Inactivated vaccine against viral hemorrhagic septicemia (VHS) emulsified with squalene and aluminum hydroxide adjuvant provides long term protection in olive flounder (Paralichthys olivaceus)

Viral hemorrhagic septicemia (VHS) in olive flounder (Paralichthys olivaceus) remains an unsolved health problem in Korean aquaculture. Vaccination plays a significant role in modern aquaculture, and the duration of protection provided is of vital importance. Here, we have demonstrated the efficacy, duration of protection and safety of an inactivated vaccine emulsified with squalene (5%) and aluminum hydroxide (0.5%). The inactivated VHS vaccine provided a moderate protection of 37% and 47% relative percent survival (RPS) at 4 and 10 weeks post vaccination (wpv). Addition of squalene and aluminum hydroxide into inactivated VHS vaccine clearly enhanced the level of protection showing 58% and 83% RPS at 4 and 10 wpv, respectively, indicating the need for adjuvants to enhance the efficacy. The vaccinated fish showed significant protection at 3, 6, 12, 18, 24, and 40 wpv (except week 57) than non-vaccinated fish to an intraperitoneal challenge of 10^7.1TCID₅₀/fish at 15 °C, with RPS of 60%, 64%, 71%, 55%, 52% and 50% (45% at 57 week), respectively, covering the duration of natural outbreak. Fish challenged at 18 wpv at 6 °C showed 56% RPS and protection at a low temperature. The antibody titer was high at 3 wpv with an OD of 1.08 ± 0.13, but decreased gradually and was undetectable by 24 wpv. The vaccine formulation was safe without injection site reactions, adhesions, or pigmentation observed at 6, 12, 18, or 24 wpv. Inflammatory reactions were observed in the spleen intestine at 6 and 12 wpv but were similar as control by 24 wpv. These results confirm that this vaccine is efficient and safe for olive flounder and could offer an appropriate strategy to prevent VHS without causing side effects.     

Production and efficacy of an Aeromonas hydrophila recombinant S-layer protein vaccine for fish

A recombinant protein for the S-layer protein of Aeromonas hydrophila was produced and its ability to protect common carp Cyprinus carpio L. against six virulent isolates of A. hydrophila was assessed. A group of 120 carp (30–40 g) were vaccinated intra-peritoneally with 0.1 ml of adjuvanted vaccine (30 μg protein per fish). Another group of 120 carp were injected with 0.1 ml of PBS-adjuvant mixture to serve as controls. Twenty fish from each group were challenged with each one of six virulent isolates of A. hydrophila 35 days post-vaccination. The fish were maintained in 12 separate tanks before terminating the experiment at 16 days post-challenge. The relative percentage survival (RPS) for the six isolates of A. hydrophila ranged from 56 to 87%. The difference in survival rate of fish challenged with four of the isolates was statistically significant in vaccinated fish compared to control fish, when analysed using a Chi-square test. The results of the study suggest that the recombinant S-layer protein of A. hydrophila could be useful as a vaccine antigen to protect fish against different isolates of this pathogenic bacterium.     

First data on Eurasian wild boar response to oral immunization with BCG and challenge with a Mycobacterium bovis field strain

The Eurasian wild boar (Sus scrofa) is considered a reservoir for bovine tuberculosis (bTB) caused by Mycobacterium bovis and closely related members of the Mycobacterium tuberculosis complex in south-central Spain. The vaccination of wildlife with BCG offers an alternative to culling and to movement restriction for the control of bTB among wildlife reservoirs. In this study, we hypothesized that oral BCG immunization of wild boar would affect the expression of immunoregulatory genes and confer protection against M. bovis. Three groups were used to describe the infection, pathological findings and gene expression profiles in wild boar: BCG-vaccinated and M. bovis-challenged (vaccinated challenged group; N = 6), non-vaccinated and M. bovis-challenged (non-vaccinated challenged group; N = 4), and non-vaccinated and mock-infected (control group; N = 2) animals. M. bovis was isolated from 50% (3/6) and 75% (3/4) of vaccinated challenged and non-vaccinated challenged animals, respectively. All four wild boar from the non-vaccinated challenged group developed bTB-compatible lesions 114 days after challenge. In contrast, only 50% of vaccinated challenged wild boar developed lesions. The PBMC mRNA levels of IL4, RANTES, C3, IFN-gamma and methylmalonyl-CoA mutase (MUT) were analyzed at several days post-vaccination (dpi). When vaccinated challenged animals were compared to controls, all five genes were significantly upregulated at the time of M. bovis infection at 186 dpi but IFN-gamma levels were also upregulated at 11 and 46 dpi. The C3 and MUT mRNA levels were higher at 46 dpi, and 11 and 186 dpi, respectively, in vaccinated protected wild boar when compared to non-vaccinated challenged animals. At the end of the experiment (300 dpi), the mRNA levels of selected genes were lower in non-vaccinated challenged animals when compared to control wild boar. Exposing wild boar to a dose of 10⁴ cfu of M. bovis by the oropharyngeal route is an adequate protocol to produce an infection model in this species. Our results suggested that oral BCG immunization of wild boar results in the upregulation of immunoregulatory genes that may be associated with protective response to M. bovis infection in this species. More studies on vaccine efficacy, delivery, and safety will be needed to confirm if oral vaccination with BCG could be used in bTB control programs for reducing M. bovis infection and clinical disease in wild boar.     

