Levamisole enhances immunity in ducklings vaccinated against Riemerella anatipestifer


  • Yuewei Zhang,

    1. Key Laboratory of Rapid Diagnostic Technology for Animal Disease, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China
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    • Yuewei Zhang and Huiling Chen contributed equally to this work.
  • Huiling Chen,

    1. Beijing General Station of Animal Husbandry and Veterinary Service, Beijing Municipal Bureau of Agriculture, Beijing, China
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    • Yuewei Zhang and Huiling Chen contributed equally to this work.
  • Xiangfan Zeng,

    1. Key Laboratory of Rapid Diagnostic Technology for Animal Disease, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China
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  • Peng Wang,

    Corresponding author
    1. Animal Health Inspection Institute, Beijing, China
    • Correspondence

      Wenxue Wu, Key Laboratory of Rapid Diagnostic Technology for Animal Disease, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China. Tel: +86-10-6273-3048; fax: +86-10-6273-3048; email: wuwenxue@cau.edu.cn

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  • Jinxiang Li,

    1. Chinese Academy of Agricultural Sciences, Beijing, China
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  • Wenxue Wu

    1. Key Laboratory of Rapid Diagnostic Technology for Animal Disease, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China
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Oil-adjuvant-inactivated vaccine is one of the most cost-effective vaccines used to protect ducklings against RA infection; however, it does not provide complete protection in very young ducklings with immature immune systems. In the current study, LMS was used as an immunopotentiator to improve the immune system in ducklings. Serum immunoglobulin (Ig)G titers and the secretions of both Th1-type (IFN-γ and IL-2) and Th2-type (IL-4 and IL-10) cytokines were higher in ducklings that had been vaccinated with LMS. In addition, a significantly higher T-lymphocyte proliferation rate was obtained with the addition of LMS. Furthermore, all of the ducklings vaccinated with LMS were protected against RA on the 9th day post-vaccination, whereas only 69.2% of the ducklings were protected in the group that did not receive LMS. These results suggest that LMS might be a useful adjuvant to enhance the immune response of ducklings. The use of LMS may also alleviate local injection lesions, caused by the oil-emulsion vaccine, by reducing the dose of the vaccine.

List of abbreviations



peripheral blood mononuclear cells


Riemerella anatipestifer


stimulation index


tryptic soy agar

Riemerella anatipestifer (RA) is a major cause of disease throughout the world. RA affects farm ducks and other species of domestic poultry and leads to economic loss through high mortality rates, low feed conversion, and high treatment costs [1, 2]. RA infection is a highly contagious disease that causes acute or chronic septicemia in ducks. The mortality rate has been reported to be as high as 70% in ducks with severe RA infection [3-7]. The age at which ducklings are becoming infected with RA has been decreasing during recent years and RA is now affecting younger ducklings than it did previously.

Antibiotics are widely used to prevent and control RA infection, but the overuse of antibiotics has contributed to the emergence of drug-resistant bacterial strains [8, 9]. Furthermore, overuse of antibiotics often leads to antibiotic residues, which can be detected in duck-related products [2]. Vaccine immunization may be an alternative way to control this disease, thus avoiding the use of antibiotics. Various vaccines for RA have been tested for the protection of farm ducks [10, 11]. Oil-emulsified bacterin has been found to be the most effective inactivated vaccine; however, it does not provide complete protection against RA in very young ducklings (those younger than 21 days old), as their immune systems are not yet well developed.

LMS is an immunomodulator that has been shown to promote the development of immune systems, restore the function of depressed phagocytes and lymphocytes, and enhance both cell-mediated and humoral immune responses [12-18]. In the current study, protection against RA was examined in ducklings inoculated with oil-emulsified, inactivated vaccine in combination with LMS, and antibody and cytokine levels, and T-lymphocyte proliferation response were examined in order to determine whether LMS enhances cell-mediated and/or humoral immunity in these ducklings.



Virulent RA strain ZZY7 was isolated from White Peking ducks from ZhangZiYing farms (Beijing, China) and characterized as serotype 2.


