Control of Salmonella Enteritidis and Salmonella Gallinarum in birds by using live vaccine candidate containing attenuated Salmonella Gallinarum mutant strain

The ideal live vaccine to control Salmonella in commercial chicken flocks should engender protection against various strains. The purpose of the present study was to confirm the attenuation of a Salmonella Gallinarum (SG) mutant strain with deletion on genes cobS and cbiA, that are involved in the biosynthesis of cobalamin. Furthermore, evaluate its use as a live vaccine against Salmonella. For the evaluation of the vaccine efficacy, two experiments were conducted separately. Birds from a commercial brown line of chickens were used to perform challenge with SG wild type strain and birds from a commercial white line of chickens were used to perform challenge with Salmonella Enteritidis (SE) wild type strain. In both experiments, the birds were separated in three groups (A, B and C). Birds were orally vaccinated with the SG mutant as the following programme: group A, one dose at 5 days of age; group B, one dose at 5 days of age and a second dose at 25 days of age; and group C, birds were kept unvaccinated as controls. At 45 days of age, birds from all groups, including the control, were challenged orally by SG wild type (brown line) or SE wild type (white line). Lastly, another experiment was performed to evaluate the use of the SG mutant strain to prevent caecal colonization by SE wild type on 1-day-old broiler chicks. Mortality and systemic infection by SG wild type strain were assessed in brown chickens; faecal shedding and systemic infection by SE wild type were assessed in white chickens and caecal colonization was assessed in broiler chicks. Either vaccination with one or two doses of SG mutant, were capable to protect brown chickens against SG wild type. In the experiment with white chickens, only vaccination with two doses of SG mutant protected the birds against challenge with SE wild type. Although, SG mutant could not prevent caecal colonization in 1-day-old broiler chicks by the challenge strain SE wild type. Overall, the results indicated that SG mutant is a promising Salmonella live vaccine candidate that demonstrated good efficacy to control the infection by two serotypes of major importance to the poultry industry.     

A Salmonella Enteritidis hilAssrAfliG deletion mutant is a safe live vaccine strain that confers protection against colonization by Salmonella Enteritidis in broilers

Consumption of contaminated poultry meat is an important cause of Salmonella infections in humans. Therefore, there is a need for control methods that protect broilers from day-of-hatch until slaughter age against infection with Salmonella. Colonization-inhibition, a concept in which a live Salmonella strain is orally administered to day-old chickens and protects against subsequent challenge, can potentially be used as control method. In this study, the safety and efficacy of a Salmonella Enteritidis ΔhilAssrAfliG strain as a colonization-inhibition strain for protection of broilers against Salmonella Enteritidis was evaluated. After administration of the Salmonella Enteritidis ΔhilAssrAfliG strain to day-old chickens, this strain could not be isolated from the gut, internal organs or faeces after 21 days of age. In addition, administration of this strain to one-day-old broiler chickens decreased faecal shedding and caecal and internal organ colonization of a Salmonella Enteritidis challenge strain administered one day later using a seeder bird model. To our knowledge, this is the first report of an attenuated Salmonella strain for which both the safety and efficacy has been shown in long-term experiments (until slaughter age) in broiler strain can potentially be used as a live colonization-inhibition strain for controlling Salmonella Enteritidis infections in broilers.     

Induction of a homologous and heterologous invasion–inhibition effect after administration of Salmonella strains to newly hatched chicks

Administration of live Salmonella strains to day-old chicks provides protection against infection within hours by intestinal colonisation–inhibition. However, the extent to which the oral application of live Salmonella wild-type or vaccine strains may induce an early invasion–inhibition effect is unknown. Potentially protective pre-treatment strains of Salmonella Enteritidis and Infantis were examined for their ability (i) to colonise the caeca, to invade the liver, to induce an influx of granulocytes in caecal mucosa and, (ii) for their capacity to inhibit the systemic invasion of homologous and heterologous Salmonella challenge organisms. The highly invasive strain Salmonella Enteritidis induced a strong influx of heterophils in the caecal mucosa followed by a complete invasion–inhibition of both homologous and heterologous Salmonella challenge organisms administered 24 h later. Pre-treatment with a less invasive Salmonella Infantis resulted in a lower influx of granulocytes in the caecal tissue followed by a complete invasion–inhibition of the homologous serovar Infantis but only an incomplete invasion–inhibition of heterologous serovars. This invasion–inhibition effect has not been described previously in chickens and should be considered in the development of novel live Salmonella vaccines to prevent an early invasion of extra-intestinal organs by Salmonella challenge organisms in young chicks.     

