Accepted Manuscript

Vaccination at different anatomic sites induces different levels of

the immune responses

Jin Haibo, Ye Xu, Fushan Shi, Hu Songhua

PII: S0034-5288(18)30044-4
DOI: https://doi.org/10.1016/j.rvsc.2018.11.005
Reference: YRVSC 3657
To appear in: Research in Veterinary Science
Received date: 15 January 2018
Revised date: 5 September 2018
Accepted date: 11 November 2018

Please cite this article as: Jin Haibo, Ye Xu, Fushan Shi, Hu Songhua , Vaccination at
different anatomic sites induces different levels of the immune responses. Yrvsc (2018),
https://doi.org/10.1016/j.rvsc.2018.11.005

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 ACCEPTED MANUSCRIPT

Vaccination at different anatomic sites induces different levels of the

immune responses

Haibo, Jin*,2 , Ye Xu*,†,2 , Fushan Shi* , Songhua, Hu*,1

*
Department of Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou,

Zhejiang 310058, PR China

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†
Animal hospital of Zhejiang University, Hangzhou, Zhejiang 310058, PR China

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1
Corresponding author. Tel.: +86 571 8898 2852; fax: +86 571 8898 2275. E-mail:

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songhua@zju.edu.cn (S. Hu).

2
These authors contributed equally to this work
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Abstract

This study was to evaluate the effects of anatomical sites for vaccination on the
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immune responses. In experiment A, rats were subcutaneously (s.c.) immunized with

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a quintuplet vaccine twice at houhai acupoint, underjaw, popliteal fossa or back with
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a two weeks interval. The serum specific antibody levels were determined 2, 4 and 6

weeks after second immunization. Splenocytes were separated for detection of

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lymphocyte proliferation and cytokine mRNA expression. In experiment B, 10 female

Rottweiler puppies at their age of 34 ± 2 days were subcutaneously injected with a

bivalent vaccine Nobivac® Puppy DP containing live attenuated canine distemper

virus (CDV) and parvovirus (CPV) for primary vaccination, and a quadrivalent

vaccine Nobivac® DHPPI containing live attenuated canine distemper virus (CDV),

adenovirus type 2 (CAV-2), parvovirus (CPV) and parainfluenza virus (CPIV) for

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subsequent vaccination at houhai acupoint (4 dogs), the shoulder (3 dogs) or the nape

(3 dogs) region. Blood samples were collected at 0, 2, 4 and 6 weeks after vaccination

for determination of serum specific antibody responses by ELISA. The results showed

that injection of a vaccine in houhai acupoint induced the highest antibody responses

in both rats and dogs. When a vaccine was injected in houhai acupoint, significantly

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increased proliferative responses to Con A and LPS as well as mRNA expression of

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IL-4, IL-10, IL-12 and IFN-γ of splenocytes were detected in rats. Therefore, houhai

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acupoint is recommended for injection of a vaccine to improve the immune response

in dogs.
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Keyword: Houhai acupoint; Vaccination; Immune response
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1. Introduction
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The aims of vaccination are to induce memory in T and/or B lymphocytes

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through injection of avirulent antigen preparations. Thus, in the event of an actual

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infection, the infectious agent is met by a secondary response and eliminated from the

body. Because of inhomogeneous distribution of lymphocytes in the body, the chance

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for antigens introduced at different places to meet lymphocytes are different, and the

immune responses provoked by the antigens may also be different. For the vaccines

injected subcutaneously (s.c.) or intramuscularly (i.m.), selection of vaccination site

should be carefully considered. For example, the studies of hepatitis B prophylaxis

revealed that injection at the deltoid was ideal and recommended for vaccination in

humans (Lindsay et al., 1985; Hellgren, 1989; Zuckerman et al., 1992). However,

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selection of a site for vaccine injection in dogs is generally based on operation habits

of veterinary staffs. Actually, canine vaccine guidelines are published every once in a

while to address on administration of vaccines (Ford, 2016). The guidelines proposed

the vaccination protocol and recommendations, but did not mention the site where the

vaccine should be injected, although the shoulder was recommended once in the Q &

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A section of 2011 AAHA (American Animal Hospital Association) Canine

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Vaccination Guidelines (Welborn et al., 2011). Dorsal surface of the neck is another

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place commonly selected for vaccination due to its convenience for operation in pets.
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The underjaw, popliteal fossa and shoulder contain lymph nodes and the antigens
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may be easily accessible to lymphocytes and induce the immune response when a

vaccine is injected in these sites. In the traditional Chinese veterinary medicine,

