Study On The Mechanism Of Salmonella Lipopolysaccharide-induced Acute Bursal Atrophy In Broiler Chicks

The domestic chicken(Gallus gallus domesticus)is economically important and its inbred lines are easily available therefore,it is mostly used in research concerning avian immunology.Bursa of Fabricius(BF)is an immune organ,which is unique and peculiar to birds and plays a crucial role in differentiation of B cells and production of antibodies.The acquired immune system in very young chicken is not completely functional until first week of age therefore the innate immunity plays an essential role in neonatal chicken.Acute atrophy of bursa of Fabricius is a common feature in different infectious diseases.Many scientific reports have described the effects of Salmonella lipopolysaccharide(LPS)on avian central immune organs.However,the molecular mechanism of Salmonella LPS induced micro-morphological alterations at transcriptional level in bursa of Fabricius is still elusive.Therefore,in the current study,oneday-old chicks were intraperitoneally(i.p.)injected with saline or Salmonella LPS(50 mg/ kg of body weight).Six tissue samples from each experimental treatment group(saline or LPS)were collected at 0,2,6,12,36,72 and 120 hours.The tissues for histo-morphological experiments were stored in 4% paraformaldehyde at room temperature and for scanning electron microscopy(SEM)in 2.5% glutaraldehyde solution at 4 ?.The part of tissues were also preserved immediately at-70 ? for RNA-seq and bio-molecular experiments.In the current investigation,transcriptional changes were detected in LPS induced micro-morphological alterations in bursa of Fabricius.The following is summary of results for the experiments conducted in this study:1.Micro-morphological and immunohistological alterations in bursa of Fabricius1.1 LPS stimulation induced acute bursal atrophy in broiler chicksAfter intra-peritoneal saline or LPS injections,LPS stimulation caused a significant loss(~ 36%)in weight of bursa of Fabricius and remarkable decrease(~ 18%)in bursal index at 36 h post treatment as compared to saline treatment.Furthermore,the decrease in weight of bursa of Fabricius was found to be strongly associated with the dosage of LPS injection.As a conclusion,these morphological observations indicates that maximum bursal atrophy occurs at 36 hours post LPS stimulation.1.2 Micro-morphological alterations in chicken Bursa under LPS stressH&E staining revealed that LPS stimulation could disrupt the structural integrity of bursa of Fabricius.As compared to saline treatment,LPS stimulation caused infiltration of heterophils along with increased number of nucleated oval shaped RBCs in or near blood vessels in chicken BF at 12 h,36 h and 72 h.Accumulated enhanced heterophilic granules were seen in the medulla of bursal follicle together with the notable disruption of outer boundary of bursal follicular cortex at 36 h post LPS stimulation.Hence,these histopathological alterations indicate that LPS stimulation not only leads to inflammatory changes but also causes bursal injury in chicken.1.3 Ultra-structural alteration in chicken bursa of Fabricius under LPS stressScanning electron micrographs from saline group showed smooth mucosal surface.As compared to saline treatment,LPS stimulation caused severe erosion and breaks at 12 h and complete exuviations of bursal mucosal epithelial cells at 36 h post treatment.Taken together,it seems that LPS stimulation causes bursal mucosal injury due to inflammatory changes at 12 h and the bursal atrophy observed due to significant decrease in bursal weight at 36 h might be partly associated with the severe loss of epithelial cells at 36 h as seen in electron micrograph.1.4 Alterations in immune-related cells in chicken bursa of Fabricius under LPS stress In comparison to the control(saline)group,Toluidine blue stained mast cells within the bursa of Fabricius illustrated no significant effect on the number of mast cells or their de-granulation,however the number of mast cells in the bursa of Fabricius showed slight increasing trend after LPS stimulation at 12 h,36 h and 72 h.As compared to the control group.the number of Chromotrope 2R stained eosinophils in chicken bursa of Fabricius were increased significantly after 12 h and 36 h post LPS stimulation.1.5 Expression of TLR4 and NF?B under LPS stress The expressions of both TLR4 and nuclear factor-?B(NF-?B)were