Salmonella Typhimurium LPS mutations for use in vaccines allowing differentiation of infected and vaccinated pigs

Contaminated pork is a major source of human salmonellosis and the serovar most frequently isolated from pigs is Salmonella Typhimurium. Vaccination could contribute greatly to controlling Salmonella infections in pigs. However, pigs vaccinated with the current vaccines cannot be discriminated from infected pigs with the LPS-based serological tests used in European Salmonella serosurveillance programmes. We therefore examined which LPS encoding genes of Salmonella Typhimurium can be deleted to allow differentiation of infected and vaccinated pigs (DIVA), without affecting the vaccine strain's protective capacity. For this purpose, deletion mutants in Salmonella strain 112910a, used as vaccine strain, were constructed in the LPS encoding genes: ΔrfbA, ΔrfaL, ΔrfaJ, ΔrfaI, ΔrfaG and ΔrfaF. Primary inoculation of BALB/c mice with the parent strain, ΔrfaL, ΔrfbA or ΔrfaJ strain but not the ΔrfaG, ΔrfaF or ΔrfaI strain protected significantly against subsequent infection with the virulent Salmonella Typhimurium strain NCTC12023. Immunization of piglets with the ΔrfaJ or ΔrfaL mutants resulted in the induction of a serological response lacking detectable antibodies against LPS. This allowed a clear differentiation between sera from pigs immunized with the ΔrfaJ or ΔrfaL strains and sera from pigs infected with their isogenic wild type strain. In conclusion, applying deletions in the rfaJ or the rfaL gene in Salmonella Typhimurium strain 112910a allows differentiation of infected and vaccinated pigs in an LPS based ELISA without reducing the strain's protective capacities in mice.     

Salmonella Enteritidis with double deletion in phoP fliC and a competitive exclusion culture elicit substantial additive protective effects against Salmonella exposure in newly hatched chicks

A live Salmonella Enteritidis vaccine (SE 147N ΔphoP fliC), able to express both a homologous intestinal colonisation-inhibition effect and a systemic invasion-inhibition effect, was tested for its potential to generate a postulated additive protective effect in case of combined application with a competitive exclusion (CE) culture against Salmonella exposure in very young chicks. Both, SE 147N ΔphoP fliC and the CE culture alone were highly protective against systemic and intestinal colonisation of the challenge strain in case of moderate Salmonella exposure, consequently, additive protective effects in combined use could not be detected. However, in case of high Salmonella Enteritidis challenge with 10⁶ cfu/bird at day 3 of life the combination of the ΔphoP fliC vaccine and the CE culture resulted in a protective effect much more pronounced than either of the single preparations and most substantial compared to untreated control birds. The term additive protective effects reflects the recognition that exclusion effects by gut flora cultures and inhibition effects by Salmonella vaccines are caused by different mechanisms.     

Deletion of a prophage-like element causes attenuation of Salmonella enterica serovar Enteritidis and promotes protective immunity

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a wide host range serovar belonging to the S. enterica genus. Worldwide, it is one of the most frequent causes of food borne disease. Similar to S. Typhimurium, some virulence genes of S. Enteritidis are located in pathogenicity islands and prophages. In this study we have generated a mutant strain of S. Enteritidis lacking a prophage-like element, denominated ?SE12. The resulting mutant strain was attenuated and promoted protective immunity in infected mice. Although S. Enteritidis strains lacking the complete prophage ?SE12 remained capable of surviving inside phagocytic cells, they showed a significantly reduced capacity to colonize internal organs and failed to cause lethal disease in mice. Consistent with these data, infection with S. Enteritidis strains lacking prophage ?SE12 promoted the production of anti-Salmonella IgG antibodies and led to protection against a challenge with virulent strains of S. Enteritidis. These results suggest that strains lacking this prophage can induce a protective immunity in mice and be considered as potential attenuated vaccines against S. Enteritidis.     

