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Article
Characterization of a Novel Cysteine Protease Inhibitor from
Poultry Red Mites: Potential Vaccine for Chickens
Sotaro Fujisawa 1, Shiro Murata 1, 2, * , Masayoshi Isezaki 1, Takuma Ariizumi 1,3, Takumi Sato 4, Eiji Oishi 4,
Akira Taneno 4, Naoya Maekawa 2, Tomohiro Okagawa 2, Osamu Ichii 5,6 , Satoru Konnai 1,2
and Kazuhiko Ohashi 1,2
Citation: Fujisawa, S.; Murata, S.;
Isezaki, M.; Ariizumi, T.; Sato, T.;
Oishi, E.; Taneno, A.; Maekawa, N.;
Okagawa, T.; Ichii, O.; et al.
Characterization of a Novel Cysteine
Protease Inhibitor from Poultry Red
Mites: Potential Vaccine for Chickens.
Vaccines 2021,9, 1472. https://
doi.org/10.3390/vaccines9121472
Academic Editors: Kim Halpin and
Charles El-Hage
Received: 24 November 2021
Accepted: 9 December 2021
Published: 13 December 2021
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Attribution (CC BY) license (https://
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4.0/).
1Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Kita 18, Nishi 9,
Kita-ku, Sapporo 060-0818, Japan; s.fujisawa@vetmed.hokudai.ac.jp (S.F.);
m-isezak@photon.chitose.ac.jp (M.I.); ariizumi@czc.hokudai.ac.jp (T.A.); konnai@vetmed.hokudai.ac.jp (S.K.);
okazu@vetmed.hokudai.ac.jp (K.O.)
2Department of Advanced Pharmaceutics, Faculty of Veterinary Medicine, Hokkaido University,
Sapporo 060-0818, Japan; maekawa@vetmed.hokudai.ac.jp (N.M.); okagawa@vetmed.hokudai.ac.jp (T.O.)
3Division of Molecular Pathology, International Institute of Zoonosis Control, Hokkaido University,
Sapporo 001-0020, Japan
4Vaxxinova Japan K.K., Tokyo 105-0013, Japan; t-sato@vaxxinova.co.jp (T.S.); e-oishi@vaxxinova.co.jp (E.O.);
a-taneno@vaxxinova.co.jp (A.T.)
5Department of Basic Veterinary Science, Faculty of Veterinary Medicine, Hokkaido University,
Sapporo 060-0818, Japan; ichi-o@vetmed.hokudai.ac.jp
6
Laboratory of Agrobiomedical Science, Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
*Correspondence: murata@vetmed.hokudai.ac.jp; Tel.: +81-11-706-5274; Fax: +81-11-706-5217
Abstract:
Poultry red mite (PRM; Dermanyssus gallinae) is a hazardous, blood-sucking ectoparasite of
birds that constitutes a threat to poultry farming worldwide. Acaricides, commonly used in poultry
farms to prevent PRMs, are not effective because of the rapid emergence of acaricide-resistant PRMs.
However, vaccination may be a promising strategy to control PRM. We identified a novel cystatin-like
molecule in PRMs: Dg-Cys.Dg-Cys mRNA expression was detected in the midgut and ovaries, in
all stages of life. The PRM nymphs that were artificially fed with the plasma from chickens that
were immunized with Dg-Cys
in vitro
had a significantly reduced reproductive capacity and survival
rate. Moreover, combination of Dg-Cys with other antigen candidates, like copper transporter 1 or
adipocyte plasma membrane-associated protein, enhanced vaccine efficacies. vaccination and its
application as an antigen for cocktail vaccines could be an effective strategy to reduce the damage
caused by PRMs in poultry farming.
Keywords: Dermanyssus gallinae; poultry red mite; cystatin; vaccine; cocktail vaccine
1. Introduction
Poultry red mites (Dermanyssus gallinae, PRM) are blood-sucking ectoparasites that
infest chickens. Mass infestation by PRMs negatively affects chickens in various ways,
causing such conditions as anemia and depression, resulting in significant losses of the
productivity in poultry farming. Since continuous use of chemical acaricides, which are
commonly used to prevent PRM infestations, could cause various problems such as the
selection of drug-resistant PRMs and the contamination of products with acaricides, an
alternative control method is needed. Over the past few years, vaccination has been in the
spotlight as a novel strategy for the control of ectoparasites, including PRMs, and several
studies have examined antigen candidates for their efficacies [
1
–
5
]. However, adequate
anti-PRM efficacy has not been reported through practical applications in the field [
6
].
Thus, vaccination strategies need to be improved, while more effective vaccine antigens
need to be explored to increase vaccine efficacy. As for the target molecules of vaccines
against ectoparasites, two types of proteins may be considered: proteins secreted into the
Vaccines 2021,9, 1472. https://doi.org/10.3390/vaccines9121472 https://www.mdpi.com/journal/vaccines
Vaccines 2021,9, 1472 2 of 17
parasite’s saliva that facilitate attachment to the host and blood sucking by mitigating host
immune responses, and proteins expressed in the parasite’s midgut, which is the middle
part of the digestive tract. Since the midgut is the reservoir for ingested blood, the latter
proteins could be frequently and efficiently exposed to the antibodies found in the blood
obtained from immunized animals. For blood feeding, tick species infest hosts for long
periods (for three to ten days or more [
7
]). Meanwhile, PRMs infest chickens within a few
minutes to one hour and do not stay on the chicken’s body after blood sucking. In addition,
PRMs are intermittent feeders that repetitively suck blood in their life cycle [
8
]. Therefore,
midgut proteins may be more appropriate as vaccine antigens against PRMs than salivary
gland proteins.
Cystatins are reversible inhibitors of papain-like cysteine proteases and legumains [
9
]
and regulate various biological processes, including innate immunity, epidermal home-
ostasis, and apoptosis in vertebrates [
10
–
12
]. Similarly, cystatins play crucial roles in the
lives of parasites, and bmcystatin isolated from Rhipicephalus microplus is involved in em-
bryogenesis, as it inhibits vitelline-degrading peptidase [
13
]. In addition, cystatins found
in Haemaphysalis longicornis and Ornithodoros moubata regulate blood digestion, heme detox-
ification, and tick innate immunity [
14
–
16
]. The knockdown of cystatins by gene silencing,
or the blockade of cystatin activities using specific antibodies, significantly reduces the
efficiency of blood feeding in Amblyomma Americanum [
17
] and increases mortality rates
in Ornithodoros moubata [
18
]. Collectively, cystatins are indispensable for hematophagous
ectoparasites; hence, these molecules could be effective antigens for vaccines.
Vaccine antigen targets should be highly and constitutively expressed or at least be
expressed in blood-fed PRMs. To investigate such molecules, we previously conducted
a comparative analysis of the transcriptome of blood-fed and starved PRMs [
19
] and
identified a novel cystatin-like transcript, Dg-Cys, that was highly expressed in both
blood-fed and starved states. In this study, we characterized the full-length sequence of
Dg-Cys and evaluated the physiological functions of Dg-Cys along with its potential as a
vaccine antigen. In addition, we assessed the acaricidal effects of mixed plasmas containing
antibodies against multiple antigens (including Dg-Cys) to evaluate the anti-PRM properties
of the “cocktail vaccine”.
2. Materials and Methods
2.1. Ethics Approval and Consent of Participants
All animal experiments were approved by the Institutional Animal Care and Use
Committee, Hokkaido University (Approval number: 20–0051). Moreover, all experiments
were performed in accordance with relevant guidelines and regulations of the Faculty of
Veterinary Medicine, Hokkaido University, which has been enacted confirming to the AR-
RIVE guidelines and fully accredited by the Association for Assessment and Accreditation
of Laboratory Animal Care International (AAALAC).
