Upload
independent
View
2
Download
0
Embed Size (px)
Citation preview
http://vet.sagepub.com/Veterinary Pathology Online
http://vet.sagepub.com/content/early/2014/01/06/0300985813516642The online version of this article can be found at:
DOI: 10.1177/0300985813516642
published online 6 January 2014Vet PatholC. Shilton, G. P. Brown, L. Chambers, S. Benedict, S. Davis, S. Aumann and S. R. Isberg
) in AustraliaCrocodylus porosusPathology of Runting in Farmed Saltwater Crocodiles (
Published by:
http://www.sagepublications.com
On behalf of:
Pathologists.American College of Veterinary Pathologists, European College of Veterinary Pathologists, & the Japanese College of Veterinary
can be found at:Veterinary Pathology OnlineAdditional services and information for
http://vet.sagepub.com/cgi/alertsEmail Alerts:
http://vet.sagepub.com/subscriptionsSubscriptions:
http://www.sagepub.com/journalsReprints.navReprints:
http://www.sagepub.com/journalsPermissions.navPermissions:
What is This?
- Jan 6, 2014OnlineFirst Version of Record >>
at University of Sydney on January 13, 2014vet.sagepub.comDownloaded from at University of Sydney on January 13, 2014vet.sagepub.comDownloaded from
Article
Pathology of Runting in Farmed SaltwaterCrocodiles (Crocodylus porosus) in Australia
C. Shilton1, G. P. Brown2, L. Chambers1, S. Benedict1, S. Davis1,S. Aumann1, and S. R. Isberg2,3
AbstractExtremely poor growth of some individuals within a birth cohort (runting) is a significant problem in crocodile farming. Weconducted a pathological investigation to determine if infectious disease is associated with runting in farmed saltwater crocodiles(Crocodylus porosus) and to look for evidence of other etiologies. In each of 2005 and 2007, 10 normal and 10 runt crocodiles, withan average age of 5.5 months and reared under identical conditions, were sampled. Laboratory testing included postmortem;histological examination of a wide variety of tissues (with quantitation of features that were noted subjectively to be differentbetween groups); hematology; serum biochemistry (total protein, albumin, globulins, total calcium, phosphorus, and iron);bacterial culture of liver and spleen (2005 only); viral culture of liver, thymus, tonsil, and spleen using primary crocodile cell lines(2007 only); and serum corticosterone (2007 only). The only evidence of infectious disease was mild cutaneous poxvirus infectionin 45% of normal and 40% of runt crocodiles and rare intestinal coccidia in 5% of normal and 15% of runt crocodiles. Bacterial andviral culture did not reveal significant differences between the 2 groups. However, runt crocodiles exhibited significant (P < .05)increases in adrenocortical cell cytoplasmic vacuolation and serum corticosterone, decreased production of bone (osteoporosis),and reduced lymphoid populations in the spleen, tonsil, and thymus. Runts also exhibited moderate anemia, hypoalbuminemia, andmild hypophosphatemia. Taken together, these findings suggest an association between runting and a chronic stress response(hyperactivity of the hypothalamic-pituitary-adrenal axis).
Keywordsadrenocortical hyperplasia, crocodile, farmed, histopathology, inanition, lymphoid atrophy, runting, stress
In the crocodilian farming industry, extremely poor growth of
some hatchlings compared with their birth cohort (runting) is a
major problem worldwide, affecting up to 30% of ani-
mals.3,21,32,33,48,49,55,58 Runting is a cause of major economic
loss, in an industry where juvenile growth and survival are
important economic factors.33
The cause of runting in farmed crocodiles is unknown.
Although various possibilities have been proposed, none have
been thoroughly investigated. Proposed factors associated with
runting include egg incubation conditions;38,69 poor yolk
absorption posthatching;22,32 inappropriate diet or problems
with diet assimilation;11,22,26,55 posthatching environment,
including temperature, stocking density, and behavior of con-
specifics;26,32,39,48,58 and failure to adapt to the captive envi-
ronment.3,64 Poor growth has been found to be clutch related
in that some clutches produce relatively high numbers of
runts.26,33,48,58 However, future crocodile runts are not identifi-
able at hatching, since body size at hatching is a poor predictor
of posthatching growth.26,34,38,69
There is limited information on diseases and pathology exhib-
ited by runt crocodiles. A few studies mention bacteremia,
mycotic dermatitis,64 hepatitis,22,64 atrophy of the liver and
intestine and ascites,21,32 hepatic lipidosis,21,49 or pancreatic
atrophy.22 There has been no in-depth pathological investigation
comparing runt crocodiles with their normal counterparts from
the same cohort and raised under identical conditions. The pur-
pose of this study was to conduct a direct and thorough patholo-
gical comparison between runt and normal crocodiles to rule out
the involvement of infectious disease and to survey for other
lesions or conditions that could suggest a cause for the runting.
Materials and Methods
The study location was a large saltwater crocodile (Crocodylus
porosus) farm 40 km south of Darwin, Australia. The weather
1Berrimah Veterinary Laboratories, Department of Primary Industry and
Fisheries, Northern Territory Government, Berrimah, Northern Territory,
Australia2The University of Sydney, Sydney, New South Wales, Australia3Porosus Pty. Ltd., Noonamah, Northern Territory, Australia
Corresponding Author:
C. Shilton, Berrimah Veterinary Laboratories, Department of Primary Industry
and Fisheries, Northern Territory Government, GPO Box 3000, Darwin, NT
0801, Australia.
Email: [email protected]
Veterinary Pathology1-13ª The Author(s) 2014Reprints and permission:sagepub.com/journalsPermissions.navDOI: 10.1177/0300985813516642vet.sagepub.com
Veterinary Pathology OnlineFirst, published on January 6, 2014 as doi:10.1177/0300985813516642
at University of Sydney on January 13, 2014vet.sagepub.comDownloaded from
in this region is tropical, with a ‘‘wet’’ season having high
humidity and monsoonal rains, from November to April, and
a ‘‘dry’’ season, characterized by low humidity and no rain,
from May to October. The daily maximum temperature is
33�C during both seasons, but the average daily minimum is
24�C in the wet season compared with 18�C in the dry season.
Saltwater crocodiles nest in northern Australia from late
October to April, corresponding to a hatching period from Feb-
ruary to June. Eggs were obtained both from the wild (13 runt
and 10 normal crocodiles; average estimated embryo age at
collection was 21 days) and from captive females (7 runt and
10 normal crocodiles; average estimated embryo age was 6
days). At the farm, eggs were incubated at 32�C and 99% to
100% humidity until hatching. Each hatchling was identified
to clutch of origin by scute cutting.30 The hatchlings used in
this study originated from 35 clutches (1 from each clutch,
except for 5 clutches from which 2 study animals originated
from each clutch).
Runt crocodiles were not identifiable at hatching. On croco-
dile farms, normal management practice involves ‘‘grading’’
animals to ensure minimal size variation within each pen to
reduce intraspecific aggression and maximize equal opportu-
nity to access food. Grading on our study farm commences
when disparity in growth starts to become apparent, at approx-
imately 2 months of age, and is performed as required but at
least every month within each pen. Thus, through systematic
grading, the runt crocodiles used in this study were continually
maintained in pens of similar sized animals and were not
selected from pens of larger animals whereby exogenous fac-
tors such as competition for food or thermoregulatory ability
could be a cause of the runting. Runt crocodiles were reared
under identical husbandry conditions to normal crocodiles of
the same birth cohort, with pens containing crocodiles of either
of the 2 groups interspersed in the same shed. Stocking density
in all pens was approximately 14 animals/m2. Within each
completely enclosed shed, pens were shaded, of concrete
construction, with shallow water (approx. 70% of pen area;
30–50 cm deep) at one end with a gradual ascent to a dry
feed-deck (approx. 30% of pen area) at the other end. Hide-
boards were provided within each pen. Water temperature in
the pens was kept at approximately 32�C. The diet consisted
of finely minced red meat (horse or buffalo) fortified with
2% vitamin-mineral mix that included vitamin D3 (25 000
IU/kg; Monsoon Crocodile Premix, Darwin, Australia) and
1.5% calcium carbonate. The animals were fed, in excess, 5
times per week until they were 3 months of age when they were
fed every second day. Food was dispersed evenly over the land
area in the afternoon and left overnight. Crocodiles were not
observed for individual food intake, and measurement of
remaining food as an indicator of how much the crocodiles in
each pen were eating was impractical, due to dispersion of the
food into the water by the crocodiles. Crocodiles were fasted
for 48 hours prior to sampling. Ten runt and 10 normal croco-
diles were sampled in each of 2 study years: on November 8 to
11, 2005 (case Nos. 1–20) and on July 10 and 12, 2007 (case
Nos. 21–40). Sampling of animals was done when the grading
process had resulted in entire pens containing subjectively
obvious runt crocodiles, which was at approximately 7 and 5
months of age in 2005 and 2007, respectively. The only differ-
ence between the ‘‘runt’’ and ‘‘normal’’ animals was their body
size and condition; all animals used in the study were subjec-
tively judged to be bright, responsive, and active.