Potential use of a recombinant replication-defective adenovirus vector carrying the C-terminal portion of the P97 adhesin protein as a vaccine against Mycoplasma hyopneumoniae in swine

Mycoplasma hyopneumoniae causes severe economic losses to the swine industry worldwide and the prevention of its related disease, enzootic porcine pneumonia, remains a challenge. The P97 adhesin protein of M. hyopneumoniae should be a good candidate for the development of a subunit vaccine because antibodies produced against P97 could prevent the adhesion of the pathogen to the respiratory epithelial cells in vitro. In the present study, a P97 recombinant replication-defective adenovirus (rAdP97c) subunit vaccine efficiency was evaluated in pigs. The rAdP97c vaccine was found to induce both strong P97 specific humoral and cellular immune responses. The rAdP97c vaccinated pigs developed a lower amount of macroscopic lung lesions (18.5 ± 9.6%) compared to the unvaccinated and challenged animals (45.8 ± 11.5%). rAdP97c vaccine reduced significantly the severity of inflammatory response and the amount of M. hyopneumoniae in the respiratory tract. Furthermore, the average daily weight gain was slightly improved in the rAdP97c vaccinated pigs (0.672 ± 0.068 kg/day) compared to the unvaccinated and challenged animals (0.568 ± 0.104 kg/day). A bacterin-based commercial vaccine (Suvaxyn^® MH-one) was more efficient to induce a protective immune response than rAdP97c even if it did not evoke a P97 specific immune response. These results suggest that immunodominant antigens other than P97 adhesin are also important in the induction of a protective immune response and should be taken into account in the future development of M. hyopneumoniae subunit vaccines.     

Development and efficacy of a novobiocin-resistant Streptococcus iniae as a novel vaccine in Nile tilapia (Oreochromis niloticus)

A novel attenuated Streptococcus iniae vaccine was developed from a virulent strain of Streptococcus iniae (ISET0901) through selection for novobiocin resistance (named ISNO). The safety of ISNO was then evaluated in Nile tilapia (Oreochromis niloticus) through intraperitoneal (IP) injection. When male tilapia (average weight 10 g) were IP injected with 2 × 10⁷ colony-forming units (CFU) of the attenuated S. iniae vaccine strain, no fish died. However, when the same age and size matched tilapia were IP injected with 2 × 10⁷ and 1 × 10⁵ CFU of the virulent parent strain of S. iniae, 100 and 90% fish died, respectively. Backpassage safety studies revealed that ISNO was unable to revert back to a virulent state. When IP vaccinated fish were challenged by the virulent ISET0901 strain of S. iniae, relative percent survival (RPS) values of vaccinated fish at 14, 28, 60, 90, and 180 days post ISNO vaccination (dpv) were 100, 100, 100, 89, and 75%, respectively, The RPS values of ISNO vaccinated fish (IP vaccination) against infections by five heterologous virulent strains of S. iniae (F3CB, 102F1K, 405F1K, IF6, and ARS60) at 60 dpv were 78, 90, 100, 100, and 100%, respectively. When tilapia were IP vaccinated by ISNO at dose of 1 × 10², 1 × 10³, 1 × 10⁴, 1 × 10⁵, 1 × 10⁶, and 1 × 10⁷ CFU/fish, RPS values at 28 dpv were 81, 94, 100, 100, 100, and 100%, respectively. At 28 dpv, RPS of vaccinated fish by ISNO through bath immersion (1 × 10⁷ CFU/ml) was 88%. ELISA results revealed that protection elicited by ISNO was due to antibody- as well as cell- mediated immunity. Our results suggest that ISNO could be used as a novel safe and efficacious vaccine to protect Nile tilapia from S. iniae infections.     