2.2. PRM Samples
PRM samples were prepared as described previously [
4
,
5
,
19
]. PRMs of mixed develop-
mental stages and sexes were obtained from an egg-laying farm in Japan. PRMs were stored
in a TubeSpin Bioreactor 600 (TPP Techno Plastic Products AG, Trasadingen, Switzerland).
A part of the dark red, round PRMs was designated as “blood-fed PRMs” and collected
using 1200
µ
L extra-long filter tips (WATSON Bio Lab, Tokyo, Japan) within
2 days
of
sample collection. The remaining PRMs were maintained at 25
◦
C in 70% humidity for
a 2-week period and designated as “starved PRMs.” Some of the blood-fed and starved
PRMs were fixed with 70% ethanol, and then eggs, larvae, protonymphs, deutonymphs,
and adults were segregated through microscopic observation. The residual PRMs were
stored at −80 ◦C until use.
Vaccines 2021,9, 1472 3 of 17
2.3. RNA Isolation and cDNA Synthesis
RNA isolation and cDNA synthesis from PRM samples were performed as described
previously [
4
,
5
]. Each collected PRM sample was suspended in 600
µ
L of Buffer RLT Plus
(RNeasy Plus Mini Kit) (Qiagen, Hilden, Germany) and homogenized thoroughly using
a 1.5-mL Homogenization Pestle for a 1.5-mL microcentrifuge tube (Scientific Specialties,
Inc., Lodi, CA, USA). Total RNA was isolated using the RNeasy Plus Mini Kit according
to the manufacturer’s instructions. cDNA was synthesized from the isolated RNA using
PrimeScript Reverse Transcriptase (Takara Bio Inc., Shiga, Japan) using 200 pmol of oligo
(dT) 18 primer (Hokkaido System Science, Hokkaido, Japan).
2.4. Identification of the Full-Length Nucleotide Sequence of Dg-Cys in PRMs
A novel cystatin-like transcript, Dg-Cys, was previously identified by RNA-Seq
analysis [
19
] (BioSample accession number: SAMD00228960, SAMD00229086). For identi-
fying the full-length nucleotide sequence of the cDNA encoding Dg-Cys transcript, rapid
amplification of cDNA ends (RACE) was performed using total RNA isolated from blood-
fed PRMs and 5
0
and 3
0
RACE systems (Invitrogen, Carlsbad, CA, USA) according to the
manufacturer’s instructions. The following primers were designed and used: For 5
0
RACE:
GSP1, 5
0
-ACC GAT CAC TAC AAA CTG CA-3
0
; GSP2, 5
0
-CAA CAG CAG CAG CAG
TAC GA-3
0
; GSP3, 5
0
-AGT ACG AAT AAA CAC GCG CG-3
0
. For 3 RACE: GSP1, 5
0
-CCA
AAC GGT CTC CGT ACT GT-3
0
; GSP2, 5
0
-CCG TAC TGT CGG ACG AAA TC-3
0
. The
signal peptide domain and the cystatin-conserved site were predicted using the SignalP
software (http://www.cbs.dtu.dk/services/SignalP/, accessed on 11 August 2019) and
InterProScan program v5.32-71.0 (https://www.ebi.ac.uk/interpro/search/sequence/,
accessed on 24 September 2019), respectively. The phylogenic tree was constructed by
MEGA software version X (https://www.megasoftware.net/show_eua, accessed on 3
October 2018 [
20
]), using a maximum likelihood of 1000 bootstrap replicates and a JTT
matrix-based model [
21
], by using a discrete Gamma distribution (+G) and assuming that
a certain fraction of sites is evolutionarily invariable (+I), to improve the tree topology.
2.5. Laser-Capture Microdissection
cDNA synthesis from salivary glands, midguts, and ovaries was performed using
laser-capture microdissection (LCM), as previously described [
22
]. Briefly, starved PRMs
of mixed developmental stages were fixed in carnoy solution, embedded in paraffin, and
cut into 5-
µ
m thick sections. Sections were mounted on glass slides precoated with LCM
films (Meiwafosis, Tokyo, Japan) and stained with 1% toluidine blue, and each tissue was
dissected using MicroBeam Rel.4.2 (Carl Zeiss, Oberkochen, Germany). Total RNA was
extracted from the tissues using the RNAqueous
®
-Micro Kit (Thermo Fisher Scientific,
Waltham, MA, USA) according to the manufacturer’s protocol, and cDNA synthesis was
performed using the SuperScript
TM
First-Strand Synthesis System for RT-PCR (Invitrogen,
Thermo Fisher Scientific, 168 Third Avenue, Waltham, MA, USA 02451), using 300 pmol of
random hexamer primer (Hokkaido System Science).
2.6. Gene Expression Analysis
The expression of Dg-Cys in each feeding state, tissue, and developmental stage was
examined by RT-PCR with Ex-Taq polymerase (Takara Bio Inc.), using the synthesized
cDNA, as described above. The specific primer sets used were as follows: Dg-Cys-for,
5
0
-GTC TTT GCC TTC CAG TCG AG-3
0
;Dg-Cys-rev, 5
0
-GGT CTA GCT TGC TCC AAA
CG-3
0
. As an internal control, elongation factor 1-alpha 1-like (Elf1a1) was amplified using the
following primer set [
23
]: Elf1a1-for, 5
0
-GTC GGT GTC ATC AAG TCC GT-3
0
; Elf1a1-rev,
5
0
-AGG GTC GAG AGT GTA GGG TC-3
0
. Since the RNA samples extracted from salivary
glands were small in quantity or of low quality, the detection of target genes in cDNA
samples synthesized from salivary glands was performed by nested PCR using the same
primer set.
Vaccines 2021,9, 1472 4 of 17
2.7. Real-Time Quantitative RT-PCR
To quantify the expression of Dg-Cys mRNA at each developmental stage and feeding
state of PRMs, quantitative PCR was performed on cDNA samples from different life
stages, except for eggs, and for different feeding states using LightCycler480
®
System II
(Roche Diagnostics, Mannheim, Germany) and TB Green Premix DimerEraser (Takara Bio
Inc.) according to the manufacturers’ instructions. To amplify the Dg-Cys gene, the primer
set Dg-Cys-for and Dg-Cys-rev was used, while the primer set Elf1a1-for and Elf1a1-rev
was used to amplify Elf1a1 as an internal control. The cycling conditions included an
initial denaturation step at 95
◦
C for 30 s, followed by 45 cycles of 95
◦
C for 5 s, 60
◦
C for
30 s
, and 72
◦
C for 30 s. To evaluate the specificities of primer pairs, a final melting curve
analysis was performed from 65
◦
C to 95
◦
C at a rate of 0.1
◦
C/s. To generate standard
curves for quantification, serial dilutions of T-vector pMD20 (Takara Bio Inc.) containing
Dg-Cys or Elf1a1 were used. Each sample was tested four times, and Dg-Cys mRNA expres-
sion was calculated as a ratio by dividing the concentration of Dg-Cys mRNA by that of
Elf1a1 mRNA.
