Antimicrobial Peptides In Housefly Larvae (Musca Domestica) Affect Intestinal Mucosal Immune In Salmonella Pullorum-infected Chickens

Pullorum disease is one of the most serious intestinal diseases in poultry production.And it is generally treated by adding antibiotics to the feed of infected chickens.Antibiotics are generally quite effective against the disease;however,the long-term use of antibiotics can undermine the positive effects of beneficial bacteria and damage the intestinal mucosal barrier.Antibiotics cause dysbacteriosis and the development of resistance to antibiotics has frequently been observed.Antimicrobial peptides?AMPs?are produced by organisms as a defense against pathogen invasion,especially bacterial invasion.The AMPs are small active peptides which are encoded by genes and synthesized in the ribosome.The bacteriostatic mechanism of AMPs is different from that of antibiotics.It is difficult for bacteria to develop resistance to AMPs.Furthermore,AMPs do not leave drug residues in animals.The objective of this experiment was to learn more about the effects of AMPs produced by maggots on Lactobacillus populations,mucosal epithelial cells numbers,and the mRNA expression levels of TGF-?₄ and TNF-?in the small intestinal mucosa of S.pullorum-infected chickens.The study included AMPs extracted from S.pullorum-infected maggot as well as non-infected ones?referred to as induced AMPs and non-induced AMPs,respectively?.Tests were then conducted to determine the activity of these AMPs against S.pullorum by the standard agar plate method.Chicks were slaughtered after 3,5,and 7 d of treatment and their intestinal tracts were dissected.The effects of the AMPs on intestinal Lactobacillus,small intestine mucosa epithelial cells,and the expression of TGF-?₄ and TNF-?mRNA were determined in S.pullorum-infected chicks.1.The results of BCA standard protein determination shown that the induced AMP had significantly greater bacteriostatic activity than non-induced AMP.There was no significant difference between the E.coli-induced AMP and S.pullorum-induced AMP?P>0.05?.The AMP concentrations in both of those treatments were significantly greater than those in the non-induced AMP treatment?24.17±4.65?g/?L??P<0.05?.The antibacterial activity tests by K-B disk method showed that the inhibition zone diameters were 21.06±0.87 mm in the S.pullorum-induced AMP group,16.41±0.51 mm in the non-induced group?P<0.05?.The MIC in the induced AMP group was 3.0 mg/L by microbroth dilution method.2.Maggot AMPs?3 ml,1 mg AMP/mL?were fed to 14-day-old chicks infected with S.pullorum.For comparison,another group of infected chicks were treated with gentamicin?100?mg/L?.The duodenum,jejunum and ileum was collected from the chicks in the gentamicin group,the maggots,the induced maggot AMP group,the non-induced maggot AMP group,and control group after 3,5,and 7 d of treatment.Measurements were done to determine Lactobacillus populations,the number of intestinal mucosal immune cells?mast cells,lymphocyte cells and goblet cell?,and the expression of TGF-?₄ and TNF-?mRNA in small intestinal mucosa.?1?The effects of the treatments on Lactobacillus populations in the duodenum,jejunum,and ileum of the chicks were determined using MRS medium.Lactobacillus populations in the duodenum were significantly less in the gentamicin treatment group and control group than in the healthy group?P<0.05?.In contrast,there was no significant difference in Lactobacillus population in any part of the small intestine between the induced maggot AMP group and the healthy group?P>0.05?.This showed that the number of Lactobacillus was below normal when the chicken after the infection S.pullorum.AMP treatment S.pullorum-infected chicken could cause Lactobacillus increase.And treatment with gentamicin generally reduced the number of Lactobacillus.?2?The number of small intestinal mucosa mast cells was determined after embedding the sections in paraffin by toluidine blue staining.The number of mast cells in the control group more than the healthy group.The number of the duodenum mucosa mast cells in the gentamicin group was significantly less than the healthy group?P<0.05?.In the ileum,there was significantly greater mast cells in the induced maggot AMP group than in the healthy group?P<0.05?.The number of lymphocyte cells in the control group less than the healthy group.The results of HE staining shown that the number of the lymphocyte cells in the gentamicin group was significantly less than the healthy group on 3 d?P<0.05?.There were no significantly difference in the induced maggot AMP group and the healthy group?P>0.05?.The results of the same method measured shown that the number of goblet cells in the control group more than the healthy group.In the dedunum,there was significantly less goblet cells in the induced maggot AMP group than in the healthy group?P<0.05?.The number of the duodenum and ileum mucosa goblet cells in the gentamicin group was significantly less than the healthy group?P<0.05?.The above results show that the number of mast cells and goblet cells were increased after S.pullorum-infected chickens.Meanwhile,the number of lymphocyte cells was reduced.The number of dedunum mast cells,dedunum and ileum goblet cells were reduced by use antibiotic treatment S.pullorum-infected chickens.The number of the lymphocyte cells was reduced on 3 d.However,the number of ileum mast cells was increased.In dedunum,the number of the goblet cells was reduced by use induced AMP.?3?The expression of two mucosal immune factors(i.e.,TGF-?₄ and TNF-?mRNA)in different sections of the small intestine of the chickens was determined by qRT-PCR after different treatment times.The results showed that the TGF-?₄ mRNA expression of duodenum was higher in this group than other groups;the TGF-?₄ mRNA expression was very low in the ileum.The TGF-?₄ mRNA expression was no significantly greater in the maggot AMP groups?induced AMP and non-induced AMP?than in the gentamicin group?P>0.05?;The TGF-?₄ mRNA expression was significantly greater in the non-induced AMP groups than in the gentamicin group?P<0.01?.The TNF-?mRNA expression of the jejunum was significantly greater in the induced AMP group than in the gentamicin group?P<0.05?.The above results show that the expression of TGF-?₄mRNA was enhanced by use antibiotic treatment S.pullorum-infected chickens in the dedunum.The TGF-?₄ mRNA expression of the ileum was promoted in the induced AMP group.And the expression of TNF-?mRNA of the jejunum was higher in induced AMP group than antibiotic group.The expression of TNF-?mRNA was significantly different in induced AMP on 5 d.In conclusion,the maggot AMPs has an inhibition effect on S.pullorum.And the AMPs from maggot offer potential for effective treatment of S.pullorum-infected chickens instead of the gentamicin application,which to deal with antibiotic abuse and antimicrobial resistance are provide a new direction.Therefore,maggot AMP may be useful for the prevention and treatment of S.pullorum when developing new chicken feed additives.And it also provides some scientific basis and theoretical basis for the treatment of bacterial infection in poultry.