Print ISSN: 2476-535X, Online ISSN: 2717-2910

Document Type : Original research paper


1 Department of Livestock Bacterial Diseases Research, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran.

2 Department of Quality Control, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran


In this study, the effect of probiotic supplementation of Pediococcus Lolii bacterial strain NGRI 0510Q (T) on the immune response to Newcastle disease virus and avian influenza virus vaccines was investigated. Sixty-four chicks from specific pathogen-free (SPF) eggs were divided into four groups. Indeed, groups 1, 2, 3, and 4 were treated with normal ration without probiotic; normal ration with probiotic as 1 g/L of water; normal ration without probiotic with 0.2 mL dual oil vaccine during the breeding period, and normal ration with probiotic with 0.2 mL dual oil vaccine during the breeding period, respectively. Blood sampling were performed eight times weekly (3rd to 10th weeks) and obtained serum samples were evaluated by HI test to determine the antibody levels. Seventh post-vaccination was significant at p <0.05, whereas this effect was not significant for the H9N2 strain. In the present study, the effect of Lactobacilli probiotic consumption on the humoral immune system of laying hens was investigated. Results showed positive effects of this probiotic on its immunogenicity along with vaccination with injectable oil vaccines against Newcastle disease strain V4 and influenza strain H9N2.


Main Subjects


The use of probiotic products has spread rapidly over recent years, improving the performance of meat chicks, laying eggs, and producing free products from any remains. Recent evidence suggests that the use of microbial probiotics can play an important role in the future of the poultry industry. Indeed, probiotics are organisms that provide benefits to host animals by improving the microbial balance of digestion (Fuller, 1992). Previous studies have shown that eating with probiotics may improve growth performance (Apata, 2008), improve nutrient uptake (Apata, 2008), improve immune response and increase mucus function (Mountzouris et al., 2009), increase the immune response against antigens (Patterson and Burkholder, 2003), production of digestive enzymes (Saarela et al., 2000); improvement of non - digestible nutrients, lactic acid production and volatile fatty acids and competition with other enzymes to bind bacteria to the digestive system. The beneficial effects of probiotics in improper health conditions and during the occurrence of stress are sensible.

For the evaluation of the poultry safety system, several criteria are used, the foremost of which can be called immunoglobulin and the weight of the lymph nodes (Yang et al., 2012). Therefore, it can be concluded that the addition of probiotics affects the safety of the chicks to the effect that these effects are observable in increasing the antibodies of the observable antibodies. The measurement of the antibody levels against the antigen SRBC and the Newcastle vaccine is the evaluation criteria of the safety system. The headline shown in feed - fed chicks is higher than in the control group (Khaksefidi and Ghoorchi, 2006).

The positive effects of prebiotics and probiotics on the poultry safety system have already been reported by different researchers (Ricke, 2018, Yang et al., 2012, Shehata et al., 2022). Recently, Yang et al. (2012) reported that in fed chicks with probiotic Clostridium botulinum, the concentrations of IgA and IgM were higher compared to the control group. Some bacteria in the probiotics, especially Lactobacilli, can improve the functioning of the immune system and macrophage activity (Yang et al., 2009). Alavi et al. (2012) studied the effects of prebiotics and probiotics seen on meat chicks, that the use of these additives caused significant safety improvement. Roller et al. (2004) reported an improved immune system performance by employing inulin and lactobacilli. In the present study, the effect of probiotic supplementation of Pediococcus Lolii bacterial strain NGRI 0510Q (T) on the immune response to both Newcastle and avian influenza vaccines was investigated.


Materials and Methods

This research was carried out at the research and development unit of Razi Vaccine and Serum Research Institute, Marand, which is located in the northwest Iran, in collaboration with the quality control unit. In this study, lactic acid bacteria Pediococcus lolii (strain NGRI 0510Q (T)) was used as a probiotic. The main objectives of this study are:

  1. Investigating the effect of probiotics (Paducococcus Lolii) on the response of the humoral immune system created against the Newcastle disease V4 vaccine.
  2. Evaluation of the effect of probiotics (Pediococcus Lolii) on the humoral immune response induced against the H9N2 influenza vaccine

Molecular identification of the bacteria

Isolation and purification of bacteria were performed in the Biotechnology Laboratory of Tabriz Agricultural Research Training Center. For DNA extraction, 20 mL of tissue buffer was poured in epindroph, and some bacterial colony was then centrifuged for 10 min at 95 ° C, then added up to 180 µL water and maintained in the fridge till next use.