The initial suite of laboratory testing for 2005 samples was
designed to be relatively broad to maximize the chance of
detection of any disease or condition that could be related to
runting. The purpose of the second sample in 2007 was to sub-
stantiate potentially significant findings from the initial 2005
sample, and therefore testing in 2007 differed slightly and was
generally not as broad (specific differences in testing between
the years is detailed in relevant sections below).
Necropsy and Histopathology
Following blood sampling (see below), crocodiles were eutha-
nized by overdose (80 mg/kg) of pentobarbitone sodium
injected into the dorsal tail vein (Lethabarb euthanasia
injection, 325 mg/ml; Virbac Animal Health, Milperra, NSW,
Australia). A full necropsy was performed on all crocodiles. In
2005, liver weights were taken on all crocodiles (not repeated
in 2007 due to no difference between groups in 2005; see
Results).
Tissues were fixed in 10% neutral buffered formalin,
processed in standard fashion for histological examination, and
5-mm sections stained with hematoxylin and eosin (HE). Sec-
tions of bone were decalcified in 10% neutral buffered formalin
with 9% formic acid prior to processing. Sections trimmed for
histological examination were standardized with respect to
location in, and orientation of, the organ/tissue. Organs/tissues
examined only in 2005 were heart, lung, trachea, kidney, gall-
bladder, esophagus, stomach, coelomic fat body, thyroid gland,
skin, femorotibial joint, spinal cord, brain, and eye. These tissues
did not display notable differences between runt and normal cro-
codile groups (see Results) and therefore were not examined
in the second set of samples taken in 2007. Organs/tissues
examined in both years were pituitary gland, adrenal gland,
spleen, thymus, tonsil, bone (mid-sagittal section of proximal
tibial metaphysis, including bone marrow), skeletal muscle,
liver, pancreas, duodenum, jejunum, colon, and gonad. Histo-
logical examination of the gonad was used to determine sex.
Sections of parathyroid gland were available for examination
in 4 normal and 9 runt crocodiles. Perls’s stain for ferric iron
was used to identify the nature of the green-brown pigment
present in splenic macrophages.9
All histology slides were examined by one person (C.S.)
unaware as to whether the tissues were from a normal or runt
crocodile. Following initial screening of all slides, aspects of
tissues that subjectively seemed to differ among crocodiles
were quantified. Features that were scored as 0 (none), 1 (mild),
2 (moderate), or 3 (marked) were degree of cytoplasmic vacuo-
lation of adrenocortical (interrenal) cells and hepatocytes,
amount of zymogen in pancreatic acinar cells, and amount of
globular green-brown pigment in splenic macrophages. Bone
2 Veterinary Pathology
at University of Sydney on January 13, 2014vet.sagepub.comDownloaded from
marrow total percent cellularity and the relative proportion of
erythroid cells (compared with myeloid cells) were subjec-
tively estimated.
Digital images were taken for the following histological
features (MicroPublisher 3.3; Q Imaging, Surrey, BC, Canada)
and the feature measured using a program calibrated to the
magnification of the microscope (analySIS Five; Soft Imaging
System GmbH, Munster, Germany). Skeletal muscle fiber
width was measured by taking the average width of 10 fibers
in the same region of cranial thigh muscle in each crocodile.
In 2005, the muscle was sectioned transversely across the
fibers, while in 2007, the muscle was sectioned longitudinally.
The amount of the primary spongiosa in the proximal tibial
growth zone was quantified by determining the proportion of
the total width of mineralizing cartilaginous trabeculae imme-
diately beneath the growth zone compared with the width of the
medulla at the same level. The area of periarteriolar lymphoid
sheaths was measured by taking the area of each of 10 lym-
phoid sheaths, minus the area of the arteriole each surrounded,
and calculating an average. The 10 sheaths were the first 10
discrete sheaths encountered in a transect of the histological
section starting from the capsule moving toward the center at
the greatest diameter of a section taken of the mid-region of the
spleen. The amount of lymphoid tissue in the tonsils was
measured as a proportion of the area of the tonsil occupied
by lymphocytes compared with the total area of the folds of the
tonsil observed at low power in a standard complete transverse
section taken from the mid-tonsil. Thymus tissue was quanti-
fied by measuring the total area of thymus lobes in a transverse
section containing all the tissues bound by the mediastinum at
the level of the proximal primary bronchi. To take into account
the smaller overall size of tissues of runt crocodiles, the area of
the thymus lobes is presented as a proportion of the area of the
adjacent primary bronchus.
Ancillary Diagnostic Testing
Blood was sampled from the occipital venous sinus and the
initial 0.5 ml of blood placed into EDTA anticoagulant for hema-
tology, with the remainder placed into serum separator gel tubes
(BD Vacutainer; Becton Dickinson, Franklin Lakes, NJ). EDTA
was used as the anticoagulant since in our experience, it does not
cause hemolysis in saltwater crocodiles and is not potentially
associated with leukocyte and platelet clumping, and thus inac-
curate cell counts, as has been reported with lithium heparin
anticoagulated blood.5 Blood samples were obtained 2 to 4 hours
after removal from the pen for crocodiles in 2005 and within 3
minutes of removal from the pen, between 0800 and 0900 in the
morning for crocodiles in 2007. The strict timing of blood sam-
pling for 2007 was to minimize any effects of circadian rhythm
or acute handling stress on serum corticosterone level.23,37,42,44
Serum corticosterone was measured in the 2007 samples in an
effort to expand on the apparent histological difference noted
in adrenal glands between the runt and normal crocodile groups
noted in 2005 (see Results). Frozen serum was not available to
retrospectively test the 2005 crocodiles. Serum corticosterone
was measured using a high-sensitivity enzyme immunoassay
according to kit directions (Corticosterone HS EIA; Immuno-
diagnostics Systems Ltd, Boldon, Tyne & Wear, UK). Values
that exceeded the 20-ng/ml upper limit of kit accuracy were set
at this limit for statistical analysis.
Selected clinical pathological parameters were determined
to assist interpretation of aspects of the gross and/or histo-
pathology. Hematology included total and differential white
blood cell counts conducted using a hemocytometer with an
eosinophil Unopette system (Becton-Dickinson, Rutherford,
NJ) and blood smear examination according to standard reptile
protocol.6,10 Packed cell volume was determined by centrifuga-
tion of blood in microhematocrit tubes. Serum biochemical
parameters were determined on an automated analyzer (Kone-
lab 20; Thermo Electron, Victoria, Australia). Parameters mea-
sured (followed in parentheses by a brief analytical basis for the
measurement provided by Thermo Electron) were total protein
(biuret method), albumin (bromcresol green dye binding
method), globulins (by subtraction of albumin from total
protein), total calcium (reaction with the metallochromogen
Arsenazo III), inorganic phosphorus (formation of phosphomo-
lybdate and subsequent reduction to molybdenum blue), and
iron (hydroxylamine hydrochloride reduction and subsequent
reaction with liquid ferrozine).
Bacterial culture of liver and spleen was performed on all
crocodiles in 2005 but not in 2007 because of a lack of signif-
icant bacteriological differences between the 2 groups in 2005
(see Results). Culture was performed on samples obtained
aseptically during necropsy. To collect samples aseptically, the
skin of the crocodile was cleaned with 100% ethanol and
incised using scalpel and forceps that had been sterilized by
dipping them in 100% ethanol and flaming them over a Bunsen
burner. Following the skin incision and reflection of the skin,
the instruments were again sterilized and the coelom opened.
Instruments were sterilized a third time prior to sampling tis-
sue. In addition, yolk was cultured from case No. 1 (runt) and
case No. 11 (normal) with enlarged internal yolk sac remnants
and a swab of the subcutis from the swollen forelimb of case
No. 16 (normal). For these samples, the tissue was entered
aseptically as described above and sampled using sterile swabs.
Bacterial culture was performed using standard veterinary bac-
teriology phenotypic and biochemical techniques. Briefly,
samples were homogenized and plated onto sheep blood agar
(Oxoid Australia, Thebarton, Australia) and MacConkey agar
(Oxoid Australia, Thebarton, Australia) and incubated at
35�C for 48 hours. The bacterial isolates were initially charac-
terized using Gram’s stain, colony morphology, and relevant
preliminary tests, including oxidase and catalase, and then the
appropriate commercial kits were used for speciation (api 20
Strep, api Coryne, bioMerieux, Marcy-l’Etoile, France;
Microbact Gram-Negative Identification System, Oxoid Ltd,
Basingstoke, Hants, UK).
