| Salmonella enterica serovar Gallinarum biovar Pullorum(Salmonella Pullorum)is the causative agent of pullorum disease,characterized by acute systemic symptoms that results in high mortality in very young chicks.In addition,Salmonella Pullorum is known to produce a persistent infection in hens,in which localization in the reproductive tract is associated with vertical transmission.Eggs contaminated with Salmonella Pullorum are the most commonly known route of transmission from one generation to the next.Pullorum disease has been eradicated from commercial poultry in many developed countries,but it still persists in China,leading to severe economic losses due to widespread outbreaks accompanied by high mortality.In recent years,a syndrome of claudication,arthritis,swelling of the tibiotarsal joint and the radial,humeral and ulnar articulations has been described in chickens with Salmonella Pullorum infection.In America and several European countries,fowl typhoid(caused by Salmonella Gallinarum)have been eliminated,elimination of pullorum disease is more difficult,due to its epidemic characteristics of vertical transmission and horizontal transmission.Identification and slaughter of the positive animals are the basic measures to control pullorum disease in chickens.However,in China,pullorum disease infection rate was usually over 30%,which is due to the diversity of breeding pattern and uneven management level.In China where prevalence rate is high,serological test and slaughter of reactors to eliminate pullorum disease may be economically unviable.For a long time,the prevention and control Salmonella Pullorum infection in chickens have been mainly depended on antibiotics,which has led to a high level of antibiotic resistance and some long-term harmful effects.Recently,the government issued the National Medium and Long Term Planning for Prevention and Control of Animal Epidemics(2012-2020).The official document suggested a control strategy of pullorum disease through the detection and purification program based on the practical conditions.Control of Salmonella Pullorum infection has depended on the level of carriage rate in the region.Serological testing and slaughter of reactors can be used where levels of infection are low or where eradication can be contemplated,while vaccination is considered where levels of infection rates are high or where eradication may be lead to severe economic losses.Previous researches indicated that Salmonella live,attenuated vaccines were much more protective and effective than inactivated vaccines,bacterial ghost vaccines and subunit vaccines although some killed vaccines have been produced and used for controlling Salmonella infection over the past few years.Sero-diagnosis of Pullorum disease is generally based on the detection of antibodies against Salmonella lipopolysaccharides(LPS)by use of a macroscopic tube agglutination test,a rapid serum plate agglutination(SPA)test,a stained antigen whole blood test,or a micro-agglutination test.Because breeding flocks are screened for specific serum antibodies against Salmonella LPS using the SPA test,antibodies produced following vaccination are indistinguishable from those produced in response to a wild type Salmonella Pullorum infection.A central goal in ideal vaccine development is that it should not interfere with this salmonellosis-monitoring program.The concept of DIVA(Differentiation of Infected and Vaccinated Animals)vaccines based on the absence of at least one immunogenic protein or antigen in the vaccine,which is,however,present in the wild type strain,has already been proposed for commercial veterinary use.LPS is a major virulence factor and the O-antigen is the immunodominant antigen in serological diagnosis tests.Truncating O-antigen and preventing its synthesis are effective strategies for attenuating Salmonella for use as a DIVA vaccine.In the present study,our results demonstrated that arthritis in chickens could be associated with highly diverse Salmonella Pullorum based on molecular and phenotypic characteristics.O-polysaccharide is important for Salmonella Pullorum virulence and colonisation in chicken embryos and survival in egg albumen.In addition,we evaluated the safety and protective capacity of a rough mutant Salmonella Pullorum vaccine candidate and the usefulness of this strain as DIVA strategy.1.Identification of a novel arthritis-causing Salmonella Pullorum in broilersLittle is known about the relationship between arthritis or joint enlargemen and Salmonella Pullorum infection in broilers.The aim of our present work is to provide data on the molecular and pathogenic characteristics of Salmonella Pullorum strains isolated from arthritis cases in broilers.Five Salmonella Pullorum strains were isolated from broilers presenting arthritis at commercial poultry farms.All of five Salmonella isolates were resistant to at least one antibiotic,and the highest resistance observed was to NAL,AMP and STR.Two CRISPR loci(CRISPR 1 and CRISPR 2)were present in Salmonella Pullorum isolate genomes.The analysis showed that the five isolates had close genetic relationship and all belonged to a single major cluster.Isolates showed a high capacity to form biofilm.Salmonella Pullorum isolates are virulent for 12-day-old chicken embryos.The LD50 was ranged from 1.7×107 CFU?7.2×107 CFU following intramuscular inoculation of 1-day-old chicks.Clinically,all of the chicks in infected group were generally emaciated and in poor physical condition.Of the broilers inoculated with SP1621,swollen tibio-metatarsal(hock)joint,arthritis and lameness were observed post infection.When the tarsal joint(hock)was dissected,there was accumulation of serous yellowish fluid in the articular cavity.Histopathological evaluation of affected tarsal joint included arthritis,besides degeneration and necrosis of overlying spinal cord.Disruptive and characteristic damages on the liver,spleen,heart and cecum parenchyma were observed.After infection,the spleen,liver and joint were the main sites of SP1621 bacterial