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houhai acupoint, the dorsal midline between the anus and the tail base, is a place for

acupuncture stimulation to treat animal’s diarrhea, constipation and rectal paralysis,

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etc. Some studies reported that injection of a vaccine at houhai acupoint induced a

higher immune response in animals (Gao and Li, 1995; Zhang and Yu, 1996; Huang
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and Meng, 2001). However, no report has been found regarding injection of a vaccine

at this site to improve the immune response in dogs. In this study, the underjaw,

popliteal fossa, back and houhai acupoint in rats and the shoulder, nape region and

houhai acupoint in dogs were investigated for their effects on the immune responses

induced by a vaccine.

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2. Material and methods

2.1 Animals

Female SD rats were purchased from Shanghai Laboratory Animal Center (SLAC)

Co. Ltd. (Shanghai, China), and housed in polypropylene cages with processed

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corncob bedding in hygienically controlled environment. The temperature was

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controlled at 24 ± 1°C and humidity at 50 ± 10%. Feed and water were supplied ad

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libitum. Ten healthy female Rottweiler puppies at their age of 34 ± 2 days were kept

in the kennels of Zhoushan Sparda’s Dog Industry Co. Ltd. Zhoushan, China. All
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animals received humane care according to the criteria outlined in the Zhejiang
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University Committee on Animal Care and Use.

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2.2 Vaccines
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A quintuplet vaccine containing live attenuated canine distemper virus (CDV),

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adenovirus type 2 (CAV-2), parvovirus (CPV), parainfluenza virus (CPIV) and rabies

virus (Lot: 20150101) was the product of Jilin Five Star Animal Health Ltd. Co. (Jilin,
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CN). Nobivac® Puppy DP (containing live attenuated CDV and CPV, Lot: A122D01)

and Nobivac® DHPPI (containing live attenuated CDV, CAV-2, CPV and CPIV, Lot:

A421B01) were the products of Schering-Plough Animal Health Ltd. Co. (Wellington,

NZ).

2.3 Experiment A

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Rats were randomly divided into five groups with eight rats in each. The

quintuplet vaccine was s.c. injected twice with No. 5 needle at 2 weeks interval at one

of following four sites (Fig. 1A): (1) houhai acupoint, the center part of depression on

the midline between the anus and the ventral base of the tail (Chrisman and Xie,

2007), marked with H, (2) under jaw, the point between the sternohyoid muscles

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under its jaw, marked with U, (3) popliteal fossa, marked with P and (4) back, the last

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thoracic vertebra on the dorsal middle line, marked with B. To make the inoculation

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exactly in houhai acupoint, the needle pierced was parallel to the spine at 0.6 - 0.8 cm

depth. The control group was subcutaneously injected with saline at the back. Blood
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samples were collected from the tail vein with No. 7 needle at 2, 4, 6, weeks after the
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booster immunization (Fig. 2A). Serum was prepared and preserved at -20 °C until

use.
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2.4 Experiment B
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This experiment was carried out in a private kennel, Zhoushan Sparda’s Dog

Industry. The use of vaccines and vaccination schedules was the owner’s routine
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practice and changes were not allowed except for vaccination sites. Dogs were first

immunized with 2 ml of vaccine Nobivac® Puppy DP and then with 2 ml of vaccine

Nobivac® DHPPI three weeks later. The vaccine was injected at one of following

three sites (Fig. 1B) : (1) houhai acupoint, the center part of depression on the midline

between the anus and the ventral base of the tail (Chrisman and Xie, 2007), marked

with H, 4 dogs, (2) the shoulder region, the depression of the medial shoulder joint,

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marked with S, 3 dogs and (3) the nape, at the fourth cervical vertebra on the dorsal

middle line, marked with N, 3 dogs. To make the vaccines injected exactly in houhai

acupoint, the needle pierced was parallel to the spine at 1 - 1.2 cm depth. Blood

samples (2 ml) were collected 3 weeks after the immunisation of Nobivac ® Puppy DP

and 2, 4 and 6 weeks after immunisation of Nobivac ® DHPPI (Fig. 2B). Serum was

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prepared and preserved at -20 °C before antibody assay.

