Dietary supplemental coated essential oils and organic acids mixture improves growth performance and gut health along with reduces Salmonella load of broiler chickens infected with Salmonella Enteritidis

Background Reducing Salmonella infection in broiler chickens by using effective and safe alternatives to antibiotics is vital to provide safer poultry meat and minimize the emergence of drug-resistant Salmonella and the spread of salmonellosis to humans.This study was to first evaluate the protective efficacy of feeding coated essential oils and organic acids mixture(EOA)on broiler chickens infected with Salmonella Enteritidis(S.Enteritidis,SE),and then its action mechanism was further explored.Methods A total of 4801-day-old Arbor Acres male chickens were randomly assigned into five treatments with six replicates,including non-challenged control fed with basal diet(A),SE-challenged control(B),and SE-infected birds fed a basal diet with 300 mg/kg of EOA(BL),500 mg/kg of EOA(BM)and 800 mg/kg of EOA(BH),respectively.All birds on challenged groups were infected with Salmonella Enteritidis on d 13.Results Feeding EOA showed a reversed ability on negative effects caused by SE infection,as evidenced by decreasing the feed conversion rate(FCR)and the ratio of villus height to crypt depth(VH/CD)(P<0.05),obviously decreasing intestinal and internal organs Salmonella load along with increasing cecal butyric acid-producing bacteria abundance(P<0.05).Moreover,supplemental different levels of EOA notably up-regulated claudin-1(CLDN-1),occludin(OCLN),zonula occludens-1(ZO-1),mucin-2(MUC-2),fatty acid binding protein-2(FABP-2),nuclear factor kappa-light-chainenhancer of activated B cells(NF-κB),myeloid differential protein-88(MyD88)and interleukin-6(IL-6)mRNA levels in the ileum of the infected chickens after challenge,whereas down-regulated toll-like receptor-4(TLR-4)mRNA levels(P<0.05).Linear discriminant analysis combined effect size measurements analysis(LEfSe)showed that the relative abundance of g_Butyricicoccus,g_Anaerotruncus and g_unclassified_f_Bacillaceae significantly was enriched in infected birds given EOA.Also,phylogenetic investigation of communities by reconstruction of unobserved states(PICRUSt)analysis showed that alpha-linolenic acid metabolism,fatty acid metabolism and biosynthesis of unsaturated fatty acids were significantly enriched in the EOA group.Conclusion Our data suggest that the essential oils and organic acids mixture can be used as an effective strategy to ameliorate and alleviate Salmonella Enteritidis infection in broilers.

Cross-protective effect of acid adaptation on ethanol tolerance in Salmonella Enteritidis

Cross protection can undermine the effectiveness of control measures on foodborne pathogens,and therefore brings major implications for food safety.In this work,the capacity of Salmonella Enteritidis to mount ethanol tolerance following acid adaptation was characterized by analysis of cell viability and cell membrane property.It was observed that preadaptation to pH 4.5 significantly(P<0.05)increased the tolerance of log-phase cells to ethanol;in contrast,stationary-phase cells displayed reduced ethanol tolerance after acid adaptation.However,acid adaptation did not cause cell leakage and morphological change in both log-phase and stationary-phase S.Enteritidis.Fatty acid analysis further revealed that the amount of C_(14:0),C_(17:0 cyclo) and C_(19:0 cyclo) fatty acids was increased,while that of C_(16:1ω7c) and C_(18:1ω7c) fatty acids was decreased,respectively,in response to acid adaptation,regardless of bacterial growth phase.Notably,acid adaptation significantly(P<0.05)increased the proportion of C_(16:0) fatty acid in log-phase cells,but this effect did not occur in stationary-phase cells.Moreover,exogenous addition of C_(16:0) fatty acid to stationary-phase acid-adapted cultures was able to enhance bacterial ethanol tolerance.Taken together,C_(16:0) fatty acid is involved in the growth-phase-dependent protective effect of acid adaptation on ethanol tolerance in S.Enteritidis.