Bacteria were grown on TSA (Becton Dickinson Company, New York, NY, USA) with 3% fetal bovine serum (ShengMaYuanHen, Beijing, China). TSA plates were incubated at 37 °C with 5% CO2. A volume of 100 mL tryptic soy broth (TSB; Becton Dickinson Company) containing 3% FBS was inoculated with three or six colonies and the colonies were incubated in a shaker bath at 37 °C for 16–24 hr. Purity was checked on TSA and viable CFU/mL were determined by the serial dilution method. The bacteria were inactivated with 0.3% formalin at 37 °C for 24 hr, and concentrated by centrifugation at 8200 g for 15 min at 4 °C and a water-soluble antigen approximately equivalent to 6.0 × 1010 CFU/mL was obtained. An oil-emulsified vaccine was produced by mixing the water-soluble antigen and white oil adjuvant (Port Jerome Gravenchon Lube Plant, Normandy, France) at a ratio of 100:200 (v/v), and the LMS-oil-emulsion vaccine was produced by adding the LMS to the oil-emulsion vaccine to a final concentration of 4 mg/mL.


A total of 57 White Peking ducklings, aged 1 day, were purchased from QianCheng farm (Beijing, China). Ducklings had no RA-specific antigens and antibodies and were maintained in a pathogen-free environment. At the age of 10 days old, the ducklings were divided into three groups of 19 birds each (n = 19). Each duckling received a subcutaneous injection, in the neck region, of 0.5 mL: (i) oil-emulsified vaccine; (ii) LMS-oil-emulsion vaccine; or (iii) 0.9% NaCl (non-vaccinated control). Animal treatments were governed by the Regulations of Experimental Animals of Beijing Authority and approved by the Animal Ethics Committee of the China Agricultural University.

Detection of adverse reactions

Adverse systemic and local injection-site reactions were observed and recorded. Sections of tissue were taken from the injection sites (nape) 2 weeks after inoculation with vaccines or 0.9% NaCl and Gram stained. Bodyweights of ducklings in all three groups were recorded 1 day before inoculation and 10 days after inoculation and the average gain in bodyweight during the observation period was calculated for each group and compared.

Detection of RA serum antibodies by ELISA

Blood samples were collected from six ducklings in each group on days 9, 16, 23, 30, 37, and 44 after vaccination, and changes in antibody levels against RA antigen were detected by ELISA as follows: plates were coated with 100 µL RA lysate antigen at a concentration of 5.5 µg/mL [19]. After overnight incubation at 4 °C, the wells were washed three times with PBST, subsequently blocked with 5% BSA–PBST for 2 hr at 37 °C, and then incubated with appropriate dilutions (determined in preliminary experiments as 1:25–1:12,800) of serum samples for 1 hr at 37 °C. The plates were subsequently incubated with HRP-labeled goat anti-duck antibody (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA) (1:10,000 dilution in PBST) at 37 °C for 1 hr and then washed with PBST. ELISA was prepared using a 10-mg 3,3′,5,5′-tetramethylbenzidine (TMB tablet; Sigma, St Louis, MO, USA), dissolved in 1 mL of 0.025 M phosphate-citrate buffer. A 100-µL volume of the resulting solution was added to each well and the wells were incubated in darkness for 10 min. A 50-µL volume of 0.2 M H2SO4 was then added to stop the reaction. The OD450 nm was determined using a model 680 microplate reader (Bio-Rad, Hercules, CA, USA). Titer values were assigned as the highest dilution at which the OD was higher than the mean of the OD value produced by the negative serum plus 2 SD. The mean titers were calculated using log conversion for each dilution.

