Intraperitoneal Challenge Research Articles

Effective Immune Protection of Mice from Murine Cytomegalovirus Infection by Oral Salmonella-Based Vaccine Expressing Viral M78 Antigen

Background: Human cytomegalovirus (CMV) is the most common cause of viral congenital infections worldwide. The development of effective vaccines against human CMV infection and disease is a high priority. Attenuated Salmonella are attractive oral vaccine vectors against human diseases because they can be administrated orally. Methods: In this study, an attenuated Salmonella strain was generated as an oral vaccine vector for the delivery and expression of the M78 protein of murine cytomegalovirus (MCMV). Using the MCMV infection of mice as the CMV infection model, we characterized the immune responses and protection induced by the constructed Salmonella-based vaccine. Results: The generated Salmonella-based vaccine, v-M78, which contained an M78 expression plasmid construct, carried out gene transfer efficiently for M78 expression and showed little pathogenicity and virulence in mice. In orally vaccinated mice, v-M78 induced anti-MCMV serum IgG and mucosal IgA responses and also elicited anti-MCMV T cell responses. Furthermore, mice immunized with v-M78 were protected from intraperitoneal and intranasal challenges with MCMV. The v-M78 vaccination reduced the titers of the challenged viruses in spleens, livers, lungs, and salivary glands. Conclusions: These results provide the first direct evidence that a Salmonella-based vaccine expressing M78 elicits strong humoral and cellular immune responses and induces immune protection against MCMV infection. Furthermore, our study demonstrates the potential of using Salmonella-based oral vaccines against CMV infection.

Evaluation of Marburg Virus Medical Countermeasures in Guinea Pigs.

Various animal models have been established to gain a better understanding of the pathogenesis of Marburg virus (MARV) and Ravn virus (RAVV), and to develop medical countermeasures (MCMs) against them. Of these models, which range from rodents to nonhuman primates (NHPs), the macaque model most closely mimics the severe disease displayed in humans. Nevertheless, rodent models mirror many key aspects of human infection and are frequently used for the initial assessment of experimental vaccines and treatments. Due to the less restrictive housing and husbandry requirements for these models, large-scale experiments can be performed to evaluate a number of test articles and/or dosing regimens.Adaptation of MARV and RAVV by serial passaging is necessary to cause disease in immunocompetent rodent species. While mice provide limited predictive value of vaccine and therapeutic efficacy against these viruses, guinea pigs have emerged as a dependable indicator of outcomes in late-stage NHP testing. Additionally, the larger size of guinea pigs compared to mice permits more frequent and substantial blood sample collection. This chapter outlines the essential procedures to conduct intraperitoneal challenge, blood collection, and the administration of MCMs in MARV and RAVV guinea pig models using biosafety level 4 practices.

Stability and Efficacy of Live-Attenuated Vibrio harveyi Vaccines Under Different Storage Conditions in Zebrafish (Danio rerio) Models

Graphical Abstract Highlight Research The LD50 (median lethal dose) of Vibrio parahaemolyticus and harveyi in zebrafish was determined to be 1 x 106 CFU/mL, while for V. alginolyticus it was found to be 1 x 105 CFU/mL. The LAVh vaccine demonstrated cross-protection against various pathogenic strains of Vibrio, leading to an average of 80% survival rate in vaccinated individuals. The analysis of the LAVh vaccine emphasized its versatility, as it can be quickly deployed and stored as a freeze-dried powder. The LAVh vaccine can be easily accessible and user-friendly in various aquaculture environments, such as offshore and remote farms. Abstract Vibriosis poses a significant threat to marine teleosts, causing substantial losses in the global aquaculture industry. Previous work in our lab led to the development of a live-attenuated V. harveyi vaccine (LAVh) candidate that targets the serine endoprotease gene with a three-point knockout and has shown promise in protecting against vibriosis. However, further investigation is necessary to evaluate the stability and efficacy of its various storage conditions for broader applications. This study aims to determine how well the three different LAVh vaccine storage (fresh, stale, and freeze-dried LAVh) worked against vibriosis. A total of 1000 adult zebrafish (Danio rerio) (mean weight: 0.20±0.5 g) were divided into four groups. Groups 1, 2, and 3 were intraperitoneally injected with different LAVh vaccine storage (fresh, stale, and freeze-dried, respectively), while Group 4 received 0.01 M phosphate-buffered saline (PBS) and served as the unvaccinated control. Fish were monitored for 21 days post-vaccination for safety, stability, efficacy, and antibody analysis. The results showed that a modest dosage of 1 x 104 CFU/mL of LAVh vaccine from all storage conditions provided 80% survival upon intraperitoneal challenge with pathogenic strains of pathogenic V. harveyi, V. alginolyticus, and V. parahaemolyticus. This dosage induced significant antibody production and conferred cross-protection against different Vibrio spp., indicating the LAVh vaccine’s potential for commercial application. The LAVh vaccine demonstrated high effectiveness and suitability for storage as a freeze-dried powder. This study might offer significant insights into practical strategies for reducing vibriosis, especially in aquaculture settings with limited infrastructure.

