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Abstract In order to study the anti??bacterial infection effect of polysaccharide from Agaricus blazei Murrill on chickens， the experimental groups were orally administrated A. blazei polysaccharide at low dose and high dose， respectively， for 14 d continuously， and then， the chickens in various groups were infected with Escherichia coli or Pasteurella pneumotropica， so as to observe the clinical symptoms of chickens and record the change in body weight. Anatomy was performed 14 d later， and the organ indices were determined， so as to study the anti??bacterial infection effect of A. blazei polysaccharide on chickens. The results showed that after bacterial infection， the high??dose A. blazei polysaccharide group was significantly differed from other groups in changes of body weight and organ indices. It indicates that oral administration of high concentration of A. blazei polysaccharide could promote the development of poultry organs， thereby improving the immunity of organisms.
Key words Polysaccharide from Agaricus blazei Murrill； Escherichia coli； Pasteurella pneumotropica； Chicken； Body weight； Immune organ index
Agaricus blazei Murrill （ABM） is native to Brazil. It tastes delicious， and could enhance the immunoregulation mechanism of organisms. A. blazei has anti??virus and anti??tumor effects and is a kind of fungus used as both food and medicine[1-3]. It was found in researches related to animal husbandry and veterinary medicine that in early growth period of AA broiler chicken， the addition of A. blazei polysaccharide in daily ration could improve the daily gain of chickens and the oxidation resistance in serum， thereby improving the immunocompetence of organisms and reducing diseases[4-5]. Duan et al.[6] found that oral administration of A. blazei polysaccharide could enhance the humoral immune response， improve the protection rate of vaccine and prolong the lasting time of immunity， and meanwhile， in the early period of vaccine immunity， a proper amount of A. blazei polysaccharide could improve RBC??C3b receptor rosette rate， and enhance the immunologic function of red blood cell[6]. A. blazei extract also has antimicrobial effect on bacteria and fungi[7]. To sum up， A. blazei polysaccharide has an important immunomodulatory effect and could help organism to effectively resist the invasion of bacteria and viruses. In this study， Escherichia coli and Pasteurella pneumotropica were used to infect chickens， respectively， and the anti??bacterial infection of A. blazei polysaccharide on chickens was studied， so as to discuss its action mechanism and protecting effect. This study will provide reference for clinical application and deep research. Materials
Experimental animals
Sixty three day??old AA broiler chickens were purchased from Tianjin Jingshengyuan Experimental Animal Sales Co.， Ltd.
Main reagents
A. blazei polysaccharide having a purity of 48.63 was purchased from Shanghai Kangzhou Fungal Polysaccharide Co.， Ltd.
Strains
E. coli and P. pneumotropica were preserved by the Basic Veterinary Medicine Laboratory， Tianjin Agricultural College.
Main instruments
Centrifuge （type LW??250A）， purchased from Shanghai Medical Instruments Factory； aseptic operation table （type SW??CJ??1D）， purchased from Jiangsu Tongjing Purification Equipment Co.， Ltd.； biochemical incubator （type 2500）， purchased from Changzhou Aohua Instruments Co.， Ltd.； medical vertical refrigerator （type DW??40L262）， purchased from Qingdao Haier Co.， Ltd.； ultra??low temperature refrigerator （type 902??ULTS）， purchased from Thermo Fisher Scientific Inc.； electronic analytical balance （type BS124S）， purchased from Sartorius Scientific Instruments （Beijing） Co.， Ltd.； oscillator （type ZW??A）， purchased from Jintan Longsheng Experimental Instruments Factory； pipettor （type 720005）， purchased from Biohit Biotech （Suzhou） Co.， Ltd.； clean bench （type BHC??1300IIA2）， purchased from Suzhou Sujie Purifying Equipment Co.， Ltd.
Methods
Grouping and treatments of experimental animals
One??day??old chickens were fed adaptively for 2 d and randomly divided into seven groups， i.e.， low??concentration polysaccharide E. coli group （group 1）， high??concentration polysaccharide E. coli group （group 2）， E. coli control group （group 3）， blank control group （group 4）， low??concentration polysaccharide P. pneumotropica group （group 5）， high??concentration polysaccharide P. pneumotropica group （group 6） and P. pneumotropica control group （group 7）.