T-lymphocyte proliferation assay

Peripheral blood samples were collected 9 days after vaccination. Ficoll-Paque (Chinese Academy of Medical Sciences, Tianjin, China) was used to separate the lymphocytes from whole blood [19]. The lymphocytes were washed three times with PBS, resuspended in RPMI-1640, diluted to 4 × 104 cells/well, and incubated at 41 °C for 2 hr. The lymphocytes were then treated with RA lysate (12.5 µg/mL, 50 µL/well) as a stimulating antigen and with BSA as a non-related antigen [18, 19]. RA-free culture solution was used as a negative control. Wells were incubated for 48 hr and then 20 µL (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)/phenazine methosulfate (MTS/PMS) was added to each well and the wells were incubated for an additional 4 hr. OD values were measured at 490 nm (Bio-Rad) according to the introduction of CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA). SI was calculated using the following formula:

display math(1)

Cytokine response to immunization

Serum samples were collected on days 9, 23, and 37 post-vaccination. Commercial ELISA kits (IFN-γ and IL-2 ELISA KIT from USCNLIFE, Wuhan, China; IL-4 and IL-10 KIT from Hermes Criterion Biotechnology, Vancouver, BC, Canada) were used to quantify levels of the cytokines in peripheral blood samples, according to the manufacturer’s instructions.


Nine days after the immunizations, the remaining 13 ducklings in each group were challenged by i.m. injection of 4.0 × 105 CFU RA serotype 2. Observations and clinical signs, such as depression, lethargy, increase in nasal discharge and ataxia were recording during the subsequent 10 days, Pathological changes, such as hepatomegaly and splenomegaly with bleeding points on the serous membrane, fibrinous pericarditis, perihepatitis, and air sacculitis were examined by necropsy. Protection index of each vaccination group was calculated based on the morbidity and mortality.


Statistical analysis of the differences between groups was done via anova test incorporating a logarithmic transformation of individual serum antibody. A one-sided Student’s t-test was done for the T-cell proliferation assays and bodyweight gains. Fisher’s exact test was done for the groups challenged with RA serotype 2. Results are expressed as mean ± SE.


Adverse reactions

No systemic adverse reactions were observed in any of the ducklings after inoculation. However, congestion and edema appeared at the injection site on the first day post-vaccination and began to subside gradually on day 4, when a small nodule could be detected by touch. All the clinical signs disappeared after 2 weeks. An obvious accumulation of inflammatory cells and hyperplasia of fibroblasts were found in pathological sections from injection sites of vaccine-group ducklings, but no abnormal changes were found at the injection sites of control-group ducklings (Fig. 1). Body temperature and ingestion were both normal in all the ducklings during the observation period. Average gain in bodyweight during the observation period was assessed by t-test in each group, and all P-values were >0.05 (Table 1), indicating that the vaccines did not significantly affect bodyweight gain in any group of ducklings.

Figure 1.

Comparison of sections from the injection site of immunized and control ducks. (a) Injection site of the immunized group. Original magnification ×10. (b) Detailed section of the injection site. Original magnification ×40. Obvious inflammatory cells had accumulated in the injection site with hyperplasia of fibroblasts, but without nodules or granulomas. (c) Control group.

Table 1. Duckling bodyweight and bodyweight gain during the observation period (mean ± SE)
GroupBodyweight before inoculation (g)Bodyweight 10 days post-inoculation (g)Bodyweight gain (g)
  1. Ducklings were immunized with the following vaccines or injected with 0.9% NaCl as control at 10 days old: 1, LMS-oil-emulsion; 2, oil-emulsion vaccine; 3, control.
1315.4 ± 16.81177.4 ± 34.9862.0 ± 39.7
2311.0 ± 14.51186.2 ± 55.4875.2 ± 45.1
3307.3 ± 15.81175.8 ± 48.8868.5 ± 36.6

Serum RA antibodies after vaccination

Blood samples were collected from the ducklings on days 9, 16, 23, 30, 37, and 44 after the immunization, and serum antibodies against RA serotype 2 were titrated by ELISA (Fig. 2). Antibody titers of the control ducklings, injected with 0.9% NaCl, were below the detection limit. However, ducklings in both groups that received the oil-emulsion or LMS-oil-emulsion vaccines had high IgG titers. Furthermore, ducklings in the LMS-oil-emulsion vaccine group had higher antibody titers than ducklings in the oil-emulsion vaccine group, especially at 37 and 44 days post-immunization (Fig. 2, P < 0.05). These results indicate that LMS can promote the efficacy of the oil-adjuvant inactivated vaccine by enhancing the secretion of IgG for a longer period of time.

