The role of rpoS,hmp, and ssrAB in Salmonella enterica Gallinarum and evaluation of a triple-deletion mutant as a live vaccine candidate in Lohmann layer chickens

Salmonella enterica Gallinarum (SG) causes fowl typhoid (FT), a septicemic disease in avian species. We constructed deletion mutants lacking the stress sigma factor RpoS, the nitric oxide (NO)-detoxifying flavohemoglobin Hmp, and the SsrA/SsrB regulator to confirm the functions of these factors in SG. All gene products were fully functional in wild-type (WT) SG whereas mutants harboring single mutations or a combination of rpoS, hmp, and ssrAB mutations showed hypersusceptibility to H₂O₂, loss of NO metabolism, and absence of Salmonella pathogenicity island (SPI)-2 expression, respectively. A triple-deletion mutant, SGΔ3 (SGΔrpoSΔhmpΔssrAB), was evaluated for attenuated virulence and protection efficacy in two-week-old Lohmann layer chickens. The SGΔ3 mutant did not cause any mortality after inoculation with either 1 × 10⁶ or 1 × 10⁸ colony-forming units (CFUs) of bacteria. Significantly lower numbers of salmonellae were recovered from the liver and spleen of chickens inoculated with the SGΔ3 mutant compared to chickens inoculated with WT SG. Vaccination with the SGΔ3 mutant conferred complete protection against challenge with virulent SG on the chickens comparable to the group vaccinated with a conventional vaccine strain, SG9R. Overall, these results indicate that SGΔ3 could be a promising candidate for a live Salmonella vaccine against FT.     

Safety evaluation and immunogenicity of arabinose-based conditional lethal Salmonella Gallinarum mutant unable to survive ex vivo as a vaccine candidate for protection against fowl typhoid

In seeking to develop a safe fowl typhoid (FT) vaccine, a novel candidate lacking cpxR, lon, and asd Salmonella Gallinarum (SG) genes was constructed with the plasmid-containing araC::P(araBAD)::asd system. A balanced-lethal host-vector system based on the essential bacterial gene for aspartate beta-semialdehyde dehydrogenase (asd) was used to construct the SG mutant strain. A plasmid (p15A ori) with an araC::P(araBAD)::asd cassette was introduced into an auxotrophic mutant to prevent ex vivo survival. The safety, immunity, and protective properties of the SG mutant were evaluated. Inoculation of the mutant at 10(6) colony-forming units (CFU) did not result in recovery in feces and internal organs, whereas inoculation at 10(8) and 10(10) CFU resulted in moderate bacterial recovery from feces and organs. Birds immunized with the mutant were challenged with a virulent SG strain at day 14 postimmunization; significantly reduced mortality and induced plasma immunoglobulin (Ig)G and mucosal IgA responses were noted. Cellular immune responses as evaluated by a peripheral lymphocyte proliferation assay were also significantly induced. The balanced-lethal host-vector system for construction of SG mutants is an effective and improved approach for safe vaccine construction against FT.     

Efficiency of Matricaria chamomilla CH12 and number of doses of rabies vaccine on the humoral immune response in cattle

This study evaluated the effect of Matricaria chamomilla and vaccination frequency on cattle immunization against rabies. Four groups (n = 15 /group) were treated with or without Matricaria chamomilla CH₁₂ and vaccinated with one or two doses of rabies vaccine (30 day interval). No effect of chamomile was found on cattle immunization against rabies; however, antibody titers were protective in cattle vaccinated twice, while 93.3% of cattle vaccinated only once had titers under 0.5 UI/ml after 60 days. In conclusion, the use of chamomile did not alter the humoral immune response in cattle, and two vaccine doses are suggested for achieving protective antibody titers.     

Sortase anchored proteins of Streptococcus uberis play major roles in the pathogenesis of bovine mastitis in dairy cattle

Streptococcus uberis, strain 0140J, contains a single copy sortase A (srtA), encoding a transamidase capable of covalently anchoring specific proteins to peptidoglycan. Unlike the wild-type, an isogenic mutant carrying an inactivating ISS1 insertion within srtA was only able to infect the bovine mammary gland in a transient fashion. For the first 24 h post challenge, the srtA mutant colonised at a similar rate and number to the wild type strain, but unlike the wild type did not subsequently colonise in higher numbers. Similar levels of host cell infiltration were detected in response to infection with both strains, but only in those mammary quarters infected with the wild type strain were clinical signs of disease evident. Mutants that failed to express individual sortase substrate proteins (sub0135, sub0145, sub0207, sub0241, sub0826, sub0888, sub1095, sub1154, sub1370, and sub1730) were isolated and their virulence determined in the same challenge model. This revealed that mutants lacking sub0145, sub1095 and sub1154 were attenuated in cattle. These data demonstrate that a number of sortase anchored proteins each play a distinct, non-redundant and important role in pathogenesis of S. uberis infection within the lactating bovine mammary gland.     