Each group included nine AA broiler chickens. From the first day of the experiment， the chickens in groups 1 and 5 were drenched 50 mg/kg A. blazei polysaccharide solutions according to body weight， chickens in groups 2 and 6 were drenched 200 mg/kg A. blazei polysaccharide solutions according to body weight， and chickens in groups 3， 4 and 7 were drenched equal amount of normal saline， for 14 d continuously. During the period， change of body weight in each group was recorded every day. On the 15th day， chickens in groups 1， 2 and 3 were orally administrated E. coli according to 0.4 ml/chicken， 2??108 cfu/ml； and chickens in groups 5， 6 and 7 were orally administrated P. pneumotropica according to 0.4 ml/chicken， 1??108 cfu/ml； and chickens in group 4 were orally administrated equal amount of normal saline. The resistance and morbidity of chickens in each group after infection were observed， and the change in body weight in each group was recorded every day. On the 28th day， organ diseases of chickens in each group were observed， and organs were weighed， followed by calculation of organ index. Statistical analysis
The experimental data were treated with SPSS 11.5， and plotting was performed with Graphad Prism software.
Results and Analysis
Body weight change
The results are shown in Fig. 1. It could be seen from Fig. 1 that the body weight of chickens in various groups was on the increase in 1-14 d， without significant differences （P>0.05）. On the 17th day of the experiment， i.e.， 2 d after infection， the body weight of chickens in the groups subjected to infection with bacteria decreased remarkably， and was recovered then. Specifically， the groups in which chickens were orally administrated A. blazei polysaccharide showed the body weights recovered to higher levels than the groups in which chickens were not given the polysaccharide， and the groups in which chickens were orally administrated A. blazei polysaccharide at the high dose exhibited the body weights recovered to higher levels than the low??dose groups， indicating that A. blazei polysaccharide could protect organisms to resist the infection with E. coli and P. pneumotropica， and high??dose A. blazei polysaccharide has a better protecting effect than low??dose A. blazei polysaccharide.
Immune organ index
Thymus index The results are shown in Fig. 2. It could be seen from Fig. 2 that there were very significant differences in thymus index between groups 3 and 4 and between groups 1 and 4 （P0.05）， while there was a significant difference in thymus index between groups 2 and 3 （P0.05）， indicating that the high concentration of A. blazei polysaccharide could promote the development of thymus， thereby effectively resisting the infection with P. pneumotropica.
Spleen index The results are shown in Fig. 3. It could be seen from Fig. 3 that there was a significant difference in spleen index between groups 3 and 4 （P0.05）， while there was a very significant difference between groups 2 and 4 （P0.05）， indicating that A. blazei polysaccharide could promote the development of spleen， thereby effectively resisting the infection of P. pneumotropica.
Bursal index The results are shown in Fig. 4. It could be seen from Fig. 4 that there was a very significant differnce in bursal index between groups 3 and 4 （P0.05）， while there was a very significant difference in bursal index between groups 2 and 3 （P0.05）， indicating that the high concentration of A. blazei polysaccharide could more significantly promote the development of bursa of fabricius than the low concentration of A. blazei polysaccharide， thereby effectively resisiting the infection of P. pneumotropica. Chao REN et al. Study on Anti??infection Effect of Polysaccharide from Agaricus blazei Murrill on ChickensDiscussion
The important immune organs of poultry are thymus， spleen and bursa of fabricius. Thymus is the occasion for the differentiation， development and maturation of T cells， and serves as an important central immune organ. Spleen as the largest peripheral immune organ of poultry participates in systemic humoral immunity and cell??mediated immunity. Bursa of fabricius is a central immune organ unique to poultry， which could produce B lymphocytes and participate in humoral immune response. The development and function of immune organs greatly affect the immune level of poultry， and the relative increase in the weight of immune organ indicates the enhancement of the immune function of organisms[8]. In this study， the infection of E. coli and P. pneumotropica affects the weight gain of chickens and the growth and development of thymus， spleen and bursa of fabricius， thereby reducing the immunity of organisms. Orally administrating the high concentration of A. blazei polysaccharide could more significantly promote the growth and development of thymus， spleen and bursa of fabricius than orally administrating the low concentration of A. blazei polysaccharide， thereby effectively resisting the infection of E. coli and P. pneumotropica （Fig. 1??Fig. 4）.
