Salmonella Typhimurium lacking the Znuabc transporter is attenuated and immunogenic in pigs

Meat contamination by Salmonella spp. is emerging as a major cause of human enteric infections in industrialized countries. The attempts to reduce human cases of salmonellosis encompass pre- and post-harvest interventions. In this context, vaccination of pigs may represent an effective instrument in eliminating/reducing Salmonella burden through the food chain. We have previously demonstrated that Salmonella Typhimurium lacking the ZnuABC transporter (S. Typhimurium ΔznuABC) is a promising candidate live vaccine in different mouse models of Salmonella Typhimurium infection. In this study, we confirmed in pigs the attenuation of S. Typhimurium ΔznuABC. Moreover, we evaluated the safety and immunogenicity of S. Typhimurium ΔznuABC administered to pigs by the oral route. We monitored clinical conditions of animals and we conducted a microbiological culture and a quantification of the humoral and cellular immune response, respectively, on fecal and blood samples of pigs. After vaccination with attenuated S. Typhimurium ΔznuABC, pigs showed a modest degree of hyperthermia. In addition, fecal shedding of S. Typhimurium ΔznuABC could not be detected 28 days after the inoculum. Furthermore, vaccination with S. Typhimurium ΔznuABC elicited a distinct production of anti-Salmonella antibodies and IFN-γ. Taken together, these results suggest that S. Typhimurium ΔznuABC is attenuated and immunogenic in pigs. Although the vaccine dosages do not guarantee complete safety there is ample margin to set up better conditions of use, suggesting that S. Typhimurium ΔznuABC could be a promising attenuated strain to be used as live mucosal vaccine for oral delivery.     

Salmonella Enteritidis with double deletion in phoPfliC--a potential live Salmonella vaccine candidate with novel characteristics for use in chickens

Salmonella Enteritidis mutants with deletions in phoP, fliC or phoP fliC were tested for their virulence and their ability to induce parameters of the innate and adaptive immunity in addition to their potential for serological differentiation between vaccinated, non-vaccinated and infected chickens. The double phoP fliC deletion mutant was sufficiently attenuated but not diminished in its capability to inhibit the caecal colonisation and systemic invasion of homologous Salmonella Enteritidis shortly after administration of the vaccine strain to very young chicks. Immunisation with the attenuated ΔphoP fliC mutant resulted in protective effects which were only slightly and insignificantly lower than after “immunisation” with a Salmonella wild-type strain, indicating the capability to induce an intense adaptive immune response and protection against Salmonella exposure in older chickens. The deletion in fliC enabled the effective the differentiation between immunised and infected chickens using a commercially available ELISA kit. The double phoP fliC deletion mutant of Salmonella Enteritidis might be a potential and promising live Salmonella vaccine candidate with novel characteristics for use in poultry.     

Parenteral administration of attenuated Salmonella Typhimurium ΔznuABC is protective against salmonellosis in piglets

A major cause of salmonellosis in humans is the contamination of pork products. Infection in pigs can be controlled using bio-security programs, but they are not sufficient in countries where a high level of infection is recorded. In this context, the use of vaccines can represent a valid supplementary method of control. Recently, we have demonstrated that an attenuated strain of Salmonella enterica serovar Typhimurium (Salmonella Typhimurium ΔznuABC) is protective against systemic and enteric salmonellosis in mouse and pig infection models, candidating this strain as an oral attenuated vaccine. In this study, we compared the efficacy of this attenuated Salmonella Typhimurium strain when administered orally or parenterally. Furthermore, in order to reproduce a pseudo-natural infection model, vaccinated pigs were allocated in the same pen with animals shedding virulent Salmonella Typhimurium. Animals were monitored weekly after vaccination and contact with infected piglets. Diarrhea and ataxia were recorded and Salmonella shedding was tested individually through bacterial culture. After four weeks of cohousing, piglets were euthanized, after which lymph nodes reactivity and gross lesions of the gut sections were scored at necropsy. Organs were submitted to microbiological and histological analyses.     