Potential for a live red seabream iridovirus (RSIV) vaccine in rock bream Oplegnathus fasciatus at a low rearing temperature

Serious economic losses have occurred in fingerlings and market-sized rock bream Oplegnathus fasciatus in Korea due to red seabream iridovirus (RSIV) infection. We demonstrated previously that viral multiplication in fish is downregulated by maintaining fish at far from optimum temperatures at the onset of disease. We applied this concept to develop a live RSIV vaccine in rock bream. Mortalities in rock bream that were inoculated with RSIV and reared at 21–30 °C were ≥90%, whereas no mortality was observed in fish that received an RSIV inoculation and were reared at ≤18 °C. RSIV kinetics revealed that RSIV multiplied rapidly in fish reared at 24.3 ± 1.3 °C, and achieved the critical level for rock bream (approximately 10^9.0 genomes/mg) within 28 days. In contrast, the RSIV genome was detected on day 10 in fish that received an RSIV inoculation at 15.5 °C, and peaked on day 28 at 10^{5.91 ± 0.54} genomes/mg, then decreasing gradually, and were then maintained under the detection level beginning on day 84 after RSIV inoculation. Furthermore, the fish surviving the RSIV infection at low rearing temperature were strongly protected from re-challenge with homologous RSIV; the threshold level of RSIV for rock bream to mount a protective immune response was ≤10^5.4 genomes/mg. Cohabitation experiments revealed that the spread of RSIV from rock bream vaccinated with a live RSIV could be low if it is limited to fish in the late stage (≥84 days of elapse) after vaccination. Thus, it was concluded that when rock bream are reared at ≤18 °C and inoculated with RSIV, the survivors can mount a protective immune response against RSIV, suggesting a positive effect of a live RSIV vaccine for rock bream.     

Comparison of carotenoid and anthocyanin profiles of raw and boiled Solanum tuberosum and Solanum phureja tubers

Tubers of the closely related species Solanum tuberosum (potatoes) and Solanum phureja belong to the staff of life for humans. The coloration of the often very colorful tubers is caused by carotenoids (especially xanthophylls) and anthocyanins. The new S. phureja cultivars Mayan Gold and Mayan Twilight, the new S. tuberosum cultivar Red Laura and the traditional S. tuberosum variety Shetland Black were analyzed for their pigment content. The dominant anthocyanin in the red-skinned cultivars Red Laura and Mayan Twilight was characterized as pelargonidin-3-coumarylrutinoside-5-glucoside by LC-ESI-MS. The blue pigment of the Shetland Black cultivar was petunidin-3-coumaroylrutinoside-5-glucoside. Red Laura contained 10 mg anthocyanins g⁻¹ dry weight and Shetland Black 31 mg anthocyanins g⁻¹ dry weight. The main carotenoids in raw tubers are 9-cis-violaxanthin, lutein and one unidentified carotenoid in all S. tuberosum and S. phureja cultivars. Minor compounds were identified as neoxanthin and neochrome. Additionally, zeaxanthin and one further unidentified carotenoid could be found in Red Laura and Mayan Gold. No differences in the carotenoid composition between peel and parenchyma could be found. Total carotenoid content ranged from 2.57 ± 0.53 μg g⁻¹ dry weight in Shetland Black to 14.77 ± 2.22 μg g⁻¹ dry weight in Red Laura (calculated as β-carotene-equivalents). In heat processed tubers, high amounts of carotenoids either changed from all-trans to 9-cis and 13-cis-isomeric form or was degraded. The total pigment content was decreased by heat processing in all cultivars. 8.23 ± 2.98 μg g⁻¹ dry weight could be found in Mayan Twilight and 1.51 ± 0.31 μg carotenoids g⁻¹ dry weight in Shetland Black.     

