Longitudinal study on the effect of autogenous vaccine application on the sequence type and virulence profiles of Escherichia coli in broiler breeder flocks.

Colibacillosis is one of the most common problems in the poultry industry. Escherichia coli strains on farms are often genetically diverse and therefore commercial vaccines provide little protection to the flocks. Here, we investigated the effect of the autogenous E. coli vaccines on the prevalence of 84 virulence-associated genes in E. coli isolated from four and five consecutive flocks on two broiler breeder farms, respectively. 115 E. coli isolates were sequenced using Illumina technologies, and compared based on both their set of housekeeping genes and their virulence profiles, defined through the composition of virulence genes. Predominantly, phylogenetic analysis showed obvious distinction between the isolates originating from different farms suggesting spatial-dependent transmission of pathogenic strains. We detected 23 sequence types, while 52.58 % of the isolates belonged to two clonal complexes. Analysis of the virulence genes showed highest prevalence (>85 %) of feoB, uspA, uspB, uspG, uspE, fimH, ompA, astA, focA, hlyE, uspC, crl, csgA, ompT and iss, of which 50 % are toxin associated genes, demonstrating the importance of competition in the pathogenesis process. Interestingly, usp genes, which are primarily associated with uropathogenic E. coli strains, were detected in all investigated isolates. The heatmap analysis demonstrated that strains belonging to same phylogenetic groups often share similar virulence profiles, confirming the usefulness of quick tests for phylogenetic typing. However, our results suggest the need to update the list of the minimal predictors used for the identification of avian pathogenic strains. Overall results indicate that continuous application of autogenous vaccines led to lower genetic diversity of E. coli housekeeping genes, but not virulence genes.

Immunogenicity study of a Streptococcus suis autogenous vaccine in preparturient sows and evaluation of passive maternal immunity in piglets.

BACKGROUND: Streptococcus suis is an important pathogen that causes severe diseases mostly in weaned piglets. Only available vaccines in the field are those composed of killed bacteria (bacterins) but data about their effectiveness are missing. We report here a field study on the immunological response induced by an autogenous vaccine applied in pre-parturient sows. Using a farm with recurrent S. suis serotype 7 problems, the study was divided in three experiments: (I) Sows received the vaccine at 7 and 3 weeks pre-farrowing. (II) Replacement gilts introduced to the herd received the vaccine at 4 and 7 weeks after their entry in quarantine and a boost 3 weeks pre-farrowing. (III) Gilts from experiment II received another boost 3 weeks pre-farrowing at their 3rd/4th parity. Levels, isotype profile and opsonophagocytosis capacity of the serum antibodies induced by vaccination were evaluated in sows and maternal immunity in piglets. RESULTS: In sows (I), the vaccine induced a slight, albeit significant, increase in anti-S. suis total antibodies after 2 doses when compare to basal levels already present in the animals. These antibodies showed a high opsonic capacity in vitro, highlighting their potential protective capacity. A gilt vaccination program of 3 doses (II) resulted in a significant increase in anti-S. suis total antibodies. Levels of maternal immunity transferred to piglets were high at 7 days of age, but rapidly decreased by 18 days of age. A gilt vaccination program ensued a higher transfer of maternal immunity in piglets compared to control animals; nevertheless duration was not improved at 18 day-old piglets. The vaccine response in both gilts and sows was mainly composed of IgG1 subclass, which was also the main Ig transferred to piglets. IgG2 subclass was also found in piglets, but its level was not increased by vaccination. Finally, a recall IgG1 response was induced by another boost vaccination at 3rd/4th parity (III), indicating that the vaccine induced the establishment of a lasting memory response in the herd. CONCLUSIONS: Overall, an optimal gilt/sow vaccination program might result in increased antibody responses; nevertheless duration of maternal immunity would not last long enough to protect post-weaned piglets.

A commercial autogenous injection vaccine protects ballan wrasse (Labrus bergylta, Ascanius) against Aeromonas salmonicida vapA type V.

