Weaned pigs experimentally infected with Salmonella display sexually dimorphic innate immune responses without affecting pathogen colonization patterns123

Abstract

Sexually dimorphic innate immune responses have been observed in several species, but have not been studied in response to a live pathogen challenge in pigs. This study aimed to elucidate sexually dimorphic innate immune responses along with Salmonella translocation patterns in newly weaned pigs orally inoculated with Salmonella. Newly weaned pigs (n = 8 gilts and 12 barrows; 6.2 ± 0.2 kg BW) were obtained from a commercial swine facility and were maintained in an environmentally-controlled facility in individual pens equipped with feeders and nipple waterers. Pigs were allowed ad libitum access to a commercial non-medicated starter ration and water throughout the study. On d 12 post-weaning, pigs were anesthetized to allow placement of a temperature measuring device in the abdominal cavity for measurement of intraperitoneal temperature (TEMP). On d 17, pigs were anesthetized and fitted with indwelling jugular vein catheters. On the following day (d 18), pigs were orally inoculated with 4.7x109 Salmonella typhimurium. Blood samples were collected at 0.5-h intervals from -2 to 8 h, and at 8-h intervals from 8 to 72 h post-challenge. Whole blood was analyzed for complete blood cell counts. Serum was isolated for measurement of cortisol. Following collection of the 72 h sample, pigs were humanely euthanized and tissues were collected for Salmonella isolation. There was a sex × time interaction (P < 0.001) for TEMP such that gilts had a greater TEMP response to the Salmonella challenge compared to barrows. There was also a sex × time interaction (P = 0.03) for serum cortisol with gilts having decreased cortisol at 16 h yet greater cortisol at 32 h than barrows. Barrows had greater total white blood cells (17.8 vs. 16.2 ± 0.4 103 cells/μL; P < 0.01; respectively) and neutrophils (7.8 vs. 6.1 ± 0.4 103 cells/μL; P < 0.01; respectively) than gilts. However, gilts had greater lymphocytes (9.6 vs. 9.0 ± 0.2 103 cells/μL; P = 0.05; respectively) than barrows. While immune parameters were influenced by sex, there was no effect of sex (P > 0.05) on Salmonella concentrations from fecal shedding 3 d post-inoculation in the cecum, mesenteric and subiliac lymph nodes, liver, spleen, gallbladder, or kidney tissues. These data demonstrate that weaned gilts appear to produce a stronger acute phase response to a Salmonella challenge compared to barrows, without affecting the tissue translocation or shedding of Salmonella.

Full text

69
Weaned pigs experimentally infected with Salmonella display sexually dimorphic
innate immune responses without aecting pathogen colonization patterns1,2,3
Nicole C. Burdick Sanchez,* Paul R. Broadway,* Jeery A.
Carroll,*4 Elena V. Gart,† Laura K. Bryan,† and Sara D. Lawhon†
*Livestock Issues Research Unit, USDA-ARS, Lubbock, TX 79403 and †Department of Veterinary
Pathology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station 77843
ABSTRACT: Sexually dimorphic innate immune
responses have been observed in several species, but
have not been studied in response to a live pathogen
challenge in pigs. This study aimed to elucidate sexu-
ally dimorphic innate immune responses along with
Salmonella translocation patterns in newly weaned
pigs orally inoculated with Salmonella. Newly
weaned pigs (n = 8 gilts and 12 barrows; 6.2 ± 0.2
kg BW) were obtained from a commercial swine
facility and were maintained in an environmentally-
controlled facility in individual pens equipped with
feeders and nipple waterers. Pigs were allowed ad
libitum access to a commercial non-medicated starter
ration and water throughout the study. On d 12 post-
weaning, pigs were anesthetized to allow placement
of a temperature measuring device in the abdominal
cavity for measurement of intraperitoneal tempera-
ture (TEMP). On d 17, pigs were anesthetized and
tted with indwelling jugular vein catheters. On the
following day (d 18), pigs were orally inoculated
with 4.7x109 Salmonella typhimurium. Blood sam-
ples were collected at 0.5-h intervals from -2 to 8 h,
and at 8-h intervals from 8 to 72 h post-challenge.
Whole blood was analyzed for complete blood cell
counts. Serum was isolated for measurement of cor-
tisol. Following collection of the 72 h sample, pigs
were humanely euthanized and tissues were collect-
ed for Salmonella isolation. There was a sex × time
interaction (P < 0.001) for TEMP such that gilts had
a greater TEMP response to the Salmonella challenge
compared to barrows. There was also a sex × time
interaction (P = 0.03) for serum cortisol with gilts
having decreased cortisol at 16 h yet greater cortisol
at 32 h than barrows. Barrows had greater total white
blood cells (17.8 vs. 16.2 ± 0.4 103 cells/μL; P < 0.01;
respectively) and neutrophils (7.8 vs. 6.1 ± 0.4 103
cells/μL; P < 0.01; respectively) than gilts. However,
gilts had greater lymphocytes (9.6 vs. 9.0 ± 0.2 103
cells/μL; P = 0.05; respectively) than barrows. While
immune parameters were inuenced by sex, there
was no eect of sex (P > 0.05) on Salmonella con-
centrations from fecal shedding 3 d post-inoculation
in the cecum, mesenteric and subiliac lymph nodes,
liver, spleen, gallbladder, or kidney tissues. These
data demonstrate that weaned gilts appear to produce
a stronger acute phase response to a Salmonella chal-
lenge compared to barrows, without aecting the
tissue translocation or shedding of Salmonella.
