| Pasteurella multocida(Pm)is a kind of gram-negative coccobacillus bacteria.According to the specificity of capsular antigen and somatic form,P.multocida can be divided into A,B,D,E and F serotypes and 1~16 serotypes,respectively.Since 2006,bovine serotype A Pasteurella multocida(bovine PmA)has become a main threat to the China's beef cattle industry.Bovine PmA is a conditional pathogen that mainly causes calf pneumonia and adult bovine respiratory syndrome.However,at present,the prevention and control measures for bovine PmA are still very limited in China.The disease caused by bovine PmA seriously jeopardizes the development of the beef cattle industry and causes huge economic losses every year.Clinically,cattle infected with bovine PmA mainly shows obvious lung lesions,probably because the bacteria colonization density was significantly higher in the lung than other organs.Differentially expressed substances between the infected lung and other infected organs may be one of the causes.Thus,by comparing the composition of the infected lung with other organs,it may be possible to find some substances associated with the disease.In order to find potential substances,this study used a bovine PmA(PmCQ2)to establish a mouse pneumonia model with different challenge ways.Based on the model,this study figured out the bacterial colonization density of each organ,identified the tissue metabolomes of the infected lung and the liver via metabolome sequencing technology,analyzed the different substances between these two tissues,and determined some substances directly affecting PmCQ2 growth in vitro.Finally,this work estimated whether exogenous these substances could influence PmCQ2infection,and then find some potential mechanisms.The results are as follows:1.Establishment of the bovine PmA-induced mouse pneumonia model and determination of bacteria colonization of different organs1.1 The bovine PmA-induced mouse pneumonia model can be successfully established via different challenge waysIn this study,mice were challenged with 10~4 CFU of PmCQ2 via natural infection route (intranasal infection)and non-natural infection route(intramuscular and intraperitoneal infection).The mortality,clinical manifestations,pathological changes of the lungs and HE staining results of the lungs were combined to determine whether the PmA-induced mouse pneumonia model was successfully established.Survival curves showed that PmCQ2 was highly lethal.Additionally,after the infection of PmCQ2,the mice showed obvious respiratory symptoms,and the lung lesions just like the bovine lung lesions.HE staining showed that PmCQ2 infection can induced the lung structural damages and inflammatory lesions under all conditions.These inflammatory lesions were mainly characterized by neutrophil,which was also found in bovine infection.The above results demonstrate that both the natural infection route and the unnatural infection route can successfully establish PmA-induced the mouse pneumonia model.1.2 Compared with other organs,the lung has the highest bacteria colonization density in bovine PmA-infected mouseThis study further determinated the PmCQ2 colonization density of various organs(lung,liver,spleen and kidney)under different challenge ways,and verified PmCQ2 by PCR.The results showed PmCQ2 infection can induce bacteremia.Under natural infection,PmCQ2 can colonize in these four organs,and the colonization density of bacteria in each organ gradually increased with time.The colonization density of lung bacteria is significantly higher than that of other organs.Similarly,under non-natural infection,PmCQ2 can also colonize in these four organs,and the colonization density of bacteria in each organ gradually increased with time.The colonization density of lung bacteria was significantly higher than that of liver and kidney,but there was no significant difference when compared with spleen.However,considering that the blood filtration function of spleen could affect the colonization result,it was still considered that the lung colonization density is higher than other organs.In summary,the lung has the highest bacteria colonization density in bovine PmA-infected mouse.2.Metabolomics sequencing and quality analysis,and screening of differentially expressed substances2.1 Analysis of metabolomicsA total of eight infected mice's lungs and livers were sequenced by metabolomics sequencing technology.Statistical software was used to analyze the quality of the metabolomics results,and then the differentially expressed substances were screened.The OPLS-DA model was analyzed by7-fold cross-validation and permutation test.Combined with the Student's t test,the differentially expressed substances were screened out.The results showed that there is a significant difference between the two groups of samples and most of the samples were in the 95%confidence interval,and the OPLS-DA model was accurate and stable,indicating that the metabolomics sequencing results were stable,reliable and