Comparative analysis of intestinal microbial community diversity between healthy and orally infected ducklings with Salmonella enteritidis by ERIC-PCR

AIM: To analyze the difference of intestinal microbial community diversity between healthy and (S. enteritidis) orally infected ducklings.METHODS: Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR was applied to analyze the intestinal microbial community diversity and dynamic change including duodenum, jejunum, ileum, cecum and rectum from healthy ducklings and 7-day-old ducklings after oral infection with S. enteritidis at different time points.RESULTS: The intestinal microbial community of the control healthy ducklings was steady and the ERIC-PCR band numbers of the control healthy ducklings were the least with rectum and were the most with caecum. ERIC-PCR bands of orally inoculated ducklings did not obviously change until 24 h after inoculation (p.i.). The numbers of the ERIC-PCR bands gradually decreased from 24 h to 72 h p.i., and then, with the development of disease, the band numbers gradually increased until 6 d p.i. The prominent bacteria changed because of S. enteritidis infection and the DNAstar of staple of ERIC-PCR showed that aerobe and facultative aerobe (Escherichia coli, Shigella, Salmonella) became preponderant bacilli in the intestine of orally infected ducklings with SE.CONCLUSION: This study has provided significant data to clarify the intestinal microbial community diversity and dynamic change of healthy and S. enteritidis orally infected ducklings, and valuable insight into the pathogenesis of S. enteritidis infection in both human and animals.

Gastrointestinal tract distribution of Salmonella enteritidis in orally in fected mice with a species-specific fluorescent quantitative polymerase chain reaction

AIM: To identify and understand the regular distribution pattern and primary penetration site for Salmonella enteritidis (S. enteritidis) in the gastrointestinal tract. METHODS: Based on the species-specific DNA sequence of S. enteritidis from GenBank, a species-specific real- time, fluorescence-based quantitative polymerase chain reaction (FQ-PCR) was developed for the detection of S. enteritidis. We used this assay to detect genomic DNA of S. enteritidis in the gastrointestinal tract, including duodenum, jejunum, ileum, cecum, colon, rectum, esophagus and stomach, from mice after oral infection. RESULTS: S. enteritidis was consistently detected in all segments of the gastrointestinal tract. The jejunum and ileum were positive at 8 h post inoculation, and the final organ to show a positive result was the stomach at 18 h post inoculation. The copy number of S. enteritidis DNA in each tissue reached a peak at 24-36 h post inoculation, with the jejunum, ileum and cecum containing high concentrations of S. enteritidis, whereas the duodenum, colon, rectum, stomach and esophagus had low concentrations. S. enteritidis began to decrease and vanished at 2 d post inoculation, but it was still present up to 5 d post inoculation in the jejunum, ileum andcecum, without causing apparent symptoms. By 5 d post inoculation, the cecum had significantly higher numbers of S. enteritidis than any of the other areas (P < 0.01), and this appeared to reflect its function as a repository for S. enteritidis. CONCLUSION: The results provided significant data for clarifying the pathogenic mechanism of S. enteritidis in the gastrointestinal tract, and showed that the jejunum, ileum and cecum are the primary sites of invasion in normal mice after oral infection. This study will help to further understanding of the mechanisms of action of S. enteritidis.

Quantitative studies of the regular distribution pattern for Salmonella enteritidis in the internal organs of mice after oral challenge by a specific real-time polymerase chain reaction

AIM： To identify and understand the regular distribution pattern for Salmonella enteritidis （S. enteritidis） in the internal organs of mice after an oral challenge over a 3 wk period. METHODS： Assays based on the serovar-specific DNA sequence of S. enteritidis from GenBank, and a serovar-specific real-time, fluorescence-based quantitative polymerase chain reaction （FQ-PCR） were developed for the detection of S. enteritidis. We used this assay to detect genomic DNA of S. enteritidis in the blood and the internal organs, including heart, liver, spleen, kidney, pancreas, and gallbladder, from mice after oral challenge at different time points respectively.RESULTS： The results showed that the spleen was positive at 12 h post inoculation （PI）, and the blood was at 14 h PI. The organism was detected in the liver and heart at 16 h PI, the pancreas was positive at 20 h PI, and the final organs to show positive results were the kidney and gallbladder at 22 h PI. The copy number of S. enteritidis DNA in each tissue reached a peak at 24-36 h PI, with the liver and spleen containing high concentrations of S. enteritidis, whereas the blood, heart, kidney, pancreas, and gallbladder had low concentrations. S. enteritidis populations began to decrease and were not detectable at 3 d PI, but were still present up to 12 d PI in the gallbladder, 2 wk for the liver, and 3 wk for the spleen without causing apparent symptoms.CONCLUSION： The results provided significant data for understanding the life cycle of S. enteritidis in the internal organs, and showed that the liver and spleen may be the primary sites for setting itself up as a commensa over a long time after oral challenge. Interestingly, it may be the first time reported that the gallbladder is a site of carriage for S. enteritidis over a 12 d period. This study will help to understand the mechanisms of action of S. enteriCdis infection in vivo.