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.     

Attenuated Salmonella enterica serovar Typhimurium lacking the ZnuABC transporter: An efficacious orally-administered mucosal vaccine against salmonellosis in pigs

We have recently demonstrated that an attenuated strain of Salmonella enterica serovar Typhimurium unable to synthesize the zinc transporter ZnuABC (S. Typhimurium ΔznuABC), is able to protect mice against systemic and enteric salmonellosis and is safe in pigs. Here, we have tested the protective effects of S. Typhimurium ΔznuABC in pigs. Resistance to challenge with the fully virulent strain S. Typhimurium ATCC 14028 was assessed in animals vaccinated with S. Typhimurium ΔznuABC (two dosages tested), in controls vaccinated with a formalin-inactivated virulent strain and in unvaccinated controls. Clinical signs of salmonellosis, faecal shedding and bacterial colonization of organs were used to assess vaccine-induced protection. After the challenge, pigs vaccinated with the attenuated S. Typhimurium ΔznuABC strain did not display clinical signs of salmonellosis (fever or diarrhoea). The vaccine also reduced intestinal tract colonization and faecal shedding of the fully virulent Salmonella strain, as compared to control groups. S. Typhimurium ΔznuABC represents a promising candidate vaccine against salmonellosis in pigs.     

Regulated delayed synthesis of lipopolysaccharide and enterobacterial common antigen of Salmonella Typhimurium enhances immunogenicity and cross-protective efficacy against heterologous Salmonella challenge

Lipopolysaccharide (LPS) O-antigen and enterobacterial common antigen (ECA) are two major polysaccharide structures on the surface of Salmonella enterica serovar Typhimurium. Previous studies have demonstrated that regulated truncation of LPS enhances the cross-reaction against conserved outer membrane proteins (OMPs) from enteric bacteria. We speculate that the regulation of both O-antigen and ECA may enhance the induction of immune responses against conserved OMPs from enteric bacteria. In this work we targeted rfbB and rffG genes which encode dTDP-glucose 4,6-dehydratases and share the same function in regulating O-antigen and ECA synthesis. We constructed a mutant, S496 (ΔrfbB6 ΔrffG7 ΔpagL73::TT araC P_BAD rfbB-3), in which rfbB gene expression was dependent on exogenously supplied arabinose during in vitro growth and achieved the simultaneous tight regulation of both LPS and ECA synthesis, as demonstrated by the LPS profile and Western blotting using antisera against LPS and ECA. When administered orally, S. Typhimurium S496 was completely attenuated for virulence but still retained the capacity to colonize and disseminate in mice. In addition, we found that oral immunization with S496 resulted in increased immune responses against OMPs from enteric bacteria and enhanced survival compared with immunization with S492 possessing ΔrfbB6 ΔrffG8 mutations when challenged with lethal doses of Salmonella Choleraesuis or Salmonella Enteritidis. These results indicate that S. Typhimurium arabinose-regulated rfbB strain S496 is a good vaccine candidate, conferring cross-protection against lethal challenge with heterologous Salmonella.     