2.8. Preparation of Recombinant Dg-Cys
Recombinant Dg-Cys was expressed as a fusion protein with a histidine tag (Dg-Cys-
his), using the BIC system (Takara Bio Inc.). Specific primers containing homologous
recombination sites were designed to express Dg-Cys-his, according to the manufacturer’s
instructions: pBIC4-Dg-Cys-for, 5
0
-GAT GAC GAT GAC AAA GGC CTT TCG GAC GTG
GCC G-30; pBIC4-Dg-Cys-rev, 50-CAT CCT GTT AAG CTT TTA ATT CTC GCA CCG CTT
C-3
0
. PCR was performed using KOD-Plus-Neo (TOYOBO Co., Ltd., Osaka, Japan) to
amplify the open reading frame (ORF) of Dg-Cys lacking the signal peptide region, and
the fragments were integrated into the cloning site of the pBIC4 vector (Takara Bio Inc.)
and transformed into Brevibacillus competent cells by homologous recombination. The
transformed bacteria were cultured in 2SY medium for 48 h at 32
◦
C, as per the manu-
facturer’s instructions. The supernatants were then collected, and proteins were purified
using Ni Sepharose 6 Fast Flow (Thermo Fisher Scientific). The buffer was replaced with
phosphate-buffered saline (PBS) using SnakeSkin
TM
Dialysis Tubing, 10K MWCO (Thermo
Fisher Scientific) overnight at 4
◦
C. To confirm protein purification, the obtained proteins
were mixed with 2
×
SDS buffer (125 mM Tris–HCl pH 6.8, 4% SDS, 10% 2-mercaptoethanol,
and 20% glycerol), boiled for 5 min, separated using 14% SDS-polyacrylamide gel elec-
trophoresis (SDS-PAGE), and stained with Coomassie brilliant blue (CBB, FUJIFILM Wako
Pure Chemical Corporation, Osaka, Japan). The protein concentration was determined
using the Pierce
TM
BCA Protein Assay Kit (GE Healthcare, Chicago, IL, USA), according to
the manufacturer’s instructions.
2.9. Enzymatic Activity of Dg-Cys-his
The inhibitory properties of Dg-Cys-his against cathepsin L, S, and B were assessed
using the SensoLyte Rh110 Cathepsin L Assay Kit (ANASPEC, Fremont, CA, USA), Sen-
soLyte 520 Cathepsin S Assay Kit (ANASPEC), and SensoLyte 520 Cathepsin B Assay Kit
(ANASPEC), respectively, according to the manufacturer’s instructions. Bovine serum
albumin (Sigma-Aldrich, St. Louis, MO, USA) (BSA) diluted in PBS was used as a negative
control. Y-axis indicates the percentage of enzymatic activities of cathepsin L, S, and B in
the presence of Dg-Cys-his compared to that in the presence of BSA.
2.10. Immunization of Chickens with Dg-Cys-his
Plasma was extracted from chickens immunized with Dg-Cys-his (Supplementary
Figure S1). Specifically, purified Dg-Cys-his was mixed with light liquid paraffin as an
adjuvant (20
µ
g/mL). An emulsion of PBS and light liquid paraffin was prepared and used
as a control. Four chickens (Hy-Line Brown) were subcutaneously immunized with 20
µ
g
of Dg-Cys-his at 3 weeks of age. Four weeks later, chickens in the immunized group were
immunized again with 20
µ
g of Dg-Cys-his in light liquid paraffin. Heparinized blood
Vaccines 2021,9, 1472 5 of 17
was collected 3 weeks after the second immunization, and the plasma was isolated by
centrifugation at 2000
×
gfor 10 min. As a control, two chickens were subcutaneously
immunized with PBS, and plasma was isolated 3 weeks after the second immunization.
2.11. Enzyme-Linked Immunosorbent Assay (ELISA)
Antibody titers in plasma samples were determined by ELISA. The purified Dg-Cys-
his was coated onto the wells of 96-well plates (Sumitomo Bakelite Co., Ltd., Tokyo, Japan)
(100 ng/well) for 16 h in a carbon-bicarbonate buffer. After washing each well three times
with PBS, PBS containing 0.05% Tween 20 (PBS-T) and 1% BSA was added and incubated
at 37
◦
C for 2 h. The wells were then washed five times with PBS-T, and plasma samples
diluted at 2000
×
, 4000
×
8000
×
, and 16,000
×
with PBS were added. After incubation for
1 h
at 25
◦
C, the wells were washed five times with PBS-T and incubated with anti-chicken
IgY peroxidase rabbit antibodies (Sigma-Aldrich, A9046) diluted at 5000
×
with PBS-T for
1 h
at 25
◦
C. Finally, the wells were washed five times with PBS-T and allowed to react with
the TMB one-component substrate (Bethyl Laboratories, Montgomery, TX, USA) for 20 min
at 25
◦
C, in the dark. The reaction was quenched with 0.18 M H
2
SO
4
, and the absorbance
was measured at 450 nm. The assay was performed in duplicate.
2.12. Western Blotting
The production of specific antibodies against Dg-Cys-his was examined by Western
blotting, as described previously [
4
,
5
]. Purified Dg-Cys-his was separated using a 14%
SDS-polyacrylamide gel and then transferred to polyvinylidene difluoride membranes
(Merck Millipore, Burlington, MA, USA). The membrane was blocked overnight at 4
◦
C
with PBS-T containing 1% skim milk. The membranes were incubated at 25
◦
C with
the isolated plasmas from the immunized chickens, washed three times with PBS-T, and
incubated at 25
◦
C with anti-chicken IgY peroxidase rabbit antibodies (Sigma-Aldrich,
A9046) diluted at 5000
×
. Finally, the membranes were incubated with Immobilon Western
Chemiluminescent HRP Substrate (Merck Millipore) to visualize the peroxidase reaction.
To analyze the specificities of antibodies, the His-tagged recombinant protein of D. gallinae
copper transporter 1 (Dg-Ctr1-N-his) was also incubated with plasma from the Dg-Cys-his-
immunized chickens [
4
] in the same manner as described above. To visualize Dg-Ctr1-N-his,
SDS-PAGE and CBB staining was also performed as described above.
2.13. Evaluation of the Potential of Dg-Cys as a Vaccine Antigen
The potential of Dg-Cys as a vaccine antigen was analyzed through an
in vitro
feeding
assay [
23
]. Fresh chicken blood was collected from healthy chickens maintained at the
Field Science Center for Northern Biosphere, Hokkaido University, and incubated at
40 ◦C
before use. After centrifugation at 2000
×
gfor 10 min, the plasma was replaced with
an equal volume of immune plasma as described above. Approximately 100 PRMs of
mixed developmental stages were collected in the artificial feeding devices as previously
described [
23
], and the devices were capped with rubber caps type 2 (GE Healthcare). For
ventilation, the rubber cap was penetrated using a 27-G needle (TERUMO CORPORATION,
Tokyo, Japan). The top of the devices were filled with 400
µ
L of blood, and blood feeding
was performed for 4 h at 40
◦
C in a dark, humid environment with modest shaking. Only
blood-fed PRMs were collected using Pasteur pipettes (day 0) and kept at 25
◦
C in 70%
humidity during the observation period. The number of dead PRMs was monitored for
1 week
, and the numbers of newborn larvae, protonymphs, and cast-off skins were counted
at day 7. The anti-PRM property of Dg-Cys-his immunization was evaluated based on the
following assessment items:
(1)
The SR:
SR (%)=1−No. of dead PRMs on day 7
No. of blood −fed PRMs ×100
(2)
The RC:
Vaccines 2021,9, 1472 6 of 17
RC =No. of new borne larvae and protonymphs on day 7
No. of blood −fed adults
(3)
The molting rate (MR):
MR (%)=No. of cast −off skins on day 7
No. of blood −fed protonymphs and deutonymphs ×100
The feeding assay was performed twice. In the first experiment, the SR and RC were
assessed; to measure the RC accurately, deutonymphs were excluded from the analysis.
In the second experiment, the SR and MR were evaluated. To precisely evaluate the MR,
adults were not included in the analysis in the second experiment. The SR, RC, and MR
were analyzed by comparing the total number of PRMs or cast-off skins in the immunized
and control groups.
2.14. Evaluation of the Efficacy of the “Cocktail Vaccine”
To assess the effects of the “cocktail vaccine”, plasmas from chickens immunized with
copper transporter 1-like molecule (Dg-Ctr1 [
4
]) or adipocyte plasma membrane-associated
protein-like molecule (Dg-APMAP [
5
]) were mixed with an equal volume of immune
plasmas of Dg-Cys. As a control, equal volumes of plasmas from chickens immunized with
PBS, as described above, were mixed with plasmas prepared in our previous studies [
4
,
5
].
