| Salmonella can cause severe economic loss in poultry deaths,and the poultry products contaminated by salmonella will seriously threaten human health.Previous researches have shown that exogenous added lysozyme can significantly inhibit chicken salmonellosis.To systematically elucidate effects of recombinant human lysozyme(rhLZ)on chicken intestinal mucosal immunity,transgenic chickens expressing rhLZ were introduced.Salmonella enteritidis counts in the cecum and spleen,levels of serum IgG,IgA secretion in the mucosa,cytokines expression in the cecum,changes of chicken body weight,as well as inflammatory damage of the heart,liver,spleen and cecum of rhLZ transgenic chickens after oral infection with Salmonella enteritidis were evaluated compared with nontransgenic chickens in the current study.The findings provide theoretical basis for reduction of the occurrence of salmonellosis in chicken breeding.Firstly,transgenic chickens of rhLZ high expression were reproduced.Detection of exogenous gene was optimized by PCR identification using two special primers,and Southern blot was implemented to further guarantee the insertion of exogenous fragments.Successfully,we generated 194 rhLZ transgenic chickens.Then the average concentration of rhLZ expressed in serum exhibited 29.90±6.50 μg/mL detected by ELISA.Additionally,the antibacterial activity of lysozyme was detected by bacteriostasis and dilution plate amplification method,lysozyme in egg white of transgenic chickens had a significantly higher antibacterial activity against Micrococcus lysodeikticus than those of nontransgenic chickens(P<0.05).Feces of transgenic and nontransgenic chickens were collected and five types of bacteria(Lactobacillus,Salmonella,Bifidobacterium,Staphylococcus aureus and Escherichia coli)were isolated and cultured.Among the five bacteria,the number of Bifidobacterium in those transgenic chickens was significantly higher than that of nontransgenic chickens(P<0.05).Moreover,6-week shank length,6-week weight and 18-week weight of transgenic chickens increased obviously than those of non-transgenic chickens.Subsequently,to verify the inhibitory effect of rhLZ transgenic chickens on salmonellosis,ten transgenic cock and nontransgenic hen of G2 from the same pedigree were mated.As a result,232 newly hatched transgenic chicken offsprings from 300 fertilized eggs were achieved and randomly distributed into two groups:infected and noninfected control.Chicks in the infected group and control group were orally inoculated with 0.2 mL 1x108 CFU/mL Salmonella enteritidis or equivalent sterile PBS,respectively.The chicks were reared in wire cages and allowed free access to water and pathogen-free feed.At 1,3,7 and 14 day postinfection,infected and noninfected chicks were slaughtering sampled to detect the infection situation.Meanwhile,growth performance,histopathological observation and serological index were determined.Results showed that:(1)Regardless of Salmonella enteritidis infection,body weight of transgenic chicks were significantly higher than that of nontransgenic chicks;(2)At 7 dpi and 14 dpi,compared with nontransgenic chickens,transgenic chickens showed slighter tissue lesions;(3)At 7 dpi and 14 dpi,Salmonella enteritidis counts in the cecum of transgenic chicks were significantly lower than those of nontransgenic chicks(P<0.05),while the counts in spleen made no difference(P>0.05);(4)Transgenic chicks infected with Salmonella enteritidis exhibited significantly higher IgG levels than their uninfected countparts at 14 dpi(P<0.05)and their significantly higher IgA contents increased to a maximum at 7 dpi and 14 dpi(P<0.05),as well as a significantly lower IFNγ expression(P<0.05).In conclusion,we indicated that rhLZ may play a certain role in regulating the intestinal flora of chickens,and can promote the growth of intestinal Bifidobacterium and postnatal weight.In addition,rhLZ can improve intestinal inflammatory lesion and induce humoral immunity in advance to enhance the inhibitory effect of Salmonella enteritidis. |
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