© 2017 American Society of Animal Science. This is an open access article distributed under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
Transl. Anim. Sci. 2017.1:69–76
doi:10.2527/tas2016.0008
1Mention of trade names or commercial products in this ar-
ticle is solely for the purpose of providing specic information
and does not imply recommendation or endorsement by the U.S.
Department of Agriculture.
2
The U.S. Department of Agriculture (USDA) prohibits discrimi-
nation in all its programs and activities on the basis of race, color,
national origin, age, disability, and where applicable, sex, marital
status, familial status, parental status, religion, sexual orientation,
genetic information, political beliefs, reprisal, or because all or part
of an individual’s income is derived from any public assistance
program. (Not all prohibited bases apply to all programs.) Persons
with disabilities who require alternative means for communication
of program information (Braille, large print, audiotape, etc.) should
contact USDA’s TARGET Center at (202) 720–2600 (voice and TDD).
To le a complaint of discrimination, write to USDA, Director, Oce
of Civil Rights, 1400 Independence Avenue, S.W., Washington, DC.
20250–9410, or call (800) 795–3272 (voice) or (202) 720–6382 (TDD).
3
The authors would like to acknowledge the technical assis-
tance of J. W. Dailey and J. R. Carroll (USDA-ARS). Elena Gart
was supported by a Texas A&M University Association of Former
Students Diversity Fellowship. Laura Bryan was supported by NIH
T32 grant 5T320D011083.
4Corresponding author: Je.Carroll@ars.usda.gov
Received October 28, 2016.
Accepted November 17, 2016.
Key words: cortisol, innate immunity, pigs, Salmonella, sexual dimorphism
Published April 27, 2017

Burdick Sanchez et al.
70
Translate basic science to industry innovation
INTRODUCTION
The responsiveness of the immune system to a
perceived challenge can be modulated by various
factors, including internal factors such as the hypo-
thalamic-pituitary-adrenal (HPA) axis and energy
availability/storage, as well as naturally-occurring
factors such as behavior, breed, and sex (Burdick et
al., 2009; Carroll et al., 2011; Carroll et al., 2015).
Specically regarding sex, studies conducted by
our lab have demonstrated sex-specic innate im-
mune responses to a lipopolysaccharide (LPS), and
to a dual corticotropin-releasing hormone (CRH)
and vasopressin challenge in cattle (Hulbert et al.,
2013; Carroll et al., 2015). Additionally, Williams et
al. (2009) observed greater baseline and peak serum
TNF-ɑ and IL-6, but decreased IL-1β concentrations
in weaned gilts in response to an LPS challenge com-
pared to barrows. Thus, sexual dimorphic eects are
found in livestock species, and particularly in pigs
utilizing exogenous challenges.
Utilizing substances such as LPS and CRH to ex-
ogenously stimulate immune and stress responses in the
body have proven to be useful models for elucidating
these complex biological systems. However, the use
of live pathogen challenge models provides a further
means of studying immune and stress responses that are
more reective of real-world scenarios. Live pathogen
challenge studies are more indicative and representa-
tive of the immunological challenges pigs encounter in
the commercial production setting; however increased
variation is introduced due to endogenous factors asso-
ciated with each animal and the specic microorganism.
The use of a live Salmonella challenge in swine is of
particular relevance due to the health issues associated
with Salmonella outbreaks in swine herds, and due to an
increasing concern within the livestock industry associ-
ated with post-harvest contamination, food safety and
human illness (Ojha and Kostrzynska, 2007). Aligned
with the observed eects of sex on the innate immune
response that have been observed in response to vari-
ous immune challenges in swine and other species, this
study sought to determine whether newly weaned pigs
exhibit sexually dimorphic innate immune responses to
an oral Salmonella typhimurium challenge.
MATERIALS AND METHODS
All experimental procedures were in compliance with
the Guide for the Care and Use of Agricultural Animals in
Research and Teaching and approved by the Institutional
Animal Care and Use Committee at the Livestock Issues
Research Unit (Protocol # 2014–04-JAC18).
Experimental Design
Newly weaned pigs (n = 8 gilts and 12 bar-
rows; 6.2 ± 0.2 kg body weight) were obtained from
a commercial swine facility and transported to the
Livestock Issues Research Unit’s Liberty Farm facil-
ity. Pigs were housed in an enclosed, environmental-
ly-controlled facility in individual pens equipped with
stainless steel feeders and nipple waterers. Pigs were
allowed ad libitum access to a commercial non-med-
icated starter ration and water throughout the study.