Blood feeding was conducted as described above using adult PRMs, and the SR was
monitored for a 10-day period.
2.15. Statistics
For gene expression analyses, differences were analyzed using the Mann–Whitney
Utest. For
in vitro
feeding assay, the SR, RC, and MR between the Dg-Cys- and PBS-
immunized groups were compared by Fisher’s exact test. The odds ratio and 95% con-
fidential interval (CI) was estimated. In addition to assessing the difference in the SR,
Kaplan–Meier curves were generated and a log-rank test was conducted. For multiple
comparisons, Fisher’s multicomparison test and a pvalue was adjusted by Holm method.
pvalues of <0.05 and <0.01 were considered statistically significant.
3. Results
3.1. Cloning and Sequence Analysis of Dg-Cys from PRMs
We obtained the transcript of the cystatin-like molecule, which showed high expression
levels in both blood-fed and starved PRMs, from the dataset of our RNA-Seq analysis [
19
]
and designated the transcript as Dg-Cys. We did not observe any significant difference in
the expression of Dg-Cys between the two feeding states of PRMs (Supplementary Table S1).
We performed 5
0
and 3
0
RACE analyses to reveal that the full-length cDNA was 669 bp, and
the ORF was 423 bp, encoding a 140 amino-acid protein. The deduced amino acid sequence
of Dg-Cys contained a putative signal peptide in positions 1–29 and a cystatin-conserved
site at positions 85–98 (Figure 1A). Phylogenetic analysis using cystatins from chickens,
mammals, ticks, and mites revealed that Dg-Cys formed a distinct cluster, but was closer to
secreted cystatins (cystatin 2, cystatin C) rather than intracellular ones (stefin, cystatins A
and B) (Figure 1B).
3.2. Dg-Cys Expression Profile
To investigate the gene expression profiles of Dg-Cys, we first analyzed the expression
of Dg-Cys mRNA at different life-stages. Real-time reverse transcription polymerase chain
reaction (RT-PCR) and real-time quantitative RT-PCR analyses revealed that Dg-Cys mRNA
was expressed in all life stages, except in eggs, and was detected regardless of the feeding
state (Figure 2A,B and Figure S3). Moreover, the expression analysis of Dg-Cys in the
midgut, salivary glands, and ovaries by LCM and RT-PCR/nested PCR showed that Dg-
Vaccines 2021,9, 1472 7 of 17
Cys was clearly expressed in the midgut and ovaries, whereas Dg-Cys was not detectable in
the salivary glands (Figure 2C and Figure S3).
Vaccines 2021, 9, x FOR PEER REVIEW 7 of 18
Figure 1. Cloning of a cystatin-like molecule from Dermanyssus gallinae. (A) Nucleotide and de-
duced amino acid sequences of cDNA encoding a D. gallinae cystatin-like molecule (Dg-Cys). Puta-
tive signal peptide sites were underlined, and the cystatin-conserved site was boxed. (B) A phylo-
genic tree based on the deduced amino acid sequence of Dg-Cys. The tree was built with the maxi-
mum likelihood method using the MEGA Ⅹ software [20]. Numbers indicate bootstrap percentage
(1000 replicates). The scale indicates the divergence time.
Figure 1.
Cloning of a cystatin-like molecule from Dermanyssus gallinae. (
A
) Nucleotide and deduced
amino acid sequences of cDNA encoding a D. gallinae cystatin-like molecule (Dg-Cys). Putative signal
peptide sites were underlined, and the cystatin-conserved site was boxed. (
B
) A phylogenic tree based
on the deduced amino acid sequence of Dg-Cys. The tree was built with the maximum likelihood
method using the MEGA X software [
20
]. Numbers indicate bootstrap percentage (
1000 replicates
).
The scale indicates the divergence time. *Stop codon.
Vaccines 2021,9, 1472 8 of 17
Vaccines 2021, 9, x FOR PEER REVIEW 8 of 18
3.2. Dg-Cys Expression Profile
To investigate the gene expression profiles of Dg-Cys, we first analyzed the expres-
sion of Dg-Cys mRNA at different life-stages. Real-time reverse transcription polymerase
chain reaction (RT-PCR) and real-time quantitative RT-PCR analyses revealed that Dg-Cys
mRNA was expressed in all life stages, except in eggs, and was detected regardless of the
feeding state (Figures 2A,B and S3). Moreover, the expression analysis of Dg-Cys in the
midgut, salivary glands, and ovaries by LCM and RT-PCR/nested PCR showed that Dg-
Cys was clearly expressed in the midgut and ovaries, whereas Dg-Cys was not detectable
in the salivary glands (Figures 2C and S3).
Figure 2. Gene expression analysis of Dg-Cys. Dg-Cys expression was examined by RT-PCR/nested
PCR at each life-stage and blood-feeding state (A), and in different tissues of PRMs (C). Elongation
factor 1-alpha 1-like gene (Ef1a1) was amplified as an internal control. (B) Real-time quantitative
RT-PCR was performed to quantify the gene expression of Dg-Cys at each life-stage and blood-
Figure 2.
Gene expression analysis of Dg-Cys.Dg-Cys expression was examined by RT-PCR/nested
PCR at each life-stage and blood-feeding state (
A
), and in different tissues of PRMs (
C
). Elongation
factor 1-alpha 1-like gene (Ef1a1) was amplified as an internal control. (
B
) Real-time quantitative
RT-PCR was performed to quantify the gene expression of Dg-Cys at each life-stage and blood-feeding
state of PRMs. The extent of Dg-Cys expression was calculated by dividing the copy numbers of
Dg-Cys by those of Elf1a1. Each experiment was repeated four times and error bars indicate SEM.
Statistical analyses were performed using Steel–Dwass test. N.S.: not significant. (
C
) The expression
of Dg-Cys in the midguts and ovaries was analyzed by RT-PCR, and the expression in the salivary
glands was analyzed by RT-nested PCR.
3.3. Dg-Cys Enzymatic Activity
By using the BIC system, we prepared the recombinant Dg-Cys as a fusion protein
with a His-tag (Dg-Cys-his). Dg-Cys-his was purified from the culture supernatant of the
transformed bacteria, and the expression and purity of Dg-Cys-his were confirmed by
Vaccines 2021,9, 1472 9 of 17
sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie
brilliant blue staining. Dg-Cys-his was detected at the predicted molecular weight (approx-
imately 13.4 kDa) (Figure 3A and Figure S4). Next, we investigated the function of Dg-Cys
protein. Specifically, we assessed the inhibitory properties of Dg-Cys-his against cysteine
proteases using commercial kits to measure the enzyme activities of cysteine proteases,
cathepsins L, B, and S. The enzymatic activity of cathepsins L and S was suppressed in
the presence of Dg-Cys-his in a dose-dependent manner (50% inhibitory concentration for
cathepsins L and S: 666.8 nM and 36.1 nM, respectively), whereas that of cathepsin B was
not inhibited (Figure 3B). These data suggest that Dg-Cys-his functions as an inhibitor of
cysteine proteases.
Vaccines 2021, 9, x FOR PEER REVIEW 9 of 18
feeding state of PRMs. The extent of Dg-Cys expression was calculated by dividing the copy num-
bers of Dg-Cys by those of Elf1a1. Each experiment was repeated four times and error bars indicate
SEM. Statistical analyses were performed using Steel–Dwass test. N.S.: not significant. (C) The
expression of Dg-Cys in the midguts and ovaries was analyzed by RT-PCR, and the expression in
the salivary glands was analyzed by RT-nested PCR.