Virus isolation was attempted on liver, thymus, tonsil, and
spleen from all crocodiles in 2007 using primary crocodile liver
and kidney cell lines developed at Berrimah Veterinary Labora-
tories.50 Culture was not performed on samples from 2005 since
Shilton et al 3
at University of Sydney on January 13, 2014vet.sagepub.comDownloaded from
the crocodile cell lines had not been developed yet and frozen
stored samples from 2005 were no longer available. In 2007,
samples were stored at –70�C until processing. Briefly, tissue
samples were homogenized, clarified by centrifugation, and the
supernatant filtered through a 0.45-mm filter. The filtered super-
natant was then inoculated into 25-cm2 flasks containing conflu-
ent primary cell line monolayers. Each sample was inoculated
into 2 different cell lines. The flasks were incubated at 28�C and
examined for viral growth every 3 days for 21 days. The cultures
were then passaged into fresh flasks for another 21 days and
examined every 3 days. This process was repeated a final time,
and if no growth was observed, the culture was deemed negative.
Viral growth was recognized by a cytopathic effect characterized
by loss of confluence of the cell monolayer, rounding up of cells,
and, in some cases, syncytia formation.50 The virus isolations
were part of a larger study investigating the viral flora of salt-
water crocodiles,50 and further characterization of isolated
viruses was beyond the scope of this study.
Statistical Analyses
To generate a single continuous variable that described overall
body size relative to age (a growth index), we first conducted
a principal component analysis to combine 2 measures of body
length (snout-vent length and vent-tail tip length) with body
weight. The resultant first principal component (PC1) incorpo-
rated 99% of the variation in the 3 measures and was used as
a single measure of overall body size. This PC1 body size
measure was then corrected for crocodile age by calculating the
residuals from a linear regression of PC1 on age. These residual
values form the growth index, as they provide a composite mea-
sure of how large each crocodile was for its age. There was no
overlap in this value for crocodiles assigned to the normal and
runt groups, verifying its validity in distinguishing runts from
normal crocodiles (Table 1). This continuous variable was used
as an explanatory covariate in all statistical analyses. However,
for ease of interpretation, summary statistics of basic measure-
ments are presented as 2 groups: normal and runt crocodiles.
Response variables that were measured as ordinal categories
(eg, degree of cytoplasmic vacuolation of adrenocortical cells)
were analyzed using logistic regression with year and growth
index as explanatory variables. Measurements made on a
continuous scale (eg, hematology parameters, size of splenic
periarteriolar lymphoid cuffs) were analyzed using multiple
regression, with year and growth index as explanatory
variables.
Table 1. Body Size, Age, Sex, Hematological, and Biochemical Parameters for All Normal and Runt Crocodiles in 2005 (Case Nos. 1–20) and2007 (Case Nos. 21–40).a
Parameter
Normal Crocodile Runt Crocodile
2005 2007 2005 2007
Total length, cm*# 56.0 + 1.5 (47.0, 64.0) 48.4 + 1.2 (43.0, 55.0) 35.4 + 0.4 (33.0, 37.5) 34.4 + 0.5 (32.5, 37.5)Body weight, g*# 460 + 40 (251, 743) 286 + 24 (185, 393) 74 + 8 (47, 110) 64 + 2 (52, 74)Age, d# 225 + 5 (208, 254) 118 + 1 (115, 121) 197 + 12 (136, 256) 125 + 4 (114, 142)Growth index,*b 0.9 + 0.2 (–0.1, 2.0) 0.8 + 0.1 (0.2, 1.4) –1.1 + 0.1 (–1.3, –0.7) –0.6 + 0.03 (–0.7, –0.5)Sex, male, female, unknown#c 8, 0, 2 8, 2, 0 9, 1, 0 6, 4, 0Hematology
Total white blood cell count,�109/L#
9.4 + 1.4 (3.1, 17.6) 12.1 + 1.0 (4.1, 15.9) 6.6 + 0.7 (3.1, 10.0) 13.1 + 2.0 (4.5, 21.6)
Heterophils, �109/L 4.1 + 0.8 (0.3, 9.1) 3.4 + 0.2 (2.0, 4.8) 3.4 + 0.3 (2.1, 5.2) 3.2 + 0.3 (2.1, 5.4)Lymphocytes, �109/L# 4.0 + 1.0 (1.0, 12.0) 7.5 + 0.7 (1.9, 9.7) 2.5 + 0.5 (0.9, 4.8) 9.2 + 1.8 (1.1, 17.3)Monocytes, �109/L 1.1 + 0.3 (0.3, 3.6) 0.9 + 0.2 (0.1, 2.1) 0.7 + 0.2 (0.1, 1.4) 0.7 + 0.2 (0.2, 1.7)Eosinophils, �109/L 0.1 + 0.04 (0, 0.4) 0.1 + 0.06 (0, 0.4) 0.1 + 0.04 (0, 0.5) 0.01 + 0.01 (0, 0.1)Packed cell volume, L/L*# 0.23 + 0.004 (0.2, 0.24) 0.18 + 0.002 (0.17, 0.19) 0.15 + 0.01 (0.09, 0.19) 0.12 + 0.01 (0.1, 0.14)
Serum biochemistryTotal protein, g/L*# 43.9 + 1.9 (31, 54) 38.7 + 1.1 (33, 43) 32.5 + 1.3 (27, 39) 32.2 + 1.2 (25, 38)Albumin, g/L*# 18.6 + 0.4 (16, 20) 16.5 + 0.4 (15, 18) 11.6 + 0.7 (8, 14) 10.1 + 0.5 (8, 13)Globulins, g/L 25.7 + 1.6 (16, 35) 22.2 + 1.0 (17, 27) 20.8 + 0.6 (18, 24) 21.9 + 1.2 (14, 27)Total calcium, mmol/L*#d 2.8 + 0.05 (2.5, 3.0) 2.7 + 0.05 (2.4, 2.9) 2.5 + 0.04 (2.3, 2.7) 2.3 + 0.02 (2.2, 2.5)Phosphorus, mmol/L* 1.6 + 0.1 (0.9, 2.3) 1.5 + 0.1 (1.3, 1.8) 1.1 + 0.1 (0.9, 1.5) 1.2 + 0.3 (0.7, 4.1)Calcium/phosphorus ratio* 1.8 + 0.2 (1.2, 3) 1.8 + 0.1 (1.5, 2.2) 2.3 + 0.1 (1.7, 2.7) 2.5 + 0.2 (0.6, 3.2)Iron, mmol/L# 11.9 + 4.5 (1, 39) 1.8 + 0.4 (1, 5) 3.9 + 0.8 (1, 7) 1.4 + 0.3 (1, 4)
aSummary statistics are presented by group, although statistics were performed using the growth index as the independent variable with year as a covariate. Exceptfor sex, values are mean + SE with minimum and maximum in parentheses. Statistically significant differences (at least P < .05 level) between runt and normalgroups are signified with an asterisk (*); differences between years of the project are signified with a hash symbol (#). All differences between groups were observedin both years and vice versa. N ¼ 10 for normal and runt groups in each year of the study.bGrowth index is a summary statistic indicating how well grown a crocodile is for its age. It was generated by combining snout-vent length, vent-tail tip length, andbody weight into 1 principal component to indicate body size, then correcting this value for body age by linear regression and using the resultant residual values toform the growth index.cThere were significantly more females in 2007 in both groups.dTotal calcium was not significantly different between the runt and normal crocodile groups when albumin level was controlled for statistically.
4 Veterinary Pathology
at University of Sydney on January 13, 2014vet.sagepub.comDownloaded from
Since 40% to 45% of total calcium is transported in serum
bound to protein (principally albumin), serum albumin level
may influence total calcium level.1,51 To correct for this, we
regressed total calcium on albumin measures. The residual
values from this regression were then compared between the
runt and normal crocodiles using analysis of covariance.
Results
Necropsy
The runt and normal crocodile groups differed significantly in
total length and body weight in both years, with runt crocodiles
being, on average, 33% shorter and 82% lighter than normal
crocodiles (Fig. 1, Table 1). There was no significant difference
in the ages of runt vs normal crocodiles; however, both groups
were older in 2005. The growth index differed significantly
between the runt and normal crocodile groups, with no overlap
in range, confirming significantly smaller body size for age
(ie, poorer growth) in runt crocodiles. Overall, there was no dif-
ference in sex ratio between the 2 groups, but in 2007, there
were more females in both groups than there had been in
2005 (Table 1). There was no significant difference in the ori-
gin of the eggs (wild vs captive nest) between the 2 groups.
