Kiku Matsuda

Evaluation of safety and protection efficacy on cpxR and lon deleted mutant of Salmonella Gallinarum as a live vaccine candidate for fowl typhoid

We evaluated a recently developed live fowl typhoid (FT) vaccine candidate, JOL916, the cpxR/lon mutant of Salmonella Gallinarum (SG), for safety and protection efficacy in 5-week-old layer chickens. Intramuscular vaccination with JOL916 revealed no or very few lesions in livers and spleens of the animals until the fourth week post-vaccination (wpv). This candidate clearly induced cellular immune responses in 5 of 5 chickens on the first and second wpv based on the peripheral lymphocyte proliferation assay. Systemic IgG responses were observed in 5 of 5 chickens from the first wpv and dramatic elevations were observed on the second and third wpv. Vaccination of chickens offered efficient protection against challenge by a wild-type SG; only slight anorexia and depression were temporarily observed after challenge in the vaccinated group while 100% mortality was observed in the positive control group. Body weight increases per day were slightly reduced between the 3rd and 6th day post challenge (dpc) compared to the negative control group; it was recovered from the 6th dpc. Collectively, these results demonstrate the safety and protective efficacy of JOL916 as a live vaccine for systemic FT.

Physiology, pathogenicity and immunogenicity of lon and/or cpxR deleted mutants of Salmonella Gallinarum as vaccine candidates for fowl typhoid

To construct a novel live vaccine candidate for fowl typhoid (FT) caused by Salmonella Gallinarum (SG), the lon and cpxR genes that are related to host-pathogen interaction were deleted from a wild type SG using the allelic exchange method. The mutants were grown normally, as was the wild type. The biochemical properties of the mutants remained very similar to those of the wild-type, while JOL914 (Δlon) and JOL916 (ΔlonΔcpxR) were mucoid. Extracellular polysaccharide increased 30.6-, 1.3-, and 46.2-fold in JOL914, JOL915 (ΔcpxR), and JOL916, respectively. Dot-blot analysis demonstrated significant increases of FimA expression at 6.77-, 2.33-, and 3.90-fold for JOL914, JOL915, and JOL916, respectively. Internalizations of JOL914, JOL915, and JOL916, in chicken abdominal macrophages, were increased at 4.65-, 0.50-, and 2.72-fold, respectively. Virulences of JOL914, JOL915 and JOL916, analyzed by LD50 using 1-week-old chickens, were attenuated approximately at 101-, 101-, and > 103-fold, respectively. The oral inoculations of 2 × 107 cfu of the wild type, JOL914, JOL915 and JOL916 caused 55.6, 16.7, 22.2, and 0.0% mortality, respectively. Significantly moderate gross lesions of the liver and spleen were observed in the JOL916 group compared to the other groups. An induced immune response and significant peripheral mononuclear proliferation reaction were observed in the JOL916 group. At the protection against the wild type challenge, JOL916 offered 100% protection. Thus, the results of this study suggest that JOL916 among the mutants studied represented the safest and most effective live vaccine candidate against FT.

Immunogenicity of a Salmonella Enteritidis mutant as vaccine candidate and its protective efficacy against salmonellosis in chickens

A novel Salmonella Enteritidis (SE) vaccine candidate strain, JOL919 was constructed by deleting the lon and cpxR genes from a wild-type SE using an allelic exchange method. The study was carried out to evaluate the strain as a vaccine candidate against salmonellosis. The strain showed the enhanced macrophage invasion, early bacterial clearance and higher immune responses as compared to the other mutants, JOL917 (Δlon) and JOL918 (ΔcpxR), and the wild type. In further analysis, the chickens immunized with JOL919 showed a significant increase in plasma IgG and intestinal secretory IgA levels, which was an indication of robust humoral and mucosal immune responses induced by the candidate. The lymphocyte proliferation response and CD45(+)CD3(+) T cells, associated with an activation of T helper and cytotoxic cells, were also significantly increased in the immunized group, which indicated that the candidate also induced cellular immune responses. The immune cell influx into caecal tissues analyzed by immunohistochemistry showed that CD8(+) T cells were predominated in the immunized group, suggesting that the candidate can clear the invaded pathogen in the intestines by a more direct way involving cytotoxic activity. By the examination of the protection efficacy measured by observations of gross lesions in the organs and bacterial recovery, the candidate can provide an efficient protection upon virulent challenge.