3.3. Dg-Cys Enzymatic Activity
By using the BIC system, we prepared the recombinant Dg-Cys as a fusion protein
with a His-tag (Dg-Cys-his). Dg-Cys-his was purified from the culture supernatant of the
transformed bacteria, and the expression and purity of Dg-Cys-his were confirmed by so-
dium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie
brilliant blue staining. Dg-Cys-his was detected at the predicted molecular weight (ap-
proximately 13.4 kDa) (Figures 3A and S4). Next, we investigated the function of Dg-Cys
protein. Specifically, we assessed the inhibitory properties of Dg-Cys-his against cysteine
proteases using commercial kits to measure the enzyme activities of cysteine proteases,
cathepsins L, B, and S. The enzymatic activity of cathepsins L and S was suppressed in the
presence of Dg-Cys-his in a dose-dependent manner (50 % inhibitory concentration for
cathepsins L and S: 666.8 nM and 36.1 nM, respectively), whereas that of cathepsin B was
not inhibited (Figure 3B). These data suggest that Dg-Cys-his functions as an inhibitor of
cysteine proteases.
Figure 3. Functional analysis of recombinant Dg-Cys. (A) The recombinant protein of Dg-Cys (Dg-
Cys-his) was prepared using BIC system. Purified Dg-Cys-his was separated by SDS-PAGE and
visualized by staining with Coomassie brilliant blue. (B) Inhibitory properties of Dg-Cys-his
against cysteine proteases were examined. Cathepsins L, S, and B were incubated with their fluo-
Figure 3.
Functional analysis of recombinant Dg-Cys. (
A
) The recombinant protein of Dg-Cys (Dg-Cys-
his) was prepared using BIC system. Purified Dg-Cys-his was separated by SDS-PAGE and visualized
by staining with Coomassie brilliant blue. (
B
) Inhibitory properties of Dg-Cys-his against cysteine
proteases were examined. Cathepsins L, S, and B were incubated with their fluorometric substrates
in the presence of Dg-Cys-his or bovine serum albumin (BSA). The X-axis indicates the concentrations
of Dg-Cys-his. The Y-axis indicates relative enzymatic activities of each cysteine proteases in the
presence of Dg-Cys-his compared to those in the presence of BSA. The assays were performed in
triplicate, and error bars indicate SEM.
3.4. Acaricidal Potential of the Plasma from Chickens Immunized with Dg-Cys-his
Chickens were subcutaneously immunized twice with Dg-Cys-his as an emulsion with
light liquid paraffin before collecting their plasma (Supplementary Figure S1). Antibody
titers in the plasma samples were determined by ELISA, and two of the immunized
chickens, IM2 and IM4, showed higher antibody titers against Dg-Cys-his (Supplementary
Vaccines 2021,9, 1472 10 of 17
Table S2). Further Western blot analysis revealed that the antibodies produced in IM2
and IM4 contained antibodies specific to Dg-Cys-his (Supplementary Figures S2 and S5).
Plasma from IM2 and IM4, and from two chickens of the control group, were subjected to
an
in vitro
feeding assay [
23
] to evaluate anti-PRM effects. After the
in vitro
feeding, we
collected the blood-fed PRMs and assessed the acaricidal potential. We monitored PRMs
that fed on the plasmas and assessed them along the following three criteria: the survival
rate (SR), the reproductive capacity (RC), and molting rate (MR). In the first experiment,
we used protonymphs and adults to evaluate the effects of plasmas on the SR and RC of
blood-fed PRMs. The RC of PRMs that fed on the plasmas from IM2 and IM4 (i.e., chickens
immunized with Dg-Cys-his) was significantly reduced (Table 1and Figure 4A), whereas
no difference was observed in the SR of PRMs that fed on the plasmas of immunized
and control chickens, throughout the observation period (Table 1and Figure 4B). In the
second experiment, we used protonymphs and deutonymphs to assess the effect of the
immune plasmas on the MR and SR. While no significant difference was observed in the
MR (Table 2and Figure 5A), the SR of PRMs that fed on the plasmas from IM2 and IM4
was significantly lower than that of PRMs that fed on the control plasma (Table 2and
Figure 5B
). These results suggest that the plasma from chickens immunized with Dg-Cys
increases the mortality of PRMs, especially of protonymphs, and affects their reproductive
activity while reducing the generation of progenies.
Table 1. Summary of the anti-PRM property of Dg-Cys-his immunization (first experiment).
Days Post-Feeding RC
1 2 3 4 5 6 7
Immunized (n= 255, nymphs: n= 38, adults: n= 219)
No. of dead PRMs 5 12 16 21 35 38 45 1.29
SR (%) 98.04 95.29 93.73 91.76 86.27 85.10 82.35
Control (n= 211, nymphs: n= 51, adults: n= 160)
No. of dead PRMs 1 4 13 15 23 26 37 2.24
SR (%) 99.53 98.10 93.84 92.89 89.10 87.68 82.46
pvalue (Fisher’s exact) 0.228 0.126 1 0.729 0.399 0.499 1 2.38 ×10−5*
Odds ratio 4.19 2.55 1.01 1.17 1.30 1.25 1.01 0.57
95% CI (lower limit) 0.46 0.76 0.45 0.56 0.72 0.71 0.61 0.44
95% CI (upper limit) 199.39 11.02 2.36 2.52 2.39 2.22 1.68 0.75
The data are compared by Fisher’s exact test between immunized and control groups. SR, survival rate; RC, reproductive capacity. * p<
0.05 was considered statistically significant.
Vaccines 2021, 9, x FOR PEER REVIEW 11 of 18
Figure 4. Anti-PRM effects of plasma from chickens immunized with Dg-Cys-his (first experiment). (A) The reproductive
capacity (RC) at seven days post-feeding was assessed. Statistical analysis was performed using Fisher’s exact test. p < 0.01
was considered statistically significant. (B) The survival rate (SR) of PRMs that were fed with the plasma from immunized
chickens was assessed every day for a one-week period. Statistical analysis was performed using Log-rank test.
Table 2. Summary of the anti-PRM property of Dg-Cys-his immunization (second experiment).
Days Post-Feeding
MR (%)
1 2 3 4 5 6 7
Immunized (n = 189, all nymphs)
48.68
No. of dead PRMs 5 6 10 13 34 57 74
SR (%) 97.35 96.83 94.71 93.12 82.01 69.84 60.85
Control (n = 150, all nymphs)
49.33
No. of dead PRMs 1 3 4 4 14 28 38
SR (%) 99.33 98.00 97.33 97.33 90.67 81.33 74.67
p value 0.233 0.736 0.280 0.085 0.028 * 0.017 * 0.008 * 1
Odds ratio 4.04 1.60 2.04 2.69 2.13 1.88 1.89 1.01
95% CI (lower limit) 0.44 0.34 0.57 0.81 1.06 1.09 1.16 0.68
95% CI (upper limit) 192.59 10.08 9.07 11.57 4.48 3.28 3.13 1.50
The data are compared by Fisher’s exact test between immunized and control groups. SR, survival rate; MR, molting rate.
* p < 0.05 was considered statistically significant.
Figure 4.
Anti-PRM effects of plasma from chickens immunized with Dg-Cys-his (first experiment). (
A
) The reproductive
capacity (RC) at seven days post-feeding was assessed. Statistical analysis was performed using Fisher’s exact test. p< 0.01
was considered statistically significant. (
B
) The survival rate (SR) of PRMs that were fed with the plasma from immunized
chickens was assessed every day for a one-week period. Statistical analysis was performed using Log-rank test.
Vaccines 2021,9, 1472 11 of 17
Table 2. Summary of the anti-PRM property of Dg-Cys-his immunization (second experiment).