Risk reduction assessment of waterborne Salmonella and Vibrio by a chlorine contact disinfectant point-of-use device

Unsafe drinking water continues to burden developing countries despite improvements in clean water delivery and sanitation, in response to Millennium Development Goal 7. Salmonella serotype Typhi and Vibrio cholerae bacteria can contaminate drinking water, causing waterborne typhoid fever and cholera, respectively. Household water treatment (HWT) systems are widely promoted to consumers in developing countries but it is difficult to establish their benefits to the population for specific disease reduction. This research uses a laboratory assessment of halogenated chlorine beads treating contaminated water to inform a quantitative microbial risk assessment (QMRA) of S. Typhi and V. cholerae disease in a developing country community of 1000 people. Laboratory challenges using seeded well water resulted in log₁₀ reductions of 5.44 (±0.98 standard error (SE)) and 6.07 (±0.09 SE) for Salmonella serotype Typhimurium and V. cholerae, respectively. In well water with 10% sewage and seeded bacteria, the log₁₀ reductions were 6.06 (±0.62 SE) and 7.78 (±0.11 SE) for S. Typhimurium and V. cholerae, respectively. When one infected individual was contributing to the water contamination through fecal material leaking into the water source, the risk of disease associated with drinking untreated water was high according to a Monte Carlo analysis: a median of 0.20 (interquartile range [IQR] 0.017–0.54) for typhoid fever and a median of 0.11 (IQR 0.039–0.20) for cholera. If water was treated, risk greatly decreased, to a median of 4.1 × 10⁻⁷ (IQR 1.6 × 10⁻⁸ to 1.1 × 10⁻⁵) for typhoid fever and a median of 3.5 × 10⁻⁹ (IQR 8.0 × 10⁻¹⁰ to 1.3 × 10⁻⁸) for cholera. Insights on risk management policies and strategies for public health workers were gained using a simple QMRA scenario informed by laboratory assessment of HWT.     

Evaluation of the adjuvant properties of Astragalus membranaceus and Scutellaria baicalensis GEORGI in the immune protection induced by UV-attenuated Toxoplasma gondii in mouse models

Human vaccines are not available and current anti-toxoplasma treatment is disappointing. To investigate the possible adjuvant effect of aqueous extracts obtained from medicinal herbs of Astragalus membranaceus (Am) and Scutellaria baicalensis GEORGI (Sb) on the immune response to Toxoplasma gondii in the mouse models induced by ultraviolet (UV)-attenuated T. gondii, this paper studies the possible vaccination strategies to help combat infections with Toxoplasma and looking towards developing new vaccine and approaches. We used UV-attenuated T. gondii (UV-T.g) of RH strain as a vaccine and the extracts of Am (AmE) and Sb (SbE) as adjuvant. Mice were infected by intraperitoneal (i.p.) injection of 10² RH tachyzoites alone (infected controls), infected and treated with AmE (T.g + AmE) and SbE (T.g + SbE), respectively; and mice immunized i.p. with UV-T.g alone, UV-T.g co-administrated with AmE (UV-T.g + AmE) or SbE (UV-T.g + SbE), and then challenged with T.g, respectively. The animal survival time, parasite burden in peritoneal lavage fluids, liver histopathological analysis, and levels of serum antibodies among the groups were compared after either infection or challenge. The results showed that, compared to infected controls, infected mice treated with AmE or SbE, or vaccinated mice and then challenged, had significantly prolonged survival time, decreased parasite burden, improved liver histopathological score, and increased Th1-type cellular immune response; furthermore, vaccinated mice co-administrated with AmE or SbE had even longer survival, lower parasite burden, lower liver histopathological score, and higher Th1 response after challenge. Our data demonstrated that the protective immunity of UV-attenuated T. gondii could be markedly enhanced by AmE or SbE co-administration, which suggests that both AmE and SbE may have the potential to be used as effective vaccine adjuvant.     

Attenuated Francisella asiatica iglC mutant induces protective immunity to francisellosis in tilapia

Francisella asiatica is a Gram-negative, facultative intracellular bacteria that causes fish francisellosis. Fish francisellosis is a severe sub-acute to chronic granulomatous disease with high mortalities and high infectivity rates in cultured and wild fish. To date, there is no approved vaccine for this widespread emergent disease. The goal of this study was to characterize the efficacy of a defined F. asiatica mutant (ΔiglC) as a live attenuated vaccine against subsequent immersion challenge with the wild-type (WT) organism. In previous work, the ΔiglC was found to be attenuated upon intraperitoneal injection and immersion challenges. In vitro, the ΔiglC exhibited reduced growth in tilapia head-kidney derived macrophages, and was significantly attenuated (p < 0.001) as demonstrated by cytopathogenic and apoptosis assays. In this study, the ΔiglC was tested to determine its ability to protect tilapia against challenge with high doses (lethal dose 80) of WT bacteria. Naïve tilapia vaccinated by immersion with a suspension of the ΔiglC and subsequently challenged with WT F. asiatica were protected (90% mean percent survival) from the lethal challenges. F. asiatica-specific antibodies produced in response to immunization with the ΔiglC were subsequently found to protect naïve tilapia against high-dose F. asiatica challenge in passive immunization experiments. Significant protection (p < 0.001) was obtained when fish were passively immunized and challenged with 10⁴ and 10⁵ CFU/fish of WT F. asiatica; but not when challenged with 10⁶ CFU/fish. This is the first report of a defined live attenuated strain providing protection against F. asiatica in fish.     