Experimental design for the chickens breeding period

This study was done on 64 SPF-laying chickens with respective steps of preparation of chickens breeding hall, hatching, vaccination, etc., in standard conditions. Chickens were randomly categorized into four groups (n=16).  Table 1 shows rations for each group prepared according to company recommendations.

Hemagglutination inhibition (HI) test

Test tubes containing blood samples were placed on an inclined plane for 2 hours to separate their serum.  Antibody titers against Newcastle and influenza viruses was measured by HA and HI test according to Sun et al. (2005).

Statistical analyses

A one-way ANOVA was used for statistical analysis of the data using SPSS software (version 22, Inc., Chicago, IL, USA) and a P ˂ 0.05 was considered significant.


Results of micro titration of blood samples within 3rd to 10th week after vaccination against influenza and Newcastle diseases by HT test are presented as mean ± SD and showed in Table 2 and Table 3, and Figures 1 and 2 (supplementary data), respectively.

Increased antibody titers against the Newcastle disease virus in the third to fifth weeks after injection into vaccinated probiotic chickens have shown positive effects on the activity of the humoral immune system of the chickens being tested, as well as high levels of serum immunoglobulins after weeks. The next step is to stabilize the immune system in these chicks if the unvaccinated probiotic birds are vaccinated after the fifth week onwards, with a decrease in serum antibody titers.

Regarding the antibody titer against the H9N2 strain of influenza virus, the results showed that the probiotic has not been able to have a significant effect on the antibody titer against the virus, but in the third to fifth weeks, it has a slightly higher ability to stimulate the immune system than other groups.



The overall high grade of the antibody can be attributed to the high age of laying hens. At the herd level, healthier animals show a more limited response, making the results appear less pronounced at the herd level and seem ineffective. Therefore, it is difficult to determine the usefulness of using probiotics in poultry farms that produce life under normal conditions. This has certainly affected the acceptance of probiotics in animal nutrition in the past. The results of working with probiotics are sometimes very varied due to the fact that the methods and conditions under which probiotics are processed are not the same, and sometimes even probiotic microorganisms may be inactive. It should also be noted that probiotics are not a single substance or a single entity, but they contain different microorganisms that may act and behave differently, even different strains of the same species may have different metabolic activities. For this reason, when these strains are used as probiotics, they will affect the results. The negative results reported so far can also be attributed to the poor viability of probiotic microorganisms. Other factors that contribute to the diversity of the results include the period (phase) of animal growth, the type of probiotic used, and the health conditions of the animal farm. Given the variations in the results of experiments with probiotics, it would not be surprising that the use of probiotics does not always produce the desired results. However, it should be noted that significant results from probiotic experiments have shown that probiotics can be used as an effective oral supplement if used correctly with probiotics in combination with management conditions and proper feeding methods.

The positive effects of probiotics on the performance of meat - fed chickens with food containing lower levels of nutrient intake are reported by Angel et al. (2005). The researchers found that reducing the amount of nutrients (protein, lysine, calcium, calcium, non - phytate) would have a negative effect on performance, but adding by maintaining and improving the efficiency of using nutrients helps the meat chicks to overcome the lack of nutrients. The reported use of probiotics is associated with the creation of some changes in the body, the immune system, food consumption, probiotic nutrient uptake, morphology, and reduction of pathogenic factors (Sun et al., 2005, Chichlowski et al., 2007). The beneficial effects of probiotics may be related to changes caused in intestinal microorganisms and body metabolism (Angel et al., 2005). Previous research has shown that the use of probiotics on meat chicks will affect the function, the ability to digest the nutrients, and the composition of the blind bowel.

As mentioned before, the use of probiotics has been associated with different results in feeding meat chicks. The instability observed in the results of the research can depend on different reasons, and many factors can affect the response of the chicks to these additives. The environment, the way of managing, nutrition, type and amount of additive, and bird characteristics (age, species, production stage and use), as an example, through water or food, can affect the response of meat chicks to additives (Yang et al., 2009).