Days Post-Feeding MR (%)
1 2 3 4 5 6 7
Immunized (n= 189, all nymphs) 48.68
No. of dead PRMs 5 6 10 13 34 57 74
SR (%) 97.35 96.83 94.71 93.12 82.01 69.84 60.85
Control (n= 150, all nymphs) 49.33
No. of dead PRMs 1 3 4 4 14 28 38
SR (%) 99.33 98.00 97.33 97.33 90.67 81.33 74.67
pvalue 0.233 0.736 0.280 0.085 0.028 * 0.017 * 0.008 * 1
Odds ratio 4.04 1.60 2.04 2.69 2.13 1.88 1.89 1.01
95% CI (lower limit) 0.44 0.34 0.57 0.81 1.06 1.09 1.16 0.68
95% CI (upper limit) 192.59 10.08 9.07 11.57 4.48 3.28 3.13 1.50
The data are compared by Fisher’s exact test between immunized and control groups. SR, survival rate; MR, molting rate. * p< 0.05 was
considered statistically significant.
Vaccines 2021, 9, x FOR PEER REVIEW 12 of 18
Figure 5. Anti-PRM effects of plasma from chickens immunized with Dg-Cys-his (second experiment). (A) The molting
rate (MR) at seven days post-feeding was assessed. Statistical analyses were performed using Fisher’s exact test. (B) The
survival rate (SR) of PRMs that were fed with the plasma from immunized chickens was assessed every day for a one-
week period. Statistical analyses were performed using Log-rank test. p < 0.01 was considered statistically significant. N.S.:
not significant.
3.5. Enhanced Acaricidal Effect of Dg-Cys-his-Immunized Plasma in Combination with Dg-
Ctr1- or Dg-APMAP-Immunized Plasma
Although plasmas from Dg-Cys-his-immunized chickens exhibited anti-PRM prop-
erties, their acaricidal effects on adults could not be observed (Table 1 and Figure 4B).
Recently, we characterized two more candidate vaccine antigens: copper transporter 1-
like molecule (Dg-Ctr1) and adipocyte plasma membrane-associated protein-like mole-
cule (Dg-APMAP). Moreover, acaricidal effects of immune plasmas containing antibodies
against these antigens were higher in nymphs than in adults [4,5]. In ticks, several studies
have suggested that the “cocktail vaccine”, which combines different vaccine antigens,
could potentially have enhanced efficacy than single-antigen vaccines [24,25]. Therefore,
to assess the potential of Dg-Cys as a cocktail vaccine antigen, we next examined whether
the combined use of immune plasmas could enhance the acaricidal effects. We combined
the plasma from Dg-Cys-his-immunized chickens with other immune plasmas and as-
sessed their acaricidal effects on adult PRMs. The chickens were immunized with the re-
combinant proteins of Dg-Ctr-1 or Dg-APMAP and exhibited the acaricidal effects against
PRMs
4,5
. Upon using immune plasma against a single antigen, the immune plasma of Dg-
Cys exhibited significant reduction in SR at days five and seven post-feeding compared to
the control plasma. Conversely, no acaricidal effect was observed in PRMs that fed on Dg-
Ctr1-immune plasma (Table 3 and Figure 6A). Notably, PRMs that fed on the combined
plasmas, which contained antibodies specific to Dg-Cys and Dg-Ctr1, showed significant
decrease in the SR at seven and nine days post-feeding compared to those that fed on
control plasmas. In addition, at ten days post-feeding, the SR of PRMs that fed on mixed
plasmas was lower than those of PRMs in the immune plasmas against a single antigen
(Dg-Cys: 10% lower; Dg-Ctr1: 18% lower); however, we did not observe a significant dif-
ference (Table 3 and Figure 6A). During the assessment of the combination of Dg-Cys with
Dg-APMAP, the use of immune plasma against a single antigen exerted no acaricidal ef-
fects. Conversely, the combined use of immune plasmas exhibited a drastic decline in the
SR from eight days post-feeding compared to those of PRMs fed Dg-Cys-immune plasma.
Furthermore, the SR of PRMs that fed on mixed plasmas were significantly lower than
those of all the other groups at day ten post-feeding (Table 4 and Figure 6B). These data
suggest that Dg-Cys may function as a cocktail vaccine antigen.
Figure 5.
Anti-PRM effects of plasma from chickens immunized with Dg-Cys-his (second experiment). (
A
) The molt-
ing rate (MR) at seven days post-feeding was assessed. Statistical analyses were performed using Fisher’s exact test.
(
B
) The survival rate (SR) of PRMs that were fed with the plasma from immunized chickens was assessed every day for a
one-week period. Statistical analyses were performed using Log-rank test. p< 0.01 was considered statistically significant.
N.S.: not significant.
3.5. Enhanced Acaricidal Effect of Dg-Cys-his-Immunized Plasma in Combination with Dg-Ctr1-
or Dg-APMAP-Immunized Plasma
Although plasmas from Dg-Cys-his-immunized chickens exhibited anti-PRM prop-
erties, their acaricidal effects on adults could not be observed (Table 1and Figure 4B).
Recently, we characterized two more candidate vaccine antigens: copper transporter 1-like
molecule (Dg-Ctr1) and adipocyte plasma membrane-associated protein-like molecule (Dg-
APMAP). Moreover, acaricidal effects of immune plasmas containing antibodies against
these antigens were higher in nymphs than in adults [
4
,
5
]. In ticks, several studies have
suggested that the “cocktail vaccine”, which combines different vaccine antigens, could
potentially have enhanced efficacy than single-antigen vaccines [
24
,
25
]. Therefore, to as-
sess the potential of Dg-Cys as a cocktail vaccine antigen, we next examined whether the
combined use of immune plasmas could enhance the acaricidal effects. We combined the
plasma from Dg-Cys-his-immunized chickens with other immune plasmas and assessed
their acaricidal effects on adult PRMs. The chickens were immunized with the recombinant
proteins of Dg-Ctr-1 or Dg-APMAP and exhibited the acaricidal effects against PRMs
4,5
.
Upon using immune plasma against a single antigen, the immune plasma of Dg-Cys ex-
hibited significant reduction in SR at days five and seven post-feeding compared to the
control plasma. Conversely, no acaricidal effect was observed in PRMs that fed on Dg-Ctr1-
Vaccines 2021,9, 1472 12 of 17
immune plasma (Table 3and Figure 6A). Notably, PRMs that fed on the combined plasmas,
which contained antibodies specific to Dg-Cys and Dg-Ctr1, showed significant decrease in
the SR at seven and nine days post-feeding compared to those that fed on control plasmas.
In addition, at ten days post-feeding, the SR of PRMs that fed on mixed plasmas was lower
than those of PRMs in the immune plasmas against a single antigen (Dg-Cys: 10% lower;
Dg-Ctr1: 18% lower); however, we did not observe a significant difference (Table 3and
Figure 6A). During the assessment of the combination of Dg-Cys with Dg-APMAP, the use
of immune plasma against a single antigen exerted no acaricidal effects. Conversely, the
combined use of immune plasmas exhibited a drastic decline in the SR from eight days
post-feeding compared to those of PRMs fed Dg-Cys-immune plasma. Furthermore, the
SR of PRMs that fed on mixed plasmas were significantly lower than those of all the other
groups at day ten post-feeding (Table 4and Figure 6B). These data suggest that Dg-Cys
may function as a cocktail vaccine antigen.
Table 3. Summary of the acaricidal effects of combined immune plasmas on adult mites (Dg-Cys and Dg-Ctr1).