Restoring catalase activity in Staphylococcus aureus subsp. anaerobius leads to loss of pathogenicity for lambs

Staphylococcus aureus subsp. anaerobius, a microaerophilic and catalase-negative bacterium, is the etiological agent of abscess disease, a specific chronic condition of sheep and goats, which is characterized by formation of necrotic lesions that are located typically in superficial lymph nodes. We constructed an isogenic mutant of S. aureus subsp. anaerobius (RDKA84) that carried a repaired and functional catalase gene from S. aureus ATCC 12600, to investigate whether the lack of catalase in S. aureus subsp. anaerobius plays a role in its physiological and pathogenic characteristics. The catalase activity had no apparent influence on the in vitro growth characteristics of RDKA84, which, like the wild-type, did not grow on aerobically incubated agar plates. Restoration of catalase activity in RDKA84 substantially increased resistance to H₂O₂ when analyzed in a death assay. The intracellular survival rates of the catalase-positive mutant RDKA84 in polymorphonuclear neutrophils (PMN) isolated from adult sheep were significantly higher than those of the wild-type, while no differences were found with PMN isolated from lambs. RDKA84 showed significantly lower survival rates in murine macrophages (J774A.1 cells) than the wild-type strains did, whereas, in bovine mammary epithelial cells (MAC-T), no differences in intracellular survival were observed. Interestingly, the virulence for lambs, the natural host for abscess disease, of the catalase-positive mutant RDKA84 was reduced dramatically in comparison with wild-type S. aureus subsp. anaerobius in two experimental models of infection.     

Evaluation of glycoproteins purified from adult and larval camel ticks (Hyalomma dromedarii) as a candidate vaccine

In order to identify antigens that can help prevent camel tick infestations, three major glycoproteins (GLPs) about 97, 66 and 40 kDa in size were purified from adult and larval Egyptian ticks, Hyalomma (H.) dromedarii, using a single-step purification method with Con-A sepharose. The purified GLPs were evaluated as vaccines against camel tick infestation in rabbits. The rabbits received three intramuscular inoculations of GLPs (20 µg/animal) on days 0, 14, and 28. In the immunoblot analysis, Sera from the immunized rabbits recognized the native GLPs and other proteins from larval and adult H. dromedarii ticks along with those from other tick species such as Rhipicephalus sanguineus but not Ornithodoros moubata. The effects of immunity induced by these GLPs were determined by exposing rabbits to adult H. dromedarii ticks. These results demonstrated that GLP immunization led to a slightly decreased reproductive index and significantly reduced rates of egg hatchability. These results demonstrated that immunization with the purified GLPs can provide protection against infestation by H. dromedarii and some other tick species. Further studies are needed to confirm the effectiveness of immunization with GLPs against other tick species.     

Efficacy of VP2 protein expressed in E. coli for protection against highly virulent infectious bursal disease virus

The ability of a heat-inactivated whole virus from a highly virulent infectious bursal disease virus (hvIBDV) and VP2 protein from hvIBDV expressed in E. coli provided protection against a hvIBDV challenge in specific-pathogen-free (SPF) chickens. Six out of seven chickens that were injected three times with crude VP2 protein developed significant antibody titer against IBDV. However, only four out of the seven chickens survived the hvIBDV challenge. Despite showing low antibody titer profiles, all chickens immunized with the heat-inactivated whole virus also survived the challenged with hvIBDV. However, all of these chickens had bursal atrophy and mild to moderate depletion of lymphocytes. Thus, antibodies raised against IBDV VP2 protein expressed in E. coli and denatured IBDV proteins induced some degree of protection against mortality but not against bursal damage following challenge with hvIBDV.     

Improvement of bacterial clearance and relief of clinical signs of Salmonella enterica serovar Typhimurium infection in pigs through upregulation of Th 1-specific responses by administration of a combination of two silicate minerals,biotite and bentonite

Biotite and bentonite are phyllosilicate minerals that were originally used inindustrial applications. Several beneficial activities of them have recently beenreported, especially regulation of the immune system and antimicrobial effects. Therefore,we investigated the immune-enhancing and bacterial clearance effects of a biotite andbentonite mixture (BBM) on experimental infection of Salmonella entericaserovar Typhimurium (S. Typhimurium) to determine whether the BBM couldbe used as an alternative antibiotic. We administered 1% or 2% BBM as a feed supplement.We then evaluated the bacterial clearance effects of the BBM against S.Typhimurium. We also evaluated the immune-enhancing effect of the BBM through severalimmunological experiments that included examination of the lysozyme activity,CD4⁺/CD8⁺ T lymphocyte ratio and the T-helper type 1 (Th 1)cytokine profile. The clinical signs of S. Typhimurium and the number ofviable bacteria in feces and tissues were significantly decreased in both BBM groups,especially in the 2% BBM group. The BBM also markedly enhanced the lysozyme activity,CD4⁺/CD8⁺ T lymphocyte ratio and expression levels of IFN-γ andIL-12 in S. Typhimurium-challenged pigs. Therefore, the BBM could be agood candidate as an alternative antibiotic that improves Th 1-specific immune responsesand the bacterial clearance effect.     