Visually, runt crocodiles exhibited reduced muscling, pro-
minence of bony protuberances, reduced amount of adipose tis-
sue in the base of the tail, and markedly reduced size of the
coelomic fat body (which was not grossly visible in many of
the runts). These subjective necropsy findings indicate poor
body condition in runt compared with normal crocodiles. Com-
pared with normal crocodiles, gallbladders of runt crocodiles
were relatively large and distended with bile. Bones were sub-
jectively judged to be of comparable strength between the 2
groups. Grossly, lobes of the thymus were generally unapparent
Figure 1. Normal crocodile Nos. 21 to 25 (left) and runt crocodile Nos. 26 to 30 (right), sampled July 10, 2007.
Shilton et al 5
at University of Sydney on January 13, 2014vet.sagepub.comDownloaded from
in runt crocodiles while they were usually obvious in normal
crocodiles. The tonsils of runt crocodiles had less prominent
folds than those of normal crocodiles. Case No. 1 (runt) and
case No. 11 (normal) had 1-cm diameter yolk sac remnants pro-
truding from the outer wall of the mid-jejunum. The remainder
of the crocodiles either had a small (1–2 mm diameter) yolk sac
remnant or the remnant was not grossly appreciable. One nor-
mal crocodile (case No. 16) had moderate diffuse enlargement
and edema involving 1 forelimb. Except for small fecal pellets
in the rectum, the gastrointestinal tracts were largely empty in
both groups. There were rare to occasional 0.3- to 1.0-mm
round white cutaneous foci, typical of mild superficial poxvirus
lesions, in case Nos. 2, 6, 13, 14, 27, 29, 30, and 32 (8 runt cro-
codiles) and case Nos. 5, 9 to 12, 16, and 38 to 40 (9 normal
crocodiles). Liver weight as a percentage of body weight aver-
aged 2.4% in both the runt and normal crocodile groups.
Histopathology
Except for features that are expanded upon below, there were
no differences in histological appearance of organs/tissues
among the crocodiles. The significance of findings between
normal and runt crocodiles applies to both years of the project
unless otherwise stated.
There were rare sporulated coccidia oocysts in the mucosal
epithelium of the jejunum and/or colon in case Nos. 2, 6, and 15
(runts) and case No. 25 (normal). Histologically, the poxvirus
lesions appeared as discrete slightly raised foci of markedly
hypertrophic epithelium containing large eosinophilic intracy-
toplasmic inclusion bodies, typical of mild crocodile poxvirus
lesions.33,35 Histologically, the single swollen leg present in
case No. 16 (normal) exhibited marked subcutaneous and intra-
muscular edema, associated moderate heterophil and macro-
phage infiltration, and numerous macrophages containing
abundant intracytoplasmic bacterial cocci.
Statistics for histological features that were quantified are
presented in Table 2. Degree of vacuolation of adrenocortical
cells was significantly greater in runt compared with normal
crocodiles and slightly greater in both groups in 2007 (Figs.
2, 3). The degree of hepatocyte vacuolation was slightly but
significantly less in runt crocodiles and less in both groups in
2005. The degree of pancreatic zymogen was significantly
decreased in runt crocodiles. Skeletal muscle fiber width was
significantly less in runt crocodiles compared with normal cro-
codiles in both years. Muscle fiber width was significantly less
in both groups in 2007, presumably artifactually, due to the
muscle being sectioned longitudinally in 2007, compared with
transversely in 2005. Bone marrow cellularity was reduced in
runt compared with normal crocodiles with no difference in the
proportion of erythroid cells between the 2 groups. There was
an increase in the proportion of erythroid cells in the bone mar-
row in both groups in 2005.
Proximal tibial bone growth zones differed subjectively in
histological appearance between runt and normal crocodiles.
While the growth zones of runt crocodiles retained all normal
histological components with no additional lesions, runts had
generally narrower and less distinct zones of proliferating,
maturing, and hypertrophic chondrocytes and decreased
production of primary spongiosa (Figs. 4, 5). Quantitation of
the amount of primary spongiosa confirmed decreased produc-
tion of bone at the growth zone in runt compared with normal
crocodiles (Table 2).
Runt crocodiles had significantly reduced lymphoid popula-
tions compared with normal crocodiles in all tissues in which
this was evaluated, including, spleen (Figs. 6, 7), tonsil, and
thymus (Figs. 8, 9). Tonsils had a greater lymphoid component
in both groups in 2005. The amount of green-brown pigment in
splenic macrophages was greater in runt compared with normal
crocodiles. The majority of this pigment was strongly positive
with Perls’s stain for ferric iron (Fig. 7, inset).
Ancillary Testing
In 2007 (testing was not performed in 2005), mean serum cor-
ticosterone levels were significantly higher in runt (mean [SE],
17.1 [1.1] ng/ml; range, 10.2–20) compared with normal croco-
diles (mean [SE], 9.4 [2.3] ng/ml; range, 0.5–20).
Hematologically, there were no significant differences
between runt and normal crocodiles in white blood cell para-
meters, although both groups had significantly higher total
white blood cell and lymphocyte counts in 2007. Mean packed
cell volume was significantly lower in runt compared with nor-
mal crocodiles and significantly lower in both groups in 2007
(Table 1). Examination of blood smears did not reveal evidence
of hemoparasites in crocodiles in either group. There were low
to moderate numbers of polychromatophilic red blood cells in
peripheral blood smears in 11 runt and 17 normal crocodiles.
Serum biochemistry (Table 1) revealed significantly lower
total protein and albumin in runt compared with normal croco-
diles in both years, and these parameters were higher in both
groups in 2005. Serum total calcium and phosphorus were sig-
nificantly lower in runt compared with normal crocodiles, with
total calcium being slightly but significantly higher in all cro-
codiles in 2005. However, when the residuals of a regression
of total calcium vs albumin were compared between the runt
and normal crocodile groups, there was no significant differ-
ence, indicating that the difference in total calcium between the
2 groups was attributable to their differing serum albumin lev-
els. The relatively greater decrease in phosphorus in runt croco-
diles compared with total calcium resulted in significantly
higher total calcium to phosphorus ratio in runt compared with
normal crocodiles (Table 1). There were no statistically signif-
icant differences between runt and normal crocodiles in serum
iron, although this parameter exhibited substantial variation
within the groups and was on average higher in both groups
in 2005.
Bacterial culture results in 2005 were negative except as
follows: Streptococcus agalactiae was isolated in 3 normal cro-
codiles (from the liver and spleen of case No. 4, the liver of
case No. 8, and the swollen forelimb of case No. 16). Edward-
siella tarda was isolated from the enlarged yolk sac remnant of
case No. 11 (normal). Corynebacterium sp was isolated from
6 Veterinary Pathology
at University of Sydney on January 13, 2014vet.sagepub.comDownloaded from
Tab
le2.
His
tolo
gica
lFe
ature
sQ
uan
tifie
dfo
rA
llN
orm
alan
dR
unt
Cro
codile
sin
2005
(Cas
eN
os.
1–20)
and
2007
(Cas
eN
os.
21–41).