Days Post-Feeding
12345678910
Control (n= 70)
No. of dead PRMs 0 1 2 2 3 5 8 19 25 34
SR (%) 100 98.57 97.14 97.14 95.71 92.86 88.57 72.86 64.29 51.43
Dg-Cys (n= 78)
No. of dead PRMs 0 5 7 11 16 18 24 32 38 46
SR (%) 100 93.59 91.03 85.90 79.49 76.92 69.23 58.97 51.28 41.03
Dg-Ctr1 (n= 57)
No. of dead PRMs 1 2 4 4 6 7 11 13 21 29
SR (%) 98.25 96.49 92.98 92.98 89.47 87.72 80.70 77.19 63.16 49.12
Combination (n= 80)
No. of dead PRMs 0 1 5 8 11 16 25 33 47 54
SR (%) 100.00 98.72 93.59 89.74 85.90 79.49 67.95 57.69 39.74 30.77
pvalue
Control vs. Dg-Cys 1 1 1 0.116 0.019 * 0.068 0.025 * 0.256 0.539 0.970
Control vs. Dg-Ctr1 1 1 1 1 0.891 0.754 0.634 1 1 0.970
Control vs. Combination 1 1 1 0.513 0.254 0.16 0.018 * 0.239 0.019 * 0.074
Dg-Cys vs. Dg-Ctr1 1 1 1 1 0.633 0.491 0.496 0.161 0.595 0.970
Dg-Cys vs. Combination 1 1 1 1 0.891 0.847 1 1 0.595 0.970
Dg-Ctr1 vs. Combination 1 1 1 1 0.891 0.754 0.468 0.161 0.045 * 0.168
The data are compared by Fisher’s exact test. SR, survival rate. * Holm-adjusted p< 0.05 was considered statistically significant.
Vaccines 2021, 9, x FOR PEER REVIEW 14 of 18
Figure 6. Acaricidal effects of combined immune plasmas on adult mites. The efficacies of “cocktail vaccine” on adult
mites were evaluated in vitro. The survival rate (SR) of adult PRMs that were fed with the combined plasmas derived
from (A) Dg-Cys- and Dg-Ctr1-immunized chickens and (B) Dg-Cys- and Dg-APMAP-immunized plasmas was assessed
every day for a ten-day period.
4. Discussion
Previous reports have demonstrated the crucial roles of cystatins in ticks’ biological
activities, such as blood digestion, egg development, and modulation of host immunity
[26,27]. Therefore, the inhibition of these molecules could be an effective control strategy
against ticks. However, little is known about the contribution of cystatins to the physiol-
ogy of PRMs. In the present study, we characterized a newly identified cystatin-like mol-
ecule, Dg-Cys, which showed marked expression in both fed and starved PRMs [19].
Based on the deduced amino acid sequence and the results of the phylogenetic anal-
ysis, Dg-Cys was predicted to be a secreted cystatin. Indeed, several studies have reported
that secreted cystatins in the salivary glands of ticks help blood sucking and facilitate
pathogen transmission by suppressing host immune responses, such as the production of
inflammatory cytokines [28–30]. In contrast, functions like blood digestion and/or egg de-
velopment have been proposed for cystatins in the midgut [13,31–33]. Here, we revealed
that Dg-Cys is expressed in the midgut and ovaries, but not in the salivary glands of PRMs,
suggesting that it plays important roles in blood digestion and embryogenesis. In addi-
tion, recombinant Dg-Cys-his inhibited the enzymatic activities of cathepsins L and S,
which mediate innate immunity and antigen processing [34,35], suggesting that it might
be associated with the modulation of host immunity. In contrast, consistent with previous
reports of several cystatins [13,15,36],
Dg-Cys did not interrupt the enzymatic activity of
cathepsin B, thus indicating the selectivity and/or specificity of Dg-Cys against its target
cysteine proteases. In addition, it implies the existence of other cystatin-like molecules in
PRMs. Moreover, in phylogenetic analysis of Dg-Cys constituted a discrete cluster from
cystatins that are previously reported, including those from ticks, suggesting that Dg-Cys
potentially has a unique function in PRMs. Therefore, further experiments, such as gene-
specific, RNA interference-mediated silencing, are required to elucidate the function of
Dg-Cys in PRM physiology.
To evaluate the potential of Dg-Cys as a vaccine antigen, we assessed the SR, RC, and
MR of PRMs. While no difference in the SR was observed in the first experiment, the RC
of PRMs that fed on the immune plasma was significantly decreased. These data indicate
that Dg-Cys plays a crucial role in the development and/or hatching of eggs, as previously
reported in cystatins in ticks [37,38], and that vaccinating with Dg-Cys-his may control the
population growth of PRMs. In the second experiment, no significant differences were
Figure 6.
Acaricidal effects of combined immune plasmas on adult mites. The efficacies of “cocktail
vaccine” on adult mites were evaluated
in vitro
. The survival rate (SR) of adult PRMs that were
fed with the combined plasmas derived from (
A
)Dg-Cys- and Dg-Ctr1-immunized chickens and
(B)Dg-Cys- and Dg-APMAP-immunized plasmas was assessed every day for a ten-day period.
Vaccines 2021,9, 1472 13 of 17
Table 4. Summary of the acaricidal effects of combined immune plasmas on adult mites (Dg-Cys and Dg-APMAP).
Days Post-Feeding
12345678910
Control (n= 71)
No. of dead PRMs 2 4 6 10 11 14 21 27 33 40
SR (%) 97.18 94.37 91.55 85.92 84.51 80.28 70.42 61.97 53.52 43.66
Dg-Cys (n= 52)
No. of dead PRMs 0 1 2 4 6 8 10 16 20 28
SR (%) 100 98.08 96.15 92.31 88.46 84.62 80.77 69.23 61.54 46.15
Dg-APMAP (n= 74)
No. of dead PRMs 0 3 4 9 9 18 23 30 38 47
SR (%) 100 95.95 94.59 87.84 87.84 75.68 68.92 59.46 48.65 36.49
Combination (n= 48)
No. of dead PRMs 0 2 2 5 8 16 20 31 34 41
SR (%) 100 95.83 95.83 89.58 83.33 66.67 58.33 35.42 29.17 14.58
pvalue
Control vs. Dg-Cys 1 1 1 1 1 1 0.855 1 0.923 1
Control vs. Dg-APMAP 1 1 1 1 1 1 0.859 1 0.923 1
Control vs. Combination 1 1 1 1 1 0.657 0.855 0.026 * 0.070 0.006 **
Dg-Cys vs. Dg-APMAP 1 1 1 1 1 1 0.772 1 0.611 1
Dg-Cys vs. Combination 1 1 1 1 1 0.357 0.104 0.007 ** 0.008 ** 0.006 **
Dg-APMAP vs. Combination 1 1 1 1 1 1 0.855 0.062 0.165 0.050 *
The data are compared by Fisher’s exact test. SR, survival rate. Holm-adjusted * p< 0.05 and ** p< 0.01 were considered statistically significant.
4. Discussion
Previous reports have demonstrated the crucial roles of cystatins in ticks’ biological
activities, such as blood digestion, egg development, and modulation of host immu-
nity
[26,27]
. Therefore, the inhibition of these molecules could be an effective control
strategy against ticks. However, little is known about the contribution of cystatins to the
physiology of PRMs. In the present study, we characterized a newly identified cystatin-like
molecule, Dg-Cys, which showed marked expression in both fed and starved PRMs [19].
Based on the deduced amino acid sequence and the results of the phylogenetic analysis,
Dg-Cys was predicted to be a secreted cystatin. Indeed, several studies have reported
that secreted cystatins in the salivary glands of ticks help blood sucking and facilitate
pathogen transmission by suppressing host immune responses, such as the production
of inflammatory cytokines [
28
–
30
]. In contrast, functions like blood digestion and/or
egg development have been proposed for cystatins in the midgut [
13
,
31
–
33
]. Here, we
revealed that Dg-Cys is expressed in the midgut and ovaries, but not in the salivary glands
of PRMs, suggesting that it plays important roles in blood digestion and embryogenesis.