Weighted gene co-expression network analysis identiffes potential regulators in response to Salmonella Enteritidis challenge in the reproductive tract of laying ducks

Salmonella Enteritidis(SE)is a zoonotic and vertically transmitted pathogen,often colonized in the reproductive tract of adult poultry,which can result in direct contamination of eggs and threaten human health.Previous studies have revealed that some pattern recognition receptors and resistance genes were involved in regulating immune responses to SE invasion in birds.However,the role of these immune response genes was not independent,and the interactions among the genes remained to be further investigated.In this study,SE burden and colonization were determined in reproductive tissue after the ducks were SE-infected,and RNA-sequencing was performed to construct co-expression networks by weighted gene co-expression network analysis(WGCNA).The result showed that SE could be isolated from 22% of infected-birds in any segment of the reproductive tract and the SE was readily colonized in the stroma,small follicle,isthmus,and vagina of the reproductive tracts in morbid ducks.The top central,highly connected genes were subsequently identified three specific modules in the above four tissues at the defined cut-offs(P<0.01),including 60 new candidate regulators and 125 transcription factors.Moreover,those 185 differentially expressed genes(DEGs)in these modules were co-expressed.Moreover,the hub genes(TRAF3,CXCR4 and IL13RA1)were identified to act with many other genes through immune response pathways including NF-kappaB,Toll-like receptor,steroid biosynthesis,and p53signaling pathways.These data provide references that will understand the immune regulatory relationships during SE infection,but also assist in the breeding of SE-resistant lines through potential biomarkers.

Acalculous cholecystitis due to Salmonella enteritidis

Acute acalculous cholecystitis (AAC) is defi ned as an acute infl ammation of the gallbladder in the absence of stones. We herein report a case of a young man who developed AAC after a Salmonella enteritidis gastrointestinal infection.

Vitamin K alleviates bone calcium loss caused by Salmonella Enteritidis through carboxylation of osteocalcin

Background:The present study aimed at evaluating the effect of vitamin K(VK)supplementation on bone health of laying hens challenged by Salmonella Enteritidis.Methods:A total of 8032-week-old double negative salmonella-free brown-egg laying hens were randomly assigned to 4 treatments with 20 replicates each(1 bird per replicate)according to a 2×2 factorial design with 2 dietary VK supplementation levels[0 mg/kg(VK0)vs 2 mg/kg VK(VK2)and 2 challenge treatments[Salmonella Enteritidis(SE)vs physiological saline solution(PS)].During the last 3 days of week 43 of age,birds of both VK treatments were either orally challenged with 1.0 mL suspension of 109 cfu/mL S.Enteritidis daily or received the same volume of PS.Results:The laying rate,daily egg mass,tibia strength,CT,cOC and cOC/(cOC+ucOC)of VK2 treatment increased(P<0.05)in contrast to VK0,however,the medullary area and ucOC of VK2 treatment decreased(P<0.05)in contrast to VK0.Mortality,medullary area,serum Ca content of SE treatments increased(P<0.05)in contrast to PS treatments.In both SE treatments,the decrease(P<0.05)in birds’tibia strength was associated with higher(P<0.05)Ca levels in serum.There is an interaction(P<0.05)between SE challenge and VK levels with regard to tibia strength and serum Ca levels.At week 42,serum CT was positively correlated with cOC(R=0.99,P=0.009);at week 44,tibia strength was positively correlated with BMD(R=0.95,P=0.045),but negatively correlated with medullary area(R=−0.98,P=0.018).Conclusions:VK(2 mg/kg)supplementation to diets of laying hens can enhance bone strength under challenge situations with Salmonella Enteritidis.Medullary area has proven to be a sensitive biomarker for bone calcium loss caused by SE infection.

Establishment of probabilistic model for Salmonella Enteritidis growth and inactivation under acid and osmotic pressure

The growth and survival characteristic of Salmonella Enteritidis under acidic and osmotic conditions were studied.Meanwhile,a probabilistic model based on the theory of cell division and mortality was established to predict the growth or inactivation of S.Enteritidis.The experimental results demonstrated that the growth curves of planktonic and detached cells showed a significant difference(p<0.05)under four conditions,including pH5.0+0.0%NaCl,pH7.0+4.0%NaCl,pH6.0+4.0%NaCl,and pH5.0+4.0%NaCl.And the established primary and secondary models could describe the growth of S.enteritis well by estimating four mathematics evaluation indexes,including determination coefficient(R2),root mean square error(RMSE),accuracy factor(Af)and bias factor(Bf).Moreover,sequential treatment of 15%NaCl stress followed by pH 4.5 stress was the best condition to inactivate S.Enteritidis in 10 h at 25◦C.The probabilistic model with Logistical or Weibullian form could also predict the inactivation of S.Enteritidis well,thus realize the unification of predictive model to some extent or generalization of inactivation model.Furthermore,the primary 4-parameter probabilistic model or generalized inactivation model had slightly higher applicability and reliability to describe the growth or inactivation of S.Enteritidis than Baranyi model or exponential inactivation model within the experimental range in this study.