Comparative evaluation of Salmonella Enteritidis ghost vaccines with a commercial vaccine for protection against internal egg contamination with Salmonella

The study was conducted for the comparative evaluation of the vaccine potential of Salmonella Enteritidis (S. Enteritidis, SE) ghost, SE ghost carrying Escherichia coli heat labile enterotoxin B subunit (LTB) protein, and a commercial vaccine. Group A chickens were used as a non-vaccinated control, group B chickens were immunized with the ghost carrying LTB protein, group C chickens were immunized with the ghost and, group D chickens were immunized with a commercial vaccine. Group D chickens showed the swelling at the injection site, while no adverse reactions were observed at injection sites of the group B and C chickens. Chickens from the immunized groups B, C, and D demonstrated significant increases in plasma IgG, intestinal secretory IgA levels, and antigen-specific lymphocyte proliferative responses. After challenge with a virulent SE strain via intravenous route, groups B, C, and D showed significantly higher egg production and lower internal egg contamination and lower recovery of the challenge strain from internal organs compared to non-immunized-challenged control group A. In conclusion, these data indicate that immunization of chickens with the ghost and ghost carrying LTB is safe, without causing any adverse reaction, and is effective as the commercial vaccine in terms of reduction in internal egg contamination and internal organ colonization of Salmonella.     

Salmonella enterica serovar Typhimurium ruvB mutant can confer protection against salmonellosis in mice

Salmonella enterica is an important pathogen that causes a variety of infectious diseases in animals and humans. Live attenuated vaccines generally confer better protection than killed or subunit vaccines; however, the former are limited by their inherent toxicity. We evaluated the potential of a novel candidate Salmonella vaccine strain that lacks the ruvB gene. The ruvB gene encodes a Holliday junction helicase that is required to resolve junctions that arise during the repair of non-arresting lesions after DNA replication. The deletion of this gene in Salmonella significantly impaired cell survival and proliferation within epithelial cells and macrophages. The defective virulence in ruvB mutant may be partially due to decreased expression of ssaG, a Salmonella pathogenicity island-2 gene, and increased sensitivity to hydrogen peroxide in the lack of ruvB gene. The virulence of the ruvB-deleted mutant was also greatly attenuated in BALB/c mice. The ruvB mutant conferred strong and durable immune-based protection against a challenge with a lethal dose of a virulent strain of Salmonella Typhimurium. Moreover, protective immunity was induced by a single dose of the vaccine, and the efficacy of protection was maintained for at least 6 months. These results suggest the use of the S. Typhimurium ruvB mutant as a novel vaccine.     

A colonisation-inhibition culture consisting of Salmonella Enteritidis and Typhimurium ΔhilAssrAfliG strains protects against infection by strains of both serotypes in broilers

Consumption of contaminated poultry meat is still an important cause of Salmonella infections in humans and there is a need for control methods that protect broilers from day-of-hatch until slaughter age against infection with Salmonella. Colonisation-inhibition, a concept in which a live Salmonella strain is orally administered to day-old chickens and protects against subsequent challenge, can potentially be used as control method. In this study, the efficacy of a Salmonella Typhimurium ΔhilAssrAfliG strain as a colonisation-inhibition strain for protection of broilers against Salmonella Typhimurium was evaluated. Administration of a Salmonella Typhimurium ΔhilAssrAfliG strain to day-old broiler chickens decreased faecal shedding and strongly reduced caecal and internal organ colonisation of a Salmonella Typhimurium challenge strain administered one day later using a seeder bird model. In addition, it was verified whether a colonisation-inhibition culture could be developed that protects against both Salmonella Enteritidis and Typhimurium. Therefore, the Salmonella Typhimurium ΔhilAssrAfliG strain was orally administered simultaneously with a Salmonella Enteritidis ΔhilAssrAfliG strain to day-old broiler chickens, which resulted in a decreased caecal and internal organ colonisation for both a Salmonella Enteritidis and a Salmonella Typhimurium challenge strain short after hatching, using a seeder bird model. The combined culture was not protective against Salmonella Paratyphi B varietas Java challenge, indicating serotype-specific protection mechanisms. The data suggest that colonisation-inhibition can potentially be used as a versatile control method to protect poultry against several Salmonella serotypes.     