Cui[9] found that the effective components of A. blazei polysaccharide could significantly enhance humoral immunity and cellular immune response， indicating that the effective components of such polysaccharide could stimulate Th1 immunity， and could be developed as a novel immunologic adjuvant. Ni et al.[10] reported that the extract of A. blazei polysaccharide could improve the phagocytic activity of macrophage and the killing ability of KN cells， promote humoral immune response and regulate cellular immune response， thereby regulating innate immune response and adaptive immune response. Sokovi [11] found that the hot water extract of A. blazei could reduce the biofilm??forming ability of P. aeruginosa， lower the production of pyocyanine， and reduce the movement ability of bacteria， thereby significantly reducing the virulence factor of P. aeruginosa. Zhang et al.[12] reported that adding effervescent tablets containing A. blazei polysaccharide in daily drinking water not only could improve the immunity of animals， but also could effectively treat or alleviate the symptoms of E. coli diseases as well as avoiding complication， and meanwhile， the quality of drinking water could be effectively improved. To sum up， A. blazei polysaccharide could serve as a potential immunological adjuvant or immunopotentiator for poultry for improving the immunity of organisms and resisting bacterial infection， used in clinical veterinary medicine and animal production. References
[1] HAKIME??SILVA RA， VELLOSA JC， KHALIL NM， et al. Chemical， enzymatic and cellular antioxidant activity studies of Agaricus blazei Murrill[J]. An Acad Bras Cienc， 2013， 85（3）： 1073-1081.
[2] SHIMIZU T， KAWAI J， OUCHI K， et al. Agarol， an ergosterol derivative from Agaricus blazei， induces caspase??independent apoptosis in human cancer cells[J]. Int J Oncol， 2016， 48（4）： 1670-1678.
[3] LIMA CU， CORDOVA CO， N¨?BREGA ODE T， et al. Does the Agaricus blazei Murill mushroom have properties that affect the immune system An integrative review[J]. J Med Food， 2011， 14（1-2）： 2-8.
[4] GE HX， HAN LM. Effect of Agaricus blazei polysaccharide on the production property of AA broiler chicken[J]. Contemporary Animal Husbandry， 2012， （4）： 35-36.
[5] GE HX， JIA XJ， HAN LM. Effect of Agaricus blazei polysaccharide on anti??oxidation effect of AA broiler chicken[J]. Chinese Journal of Animal Husbandry and Veterinary Medicine， 2012， （6）： 23-24.
[6] DUAN XP， ZHAO SH， MA JF， et al. The Study of ABM polysaccharide by oral administration on immunity of ND vaccine and red cell immune fuction[J]. Animal Husbandry and Feed Science， 2006， 33（6）： 17-19.
[7] GE HX. Advances in Agaricus blazei and its polysaccharide[J]. Animal Husbandry and Feed Science， 2011， 32（11）： 77-78.
[8] LI YJ， ZHAO XJ. Effects of microcapsule of profitable microbe and Astragalus polysaccharide on growth performance， immune organ index and blood biochemical index of chicks[J]. Journal of Anhui Agricultural Sciences， 2007， 35（1）： 100-103.
[9] CUI L， SUN Y， XU H， et al. A polysaccharide isolated from Agaricus blazei Murill （ABP??AW1） as a potential Th1 immunity??stimulating adjuvant[J]. Oncol Lett， 2013， 6（4）： 1039-1044.
[10] NI WY， WU MF， LIAO NC， et al. Extract of medicinal mushroom Agaricus blazei Murill enhances the non??specific and adaptive immune activities in BALB/c mice[J]. In Vivo， 2013， 27（6）： 779-786.
[11] SOKOVI M， IRI A， GLAMO?gLIJA J， et al. Agaricus blazei hot water extract shows anti quorum sensing activity in the nosocomial human pathogen Pseudomonas aeruginosa[J]. Molecules， 2014， 19（4）： 4189-4199.