A DIVA vaccine for cross-protection against Salmonella

Swine are often asymptomatic carriers of Salmonella spp., a leading cause of human bacterial foodborne disease. Vaccination against Salmonella is effective for protecting animal health and enhancing food safety. However, with >2500 Salmonella serovars, current vaccines for swine offer limited cross-protection against heterologous serovars. Also, existing vaccines can interfere with surveillance programs that monitor the Salmonella status of swine herds. To overcome Salmonella vaccine limitations, we rationally designed and constructed an attenuated Salmonella enterica serovar Typhimurium vaccine (BBS 866) by deleting multiple small regulatory RNA (sRNA) genes (omrA, omrB, rybB, micA, and invR) in combination with an rfaH mutation. We vaccinated swine intranasally at 3-weeks of age with PBS (mock-vaccinated), BBS 866 or BBS 202 (S. Typhimurium rfaH, Bearson et al., Front Vet Sci 2014;1:9.) and challenged at 7-weeks of age with virulent S. Choleraesuis, a swine pathogen. Vaccination with BBS 866 enhanced protection against S. Choleraesuis by significantly limiting the duration of fever, weight loss, the levels of circulating INFγ, and the total number of swine with S. Choleraesuis septicemia. Vaccination with either BBS 866 or BBS 202 significantly reduced S. Choleraesuis colonization of both systemic (spleen and liver) and gastrointestinal (Peyer's Patch, Ileocecal lymph nodes, and cecum) tissues. Similar to our earlier report for BBS 202, the BBS 866 vaccine strain can be used in swine without compromising the differentiation of infected from vaccinated animals (DIVA). Therefore, the attenuated S. Typhimurium BBS 866 strain, containing mutations in rfaH and multiple sRNAs, addresses the limitations of current Salmonella vaccines by providing cross-protection against Salmonella serovars in swine without interfering with established monitoring programs for Salmonella surveillance.     

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.     

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.     

Reversible synthesis of colanic acid and O-antigen polysaccharides in Salmonella Typhimurium enhances induction of cross-immune responses and provides protection against heterologous Salmonella challenge

Colanic Acid (CA) and lipopolysaccharide (LPS) are two major mannose-containing extracellular polysaccharides of Salmonella. Their presence on the bacterial surface can mask conserved protective outer membrane proteins (OMPs) from the host immune system. The mannose moiety in these molecules is derived from GDP-mannose, which is synthesized in several steps. The first two steps require the action of phosphomannose isomerase, encoded by pmi (manA), followed by phosphomannomutase, encoded by manB. There are two copies of manB present in the Salmonella chromosome, one located in the cps gene cluster (cpsG) responsible for CA synthesis, and the other in the rfb gene cluster (rfbK) involved in LPS O-antigen synthesis. In this study, it was demonstrated that the products of cpsG and rfbK are isozymes. To evaluate the impact of these genes on O-antigen synthesis, virulence and immunogenicity, single mutations (Δpmi, ΔrfbK or ΔcpsG) and a double mutation (ΔrfbK ΔcpsG) were introduced into both wild-type Salmonella enterica and an attenuated Δcya Δcrp vaccine strain. The Δpmi, ΔrfbK and ΔcpsG ΔrfbK mutants were defective in LPS synthesis and attenuated for virulence. In orally inoculated mice, strain S122 (Δcrp Δcya ΔcpsG ΔrfbK) and its parent S738 (Δcrp Δcya) were both avirulent and colonized internal tissues. Strain S122 elicited higher levels of anti-S. Typhimurium OMP serum IgG than its parent strain. Mice immunized with S122 were completely protected against challenge with wild-type virulent S. Typhimurium and partially protected against challenge with either wild-type virulent S. Choleraesuis or S. Enteritidis. These data indicate that deletions in rfbK and cpsG are useful mutations for inclusion in future attenuated Salmonella vaccine strains to induce cross-protective immunity.     

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.     

Characterisation of a live Salmonella vaccine stably expressing the Mycobacterium tuberculosis Ag85B–ESAT6 fusion protein

A recombinant Salmonella enterica serovar Typhimurium (S. Typhimurium) vaccine strain was constructed that stably expressed the Mycobacterium tuberculosis fusion antigen Ag85B–ESAT6 from the chromosome. Live oral vaccination of mice with the Salmonella/Ag85B–ESAT6 strain generated a potent anti-Ag85B–ESAT6 T_H1 response with high antibody titres with a IgG2a-bias and significant IFN-γ production lasting over a 120-day period. When mice primed with the Salmonella/Ag85B–ESAT6 vaccine were mucosally boosted with the Ag85B–ESAT6 antigen and adjuvant the IFN-γ responses increased markedly. To determine the protective efficacy of this vaccine strain, guinea pigs were immunised and followed for a 30-week period after aerosol challenge with M. tuberculosis. The heterologous prime-boost strategy of live Salmonella vaccine followed by a systemic boost of antigen and adjuvant reduced the levels of M. tuberculosis bacteria in the lungs and spleen to the same extent as BCG. Additionally, this vaccination regimen was observed to be statistically equivalent in terms of protection to immunisation with BCG. Thus, live oral priming with the recombinant Salmonella/Ag85B–ESAT6 and boosting with Ag85B–ESAT6 plus the adjuvant LTK63 represents an effective mucosal vaccination regimen.     