The typical processes in raising new chicks, like the sudden change of rations, rations, transportation, operations carried out at the incubation site, and the high fertility of the bird at the time of breeding cause stress (Spreeuwenberg et al., 2001, St-Pierre et al., 2003, Humphrey, 2006). This can undermine the safety of the immune system, thus providing the ground for pathogenic bacteria that can be propagated within the digestive tract and harm host health as well as food products (Gaggia et al., 2010, Vilà et al., 2010).

Zulkifli et al. (2000) reported that adding probiotics to the diet of broilers under heat stress significantly increased the antibody titer. The findings of Huang et al. (2005) showed that feeding broilers with fructooligosaccharides could improve the integrity of the intestinal mucosal layer and thus be effective in improving the immune response. Kabir et al. (2004), in another experiment, by adding probiotics containing several strains of bacteria to the drinking water of chickens showed that antibody production against SRBC increased significantly compared to the control group, as well as the weight of stock and spleen increased significantly. Koenen et al. (2004) reported that feeding broiler chickens with the probiotic Lactobacillus casei increased the alienation activity of intestinal cells. Lactobacillus-based probiotics have been reported to improve the function of the immune system against certain river pathogens by activating and increasing cell-mediated immunity (Dalloul et al., 2003). The use of probiotics increases macrophage activity and enhances immune function (Panda et al., 2005). For example, in a recent study, Kim et al. (2011) showed that adding prebiotics to the diet of broilers did not affect plasma immunoglobulin, immunoglobulin A, and G concentrations. The findings of Houshmand et al. (2011) also showed that the addition of prebiotics did not have a significant effect on immunity.

The exact mechanisms of action of probiotics to increase the function of the immune system remain largely unknown. Experimental results have shown that the effect of probiotics on improving poultry immunity has not been the same (Yang et al., 2009). Probiotics are associated with the activation of innate immunity by acting on foreign cells (Higgins et al., 2007). Nutrition with probiotics has been reported to increase the proliferative and functional activity of antibody-producing B cells (Panda et al., 2008). Special regulatory effects of probiotics on the immune system to strains or bacterial species depend on the type of the probiotics (Hooge, 2004). In addition, Zulkifli et al. (2000) showed that the effect of probiotics on antibody titers could be affected by the age and direction of broilers. Murry et al. (2006) reported that the digestive tract of birds receiving lactobacilli-containing probiotic supplements had higher lactobacilli than the control group. Mountzouris et al. (2010) reported that the use of probiotics had a significant effect on the natural flora of the appendix, increasing the population of lactobacilli and bifidobacteria.

Ng et al. (2009) showed in their study that probiotics might stimulate the natural flora of the intestine in various ways, including mucosal cellular immune responses, facilitate antibody production, improve the integrity of epithelial cell barriers, and reduce epithelial cell mortality. Zhang et al. (2011)  reported that adding probiotics containing Clostridium butyrium to the diet of chickens increased the population of lactic acid bacteria in the appendix. The researchers said that a diet containing probiotics increased the production of acetic acid, butyric acid, valeric acid, and all short-chain fatty acids in the chicks' intestines. This condition reduces the acidity of the contents of the appendix, and inhibits the growth of pathogenic bacteria and stimulates the growth of beneficial bacteria.


The results of this study suggest that since the development of Newcastle disease can affect the respiratory, gastrointestinal, and nervous systems of poultry, this is necessary for the occurrence of secondary poultry diseases, including microbial and viral diseases. Hence, the use of probiotic compounds such as Pediococcus Lulii bacteria studied in this study aims to increase cellular and humoral levels of the poultry immune system, it can prevent many economic losses, along with other preventive factors, including vaccination.



The authors want to acknowledge Razi Vaccine and Serum Research Institute for its financial support for doing this study.

Ethical approval

The used data is extracted from the approved project of the Razi Vaccine and Serum Research Institute with research code 3-35-1851-114-961101.

Conflict of interest statement

The authors have no conflicts of interest to declare.