Current vaccination strategy against Piscirickettsia salmonis in Chile based only on the EM-90 genogroup shows incomplete cross-protection for the LF-89 genogroup

Piscirickettsia salmonis, the primary bacterial disease in Chilean salmon farming, necessitates a constant refinement of control strategies. This study hypothesized that the current vaccination strategy for SRS control in the Chilean Atlantic salmon aquaculture industry, which has been in place since 2017 (ALPHA JECT® 5.1 plus LiVac®), solely relies on vaccines formulated with the EM-90 genogroup of P. salmonis (PS-EM-90), triggering a partial cross-immunity response in fish infected with the LF-89 genogroup (PS-LF-89). Relative Percent Survival (RPS) and cell-mediated immune (CMI) response were evaluated in Atlantic salmon post-smolts vaccinated with the standard vaccination strategy but challenged with both PS-EM-90 and PS-LF-89, in addition to other vaccination strategies considering primo vaccination and booster with other commercial vaccines and the possible enhancing effects of the combination with a natural immunomodulator (PAQ-Xtract®) administered orally. The intraperitoneal (I.P.) challenge was performed after 2395°-days (DD) after the start of the immunostimulant delivery, 1905 DD after the primo vaccination, and 1455 DD after the booster vaccination. Unvaccinated fish showed 73.6 and 41.7 % mortality when challenged with PS-EM-90 and PS-LF-89, respectively. Fish infected with PS-LF-89 died significantly faster (21 days post-infection, dpi) than fish challenged with PS-EM-90 (28 dpi) (p = 0.0043) and had a higher probability of death (0.4626) than fish challenged with PS-EM-90. RPS had a significant positive correlation with the PS-EM-90 load of the P. salmonis genogroup (r = 0.540, p < 0.01) but not with the PS-LF-89 load (r = 0.155, p > 0.05). This demonstrated that the immunization strategies were more effective in lowering PS-EM-90 loads, resulting in higher survival rates in fish challenged with PS-EM-90. The current industry vaccination strategy recorded a 100 % RPS when fish were challenged with PS-EM-90, but the RPS dropped significantly to 77 % when fish were challenged with PS-LF-89, meaning that the strategy did not show complete cross-protection. But after adding PAQ-Xtract®, the RPS improved from 77 % to 92 % in fish that were vaccinated with the standard method but then challenged with PS-LF-89. The most effective vaccination strategy was based on LiVac® as primo vaccination and ALPHA JECT® 5.1 plus LiVac® as booster vaccination, with or without PAQ-Xtract®, in both PS-EM-90 (100 %) and PS-LF-89 (96 %) challenged fish. The serum concentration of anti-P. salmonis IgM did not show a correlation with the protection of immunization strategies expressed in survival. Low serum IL-12 and high serum IFNγ concentrations showed a correlation with higher bacterial loads and lower survival. Aggregate analysis showed a significant correlation between higher numbers of CD8+ cells in the head-kidney, higher fish survival, and a lower bacterial load. The immunization strategies were safe for fish and induced only mild microscopic lesions in the gut. Taken together, our results help to better understand the biological interaction between P. salmonis and post-smolt vaccinated Atlantic salmon to deepen the knowledge on vaccine-induced protection, CMI immune response, and cross-immunity applied to improve the current immunization strategy for SRS control in the Chilean salmon industry.

Salmonid Rickettsial Septicemia (SRS) disease dynamics and Atlantic salmon immune response to Piscirickettsia salmonis LF-89 and EM-90 co-infection