In addition, recombinant Dg-Cys-his inhibited the enzymatic activities of cathepsins L
and S, which mediate innate immunity and antigen processing [
34
,
35
], suggesting that it
might be associated with the modulation of host immunity. In contrast, consistent with
previous reports of several cystatins [
13
,
15
,
36
], Dg-Cys did not interrupt the enzymatic
activity of cathepsin B, thus indicating the selectivity and/or specificity of Dg-Cys against
its target cysteine proteases. In addition, it implies the existence of other cystatin-like
molecules in PRMs. Moreover, in phylogenetic analysis of Dg-Cys constituted a discrete
cluster from cystatins that are previously reported, including those from ticks, suggesting
that Dg-Cys potentially has a unique function in PRMs. Therefore, further experiments,
such as gene-specific, RNA interference-mediated silencing, are required to elucidate the
function of Dg-Cys in PRM physiology.
To evaluate the potential of Dg-Cys as a vaccine antigen, we assessed the SR, RC, and
MR of PRMs. While no difference in the SR was observed in the first experiment, the RC
of PRMs that fed on the immune plasma was significantly decreased. These data indicate
that Dg-Cys plays a crucial role in the development and/or hatching of eggs, as previously
reported in cystatins in ticks [
37
,
38
], and that vaccinating with Dg-Cys-his may control the
population growth of PRMs. In the second experiment, no significant differences were
observed in the MR. Moreover, in contrast to the data observed in the first experiment, the
SR of PRMs that fed on immune plasma was significantly reduced. While the majority
of PRMs used in the first experiment were adults, only protonymphs and deutonymphs
Vaccines 2021,9, 1472 14 of 17
were used in the second experiment. Although the difference in acaricidal effects observed
among developmental stages is unclear, it is possible that the expression pattern of Dg-Cys
may be different at each developmental stage. Therefore, the expression profile of Dg-Cys
should be investigated through LCM and RT-PCR analyses at each developmental stage in
more detail. Furthermore, in our dataset of RNA-seq analysis, several other cystatin-like
transcripts were identified, although their expression intensities were lower than that
of Dg-Cys [
19
]. Therefore, the reduced function of Dg-Cys conferred by the antibodies
may be compensated by other cystatin-like molecules present in the midgut of PRMs and
thus, the gene expression analyses and the functional characterization of those molecules
should be performed. In addition, the SR of PRMs in the second experiment were relatively
lower, even in the control groups, compared with those in the first experiment, suggesting
that the inhibition of Dg-Cys more critically affects the enfeebled PRMs. Alternatively,
adult PRMs may be more resistant against an immunological approach. Recently, we
identified two other antigen candidates: Dg-Ctr1 and Dg-APMAP. We found that acaricidal
effects of immune plasmas against these antigens were stronger against adults than against
nymphs [4,5].
In ticks, a growing number of studies has indicated that combining antigens enhances
the efficacies of vaccine [
24
,
25
,
39
]. Here, we demonstrated that the combination of plasmas
containing antibodies against Dg-Cys and Dg-Ctr1 or Dg-APMAP can augment anti-PRM
efficacy. It is noteworthy that the acaricidal effects of the use of combined immune plasmas
surpassed those of immune plasma against a single antigen, even though the mixed
immune plasmas contained only half the quantity of antibodies against each antigen
compared to immune plasma against single antigens, suggesting a prominent potential
for a cocktail vaccine including Dg-Cys. Although the mechanisms by which the use of
combined immune plasmas enhanced anti-PRM properties remains unclear, the disruption
of the homeostasis at two different points were more critical to PRMs. Importantly, it may
contribute to the reduction in the risk of the selection of PRMs with resistance to anti-PRM
effects induced by vaccination [
39
]. To evaluate the efficacies of the cocktail vaccine in
detail, further analyses are needed; including elucidation of the minimal antibody titers
of each antigen required to elicit acaricidal effects. Moreover, previous studies indicated
that an inadequate combination of antigens could impair vaccine efficacy as there may
be antigenic competition [
39
–
41
]. Therefore, the selection of antigens must be carefully
considered, and vaccine antigens should be continuously explored.
Thus, our data suggest that vaccination with Dg-Cys in combination with other anti-
gens could be an effective strategy to control PRMs in poultry farms. In this study, however,
one-shot immunization did not induce sufficient antibody responses, and only half of
the chickens immunized with Dg-Cys twice could produce specific antibodies. Therefore,
considering the practical applications, further investigation on the appropriate dose, routes,
and adjuvants is needed to improve the immunogenicity of the Dg-Cys-based vaccine
and to efficiently induce antibody responses. In addition, PRM-infestation trials using
immunized chickens should be conducted to further evaluate vaccine efficacies.
5. Conclusions
In the present study, a novel cystatin-like molecule (Dg-Cys) was identified and
characterized
in vitro
.Dg-Cys mRNA was expressed in the midgut and ovaries of PRMs,
regardless of the life stages and feeding states. Notably, PRMs fed on plasmas from
chickens immunized with the recombinant Dg-Cys exhibited a significant reduction in
the survival rates of nymphs and the reproductive capacity. Moreover, the combined use
of Dg-Cys-immunized plasmas with either plasmas from D. gallinae copper transporter
1 (Dg-Ctr1)-immunized chickens or those from D. gallinae adipocyte plasma membrane-
associated protein (Dg-APMAP) enhanced acaricidal effects of vaccines. Our data suggest
that the immunization with cocktail vaccines including Dg-Cys could potentially be effective
methods to control PRMs in the poultry industry.
Vaccines 2021,9, 1472 15 of 17
Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10
.3390/vaccines9121472/s1, Figure S1: The schedule of immunization with Dg-Cys-his and sample
collection, Figure S2: Dg-Cys-his-specific antibody production of immunized chickens, Figure S3:
The original uncropped image related to Figure 2, Figure S4:The original uncropped image related to
Figure 3, Figure S5:The original uncropped image related to Figure S2, Table S1: Gene expression
profiles of a cystatin-like molecule in poultry red mites (PRMs), Table S2: Antibody titers in the
plasmas from chickens immunized with Dg-Cys-his.
Author Contributions:
Conceptualization, S.F., S.M. and K.O.; Methodology, S.F., S.M., M.I., O.I. and
K.O.; Software, S.M. and K.O.; Validation; S.M., N.M., T.O., S.K. and K.O.; Formal analysis, S.M.,
N.M., T.O., S.K. and K.O.; Investigation, S.F., S.M., M.I., T.A., T.S. and O.I.; Resources, S.F., S.M., N.M.,
T.O. and S.K.; Data curation, S.F., S.M., N.M., T.O., S.K. and K.O.; Writing–original draft preparation,
S.F.; Writing–review and editing, S.M., E.O., A.T., N.M., T.O., O.I., S.K. and K.O.; Visualization, S.F.
and S.M.; Supervision, K.O.; Project administration, S.M. and K.O.; Funding acquisition, S.F., S.M.
and K.O. All authors have read and agreed to the published version of the manuscript.
Funding:
This work was partially supported by Grants-in-Aid for Scientific Research (B: 18H02332
and B: 20H03137); a Grant-in-Aid for Challenging Research (Exploratory) (20K21357); and a Grant-in-
Aid for the Japan Society for the Promotion of Science Research Fellow (grant no. 20J22235) from the
Japan Society for the Promotion of Science.
Data Availability Statement:
The data supporting the findings of this study are present within the
article and the Supplementary Materials.
Acknowledgments:
We would like to thank Yukiko Uno, Ryo Ogawa, and Eiji Oishi, Vaxxinova
Japan K.K., Tokyo, Japan, for their support during the experiments and their suggestions during the
manuscript editing process. We thank all farmers, Wataru Hashimoto, Japan Layer K.K., Gifu, Japan,
and Akio Enya for their assistance in the collection of samples.
Conflicts of Interest:
T.S., E.O., and A.T. are employed by Vaxxinova Japan K.K., Tokyo, Japan.
S.F., S.M., T.A., T.S., E.O., N.M., T.O., S.K., and K.O. are authors of patent-pending materials and
techniques described in this manuscript (2021-080691). The other authors have no competing financial
interests and all the authors declare no competing non-financial interests.
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