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Assessment of the cytokine profile in peripheral blood mononuclear cells of

naturally Sarcoptes scabiei var. canis infested dogs

Article  in  Veterinary Parasitology · October 2014

DOI: 10.1016/j.vetpar.2014.10.024

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 Veterinary Parasitology 206 (2014) 253–257

Contents lists available at ScienceDirect

Veterinary Parasitology
journal homepage: www.elsevier.com/locate/vetpar

Assessment of the cytokine profile in peripheral blood

mononuclear cells of naturally Sarcoptes scabiei var. canis
infested dogs
Shanker K. Singh a,∗ , Umesh Dimri a , Bhaskar Sharma b , Meeta Saxena b ,
Priyambada Kumari a
a
Division of Medicine, Indian Veterinary Research Institute, Izatnagar, 243 122 Bareilly, U.P., India
b
Division of Biochemistry, Indian Veterinary Research Institute, Izatnagar, 243 122 Bareilly, U.P., India

a r t i c l e i n f o a b s t r a c t

Article history: The mechanism of cytokine secretion from T lymphocytes plays an important role in the
Received 6 August 2014 immune response of dogs and parasitic skin infestations. Assessment of the cytokine profile
Received in revised form 5 October 2014 of naturally S. scabiei var. canis infested dogs could augment understanding of the patho-
Accepted 7 October 2014
biology of canine sarcoptic mange. Therefore, the present study examined the cytokines in
peripheral blood mononuclear cells of dogs suffering from sarcoptic mange. Thirteen dogs
Keywords: naturally infected with sarcoptic mange participated in the study. The dogs were found
Cytokine
positive for S. scabiei var. canis mites in skin scraping examinations and revealed at least
Dogs
three clinical inclusion criteria. Another five clinically healthy dogs were kept as healthy
IL-4
IL-5 controls. Peripheral blood mononuclear cells were isolated from heparinized blood sam-
Sarcoptic mange ples and used for extraction of mRNA. Further, cDNA was synthesized by using 1 mg of
TGF-␤ mRNA by reverse transcription using oligonucleotide primers. Relative levels of cytokine
expression were compared with normalized glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) transcripts. The levels of interleukin-4, interleukin-5 and transforming growth
factor beta (TGF-␤) mRNA expression in dogs with sarcoptic mange were significantly
higher (P ≤ 0.01), whereas the level of tumor necrosis factor alpha (TNF-␣) was signifi-
cantly lower (P ≤ 0.01) in comparison with the healthy dogs. No remarkable difference was
seen for interleukin-2 mRNA expression between these animals. An overproduction IL-4
and IL-5 might be involved in immuno-pathogenesis of canine sarcoptic mange. S. scabiei
var. canis mites possibly induce an overproduction of TGF-␤ and reduced expression of
TNF-␣ and thus could be conferring the immune suppression of infested dogs.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction including dogs. In most animal species, prevalence of sar-

Sarcoptes scabiei mite infestation is a highly contagious
and pruritic skin disease, affecting a broad host range
∗ Corresponding author. Present address: Department of Veterinary
Clinical Medicine, College of Veterinary Science and Animal Husbandry,
DUVASU, Mathura 281 001, U.P., India.
E-mail address: pshankervet@gmail.com (S.K. Singh).
coptic mange is very high and untreated animals often
succumb (Giadinis et al., 2011). Presently it is considered as
an emerging/re-emerging parasitic disease that threatens
human and animal health globally (Alasaad et al., 2013).
It is mainly characterized by intense pruritus associated
with a vesiculo-papular eruption and pinpoint crusts in
combination with alopecia (Pin et al., 2006). The parasite
has had a long co-evolution with its mammalian hosts
and is capable of modulating many aspects of the innate