Alavi S., Zakeri A., Kamrani B. & Pourakbari Y. Effect of prebiotics, probiotics, acidfire, growth promoter antibiotics and synbiotic on humural immunity of broiler chickens. Global Veterinaria, 2012, 8612-7.
Angel R., Dalloul R.A. & Doerr J. Performance of broiler chickens fed diets supplemented with a direct-fed microbial. Poultry Science, 2005, 84(8), 1222-31.
Apata D.F. Growth performance, nutrient digestibility and immune response of broiler chicks fed diets supplemented with a culture of Lactobacillus bulgaricus. Journal of the Science of Food and Agriculture, 2008, 88(7), 1253-8.
Chichlowski M., Croom W.J., Edens F.W., McBride B.W., Qiu R., Chiang C.C., Daniel L.R., Havenstein G.B. & Koci M.D. Microarchitecture and spatial relationship between bacteria and ileal, cecal, and colonic epithelium in chicks fed a direct-fed microbial, PrimaLac, and salinomycin. Poultry Science, 2007, 86(6), 1121-32.
Dalloul R.A., Lillehoj H.S., Shellem T.A. & Doerr J.A. Enhanced mucosal immunity against Eimeria acervulina in broilers fed a Lactobacillus-based probiotic. Poultry Science, 2003, 82(1), 62-6.
Fuller R., Wallace R.J. & Newbold C.J. Probiotics for ruminants. Probiotics: the Scientific Basis, 1992,317-53.
Gaggia F., Mattarelli P. & Biavati B. Probiotics and prebiotics in animal feeding for safe food production. International Journal of Food Microbiology, 2010, 141 Suppl 1S15-S28.
Higgins S.E., Erf G.F., Higgins J.P., Henderson S.N., Wolfenden A.D., Gaona-Ramirez G. & Hargis B.M. Effect of probiotic treatment in broiler chicks on intestinal macrophage numbers and phagocytosis of Salmonella enteritidis by abdominal exudate cells. Poultry Science, 2007, 86(11), 2315-21.
Hooge D. Meta-analysis of Broiler Chicken Pen Trials Evaluating Dietary Mannan Oligosaccharide, 1993-2003. International Journal of Poultry Science, 2004, 3163-74.
Houshmand M., Azhar K., Idrus Z., Bejo M.H. & Kamyab A. Effects of nonantibiotic feed additives on performance, nutrient retention, gut pH, and intestinal morphology of broilers fed different levels of energy. The Journal of Applied Poultry Research, 2011, 20121-8.
Huang R., Yin Y., Wu G., Zhang Y., Li T., Li L., Li M., Tang Z., Zhang J. & Wang B. Effect of dietary oligochitosan supplementation on ileal digestibility of nutrients and performance in broilers. Poultry Science, 2005, 84(9), 1383-8.
Humphrey T. Are happy chickens safer chickens? Poultry welfare and disease susceptibility. British Poultry Science, 2006, 47(4), 379-91.
Kabir S.L., Rahman M., Rahman M., Rahman M. & Ahmed S. The dynamics of probiotics on growth performance and immune response in broilers. International Journal of Poultry Science, 2004, 3(5), 361-4.
Khaksefidi A. & Ghoorchi T. Effect of Probiotic on Performance and Immunocompetence in Broiler Chicks. The Journal of Poultry Science, 2006, 43(3), 296-300.
Kim G.-B., Seo Y., Kim C. & Paik I. Effect of dietary prebiotic supplementation on the performance, intestinal microflora, and immune response of broilers. Poultry Science, 2011, 90(1), 75-82.
Koenen M., Kramer J., Van Der Hulst R., Heres L., Jeurissen S. & Boersma W. Immunomodulation by probiotic lactobacilli in layer-and meat-type chickens. British Poultry Science, 2004, 45(3), 355-66.
Mountzouris K., Tsitrsikos P., Palamidi I., Arvaniti A., Mohnl M., Schatzmayr G. & Fegeros K. Effects of probiotic inclusion levels in broiler nutrition on growth performance, nutrient digestibility, plasma immunoglobulins, and cecal microflora composition. Poultry Science, 2010, 89(1), 58-67.