[12] ZHANG SW， CHEN YJ， LI JG， et al. Experimental study on Agaricus blazei polysaccharide effervescent tablets on colibacillosis of broiler chickens[J]. Today Animal Husbandry and Veterinary Medicine， 2012， （5）： 43-45.
Key words Polysaccharide from Agaricus blazei Murrill； Escherichia coli； Pasteurella pneumotropica； Chicken； Body weight； Immune organ index
Agaricus blazei Murrill （ABM） is native to Brazil. It tastes delicious， and could enhance the immunoregulation mechanism of organisms. A. blazei has anti??virus and anti??tumor effects and is a kind of fungus used as both food and medicine[1-3]. It was found in researches related to animal husbandry and veterinary medicine that in early growth period of AA broiler chicken， the addition of A. blazei polysaccharide in daily ration could improve the daily gain of chickens and the oxidation resistance in serum， thereby improving the immunocompetence of organisms and reducing diseases[4-5]. Duan et al.[6] found that oral administration of A. blazei polysaccharide could enhance the humoral immune response， improve the protection rate of vaccine and prolong the lasting time of immunity， and meanwhile， in the early period of vaccine immunity， a proper amount of A. blazei polysaccharide could improve RBC??C3b receptor rosette rate， and enhance the immunologic function of red blood cell[6]. A. blazei extract also has antimicrobial effect on bacteria and fungi[7]. To sum up， A. blazei polysaccharide has an important immunomodulatory effect and could help organism to effectively resist the invasion of bacteria and viruses. In this study， Escherichia coli and Pasteurella pneumotropica were used to infect chickens， respectively， and the anti??bacterial infection of A. blazei polysaccharide on chickens was studied， so as to discuss its action mechanism and protecting effect. This study will provide reference for clinical application and deep research. Materials
Experimental animals
Sixty three day??old AA broiler chickens were purchased from Tianjin Jingshengyuan Experimental Animal Sales Co.， Ltd.
Main reagents
A. blazei polysaccharide having a purity of 48.63 was purchased from Shanghai Kangzhou Fungal Polysaccharide Co.， Ltd.
Strains
E. coli and P. pneumotropica were preserved by the Basic Veterinary Medicine Laboratory， Tianjin Agricultural College.
Main instruments
Centrifuge （type LW??250A）， purchased from Shanghai Medical Instruments Factory； aseptic operation table （type SW??CJ??1D）， purchased from Jiangsu Tongjing Purification Equipment Co.， Ltd.； biochemical incubator （type 2500）， purchased from Changzhou Aohua Instruments Co.， Ltd.； medical vertical refrigerator （type DW??40L262）， purchased from Qingdao Haier Co.， Ltd.； ultra??low temperature refrigerator （type 902??ULTS）， purchased from Thermo Fisher Scientific Inc.； electronic analytical balance （type BS124S）， purchased from Sartorius Scientific Instruments （Beijing） Co.， Ltd.； oscillator （type ZW??A）， purchased from Jintan Longsheng Experimental Instruments Factory； pipettor （type 720005）， purchased from Biohit Biotech （Suzhou） Co.， Ltd.； clean bench （type BHC??1300IIA2）， purchased from Suzhou Sujie Purifying Equipment Co.， Ltd.
Methods
Grouping and treatments of experimental animals
One??day??old chickens were fed adaptively for 2 d and randomly divided into seven groups， i.e.， low??concentration polysaccharide E. coli group （group 1）， high??concentration polysaccharide E. coli group （group 2）， E. coli control group （group 3）， blank control group （group 4）， low??concentration polysaccharide P. pneumotropica group （group 5）， high??concentration polysaccharide P. pneumotropica group （group 6） and P. pneumotropica control group （group 7）.