A genetically engineered derivative of Salmonella Enteritidis as a novel live vaccine candidate for salmonellosis in chickens

To construct a novel live Salmonella Enteritidis (SE) vaccine candidate, SE was genetically engineered using the allelic exchange method to delete two virulence genes, lon and cpxR. The lon gene deletion is essential to impair Salmonella replication and avoid overwhelming systemic disease in the host. The cpxR gene deletion is needed to enhance the ability of bacteria to adhere and invade the host cell. Scanning electron microscopy revealed that the derivatives JOL917 (Δlon), JOL918 (ΔcpxR), and JOL919 (Δlon/ΔcpxR) had increased surface fimbrial filamentous structures. Significant elevations of extracellular polysaccharide and FimA expression were observed for the derivatives compared to the parental wild type JOL860, while biochemical properties of the derivatives were not altered. In the safety examination by inoculation of the derivatives in chickens, gross lesion scores of the liver, spleen, kidney, small intestine and caecal tonsils were moderate in the JOL917 and JOL918 groups, and significantly lower in the JOL919 group than those of the JOL860. Bacterial counts from the spleen and caeca of the JOL917 and JOL918 groups were moderate, and significantly reduced in the JOL919 group compared to the JOL860 group. In addition, only the JOL919 group showed significantly lower bacterial counts in the faecal samples than those of the JOL860 group. Significant elevations of IgG and secretory IgA levels observed in the derivative groups, while the JOL919 and JOL860 groups showed a potent lymphocyte proliferation response as compared to those of the control group. In the protection efficacy examination, JOL919 immunized group showed significantly lower depression, lower gross lesion in the liver and spleen, and lower number of the SE positive internal organs than those of the control group against a virulent wild type SE challenge.

Comparison of the Safety and Efficacy of a New Live Salmonella Gallinarum Vaccine Candidate, JOL916, with the SG9R Vaccine in Chickens

SUMMARY. We evaluated a recently developed live vaccine candidate for fowl typhoid (FT)—JOL916, a lon/cpxR mutant of Salmonella Gallinarum (SG)—by comparing its safety and efficacy with that of the well-known rough mutant strain SG9R vaccine in 6-wk-old Hy-Line hens. Forty-five chickens were divided into three groups of 15 chickens each. The chickens were then intramuscularly inoculated with 2 × 107 colony-forming units (CFUs) of JOL916 (JOL916 group), 2 × 107 CFUs of SG9R (SG9R group), or phosphate-buffered saline (control group). After vaccination, no clinical symptoms were observed in any of the groups. No differences in body weight increase were detected among the three groups postvaccination. A cellular immune response was observed at 2 wk postvaccination (wpv) in the JOL916 group with the peripheral lymphocyte proliferation assay, whereas no response was detected in the SG9R group. Elevation of SG antigen-specific plasma immunoglobulin was observed 2 and 3 wpv in the JOL916 and SG9R vaccine groups, respectively. After virulent challenge on day 25 postvaccination, 0, 1, and 15 chickens in the JOL916 group, SG9R group, and control group, respectively, died by 12 days postchallenge; the death rate of the SG9R vaccine group was statistically similar to that of the JOL916 group. Postmortem examination revealed that the JOL916 vaccine offered more efficient protection than the SG9R vaccine, with significantly decreased hepatic necrotic foci scores, splenic enlargement scores, necrotic foci scores, and recovery of the challenge strain from the spleen. Vaccination with JOL916 appears to be safe and offers better protection than SG9R against FT in chickens.

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

Abstract 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::ParaBAD::asd system. A balanced-lethal host-vector system based on the essential bacterial gene for aspartate β-semialdehyde dehydrogenase (asd) was used to construct the SG mutant strain. A plasmid (p15A ori) with an araC::ParaBAD::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 106 colony-forming units (CFU) did not result in recovery in feces and internal organs, whereas inoculation at 108 and 1010 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.

Efficacy for a New Live Attenuated Salmonella Enteritidis Vaccine Candidate to Reduce Internal Egg Contamination

To evaluate the efficacy of a novel attenuated Salmonella Enteritidis (△lon△cpxR) vaccine candidate (JOL919), chickens were immunized through oral and intramuscular routes to reduce egg contamination against S. Enteritidis challenge. Birds were orally immunized with JOL919 on the first day of life and were subsequently boosted in the 6th and 16th weeks through oral (group B) or intramuscular (group C) route, while control birds were unimmunized (group A). The chickens of all groups were challenged intravenously with the virulent S. Enteritidis strain in the 24th week. The immunized groups B and C showed significantly higher plasma IgG and intestinal secretory IgA levels as compared to those of the control group. The lymphocyte proliferation response and CD45+CD3+ T‐cell number in the peripheral blood of the groups B and C were significantly increased. In addition, the egg contamination rates were significantly lower in the group B (0%, 10.7% and 0%) and the group C (3.6%, 14.3% and 3.6%) as compared to the group A (28.6%, 42.8% and 28.6%) in the 1st, 2nd and 3rd weeks post‐challenge. All animals in the groups B and C showed lower organ lesion scores in the liver and spleen and lower bacterial counts in the liver, spleen and ovary at the 3rd week post‐challenge. These results indicate that this vaccine candidate can be an efficient tool for prevention of Salmonella infections by inducing protective humoral and cellular immune responses. In addition, this vaccine did not prevent egg contamination, but did appear to reduce incidence. Booster immunizations, especially via oral administration route, showed an efficient protection against internal egg contamination with S. Enteritidis.

