling response to PHA 12 h after injection in red-eared
sliders (Trachemys scripta) and Turmelle et al.(2010) observed
peak swelling response and peak lymphocyte infiltration 12 h
after injection in Brazilian free-tailed bats (Tadarida
brasiliensis). Within Aves, mixed results have been obtained,
with several studies showing peak swelling responses between
12 and 24 h (Grasman 2002). By contrast, Martin et al.(2006)
observed the peak swelling response at 48 h after injection in
house sparrows (Passer domesticus), although the maximum
lymphocyte and heterophil counts were detected after 6 and 12 h,
respectively. In ostriches (Struthio camelus), swelling at 6 h after
injection was not significantly different from that at 24 h (Bonato
et al. 2009), albeit measurements were not taken at 12 h. In cane
toads, Brown et al.(2011) observed both peak lymphocytic
infiltration and peak swelling 24 h after injection, although peak
numbers of granulocytes and macrophages were observed 12 h
after injection, similar to our findings. Interestingly, the greatest
swelling response in another anuran, the southern leopard frog
(Rana sphenocephala), was observed 48 h after injection but
no histological examinations were undertaken to quantify the
cell type infiltration (Venesky et al. 2012). These examples
show the lack of interspecifi
c standardisation in peak swelling
response
and leukocyte infiltration, with such high variability
probably attributable to several factors. As such, direct
comparisons in swelling response may prove unwise (see
Matson et al. 2006), requiring independent validation for the
study species of interest.
Whilst demonstrating the use of this technique as a measure of
immune function in a crocodilian species, the response elicited
may be affected by multiple biotic, such as diet or disease status
(Tschirren et al. 2007; Venesky et al. 2012), and abiotic factors.
As such, results obtained in this study using farmed saltwater
crocodiles raised within temperature-controlled sheds may be
different from using crocodiles obtained from wild habitats,
from other farms with different management regimes, or at
differing ages or seasons (Zimmerman et al. 2010; Schwanz
et al. 2011). Efficacy of ectothermic immune response is
Fig. 6. Photomicrograph (200) of transverse section of PHA-injected web at 12 h after injection. Scale
bar = 50 mm. Examples of representative cell types are indicated by arrowheads for macrophages, dotted arrows
for granulocytes, and solid arrows for lymphocytes.
Validation of PHA in crocodiles Australian Journal of Zoology G
dependent on temperature and, as such, seasonality may affect
swelling response (Zapata et al. 1992; Martin et al. 2008;
Zimmerman et al. 2010). Seasonality (and temperature) may
also affect levels of endogenous sex steroids even in sexually
immature crocodilians (Rooney et al. 2004) and, as sex steroids
affect immune function (Lutton and Callard 2006), variation in
response to PHA challenge may be observed depending on
season of administration. Furthermore, animals in farming
situations as opposed to natural environments may be exposed to
different types of stressors (Elsey et al. 1990). Thus, differences
in swelling response may arise due to corticosterone-mediated
immunosuppression (Martin et al. 2005). Indeed, saltwater
crocodiles housed at higher stocking densities demonstrated
elevated corticosterone, with subsequent increased perturbations
in immune function and increased susceptibility to opportunistic
infection (Turton et al. 1997). Moreover, circadian cycles of
dark and light are also known to affect corticosterone secretion
in crocodilians (Lance and Lauren 1984) and the crocodiles
used herein were housed in temperature-controlled sheds,
which may restrict natural circadian rhythms, possibly affecting
corticosterone levels and, consequently, swelling response.
Furthermore, it is important to emphasise that no one
technique can serve as a quintessential immunological
measure; instead, PHA injection should be coupled with
other immunological assays for better assessment of immune
responsiveness (Norris and Evans 2000; Demas et al. 2011), as
an individual’s responsiveness to one antigen may not be
indicative of its responsiveness to another. In fact, many studies
incorporate measures of multiple immune parameters to aid in
elucidation of immunity in an individual (e.g. Fair et al. 1999;
Schwanz et al. 2011). When interpreting results from PHA
injection, cautionary analysis must be employed (Martin et al.
2006). For example, as primary injections are composed of
both innate and adaptive components, an enhanced swelling
response to primary injection may not be indicative of an
enhanced cell-mediated (adaptive) response (Martin et al. 2006).
In fact, subsequent injections are necessary for evaluation of
the adaptive (acquired) response (Demas et al. 2011 ), as the
adaptive immune response changes (adapts) with repeated
exposures to the same antigen (Abbas et al. 2010). Furthermore,
higher-quality individuals may not necessarily develop a greater
response compared with lower-quality individuals (Vinkler et al.
2012). The type of PHA lectin utilised in experimentation may
also infl
uence the response generated and, thus, confound
results
(see Vinkler et al. 2010). Therefore it is important to be
aware of these and other confounding variables when conducting
experimentation using PHA injection.
PHA administration has been utilised in many disciplines
to understand different variables affecting immunity, including
immunotoxicological (Smits and Williams 1999; Grasman 2002;
Fairbrother et al. 2004; Muller et al. 2005; Markman et al. 2011),
agricultural (Hernandez et al. 2005; Boughton et al. 2007; Bonato
et al. 2009), and ecological studies (Smits et al. 1999; Martin et al.
2006; Tschirren et al. 2007; Brzek and Konarzewski 2007 ). Thus,
we propose similar use to determine immune status in studies
relating to crocodilians. Crocodilians are important components
of their ecosystem, acting as top trophic carnivores, serving as
sentinels of environmental quality (Milnes and Guillette 2008)
and, in some species, modifying ecosystems for the benefitof
other species (Craighead 1968; Magnusson and Taylor 1982).
Furthermore, use of crocodilians is important in several countries,
including Australia (Isberg et al. 2004), where leather products
made from the skins provide employment and economic
incentives for sustainable use. Thus, the utilisation of a technique
Fig. 7. Photomicrograph (200) of PBS injected web 12 h after injection. Scale bar = 50 mm.
H Australian Journal of Zoology J. W. Finger Jr et al.
to measure immune responsiveness, such as PHA administration,
in crocodilians may enable further investigation throughout
several wide-ranging disciplines and provide greater
understanding of the immune systems of these organisms.
Acknowledgements
We thank Porosus Pty Ltd for providing access to crocodiles used in this
study and Berrimah Veterinary Laboratories for processing biopsies and
providing JWF and ALA access to facilities for examining biopsies. This
project was funded by a grant from the Rural Industries Research and
Development Corporation, Australia. All experimental protocols were
approved by the University of Sydney Animal Ethics committee (approval
no.: N00/5-2012/3/5729).
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