http://dx.doi.org/10.1016/j.vetpar.2014.10.024
0304-4017/© 2014 Elsevier B.V. All rights reserved.
254 S.K. Singh et al. / Veterinary Parasitology 206 (2014) 253–257
and adaptive protective responses of their host enabling
them to survive and thrive (Morgan et al., 2013). An abun-
dance of macrophage migration inhibitory factor in scabies
mites has been recently demonstrated and considered to
aid the parasite to evade the innate and adaptive immune
responses in the host (Cote et al., 2013). Recently, it has
been demonstrated that S. scabiei is the source of molecules
that can downregulate the secretion of selected cytokines
and chemokines and expression of adhesion molecules by
resident and infiltrating cells of the epidermis and dermis,
and this may contribute to the initial delay in inflammatory
and immune responses to the scabies mite (Arlian et al.,
1996, 2009; Elder et al., 2006; Walton, 2010). Moreover,
the burrowing of live scabies mites in keratinocytes and
fibroblasts in human skin equivalents regulate large num-
bers of genes that are related to host protective responses
including those involved in the immune response, defense
response, cytokine activity, taxis, response to other orga-
nisms, and cell adhesion (Morgan et al., 2013).
Functional integration of the immune system is accom-
plished mainly by cell-to-cell communication that relies
on small molecules cytokines (Kidd, 2003). Based on the
kind of cytokine production, fully differentiated T helper
(Th) lymphocytes are divided into at least two distinct
subsets. Th1 lymphocytes were characterized by the pro-
duction of interferon-gamma (IFN-␥) and interleukin-2
(IL-2) cytokines. On the other hand, Th2 lymphocytes are
characterized by the production of interleukin-4 (IL-4) and
interleukin-5 (IL-5) concerned with the humoral immune
response. The mechanism of cytokine secretion from T lym-
phocytes plays an important role in the immune response
of the dogs against parasitic skin infestations (Arlian et al.,
1996; Tani et al., 2002; Felix et al., 2013; Yarim et al., 2013;
Singh and Dimri, 2014). Scientific reports demonstrating
the association of the host’s cytokine expression to S. scabiei
var. canis mite infestation in dogs are meager. An assess-
ment of the cytokine profile of naturally S. scabiei var. canis
infested dogs could augment understanding of the patho-
biology of canine sarcoptic mange. Thus, in the present
study we aimed to evaluate the cytokine profile in periph-
eral blood mononuclear cells of dogs naturally suffering
from sarcoptic mange.
2. Materials and methods
2.1. Animal selection criteria and design of the study
The dogs enrolled in the study were recruited among the
patients presented for clinical and dermatological exami-
nation. Thirteen dogs (1–4 years of age) naturally infested
with S. scabiei var. canis participated in the study. Dogs
were reported to be suffering from the clinical disease
for at least 15 days before presentation. None of the
dogs was treated with ectoparasiticides or steroidal anti-
inflammatory drugs in the last 30 days before obtaining
blood samples. The diseased dogs were also free from mites
apart from the S. scabiei var. canis mite infestation. Another
five dogs (1–4 years of age) clinically healthy and free of
mites in microscopic examination of material from up to
six deep skin scrapings were kept as healthy controls.
Clinical diagnosis of sarcoptic mange was based on the
following six clinical inclusion criteria: (1) intense pruri-
tus, (2) papular eruption (abdomen, inner leg region and
thorax), (3) self-trauma, (4) excoriations, (5) alopecia, and
(6) crusting of the elbow, ear margins and hocks. All dis-
eased dogs participated in the study were found positive
for S. scabiei var. canis mite in microscopic examination of
material from up to six deep skin scrapings and revealed at
least three clinical inclusion criteria. Approximately 5 ml of
blood samples were obtained from each dog in a tube con-
taining heparin and used for isolation of peripheral blood
mononuclear cells (PBMC).
2.2. Extraction of mRNA from PBMC and cDNA synthesis
PBMC were isolated from heparinized collected blood
samples diluted twofold with Dulbecco’s phosphate-
buffered saline (PBS) using HISTOPAQUE® solution
(s.g. = 1.077 g ml−1 , Sigma–Aldrich Co., UK) density gradi-
ent centrifugation at 350 × g for 45 min. Isolated cells were
treated for 10 min with 17 mM Tris-HCL, containing 0.75%
ammonium chloride (pH 7.2) to lyse any contaminating
erythrocytes, and then were washed twice with PBS (pH
7.4). Total mRNA was extracted from PBMC according to
the manufacturer’s recommendations using TRIzol® (Invi-
trogen). Synthesis of cDNA was performed by using 1 mg of
mRNA that was reverse transcribed with oligonucleotide
primers according to the manufacturer’s instructions.
2.2.1. Oligonucleotide primers
The primers of canine IL-2, IL-4, IL-5, TNF-␣, TGF-␤ and
the housekeeping protein glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) used as reported by Tani et al.
(2002) were as follows:
IL-2 Sense 5 -GTGCGCCTATTACTTCAAGCTC-3 405 bp
Anti-sense 5 -GCTGTCTCGTCATCATATTC-3
IL-4 Sense 5 -ATGGGTCTCACCTCCCAACTG-3 399 bp
Anti-sense 5 -TCAATGCCTGTAGTATTTCTTC-3
IL-5 Sense 5 -GGTGAAAGAGACCTTGGCAC-3 305 bp
Anti-sense 5 -CACTCGGTGTTCATTACACC-3
TNF-␣ Sense 5 -CCAAGTGACAAGCCAGTAGC-3 274 bp
Anti-sense 5 -CCAAGTGACAAGCCAGTAGC-3
TGF-␤ Sense 5 -TTCCTGCTCCTCATGGCCAC-3 393 bp
Anti-sense 5 -GCAGGAGCGCACGATCATGT-3
GAPDH Sense 5 -GCCAAAAGGGTCATCATCTC-3 229 bp
Anti-sense 5 -GGCCATCCACAGTCTTCT-3
2.2.2. Reverse transcription polymerase chain reaction
(RT-PCR)
10 ␮l of mRNA was reversed with murine reverse
transcriptase following the manufacturer’s protocol (Fer-
mentas) and method suggested by Tani et al. (2002). The
stability of primers for PCR, and the appropriate conditions
were established using RNA isolated from PBMC. Primers
used for PCR are summarized above. PCR was performed
using the appropriate cycles (1 min at 94 ◦ C, 1 min at 59.5 ◦ C
for TNF-␣ and TGF-␤, or 1 min at 94 ◦ C, 1 min at 57 ◦ C, fol-
lowed by 1.5 min at 72 ◦ C for the other cytokines) within the
linear amplification range as follows: 30 cycles for GAPDH,
40 cycles for TNF-␣ and TGF-␤ and 45 cycles for the other
cytokines. Samples from PCR reactions were separated by
electrophoresis through a 2.5% TAE agarose gel. The gel was
 S.K. Singh et al. / Veterinary Parasitology 206 (2014) 253–257 255