suitable for further analysis.This study totally figured out 253differentially expressed substances,and 99 of them could matched to the database.2.2 L-ascorbic acid and L-aspartate can directly affect bovine PmA growthBactrac 4300 was used to screen potential effective substances that can affect the growth of PmCQ2 from those differentially expressed substances.The function and strength of these potential effective substances via measuring PmCQ2 growth curve and plate count method respectively.The results showed that only L-ascorbic acid and L-aspartic acid can affect the growth of PmCQ2.In the metabolomics results,L-aspartate is more abundant in the infected lung,and promotes PmCQ2growth in a dose-dependent manner.On the contrary,L-ascorbic acid is more abundant in the infected liver,and inhibits PmCQ2 growth in a dose-dependent manner.Notably,L-ascorbic acid played a bactericidal effect at 2mg/mL.The above results show that L-ascorbic acid has a strong inhibitory effect on the growth of bovine PmA,while L-aspartic acid has a positive effect on bovine PmA.The content of both in their organs is correlated with bacteria colonization density.3.The role of exogenous L-ascorbic acid and L-aspartate in bovine PmA infection3.1 L-ascorbic acid and L-aspartic acid can influence the virulence of bovine PmATo investigate the effects of L-ascorbic acid and L-aspartate on bacterial virulence,some virulence genes and biofilm formation of PmCQ2 were examined in this study.The results showed that low concentration of L-aspartic acid could promote the expression of PmCQ2 partial virulence gene and biofilm formation,while L-ascorbic acid could inhibit the expression of OmpA and oma87 and biofilm formation.In addition,the expression level of OmpA in the infected lungs was higher than that of the infected liver.Thus,L-aspartate may enhance the virulence of bovine PmA,while L-ascorbic acid can inhibit the virulence of bovine PmA.3.2 L-ascorbic acid and L-aspartate metabolism were influenced in infectionTo investigate whether the metabolism of L-ascorbic acid and L-aspartate was changed during infection.This study determined the expression of endogenous L-ascorbate-producing gene Gulo,the content of L-ascorbic acid in the infected and the expression of L-aspartate metabolic pathway-related genes in the infected lung.The expression of Gulo gene was down-regulated under different infection pathway,and L-ascorbic acid in the infected lungs were down-regulated compared with health.L-aspartate metabolism-related genes was enriched.The above results indicated that the synthesis of endogenous L-ascorbic acid was inhibited in mice after infection and the L-aspartate metabolism in the infected lung was changed.3.3 Exogenous L-ascorbic acid can partly prevent murine from deathTo investigate the effects of exogenous L-ascorbic acid and L-aspartate on PmCQ2 infection at the animal level,this study determined the colonization of the lung and murine survival curves after addition of L-ascorbic acid and L-aspartate.The results showed that compared with the control group,addition of L-ascorbic acid reduced the bacteria colonization density of the lung and increased the survival rate of the mice.However,addition of L-aspartic acid can not affect the bacteria colonization density of the lung and survival rate of the mice.The above results showed that exogenously added L-ascorbic acid can reduce PmCQ2 colonization and is protective to mice3.4.L-ascorbic acid enhanced macrophages phagocytic killing on PmCQ2To investigate the effect of L-ascorbic acid and L-aspartate on the macrophages phagocytic killing on PmCQ2,this study first determined whether exogenous L-ascorbic acid and L-aspartate had an effect on the viability of mouse primary peritoneal macrophages by adding different doses of L-ascorbic acid and L-aspartate.Based on the result,an optimal concentration was chosen to determine the phagocytic killing effect of macrophages on PmCQ2.The results showed that low concentration of L-ascorbic acid could increase the viability of mouse peritoneal macrophages,and100?g/mL L-ascorbic acid could significantly increase the phagocytic killing ability of macrophages on PmCQ2.L-aspartate did not inhibit cell viability It also does not affect the phagocytic killing ability of macrophages on PmCQ2.This result indicated that L-ascorbic acid can enhance the phagocytic killing effect of macrophages on PmCQ2.In summary,this study found that PmA can colonize multiple organs,with the highest colonization density in the lungs.Both L-ascorbic acid and L-aspartic acid were found affecting the growth of bovine PmA by comparing organs with different colonization densities.Exogenously added L-ascorbic acid can inhibit the virulence of bacteria,increase the phagocytic killing ability of macrophages,and improve murine survival rate.Thus,L-ascorbic acid can be used as a potential target for the prevention and control of bovine PmA infection. |
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