Effect of Se-enriched Lactobacillus on Antibody of NDV of Broilers

Yaowei LIN Yanfei LIU Jiande YANG Shunyi QIN

Abstract This study was conducted to investigate the effect of Se-enriched lactobacillus on antibody of Newcastle Disease Virus （NDV） of broilers. One thousand day-old AA broiler chickens were selected from a chicken farm of Jinghai County， and the chickens were divided randomly into two groups， which respectively had five repeat groups with 100 chickens each， one named basal diet group， and the other named Se-enriched lactobacillus group. Corn soybean meal diet was selected for basal diet group， while 2% se-enriched lactobacillus was added into corn soybean meal diet for the other group. The method of feeding was free diet， drinking， and routine immunization program was adopted. This test period was 42 d. Two chickens were selected randomly in each repeat for slaughtering and collecting serums on the 14th， 28th and 42nd d. Assessments on the effect of Se-enriched lactobacillus in antibody titer of NDV of broilers were made by hemagglutination-inhibition test. The results indicated that antibody titer of NDV was increased significantly by Se-enriched lactobacillus.

Key words Se-enriched lactobacillus; NDV; Hemagglutination-inhibition test; Antibody titers

In recent years， scientists have studied Se and lactobacillus more and more widely， ranging from single biological activity to the biological activity of their complexes. The trace element Se is lacking in many cities in our country， and the breeding industry often needs to pay more attention to Se. The lack of Se in chickens can lead to decreased immunity， decreased appetite or desuetude， and even death[1]. The biological activity of the trace element Se is mainly anti-oxidation and growth promotion[2-3]. Lactobacillus can assist the body to digest and absorb food and inhibit the growth of harmful bacteria[4-5]. Se-enriched lactobacillus has also been extensively studied because of their common advantages. Adding Se-containing compounds （sodium selenite） directly to the feed is prone to Se poisoning without controlling the diet. After the biological conversion by lactobacillus， Se can be effectively supplemented without causing poisoning. Se-enriched lactobacillus is a new type of feed， which needs to be tested first in the application of aquaculture， mainly for meat and animals with production performance. Relevant studies have shown that the addition of Se-enriched lactobacillus in the diet can effectively reduce the toxicity of aflatoxin B1 to broilers[6]， increase weight and effectively reduce the feed-to-meat ratio， improve the antioxidant function of broilers[7]， and increase egg production and Se content of laying hens[8].

The Newcastle Disease Virus （NDV） mainly harms domestic chickens and turkeys， and is a huge harm to the breeding industry. In different regions， the symptoms of the disease caused by the Newcastle disease virus strain are different， and the clinical symptoms shown by the same species are also different， often accompanied by immune failure. Sometimes， routine immunization cannot avoid the infection of the virulent strain[9]. In this study， the effect of Se-enriched lactobacillus on NDV antibodies in broilers was investigated， aiming to provide a scientific basis for clarifying the immunity-improving effect of Se-enriched probiotics.

Materials and Methods

Materials

1 000 one-day-old AA broilers， 1 healthy adult chicken， corn-soybean meal diet and NDV attenuated vaccine were all provided by Dacheng Wanda Tianjin Co.， Ltd. The number of viable Se-enriched lactobacillus was not less than 5×1011 cfu/L， and the Se content was 10.00 mg/L; and standard Se[GBW（E）080215]was provided by the National Standard Material Research Center. The NDV antigen was provided by the College of Animal Science and Veterinary Medicine， Tianjin Agricultural University. Chicken 0.5% red blood cells were prepared according to the method provided in the literature[10].

Methods

Animal feeding and serum collection

1 000 one-day-old chicks were randomly divided into two experimental groups， the first of which was the basal diet group， and the second group was the Se-enriched lactobacillus group. Each experimental group was randomly divided into 5 repeated control groups. Corn soybean meal type diet was selected for the basal diet group as feed， and the Se-enriched lactobacillus group feed was supplemented with 2% Se-enriched lactobacillus in the corn soybean meal feed. The chicks were immunized with NDV attenuated vaccine intranasally at the age of 7 d， and the total test days were 42 d. On the 14th， 28th， and 42nd d， 2 chicks were randomly selected from each replicate control group for slaughtering， and the serum was collected and stored in a refrigerator for use.

Hemagglutination test （HA） and hemagglutination inhibition test （HI）

NDV antigen titer detection used the conventional HA method， and the antibody titer detection used the HI method， referring to literatures[11-12].

HA was carried out on a 96-well micro-reaction plate. Then， 50 μl of normal saline was added to each well from left to right. Next， 50 μl of NDV virus solution was added to the first well on the left， followed by mixing well， and 50 μl was pipetted from the first well to the second well. And dilution was performed sequentially to the 11th well， from which 50 μl was discarded， as the 12th hole was the red blood cell control. Next， 50 μl of 0.5% chicken red blood cell suspension was added to each hole from right to left， followed by shaking on a shaker. Finally， the results were observed after standing at room temperature.