Table 1
The ratios of cytokine to GADPH expression in peripheral blood of healthy and infested dogs.

Groups Dogs IL-2 IL-4 IL-5 TNF-␣ TGF-␤

Diseased dogs DD1 0.92 0.18 0.43 0.45 1.74

(DD) DD2 0.93 0.17 0.41 0.54 1.79
DD3 0.95 0.17 0.44 0.49 1.73
DD4 0.89 0.16 0.45 0.48 1.58
DD5 0.98 0.14 0.46 0.49 1.82
DD6 0.94 0.17 0.50 0.46 1.83
DD7 0.99 0.18 0.44 0.53 1.88
DD8 0.94 0.19 0.43 0.53 1.85
DD9 0.93 0.17 0.49 0.48 1.75
DD10 0.89 0.19 0.48 0.52 1.77
DD11 0.94 0.16 0.48 0.51 1.74
DD12 0.97 0.17 0.49 0.55 1.79
DD13 0.94 0.18 0.48 0.44 1.73

Mean ± S.D 0.94 ± 0.03 0.17 ± 0.013a 0.46 ± 0.029a 0.498 ± 0.035b 1.76 ± 0.07a

Healthy dogs HD1 0.85 0.08 0.21 1.31 1.19

(HD) HD2 0.87 0.09 0.24 1.29 1.19
HD3 0.88 0.07 0.23 1.19 1.17
HD4 0.89 0.09 0.22 1.23 1.17
HD5 0.86 0.08 0.22 1.14 1.18

Mean ± S.D 0.87 ± 0.02 0.08 ± 0.008 0.22 ± 0.011 1.23 ± 0.07 1.18 ± 0.01
a
Significantly higher (P ≤ 0.01), when compared with the values of healthy dogs.
b
Significantly lower (P ≤ 0.01), when compared with the values of healthy dogs.

stained with ethidium bromide and DNA was quantified
by densitometry. Relative levels of the cytokine expres-
sion were compared with normalized GAPDH transcripts
and presented as ratio of the band intensities of cytokine
RT-PCR amplification products.
2.3. Statistical analysis
For statistical analysis, the t-test was used to determine
the difference between the diseased and healthy dogs. The
level of statistical significance for all the comparisons was
established at P ≤ 0.05. All data were analyzed by means
of the statistical package SPSS 15 (SPSS Sciences, Chicago,
USA).
3. Results
The ratios of cytokine to GADPH expression in periph-
eral blood of healthy and infested dogs are summarized in
Table 1. All infested and healthy dogs used in the study
revealed positive expression for all the studied cytokines
in their PBMC (Fig. 1). The mean values of IL-4, IL-5 and
TGF-␤ mRNA levels in dogs with sarcoptic mange were sig-
nificantly higher (P ≤ 0.01) than the mean values of theses
cytokines in PBMC of healthy dogs. In contrast, the mean
values of mRNA expression for TNF-␣ levels in dogs with
sarcoptic mange were significantly lower (P ≤ 0.01) than
the mean values of healthy dogs. The values of IL-2 levels
of diseased dogs did not differ significantly from healthy
dogs.
not treated with corticosteroid before taking the blood
sample; thus the results of the current study provide S.
scabiei var. canis induced cytokine expression solely. The
mechanism of cytokine secretion from T lymphocytes plays
important roles in immune response of the host against
the mange mites (Arlian et al., 1996, 2009; Elder et al.,
2006; Walton, 2010; Singh and Dimri, 2014). Th1 and
Th2 cells seem to moderate virtually all the known pat-
terns of the immune response. Th1 cells are hypothesized
to lead the attack against intracellular pathogens such
as viruses, raise the classic delayed-type hypersensitivity
(DTH) skin response to viral and bacterial antigens, and
fight cancer cells. Th2 cells are believed to provide protec-
tion against extracellular pathogens such as multicellular
parasites (Kidd, 2003).
It has been demonstrated that live scabies mites pro-
duce substances that can down-regulate expression of
adhesion molecules, cytokines, chemokines, chemokine

