| Campylobacter jejuni(C.jejuni)is a highly infectious zoonotic pathogen,which has been listed by the World Health Organization as one of the most common sources of foodborne infections.Chickens are the natural hosts and important sources of infection for C.jejuni,and poor farming practices can lead to the rapid multiplication of this bacterium.Therefore,the prevention and control of this pathogen has become a research hotspot in poultry breeding industry.Studies have shown that circadian rhythms play a key role in the host’s response to colonization by C.jejuni,and genes such as CLOCK and SOCS3 related to immunity that are differentially expressed after C.jejuni infection are regulated by circadian rhythms.However,it is unclear whether changes in circadian rhythms occur in intestinal microbiota due to host infection with C.jejuni.This paper discusses this issue using SPF chicks as the experimental animals.In this experiment,144 3-day-old specific pathogen-free(SPF)White Leghorn chickens were randomly divided into three groups with 48 chickens in each group.The three groups of chickens were inoculated at 10:00 am(treatment A),14:00 pm(treatment B)and 18:00 pm(treatment C)respectively.In each group,24 chickens were randomly selected as the infected group and orally administered 0.5m L of 1.65×10~8cfu/m L C.jejuni bacterial solution,while the other 24 chickens were designated as the control group and orally administered 0.5m L of sterile PBS buffer.At 4,8,12,16,and 24 hours after inoculation,four chickens were selected from the control group and the infection group,respectively,and euthanized by cervical dislocation.The weight of each chicken and the weight of immune organs were measured,and the cecal contents,liver,cecum,and other tissues were collected.The cecal contents were used for 16S r RNA microbial diversity sequencing and metagenomic sequencing to analyze changes in gut microbiota.The liver and cecum tissues were analyzed for pathological changes using HE staining,and the sequencing results were validated by q PCR.The main conclusions are as follows:(1)The body weight and immune organ index of SPF chicks infected with C.jejuni at different times showed rhythmic changes from 4 to 24 hours,and there were significant differences in the resistance of SPF chicks at different times.The results of weight changes showed that the chickens treated with A showed a significant increase in weight and had the strongest immunity,while the chickens treated with C showed a significant trend of weight loss during the experiment and had the weakest immunity.The immune organ index results showed that chicks exhibited strong immune responses during the nighttime period of 22:00to 02:00.(2)After infection with C.jejuni at three different times,chicks were tested for the excretion of C.jejuni by stool coating plate.The results showed that the detection rate of C.jejuni in stool was the highest at 4 hours post-infection(hpi),and the bacterial content decreased with the delay of time.(3)The results of OTU clustering,dilution curve,and rank-abundance curve all showed that the treatment C had the lowest OTU content,and infection with C.jejuni at 18:00significantly reduced the species richness of cecal microbiota.(4)The species richness and diversity results in the Alpha diversity index after infection with Campylobacter jejuni at three different times showed that treatment A had the highest values,followed by treatment B and then C.In the 4-24 hours Alpha diversity results after infection at three different times,there was a significant difference in species richness between treatments A and B at 12hpi compared to the other groups.Furthermore,the infected group in treatment A was significantly higher than the control group,while the results were the opposite for treatments B and C.The PCA results showed differences between the treatments at the three different times after C.jejuni infection.(5)The analysis of the microbial community structure of the cecum after infection with C.jejuni at three different times showed that the dominant phyla were Firmicutes and Proteobacteria,both of which showed a fluctuating trend.Firmicutes had the highest relative abundance at the end of the day(18:00)and decreased relative abundance at night(22:00～02:00).The relative abundance of Proteobacteria in treatment C was higher than that in treatment A and B.At the class level,Clostridia,Gammaproteobacteria,and Bacilli were the dominant classes.There are differences in species composition among the three groups at the genus level.(6)The infection of C.jejuni has an impact on the microbial function of the cecum.Through metagenomic sequencing,the expression of genes related to carbohydrate metabolism,membrane transport and nucleotide metabolism,as well as replication and repair,increased after C.jejuni infection.In contrast,the levels of GH130,GH76,GH111 in the GH family and CBM38 in the CBM family decreased,while the PL1 level increased.In addition,the expression of antibiotic resistance genes such as multidrug,tetracycline,and aminoglycosides increased.(7)The results of fluorescence quantitative PCR validation for the dominant phyla Firmicutes,Bacteroides,and randomly selected gut microbiota Clostridium,Enterococcus,and Klebsiella were consistent with the sequencing results.(8)H&E staining revealed that chickens infected with C.jejuni increased the thickness of the mucosa and submucosa of the cecum wall.This study provides a theoretical basis for grasping the treatment time of C.jejuni by revealing the impact of infection with C.jejuni at different times on host resistance and changes in intestinal microbial rhythm. |