a
Feat
ure
Norm
alC
roco
dile
Runt
Cro
codile
2005
2007
2005
2007
Adre
noco
rtic
alce
llva
cuola
tion*#
b1.0
+0.0
(1,1)
1.1
+0.1
(1,2)
2.0
+0.2
(1,3)
2.2
+0.1
(2,3)
Skel
etal
musc
lefib
erw
idth
,mm
*#31.3
+1.3
(25.6
,40.1
)11.3
+0.3
(9.4
,12.9
)12.2
+0.9
(8.9
,17.3
)6.8
+0.3
(5.2
,8.6
)H
epat
ocy
teva
cuola
tion*#
b1.7
+0.2
(1,2)
2.0
+0.0
(2,2)
1.3
+0.2
(0,2)
1.9
+0.1
(1,2)
Pan
crea
tic
zym
oge
n*b
3.0
+0.0
(3,3)
3.0
+0.0
(3,3)
2.0
+0.3
(1,2)
2.9
+0.1
(2,3)
Sple
nic
mac
rophag
egr
een-b
row
npig
men
t*b
1.0
+0.2
(0,2)
0.9
+0.3
(0,2)
2.5
+0.2
(2,3)
2.9
+0.2
(2,3)
Bone
mar
row
per
cent
cellu
lari
ty*
46+
2(4
0,60)
53+
2(5
0,60)
32+
4(2
0,50)
25+
3(1
5,40)
Bone
mar
row
eryt
hro
idpro
port
ion
#0.5
1+
0.0
3(0
.33,0.7
5)
0.4
5+
0.0
3(0
.33,0.5
0)
0.5
3+
0.0
2(0
.50,0.6
6)
0.3
9+
0.0
3(0
.25,0.5
0)
Pri
mar
ysp
ongi
osa
pro
port
ion
ofm
edulla
ryw
idth
intibia
lgr
ow
thzo
ne*
#0.4
9+
0.0
2(0
.37,0.5
6)
0.4
1+
0.0
2(0
.31,0.5
2)
0.2
8+
0.0
3(0
.16,0.4
3)
0.2
5+
0.0
2(0
.17,0.3
2)
Sple
nic
peri
arte
riola
rly
mph
oid
cuff
mea
nar
ea,
mm2*
19
078+
1560
(11
507,26
052)
15
952+
2114
(9328,32
816)
9736+
1431
(2763,16
755)
11
727+
1220
(8177,20
441)
Tonsi
lar
eapro
port
ion
popula
ted
by
lym
phocy
tes*
#0.6
7+
0.0
3(0
.52,0.8
2)
0.4
4+
0.0
4(0
.23,0.6
5)
0.2
9+
0.0
7(0
.00,0.6
6)
0.2
1+
0.0
6(0
.00,0.4
9)
Thym
us
lobe
area
aspro
port
ion
ofpri
mar
ybro
nch
us
area
*3.6
+0.6
(1.8
,7.2
)3.7
+0.7
(1.4
,7.7
)0.6
+0.3
(0.1
,2.9
)0.5
+0.1
(0.0
5,1.2
)
a Sum
mar
yst
atis
tics
are
pre
sente
dby
group,a
lthough
stat
istics
wer
eper
form
edusi
ng
agr
ow
thin
dex
asth
ein
dep
enden
tva
riab
lew
ith
year
asa
cova
riat
e.V
alues
are
mea
n+
SEw
ith
min
imum
and
max
imum
inpar
enth
eses
.St
atis
tica
llysi
gnifi
cantdiff
eren
ces
(P<
.05
leve
l)bet
wee
nnorm
alan
dru
ntgr
oups
are
sign
ified
with
anas
teri
sk(*
);diff
eren
ces
bet
wee
nye
ars
oft
he
pro
ject
are
sign
ified
with
ahas
hsy
mbol(
#).
All
diff
eren
ces
bet
wee
nye
ars
wer
eobse
rved
inboth
norm
alan
dru
nt
groups
and
vice
vers
a.N¼
10
for
norm
alan
dru
nt
groups
inea
chye
arofth
est
udy.
bFe
ature
was
grad
edas
0(n
one)
,1
(mild
),2
(moder
ate)
,or
3(m
arke
d).
c Gro
wth
index
isa
sum
mar
yst
atis
tic
indic
atin
ghow
wel
lgro
wn
acr
oco
dile
isfo
rits
age.
Itw
asge
ner
ated
by
com
bin
ing
snout-
vent
lengt
h,v
ent-
tail
tip
lengt
h,a
nd
body
wei
ght
into
1pri
nci
pal
com
ponen
tto
indic
ate
body
size
,th
enco
rrec
ting
this
valu
efo
rbody
age
by
linea
rre
gres
sion
and
usi
ng
the
resu
ltan
tre
sidual
valu
esto
form
the
grow
thin
dex
.
7 at University of Sydney on January 13, 2014vet.sagepub.comDownloaded from
the liver and spleen of case No. 14 (runt) and Morganella
morganii from the liver and Salmonella sp from the enlarged
yolk sac remnant in case No. 1 (runt).
Cytopathic effect in viral culture suggestive of viral growth
was observed in 2007 from the liver and tonsil of case No. 22
(normal) and from the tonsil of case Nos. 21, 24, and 25 (nor-
mal). No cytopathic effect in viral culture was detected from
runt crocodiles.
Discussion
Runt crocodiles in this study exhibited markedly smaller body
size and weight for their age compared with normal crocodiles,
indicating very poor growth as noted in other stud-
ies.3,21,32,48,49,55,58 In addition, multiple parameters indicated
inanition in runt crocodiles, including their moderately
decreased serum albumin, smaller skeletal muscle fiber width,
and decreased pancreatic zymogen. Liver weight as a percent-
age of body weight was the same in runt and normal crocodiles,
suggesting that a significant degree of hepatic atrophy was not
present in runt crocodiles. Histologically, runt crocodiles had
slightly decreased hepatocyte cytoplasmic vacuolation com-
pared with normal crocodiles. In reptiles, mild to moderate
hepatocyte vacuolation, usually due to lipid, is commonly pres-
ent in reptiles in good body condition and is considered within
physiological normal, rather than a degenerative change.30,59
Thus, the mildly decreased hepatocyte vacuolation in runt com-
pared with normal crocodiles likely reflects their generalized
paucity of body fat and is another parameter supporting inani-
tion in the runt crocodiles.
Runt crocodiles had significantly lower packed cell volumes
than normal crocodiles. The anemia in runts was nonregenera-
tive, being unaccompanied by evidence of erythroid hyperpla-
sia in bone marrow or evidence of regeneration in peripheral
Figure 2. Adrenal gland; normal crocodile (case No. 36). Mild vacuolation of the corticosterone-secreting cells of the interrenal cords (deli-neated by double-headed arrow). Hematoxylin and eosin (HE). Figure 3. Adrenal gland; runt crocodile (case No. 32). Marked vacuolation of thecorticosterone-secreting cells of the interrenal cords (delineated by double-headed arrow). HE. Figure 4. Proximal tibial growth zone; normalcrocodile (case No. 5). Note layer of proliferating chondrocytes (P) and abundant primary spongiosa (PS). Decalcified, HE. Figure 5. Proximaltibial growth zone; runt crocodile (case No. 3). Note indistinct, poorly cellular layer of proliferating chondrocytes (P) and paucity of primaryspongiosa (PS). Decalcified, HE.
8 Veterinary Pathology
at University of Sydney on January 13, 2014vet.sagepub.comDownloaded from
blood beyond the mild to moderate red blood cell polychroma-
sia that was observed in both runt and normal crocodiles. This
may be at least in part due to the tendency of young reptiles to
exhibit a greater degree of polychromasia than adults.5 Serum
iron was inconsistent between the 2 years, with 2005 crocodiles
having significantly higher serum iron levels than 2007 croco-
diles regardless of whether they were runt or normal animals.
Both groups also had lower packed cell volumes in 2007, pos-
sibly reflecting lower serum iron. In the runt crocodiles, there
appeared to be sequestration of iron in the form of splenic
hemosiderin. Splenic hemosiderosis occurs with starvation or
other catabolic states in all species and, in reptiles, is generally
considered secondary to other conditions, such as chronic
inanition.46 Thus, it is likely that inanition is the main contribu-
tor to the anemia in runt crocodiles.62,67
One of the main objectives of this study was to investigate
whether chronic infectious disease could be associated with
runting. Despite a thorough examination of all organ systems
in runt vs normal crocodiles, there were no gross or histological
lesions to suggest a significant presence of infectious disease.
The only parasite detected in the study was rare sporulated
oocysts in the intestinal epithelium of 3 runt crocodiles and 1
normal crocodile, consistent with the Goussia-like coccidia
described in saltwater crocodiles in Australia.25,40 The pres-
ence of this parasite as a contributing factor to runting is
dubious, given that the coccidia were evident in only 3 of 20
runt crocodiles, and then only rarely in the intestinal epithelium
with no associated histological lesions.
Bacterial culture of 2 filtering organs (liver and spleen) was
conducted to detect bacterial sepsis, since this may be difficult
Figure 6. Spleen; normal crocodile (case No. 12). Large discrete lymphoid cuffs (arrows) surround arterioles (A). Hematoxylin and eosin (HE).Figure 7. Spleen; runt crocodile (case No. 13). Relatively small lymphoid cuffs (arrows) surround arterioles (A). Note yellow-brown tinge tored pulp due to pigment within macrophages. HE. Inset: Pigment within macrophages stains positive for ferric iron indicative of hemosiderin.Perls’s stain. Figure 8. Thymus; normal crocodile (case No. 12). Thymus lobes are densely populated with lymphocytes with clear distinctionbetween cortex (C) and medulla (M). For perspective, a section of the primary bronchus (lumen, B) and thyroid gland (T) is noted. Same mag-nification as Fig. 9. HE. Figure 9. Thymus; runt crocodile (case No. 1). Thymus lobes (arrows) are small with poor distinction between cortexand medulla. For perspective, a section of the primary bronchus (lumen, B) and thyroid gland (T) is noted. Same magnification as Fig. 8. HE.
Shilton et al 9
at University of Sydney on January 13, 2014vet.sagepub.comDownloaded from
to detect histologically and is a common cause of morbidity and
mortality in juvenile crocodiles.3,41 Bacterial isolates were
limited to a total of 5 species in 2 runt crocodiles and 4 normal
crocodiles, with no particular pattern to suggest a relationship
with runting.