In Chile, Piscirickettsia salmonis contains two genetically isolated genogroups, LF-89 and EM-90. However, the impact of a potential co-infection with these two variants on Salmonid Rickettsial Septicemia (SRS) in Atlantic salmon (Salmo salar) remains largely unexplored. In our study, we evaluated the effect of P. salmonis LF-89-like and EM-90-like co-infection on post-smolt Atlantic salmon after an intraperitoneal challenge to compare changes in disease dynamics and host immune response. Co-infected fish had a significantly lower survival rate (24.1%) at 21 days post-challenge (dpc), compared with EM-90-like single-infected fish (40.3%). In contrast, all the LF-89-like single-infected fish survived. In addition, co-infected fish presented a higher presence of clinical lesions than any of the single-infected fish. The gene expression of salmon immune-related biomarkers evaluated in the head kidney, spleen, and liver showed that the EM-90-like isolate and the co-infection induced the up-regulation of cytokines (e.g., il-1β, ifnγ, il8, il10), antimicrobial peptides (hepdicin) and pattern recognition receptors (PRRs), such as TLR5s. Furthermore, in serum samples from EM-90-like and co-infected fish, an increase in the total IgM level was observed. Interestingly, specific IgM against P. salmonis showed greater detection of EM-90-like antigens in LF-89-like infected fish serum (cross-reaction). These data provide evidence that P. salmonis LF-89-like and EM-90-like interactions can modulate SRS disease dynamics in Atlantic salmon, causing a synergistic effect that increases the severity of the disease and the mortality rate of the fish. Overall, this study contributes to achieving a better understanding of P. salmonis population dynamics.

Characterization of Histophilus somni sialic acid uptake mutant (ΔnanP-ΔnanU) using a mouse septicemia and mortality model

Histophilus somni is an important pathogen of the bovine respiratory disease complex, yet the mechanisms underlying its virulence remain poorly understood. It is known that H. somni can incorporate sialic acid into lipooligosaccharide (LOS), and sialylated H. somni is more resistant to phagocytosis and complement-mediated killing by serum compared to non-sialylated bacteria in vitro. However, the virulence of non-sialylated H. somni has not been evaluated in vivo using an animal model. In this study, we investigated the contribution of sialic acid to virulence by constructing an H. somni sialic acid uptake mutant (ΔnanP-ΔnanU) and comparing the parent and mutant strains in a mouse septicemia and mortality model. Intraperitoneal challenge of mice with wildtype H. somni (1 × 108 colony forming units/mouse, CFU) was lethal to all animals. Mice challenged with three different doses (1, 2, or 5 × 108 CFU/mouse) of an H. somni ΔnanP-ΔnanU sialic acid uptake mutant exhibited survival rates of 90 %, 60 %, and 0 % respectively. High-performance anion exchange chromatography analyses revealed that LOS prepared from both parent and the ΔnanP-ΔnanU mutant strains of H. somni were sialylated. These findings suggest the presence of de novo sialic acid synthesis pathway, although the genes associated with de novo sialic acid synthesis (neuB and neuC) were not identified by genomic analysis. The lower attenuation in mice is most likely attributed to the sialylated LOS of H. somni nanPU mutant.

Immune correlates of protection of the four-segmented Rift Valley fever virus candidate vaccine in mice

ABSTRACT Rift Valley fever (RVF) is a mosquito-borne zoonotic disease caused by RVF virus (RVFV). RVFV infections in humans are usually asymptomatic or associated with mild febrile illness, although more severe cases of haemorrhagic disease and encephalitis with high mortality also occur. Currently, there are no licensed human vaccines available. The safety and efficacy of a genetically engineered four-segmented RVFV variant (hRVFV-4s) as a potential live-attenuated human vaccine has been tested successfully in mice, ruminants, and marmosets though the correlates of protection of this vaccine are still largely unknown. In the present study, we have assessed hRVFV-4s-induced humoral and cellular immunity in a mouse model of RVFV infection. Our results confirm that a single dose of hRVFV-4s is highly efficient in protecting naïve mice from developing severe disease following intraperitoneal challenge with a highly virulent RVFV strain and data show that virus neutralizing (VN) serum antibody titres in a prime-boost regimen are significantly higher compared to the single dose. Subsequently, VN antibodies from prime-boost-vaccinated recipients were shown to be protective when transferred to naïve mice. In addition, hRVFV-4s vaccination induced a significant virus-specific T cell response as shown by IFN-γ ELISpot assay, though these T cells did not provide significant protection upon passive transfer to naïve recipient mice. Collectively, this study highlights hRVFV-4s-induced VN antibodies as a major correlate of protection against lethal RVFV infection.

A first report of Streptococcus iniae infection of the spotted sea bass (Lateolabrax maculates).