Genetically modified Bifidobacterium displaying Salmonella-antigen protects mice from lethal challenge of Salmonella Typhimurium in a murine typhoid fever model

We developed a novel vaccine platform utilizing Bifidobacterium as an antigen delivery vehicle for mucosal immunization. Genetically modified Bifidobacterium longum displaying Salmonella-flagellin on the cell surface was constructed for the oral typhoid vaccine. The efficiency of this vaccine was evaluated in a murine model of typhoid fever. We then orally administered 2.5 × 10⁷ CFU of the recombinant Bifidobacterium longum (vaccine) or parental Bifidobacterium longum, or PBS to BALB/C mice every other day for 2 weeks. After the administration, a total of 42 mice (14 mice in each group) were challenged with Salmonella Typhimurium (1.0 × 10⁷ CFU/mouse). While 12 mice in the PBS group, and 9 in the parental Bifidobacterium longum group died (median survival: 14 and 25 days), only two in the vaccine group died. These data support that our genetically modified Bifidobacterium antigen delivery system offers a promising vaccine platform for inducing efficient mucosal immunity.     

Delivery of woodchuck hepatitis virus-like particle presented influenza M2e by recombinant attenuated Salmonella displaying a delayed lysis phenotype

The use of live recombinant attenuated Salmonella vaccines (RASV) is a promising approach for controlling infections by multiple pathogens. The highly conserved extracellular domain of the influenza M2 protein (M2e) has been shown to provide broad spectrum protection against multiple influenza subtypes sharing similar M2e sequences. An M2e epitope common to a number of avian influenza subtypes was inserted into the core antigen of woodchuck hepatitis virus and expressed in two different recombinant attenuated Salmonella Typhimurium strains. One strain was attenuated via deletion of the cya and crp genes. The second strain was engineered to exhibit a programmed delayed lysis phenotype. Both strains were able to produce both monomeric fusion proteins and fully assembled core particles. Mice orally immunized with the strain exhibiting delayed lysis induced significantly greater antibody titers than the Δcya Δcrp strain and provided moderate protection against weight loss to a low level challenge with the influenza strain A/WSN/33 modified to express the M2e sequence common to avian viruses. Further studies indicated that the Salmonella expressed core antigen induced comparable antibody levels to the purified core antigen injected with an alum adjuvant and that both are able to reduce viral replication in the lungs. To our knowledge this is the first report demonstrating Salmonella-mediated delivery of influenza virus M2e protein in a mammalian host to induce a protective immune response against viral challenge.     

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.     

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.     

Oral delivery of a DNA vaccine against tuberculosis using operator-repressor titration in a Salmonella enterica vector

Attenuated Salmonella enterica offers a vaccine delivery route that has the benefits of enhanced immunogenicity and oral delivery. The majority of immunization studies have been conducted to deliver recombinant proteins, expressed from a gene that is either chromosomally integrated or carried on a low- or medium-copy number plasmid. There are, however, an increasing number of reports demonstrating the delivery of DNA vaccines, but the high-copy number plasmids that are preferentially used for this application are unstable in Salmonella. Here, we use the Operator-Repressor Titration (ORT) plasmid maintenance system in Salmonella enterica serovar Typhimurium to deliver a high-copy number plasmid expressing the Mycobacterium tuberculosis gene mpt64 to mice. MPT64 expression was detected in phagocytes using immunofluorescence microscopy following Salmonella-mediated delivery of the DNA vaccine. The indicative CD8+ responses measured by antigen-specific IFN-γ were higher from the live bacterial vector than from injected plasmid DNA, and a reduction in the pulmonary bacterial load was seen following an aerogenic challenge. This illustrates the potential of live attenuated Salmonella as oral tuberculosis vaccine vectors.     