On d 12 post-weaning, pigs were anesthetized and a
small incision (2 to 2.5 cm) was made in the lower
abdominal region for the placement of an indwell-
ing temperature recording device (25.4 mm in length,
8.3 mm in diameter, 3.3 g; Star Oddi DST micro-T;
MeterMall USA, Marysville, OH) into the peritoneal
cavity. Intraperitoneal temperature was measured at
5-min intervals from the time of the placement of the
temperature device until the end of the study. On d
17, pigs were anesthetized and non-surgically tted
with indwelling jugular vein catheters (Carroll et al.,
1999). On d 18, pigs were orally inoculated with 4.7
× 109 cfu/pig of a nalidixic acid-resistant Salmonella
typhimurium in 10 mL of culture media. One 4.5-mL
whole blood sample was collected in Starstedt tubes
containing no additive (Sarstedt Inc., Newton, NC) at
0.5 h intervals from -2 to 8 h, and at 8-h intervals from
8 to 72 h post-challenge. Samples were allowed to clot
at room temperature for 30 min prior to centrifuga-
tion at 1,500 g for 20 min at 4°C. Isolated serum was
stored at -80°C until analyzed for cortisol concentra-
tions. A second 4-mL sample was collected in a vacu-
tainer containing EDTA for determination of complete
blood counts (CBC) using a ProCyte Dx Hematology
Analyzer (IDEXX, Westbrook, ME). Sickness behav-
ior scores were recorded for each pig following the
collection of each blood sample. Additionally, indi-
vidual pig feeders were weighed on d 0, 12, 18, and
22 to measure average daily feed disappearance. Fecal
samples were collected on d 19 (0 h), 20 (24 h), 21 (48
h) and 22 (72 h) after the challenge to determine fe-
cal shedding of the inoculated Salmonella. Following
collection of the 72 h sample, pigs were humanely eu-
thanized and samples of the cecum, mesenteric lymph
node, subiliac lymph node, liver, spleen, gallbladder
and kidney were collected to detect any Salmonella
translocation into these tissues.
Sickness Behavior Scores
Sickness behavior scores were assigned to each
pig by a single observer skilled at assessing sickness
behavior in cattle and pigs. Pigs assigned a score of
1 exhibited normal maintenance behaviors. Pigs re-

Sexual dimorphic immune response in pigs 71
Translate basic science to industry innovation
ceiving a score of 2 were calm, but were lying down
with increased respiration. A score of 3 was given to
pigs exhibiting clinical signs of sickness including in-
creased respiration with drool, scours, and(or) cough-
ing. If a pig was observed to be lying on its side with
labored breathing and frothing at the mouth it was
given a score of 4, and then was humanely euthanized.
Cortisol Analysis
Serum cortisol concentrations were determined
in duplicate using a commercially available, porcine-
specic enzyme immunoassay kit (Abnova, Walnut,
CA) according to the manufacturer’s directions by
comparison of unknowns to standard curves generated
with known concentrations of cortisol. The minimum
detectable cortisol concentration was 0.2 ng/mL, and
the intra- and inter-assay coecients of variation were
10.1% and 8.2%, respectively.
Salmonella Isolation
Prior to experimental infection, fecal samples
from all pigs were collected to conrm the absence of
Salmonella or the presence of bacteria with similar mor-
phology resistant to nalidixic acid. All fecal and tissue
samples were aseptically collected, weighed (10 g), ho-
mogenized, and placed in a 1:10 dilution of phosphate
buered saline (PBS). Overnight cultures were serially
diluted in PBS and plated on Luria-Bertani (LB) agar
with 50 µg/mL nalidixic acid and manually counted the
following day after incubation for 18 to 24 h at 37°C.
Isolates were conrmed as Salmonella via biochemical
reactions on 4 bacterial test media: Triple Sugar Iron
Slants, Indole Test, Lysine Iron Agar, and Urease Test.
Negative cultures were enriched overnight in tetrathi-
onate broth and re-plated.
Statistical Analysis
Prior to analysis, intraperitoneal tempera-
ture (TEMP) was averaged into 1-h intervals.
Intraperitoneal temperature, serum cortisol, and CBC
data were analyzed using the MIXED procedure of
SAS specic for repeated measures (SAS Inst. Inc.,
Cary, NC). Sex, time and their interaction were in-
cluded as xed eects with pig within sex as the ex-
perimental unit. Specic treatment comparisons were
made using the PDIFF option in SAS, with P ≤ 0.05
considered signicant and P ≤ 0.10 considered a ten-
dency. Salmonella tissue and fecal count data were
analyzed in SAS using Proc Glimmix of SAS at an ɑ
= 0.05 utilizing the Tukey option for mean separation.
Positive/negative binomial data to indicate the pres-
ence or absence of Salmonella in a tissue were ana-
lyzed using logistics regression in Proc Glimmix. All
data are presented as the LSM ± SEM.
RESULTS
There was a sex × time interaction (P < 0.001)
for TEMP. Specically, gilts had greater TEMP than
barrows from 36 to 63 h and at 72 h post-Salmonella
challenge (Fig. 1). Additionally, there were also sex
(P < 0.001) and time (P < 0.001) eects for TEMP.
Intraperitoneal temperature was greater in gilts
(39.72 ± 0.01°C) than barrows (39.61 ± 0.01°C).