Antibacterial Effects of Ethanolic Extract of Zehneria scabra on Quails Artificially Infected with Salmonella Enteritidis

Antimicrobial use in livestock is faced with various challenges including emergence of antimicrobial resistance and presence of drug residues in meat products,hence the need for alternatives.The aim of this work was to assess the effect a plant(Zehneria scabra)extract on Salmonella infected quails,as an alternative to antibiotic therapy.Quails were randomly assigned into six groups each containing twelve birds.The neutral control(T0)group was not infected and received tap water whereas other groups were infected.The negative control(T-)received tap water.The positive control(T+)received a single dose of oxytetracycline(20 mg/kg).T1,T2 andT3 orally received the plant extract at the following respective doses:9,18 and 37 mg/kg.Quails were infected by oral administration of a single dose of Salmonella Enteritidis(10^(5)CFU).Haematological and biochemical parameters were evaluated.From day 2 to day 9 to day 16,the bacterial load of all treatment groups(T+,T1,T2,T3)decreased.The infection resulted in a significant(p<0.05)increase in serum levels of alanine aminotransferase,triglycerides,total cholesterol and white blood cells,and a significant decrease in the liver and kidney protein content.The treatment resulted in the correction of the aforementioned effects.The plant extract(18 mg/kg)is as effective as oxytetracycline,and can be safely used in phytomedicine for the treatment of Salmonella Enteritidis infection without kidney and liver damage.

Live and Inactivated Salmonella Enteritidis Vaccines:Immune Mechanisms in Broiler Breeders

Salmonella is a ubiquitous pathogen which, in addition to causing poultry diseases, has a growing zoonotic impact. It has demanded the implementation of diverse control strategies, in which vaccines play a major role. The understanding of the immune pathways elicited by the different vaccines is important, contributing for the establishment of strong immune correlates of protection, for instance. With the purpose of determining the dynamics of the humoral and cellular immune responses to vaccination, broiler breeders (Cobb Slow) were immunized with live or inactivated vaccines against Salmonella Enteritidis. Lymphocyte and macrophage subsets were analyzed in the peripheral blood by flow cytometry and antigen-specific circulating IgY and mucosal IgA were quantified. The markers analyzed by flow cytometry were CD8/CD28, CD4/TCRVβ1, Kul/ MHC II and Bu-1. Both live and inactivated vaccines induced an increase in the proportion of circulating monocytes (Kul+MHCII+) in some time points compared to non-vaccinated controls. However, whereas the live vaccine leads to an increase in CD8-CD28+ and Bu-1+ lymphocytescompared to the control group, the inactivated vaccine prompteda reduction in the percentage of severalleucocyte subsets (Kul-MHCII+, Bu-1+, CD8+CD28+, CD8-CD28+, CD4+TCRVβ1-, CD4+TCRVβ1+, CD4-TCRVβ1+) after the boost dose. Both vaccines induced specific serum IgY and mucosal IgA production;however, the inactivated vaccine stimulated higher titers in a shorter period. These results contribute to the understanding of mechanisms of action of live and inactivated Salmonella vaccines in chickens.

The influences of SE infection on layers' production performance,egg quality and blood biochemical indicators

Background： Salmonella enterica serovar Enteritidis （SE）, as a major cause of foodborn illness, infects humans mainly through the egg. However, the symptom of laying hens usually is not typical and hard to diagnosis. In the present study, it is studied that the influences of SE infection on layers＇ performance, egg quality and blood biochemical indicators. It will help us to improve the strategy to control SE infection in commercial layers. One hundred layers at 20 wk of age were divided into 2 groups, 60 hens for experiment and others for control. The experiment group was fed with the dosage of 108 CFU SE per hen. The specific PCR was used to detect the deposition of SE. On the 8 d after SE infection, 10 hens from the control group and 30 hens from the experimenta group were slaughtered to detect the SE colonization. The production performance, egg quality and blood biochemical indices were also analyzed. Results： The results showed that the colonization rate of SE was highest in caecum contents （55.17%） and lowest in vagina （17.24%）. For the eggs the detection rate of SE was highest on the eggshell （80.00%） and lowest in yolk （18.81%）. SE infection had no significant influence on production performance and egg qualities （P 〉 0.05）. The difference of laying rate between the experimental and control groups was less than 0.30%, and both were approximately equal to 82.00%. The blood analysis showed that the aspartic aminotransferase （AST） and alanine aminotransferase （ALT） of experimental group was significantly higher than those of control group （P 〈 0.05）. For experimental and control groups AST values were 236.22 U/I and 211.84 U/I respectively, and ALT values were 32.19 U/I and 24.55 U/I. All of coefficients were less than 20%. The colonization of SE in organs increases the enzyme activities of AST and ALT in blood. Conclusions： SE in feed could invade the oviduct and infect the forming eggs. It significantly increased the concentration of ALT and AST in blood. However,SE infection was hard to be observed from the appearances of layer and egg. It might be a dangerous risk to human health.

