Establishment Of Salmonella Pullorum Model And Its Treatment Based On Syndrome DifferentiationA Thesis Submitted

Salmonella pullorum disease is a deadly infectious disease caused by Salmonella pullorum infection that has wreaked havoc on the poultry industry.Antibiotic use has also led in the emergence of drug-resistant strains of Salmonella pullorum.As an important alternative antibody drug,Chinese herbal medicine is widely used in the treatment of poultry illnesses.This experiment created the Salmonella pullorum infection model in chicken embryos.By the observation of model chicken clinical symptoms,Chinese veterinarians’ syndrome differentiation,the establishment of treatment principles,prescription screening tests,and preliminary exploration of its mechanism,the experimental data and theoretical foundation can be seen for finding new Chinese veterinary drugs which has effective prevention and treatment of Pullorum disease.1.The Salmonella pullorum C79-3 strain was used to infect 11-day-old,14-day-old,and 18-day-old SPF chicken embryos via allantoic cavity exposure,air chamber exposure,and eggshell contact exposure,respectively,to establish the Salmonella pullorum chicken embryo infection model.Three different infection doses（100 CFU,300 CFU,and 900 CFU）were set up at each day of age.The rate of shelling,as well as clinical signs and chick mortality,were studied.After being infected with allantois cavity inoculation,the 11 and 14-day-old chicken embryos were dead,while the 18-day-old chicken embryos were hatched.However,there were no symptoms of pullorum disease in chicks which were hatched.Chicken embryos infected by air chamber and eggshell contact did not die,and the shelling rate was more than 65 percent.Chicken embryos infected by air chamber and shelled chicks showed diarrhea symptoms of continuous discharge of gray and thin feces,as well as some cases of death during the 7-day observation period.Only 14-day-old chicks with eggshell infection experienced recurring diarrhea.More modeling and screening tests will be undertaken based on the preliminary screening results.90 SPF chicken embryos were formed,including one blank control group and eight model groups.The model group’s experimental objects were 11-day-old and 14-dayold chicken embryos infected with 5 matched doses of Salmonella pullorum via air chamber exposure and eggshell contact exposure.According to the findings,there were two infected ways that can cause obvious symptoms of pullorum disease in chicks.One of them was using 9×105 CFU/mL of C79-3 bacterial solution to contact 11-day-old chicken embryo eggshell which can lead to a 100%incidence rate,another was using 3×103 CFU/mL C79-3 bacteria solution to infect 14-day-old chicken embryo air chamber which can lead to an 80%incidence rate.And a positive rate of Salmonella pullorum of 70%and 80%respectively.Finally,these two methods can be used to establish the model of Salmonella pullorum in chicken embryo stably.2.Pullorum disease is classified as cold dampness trapped spleen diarrhea based on its clinical features and the syndrome differentiation standards of traditional Chinese medicine for children’s diarrhea and Chinese veterinary plague.Warming Yang and strengthening the spleen,dispersing moisture and halting dysentery,or regulating qi and strengthening the spleen,drying dampness and preventing dysentery,etc.are the therapy concepts.To investigate the therapeutic impact of chicken pullorum salmonellosis,three traditional Chinese medicine compounds with Supplementing Qi and strengthening the spleen,warming the center and strengthening the spleen,drying moisture and preventing dysentery,or detoxifying and drying dampness are prescribed.Sixty diseased chicks with typical symptoms of 1 pullorum and diarrhea within 24 hours after modeling by air chamber infection of 14-day-old chicks were randomly and evenly divided into four groups.Three of them were the traditional Chinese medicine compound treatment group,one was the positive control group.Fifteen healthy chicks were chosen as the blank group.For 7 days,each chick in the three traditional Chinese medicine compound treatment groups received 1 mL of decoction of traditional Chinese medicine compound Ⅰ,Ⅱ,and Ⅲ（1 g crude medication/mL）.Clinical symptoms,diarrhea rate,and mortality were used to assess the curative impact.The results indicated that the treatment groups’ overall effective rates were 53.3%and 60%,respectively,and their mortality rates were 33.3%and 40%,both of which were considerably lower than the positive control group（P<0.05）.The total effective rate of the second treatment group of traditional Chinese medicine was only 26.7%,with no obvious curative effect when compared to the positive control group’s self-healing rate of 13.3%.The treatment test results showed that the treatment formula based on Supplementing Qi and warming Yang,regulating qi,drying dampness,and stopping dysentery works significantly better than the treatment formula based on clearing heat and drying dampness,indicating that the disease of chicken white dysentery can be distinguished from the disease of cold dampness trapped spleen diarrhea.3.The 14-day-old chicken embryo air chamber infection was used to investigate the mechanism of compound traditional Chinese medicine in the treatment of chicken pullorum.75 chicks with characteristic symptoms of chicken pullorum diarrhea after shelling were randomly and meanly split into three groups（compound traditional Chinese medicine therapy,doxycycline hydrochloride treatment,and positive control）.The chicks in the traditional Chinese medicine group were given solution of the first formula（1mL/chick）24 hours after shelling,and then solution of the third formula（1mL/chick）every 24 hours;the chicks in the Doxycycline Hydrochloride group were given doxycycline hydrochloride（5mg/chick）,once a day for 7 days.Chick liver,cecum,and spleen were taken at 12h,36h,60h,and 108h,respectively.Real-time fluorescence quantitative PCR was used to determine the relative expression of Toll-like receptor（TLR2,TLR4,and TLR15）gene mRNA in the spleen and cecum.Immunohistochemistry was used to assess the expression of CD4+and CD8+T lymphocytes in the spleen.HE staining revealed pathological changes in the liver.Highthroughput sequencing was used to assess the microbial community of cecal contents.The results showed that TLR2,TLR4 and TLR15 expression levels in chick spleen increased continuously from 12 to 108h after treatment with traditional Chinese medicine,and were significantly higher than those in the other three groups at 108h（P<0.01）;TLR2,TLR4 and TLR15 expression levels in cecum were significantly higher than those in the other three groups at 36h（P<0.01）.Overall,Toll-like receptor expression was up-regulated in the spleen and cecum;the ratio of CD4+/CD8+in the spleen was substantially greater（P<0.01）than that in the Doxycycline Hydrochloride group and was greater（P<0.05）and substantially greater（P<0.01）at 36h and 60h than positive control group.It improved the body’s immune function by increasing the ratio of CD4+/CD8+ in the spleen;compared to the positive control group,the abundance of Campylobacter in cecal microorganisms was significantly reduced（abundance value of compound traditional Chinese medicine group:0.0000000,abundance value of positive control group:0.0000917）（P<0.01）,and the pathogenicity in cecal microorganisms was reduced.The mechanism analysis reveals that the mechanism of compound traditional Chinese medicine in the treatment of chicken white dysentery,also known as cold dampness trapped spleen diarrhea syndrome,is related to improving immunity,reducing the number of harmful microorganisms in the cecum,and lowering the pathogenic risk.The above experimental results indicate that inoculating chicken white dysentery in the air chamber of a chicken embryo can establish a disease infection model,and that chicken white dysentery can be dialectically defined as diarrhea of cold and damp imprisoned spleen.The compound traditional Chinese medicine of Supplementing Qi,warming Yang,invigorating spleen,and drying dampness can improve the structure of cecal microbiota,reduce the intestinal pathogenicity of harmful bacteria,and treat chick dysentery by increasing the ratio of CD4+/CD8+and changing the expression of Toll like receptor.