Evaluation of the immunogenicity and safety of Brucella melitensis B115 vaccination in pregnant sheep

In spite of its limitations, Rev.1 is currently recognized as the most suitable vaccine against Brucella melitensis (the causative agent of ovine and caprine brucellosis). However, its use is limited to young animals when test-and-slaughter programs are in place because of the occurrence of false positive-reactions due to Rev.1 vaccination. The B. melitensis B115 rough strain has demonstrated its efficacy against B. melitensis virulent strains in the mouse model, but there is a lack of information regarding its potential use in small ruminants for brucellosis control. Here, the safety and immune response elicited by B115 strain inoculation were evaluated in pregnant ewes vaccinated at their midpregnancy. Vaccinated (n = 8) and non-vaccinated (n = 3) sheep were periodically sampled and analyzed for the 108 days following inoculations using tests designed for the detection of the response elicited by the B115 strain and routine serological tests for brucellosis [Rose Bengal Test (RBT), Complement Fixation Test (CFT) and blocking ELISA (ELISAb)]. Five out of the 8 vaccinated animals aborted, indicating a significant abortifacient effect of B115 inoculation at midpregnancy. In addition, a smooth strain was recovered from one vaccinated animal, suggesting the occurrence of an in vivo reversion phenomenon. Only one animal was positive in both RBT and CFT simultaneously (91 days after vaccination) confirming the lack of induction of cross-reacting antibody responses interfering with routine brucellosis diagnostic tests in most B115-vaccinated animals.     

Evaluation of recombinant Mycoplasma hyopneumoniae P97/P102 paralogs formulated with selected adjuvants as vaccines against mycoplasmal pneumonia in pigs

Pig responses to recombinant subunit vaccines containing fragments of eight multifunctional adhesins of the Mycoplasma hyopneumoniae (Mhp) P97/P102 paralog family formulated with Alhydrogel^® or Montanide™ Gel01 were compared with a commercial bacterin following experimental challenge. Pigs, vaccinated intramuscularly at 9, 12 and 15 weeks of age with either of the recombinant formulations (n = 10 per group) or Suvaxyn^® M. hyo (n = 12), were challenged with Mhp strain Hillcrest at 17 weeks of age. Unvaccinated, challenged pigs (n = 12) served as a control group. Coughing was assessed daily. Antigen-specific antibody responses were monitored by ELISA in serum and tracheobronchial lavage fluid (TBLF), while TBLF was also assayed for cytokine responses (ELISA) and bacterial load (qPCR). At slaughter, gross and histopathology of lungs were quantified and damage to epithelial cilia in the porcine trachea was evaluated by scanning electron microscopy. Suvaxyn^® M. hyo administration induced significant serological responses against Mhp strain 232 whole cell lysates (wcl) and recombinant antigen F3_P216, but not against the remaining vaccine subunit antigens. Alhydrogel^® and Montanide™ Gel01-adjuvanted antigen induced significant antigen-specific IgG responses, with the latter adjuvant eliciting comparable Mhp strain 232 wcl specific IgG responses to Suvaxyn^® M. hyo. No significant post-vaccination antigen-specific mucosal responses were detected with the recombinant vaccinates. Suvaxyn^® M. hyo was superior in reducing clinical signs, lung lesion severity and bacterial load but the recombinant formulations offered comparable protection against cilial damage. Lower IL-1β, TNF-α and IL-6 responses after challenge were associated with reduced lung lesion severity in Suvaxyn^® M. hyo vaccinates, while elevated pathology scores in recombinant vaccinates corresponded to cytokine levels that were similarly elevated as in unvaccinated pigs. This study highlights the need for continued research into protective antigens and vaccination strategies that will prevent Mhp colonisation and establishment of infection.     