This study marks the first occasion that Streptococcus iniae has been isolated, identified, and characterized as the causative pathogen in spotted sea bass (Lateolabrax maculates). Infected fish exhibited a range of external symptoms, including scale loss, bleeding from the jaw, anus, and tail, among other signs, as well as internal manifestations such as congested liver, splenomegaly, branchial anemia, yellow fat syndrome, and intestinal edema. Notably, exophthalmia and meningoencephalitis-typical symptoms associated with previous S. iniae infections-were not observed. A predominant bacterial isolate (designated 10S01) was recovered from the pure culture of spleen of a diseased spotted sea bass in Zhuhai, China. The strain was then subjected to Gram staining, biochemical profiling, and molecular confirmation through 16S rRNA and gyrB gene, corroborating its identity as S. iniae. Pathogenicity was assessed by intraperitoneal injection challenge in spotted sea bass weighing approximately 13 g/fish, revealing a LD50 of 74 cfu/g-fish. The 10S01 strain demonstrated the ability to colonize various organs, including the spleen, liver, kidney, and brain, with a relatively higher affinity for the spleen. Furthermore, antimicrobial susceptibility testing indicated that the 10S01 strain was sensitive to 14 tested antibiotics, particularly chloramphenicol, ciprofloxacin, clarithromycin, florfenicol, ofloxacin, rifampicin, and trimethoprim/sulfamethoxazole, highlighting these as preferred treatments for S. iniae infections in spotted sea bass. These findings contribute significantly to our understanding of S. iniae pathogenesis and inform the prompt and appropriate antibiotic treatment of S. iniae infections.

Oral mucosa effectively protects against peanut allergy in mice

Oral consumption of peanut products early in life reduces the incidence of peanut allergy in children. However, little is known about whether exposure via the oral mucosa alone is sufficient or whether the gastrointestinal tract must be engaged to protect against peanut allergy. We used a mouse model and examined the effects of peanut allergen administration to only the oral cavity on allergy development induced by environmental exposure. Naive BALB/c mice were administered peanut flour (PNF) sublingually, followed by epicutaneous exposure to PNF to mimic a human condition. The sublingual volume was adjusted to engage only the oral cavity and prevent it from reaching the esophagus or gastrointestinal tract. The efficacy was evaluated by examining the anaphylactic response, antibody titers, and T follicular helper cells. The mice exposed epicutaneously to PNF developed peanut allergy, as demonstrated by increased plasma levels of peanut-specific IgE and the manifestation of acute systemic anaphylaxis following intraperitoneal challenge with peanut extract. The development of peanut allergy was suppressed when mice had been given PNF sublingually before epicutaneous exposure. There were fewer T follicular helper cells in the skin-draining lymph nodes of mice that received sublingual PNF than in the mice that received PBS. Suppression of IgE production was observed with sublingual PNF at 1/10 of the intragastric PNF dose. Administration of peanut allergens only to the oral cavity effectively prevents the development of peanut allergy. The capacity of the oral mucosa to promote immunologic tolerance needs to be evaluated further to prevent food allergy.

Characterization of AMEV2, A Multi-Peptide Vaccine Against Acinetobacter baumannii

Abstract Multi-drug resistant (MDR) Acinetobacter baumannii is an opportunistic pathogen associated with hospital-acquired infections. This pathogen’s extensive MDR phenotype offers limited treatment options and requires alternative immunotherapeutic interventions, such as prophylactic vaccination in susceptible populations. In this study, we employed immunoinformatics to identify peptides containing both putative B and T cell epitopes from proteins associated with A. baumannii pathogenesis. An Acinetobacter Multi-Epitope Vaccine (AMEV2) comprising an A. baumannii thioredoxin A leading protein sequence followed by 5 identified peptide antigens was created. Subcutaneous immunization of mice with AMEV2 plus AddaS03 adjuvant demonstrates robust humoral and cellular immunogenicity. AMEV2 immunization results in high antibody titers to the antigen and the constitutional peptide components in addition to high frequencies of antibody-secreting cells in the spleen and bone marrow of the mice. In vitro opsonophagocytosis assays demonstrate anti-AMEV2 serum enhances the killing of opsonized bacteria by bone marrow-derived macrophages. Additionally, AMEV2 immunized mice were protected from both a lethal intraperitoneal and intranasal challenge with hypervirulent strains of A. baumannii, 4 weeks post-final vaccination, and achieved survival rates of 80% and 60%, respectively. AMEV2 vaccination affords antibody-mediated protection against hypervirulent A. baumannii infections.