Atypical Aeromonas salmonicida (aAs) and Vibrionaceae related species are bacteria routinely recovered from diseased ballan wrasse used as cleaner fish in the Atlantic salmon farming industry. Autogenous (i.e. farm specific inactivated) multivalent vaccines formulated from these microorganisms are widely used to protect farmed wrasse despite limited experimental proof that they are primary pathogens. In this study, the components of a commercial multivalent injection vaccine containing four strains of Aeromonas salmonicida and one strain of Vibrio splendidus previously isolated from ballan wrasse in Scotland, were tested for infectivity, pathogenicity and virulence via intra peritoneal injection at pre-deployment size (25-50 g) and the efficacy of the vaccine for protection against aAs assessed. Injection with 3.5 × 109, 8 × 109 1.8 × 109 and 5 × 109 cfu/fish of Vibrio splendidus, V. ichthyoenteri, Aliivibrio logeii and A. salmonicida, respectively, did not cause significant mortalities, lesions or clinical signs after a period of 14 days. IP injection with both aAs and Photobacterium indicum successfully reproduced the clinical signs and internal lesions observed during natural outbreaks of the disease. Differences in virulence (LD50 at day 8-post infection of 3.6 × 106 cfu/fish and 1.6 × 107 cfu/fish) were observed for two aAs vapA type V isolates. In addition, the LD50 for Photobacterium indicum was 2.2 × 107 cfu/fish. The autogenous vaccine was highly protective against the two aAs vapA type V isolates after 700-degree days of immunisation. The RPSFINAL values for the first isolate were 95 and 91% at 1 × 106 cfu/fish and 1 × 107 cfu/fish, respectively, and 79% at 1 × 107 cfu/fish for the second isolate tested. In addition, significantly higher anti aAs seral antibodies (IgM), were detected by ELISA in vaccinated fish in contrast with control (mock vaccinated) fish. These results suggest wrasse can be effectively immunised and protected against aAs infection by injection with oil adjuvanted vaccines prepared with inactivated homologous isolates.

Whole cell inactivated autogenous vaccine effectively protects red Nile tilapia (Oreochromis niloticus) against francisellosis via intraperitoneal injection.

Francisella noatunensis subsp. orientalis is a pathogen of tilapia and other warm-water fish for which no vaccines are commercially available. In this study, a whole cell formalin-inactivated vaccine was developed for the first time using the highly virulent isolate STIR-GUS-F2f7 and the oil-based adjuvant Montanide™ ISA 763A VG. The efficacy of the vaccine was assessed in red Nile tilapia via intraperitoneal (i.p.) injection using homologous experimental infection and correlates of protection such as seral antibody production and bacterial loads in the spleen. For immunization, fish were i.p. injected with 0.1 ml of the vaccine, the adjuvant alone or PBS. At 840 degree days post-vaccination, all fish were i.p. injected with 4.0 × 103 CFU/fish of pathogenic bacteria. The RPS at the end of the trial was 100% in the vaccinated group with significantly higher survival than in the adjuvant and control groups. The RPS in the adjuvant group was 42%, and no significant difference was seen in survival between this and the PBS group. Moreover, significantly higher antibody titres in the serum and significantly lower bacterial loads in the spleen were detected in the vaccinated fish by ELISA and qPCR, respectively. These findings highlight the potential of autogenous vaccines for controlling francisellosis in tilapia.

Antibody responses in furunculosis patients vaccinated with autologous formalin-killed Staphylococcus aureus.

Autologous vaccines (short: autovaccines) have been used since the beginning of the 20th century to treat chronic staphylococcal infections, but their mechanisms of action are still obscure. This prospective pilot study involved four patients with furunculosis who were vaccinated with autologous formalin-killed Staphylococcus aureus cells. Vaccines were individually prepared from the infecting S. aureus strain and repeatedly injected subcutaneously in increasing doses over several months. We characterized the virulence gene repertoire and spa genotype of the infecting and colonising S. aureus strains. Serum antibody responses to secreted and surface-bound bacterial antigens were determined by two-dimensional immunoblotting and flow-cytometry based assays (Luminex). All patients reported clinical improvement. Molecular characterization showed that all strains isolated from one patient over time belonged to the same S. aureus clone. Already before treatment, there was robust antibody binding to a broad range of staphylococcal antigens. Autovaccination moderately boosted the IgG response to extracellular antigens in two patients, while the antibody response of the other two patients was not affected. Similarly, vaccination moderately enhanced the antibody response against some staphylococcal surface proteins, e.g. ClfA, ClfB, SdrD and SdrE. In summary, autovaccination only slightly boosted the pre-existing serum antibody response, predominantly to bacterial surface antigens.

Increased pulmonary tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-17A responses compensate for decreased gamma interferon production in anti-IL-12 autovaccine-treated, Mycobacterium bovis BCG-vaccinated mice.