Construction of a recombinant-attenuated Salmonella Enteritidis strain secreting Escherichia coli heat-labile enterotoxin B subunit protein and its immunogenicity and protection efficacy against salmonellosis in chickens

A live attenuated Salmonella Enteritidis (SE) strain secreting Escherichia coli heat-labile enterotoxin B subunit (LTB) protein was constructed as a new vaccine candidate. The comparative effect of this vaccine candidate was evaluated with a previously reported SE vaccine, JOL919. An asd+, p15A ori plasmid containing eltB-encoding LTB was introduced into a ΔlonΔcpxRΔasd SE strain, and designated as JOL1364. In a single immunization experiment, group A chickens were orally inoculated with phosphate-buffered saline as a control, group B chickens were orally immunized with JOL919, and group C chickens were orally immunized with JOL1364. The immunized groups B and C showed significantly higher systemic, mucosal and cellular immune responses as compared to those of the control group. In addition, the immunized group C showed significantly higher mucosal and cellular immune responses as compared to those of the immunized group B at the 1_st week post-immunization. In the examination of protection efficacy, the immunized groups B and C showed lower gross lesion scores in the liver and spleen, and lower bacterial counts of SE challenge strain in the liver, spleen, and caeca as compared to those of the control group. The number of SE-positive birds was significantly lower in the immunized group C as compared to that of the control group at the 14th day post-challenge. In addition, the number of birds carrying the challenge strain in the caeca was significantly lower in the immunized group C than those in the immunized group B and control group at the 7th and 14th day post-challenge. These results indicate that immunization with the JOL1364 vaccine candidate can induce higher mucosal and cellular immune responses than those of the JOL919 for efficient protection against salmonellosis.     

An attenuated Salmonella enterica serovar Typhimurium strain lacking the ZnuABC transporter induces protection in a mouse intestinal model of Salmonella infection

Salmonella enterica serovar Typhimurium has long been recognised as a zoonotic pathogen of economic significance in animals and humans. Attempts to protect humans and livestock may be based on immunization with vaccines aimed to induce a protective response. We recently demonstrated that the oral administration of a Salmonella enterica serovar Typhimurium strain unable to synthesize the zinc transporter ZnuABC is able to protect mice against systemic salmonellosis induced by a virulent homologous challenge. This finding suggested that this mutant strain could represent an interesting candidate vaccine for mucosal delivery. In this study, the protective effect of this Salmonella strain was tested in a streptomycin-pretreated mouse model of salmonellosis that is distinguished by the capability of evoking typhlitis and colitis. The here reported results demonstrate that mice immunized with Salmonella enterica serovar Typhimurium (S. Typhimurium) SA186 survive to the intestinal challenge and, compared to control mice, show a reduced number of virulent bacteria in the gut, with milder signs of inflammation. This study demonstrates that the oral administration a of S. Typhimurium strain lacking ZnuABC is able to elicit an effective immune response which protects mice against intestinal S. Typhimurium infection. These results, collectively, suggest that the streptomycin-pretreated mouse model of S. typhimurium infection can represent a valuable tool to screen S. typhimurium attenuated mutant strains and potentially help to assess their protective efficacy as potential live vaccines.     

Immunisation of chickens with live Salmonella vaccines – Role of booster vaccination

It is accepted that booster vaccinations of chickens with live Salmonella vaccines are essential part of vaccinations schemes to induce an effective adaptive immune response. As manufacturer of registered live Salmonella vaccines recommend different times of booster the question raises whether the duration between the first and second immunisation might influence the protective effect against Salmonella exposure. Chickens were immunised with a live Salmonella Enteritidis vaccine on day 1 of age followed by a booster vaccination at different intervals (day 28, 35 or 42 of age) to study the effects on the colonisation and invasion of the Salmonella vaccine strain, the humoral immune response and the efficacy against infection with Salmonella Enteritidis on day 56 of age. Immunisation of all groups resulted in a very effective adaptive immune response and a high degree of protection against severe Salmonella exposure, however, the time of booster had only an unverifiable influence on either the colonisation of the vaccine strain, the development of the humoral immune response or the colonisation of the Salmonella challenge strain. Therefore, the first oral immunisation of the chicks on day 1 of age seems to be of special importance and prerequisite for the development of the effective immune response. A booster immunisation should be carried out, however, the time of booster may vary between week 3 and week 7 of age of the chickens without adversely impact on the efficacy of the adaptive immune response or the protective effects.     

