Investigation Of Salmonella Contamination In Guangxi Quality-chicken Breeding Farms And Research Of The Antimicrobials Alternatives

Salmonella,as the common pathogenic bacteria in the chicken industry,can enter the chicken flock through the contamination of the whole producing chain of the breeding farms and resulting in infection,disease,reduced performance,death and contamination of the hatching eggs.The serotype/specie of Salmonella is a key factor determining the susceptibility of infected birds and the severity of disease,and its virulence is closely related to the virulence proteins encoded by the virulence genes.Therefore,it is of great importance to investigate and understand the contamination of Salmonella in the whole production chain of the breeding farms,and to confirm the serotype/specie of the Salmonella contaminated in the process of production,for the purpose of preventing Salmonella contamination and developing effective prevent and control measures.At present,the conventional measure to control salmonellosis has been mainly based on the usage of antibiotics,but with their long-term use and even abuse,the negative issues of drug residue and antimicrobial multi-resistance（AMR）became more and more serious.Therefore,it has been become an urgent issue to find antimicrobial alternatives for the solution.Oregano essential oil,as one of the antimicrobial alternatives,from the natural plants,and composed of the antimicrobial components carvacrol and thymol,has a broad application prospect for its properties of antibacterial,antioxidant,growth promotion and immunity enhancement.Phage is a kind of virus that can specifically infect specific host bacteria.It has been given intensive attention for the characteristics of wide distribution,high specificity,and difficult to induces resistance and may provide new ways for the prevention and treatment of Salmonella infection.In the study,samples collected from the whole producing chain of the large-scale quality-chicken breeding farms in Guangxi were used for the culture,isolation and identification of Salmonella contaminated in the major parts of the whole producing chain and the dominant serotypes of the Salmonella isolates,as well as the characteristics of antibiotic resistance and the carrying of virulence genes were investigated.Through the software simulation,in vitro antimicrobial test and animal challenge-protection experiment,the prevention and treatment effects of oregano essential oil for the Salmonella dominating serotype in Guangxi quality-chickens were performed.The virulent phage SP19,isolated,identified and preserved in the laboratory was used for the challenge-protection experiment against SP,to explore the potential of the phage SP19 in the prevention and treatment of PD in chickens and provide the experimental basis for the development of phage product targeting SP.A total of 1 060 samples collected from the whole producing chain of 6large-scale quality-chicken breeding farms were used for the culture,isolation and identification of Salmonella.The samples were obtained from 8 different major parts/items including the eggshell surface of hatching eggs at different phases（pre-disinfection,post-disinfection and piped）,the environment of the hatchers,the dead embryos at the late hatching period,day-old weak chicks,breeding hens of the pullorum disease-fowl typhoid（PD-FT）antibody positive and negative.A total of 158 samples were Salmonella positive（14.91%,158/1060）.Company DGXX13 had the highest positive rate of 22.64%（60/265）,while company DGGX04 had the lowest（8.15%,11/135）.The PD-FT antibody positive breeding hens had the highest Salmonella detection rate（41.67%,25/60）,followed by the eggshell surface of the piped eggs（22.22%,20/90）,the dead embryos at the late hatching period（17.33%,52/300）,day-old weak chicks（16.67%,50/300）,the environment of the hatchers（6%,6/100）and the eggshell surface before disinfection（5.56%,5/90）.In the 25 Salmonella-positive breeding hens of PD-FT antibody positive,the Salmonella-positive rate of the ovary was the highest（40%,10/25）,followed by the oviduct（32%,8/25）,while the cloacal swab had the lowest（4%,1/25）.SP was found to be the most dominating serotype（80.33%,147/183）by the serotyping of the isolates,followed by S.kentucky（14.75%,27/183）,S.give（3.28%,6/183）and S.derby（1.64%,3/183）.The results of the drug sensitivity tests showed that all the SP isolates have showed resistant of varying degrees against the 17 commonly used antibiotics.The resistances to SXT（97.3%）,SIZ（96.6%）and TMP（88.4%）were the highest,while the isolates were sensitive to CTX,CAZ,CRO,CIP,GEN and NIT.AMR was serious and the proportion of SP isolates with triple and more antimicrobials resistance reached 100%.The positive of carrying the common virulence genes including stn,fim A,vir K,sipA,sse L,mgt C,sii E,sop B and spv C of Salmonella was more than 80%.Auto Dock was used for the protein-ligand simulation docking targeting the most important virulence proteins SipA and Sse L of SP interacted to total 19small molecules of the natural plant essential oils with antimicrobial effect.Among them,the lowest binding affinity with proteins SipA and Sse L were the molecules carvacrol,menthol,zingerone and thymol.The binding affinity of carvacrol to the proteins were-22.5936 k J/mol and-23.012 k J/mol,respectively,and those of thymol were-21.3384 k J/mol and-19.6648 k J/mol,respectively.DS Visualizer was used to analyze the molecular interactions of carvacrol and thymol with SipA and Sse L.It was found that carvacrol and thymol could form hydrogen