Intra-islet glucagon signalling regulates beta-cell connectivity, first-phase insulin secretion and glucose homoeostasis

ObjectiveType 2 diabetes (T2D) is characterised by the loss of first-phase insulin secretion. We studied mice with β-cell selective loss of the glucagon receptor (Gcgrfl/fl X Ins-1Cre), to investigate the role of intra-islet glucagon receptor (GCGR) signalling on pan-islet [Ca2+]I activity and insulin secretion. MethodsMetabolic profiling was conducted on Gcgrβ-cell−/− and littermate controls. Crossing with GCaMP6f (STOP flox) animals further allowed for β-cell specific expression of a fluorescent calcium indicator. These islets were functionally imaged in vitro and in vivo. Wild-type mice were transplanted with islets expressing GCaMP6f in β-cells into the anterior eye chamber and placed on a high fat diet. Part of the cohort received a glucagon analogue (GCG-analogue) for 40 days and the control group were fed to achieve weight matching. Calcium imaging was performed regularly during the development of hyperglycaemia and in response to GCG-analogue treatment. ResultsGcgrβ-cell−/− mice exhibited higher glucose levels following intraperitoneal glucose challenge (control 12.7 mmol/L ± 0.6 vs. Gcgrβ-cell−/− 15.4 mmol/L ± 0.0 at 15 min, p = 0.002); fasting glycaemia was not different to controls. In vitro, Gcgrβ-cell−/− islets showed profound loss of pan-islet [Ca2+]I waves in response to glucose which was only partially rescued in vivo. Diet induced obesity and hyperglycaemia also resulted in a loss of co-ordinated [Ca2+]I waves in transplanted islets. This was reversed with GCG-analogue treatment, independently of weight-loss (n = 8). ConclusionThese data provide novel evidence for the role of intra-islet GCGR signalling in sustaining synchronised [Ca2+]I waves and support a possible therapeutic role for glucagonergic agents to restore the insulin secretory capacity lost in T2D.

Identification and florfenicol-treatment of pseudomonas putida infection in gilthead seabream (Sparus aurata) fed on tilapia-trash-feed

The present study aimed to determine the major cause of the high mortality affecting farmed gilthead seabream (Sparus aurata) and controlling this disease condition. Fifteen diseased S. aurata were sampled from a private fish farm located at Eldeba Triangle, Damietta, fish showed external skin hemorrhages, and ulceration. Bacterial isolates retrieved from the diseased fish were identified biochemically as Pseudomonas putida and then confirmed by phylogenetic analysis of the 16 S rRNA gene sequence. P. putida was also isolated from three batches of tilapia-trash feed given to S. aurata. Biofilm and hemolytic assay indicated that all P. putida isolates produced biofilm, but 61.11% can haemolyse red blood cells. Based on the antibiotic susceptibility test results, P. putida was sensitive to florfenicol with minimum inhibitory concentrations ranging between 0.25 and 1.0 µg mL− 1, but all isolates were resistant to ampicillin and sulfamethoxazole-trimethoprim. Pathogenicity test revealed that P. putida isolate (recovered from the tilapia-trash feed) was virulent for S. aurata with LD50 equal to 4.67 × 107 colony forming unit (CFU) fish− 1. After intraperitoneal (IP) challenge, fish treated with 10 mg kg− 1 of florfenicol showed 16.7% mortality, while no mortality was recorded for the fish group that received 20 mg kg− 1. The non-treated fish group showed 46.7% mortality after bacterial challenge. HPLC analysis of serum florfenicol levels reached 1.07 and 2.52 µg mL− 1 at the 5th -day post-drug administration in the fish groups received 10 and 20 mg kg− 1, respectively. In conclusion, P. putida was responsible for the high mortality affecting cultured S. aurata, in-feed administration of florfenicol (20 mg kg− 1) effectively protected the challenged fish.

Evaluation of Immune Protection of a Bivalent Inactivated Vaccine against Aeromonas salmonicida and Vibrio vulnificus in Turbot

The Aeromonas salmonicida is responsible for causing furunculosis in various fish species. Furunculosis is a ubiquitous disease that affects the aquaculture industry and causes the mass mortality of turbot. Vibrio vulnificus is a pathogen that causes skin ulcers and hemorrhagic septicemia in fish, resulting in significant mortality in aquaculture. In this study, we have established a bivalent inactivated vaccine against A. salmonicida and V. vulnificus with Montanide™ ISA 763 AVG as an adjuvant. This bivalent inactivated vaccine was used to immunize turbot by intraperitoneal injection, and the relevant immune indexes were detected. The results demonstrate that the bivalent inactivated vaccine exhibited a relative percent survival (RPS) of 77% following A. salmonicida and V. vulnificus intraperitoneal challenge. The vaccinated group exhibited higher levels of acid phosphatase activity and lysozyme activity compared to the control group. ELISA results showed a significant increase in serum antibody levels in immunized turbot, which was positively correlated with immunity. In the kidney tissue, related immune genes (TLR5, CD4, MHCI and MHCII) were up-regulated significantly, showing that the vaccine can induce cellular and humoral immune responses in turbot. In conclusion, the bivalent inactivated vaccine against A. salmonicida and V. vulnificus was immunogenic, efficiently preventing turbot from infection, which has the potential to be applied in aquaculture.