Interleukin-12 (IL-12) and IL-23 (which share a p40 subunit) are pivotal cytokines in the generation of protective Th1/Th17-type immune responses upon infection with the intracellular pathogen Mycobacterium tuberculosis. The role of IL-12 and IL-23 in protection conferred by the tuberculosis vaccine Mycobacterium bovis bacillus Calmette-Guérin (BCG) is, however, less well documented. By using an autovaccine approach, i.e., IL-12p70 cross-linked with ovalbumin and PADRE peptide formulated with the GSK proprietary adjuvant system AS02(V), we could specifically neutralize IL-12 while leaving the IL-23 axis intact. Neutralization of IL-12 before M. tuberculosis challenge rendered C57BL/6 mice highly susceptible, resulting in 30-fold-higher CFU in spleen and lungs and accelerated mortality. In contrast, neutralization of IL-12 in BCG-vaccinated mice prior to M. tuberculosis challenge only marginally affected vaccine-mediated protection. Analysis of cytokine production in spleen and lungs 3 weeks post-TB challenge by enzyme-linked immunosorbent assay and functional and flow cytometric assays showed significantly reduced mycobacterium-specific gamma interferon (IFN-γ) responses in M. tuberculosis-infected and BCG-vaccinated mice that had been treated with the autovaccine. Purified protein derivative-induced tumor necrosis factor alpha (TNF-α), IL-6, and IL-17A levels, however, were highest in lungs from BCG-vaccinated/IL-12-neutralized animals, and even unstimulated lung cells from these mice produced significant levels of the three cytokines. Mycobacterium-specific IL-4 and IL-5 production levels were overall very low, but IL-12 neutralization resulted in increased concanavalin A-triggered polyclonal secretion of these Th2-type cytokines. These results suggest that TNF-α, IL-6, and IL-17A may be more important pulmonary effector molecules of BCG-mediated protection than IFN-γ in a context of IL-12 deficiency.

A randomized and blinded field trial to assess the efficacy of an autogenous vaccine to prevent naturally occurring infectious bovine keratoconjunctivis (IBK) in beef calves.

A randomized and blinded 2-arm parallel trial was conducted to assess the efficacy of an autogenous vaccine to prevent naturally occurring infectious bovine keratoconjunctivis (IBK) in beef calves. The trial was managed between May and November 2008 on university owned farms in Iowa and Wisconsin. The vaccine at Iowa contained Moraxella bovoculi (M. bovoculi) while the organism used in the Wisconsin herds vaccine was Branhemella ovis (B. ovis renamed M. ovis). Calves born between January and May 2008 without visible corneal lesions were randomized to receive an autogenous vaccine or placebo vaccine using a computer generated sequence. Two subcutaneous doses were administered 21-28 days apart. Allocation to treatment was concealed using bottles marked A or B. Staff were blind to the treatment allocation. The primary outcome was IBK cumulative incidence over the study period. The secondary outcome was weaning weight. Only the Iowa herd met the criteria for an "at-risk" herd i.e. >15% IBK in unvaccinated calves and M. bovoculi isolation from IBK cases. Analysis was "per-protocol". The cumulative incidence of IBK was 47/105 in vaccinated calves and 49/109 in unvaccinated calves (unadjusted odds ratio=0.99, 95% CI: 0.58-1.70). Weight at weaning did not differ between the vaccinated cohort 148kg (SD: +/-27) and unvaccinated cohort 146kg (SD: +/-26) (unadjusted beta=1.5 and 95% CI: -5.5 to 8.6). Results indicate that the autogenous vaccine was ineffective in this study population.

The preventive effect of adjuvant-free administration of TNF-PADRE autovaccine on collagen-II-induced rheumatoid arthritis in mice.

Adjuvants are necessary to elicit high titers of antibodies in vaccine-immunization procedures. We previously developed a mouse tumor necrosis factor-alpha (TNF-alpha) autovaccine (mTNF-PADRE) capable of inducing anti-TNF-alpha antibodies. In this study, we investigated the therapeutic effect of adjuvant-free administration of the autovaccine on collagen-type-II-induced rheumatoid arthritis (CIA) in mice. Our results showed that the vaccine could ameliorate the symptoms of CIA in mice. In addition, this study suggests that it is possible to control the antibody levels in mice immunized with mTNF-PADRE without adjuvant.