Figure 2.

Immunoglobulin (Ig)G titers in ducklings. Serum from ducklings was obtained on days 9, 16, 23, 30, 37, and 44 after immunization and analyzed by ELISA. Results represent the average of six serum samples for each group. Antibody titers of the control group (0.9% NaCl) were below the detection limit. Values are expressed as mean   standard error. *Significant differences (P < 0.05). mim12169-gra-0001, LMS-oil-emulsion vaccine; □, oil-emulsion vaccine.

Cytokine response

ELISA kits were used to quantify cytokine concentrations in peripheral blood of ducks treated with different immunization strategies. As shown in Figure 3, after 37 days post-immunization, the level of IFN-γ in the oil-emulsion vaccine group was 88.11 ± 33.24 pg/mL and was not significantly different from the level of IFN-γ in the control group (75.14 ± 8.71 pg/mL). However, the level of IFN-γ was >200 pg/mL when 4 mg/mL LMS was added to the vaccine. In addition, a greater than twofold increase in IL-2 was detected in the LMS-oil-emulsion vaccine group at 23 and 37 days post-immunization (P < 0.05). Furthermore, ducklings in the LMS-oil-emulsion vaccine group produced significantly larger amounts of both IL-4 and IL-10 (Th2 cytokines) at 37 days post-immunization than did those in the non-LMS group.

Figure 3.

Cytokines induced by immunization with the vaccines. Serum IFN-γ, IL-2, IL-4, and IL-10 were detected via ELISA. IL-2 in the control group was below the detection limit. The tests were done on days 9, 23, and 37 post-vaccination. The results represent the average of six serum samples from each group and are expressed as mean ± standard error. *Significant differences (P < 0.05). ■, LMS-oil-emulsion vaccine; mim12169-gra-0001, oil-emulsion vaccine; □, control.

T-lymphocyte proliferation

PBMC from peripheral blood samples were collected 9 days after immunization and used for lymphocyte proliferation assays. As shown in Figure 4, T-lymphocyte proliferation responses were high in ducklings of the groups that received vaccines with or without LMS. However, the SI value in the LMS-oil-emulsion vaccine group was significantly higher than that in the oil-emulsion vaccine group (2.87 vs 1.76; P < 0.01). In addition, the SI value was low (0.47) when BSA was used as the non-related stimulating antigen, suggesting that the immune response was specifically caused by the bacterial protein in the vaccine.

Figure 4.

T-cell proliferation in response to RA vaccines. BSA, T cells from the LMS-oil-emulsion vaccine group after stimulation with BSA; Control, T cells from the control group after stimulation with RA lysates; LMS, T cells from the LMS-oil-emulsion vaccine group stimulation with RA lysates; Oil, T cells from the oil-emulsion vaccine group after stimulation with RA lysates. *Significant differences (P < 0.05) compared with the oil-emulsion vaccine group.

Protection of the LMS-oil-emulsion vaccine

Protection efficacies of the vaccine with and without LMS are summarized in Table 2. All of the ducks in the control group (0.9% NaCl) died within 10 days of the RA challenge. The major lesions observed in the control group were fibrinous serositis, caseous salpingitis, and arthritic septicemia. Ducks immunized with the LMS-oil-emulsion vaccine survived and were completely protected against subsequent challenge with the homologous RA strain; however, only 69.2% of the ducklings in the oil-emulsion group were protected. These results suggest that LMS significantly improves the protection efficacy of the oil-emulsion vaccine.