Avian colibacillosis caused by an intestinal pathogenic Escherichia coli isolate from calf diarrhea.

An intestinal pathogenic Escherichia coli isolate from calf diarrhea, containing the iutA, f17A, afa-8D, and cnf2 genes, was able to cause avian colibacillosis after experimental infection in chickens. Intra-tracheal inoculation and spray of this strain caused 10% of mortality and gross lesions, including airsacculitis, pericarditis, and perihepatitis. These results suggest that some bovine pathogenic E. coli can cause extra-intestinal infections in other animal species.

Construction of an Attenuated Salmonella Delivery System Harboring Genes Encoding Various Virulence Factors of Avian Pathogenic Escherichia coli and Its Potential as a Candidate Vaccine for Chicken Colibacillosis

SUMMARY. An attenuated Salmonella (&Dgr;lon, &Dgr;cpxR, and &Dgr;asdA16) delivery system containing the genes encoding P-fimbriae (papa and papG), aerobactin receptor (iutA), and CS31A surface antigen (clpG) of avian pathogenic Escherichia coli (APEC) was constructed, and its potential as a vaccine candidate against APEC infection in chickens was evaluated. The birds were divided into three groups designated group A (nonvaccinated control), group B (given a single immunization), and group C (administered prime and boost immunizations). Prime and booster vaccinations with the constructions were administered to 1-day-old and 14-day-old birds, respectively. Immune responses were measured postimmunization, and the birds were challenged via an intra–air sac route with a virulent APEC strain at the second, third, and fourth weeks of age. Group B birds were partially protected against the challenge and showed increased levels of plasma immunoglobulin (Ig)G, mucosal IgA antibodies, and lymphocyte proliferation. Group C birds showed greater protection against the challenge, with significantly stronger immune responses compared with the birds in the other groups. Overall, our data suggest that the Salmonella delivery system with recombinant constructs is capable of inducing robust immune responses and induces effective protection against colibacillosis caused by APEC. RESUMEN. Construcción de un sistema de aplicación de vacunas utilizando una Salmonella atenuada que contiene los genes que codifican varios factores de virulencia de Escherichia coli y su potencial como un candidato de vacuna para la colibacilosis del pollo. Se construyó un sistema de aplicación utilizando una Salmonella atenuada (&Dgr;lon, &Dgr;cpxR, y &Dgr;asdA16) que contiene los genes que codifican las fimbrias P (papa y papG), el gene del receptor de la aerobactina (iutA), y el antígeno de superficie CS31A (clpG) de cepas de Escherichia coli patógenas para las aves (con las siglas en inglés APEC), y se evaluó su potencial como una vacuna contra la infección por APEC en pollos. Las aves se dividieron en tres grupos designados como grupo A (control no vacunados), grupo B (con una sola inmunización), y el grupo C (con primoinmunizaciones e inmunizaciones de refuerzo). Las primovacunaciones y las vacunaciones de refuerzo con los constructos se administraron en aves de un día y de 14 días de edad, respectivamente. Las respuestas inmunes se midieron después de la inmunización, y las aves fueron desafiadas a través de una ruta de inoculación directa en el saco aéreo con una cepa virulenta de APEC en la segunda, tercera, y cuarta semana de edad. Las aves del Grupo B estuvieron parcialmente protegidas contra el desafío y mostraron niveles elevados de inmunoglobulinas plasmáticas (Ig) G, anticuerpos de mucosas tipo IgA y proliferación de linfocitos. Las aves del Grupo C mostraron una mayor protección contra el desafío, con respuestas inmunes significativamente más fuertes en comparación con las aves de los otros grupos. En general, estos datos sugieren que el sistema de aplicación utilizando un constructo recombinante de Salmonella es capaz de inducir respuestas inmunitarias robustas e induce una protección eficaz contra la colibacilosis causada por cepas de E. coli patógenas para las aves.

EURASIAN OTTER (LUTRA LUTRA), A DEFINITIVE HOST FOR DIROFILARIA IMMITIS

Abstract A mature male and a mature female Dirofilaria immitis were found in the right ventricle of the heart of a naturally infected 2-yr-old male Eurasian otter (Lutra lutra) that had died of severe lung congestion at a zoo in South Korea. Both developing embryos and microfilariae were present in the uterus of the female D. immitis. Although circulating microfilariae were not detected in blood or tissue, the Eurasian otter may serve as a definitive host for D. immitis.