Intraperitoneal temperature generally increased from
-144 h (insertion of temperature probes) to 0 h (imme-
diately prior to oral Salmonella challenge; P < 0.001;
-144 h vs. 0 h), and subsequently increased in response
to the Salmonella challenge beginning at 12 h which
persisted through the end of the study (P ≤ 0.02).
Sickness behavior score was measured on pigs
throughout the study. Interestingly, all pigs exhibit-
ed normal maintenance behaviors (i.e., a score of 1)
throughout the entire study. Therefore, no dierences
in sickness behaviors were observed. However, dier-
ences in feed disappearance were observed during the
study. Specically, average daily feed disappearance
increased from d 0 to 12 (0.21 ± 0.02 kg/d) to d 12 to
18 (0.51 ± 0.02 kg/d; P < 0.001), but decreased fol-
lowing Salmonella challenge to the end of the study (d
18 to 22; 0.38 ± 0.02; P < 0.001). There was no eect
of sex (P = 0.38) or sex × time (P = 0.75) on average
daily feed disappearance.
There was a sex × time interaction (P = 0.03) for
serum cortisol concentrations. Specically, cortisol
concentrations were greater in barrows than gilts at
Figure 1. Eect of sex on the intraperitoneal temperature (TEMP)
response to an oral Salmonella challenge (4.7 × 109 cfu/pig Salmonella ty-
phimurium) in weaned pigs. Gilts n = 8, Barrows n = 12. Data presented as
the LSM. SEM is ± 0.13 for Gilts and ± 0.10 for Barrows. There was a sex
× time interaction (P < 0.001) with gilts having greater TEMP response
than barrows from 36 to 63 and at 72 h post-Salmonella challenge.

Burdick Sanchez et al.
72
Translate basic science to industry innovation
16 h (5.6 ± 0.4 vs. 3.8 ± 0.5 ng/mL; P = 0.009), yet
cortisol was greater in gilts than barrows 32 h post
Salmonella challenge (6.4 ± 0.5 vs. 2.9 ± 0.4 ng/mL;
P < 0.001; Fig. 2). Overall, there was no eect of sex
(P = 0.16) but there was an eect of time (P < 0.01)
such that cortisol concentrations decreased below -2 h
values at 3.5 h post-challenge (P = 0.01) and remained
there before increasing above -2 h values at 16 h post-
challenge (P < 0.001) and returning to baseline values
by 64 h post-challenge (P = 0.87; -2 h vs. 64 h).
There was no eect of sex on red blood cells, he-
moglobin, monocytes, or eosinophils (Table 1). There
were sex (P = 0.02) and time (P < 0.001) eects for
hematocrit such that barrows had greater hematocrit
than gilts, and hematocrit decreased over time (Table
1). Also, similar results were found for platelet counts,
such that barrows had greater platelets than gilts (P <
0.001), with counts uctuating over time (P = 0.003;
Table 1). Total white blood cells (WBC) were aect-
ed by sex (P = 0.003; Fig. 3). Specically, barrows
had greater total WBC counts than gilts. There was
a tendency (P = 0.06) for a decrease in WBC follow-
ing Salmonella challenge, and there was no sex × time
interaction (P = 0.49). There was also a sex eect for
neutrophils (P < 0.001) such that barrows had greater
neutrophils than gilts (Fig. 4). Neutrophil counts de-
creased following Salmonella challenge (P < 0.001),
but there was no sex × time interaction (P = 0.55). In
contrast to total WBC and neutrophils, lymphocytes
were greater in gilts than barrows (P = 0.05), yet there
was no eect of time (P = 0.27) or sex × time interac-
tion (P = 0.71; Fig. 5). There was a sex × time interac-
tion (P = 0.002) for basophil counts, with gilts having
greater basophils from 56 to 72 h (P ≤ 0.05; Fig. 6).
There was also an eect of time, with basophil counts
increasing from 56 to 72 h post-Salmonella challenge,
and there was a tendency (P = 0.10) for gilts to have
greater basophil counts than barrows.
Figure 2. Eect of sex on the serum cortisol response to an oral
Salmonella challenge (4.7 × 10
9
cfu/pig Salmonella typhimurium) in weaned
pigs. Gilts n = 8, Barrows n = 12. Data presented as the LSM ± SEM. Sex ×
time interaction (P = 0.03). *Gilts and Barrows dier P ≤ 0.009.
Table 1. Summary of hematology variables measured in gilts and barrows in response to an oral Salmonella
typhimurium challenge
Variable
Sex1
SEM
P-value
Gilts Barrows Sex Time Sex × time
Red blood cells, 106/µL 4.83 4.82 0.07 0.95 < 0.001 1.00
Hemoglobin, g/dL 8.22 8.35 0.06 0.11 < 0.001 0.99
Hematocrit, % 26.91 27.58 0.23 0.02 < 0.001 0.99
Platelets, 103/µL 402.44 446.11 6.41 < 0.001 0.003 0.99
White blood cells, 103/µL 16.20 17.78 0.40 0.003 0.06 0.49
Neutrophils, 103/µL 6.05 7.80 0.40 < 0.001 < 0.001 0.55
Lymphocytes, 103/µL 9.62 9.03 0.24 0.05 0.27 0.71
Monocytes, 103/µL 1.10 1.09 0.05 0.80 < 0.001 0.98
Eosinophils, 103/µL 0.23 0.25 0.01 0.22 < 0.001 0.77
Basophils, 103/µL 0.02 0.01 0.00 0.10 < 0.001 0.002
1Values for sex represent average values for each sex pooled over time.