Histological evidence of virus infection was limited to mild
cutaneous poxvirus infection present in several crocodiles, both
normal and runts. Virus isolation was attempted for multiple
internal organs in all crocodiles in 2007 to further substantiate
lack of virus involvement and because of the association of
viral infection with atrophy of lymphoid tissues and stunted
growth in other species. Examples include mammalian
parvoviruses and pestiviruses,47 as well as retroviruses of the
reticuloendotheliosis virus group in poultry.18 However, our
isolation attempts yielded only a cytopathic effect suggestive
of 5 isolates from 4 normal crocodiles. This, as well as the lack
of histological lesions such as necrosis or inflammation that
could substantiate a significant virus infection, suggests that
viral infection was not related to runting in our study.
A second objective of the study, to look for histological clues
of other disease processes, revealed some associations with runt-
ing and could help suggest an alternate etiology to infectious dis-
ease. One notable finding was the increased cytoplasmic
vacuolation of adrenocortical cells in runt compared with normal
crocodiles in both phases of the study. Variation in the degree of
vacuolation is evidence of altered activity of the adrenocortical
cells in reptilians and is evidence of increased activity of the
cells in mammals.7,24 To further investigate this finding, in
2007, serum level of corticosterone, the main glucocorticoid
stress hormone in crocodilians,37,43 was measured and found
to be significantly elevated in runt crocodiles. Chronic elevation
of corticosteroids results in a general catabolic state7,61 and anor-
exia in crocodiles,32 and it inhibits both growth hormone secre-
tion and action in mammals.31 A negative correlation between
corticosterone level and growth rate has been noted previously
in saltwater crocodiles58,65 and alligators,15 and extremely poor
growth has been experimentally reproduced in alligators by
chronic elevation of corticosterone.43,53
Another notable histological finding in runt crocodiles was
the presence of bone growth zones that had all the components
of normal growth zones but were relatively quiescent and
exhibit decreased production of primary spongiosa. The runt
crocodiles were mildly hypocalcemic and moderately
hypophosphatemic compared with normal crocodiles. Since
approximately 40% to 45% of total calcium is transported in
serum bound to protein (principally albumin), the mild hypo-
calcemia in the runt crocodiles was suspected to be a reflection
of their concurrent substantial hypoalbuminemia (runt
crocodiles had on average 38% lower albumin than normal
crocodiles).1,51 This was confirmed by statistically correcting
for the level of albumin. Measurement of the ionized (unbound,
biologically active) portion of total calcium would have been
an additional parameter we could have used to investigate
serum calcium in this study1,51 but was judged to not be worth
pursuing given the lack of hypocalcemia in the runts once
albumin was taken into account.
The mild to moderate hypophosphatemia in runt crocodiles
is likely at least in part attributable to prolonged lack of dietary
intake, a common cause in reptiles.4 Since runt crocodiles were
offered the same diet as the normal crocodiles, the deficit may
be due to a lack of food intake rather than a deficient diet per se.
Also, increased glucocorticoid hormone has been shown in
humans to result in decreased renal resorption and gastrointest-
inal absorption of phosphate.57 A final possible cause for the
relative hypophosphatemia in runt crocodiles may be that it
is simply reflecting decreased bone formation, as serum
phosphorus tends to be higher in growing animals.1
The formation of endochondral bone in growth zones is
essentially similar in mammals, birds, and reptiles.28 In mam-
mals, abnormal bone growth in young animals due primarily to
severe prolonged lack of dietary phosphorus generally results
in rickets or osteomalacia, characterized by deformities of bone
shape with excessive production of unmineralized osteoid and
cartilaginous matrix,63 lesions that were not evident in runt
crocodiles. The histology of the poor bone growth in the runt
crocodiles is best described as osteoporosis, with relative inac-
tivity of the growth zone and decreased production of bone
being typical findings in mammals or reptiles undergoing
inanition.28,63 Another factor in reduced activity of growth
zones in runt crocodiles may be elevated corticosterone, since
elevated glucocorticoid hormones leads to osteoporosis and
decreased bone formation in some mammals.56,57,63
The decreased lymphoid populations in the major immune
tissues of crocodiles,19 including spleen, tonsil (gut-
associated lymphoid tissue), and thymus in runt crocodiles,
indicate generalized lymphoid hypoplasia or atrophy. Marked
lymphoid involution in reptiles can be seasonal or may result
from stressors such as starvation or disease.10 Corticosterone
has been shown to be associated with lymphoid involution and
immunosuppression in a wide variety of species, including cro-
codilians.43,54,60,70 Despite the lymphoid involution evident in
runt crocodiles, there was no overt evidence of immunosup-
pression in the form of an increased prevalence of infectious
disease. This may be partly related to the sampling procedure,
in that only bright, responsive, and active runt crocodiles were
selected for the study. Runt crocodiles may be predisposed to
eventual morbidity/mortality due to infectious disease, but this
would have gone undetected in the present study and would be
a useful avenue for further research. Perhaps even better would
be research aimed at detecting subtle evidence of immunosup-
pression in runt crocodiles, such as response to phytohemag-
glutinin injection or other immunoassays.20,27
The marked histological difference in lymphoid tissues of
runt vs normal crocodiles was not reflected in numbers of
circulating lymphocytes in this study. This is in contrast to
other studies in reptiles in which tissue lymphoid depletion was
correlated with a decrease in circulating lymphocytes.62
Furthermore, the stress response, characterized by increased
activity of the adrenocortical cells, has been found to be asso-
ciated with a decrease in circulating lymphocytes along with an
increase in heterophils/neutrophils in animals generally,
including crocodilians.5,29,53,56 Despite the histologically
10 Veterinary Pathology
at University of Sydney on January 13, 2014vet.sagepub.comDownloaded from
appreciable adrenocortical hyperplasia and significantly
elevated corticosterone in runt crocodiles in this study, this was
not correlated with alterations in circulating leukocytes com-
pared with normal crocodiles. However, a few other studies
have noted variable or absent alterations in numbers of circulat-
ing lymphocytes and heterophils with stress in crocodilians,
indicating that measurement of circulating leukocytes may be
an unreliable indicator of adrenocortical activity in these
species.43,65
In summary, there is no significant evidence of infectious
disease in runt crocodiles in this study. Rather, there is evi-
dence of inanition, lymphoid atrophy, and quiescent growth
zones in bones. Regarding inanition and poor growth, although
food intake of the runt crocodiles in this study was not mea-
sured, because we found no evidence of diseases or conditions
that might result in poor food conversion, it seems likely that
the runt crocodiles, while being offered equal access to food,
were not eating as much as the normal crocodiles. As discussed
previously, decreased appetite could be a consequence of a
stress response.32 Alternatively, it is possible that the food was
inherently unacceptable to runt crocodiles (eg, they required
the additional stimulation of movement of prey to eat), they
were unable to learn to eat the captive diet, or they had altered
behavior that prevented them from approaching the food (eg,
excessively fearful disposition). Supporting these as possibili-
ties is the finding by some investigators that changing the diet
will ameliorate runting in some circumstances.11,22,26,55 One
avenue for further research into runting in crocodiles could thus
be confirmation that runt crocodiles do eat less than their
normal conspecifics and alteration of the diet or feeding envi-
ronment in an attempt to ameliorate the runting.
Another possibility is that the inanition, lymphoid atrophy,
and quiescent growth zones of bones are a result of a chronic
adrenocortical stress response, which was demonstrated by the
adrenocortical cell vacuolation and elevation of corticosterone
in runt crocodiles. Chronic stress could be a secondary
response to some unknown stressor. For example, one possible
cause of secondary stress could be related to a perceived lack of
suitable food in runt crocodiles, as discussed above. Since runt
crocodiles were reared in pens containing other similarly sized
runts, a chronic stress response is unlikely to be secondary to
aggression from larger pen-mates. Although runt crocodiles
were reared under identical husbandry conditions as normal
crocodiles, it is possible that there could be something inher-
ently different in runt crocodiles that results in them being
stressed by a feature of the husbandry or environment that is
not stressful to normal crocodiles. Further research along these
lines could involve trials altering the features of runt pens, such
as design (eg, provide more hides, variation in water depth),
lowering stocking density, or providing a wider spectrum of
available temperature (eg, provide heat lamps for basking).