Protective immunity conferred by a DNA adenine methylase deficient Salmonella enterica serovar Typhimurium vaccine when delivered in-water to sheep challenged with Salmonella enterica serovar Typhimurium

Stimulation of acquired immunity to Salmonella in livestock is not feasible in neonates (which can be infected within 24 h of birth) and is challenging in feedlots, which typically source animals from diverse locations and vendors. Induction of innate immune mechanisms through mass vaccination of animals upon arrival to feedlots is an alternative approach. Transport, environmental conditions, changes in social grouping, and further handling during feedlot assembly are significant stressors. These factors, as well as concurrent exposure to a diversity of pathogens, contribute to the risk of disease. We have shown that oral immunization of calves with a modified live Salmonella enterica serovar Typhimurium vaccine strain, which lacks the DNA adenine methylase gene (S. Typhimurium dam), attenuates the severity of clinical disease, reduces fecal shedding, and promotes clearance of salmonellae following virulent homologous and heterologous challenge. This study examines the safety and efficacy of a S. Typhimurium dam vaccine in sheep via oral delivery in drinking water (ad libitum), as a means to effectively vaccinate large groups of animals. Adult merino sheep were vaccinated in drinking water −28 days, −7 days and 24 h pre and 24 h post-virulent Salmonella Typhimurium challenge which was administered via the oral route. Significant attenuation of clinical disease (temperature, appetite, and attitude) and reduction in mortality and virulent Salmonella Typhimurium fecal shedding and tissue colonization was observed in animals that received the vaccine 28 and 7 days pre-challenge. Further, vaccination did not pose a risk to stock previously infected with virulent salmonellae as mortalities and clinical disease in sheep vaccinated prior to or following virulent challenge did not differ significantly from the non-vaccinated controls. The capacity of S. Typhimurium dam vaccines delivered in drinking water to protect livestock from virulent Salmonella challenge offers an effective, economical, stressor free Salmonella prophylaxis for intensive livestock production systems.     

Vaccination of attenuated EIS-producing Salmonella induces protective immunity against enterohemorrhagic Escherichia coli in mice

There is an urgent need for vaccine against enterohemorrhagic Escherichia coli (EHEC), which causes a wide range of life-threatening diseases in human and animals. E. coli secreted protein A (EspA), intimin and shiga toxin (Stx) are important pathogenic factors and protective antigens of EHEC. In our previous study, we found that recombinant trivalent protein EIS, which is composed of EspA (E), the 300 amino acids of the carboxyl terminus of intimin (I) and the B subunit of Stx2 (S), was able to efficiently elicit protective immunity against EHEC. The application of live attenuated Salmonella as a carrier for vaccine against mucosal pathogens provided unparalleled merits. Therefore, in this study we constructed live attenuated EIS-producing Salmonella vaccine and tested it as vaccine in mice model. We found that the vaccination of EIS-producing recombinant Salmonella was able to induce significant increases of EspA, intimin and Stx2 specific IgG in serum and secretory IgA in feces. Antigen specific T cell proliferation was also observed in the mice immunized with recombinant EIS-producing Salmonella. In addition, this immunity was able to protect mice from a challenge of a lethal dose of EHEC, even after a period of 70 days. Moreover, the EIS-producing Salmonella induced immunity can be boosted by a single subcutaneous injection of purified EIS protein, even after an interval of 70 days. This EIS-producing Salmonella vaccine provides an alternative approach for the prevention of EHEC infection.     

Therapeutic efficacy of oral immunization with attenuated Salmonella typhimurium expressing Helicobacter pylori CagA, VacA and UreB fusion proteins in mice model

Therapeutic vaccination is a desirable alternative for controlling Helicobacter pylori (H. pylori) infection. In the present study, attenuated Salmonella vector vaccines were constructed that expressed fusion proteins complexed with H. pylori CagA, VacA and UreB in different arrangements, and their therapeutic efficacy was evaluated in H. pylori-infected mice. Oral therapeutic immunization with attenuated Salmonella, which expressed the fused protein CVU, significantly decreased H. pylori colonization in the stomach; protection was related to specific CD4⁺ T cell Th1 type responses and serum IgG and mucosal sIgA antibody responses. These findings suggested that therapeutic efficacy was related to the arrangement of the fusion protein. It is possible that arrangement decides the expression of recombinant antigen in mice, and the latter results in different therapeutic efficacy. The attenuated Salmonella vector vaccine, which expressed the fused protein arrangement CVU, is superior to others, and could be a candidate vaccine against H. pylori.     