4. Discussion

The present study accurately demonstrates mRNA Fig. 1. mRNA expression of cytokines in PBMC of a dog with sarcoptic
expression of cytokine in PBMC of dogs directly ex vivo mange. M: Marker, Lane 1: GAPDH, Lane 2: IL-2, Lane 3: IL-4, Lane 4: IL-5,
without in vitro stimulation. Moreover, diseased dogs were Lane 5: TGF-␤, Lane 6: TNF-␣.
256 S.K. Singh et al. / Veterinary Parasitology 206 (2014) 253–257
receptors, and lymphocyte surface molecules involved in
leukocyte sequestering and the interaction of B and T cells
during activation of an immune response in the spleen
(Arlian et al., 2007). An over-secretion of IL-4 and IL-5
cytokines in dogs with sarcoptic mange clearly indicates a
Th2 mediated immune response against S. scabiei var. canis
infestation in dogs. Interleukin-4 and probably other Th2
cytokines, help recruit B cells, mast cells and eosinophils, all
of which can produce IgE antibodies, which play a central
role in induction of allergic symptomatology (Maggi, 1998).
Additionally, IL-5 generated by allergen-reactive Th2 cell
attracts and activates eosinophils. Thus, severe pruritus in
canine sarcoptic mange could be the result of overproduc-
tion of IL-4 and IL-5 cytokines, conferring recruitment of
these immune cells and thus elevated IgE antibodies in the
affected dogs. Moreover, IL-4 produced from mature Th2
cells in collaboration with the other participating cell types
generates an autocrine loop to the naive T cells to make
more Th2 cells. In accordance with the result of the cur-
rent study, an overproduction of Th2 specific cytokines and
increased allergic immune response have been reported by
the previous scientific works in human patients infested
with sarcoptes mites (Mounsey et al., 2013; Walton et al.,
2008, 2010; Walton, 2010). Thus, Th2 cells dominance
could be a major factor in immunopathology of canine sar-
coptic mange.
The results of the present study noticeably demon-
strate regulated expression of TNF-␣ in dogs with sarcoptic
mange. TNF-␣ is a pleiotropic pro-inflammatory cytokine
that exerts multiple biological effects. At low level expres-
sion of TNF-␣ participates in beneficial tissue remodeling
and host defense response. Reduces expression of TNF-
␣ in dogs with sarcoptic mange could be helping over
proliferation of mite and flare-up of the clinical disease.
Some of the immune cells said to be regulatory T cells (Tr
cells) can intervene to block either Th1 or Th2 activity or
both. Type 3 (Tr3) cells primarily secrete TGF-␤ and inhibit
immune responses through cell-to-cell contact. Results of
the current study demonstrate an over production of TGF-
␤ in dogs with sarcoptic mange. TGF-␤ is the prototypical
member of a superfamily of pleiotropic cytokines, which
regulate a multitude of biological processes including
tissue homeostasis, angiogenesis, migration and differ-
entiation. TGF-␤ acts as a potent immunosuppressor by
regulating the proliferation and survival of many cells of
the immune system. Part of the homeostatic function of
TGF-␤ is the cell type-specific induction of apoptosis, which
occurs in several cell types including B cells. In normal
circumstances, TGF-␤ is a cytokine responsible for homeo-
stasis preservation, inducing cell cycle arrest and apoptosis
in different organs including the liver (Siegel and Massagué,
2003). Recently, mites were shown to induce an over pro-
duction of immunosuppressive cytokines such as IL-10 and
TGF-␤ and hastened apoptosis of immune cells, responsi-
ble for depressing the host’s protective response allowing
these mites to survive in the skin (Singh et al., 2011;
Cote et al., 2013; Morgan et al., 2013; Shang et al., 2014).
The present study clearly demonstrates an over produc-
tion of TGF-␤ and thus its role in Sarcoptes mite induced
immune dysfunction conferring a state of immunosuppres-
sion could be anticipated.
5. Conclusion
An overproduction of Th2 specific cytokines, IL-4 and
IL-5, might be involved in immuno-pathogenesis of canine
sarcoptic mange. Additionally, S. scabiei var. canis mites
possibly induce an over expression of TGF-␤ and reduced
expression of TNF-␣ and thus can confer immuno suppres-
sion of the infested dogs. This could be the strategies of
mites to evade the immune activities of the host and thus
flare-up of the clinical condition.
Acknowledgment
We are highly thankful to the Director of IVRI for grant-
ing funds and facilities for conducting this study.
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