Each group included nine AA broiler chickens. From the first day of the experiment， the chickens in groups 1 and 5 were drenched 50 mg/kg A. blazei polysaccharide solutions according to body weight， chickens in groups 2 and 6 were drenched 200 mg/kg A. blazei polysaccharide solutions according to body weight， and chickens in groups 3， 4 and 7 were drenched equal amount of normal saline， for 14 d continuously. During the period， change of body weight in each group was recorded every day. On the 15th day， chickens in groups 1， 2 and 3 were orally administrated E. coli according to 0.4 ml/chicken， 2??108 cfu/ml； and chickens in groups 5， 6 and 7 were orally administrated P. pneumotropica according to 0.4 ml/chicken， 1??108 cfu/ml； and chickens in group 4 were orally administrated equal amount of normal saline. The resistance and morbidity of chickens in each group after infection were observed， and the change in body weight in each group was recorded every day. On the 28th day， organ diseases of chickens in each group were observed， and organs were weighed， followed by calculation of organ index. Statistical analysis
The experimental data were treated with SPSS 11.5， and plotting was performed with Graphad Prism software.
Results and Analysis
Body weight change
The results are shown in Fig. 1. It could be seen from Fig. 1 that the body weight of chickens in various groups was on the increase in 1-14 d， without significant differences （P>0.05）. On the 17th day of the experiment， i.e.， 2 d after infection， the body weight of chickens in the groups subjected to infection with bacteria decreased remarkably， and was recovered then. Specifically， the groups in which chickens were orally administrated A. blazei polysaccharide showed the body weights recovered to higher levels than the groups in which chickens were not given the polysaccharide， and the groups in which chickens were orally administrated A. blazei polysaccharide at the high dose exhibited the body weights recovered to higher levels than the low??dose groups， indicating that A. blazei polysaccharide could protect organisms to resist the infection with E. coli and P. pneumotropica， and high??dose A. blazei polysaccharide has a better protecting effect than low??dose A. blazei polysaccharide.
Immune organ index
Thymus index The results are shown in Fig. 2. It could be seen from Fig. 2 that there were very significant differences in thymus index between groups 3 and 4 and between groups 1 and 4 （P0.05）， while there was a significant difference in thymus index between groups 2 and 3 （P0.05）， indicating that the high concentration of A. blazei polysaccharide could promote the development of thymus， thereby effectively resisting the infection with P. pneumotropica.
Spleen index The results are shown in Fig. 3. It could be seen from Fig. 3 that there was a significant difference in spleen index between groups 3 and 4 （P0.05）， while there was a very significant difference between groups 2 and 4 （P0.05）， indicating that A. blazei polysaccharide could promote the development of spleen， thereby effectively resisting the infection of P. pneumotropica.
Bursal index The results are shown in Fig. 4. It could be seen from Fig. 4 that there was a very significant differnce in bursal index between groups 3 and 4 （P0.05）， while there was a very significant difference in bursal index between groups 2 and 3 （P0.05）， indicating that the high concentration of A. blazei polysaccharide could more significantly promote the development of bursa of fabricius than the low concentration of A. blazei polysaccharide， thereby effectively resisiting the infection of P. pneumotropica. Chao REN et al. Study on Anti??infection Effect of Polysaccharide from Agaricus blazei Murrill on ChickensDiscussion
The important immune organs of poultry are thymus， spleen and bursa of fabricius. Thymus is the occasion for the differentiation， development and maturation of T cells， and serves as an important central immune organ. Spleen as the largest peripheral immune organ of poultry participates in systemic humoral immunity and cell??mediated immunity. Bursa of fabricius is a central immune organ unique to poultry， which could produce B lymphocytes and participate in humoral immune response. The development and function of immune organs greatly affect the immune level of poultry， and the relative increase in the weight of immune organ indicates the enhancement of the immune function of organisms[8]. In this study， the infection of E. coli and P. pneumotropica affects the weight gain of chickens and the growth and development of thymus， spleen and bursa of fabricius， thereby reducing the immunity of organisms. Orally administrating the high concentration of A. blazei polysaccharide could more significantly promote the growth and development of thymus， spleen and bursa of fabricius than orally administrating the low concentration of A. blazei polysaccharide， thereby effectively resisting the infection of E. coli and P. pneumotropica （Fig. 1??Fig. 4）.