Figure 3. Eect of sex on the total white blood cell (WBC) response
to an oral Salmonella challenge (4.7 × 109 cfu/pig Salmonella typhimurium)
in weaned pigs. Gilts n = 8, Barrows n = 12. Data presented as the LSM ±
SEM. Barrows had greater WBC counts than gilts (sex: P = 0.003).

Sexual dimorphic immune response in pigs 73
Translate basic science to industry innovation
While many immune parameters were inuenced
by sex, there were no eects of sex (P > 0.05) on
Salmonella concentrations from fecal shedding 4 d
post-inoculation, or within the tissues of the cecum,
mesenteric and subiliac lymph nodes, liver, spleen,
gallbladder, or kidney tissues (Table 2). The only ex-
ception was fecal shedding at 48 h post-inoculation,
which was greater in barrows than gilts (P = 0.05).
DISCUSSION
Sexual dimorphism has been reported in many ani-
mal species, including humans, cattle, dogs and pigs
(Glucksmann, 1974; Williams et al., 2009; Carroll et al.,
2015). Understanding the sexually dimorphic responses
exhibited in livestock, such as pigs, is important as it
may lead to sex-specic treatment or management to
address the potential impacts of sexual dimorphism on
health and subsequently growth. After thorough review
of the literature, we believe this is the rst study to dem-
onstrate sexually dimorphic innate immune responses
to an oral Salmonella typhimurium challenge in pigs.
Body temperature is an important physiological vari-
able that can be used to detect an inammatory response,
and has been used by industry as an indicator of illness
along with behavioral observations. Use of an intraperi-
toneal temperature device in the current study allowed
for the measurement of body temperature at a constant
interval throughout the study without having to handle
the pigs, which can be stressful and further alter body
temperature. Intraperitoneal temperature increased in re-
sponse to the oral Salmonella challenge, but this increase
was not observed until 12 h following the challenge. The
temporal response to Salmonella is similar to what has
been observed previously in pigs, with an increase in body
temperature occurring approximately 12 to 24 h follow-
Figure 4. Eect of sex on the neutrophil response to an oral
Salmonella challenge (4.7 × 109 cfu/pig Salmonella typhimurium) in
weaned pigs. Gilts n = 8, Barrows n = 12. Data presented as the LSM ±
SEM. Barrows had greater neutrophils than gilts (sex: P < 0.001).
Figure 6. Eect of sex on the basophil response to an oral Salmonella
challenge (4.7 × 109 cfu/pig Salmonella typhimurium) in weaned pigs.
Gilts n = 8, Barrows n = 12. Data presented as the LSM ± SEM. Sex ×
time interaction (P = 0.002). *Gilts and Barrows dier P ≤ 0.05.
Figure 5. Eect of sex on the lymphocyte response to an oral
Salmonella challenge (4.7 × 109 cfu/pig Salmonella typhimurium) in
weaned pigs. Gilts n = 8, Barrows n = 12. Data presented as the LSM
± SEM. Lymphocytes were greater in gilts than barrows (sex: P = 0.05).
Table 2. Summary of fecal shedding and tissue
Salmonella counts (log10) in response to an oral
Salmonella typhimurium challenge in weaned pigs
Tissue
Sex1
SEM
Sex eectGilts Barrows
Fecal Shedding 24 h 3.9 4.9 0.64 0.34
Fecal Shedding 48 h 4.8 6.0 0.35 0.05
Fecal Shedding 72 h 4.1 5.2 0.45 0.12
Cecum 4.7 4.2 0.26 0.33
Mesenteric lymph node 3.5 3.5 0.11 0.45
Subiliac lymph node 0.5 0.4 0.29 0.88
Liver 0.5 0.2 0.22 0.49
Spleen 0.6 0.4 0.25 0.60
Gallbladder 0.8 0.4 0.16 0.12
Kidney 0.3 0.2 0.18 0.78
1Values for sex represent average Salmonella counts (log10; log/g) for
each sex.

Burdick Sanchez et al.
74
Translate basic science to industry innovation
ing inoculation (Balaji et al., 2000; Jenkins et al., 2004;
Price et al., 2010). In contrast, there was no change in
body temperature in nishing barrows nasally inoculated
with Salmonella compared to non-challenged controls
(Rostagno et al., 2011), which may be a result of route
of administration or the sampling timeline. This appears
to be the rst study to report a sexually dimorphic tem-
perature response to Salmonella challenge in pigs, with
gilts producing a greater temperature response compared
to barrows. Studies in cattle in response to LPS and CRH
challenges have reported dierences in rectal tempera-
ture responses between heifers and bulls, such that rectal
temperature was greater in heifers than bulls (Hulbert et
al., 2013; Carroll et al., 2015). Additionally, greater corti-
costerone responses to LPS have been reported in female
mice (Spinedi et al., 1992). Changes in body temperature
in response to an infectious agent are regulated by cy-
tokines, including TNF-ɑ, IL-6 and IL-1β. The current
study did not measure serum cytokine concentrations
in response to Salmonella challenge. Although, a study
by Williams et al. (2009) utilizing an LPS challenge in
weaned pigs found greater peak TNF-ɑ and IL-6 concen-
trations in gilts compared to barrows. Perhaps the greater
peak concentrations of these 2 thermogenic cytokines
observed in response to LPS partially explains the greater
intraperitoneal temperature response to Salmonella chal-
lenge in the current study. However, further research is
warranted to determine if sex alters Salmonella-induced
cytokine concentrations in weaned pigs.