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2.5 Determination of antibody responses

The serum anti-rabies antibodies in rats were determined by a commercial ELISA

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kit (SERELISA® Rabies Ab Mono Indirect, Zoetis Inc., FR). Briefly, all rat sera were
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diluted 1:10 in Sample Diluent. To make a standard curve, OIE reference serum was

diluted in Sample Diluent 1:10, 1:25, 1:60, 1:80, 1:170, 1:400, 1:800, respectively.
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Sample Diluent (90 μl) was added to pre-coated 96-well plates. Ten microliter of
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negative control, positive control, pre-diluted OIE reference sera and pre-diluted rat
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sera were added to the plates orderly. The plates were incubated for 1 h at 37°C and

then washed four times. After that, 100 μl of 1:10 diluted Horseradish Peroxidase
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conjugate protein A was added to the plates and incubated for 1 h at 37°C. After

another washing, 100 μl of substrate solution was added and further incubated for 20

min at 20°C. The reaction was stopped by adding 50 μl of stop solution. OD value

was measured by a microtiter plate reader at 450 nm and 630 nm. Each sample was

performed in duplicate. The concentrations of serum antibody were calculated from

interpolation of the standard curve and expressed as IU ± SD/ml.

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Serum specific antibody responses to CDV, CPV, CAV-2 and CPIV were

measured by ELISA kits (Rapidbio Inc., US A) according to the manufacturer ’s

protocol. Briefly, 10 μl of serum and 40 μl of diluent solution buffer were added to the

antigen-coated wells. Then, 100 μl of conjugate was added to each well and incubated

for 1h at 37 °C. After that, the plates were washed with a washing solution followed

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by addition of Chromogen Solution A (50 μl) and B (50 μl). The plates were then

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incubated for 15 min at 37 °C and reaction was terminated by adding 50 μl of stop

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solution (2M H2 SO4 ). OD value was measured by a microtiter plate reader at 450 nm.

The serum antibody responses in dogs were expressed as OD value ± SD. The
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concentration of serum antibody in rats was calculated from interpolation of a
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standard curve and expressed as μg/ml.

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2.6 Splenocyte proliferation assay

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Rat spleen was aseptically collected 6 weeks after the last immunization in HBSS
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(Hank’s balanced salt solution), and was grinded on a 200 nylon mesh to obtain a

homogeneous cell suspension. The pelleted cells were washed three times in HBSS
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and re-suspended in complete medium (RPMI 1640 supplemented with 100 IU/ml

penicillin, 100 μg/ml streptomycin and 10 % heat inactivated FBS). Cell viability and

numbers were counted by trypan blue dye. Splenocyte proliferation was assayed as

described previously with some modification (Song et al., 2009). Briefly, splenocyte

were seeded into a 96-well flat-bottom microtiter plate at 5.0 × 106 cell/ml in 100 μl

complete medium, thereafter concanavalin A (Con A, final concentration 5 μg/ml),

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LPS (final concentration 8 μg/ml) or medium were added giving a final volume of

200 μl. The plates were incubated at 37 °C in a humid atmosphere with 5 % CO 2 for 2

days. Each sample was performed in triplicate. The cell proliferation was evaluated

using MTT methods. Briefly, 50 μl of MTT solution (2 mg/ml) were added to each

well 4 h before the end of incubation. The plates were then centrifuged (1400 g, 5 min)

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and the untransformed MTT were removed carefully by pipetting. To each well 150 μl

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of a DMSO working solution (192 μl DMSO with 8 μl 1 N HCl) was added, and the

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absorbance was evaluated in a microtiter plate reader at 570 nm with a 630 nm

reference after 15 min. The stimulation index (SI) was calculated based on the
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following formula: SI = the absorbance value for mitogen cultures divided by the
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absorbance value for non-stimulated cultures.

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2.7 Cytokine mRNA expression

Total RNA was isolated from splenocytes by a commercial kit (RNAisoT M Plus,
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TaKaRa Co., Ltd) according to the manufacture’s protocol and the concentration of

total RNA was quantified by determining the optical density at 260 nm. The reverse
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transcription was performed by iScript cDNA Synthesis Kit (Bio-Rad, USA) as

described previously (Su et al., 2014).