Constitutive Innate Immunity and Systemic Responses to Infection of the American Alligator (Alligator mississippiensis).

Uninfected alligators (Alligator mississippiensis) exhibited high constitutive levels of hepatic gene expression related to immune function, whereas the highest-expressed hepatic genes of uninfected mice were related to metabolism. Intraperitoneal challenge of mice with bacterial lipopolysaccharide results in dramatic inflammatory effects including peritoneal ascites, febrile response, dramatic alterations in electrophoretic serum profile, and mortality. In contrast, coelomic injection of alligators with 200× the murine LD50 of intraperitoneal bacterial lipopolysaccharide resulted in no changes in serum protein profiles, behavioral effects, mortality, and no coelomic ascites. However, injection of juvenile alligators with live bacteria resulted in a titer-dependent decrease in metabolic rate, as measured by oxygen consumption. These results are the opposite of those observed for mammalian and avian species. The decreased oxygen consumption was not accompanied by changes in heart or respiration rate, indicating that this phenomenon was not due to bradycardia or bradypnea. Interestingly, challenge of alligators with bacteria resulted in the complete expulsion of digestive tract contents within four hours. We interpret these activities as temporary minimization of other biological systemic activities to redirect and devote energy to immune function. The reallocation of resources within an organism to fight infection without increases in metabolic rate has not been described in other animals.

A TonB dependent transporter YncD of Salmonella entericaSerovar Typhi possesses vaccine potential.

Multiple TonB dependent transporters (TBDTs) contribute to bacterial virulence due to the importance roles that their substrates play in bacterial growth, and possess vaccine potential. A putative TBDT, YncD, had been identified as one of in vivo induced antigens during human infection of typhoid fever, and is required for the pathogenicity of Salmonella enterica Serovar Typhi. The present study was aimed to determine the function and immunogenicity of YncD. Homologous recombination method was used to construct an yncD-deletion mutant and cirA-iroN-fepA-deletion mutant from the wild-type S. Typhi Ty2. The growth of mutants and the wild-type strain were assessed in iron-deficient medium, as well as in human macrophage cells. Recombinant YncD protein was expressed and purified using Ni-NTA affinity chromatography and anion exchange. A mouse model was then used to evaluate the immunogenicity and protection efficacy of the recombinant YncD. Antibody levels, serum bactericidal efficiency, passive immune protection, opsonophagocysis were assayed to analyse the immunoprotection mechanism of the recombinant YncD. Our results showed that YncD is associated with the iron-uptake of S. Typhi. The yncD-deletion mutant displayed impaired growth in iron-deficient medium, comparable to that the cirA-iroN-fepA-deletion mutant did. The mutation of yncD markedly decreased bacterial growth within human macrophage cells. Moreover, subcutaneous immunization of mice with recombinant YncD elicited high levels of specific anti-YncD IgG, IgG1 and IgG2a, which protected the immunized mice against the intraperitoneal challenge of S. Typhi, and decreased bacterial burdens in the livers and spleens of the infected mice. Passive immunization using the immunized sera also efficiently protected the mice from the challenge of S. Typhi. Moreover, the immunized sera enhanced in vitro bactericidal activity of complement, and opsonophagocytosis. Our results showed that YncD displays a role in the iron-uptake of S. Typhi and possesses immunogenicity.

The O-glycan is essential for the induction of protective antibodies against lethal infection by flagella A-bearing Pseudomonas aeruginosa.

To address the problem of increased antimicrobial resistance, we developed a glycoconjugate vaccine comprised of O-polysaccharides (OPS) of the four most prevalent serotypes of Klebsiella pneumoniae (KP) linked to recombinant flagellin types A and B (rFlaA and rFlaB) of Pseudomonas aeruginosa (PA). Flagellin is the major subunit of the flagellar filament. Flagella A and B, essential virulence factors for PA, are glycosylated with different glycans. We previously reported that while both rFlaA and rFlaB were highly immunogenic, only the rFlaB antisera reduced PA motility and protected mice from lethal PA infection in a mouse model of thermal injury. Since recombinant flagellin is not glycosylated, we examined the possibility that the glycan on native FlaA (nFlaA) might be critical to functional immune responses. We compared the ability of nFlaA to that of native, deglycosylated FlaA (dnFlaA) to induce functionally active antisera. O glycan was removed from nFlaA with trifluoromethanesulfonic acid. Despite the similar high-titered anti-FlaA antibody levels elicited by nFlaA, rFlaA, and dnFlaA, only the nFlaA antisera inhibited PA motility and protected mice following lethal intraperitoneal bacterial challenge. Both the protective efficacy and carrier protein function of nFlaA were retained when conjugated to KP O1 OPS. We conclude that unlike the case with FlaB O glycan, the FlaA glycan is an important epitope for the induction of functionally active anti-FlaA antibodies.