It is interesting to note that no dierences were ob-
served in sickness behavior. In fact, there were no chang-
es in behavior outside of normal maintenance behaviors
observed in any of the pigs throughout the post-challenge
period. Typically, animal behavior is used by producers
as an objective measurement when selecting animals for
treatment of an illness or injury. These behaviors include
decreased movement in the pen, decreased feeding and
drinking behavior, and lethargy (Ahmed et al., 2015).
Ahmed et al. (2015) observed decreases in pig move-
ment and feeding and drinking behavior in response to
a Salmonella challenge in growing pigs observed over
a 4 wk period using video monitoring. While the cur-
rent study observed a measureable decrease in average
daily feed disappearance following the challenge, the de-
creased from pre-challenge to post-challenge was mini-
mal (0.13 kg/d), which may not be signicant enough to
be detected by an observer. Yet, feed disappearance was
only measured for a short period following Salmonella
challenge, as pigs were euthanized at 72 h post-challenge,
and sickness behavior was measured at specic time
points throughout the study and did not utilized video
surveillance. However, in the current study it could be
suggested, based on the lack of sickness behaviors, that
these pigs would not be selected for treatment based on
periodically-observed behaviors alone, similar to that
performed in industry, regardless of the variables that in-
deed indicate an active infection (i.e., elevated body tem-
perature and changes in blood cell parameters).
Cortisol concentrations varied between barrows
and gilts at 2 time points in response to Salmonella,
suggesting that barrows had an earlier peak in cortisol
compared to gilts. However, overall there was no sex
eect on serum cortisol concentrations. This is similar
to what was observed by Williams et al. (2009) in re-
sponse to an LPS challenge in pigs. A similar temporal
response was observed in barrows challenged orally
with Salmonella (Balaji et al., 2000). Studies in cattle
did not nd a dierence in serum cortisol between heif-
ers and bulls in response to an LPS challenge (Carroll et
al., 2015), but a greater cortisol response was observed
in response to a CRH challenge (Hulbert et al., 2013).
The temporal pattern relative to the increase in cortisol
concentration follows that of intraperitoneal tempera-
ture such that cortisol concentrations increased at 16 h
post-challenge. Larzul et al. (2015) observed dierenc-
es in cortisol binding globulin (CBG) capacity in pigs,
with a correlation between basal cortisol concentrations
and CBG in males but not in females. Additionally, fe-
male mammals have been noted to have greater adrenal
gland weights (Glucksmann, 1974). However, the dif-
ferences observed in the cortisol response to Salmonella
challenge in the current study were minimal.
White blood cells are the cellular component of the
immune response, and thus changes in these cell popu-
lations within the blood can suggest changes in the re-
sponse to pathogens. In the current study, various dier-
ences in blood cell parameters were observed between
gilts and barrows. This is in contrast to Williams et al.
(2009) who found no dierences in total white blood cell
or dierential counts in response to LPS between bar-
rows and gilts. Perhaps the nature of the live challenge
compared to a more provocative LPS challenge resulted
in the dierences observed between the 2 studies. Similar
results have been observed in cattle, such that Brahman
bulls had greater total leukocytes and neutrophils com-
pared to heifers (Hulbert et al., 2013; Carroll et al., 2015).
Females have been reported to have a heavier spleen and
thymus (Glucksmann, 1974); however, this does not cor-
respond with the decreased total white blood cells and
neutrophils observed in gilts compared to barrows. It is
possible that the decrease in neutrophils is a result of a
greater number of neutrophils leaving the periphery and
entering the gut. A study of neutrophil activity and ad-
hesion molecules would help elucidate if this is indeed
happening. A study in rats and mice found a greater
neutrophil accumulation in tissues following ischemia/
reperfusion, and was believed to be regulated by the che-
mokine Cxcl5, which was able to increase systemic neu-

Sexual dimorphic immune response in pigs 75
Translate basic science to industry innovation
trophils in the absence of ischemia/reperfusion (Madalli
et al., 2015). Thus, perhaps dierences in cytokine and
chemokine concentrations are also driving the dierenc-
es in various immune cell parameters.
Observing almost no dierences in fecal shedding
and tissue Salmonella content was surprising due to
the dierences observed in intraperitoneal temperature,
serum cortisol, and the complete blood counts. It is
possible that although there were dierences in blood
parameters, this did not inuence colonization within
the gastrointestinal system and did not inuence migra-
tion of Salmonella from the gut to peripheral tissues.
It is unclear why fecal shedding was inuenced by sex
48 h following the challenge, but not at any other time
point measured. Further, evidence of sexual dimor-
phism regarding fecal shedding of bacteria is limited.