rOmpF and OMVs as efficient subunit vaccines against Salmonella enterica serovar Enteritidis infections in poultry farms

Salmonella enterica serovar Enteritidis remains the most prevalent serotype causing human salmonellosis through the consumption of contaminated foods, especially poultry products. The development of a subunit vaccine against S. Enteritidis can not only protect chickens against Salmonella infection in the poultry industry but also cut the transmission sources. In this study, both the expressed recombinant outer membrane protein F (rOmpF) and extracted outer membrane vesicles (OMVs) were developed as subunit vaccines against S. Enteritidis challenge in chickens. Immunization with the subunit vaccine could induce not only antibody production but also strong cell-mediated immune response. Both rOmpF plus QuilA adjuvant and OMVs alone had highly protective efficacy against S. Enteritidis challenge and rapidly decreased the colonization of bacteria in chicken. These findings revealed the potential application of rOmpF and OMVs as subunit vaccines in the poultry industry.     

Salmonella Gallinarum field isolates from laying hens are related to the vaccine strain SG9R

Salmonella enterica subspecies enterica serotype Gallinarum can cause severe systemic disease in chickens and a live Salmonella Gallinarum 9R vaccine (SG9R) has been used widely to control disease. Using whole-genome sequencing we found point mutations in the pyruvate dehydrogenase (aceE) and/or lipopolysaccharide 1,2-glucosyltransferase (rfaJ) genes that likely explain the attenuation of the SG9R vaccine strain. Molecular typing using Pulsed Field Gel Electrophoresis and Multiple-Locus Variable number of tandem repeat Analysis showed that strains isolated from different layer flocks in multiple countries and the SG9R vaccine strain were similar. The genome of one Salmonella Gallinarum field strain, isolated from a flock with a mortality peak and selected on the basis of identical PFGE and MLVA patterns with SG9R, was sequenced. We found 9 non-silent single-nucleotide differences distinguishing the field strain from the SG9R vaccine strain. Our data show that a Salmonella Gallinarum field strain isolated from laying hens is almost identical to the SG9R vaccine. Mutations in the aceE and rfaJ genes could explain the reversion to a more virulent phenotype. Our results highlight the importance of using well defined gene deletion mutants as vaccines.     

Characterization of rationally attenuated Francisella tularensis vaccine strains that harbor deletions in the guaA and guaB genes

Francisella tularensis, the etiologic agent of tularemia, can cause severe and fatal infection after inhalation of as few as 10–100 CFU. F. tularensis is a potential bioterrorism agent and, therefore, a priority for countermeasure development. Vaccination with the live vaccine strain (LVS), developed from a Type B strain, confers partial protection against aerosal exposure to the more virulent Type A strains and provides proof of principle that a live attenuated vaccine strain may be efficacious. However LVS suffers from several notable drawbacks that have prevented its licensure and widespread use. To address the specific deficiencies that render LVS a sub-optimal tularemia vaccine, we engineered F. tularensis LVS strains with targeted deletions in the guaA or guaB genes that encode critical enzymes in the guanine nucleotide biosynthetic pathway. F. tularensis LVSΔguaA and LVSΔguaB mutants were guanine auxotrophs and were highly attenuated in a mouse model of infection. While the mutants failed to replicate in macrophages, a robust proinflammatory cytokine response, equivalent to that of the parental LVS, was elicited. Mice vaccinated with a single dose of the F. tularensis LVSΔguaA or LVSΔguaB mutant were fully protected against subsequent lethal challenge with the LVS parental strain. These findings suggest the specific deletion of these target genes could generate a safe and efficacious live attenuated vaccine.     

Regulated expression of virulence gene mviN provides protective immunity and colonization control of Salmonella in poultry

Current live attenuated vaccines for control of Salmonella in poultry persist in the ceca and may persist in the environment. In this paper we report the construction and characterization of the vaccine efficacy of a Salmonella mutant strain with inducible mviN expression and rapid clearance from the host. The mutant was effective in oral immunization of the broiler chicken host against a virulent Salmonella oral challenge strain, having a mean 7 × 10⁶ CFU/g in the ceca of unvaccinated controls compared to a mean 2 × 10³ CFU/g in the ceca of vaccinated chickens at 4 weeks post-challenge (6 weeks of age). The mutant strain also demonstrated immunogenicity, reduced organ colonization, and rapid clearance in broiler chickens within 3 weeks of inoculation.