Production and immunological characterisation of recombinant flagellin C of &lt;i&gt;Pseudomonas aeruginosa&lt;/i&gt;

SCIENTIFIC RELEVANCE. Pseudomonas aeruginosa is one of the main causative agents of focal and diffuse suppurative inflammations in immunocompromised individuals. The multiple antimicrobial resistance of Р. aeruginosa has created an urgent need to develop effective preventive vaccines against this bacterium and to enhance their protective properties by selecting adjuvants. A promising strategy is to use flagellin, a P. aeruginosa flagellum component inducing the innate immune system through interaction with toll-like receptor 5 (TLR5) and activating the T-cell immune response, as a component or adjuvant in vaccine development.AIM. This study aimed to produce recombinant flagellin C (FliC) of P. aeruginosa and investigate its immunogenicity, adjuvanticity, and protective properties.MATERIALS AND METHODS. The fliC nucleotide sequence was obtained by PCR on template DNA of P. aeruginosa PA-103 and was inserted into the pQE-30 plasmid for subsequent expression in Escherichia coli M15. Recombinant FliC purification involved two stages: isolation of inclusion bodies and their dissolution in buffers containing urea and guanidine hydrochloride. Mice were immunised by two intraperitoneal injections with a two-week interval. The immunisation used purified recombinant FliC at a dose of 50 μg per animal and its combination with the Klebsiella pneumoniae surface antigen in a 1:1 ratio. Serum samples from immunised mice were tested for specific antibodies to recombinant FliC by enzyme-linked immunosorbent assay (ELISA). The protective properties of FliC were assessed by intraperitoneal challenge of mice with cultures of P. aeruginosa PA-103 and K. pneumoniae 204.RESULTS. The authors obtained the producing strain, generated recombinant FliC, and purified the protein to a 97.6% purity. The analysis of serum samples from immunised mice by ELISA and the protection assessment in challenge experiments showed that the purified recombinant FliC protein had immunogenic properties. Furthermore, the experimental challenge of FliC-immunised mice with P. aeruginosa confirmed that recombinant FliC had protective properties, as evidenced by the protection index of 3.0. Recombinant FliC exhibited adjuvant properties, as demonstrated by the effectiveness index of 6.1 observed in the experimental challenge of mice immunised with the combination of recombinant FliC and the surface antigen of K. pneumoniae.CONCLUSIONS. The purified recombinant FliC protein of P. aeruginosa demonstrated protective activity in mice challenged with P. aeruginosa and adjuvant properties when combined with the K. pneumoniae surface antigen, increasing the immunogenicity of the latter. The use of recombinant FliC holds promise for the creation of a candidate vaccine against P. aeruginosa infection.

Development of human innate immune responses in a humanized mouse model expressing four human myelopoiesis transgenes.

Dysregulated innate immune responses underlie multiple inflammatory diseases, but clinical translation of preclinical innate immunity research in mice is hampered by the difficulty of studying human inflammatory reactions in an in vivo context. We therefore sought to establish in vivo human inflammatory responses in NSG-QUAD mice that express four human myelopoiesis transgenes to improve engraftment of a human innate immune system. We reconstituted NSG-QUAD mice with human hematopoietic stem and progenitor cells (HSPCs), after which we evaluated human myeloid cell development and subsequent human responses to systemic and local lipopolysaccharide (LPS) challenges. NSG-QUAD mice already displayed engraftment of human monocytes, dendritic cells and granulocytes in peripheral blood, spleen and liver at 6 weeks after HSPC reconstitution, in which both classical, intermediate and non-classical monocytes were present. These huNSG-QUAD mice responded to intraperitoneal and intranasal LPS challenges with production of NF-κB-dependent human cytokines, a human type I interferon response, as well as inflammasome-mediated production of human IL-1β and IL-18. The latter were specifically abrogated by the NLRP3 inhibitor MCC950, while LPS-induced human monocyte death was not altered. Besides providing proof-of-principle for small molecule testing of human inflammatory reactions in huNSG-QUAD mice, this observation suggests that LPS-induced in vivo release of human NLRP3 inflammasome-generated cytokines occurs in a cell death-independent manner. HuNSG-QUAD mice are competent for the NF-κB, interferon and inflammasome effectors of human innate immunity, and can thus be utilized to investigate signaling mechanisms and pharmacological targeting of human inflammatory responses in an in vivo setting.