Reports have indicated a greater incidence of bacterial
infections in males compared to females (Green, 1992;
Strachan et al., 2008). Yet, when experimentally intro-
duced, it appears there is no dierence between weaned
gilts and barrows in the tissue Salmonella content.
There have been many studies in several species
aimed at elucidating sexually dimorphic responses. From
these studies it appears that females are more resilient
to infection, yet are more prone to autoimmune diseases
(Homodelarche et al., 1991; Spitzer, 1999). For example,
female mice of reproductive age appear to have more
active polymorphonuclear leukocytes than male coun-
terparts (Spitzer, 1999). Sex hormones, estrogens and
androgens, have been implicated in these eects. For ex-
ample, Kahl and Elsasser (2006) reported greater serum
TNF-ɑ and serum amyloid A concentrations in steers
administered testosterone 2 d prior to an LPS challenge.
Additionally, heifers in estrus (i.e., low progesterone con-
centrations) had greater mean TNF-ɑ concentrations after
LPS administration compared to heifers in diestrus (i.e.,
high progesterone concentrations). In humans, Dosiou et
al. (2004) reported a decrease in the Th1 response dur-
ing the luteal phase of the menstrual cycle. However it is
interesting to note that there are several studies in which
sexually dimorphic eects were observed in pre-pubertal
animals. Thus, the observed sexually dimorphic eects
are likely initiated earlier in life, and likely in utero and/
or in the early perinatal period. This is supported by re-
search that reports a change in thymus and T lympho-
cyte development when female mice were administered
testosterone early in life (Leposavic et al., 2009). Based
on the ndings within our current study, and that of prior
reports, it is clear that pigs exhibit a sexually dimorphic
immune response which warrants further study.
Conclusion
These data provide further proof that sex can inu-
ence the acute phase immune response to a provocative
immune challenge. Specically, gilts had a greater in-
traperitoneal temperature response, greater lymphocyte
and basophil counts, yet decreased hematocrit, platelets,
total white blood cell, and neutrophil counts than bar-
rows. These data demonstrate that weaned gilts appear to
produce a stronger acute phase response to a Salmonella
challenge compared to barrows, without aecting sick-
ness behavior or the tissue translocation or shedding of
Salmonella. Based on the dierences in cellular immune
and serum variables, but the lack of dierences in sick-
ness behavior, managing gilts and barrows separately
may have signicant impacts on herd health manage-
ment during the weaning/early post-weaning period.
LITERATURE CITED
Ahmed, S. T., H. S. Mun, H. Yoe, and C. J. Yang. 2015. Monitoring
of behavior using a video-recording system for recognition of
Salmonella infection in experimentally infected growing pigs.
Animal 9:115–121. doi:10.1017/S1751731114002213
Balaji, R., K. J. Wright, C. M. Hill, S. S. Dritz, E. L. Knoppel, and J.
E. Minton. 2000. Acute phase responses of pigs challenged oral-
ly with Salmonella typhimurium. J. Anim. Sci. 78:1885–1891.
doi:10.2527/2000.7871885x
Burdick, N. C., J. P. Banta, D. A. Neuendor, J. C. White, R. C.
Vann, J. C. Laurenz, T. H. Welsh, and R. D. Randel. 2009.
Interrelationships among growth, endocrine, immune, and tem-
perament variables in neonatal Brahman calves. J. Anim. Sci.
87:3202–3210. doi:10.2527/jas.2009-1931
Carroll, J. A., N. C. Burdick, R. R. Reuter, C. C. Chase, Jr., D. E.
Spiers, J. D. Arthington, and S. W. Coleman. 2011. Dierential
acute phase immune responses by Angus and Romosinuano
steers following an endotoxin challenge. Domest. Anim.
Endocrinol. 41:163–173. doi:10.1016/j.domaniend.2011.06.002
Carroll, J. A., N. C. Burdick Sanchez, L. E. Hulbert, M. A. Ballou, J.
W. Dailey, L. C. Caldwell, R. C. Vann, T. H. Welsh Jr., and R.
D. Randel. 2015. Sexually dimorphic innate immunological re-
sponses of pre-pubertal Brahman cattle following an intravenous
lipopolysaccharide challenge. Vet. Immunol. Immunopathol.
166:108–115. doi:10.1016/j.vetimm.2015.06.009
Carroll, J. A., J. A. Daniel, D. H. Keisler, and R. L. Matteri. 1999.
Non-surgical catheterization of the jugular vein in young pigs.
Lab. Anim. 33:129–134. doi:10.1258/002367799780578345
Dosiou, C., R. B. Lathi, S. Tulac, S. T. J. Huang, and L. C. Giudice.
2004. Interferon-related and other immune genes are down-
regulated in peripheral blood leukocytes in the luteal phase of
the menstrual cycle. J. Clin. Endocrinol. Metab. 89:2501–2504.
doi:10.1210/jc.2003-031647
Glucksmann, A. 1974. Sexual Dimorphism in Mammals. Biol.