Duration of immunity, efficacy and safety in sheep of a recombinant Taenia ovis vaccine formulated with saponin or selected adjuvants

The efficacy and safety of a recombinant Taenia ovis protein was tested in sheep using 13 different adjuvant formulations, including oil adjuvants, aluminium salts, saponin, Iscoms and DEAE-dextran. The oil adjuvants, saponin and DEAE-dextran gave the highest antibody responses and greatest degree of protection against challenge infection with T. ovis eggs. Duration of immunity studies with a saponin based vaccine showed that highly significant protection (>90% reduction of cyst numbers) was achieved when sheep were challenge infected one month after immunisation. Significant protection (79%) was still present when sheep were challenged 6 months after immunisation. The optimum dose for this batch of saponin was 10 mg, which stimulated a peak antibody titre of 38,400, 4 weeks after immunisation and did not cause injection site reactions. Dialysed saponin was shown to retain its adjuvant properties and allowed an increase in dose to 30 mg without site reaction, resulting in a peak antibody titre of 51,200.     

Development and evaluation of a multiplex PCR assay for simultaneous detection of Flavobacterium psychrophilum, Yersinia ruckeri and Aeromonas salmonicida subsp. salmonicida in culture fisheries

Bacterial cold water disease, enteric red mouth disease and frunculosis are the common bacterial diseases of fish worldwide. The etiologic agents of these diseases are Flavobacterium (F.) psychrophilum, Yersinia (Y.) ruckeri and Aeromonas (A.) salmonicida subsp. salmonicida, respectively. In this study, a multiplex polymerase chain reaction (m-PCR) method with YER8/10-Fer3/4-FP1/3 primer pairs which can identify these fish pathogens simultaneously was developed and optimized. In optimized conditions, neither false specific nor nonspecific amplification occurred. The detection limits of the m-PCR method using DNA extracts from dilutions of pure cultures of bacteria were 35 pg for Y. ruckeri and F. psychrophilum and 70 pg for A. salmonicida subsp. salmonicida. It was determined that 15 CFU Y. ruckeri and F. psychrophilum and 30 CFU A. salmonicida subsp. salmonicida could be detected by m-PCR developed using genomic DNA extracted from dilutions of the suspensions. The detection limits in the presence of tissue debris were 125 CFU for Y. ruckeri and F. psychrophilum and 250 CFU for A. salmonicida subsp. salmonicida. In conclusion, we submit that the m-PCR method developed and optimized in this study can be used for accurate and rapid identification of these bacteria.     

Molecular typing of Salmonella enterica serovar 4,[5],12:i:- isolates from humans,animals and river water in Japan by multilocus variable-number tandem repeat analysis and pulsed-field gel electrophoresis

Fifty-one Salmonella enterica serovar 4,[5],12:i:- (S. 4, [5],12:i:-) isolates (14 human strains, 34 animal strains and 3 river water strains) which are assumed to be monophasic variants of S. Typhimurium were analyzed using pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem repeat analysis (MLVA) in order to investigate their genetic diversities and relationships. PFGE, MLVA and combination of them identified 28, 27 and 34 profiles (Simpson’s diversity indices [DI]=0.94, 0.96 and 0.97), respectively. No correlations were detected between MLVA clustering and PFGE clustering or phage typing. These results suggested that S. 4,[5],12:i:- originated from multiple S. Typhimurium ancestors. Two cattle and one pig isolates showing identical phage types as well as PFGE and MLVA profiles to human isolates S. 4,[5],12:i:- suggested the existence of the links between human infections and animal reservoirs.     

Potent immune responses induced by a Salmonella ghost delivery system that expresses the recombinant Stx2eB,FedF, and FedA proteins of the Escherichia coli-producing F18 and Shiga toxin in a murine model and evaluation of its protective effect as a porcine vaccine candidate

Background: In the pathogenicity of porcine edema disease (ED), which is caused by the Escherichia coli-producing F18 and Shiga toxin, F18⁺ fimbrial adhesins and Shiga toxin 2e (Stx2e) play pivotal roles in the colonization and enterotoxicity of this pathogen.

Objective: To develop a vaccine candidate against ED by combining three selected antigens of F18⁺ E. coli.

Methods: Genetically engineered Salmonella Typhimurium (ST) ghosts that express Stx2eB, FedF, and FedA were individually inserted in a ghost plasmid cassette, and the resultant plasmids were transformed into an attenuated ST (JOL912). The individual expression of Stx2eB, FedF, and FedA in JOL912 was validated by using an immunoblotting assay.

Results: Immunization of the ghosts in BALB/c mice led to a significant increase in antigen-specific secretory IgA and serum IgG. Significantly marked elevation of the CD3⁺CD4⁺ T cell subpopulation and lymphocyte proliferating activity in the primed splenocytes were also observed. Furthermore, mRNA of IL-4 and IFN-γ were highly upregulated in in vitro stimulated splenic T cells. Subsequently, the immunized mice showed significant protection efficacy against a lethal dose 50 of a virulent strain, resulting in approximately 85% and 92% survival rates in mice with a single- and double-dose immunization, respectively, compared to only 40% of the non-immunized controls.

Conclusion: A mixture of the ghosts expressing these three antigens is a potential vaccine candidate for protection against the porcine edema disease.