Comparative genomics analysis of Streptococcus iniae isolated from Trachinotus ovatus: novel insight into antimicrobial resistance and virulence differentiation

BackgroundStreptococcus iniae is an important fish pathogen that cause significant economic losses to the global aquaculture industry every year. Although there have some reports on the genotype of S.iniae and its relationship with virulence, no genome-scale comparative analysis has been performed so far. In our previous work, we characterized 17 isolates of S.iniae from Trachinotus ovatus and divided them into two genotypes using RAPD and rep-PCR methods. Among them, BH15-2 was classified as designated genotype A (in RAPD) and genotype 1 (in rep-PCR), while BH16-24 was classified as genotype B and genotype 2. Herein, we compared the differences in growth, drug resistance, virulence, and genome between BH15-2 and BH16-24.ResultsThe results showed that the growth ability of BH16-24 was significantly faster than that of BH15-2 at the exponential stage. Antimicrobial tests revealed that BH15-2 was susceptible to most of the tested antibiotics except neomycin and gentamycin. In contrast, BH16-24 was resistant to 7 antibiotics including penicillin, sulfasomizole, compound sulfamethoxazole tablets, polymyxin B, spectinomycin, rifampin and ceftazidime. Intraperitoneal challenge of T.ovatus, showed that the LD50 value of BH15-2 was 4.0 × 102 CFU/g, while that of BH16-24 was 1.2 × 105 CFU/g. The genome of S.iniae BH15-2 was 2,175,659 bp with a GC content of 36.80%. Meanwhile, the genome of BH16-24 was 2,153,918 bp with a GC content of 36.83%. Comparative genome analysis indicated that compared with BH15-2, BH16-24 genome had a large-scale genomic inversion fragment, at the location from 502,513 bp to 1,788,813 bp, resulting in many of virulence and resistance genes differentially expression. In addition, there was a 46 kb length, intact phage sequence in BH15-2 genome, which was absent in BH16-24.ConclusionComparative genomic studies of BH15-2 and BH16-24 showed that the main difference is a 1.28 Mbp inversion fragment. The inversion fragment may lead to abnormal expression of drug resistant and virulence genes, which is believed to be the main reason for the multiple resistance and weakened virulence of BH16-24. Our study revealed the potential mechanisms in underlying the differences of multidrug resistance and virulence among different genotypes of S.iniae.

Performance, immune response, disease resistance, and gut microbiota of rainbow trout, Oncorhynchus mykiss (Walbaum, 1792) juveniles fed ground leonardite with a high humic substance content

In the present study, the effects of feeding ground leonardite (LE), which contained 68% of humic substances, to juvenile Oncorhynchus mykiss were examined on overall growth and production performance, feed nutrient digestibility, gut microbiota, health condition, antioxidant status, immune response, and disease resistance. During two consecutive experiments, fish were fed four experimental diets with different LE supplementary levels: 0, 0.5, 1, and 3% of feed mass (LE0, LE0.5, LE1, and LE3, respectively). Firstly, the fish were tested in a growing experiment (GE, n = 300 fish/group, body weight 15.4 ± 2.5 g) for 56 days. Thereafter, these fish (n = 75 fish/group, body weight 51.9 ± 8.7 g) were exposed to an intraperitoneal infection challenge experiment (IC) with Aeromonas salmonicida subsp. salmonicida for 28 days.In the GE, dietary LE did not affect the growth, production, or health performance of O. mykiss juveniles, although the fish more than tripled their initial body weight. The LE dose had an impact on the digestibility of feed nutrients (dry matter, gross energy, and ash), but not on the protein and lipid digestibility or solid waste outputs. The gut microbial communities were highly diverse across the experimental groups and remained distinct between the control and LE groups. The LE had a dose-dependent effect on the cell composition of the head kidney and peripheral blood leukocytes. The highest LE addition exerted the immune suppressive effect, and a decreased count of immune cells was observed in the LE3.The peritoneal cavity infection in the IC trial induced the rapid recruitment of myeloid cells in all groups shortly after the infection, except for the LE3, which had an apparent delayed response. The cumulative mortality and specific trout antibody levels against A. salmonicida did not significantly vary among tested groups, although their average initial level dropped by half at 3 days post-infection (dpi), and subsequently doubled at 28 dpi. The plasma biochemical parameters (TP, TAG, and CHOL) showed an overall decrease at 0–3 dpi, while increasing back to the initial levels at 3–6 dpi. Only LDH negatively correlated with LE inclusion, this can reflect the oxidative stress in the highest LE inclusion.Overall, our results support that there is a dose-dependent effect that should be optimised for ground LE dietary use in aquaculture.