Cui[9] found that the effective components of A. blazei polysaccharide could significantly enhance humoral immunity and cellular immune response， indicating that the effective components of such polysaccharide could stimulate Th1 immunity， and could be developed as a novel immunologic adjuvant. Ni et al.[10] reported that the extract of A. blazei polysaccharide could improve the phagocytic activity of macrophage and the killing ability of KN cells， promote humoral immune response and regulate cellular immune response， thereby regulating innate immune response and adaptive immune response. Sokovi [11] found that the hot water extract of A. blazei could reduce the biofilm??forming ability of P. aeruginosa， lower the production of pyocyanine， and reduce the movement ability of bacteria， thereby significantly reducing the virulence factor of P. aeruginosa. Zhang et al.[12] reported that adding effervescent tablets containing A. blazei polysaccharide in daily drinking water not only could improve the immunity of animals， but also could effectively treat or alleviate the symptoms of E. coli diseases as well as avoiding complication， and meanwhile， the quality of drinking water could be effectively improved. To sum up， A. blazei polysaccharide could serve as a potential immunological adjuvant or immunopotentiator for poultry for improving the immunity of organisms and resisting bacterial infection， used in clinical veterinary medicine and animal production. References
[1] HAKIME??SILVA RA， VELLOSA JC， KHALIL NM， et al. Chemical， enzymatic and cellular antioxidant activity studies of Agaricus blazei Murrill[J]. An Acad Bras Cienc， 2013， 85（3）： 1073-1081.
[2] SHIMIZU T， KAWAI J， OUCHI K， et al. Agarol， an ergosterol derivative from Agaricus blazei， induces caspase??independent apoptosis in human cancer cells[J]. Int J Oncol， 2016， 48（4）： 1670-1678.
[3] LIMA CU， CORDOVA CO， N¨?BREGA ODE T， et al. Does the Agaricus blazei Murill mushroom have properties that affect the immune system An integrative review[J]. J Med Food， 2011， 14（1-2）： 2-8.
[4] GE HX， HAN LM. Effect of Agaricus blazei polysaccharide on the production property of AA broiler chicken[J]. Contemporary Animal Husbandry， 2012， （4）： 35-36.
[5] GE HX， JIA XJ， HAN LM. Effect of Agaricus blazei polysaccharide on anti??oxidation effect of AA broiler chicken[J]. Chinese Journal of Animal Husbandry and Veterinary Medicine， 2012， （6）： 23-24.
[6] DUAN XP， ZHAO SH， MA JF， et al. The Study of ABM polysaccharide by oral administration on immunity of ND vaccine and red cell immune fuction[J]. Animal Husbandry and Feed Science， 2006， 33（6）： 17-19.
[7] GE HX. Advances in Agaricus blazei and its polysaccharide[J]. Animal Husbandry and Feed Science， 2011， 32（11）： 77-78.
[8] LI YJ， ZHAO XJ. Effects of microcapsule of profitable microbe and Astragalus polysaccharide on growth performance， immune organ index and blood biochemical index of chicks[J]. Journal of Anhui Agricultural Sciences， 2007， 35（1）： 100-103.
[9] CUI L， SUN Y， XU H， et al. A polysaccharide isolated from Agaricus blazei Murill （ABP??AW1） as a potential Th1 immunity??stimulating adjuvant[J]. Oncol Lett， 2013， 6（4）： 1039-1044.
[10] NI WY， WU MF， LIAO NC， et al. Extract of medicinal mushroom Agaricus blazei Murill enhances the non??specific and adaptive immune activities in BALB/c mice[J]. In Vivo， 2013， 27（6）： 779-786.
[11] SOKOVI M， IRI A， GLAMO?gLIJA J， et al. Agaricus blazei hot water extract shows anti quorum sensing activity in the nosocomial human pathogen Pseudomonas aeruginosa[J]. Molecules， 2014， 19（4）： 4189-4199.
[12] ZHANG SW， CHEN YJ， LI JG， et al. Experimental study on Agaricus blazei polysaccharide effervescent tablets on colibacillosis of broiler chickens[J]. Today Animal Husbandry and Veterinary Medicine， 2012， （5）： 43-45.