Oral delivery of DNA vaccine encoding VP28 against white spot syndrome virus in crayfish by attenuated Salmonella typhimurium

Protective immune responses in shrimp induced by DNA vaccines against white spot syndrome virus (WSSV) with intramuscular injection have been reported in recent reports. In this study, we investigated the utilities of attenuated Salmonella enterica serovar Typhimurium (Salmonella typhimurium) as a bactofection vehicle for the oral delivery of a DNA vaccine plasmid to crayfish (Cambarus clarkii). The DNA vaccine plasmid pcDNA3.1-VP28, encoding viral envelope protein VP28, was transformed to an attenuated S. typhimurium strain SV4089 and the resulting recombinant bacteria named SV/pcDNA3.1-VP28 were used to orally immunize crayfish with coated feed. Successful delivery of the DNA vaccine plasmid was shown by the isolation of recombinant bacteria SV/pcDNA3.1-VP28 from the vaccinated crayfish. The distribution analysis of plasmid pcDNA3.1-VP28 in different tissues revealed the effective release of DNA vaccine plasmid into crayfish. RT-PCR and immunoflurescence results confirmed the expression of protein VP28 in the vaccinated crayfish. Challenge experiments with WSSV at 7, 15, 25 days post-vaccination demonstrated significant protection in immunized crayfish with relative survival rate 83.3%, 66.7% and 56.7%, respectively. Studies on stability and safety of SV/pcDNA3.1-VP28 showed the recombinant bacteria could exist in crayfish at least 7 days but not more than 10 days and without any observable harm to the host. Our study here demonstrates, for the first time, the ability of attenuated Salmonella as a live vector to orally deliver a DNA vaccine against WSSV into the arthropod crayfish and provides a new way to design more practical strategies for the control of WSSV and other invertebrate pathogens.     

Positive role for rApxIVN in the immune protection of pigs against infection by Actinobacillus pleuropneumoniae

The role of in vivo-induced ApxIV toxin of Actinobacillus pleuropneumoniae in protective immunity was evaluated in pigs by administering it alone or added to a multicomponent recombinant subunit vaccine composed of recombinant ApxI, ApxII, ApxIII toxin, and 42-kDa outer membrane protein (OMP). The pigs were immunized with vaccine I (rApxIVN), vaccine II (rApxI + rApxII + rApxIII + rApxIVN + rOMP), vaccine III (rApxI + rApxII + rApxIII + rOMP), or placebo (phosphate-buffered saline + adjuvant). A. pleuropneumoniae serovar 1 field isolate JMS 06 and serovar 2 field strain FX 01 were used as the challenge strains. Pigs that were immunized with vaccine I or vaccine II all developed high antibody titers against rApxIVN. The antibody titers against rApxI, rApxII, rApxIII, and rOMP in pigs immunized with vaccine II were higher than those in pigs vaccinated with vaccine III. Following the challenge, the pigs immunized with rApxIVN alone showed similar results to the pigs in the control group, such as severe respiratory symptoms and severe lung lesions. Pigs that had been immunized with vaccine II or vaccine III were protected against challenge with A. pleuropneumoniae serovar 1 and serovar 2. The pigs immunized with vaccine II had slighter lung lesions and fewer bacterial recovery than those of pigs immunized with vaccine III. These results indicate that rApxIVN contributes to the production of high level of antibodies directed against the vaccination antigens, and thus confers strong protection against challenges with different serovars of A. pleuropneumoniae.