Effects of Salmonella infection on hepatic damage following acute liver injury in rats

BACKGROUND： Acute liver injury is a common clinical disorder associated with intestinal barrier injury and disturbance of intestinal microbiota. Probiotic supplementation has been reported to reduce liver injury; however, it is unclear whether enteropathogen infection exacerbates liver injury. The purpose of this study was to address this unanswered question using a rat model. METHODS： Oral supplementation with Salmonella enterica serovar enteritidis（S. enteritidis） was given to rats for 7 days. Different degrees of acute liver injury were then induced by intraperitoneal injection of D-galactosamine. The presence and extent of liver injury was assayed by measuring the concentrations of serum alanine aminotransferase, aspartate aminotransferase, and total bilirubin. Histology was used to observe liver tissue damage. Additionally, we measured the changes in plasma endotoxin, serum cytokines and bacterial translocation to clarify the mechanisms underlying intestinal microbiota associated liver injury.RESULTS： The levels of liver damage and endotoxin were significantly increased in the Salmonella infected rats with severe liver injury compared with the no infection rats with severe liver injury（P〈0.01）; The peyer＇s patch CD3＋ T cell counts were increased significantly when the Salmonella infection with severe injury group was compared with the normal group（P〈0.05）. S. enteritidis pretreatment enhanced intestinal barrier impairment and bacterial translocation.CONCLUSIONS： Oral S. enteritidis administration exacerbates acute liver injury, especially when injury was severe.Major factors of the exacerbation include inflammatory and oxidative stress injuries induced by the translocated bacteria and associated endotoxins, as well as over-activation of the immune system in the intestine and liver.

Nanozyme Enhanced Colorimetric Immunoassay for Naked-Eye Detection of Salmonella Enteritidis

Salmonella Enteritidis is a major public health threat of global proportions,while it is still a serious challenge to develop point-of-care detection assay.In this study,nanozymes(Fe-MOF nanoparticles)were successfully synthesized and applied as signal in a colorimetric immunoassay for naked-eye detection of Salmonella Enteritidis.After optimization,the proposed assay was able to detect Salmonella Enteritidis with a detection limit of 34 CFU/mL.The coefficients of variation(CV)of the test were less than 7.0%after 30 days storage at 4°C.The estimated recoveries in milk samples of the colorimetric immu-noassay range from 94.68 to 124%,which indicated the developed method is capable of detecting Salmonella Enteritidis in real samples.This method provides a potential platform for Salmonella detection with naked eyes,which has a significant application value for foodborne pathogen analysis at the point-of-care.

Meat juice contributes to the stability of ethanol adaptation in Salmonella enterica serovar Enteritidis

Stability assessme nt of observed tolerance phe no types is integral in un dersta nding stress adaptati on in food-borne pathogens.Therefore,the current work was carried out to determine whether ethanol adaptation induced by exposure to 5 percent ethanol for 60 min is a stable phenomenon in Salmonella enterica serovar Enteritidis.The capacity of Salmonella Enteritidis(S.Enteritidis)to maintain the acquired ethanol adaptation in the absenee of sublethal ethanol stress was investigated at 37℃,25℃ or 4℃ in Luria-Bertani broth and two types of meat juice.It was found that ethanol adaptation was completely reversed within 40 min at 37℃ or within 60 min at 25℃,but was stable at 4℃ for at least 48 h in the broth assay.Ethanol adaptation was retained in chicken juice during 60-min incubation at 25℃ or 48h incubation at 4℃.Moreover,exposure to pork juice stored at either 25℃ or 4℃ sigrdficantly(P<0.05)increased the ethanol toleranee of ethanol-adapted cells.Collectively,these fin dings suggest that ethanol adaptation stability in S.En teritidis under cold conditi ons and in meat juices should be take n into acco unt whe n con ducting a comprehensive risk analysis during food processing.