HI was carried out on a 96-well micro-reaction plate. According to the fixed virus-diluted serum method， 50 μl of normal saline was added from well 1 to well 11. Then， 50 μl of the tested chicken serum was added to the first hole， followed by mixing evenly through suction and blowing. Next， 50 μl was pipetted to the second hole， and dilution was performed to well 10， from which 50 μl was discarded. The dilutions were 1∶2， 1∶4， 1∶8…. To well 12， 50 μl of Newcastle disease positive serum was added as a serum control. Next， 50 μl of 4 hemagglutination units of Newcastle disease virus solution was added to wells 1 to 12 each， of which well 11 was the control of 4 units of Newcastle disease virus solution， and after mixing by shaking， the solutions were stood at room temperature for 10 min. Next， 50 μl of 0.5% chicken red blood cell suspension was added to wells 1 to 12 each. After mixing well by shaking， the results were observed after standing at room temperature.

Statistical analysis

The HI titers of the ordinary diet group and the Se-enriched lactobacillus group were analyzed by the t test in Excel.

Results and Analysis

Determination of NDV antigen titer

After 3 repeated tests， the HA titer of NDV antigen detected by the hemagglutination test was 29， as shown in Fig. 1. According to the measured NDV antigen titer， the antigen should be diluted 27 times for the preparation of 4 units of the virus.

Effect of Se-enriched lactobacillus on NDV antibody in broilers

After HI testing， the NDV antibody titer in broilers fed with the basal diet was between 21 and 22 on day 14， between 21 and 26 on day 28， and between 23 and 27 on day 35. The test results are shown in Table 1. The NDV antibody titer in broilers fed with Se-enriched lactobacillus was between 21 and 24 on day 14， between 23 and 27 on day 28， and between 24 and 28 on day 35. The test results are shown in Table 2.

The comparative data showed that the antibody titer range of the broiler chicken group supplemented with Se-enriched lactobacillus was higher than that of the basal diet group， and was more stable. From Fig. 2， it was concluded that the NDV antibody titer of the Se-enriched lactobacillus group was higher than that of the basal diet group， and the antibody titers of the two groups both showed an upward trend. Through the determination of antibody level and statistical analysis， the NDV antibody of the Se-enriched lactobacillus group was significantly higher than that of the ordinary diet group （P<0.01）.

Discussion

In recent years， Se-enriched probiotics have gradually attracted people's attention in food and feed. Se is an essential trace element for humans and animals， which has many functions such as improving immunity， resisting free radicals， delaying aging， resisting cancer， and antagonizing toxic elements[2-3]. In many countries， dietary Se supplementation has been a routine measure adopted by the aquaculture industry. The commonly used method of Se supplementation is to add sodium selenite to the diet， but in view of its low binding rate， high toxicity， and potential pollution to the environment， it has been used in limited quantities. Probiotics are a kind of beneficial bacteria that promote the health and growth of animals by improving the balance of the animal's gastrointestinal microflora. Se-enriched probiotics are organic Se preparations which are prepared by converting inorganic Se into organic Se according to the principle of biological transformation using probiotics， which can exert the dual effects of organic Se and probiotics[4-5]. Qin et al.[6] studied the effects of adding Se-enriched lactobacillus in the diet on broiler performance， and the activity of serum total superoxide dismutase （T-SOD）， malondialdehyde （MDA） and glutathione peroxidase （GSH-Px）. The results showed that feeding Se-enriched lactobacillus could significantly increase the daily weight gain of broilers， significantly reduce the feed-to-meat ratio， and significantly increase the serum GSH-Px and T-SOD activity of broilers， and the MDA level was significantly reduced. Se-enriched lactobacillus can also increase the activity of mouse NK cells and reduce the adhesion and colonization of pathogenic E. coli in the intestine[13-14]， but there is no research on the effect of Se-enriched probiotics on virus immunity. In this study， it was found from the investigation on the effect of Se-enriched lactobacillus on the antibody of NDV virus in broilers that from the beginning to the end of the experiment， the antibody of NDV in the ordinary diet group and the Se-enriched lactobacillus group were on an upward trend， and the Se-enriched lactobacillus group showed a wide range and a higher value of the NDV antibody titer， and more stable immunity. There were values slightly lower or higher in the test， which might be related to factors such as autoimmunity， immune procedures and water intake. On the 42nd day of the experiment， the lowest NDV antibody titer detected in the basal diet group was 23， which was inferred to belong to individuals with poor immune levels. This study found that the addition of Se-enriched lactobacillus in the diet could significantly increase the level of NDV antibody in broilers， which is the first time that Se-enriched probiotics affect virus immunity. It is speculated that the possible reason was that Se and lactobacillus played a role together， which is consistent with the results of Chuang et al.[15]， confirming that these inactivated lactobacillus could stimulate cell proliferation and the production of interleukin IL-10， IL-12 and IFN-γ and increase Th cell secretion. At present， the control of Newcastle disease is the primary issue of the poultry industry， and immunization failures occur from time to time. The results of this study showed that the addition of Se-enriched lactobacillus to the feed diet could increase the NDV immune antibody level by 1.0-1.3 log2 titer， and significantly assist the vaccine to produce immune protection， thus playing an important role in resisting the disease.

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