Relative quantitation of IL-4, IL-10, IL-12 and IFN-γ cDNA and β-actin message

was conducted on ABI 7500 (PE Applied Biosystems, USA). Real-time RT-qPCR was

performed using the StepOnePlus Real- Time PCR System (Applied Biosystems; USA)

and the EvaGreen qPCR Mastermix- ROX (Applied Biological Materials Inc., Canada)

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on 2 μl of cDNA from the 20 μl reaction volumes, containing 300 nM of each

gene-specific primer (Table 1). Specificity of the amplified products was confirmed

by melting curve analysis. For each sample, three independent repetitions were

performed. Relative quantification between samples was achieved by the 2−ΔΔCt

method, and calculated by the software REST 2005 (provided by Eppendorf Company,

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German). It is reported as the n-fold difference relative to the target gene mRNA

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expression of the calibrator group (group of rats injected with saline solution).

2.8 Statistical analysis

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One-Way ANOVA and Duncan analysis were performed using SPSS 20.0. The
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data were expressed as mean ± SD. p < 0.05 was considered significant.
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3. Result
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3.1 Serum antibody responses

Fig. 3 showed the serum antibody responses in rats injected with a quintuplet
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vaccine in houhai acupoint, underjaw, popliteal fossa or back. At 4 weeks post

immunization, the antibodies reached their high levels. Serum concentrations of the

antibody to parvovirus were 174.7 ± 4.4, 154.4 ± 4.2, 147.2 ± 3.9 and 121.3 ± 3.8

μg/ml, respectively; the concentrations of the antibody to distemper virus were 241.0

± 7.8, 192.8 ± 6.0, 198.1 ± 7.4 and 141.6 ± 4.8 μg/ml, respectively; the concentrations

of the antibody to adenovirus type-2 were 216.3 ± 8.1, 201.6 ± 3.1, 190.0 ± 2.9 and

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159.3 ± 3.6 μg/ml, respectively; the concentrations of the antibody to parainfluenza

virus were 193.4 ± 5.6, 167.1 ± 5.0, 155.6 ± 5.0 and 120.7 ± 4.8 μg/ml, respectively;

the concentration of antibody to rabies virus were 2.157 ± 0.189, 1.665 ± 0.226, 1.657

± 0.259, 0.659 ± 0.138 IU/ml, respectively. The highest antibody level was found in

rats immunized in houhai acupoint and lowest level was in rats immunized in back.

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Fig. 4 showed the serum antibody responses in dogs vaccinated in houhai

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acupoint, shoulder or nape with cut off values (OD450 ) of positive serum antibodies to

parvovirus, distemper virus, adenovirus type-2 and parainfluenza virus. The serum
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antibody levels were found progressively increased in each group of dogs. Importantly,
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Fig. 4B showed that 3 weeks after primary immunization, 3 of 4 dogs in group H had

positive serum antibody to distemper virus, only 1 of 3 dogs in group S while no

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positive antibody was detected in group N; Fig. 4C showed that at 2 weeks post
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immunization, all dogs were detected to have positive serum antibody to adenovirus
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type-2 in groups H and S but only 2 of 3 dogs had positive serum antibody in group N;

Fig. 4D showed that 2 of 4 dogs were detected to have positive serum antibody to
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parainfluenza virus in group H while serum antibody from dogs in groups S and N

were negative. Similar to the results in rats, highest antibody response was found in

group H, the second was in group S, and the lowest was in group N.

Besides, no side effects were observed when vaccines were injected in houhai

acupoint, underjaw, popliteal fossa or back areas of rats or houhai acupoint, shoulder

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or nape areas of dogs.

3.2 Splenocyte proliferative responses

Fig. 5 showed the splenocyte proliferation in rats immunized at houhai acupoint,

underjaw, popliteal fossa or back. The proliferative responses to Con A and LPS of

splenocytes isolated from the rats which were immunized in houhai, underjaw and

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popliteal fossa were significantly higher than those of the rats immunized in the back

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site.

3.3 Cytokine mRNA expression

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Fig. 6 showed the cytokine mRNA expression by splenocytes of the rats
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immunized in different sites. According to the levels in cytokine mRNA expression,

the injection sites exhibited in the following proper order: houhai acupoint >
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underjaw > popliteal fossa > back group.

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4. Discussion and conclusion

The present study demonstrated that injection of a vaccine at anatomically

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different sites induced different levels of antibody responses. In rats immunized in the

areas of houhai acupoint, underjaw, popliteal fossa and back, the highest antibody

responses, mRNA expression of IL-4, IL-10, IL-12 and IFN-γ by splenocytes and

splenocyte proliferative responses to Con A and LPS were found when a vaccine was

injected in houhai acupoint. In addition, the highest antibody response was also

detected in dogs when a vaccine was injected in houhai acupoint.