bonds and hydrophobic forces with multiple amino acid residues of the proteins SipA and Sse L.The minimum inhibitory concentration（MIC）of carvacrol and thymol on SP in vitro was tested,and the growth curves of SP under the action of carvacrol,thymol and oregano essential oil were mapped.Also the effects of carvacrol and thymol on the expression of SP virulence-related genes sipA and sse L were tested by q PCR.The results showed that the MIC of carvacrol and thymol on SP were 512μg/m L and 2 048μg/m L,respectively.Carvacrol,thymol and oregano essential oil all can effectively inhibit the growth of SP,and the logarithmic phase of growth was delayed with the increase of the concentrations of carvacrol,thymol and oregano essential oil.It was also found that carvacrol and thymol could down-regulate the expression level of sipA and sse L genes.60 day-old Three-yellow chicks from the PD-FT eradicated breeder farm were randomly divided into four groups:prevention-challenge,challenge-treatment,challenge control and blank control,with 15 chicks per group.The prevention-challenge group was supplemented with 200μL/L oregano essential oil in the drinking water during the whole experiment and the challenge-treatment group was supplemented with 400μL/L oregano essential oil during 8-12 days of age,while the challenge control group and the blank control group were kept as untreated controls.At 7 days of age,birds of the prevention-challenge,the challenge-treatment and the challenge control groups were orally challenged with 0.5 m L/bird of the suspension containing 1×10⁸CFU/m L of SP after fasting and water deprivation for 6 hours.Serum antibodies against PD-FT were detected by the plate agglutinate test（PAT）.At 35 days of age,all birds were weighed and necropsied,lesions were recorded and the challenged SP were recovered.The results showed that the average body weight（BW）of the challenge control group was significantly lower（P<0.05）than those of the prevention-challenge group and the blank control group,while the prevention-challenge group was significantly higher（P<0.05）than those of the challenge-treatment group and the challenge control group,respectively.The positive rates of PD-FT antibody and the SP recovery in the prevention-challenge group were significantly lower（P<0.05）than those of the challenge control group,but there were no significant differences（P>0.05）between the prevention-challenge group and the challenge-treatment group.The blank control group was all negative at these two parameters.The phage SP19 targeting SP,a strain isolated and identified by the laboratory was used to lyse a total of 147 SP isolates from different breeding farms in Guangxi.The results showed that the total lysis rates on SP isolates was71.43%and the lysis rates from all the breeding farms were 66.67%-77.78%.40day-old Three-yellow chicks from the PD-FT eradicated breeder farm were randomly divided into four groups:the prevention-challenge,the challenge-treatment,the challenge control and the blank control,with 10 chicks per group.The prevention-challenge group was orally supplemented with 0.5m L/bird of the phage SP19 with a titer of 7.2×10¹⁰PFU/m L during the whole experiment and the challenge-treatment group was orally supplemented with 0.5m L/chick of the phage SP19 with a titer of 1.86×10¹¹PFU/m L during the age of8-14 days,while the challenge control and the blank control groups were kept as untreated with phage SP19.At the age of 7 days,birds of the prevention-challenge,the challenge-treatment and the challenge control groups were orally challenged with 0.5 m L/group of SP with a concentration of 1.8×10⁹CFU/m L after fasting and water deprivation for 6 hours.Serum antibodies against PD-FT were detected by PAT.At the age of 35 days,all birds were weighed and necropsied,lesions were recorded and the challenged SP were recovered.The results showed that the average BW of the challenge-treatment group was higher than those of the prevention-challenge and the challenge control groups,and there was a significant difference（P<0.01）between the challenge-treatment and the challenge control groups.Also,the BW of the prevention-challenge group was significantly higher（P<0.05）than that of the challenge control group,but there was no significant difference（P>0.05）with those of other groups.The positive rates of the PD-FT antibody and the SP recovery in the challenge-treatment group were significantly lower（P<0.05）than those of the challenge control group,but there was no significant difference（P>0.05）between the challenge-treatment and the prevention-challenge groups.The blank control group was all negative at these two parameters.The study demonstrated the Salmonella contamination major status in the whole producing chain of the large-scale quality-chicken breeding farms and the dominating serotypes.The AMR of the isolates was serious and the virulence gene-carrying rate was high.Carvacrol,thymol,and oregano essential oil could inhibit the growth of SP.Carvacrol and thymol inhibited the activity of the important virulence proteins SipA and Sse L by forming hydrophobic forces and hydrogen bonds with the multiple amino acid residues of SipA and Sse L,and down-regulating the expression of sipA and sse L genes.The oregano essential oil supplemented in the drinking water could effectively decrease infection of SP and could also significantly reduce the BW decline of birds challenged with SP.The phage SP19 had a high ability to lyse SP isolates and could significantly reduce the SP infection in Three-yellow chickens by the oral administration.