Thus, runt crocodiles could be suffering from a ‘‘maladaptation
syndrome’’ suggested by a few other investigators and recog-
nized as a feature of stress in reptiles.3,12,65
Rather than being a secondary response in individual hatchl-
ings to some aspect of the farming situation, the stress response
could be a primary feature inherent in a runt crocodile. It has
been established in many animals, from fish to birds and mam-
mals, that maternal stress may influence the hypothalamic-
pituitary-adrenal axis of the offspring, resulting in altered beha-
vioral development and/or ill thrift in the offspring for several
months after birth.2,8,13,16,17,36,45 In lizards, treatment of
reproductive females or eggs with corticosterone can affect
hatchling body size, growth rate, behavior (eg, dispersal and
antipredator responses), and even sex.52,66,68 Likewise, in
crocodilians, limited studies suggest that high plasma corticos-
terone in females may result in relatively poor egg quality and
decreased hatchling survival.14,61
The clutch effect in crocodile runting26,33,48,58 suggests that
crocodiles in specific clutches are hatched with a tendency to
become runts, although more research needs to be done on the
degree of clutch effect and variation within a clutch. A clutch
effect could be due to genetics, maternal circumstances, egg
incubation conditions, or, where members of a clutch are reared
together in some degree of isolation from other clutches, their
rearing environment. These considerations are beyond the
scope of this study, given the large number of clutches the
study animals originated from and the mixture of eggs from
wild vs captive nests, but may be valuable avenues for further
research. Exploration of the possible role of genetics in causing
runting would require controlled breeding experiments in cap-
tive pairs over multiple years. Maternal circumstances, includ-
ing size and body condition, geographic location, diet, food
availability, and stress level, would also be useful avenues for
further research. Egg incubation conditions in this study were
uniform and controlled at the farm once eggs were collected but
not necessarily prior to collection, particularly for eggs
originating from wild nests. An effect of incubation conditions
on posthatching growth has been noted in a few studies of
crocodilians,38,69 but its role in runting is an area that requires
further research. Finally, if it is established that there is a strong
clutch effect to runting and predictors discovered, investigation
into either preventative measures or altered rearing practices
for at-risk clutches might ameliorate the runting.
Acknowledgements
This research was approved by the University of Sydney Animal
Ethics Committee (reference number N00/9-2005/3/4204).
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 project
was funded in part by the Australian Government, Rural Industries
Research and Development Corporation and findings briefly summar-
ized in RIRDC Publication No. 09/135 Improving Australia’s Croco-
dile Industry Productivity—Understanding Runtism and Survival.
Shilton et al 11
at University of Sydney on January 13, 2014vet.sagepub.comDownloaded from
References
1. Bohn AA. Laboratory evaluation of electrolytes. In: Thrall MA,
Weiser G, Allison R, et al, eds. Veterinary Hematology and Clin-
ical Chemistry. 2nd ed. Ames, IA: John Wiley; 2012:378–392.
2. Braastad BO. Effects of prenatal stress on behaviour of offspring
of laboratory and farmed mammals. Appl Anim Behav Sci. 1998;
61(2):159–180.
3. Buenviaje GN, Ladds PW, Melville L, et al. Disease-husbandry
associations in farmed crocodiles in Queensland and the Northern
Territory. Aust Vet J. 1994;71(6):165–173.
4. Campbell TW. Clinical chemistry of reptiles. In: Thrall MA,
Weiser G, Allison R, et al, eds. Veterinary Hematology and Clin-
ical Chemistry. 2nd ed. Ames, IA: John Wiley; 2012:599–606.
5. Campbell TW. Hematology of reptiles. In: Thrall MA, Weiser G,
Allison R, et al. Veterinary Hematology and Clinical Chemistry.
2nd ed. Ames, IA: John Wiley; 2012:277–297.
6. Canfield PJ. Characterization of the blood cells of Australian cro-
codiles (Crocodylus porosus [Schneider] and C. johnstoni
[Krefft]). Zbl Vet Med C Anat Histol Embryol. 1985;14:269–288.
7. Capen CC. Endocrine glands. In: Maxie MG ed. Jubb, Kennedy,
and Palmer’s Pathology of Domestic Animals. 5th ed. Vol 3.
Philadelphia, PA: Elsevier Saunders; 2007:325–428.
8. Chadio SE, Kotsampasi B, Papadomichelakis G, et al. Impact of
maternal undernutrition on the hypothalamic-pituitary-adrenal
axis responsiveness in sheep at different ages postnatal. J Endo-
crinol. 2007;192:495–503.
9. Churukian CJ. Pigments and minerals. In: Bancroft JD, Gamble
M, eds. Theory and Practice of Histological Techniques. Philadel-
phia, PA: Churchill Livingston Elsevier; 2008:233–259.
10. Cooper EL, Klempau AE, Zapata AG. Reptilian immunity. In:
Gans C, ed. Biology of the Reptilia. Vol 14, Development A. New
York, NY: John Wiley; 1985:599–678.
11. Davenport J, Grove DJ, Cannon TR, et al. Food capture, appetite,
digestion rate and efficiency in hatchling and juvenile Crocodylus
porosus. J Zool Lond. 1990;220:569–592.
12. Denardo D. Stress in captive reptiles. In: Mader DR, ed. Reptile
Medicine and Surgery. 2nd ed. St Louis, MO: Saunders Elsevier;
2006:119–134.
13. Edwards HE, McIntyre Burnham W. The impact of corticoster-
oids on the developing animal. Pediatr Res. 2001;50(4):433–440.
14. Elsey RM, Joanen T, McNease L, et al. Stress and plasma corti-
costerone levels in the American alligator—relationships with
stocking density and nesting success. Comp Biochem Phys A.
1990;95(1):55–63.
15. Elsey RM, Joanen T, McNease L, et al. Growth rate and plasma
corticosterone levels in juvenile alligators maintained at different
stocking densities. J Exp Zool. 1990;255:30–36.
16. Entringer S, Epel ES, Kumsta R, et al. Stress in intrauterine life is
associated with shorter telomere length in young adulthood. Proc
Natl Acad Sci U S A. 2011;108(33): E513–E518.
17. Eriksen MS, Bakken M, Espmark A, et al. Prespawning stress in
farmed Atlantic salmon Salmo salar: maternal cortisol exposure
and hyperthermia during embryonic development affect offspring
survival, growth and incidence of malformations. J Fish Biol.
2006;69:114–129.
18. Fadley AM, Zaval G, Witter RL. Reticuloendotheliosis. In: Saif
YM, ed. Diseases of Poultry. 12th ed. Ames, IA: Blackwell;
2008:568–588.
19. Finger JW, Isberg SR. A review of innate immune functions in
crocodilians. CAB Rev. 2012;7(67):1–11.
20. Finger JW, Adams AL, Thomson PC, et al. Using phytohaemag-
glutinin to determine immune responsiveness in saltwater croco-
diles (Crocodylus porosus). Aust J Zool. 2013;61(4):301–311.
21. Foggin CM. Disease and disease control on crocodile farms in
Zimbabwe. In: Webb GJW, Manolis SC, Whitehead PJ, eds.
Wildlife Management: Crocodiles and Alligators. Chipping Nor-
ton, Australia: Surrey Beatty & Sons; 1987:351–362.
22. Foggin CM. Diseases of farmed crocodiles. In: Smith GA, Marais
J, eds. Conservation and Utilization of the Nile Crocodile in
Southern Africa: Handbook on Crocodile Farming. Pretoria: Cro-
codilian Study Group of Southern Africa; 1992:107–140.
23. Franklin CE, Davis BM, Peucker SKJ, et al. Comparison of stress
induced by manual restraint and immobilization in the estuarine
crocodile, Crocodylus porosus. J Exp Zool. 2003;298A:86–92.
24. Gabe M. The adrenal. In: Gans C, ed. Biology of the Reptilia. Vol
3, Morphology C. New York, NY: Academic Press; 1970:
263–318.
25. Gardiner CH, Imes GD, Jacobson ER, et al. Sporulated coccidian
oocysts resembling Goussia Labbe, 1896 in the viscera of Nile
crocodiles. J Wildl Dis. 1986;22(4):575–577.
26. Garnett ST, Murray RM. Parameters affecting the growth of the
estuarine crocodile, Crocodylus porosus, in captivity. Aust J Zool.
1986;34:211–223.
27. Grasman KA. Assessing immunological function in toxicological
studies of avian wildlife. Integ Comp Biol. 2002;42:34–42.
28. Haines RW. Epiphyses and sesamoids. In: Gans C, ed. Biology of
the Reptilia. Vol 1, Morphology A. New York, NY: Academic
Press; 1969:81–115.
29. Hakim AH. Corticosteroids and immune systems of non-
mammalian vertebrates: a review. Dev Comp Immunol. 1988;
12:481–494.
30. Hernandez-Divers SJ, Cooper JE. Hepatic Lipidosis. In: Mader
DR, ed. Reptile Medicine and Surgery. 2nd ed. St Louis, MO:
Saunders Elsevier; 2006:806–813.
31. Hochberg A. Mechanisms of steroid impairment of growth. Horm
Res. 2002;58(suppl 1):33–38.