assessed at protein levels.Immuno-staining and western blotting revealed that LPS stimulation led to enhanced TLR4 expression in chicken bursa of Fabricius at 12 h and 36 h after LPS stimulation.Similarly increased expression of NF?B was detected through immuno-staining at 12 h and 36 h post LPS stimulation as compared to saline treatment.Hence,it indicates that LPS stimulation not onlycauses activation of TLR4 expression but also leads to activation of its downstream transcriptional factor NF-?B in bursa of Fabricius in broiler birds.1.6 Alterations in apoptotic rate in chicken bursa of Fabricius under LPS stress Apoptosis in chicken bursa of Fabricius was evaluated by terminal-deoxynucleoitidyl transferase mediated nick end labeling method(TUNEL method).The bursal sections exhibited enhanced expression at 36 h post LPS stimulation as compared to saline group.On the basis of these observations,it is speculated that LPS stimulation may induce apoptotic changes in bursa microenvironment.1.7 Alterations in expressions of PCNA and ssDNA in bursa of Fabricius under LPS stress Cellular expression of proliferative cell nuclear antigen(PCNA)in chicken bursal tissue sections was down-regulated at 12 h and 36 h post LPS stimulation.In-situ cell apoptosis/ cellular expression of single stranded DNA(ssDNA)showed up-expression in LPS group at 12 h and 36 h post LPS stimulation.These results showed noticeable decrease in proliferation and remarkable increase in apoptosis in bursa of Fabricius after intra-peritoneal LPS stimulation.2.Transcriptome analysis of LPS induced micro-morphological changes in bursa2.1 RNA-sequencing and identification and validation of DEGs RNA-seq was performed using SE50 sequencing strategy in group-wise comparisons(saline vs LPS)at 12 h,36h and 72 h.NOISeq method was used for gene expression profiling of the groupwise comparisons explored the differentially expressed genes(DEGs)at each time point compared to control(saline)vs.infected(LPS)samples.A few of randomly selected candidates DEGs from RNA-seq data were validated by RT-qPCR.2.2 Bioinformatics analysis of transcriptome data GO and KEGG analysis at early time point showed that maximum number of DEGs were enriched in metabolic,immune and genetic information systems.In agreement with these results,Reactome analysis also found maximum number of DEGs enrichment in immune system,signal transduction and metabolism.Moreover,the interaction among immune system and signal transduction was also found significant at early time point post LPS stimulation in bursa.Hence,this comparative mapping of DEGs with KEGG and Reactome data bases showed the involvement of DEGs in almost similar pathways.Subsequently,string protein functional association network analysis was performed for the understanding of the putative interaction and relationship among the genes at protein level.Many of the candidate genes and genes validated by RT-qPCR in this study such as MAPK 9 and TGFBR1,HSPB1,SASH3 and E2F1 showed strong interaction within this protein-protein interaction(PPI)network at early time point post LPS stimulation in the bursa.2.3 Integrated Functional Pathway Analysis Integrated pathway analysis indicated that most of the candidate genes were enriched in mitogenactivated protein kinase(MAPK)transduction signaling pathways.Transcriptional factors such as Ras,FGFR,c-Jun(JUN),p53 and JNK exhibited up-expression while HSP27,MEK2,crk2 and Akt1 showed down-expression in MAPK pathways.The second most enriched pathway was toll like receptor signaling that showed up-regulation of p105,JNK,NF-?B,CD80 and downexpression of AKT and MEKI/II.Taken together,it seems that LPS stimulation causes acute bursal atrophy and disrupts the structural integrity of bursa of Fabricius and leads to infiltrations of heterophils,mast cells and eosinophils along with decreased cell proliferation and increased cell apoptosis at early time points.Conclusively,the findings of current investigation shed light on a novel transcriptionbased molecular mechanism that first time explored the underlying phenomenon of LPS induced acute inflammatory injury in chicken bursa of Fabricius.Hence,transcriptional factors such as CNN1,E2F1 and HRAS played critical role in acute bursal atrophy through LPS-TLR/JNKMAPK functional pathway along with a few anti-inflammatory factors such as BPI,AVD and avidin related antimicrobial agents under salmonella LPS stress.