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Different immune response levels induced by injection of a vaccine in different

sites may be attributed to uneven distribution of the immunocytes in the body. Once a

vaccine was introduced, immunocytes were recruited into the injection site from other

places. After that the APCs were activated and drained to lymph nodes where a typical

immune process occurred. The fat tissues in the injection site may hamper migration

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of the immune cells (Centers for Disease Control, 1985; Shaw et al., 1989). As the

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nape in dogs and the back region in rats have thicker fat layer and looser skin, antigen

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injected at these areas were supposed to have less opportunity to encounter

lymphocytes and induce lower immune response. Compared to the nape and back
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regions, the shoulder in dogs and the underjaw and popliteal fossa regions in rats are
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the places where draining lymph nodes are located nearby, and have compact skin and

more muscular movement. These factors might facilitate the recruitment of

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immunocytes to the draining lymph nodes cause higher immune responses when a
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vaccine was introduced in these sites. Interestingly, a vaccine injected in houhai

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acupoint induced the highest immune responses in both rats and dogs although the

exact mechanism is unknown. This may relate to its anatomical location where the
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caudal mesenteric, iliosacral, iliofemoral, femoral and inguinal lymph nodes are

nearby (König and Liebich, 2014). We recently found that a blue dye could be rapidly

transported into the inguinal, popliteal fossa, sciatic, para-aortic and iliac lymph nodes

when the dye was injected in houhai acupoint in rats (unpublished data).

Lymphocyte proliferation assay revealed the capacity of eliciting an effective

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cellular immunity by vaccinating at different sites. Depending on the mitogen used,

Con A stimulates T cell proliferation, whereas LPS stimulates B cells (Tizard, 1992).

As shown in Fig. 5, compared to the back site, significantly higher splenocyte

proliferative responses to both Con A and LPS were found when a vaccine was

injected in houhai acupoint, underjaw or popliteal fossa. To induce antibody

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production, both triggered T and B lymphocytes are required. The splenocyte

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proliferative induced by ConA and LPS presented a similar tendency which were

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observed in serum antibody assay. Therefore, the different level of serum antibody in

rat vaccinated with the quintuplet vaccine at different anatomic sites (Fig. 1A), at least
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in part, may be attributed to the activation of T and B-cells.
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To resist intracellular pathogen infections, Th1 type immune responses dominated

by the production of IL-12, IFN-γ, IgG2a and cytotoxic T lymphocytes are needed,
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while protecting from extracellular pathogen infections is often associated with

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humoral immune responses dominated by the production of IgG1, IL-4 and IL-10
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(Constant and Bottomly, 1997). IFN-γ, a key cytokine secreted by Th1-CD4 and

Th1-CD8 lymphocytes as well as natural killer cells, is essential for the activation of
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macrophages which can destroy intracellular pathogens by producing NO and O3

(Feria-Romero et al., 2011). IL-12 is a powerful inducer of the differentiation of naïve

T-helper cells to Th1-type cells that generally produce specific sets of cytokines, such

as IFN-γ and TNF-β. On the other hand, IL-4 plays an important role in Th2

differentiation and potently induces IL-10, IL-5 and IL-9 production (Constant and

Bottomly, 1997). The ideal anatomic sites for vaccination should be able to enhance

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both Th1 and Th2 type immune responses to protect animals from viral infections. In

this study, we investigated IL-12, IFN-γ (Th1 type cytokines) and IL-4, IL-10 (Th2

type cytokines) mRNA expression of rat splenocyte. As shown in Fig. 6, the paralleled

tendency (houhai acupoint group > under jaw group > popliteal fossa group > back

group) were observed in all investigated cytokines (p < 0.05). This result suggested

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that both Th1 and Th2 immune responses were enhanced by vaccinating at houhai

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acupoint. Furthermore, IL-4, IL-5, IL-10 were associated with B cell activation and

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IgG production (Pecanha et al., 1992), which indicated that the different level of rat

serum antibody may be attributed to the Th2 cell excitation and the related cytokine
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production.
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Therefore, the present study demonstrated that administration of a vaccine in

anatomically different sites induced different levels of antibody production and the
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houhai acupoint induced the highest antibody production. Immunized at houhai

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acupoint provided a balance enhancement on immunoresponses. For these reason,

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houhai acupoint should be recommended for vaccine injection site in order to

improve the immune response in dogs.

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Acknowledgements

This study was supported by the American Academy of Veterinary Acupuncture

(AAVA).The authors wish to thank the staffs of Zhoushan Sparda’s Dog Industry Co.