32. Huchzermeyer FW. Crocodiles: Biology, Husbandry and
Diseases. Oxon, UK: CABI Publishing; 2003.
33. Isberg S, Shilton C, Thomson P. Improving Australia’s Crocodile
Industry Productivity—Understanding Runtism and Survival.
Barton, Australia: Rural Industries Research and Development
Corporation; 2009.
34. Isberg SR, Thomson PC, Nicholas FW, et al. Quantitative analysis
of production traits in saltwater crocodiles (Crocodylus porosus):
II. Age at slaughter. J Anim Breed Genet. 2005;122:370–377.
35. Jacobson ER. Viruses and viral diseases of reptiles. In: Jacobson
ER, ed. Infectious Diseases and Pathology of Reptiles. Boca
Raton, FL: CRC Press; 2007:395–460.
36. Janczak AM, Braastad BO, Bakken M. Behavioural effects of
embryonic exposure to corticosterone in chickens. Appl Anim
Behav Sci. 2006;96:69–82.
12 Veterinary Pathology
at University of Sydney on January 13, 2014vet.sagepub.comDownloaded from
37. Jessop TS, Tucker AD, Limpus CJ, et al. Interactions between
ecology, demography, capture stress, and profiles of corticoster-
one and glucose in a free-living population of Australian fresh-
water crocodiles. Gen Comp Endocr. 2003;132:161–170.
38. Joanen T, McNease L, Ferguson MWJ. The effects of egg incuba-
tion temperature on post-hatching growth of American alligators.
In: Webb GJW, Manolis C, Whitehead PJ, eds. Wildlife Manage-
ment: Crocodiles and Alligators. Chipping Norton, Australia:
Surrey Beatty & Sons; 1987:533–537.
39. Kanui T, Mwenda C, Aulie A, et al. Effects of temperature on growth,
food uptake and retention time of juvenile Nile crocodiles (Crocody-
lus niloticus). Comp Biochem Physiol A. 1991;99(3):453–456.
40. Ladds PW. Pathology of Australian Native Wildlife. Colling-
wood, Australia: CSIRO Publishing; 2009.
41. Ladds PW, Bradley J, Hirst RG. Providencia rettgeri meningitis in
hatchling saltwater crocodiles (Crocodylus porosus). Aust Vet J.
1996;74:397–398.
42. Lance VA, Lauren D. Circadian variation in plasma corticoster-
one in the American alligator, Alligator mississippiensis, and the
effects of ACTH injections. Gen Comp Endocr. 1984;54:1–7.
43. Lance VA, Morici LA, Elsey RM. Physiology and endocrinology
of stress in crocodilians. In: Grigg CG, Seebacher F, Franklin CE,
eds. Crocodilian Biology and Evolution. Chipping Norton,
Australia: Surrey Beatty & Sons; 2000:327–340.
44. Lauren DJ. The effect of chronic saline exposure on the electro-
lyte balance, nitrogen metabolism and corticosterone titer in the
American alligator, Alligator mississippiensis. Comp Biochem
Physiol. 1985;81A(2):217–223.
45. Lesage J, Sebaai N, Leonhardt M, et al. Perinatal maternal under-
nutrition programs the offspring hypothalamo-pituitary-adrenal
(HPA) axis. Stress. 2006;9(4):183–198.
46. Lowenstine LJ, Munson L. Iron overload in the animal kingdom.
In: Fowler ME, Miller RE, eds. Zoo and Wild Animal Medicine,
Current Therapy 4. Philadelphia, PA: Saunders; 1999:260–268.
47. MacLachlan NJ, Dubovi EJ. Fenner’s Veterinary Virology. 4th
ed. San Diego, CA: Academic Press; 2011.
48. Mayer R. Crocodile Farming: Research, Development and On-
Farm Monitoring, 1995–1998. Barton: Australian Government
Rural Industries Research and Development Corporation; 1998.
49. McInerney J. Liver enzymes and pathology in runt crocodiles (C.
porosus). Proc Assoc Reptilian Amphibian Vet. 1994:57–58.
50. Melville L, Davis S, Shilton C, et al. Hunting for Viruses in
Crocodiles: Viral and Endogenous Retroviral Detection and
Characterization in Farmed Crocodiles. Barton: Australian
Government Rural Industries Research and Development Corpo-
ration; 2012.
51. Meuten D. Parathyroid glands and calcium and phosphorus meta-
bolic pathology: Thrall MA, Weiser G, Allison R, et al. Veterin-
ary Hematology and Clinical Chemistry. Ames, IA: John Wiley;
2012:545–568.
52. Meylan S, Belliure J, Clobert J, et al. Stress and body condition
as prenatal and postnatal determinants of dispersal in the com-
mon lizard (Lacerta vivipara). Horm Behav. 2002;42:319–326.
53. Morici LA, Elsey RM, Lance V. Effects of long-term corticoster-
one implants on growth and immune function in juvenile alliga-
tors, Alligator mississippiensis. J Exp Zool. 1997;279:156–162.
54. Origgi FC. Reptile immunology. In: Jacobson ER, ed. Infectious
Diseases and Pathology of Reptiles. Boca Raton, FL: CRC Press;
2007:131–166.
55. Peucker S, Mayer R. Runt observation studies. Crocodile Res
Bull. 1995;1:57–62.
56. Poetker DM, Reh DD. A comprehensive review of the adverse
effects of systemic corticosteroids. Otolaryngol Clin North Am.
2010;43:753–768.
57. Reid IR. Glucocorticoid osteoporosis—mechanisms and manage-
ment. Eur J Endocrinol. 1997;137:209–217.
58. Riese G. Factors Affecting Food Intake and Growth in Captive
Saltwater Crocodile (Crocodylus porosus) [MSc thesis]. Brisbane:
Department of Zoology, University of Queensland, Australia;
1995.
59. Schaffner F. The liver. In: Gans C, Gaunt AS, eds. Biology of the
Reptilia, Volume 19, Morphology G, Visceral Organs. Ithaca, NY:
Society for the Study of Amphibians and Reptiles; 1998:485–531.
60. Schobitz B, Reul JMHM, Holsboer F. The role of the
hypothalamic-pituitary-adrenocortical system during inflamma-
tory conditions. Crit Rev Neurobiol. 1994;8(4):263–291.
61. Smith GA, Marais J. Stress in crocodilians—the impact of nutri-
tion: Proceedings of the 12th Working Meeting of the Crocodile
Specialist Group. Vol 2. Gland, Switzerland: IUCN—The World
Conservation Union; 1994:2–38.
62. Sypek J, Borysenko M. Reptiles. In: Rowley AF, Ratcliffe NA,
eds. Vertebrate Blood Cells. Cambridge, UK: Cambridge Univer-
sity Press; 1988:211–256.
63. Thompson K. Bones and joints. In: Maxie MG, ed. Jubb, Ken-
nedy, and Palmer’s Pathology of Domestic Animals. 5th ed. Vol
1. Philadelphia, PA: Elsevier Saunders; 2007:1–184.
64. Turton JA. Preliminary report on stress in farmed Crocodylus poro-
sus hatchlings. In: Proceedings of the 2nd Regional Meeting of the
Crocodile Specialist Group of the Species Survival Commission of
IUCN—The World Conservation Union. International Union for the
Conservation of Nature and Natural Resources in conjunction with
the Conservation Commission of the Northern Territory, Darwin,
Australia; 1993.
65. Turton JA, Ladds PW, Manolis SC, et al. Relationship of blood
corticosterone, immunoglobulin and haematological values in
young crocodiles (Crocodylus porosus) to water temperature,
clutch of origin and body weight. Aust Vet J. 1997:75(2):114–119.
66. Uller T, Olsson M. Direct exposure to corticosterone during
embryonic development influences behaviour in an ovovivipar-
ous lizard. Ethology. 2006;112(4):390–397.
67. Valli VEO. Hematopoietic system. In: Maxie MG, ed. Jubb, Ken-
nedy, and Palmer’s Pathology of Domestic Animals. 5th ed. Vol
3. Philadelphia, PA: Elsevier Saunders; 2007:107–324.
68. Warner D, Radder RS, Shine R. Corticosterone exposure during
embryonic development affects offspring growth and sex ratios
in opposing directions in two lizard species with environmental
sex determination. Physiol Biochem Zool. 2009;82(4):363–371.
69. Webb GJ, Cooper-Preston H. Effects of incubation temperature
on crocodiles and the evolution of reptilian oviparity. Am Zool.
1989;29:953–971.
70. Webster Marketon JI, Glaser R. Stress hormones and immune
function. Cell Immunol. 2008;252:16–26.
Shilton et al 13
at University of Sydney on January 13, 2014vet.sagepub.comDownloaded from