localization.The general characteristics of Salmonella Pullorum isolates genome were analyzed with whole genome sequencing.The results indicated that the genome contains 5006 genes with a length of 4855288 bp and a GC content of 52.1%.Comparative genomic analysis of Salmonella Pullorum strains showed,there are 46 ORFs and 2213 single nucleotide polymorphism sites specific in 2 different pathotype.2.O-polysaccharide is an important virulence factor for Salmonella PullorumSalmonella Pullorum is the causative agent of persistent systemic infection of poultry,leading to economic losses in developing countries due to morbidity,mortality and reduction in egg production.These infections may result in vertical transmission to eggs or progeny.Limited information is available regarding to the mechanisms involved in the survival of Salmonella Pullorum in egg albumen and developing chicken embryos.Hence,we investigated the role of O-polysaccharide in the contamination of eggs and the colonisation of chicken embryos.We constructed a rough mutant lacking O-polysaccharide chains by deleting the waaL(or rfaL)gene encoding the unique O-antigen ligase from the well characterized Salmonella Pullorum strain S06004.Compared with the wild type strain,the isogenic waaL mutant exhibited an O-antigen-deficient rough phenotype,and increased sensitivity to egg albumen and chicken serum,as well as reduced adherence to DF-1 cells.Infection with Salmonella Pullorum lacking O-polysaccharide resulted in significantly reduced embryo lethality and bacterial colonisation.These results suggest that O-polysaccharide is essential for Salmonella Pullorum colonization in eggs,both postlay and developing embryos.The chicken embryo infection model could be used to characterize the interaction between Salmonella Pullorum and developing embryos,and will also contribute to the development of more rational vaccines to protect laying hens and embryos.3.Effect of mutation of genes involved in LPS synthesis on virulence and immunogenicity of Salmonella PullorumLPS is a recognized virulence factor of Salmonella and is essential for various functions.Truncating the LPS and ceasing O-antigen polysaccharide or core oligosaccharide synthesis are strategies for constructing live attenuated Salmonella vaccines.The potential of Salmonella Pullorum mutants with truncated O-antigen and core oligosaccharide for use as attenuated DIVA vaccines has not been systematically studied.In this study,we constructed strains with a defined mutation in rfaL,rfaJ,rfal,rfaH,wzy,wzzB and fepE of Salmonella Pullorum and evaluated these mutant strains for virulence,immunogenicity and DIVA capability.Results showed that,Salmonella Pullorum deletion with the above-mentioned genes had minimal effect on the growth and biochemical characteristics.The mutants with the rfaL,rfaJ,rfal,rfaH and wzy genes had a rough type A(Ra)LPS structure.All rough mutants were significantly attenuated,with the LD50 being 400 fold higher than that of wild type when administered intramuscularly to 1-day-old chickens.Salmonella Pullorum rough strains were less invasive in host tissues.In the early stages of infection,strains with rfaJ and rfaI deletion had reduced colonization in the liver and spleen.All samples taken 28 days after inoculation were negative,indicating that the mutant strains were efficiently cleared in internal organs.Chickens inoculated with the rough mutants showed significantly higher protection against Salmonella Pullorum challenge,while only the S06004?spiC?rfaL,S06004?spiC?rfaH and S06004?spiC?wzy induced effective cross-protection against virulent Salmonella Gallinarum.Inoculation of chicks with the ?rfaL,?rfaJ,?rfal or ?rfaH mutants resulted in the induction of a serological response lacking detectable antibodies against O-antigen.This allowed a clear differentiation between serum from chickens inoculated with the ?rfaL,?rfaJ,?rfaI and ?rfaH strains and serum from chicken infected with wild type strain.In conclusion,deletion in the rfaL or the rfaH gene in Salmonella Pullorum is a viable option for developing a live attenuated vaccine allows differentiation of infected and vaccinated chickens in an LPS based serum agglutination test.4.Evaluation of a rough mutant Salmonella Pullorum as a DIVA vaccine candidate in broilersVaccination represents one approach for promoting animal health,food safety and reducing environmental persistence in Salmonella control.An important consideration is that Salmonella vaccination in poultry should not interfere with the salmonellosis monitoring program.This is the basis of the DIVA program.In order to achieve this goal,rfaL mutant was constructed on a spiC mutant that was developed previously.The safety,efficacy,and DIVA features of this vaccine candidate(Salmonella Pullorum ?spiC?rfaL)were evaluated in broilers.Our results showed that the truncated LPS in the vaccine strain has a differentiating use as both a bacteriological marker(rough phenotype)and also as a serological marker facilitating the differentiation between infected and vaccinated chickens.The rough mutant showed adequate safety being avirulent in the host chicks and showed increased sensitivity to environmental stresses.Single intramuscular immunization of day-old broiler chicks with the mutant confers ideal protection against lethal wild type challenge by significantly stimulating both humoral and cellular immune responses as well as reducing the colonization of the challenge strain.Significantly lower mean pathology scores were observed in the vaccination group compared to the control group.Additionally,the mutant strain generated cross-protection against challenge with the wild type Salmonella Gallinarum thereby improving survival and with the wild type Salmonella Enteritidis thereby reducing colonization.These results suggest that the double-mutant strain may be a safe,effective,and cross-protective vaccine against Salmonella infection in chicks while conforming to the requirements of the DIVA program. |
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