Table 2. Mortality and PI of ducklings when challenged with RA strain zzy-7
GroupInoculation time (days old)Protection efficacy after challenge
Mortality (%)PI (%)
  1. Significantly different from Group 1 (*P < 0.05). Group 1, LMS-oil-emulsion; Group 2, oil-emulsion vaccine; Group 3, control. PI, protection index.
1100/13 (0)100
2104/13 (30.8)*69.2*
31013/13 (100)0


China has the largest duck industry in the world, accounting for nearly 80% of the global duck market at more than 2 billion dollars per year. RA is one of the most hazardous contagious diseases and causes economic losses of approximately 200 million dollars each year. The first case of infectious serositis caused by RA infection was reported in 1987 and, since then, a total of 25 RA serotypes have been reported [20]. Approximately 37% and 29% of RA cases are of serotypes 1 and 2, respectively.

RA always infects the duckling between 1 and 8 weeks of age and is especially prevalent in ducklings that are 2–3 weeks old; adult ducks are not susceptible to the infection. Therefore, 10-day-old ducklings were chosen for the current study. The high susceptibility of ducklings to RA may be as a result of the immature immune systems of young ducks. For example, ducks begin to secrete IgA at 3–4 weeks of age, and the T-cell immune system does not mature until 3 weeks of age [10, 21, 22]. Maternal immunization can provide efficacious protection from RA infections in ducklings younger than 1 week of age; however, when maternal antibody titers decrease, ducklings become susceptible to RA infection. Thus, effective vaccines for young ducklings are particularly important. It should be confirmed that ducklings have no maternal antibody to RA prior to immunization. In the current study, at the time of purchase, all the ducklings were identified to have no RA-specific antigens and antibodies.

Some oil-emulsified killed vaccines have been registered and approved for use in China for the control of RA infection. However, a lower protection efficacy was obtained when the ducklings (immunization at the age of 10 days) were challenged 1 week after immunization [23]. This was confirmed in the present study; the immune protection index (69.2%) was low when the ducklings were challenged at 9 days after the immunization using oil-emulsified killed vaccines. LMS can be used to improve the protection efficacy of the vaccines when given to ducklings at an early age.

LMS promotes the development of the immune system through binding to the thymopoietin receptor [13, 24] and has a thymopoietin-like function. It increases numbers of lymphocytes, promotes differentiation, transformation, and proliferation of lymphocytes and causes an elevated immune response to antigen stimulation [16-19]. The increased immune function as a result of LMS makes it a suitable immunopotentiator. It has been used in chicken vaccines [25-27], a DNA vaccine against foot-and-mouth disease virus (FMDV) [28] and a binary inactivated vaccine of Pasteurella anatipestifer and Escherichia coli in ducks [11].

It had been reported that the RA inactivated vaccine can stimulate vaccinated ducks to produce larger amounts of both Th1 and Th2 cytokines [29], and a similar phenomenon was observed in the present study. The production of IL-2, IL-4, and IL-10 was higher in the oil-emulsion vaccine group compared with the control group and the addition of LMS to the inactivated vaccine further promoted secretion of both Th1 and Th2 cytokines. It has been well documented that LMS is able to promote Th1-type immune response [18, 30]. In the present study, an over twofold increase of IFN-γ and IL-2 was detected in the LMS-oil-emulsion vaccine group compared with the oil-emulsion vaccine group, supporting previous findings regarding the effects of LMS on Th1-type immune response. Moreover, significantly higher levels (P < 0.05) of IL-4 and IL-10 (Th2-type cytokines) were detected when the LMS-oil-emulsion vaccine was used, indicating that vaccination with LMS can induce both the Th1-type and the Th2-type immune response. Finally, promotion of the immune system by LMS enabled the LMS-oil-emulsion vaccine to completely protect the ducklings from the RA challenge. This may be as a result of some characteristics of the duck immune system; more research into the effect of LMS on the duck immune system is needed.

In summary, LMS is an effective immunoadjuvant and improves development of the immune system. When LMS is incorporated into an oil-emulsion inactivated RA vaccine, it protects ducklings from RA infection.


Authors declare no conflicts of interest.