Mountzouris K.C., Balaskas C., Xanthakos I., Tzivinikou A. & Fegeros K. Effects of a multi-species probiotic on biomarkers of competitive exclusion efficacy in broilers challenged with Salmonella enteritidis. British Poultry Science, 2009, 50(4), 467-78.
Murry A.C., Hinton A. & Buhr R.J. Effect of Botanical Probiotic Containing Lactobacilli on Growth Performance and Populations of Bacteria in the Ceca, Cloaca, and Carcass Rinse of Broiler Chickens. International Journal of Poultry Science, 2006, 5344-50.
Ng S., Hart A., Kamm M., Stagg A. & Knight S.C. Mechanisms of action of probiotics: recent advances. Inflammatory Bowel Diseases, 2009, 15(2), 300-10.
Panda A.K., Raju M.V.L.N., Rao S.V.R. & Sharma S.R. The influence of supplementation of lactobacillus sporogenes on the performance of broilers. Indian Journal of Animal Nutrition, 2005, 2237-40.
Panda A.K., Rama Rao S.S., Raju M.V.L.N. & Sharma S.S. Effect of probiotic (Lactobacillus sporogenes) feeding on egg production and quality, yolk cholesterol and humoral immune response of White Leghorn layer breeders. Journal of the Science of Food and Agriculture, 2008, 88(1), 43-7.
Patterson J.A. & Burkholder K.M. Application of prebiotics and probiotics in poultry production. Poultry science, 2003, 82(4), 627-31.
Ricke S. Impact of Prebiotics on Poultry Production and Food Safety. The Yale Journal of Biology and Medicine, 2018, 91151-9.
Roller M., Rechkemmer G. & Watzl B. Prebiotic inulin enriched with oligofructose in combination with the probiotics Lactobacillus rhamnosus and Bifidobacterium lactis modulates intestinal immune functions in rats. The Journal of Nutrition, 2004, 134(1), 153-6.
Saarela M., Mogensen G., Fonden R., Matto J. & Mattila-Sandholm T. Probiotic bacteria: safety, functional and technological properties. Journal of Biotechnology, 2000, 84(3), 197-215.
Shehata A.A., Yalçın S., Latorre J.D., Basiouni S., Attia Y.A., Abd El-Wahab A., Visscher C., El-Seedi H.R., Huber C., Hafez H.M., Eisenreich W. & Tellez-Isaias G. Probiotics, Prebiotics, and Phytogenic Substances for Optimizing Gut Health in Poultry. Microorganisms, 2022, 10(2).
Spreeuwenberg M.A., Verdonk J.M., Gaskins H.R. & Verstegen M.W. Small intestine epithelial barrier function is compromised in pigs with low feed intake at weaning. Journal of Nutrition, 2001, 131(5), 1520-7.
St-Pierre N.R., Cobanov B. & Schnitkey G. Economic Losses from Heat Stress by US Livestock Industries1. Journal of Dairy Science, 2003, 86E52-E77.
Sun X., McElroy A., Webb K.E., Jr., Sefton A.E. & Novak C. Broiler performance and intestinal alterations when fed drug-free diets. Poultry Science, 2005, 84(8), 1294-302.
Vilà B., Esteve-Garcia E. & Brufau J. Probiotic micro-organisms: 100 years of innovation and efficacy; modes of action. World's Poultry Science Journal, 2010, 66(3), 369-80.
Yang C.M., Cao G.T., Ferket P.R., Liu T.T., Zhou L., Zhang L., Xiao Y.P. & Chen A.G. Effects of probiotic, Clostridium butyricum, on growth performance, immune function, and cecal microflora in broiler chickens. Poultry Science, 2012, 91(9), 2121-9.
Yang Y., Iji P.A. & Choct M. Dietary modulation of gut microflora in broiler chickens: a review of the role of six kinds of alternatives to in-feed antibiotics. World's Poultry Science Journal, 2009, 65(1), 97-114.
Zhang B., Yang X., Guo Y. & Long F. Effects of dietary lipids and Clostridium butyricum on the performance and the digestive tract of broiler chickens. Archives of Animal Nutrition, 2011, 65(4), 329-39.
Zulkifli I., Abdulllah N., Azrin N.M. & Ho Y.W. Growth performance and immune response of two commercial broiler strains fed diets containing Lactobacillus cultures and oxytetracycline under heat stress conditions. British Poultry Science, 2000, 41(5), 593-7.