Rev. Camb. Philos. Soc. 49:423–475. doi:10.1111/j.1469-
185X.1974.tb01171.x
Green, N. S. 1992. The Male Predominance in the Incidence of
Infectious-Disease in Children: A Postulated Explanation for
Disparities in the Literature. Int. J. Epidemiol. 21:381–386.
doi:10.1093/ije/21.2.381

Burdick Sanchez et al.
76
Translate basic science to industry innovation
Homo-Delarche, F., F. Fitzpatrick, N. Christe, E. A. Nunez, J. F.
Bach, and M. Dardenne. 1991. Sex Steroids, Glucocorticoids,
Stress and Autoimmunity. J. Steroid Biochem. Mol. Biol.
40:619–637. doi:10.1016/0960-0760(91)90285-D
Hulbert, L. E., J. A. Carroll, M. A. Ballou, N. C. Burdick, J. W. Dailey,
L. C. Caldwell, A. N. Loyd, R. C. Vann, T. H. Welsh, Jr., and R.
D. Randel. 2013. Sexually dimorphic stress and pro-inammato-
ry cytokine responses to an intravenous corticotropin-releasing
hormone challenge of Brahman cattle following transportation.
Innate Immun. 19:378–387. doi:10.1177/1753425912462752
Jenkins, N. L., J. L. Turner, S. S. Dritz, S. K. Durham, and J. E.
Minton. 2004. Changes in circulating insulin-like growth
factor-I, insulin-like growth factor binding proteins, and
leptin in weaned pigs infected with Salmonella enterica se-
rovar Typhimurium. Domest. Anim. Endocrinol. 26:49–60.
doi:10.1016/j.domaniend.2003.09.001
Kahl, S., and T. H. Elsasser. 2006. Exogenous testosterone modulates
tumor necrosis factor-alpha and acute phase protein respons-
es to repeated endotoxin challenge in steers. Domest. Anim.
Endocrinol. 31:301–311. doi:10.1016/j.domaniend.2005.11.005
Larzul, C., E. Terenina, A. Foury, Y. Billon, I. Louveau, E. Merlot,
and P. Mormede. 2015. The cortisol response to ACTH in pigs,
heritability and inuence of corticosteroid-binding globulin.
Animal 9:1929–1934. doi:10.1017/S1751731115001767
Leposavić, G., M. Perisic, D. Kosec, N. Arsenovic-Ranin, K.
Radojevic, Z. Stojic-Vukanic, and I. Pilipovic. 2009. Neonatal
testosterone imprinting aects thymus development and leads
to phenotypic rejuvenation and masculinization of the periph-
eral blood T-cell compartment in adult female rats. Brain Behav.
Immun. 23:294–304. doi:10.1016/j.bbi.2008.11.002
Madalli, S., M. Beyrau, J. Whiteford, J. Duchene, I. S. Nandhra, N.
S. A. Patel, M. P. Motwani, D. W. Gilroy, C. Thiemermann, S.
Nourshargh, and R. S. Scotland. 2015. Sex-specic regulation
of chemokine Cxcl5/6 controls neutrophil recruitment and tis-
sue injury in acute inammatory states. Biol. Sex Dier. 6:27.
doi:10.1186/s13293-015-0047-5
Ojha, S., and M. Kostrzynska. 2007. Approaches for reducing
Salmonella in pork production. J. Food Prot. 70:2676–2694.
Price, K. L., H. R. Totty, H. B. Lee, M. D. Utt, G. E. Fitzner, I. Yoon,
M. A. Ponder, and J. Escobar. 2010. Use of Saccharomyces cere-
visiae fermentation product on growth performance and micro-
biota of weaned pigs during Salmonella infection. J. Anim. Sci.
88:3896–3908. doi:10.2527/jas.2009-2728
Rostagno, M. H., S. D. Eicher, and D. C. Lay. 2011. Immunological,
Physiological, and Behavioral Eects of Salmonella enterica
Carriage and Shedding in Experimentally Infected Finishing
Pigs. Food. Pathog. Dis. 8:623–630. doi:10.1089/fpd.2010.0735
Spinedi, E., M. O. Suescun, R. Hadid, T. Daneva, and R. C. Gaillard.
1992. Eects of Gonadectomy and Sex-Hormone Therapy on
the Endotoxin-Stimulated Hypothalamo-Pituitary-Adrenal
Axis- Evidence for a Neuroendocrine-Immunological Sexual
Dimorphism. Endocrinology 131:2430–2436. doi:10.1210/
endo.131.5.1330501
Spitzer, J. A. 1999. Gender dierences in some host defense mecha-
nisms. Lupus 8:380–383. doi:10.1177/096120339900800510
Strachan, N. J. C., R. O. Watson, V. Novik, D. Hofreuter, I. D.
Ogden, and J. E. Galan. 2008. Sexual dimorphism in campy-
lobacteriosis. Epidemiol. Infect. 136:1492–1495. doi:10.1017/
S0950268807009934
Williams, P. N., C. T. Collier, J. A. Carroll, T. H. Welsh, and J. C.
Laurenz. 2009. Temporal pattern and eect of sex on lipopoly-
saccharide-induced stress hormone and cytokine response in
pigs. Domest. Anim. Endocrinol. 37:139–147. doi:10.1016/j.
domaniend.2009.04.004