Ltd for keeping the dogs as well as the students of TCVML (Laboratory of Traditional

Chinese Veterinary Medicine) for their assistance in sample collection.

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Table 1
Sequences of primers for quantitative RT-PCR of cytokines.

Gene Primer sequence

β-actin forward:5’-CGACGAGGCCCAGAGCAAGAGAGG-3’
reverse:5’-CGTCAGGCAGCTCATAGCTCTTCTCCAG
GG-3’
IL-4 forward:5’-GCCCCCACCTTGCTGTCACCCTG-3’

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reverse:5’-TTCAGTGTTGTGAGCGTGGACTACTTCA-
3’

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IL-10 forward:5’-GGCCATTCCATCCGGGGTGACAATA-3’
reverse:5’-GGCCTTGTAGACACCTTTGTCTTGG-3’

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IL-12 forward:5’-TGGTTTGCCATGGTTTTGCTGG-3’
reverse:5’-AGGAATGGGGAGTGCTCCAGGA-3’
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IFN-γ forward:5’-GCTCTGCCTCATGGCCCTCTCTGGC-3’
reverse:5’-GCACCGACTCCTTTTCCGCTTCCTT-3’
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Legends

Fig.1. Injection sites for vaccination in rats (A) and dogs (B). H, houhai acupoint; U,

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under jaw; P, popliteal fossa; B, back; S, the shoulder region; N, the nape of neck.

Fig.2. Timeline of vaccination and sample collection schedule for rats (A) and dogs

(B).

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Fig.3. Levels of serum specific antibodies in rats. Rats (n = 8/group) were injected

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(s.c.) with the quintuplet vaccine (containing canine distemper virus, adenovirus type

SC
2, parvovirus, parainfluenza virus and rabies virus) at houhai acupoint, under jaw,

popliteal fossa or back. After that, sera were collected for analysis of serum specific
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IgG using ELISA method. The values are presented as the mean ± S.D. Bars with
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different letters are statistically different (p < 0.05).

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Fig.4. Levels of serum specific antibodies in dogs. Rottweiler puppies were

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vaccinated (s.c.) against canine distemper virus (CDV), canine parvovirus (CPV),
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canine adenovirus type 2 (CAV-2) and canine parainfluenza virus (CPIV) in houhai

acupoint (H, 4 dogs), the shoulder region (S, 3 dogs) and the nape of the neck (N, 3
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dogs). After that, blood samples were collected for determination of serum specific

antibody levels by ELISA kits. * p < 0.05, ** p < 0.01, NS, no significant.

Fig.5. Splenocyte proliferative responses to Con A and LPS. Rats (n = 8/group) were

injected (s.c.) with the quintuplet vaccine (containing canine distemper virus,

adenovirus type 2, parvovirus, parainfluenza virus and rabies virus) in houhai

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acupoint, under jaw, popliteal fossa or back. After that, splenocytes were prepared and

cultured for 48 h with Con A or LPS. Cell proliferation was measured by the MTT

method as described in the text, and shown as a stimulation index (SI). The values are

presented as the mean ± S.D. Bars with different letters are statistically different (p <

0.05).

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RI
Fig.6. Expression of IL-4, IL-10, IL-12 and IFN-γ mRNA by splenocytes. Rats (n =

SC
8/group) were injected (s.c.) with the quintuplet vaccine (containing canine distemper

virus, adenovirus type 2, parvovirus, parainfluenza virus and rabies ) in houhai

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acupoint, under jaw, popliteal fossa or back. After that, splenocytes were prepared for
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analysis of mRNA expression of IL-12, IL-10, IL-4 and IFN-γby real- time PCR. The

values are presented as the mean ± S.D. Bars with different letters are statistically
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different (p < 0.05).

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C EP
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Highlights

 Immune responses were detected after a vaccine was injected at anatomically

different sites (Houhai, underjaw, popliteal fossa or back in rats, Houhai,

shoulder or nape in dogs).

 Vaccination at Houhai acupoint induced the highest antibody responses in both

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rats and dogs.

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 Vaccination at Houhai acupoint significantly increased proliferative responses to

SC
Con A and LPS as well as mRNA expression of IL-4, IL-10, IL-12 and IFN-γ of

splenocytes in rats.
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 Houhai acupoint is recommended for injection of a vaccine to improve the
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immune response in dogs.

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C EP
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