Local and systemic immune responses in pigs intramuscularly injected with an inactivated Mycoplasma hyopneumoniae vaccine

The immune response induced by intramuscular administration of a commercial inactivated Mycoplasma hyopneumonie whole-cell vaccine (Suvaxyn^®MH One) was investigated in conventional M. hyopneumoniae-free pigs. The animals were assigned randomly to two groups: non-vaccinated and vaccinated. Pigs in the vaccinated group were injected intramuscularly with the vaccine at 7 days of age, whereas non-vaccinated pigs received physiological saline solution (PBS). Pigs were euthanized and necropsied at 30, 36 and 58 days of age. Blood, bronchoalveolar lavage (BAL) fluid, spleen, lung and bronchial lymph nodes (BLN) were collected. Serum and BAL fluid were tested for the presence of antibodies by ELISA. Monomorphonuclear cells from the peripheral blood and tissues were isolated to quantify the T cell subsets by flow cytometry, and cytokine production by ELIspot and ELISA. Antibodies against M. hyopneumoniae were detected in serum of most vaccinated pigs at 30 days of age. M. hyopneumoniae specific IgG, IgM and IgA were detected in BAL fluid from vaccinated animals, but not from control animals. Significantly higher numbers of IL-12 secreting cells were observed in the lung at day 58 in the vaccinated than in the non-vaccinated group (p < 0.05). The number of IL-10 secreting cells from BLN was also higher in the vaccinated group at day 58 (p < 0.05). After restimulation in vitro, lymphocytes from BLN and lungs secreted significantly higher levels of IL-12 in the vaccinated group at day 58. These results show that the vaccine induced both systemic and mucosal cellular and humoral immune responses.     

Efficiency of inactivated vaccines against heartwater in Burkina Faso: Impact of Ehrlichia ruminantium genetic diversity

In order to identify the appropriate strains to use in vaccination trials against heartwater in Burkina Faso, the protective effect of Gardel and Welgevonden strains was assessed against local strains on sheep vaccinated by infection-and-treatment method: Gardel protected significantly against Burkina Faso strains tested (survival rate 59% for immunised sheep vs 13% for control sheep) while Welgevonden did not (survival rate 45% for immunised sheep vs 25% for control sheep). The efficacy of the ISA50 inactivated vaccine, produced under industrial process, was evaluated in sheep during field challenges in two successive years. During year 1, there was a limited protective effect of the Gardel vaccine with 65% of survival rate for the vaccinated group compared to 49% for the control group (N = 153, p = 0.053). During year 2, the vaccine containing Gardel and a local strain gave an increased protective effect compared to the first trial: 72% of the vaccinated animals survived compared to 47% of the naïve animals (N = 173, p < 0.001). There was an important genetic diversity of strains in the field with detection of 11 different map1 genotypes in brains from control and vaccinated sheep post mortem. Map1 genotyping of strains detected in brains from control sheep showed that genotype distribution varied according to time and study areas, which could explain the difference in efficacy of the vaccine.     

Mercury and lead: Assessing the toxic effects on growth and metal accumulation by Lemna minor

Lemna minor L. was used to investigate the toxic effects of Pb and Hg either alone or in various binary mixtures under the static test conditions. A full-strength Jacob culture medium was used for the long-term cultivation of duckweeds and the experiments. Tests were run for 4 and 7 days. The EC₅₀ values for Pb was estimated as 6.8±0.2 mg L⁻¹ and 5.5±0.1 mg L⁻¹ for a 4 and a 7-day test periods, respectively. Hg was much more toxic than Pb with the EC₅₀ of 0.64±0.03 mg L⁻¹ (4 days) and 0.48±0.02 mg L⁻¹ (7 days). The amounts of Pb uptake by the plants were determined by atomic absorption spectrometer (AAS). The cold vapor AAS-technique (CV-AAS) was used for Hg determination. The interactive effect between Pb and Hg on growth was evaluated as additive on the basis of statistical data analysis. However, an antagonistic interaction was observed on the metal accumulation efficiency.     

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.     

Nasal vaccination with attenuated Salmonella expressing VapA: TLR2 activation is not essential for protection against R. equi infection

Virulent strains of Rhodococcus equi have a large plasmid of 80–90 kb, which encodes several virulence-associated proteins (Vap), including VapA, a lipoprotein highly associated with disease. We have previously demonstrated that oral immunisation with attenuated Salmonella enterica Typhimurium strain expressing the antigen VapA (STM VapA+) induces specific and long-term humoral and cellular immunity against R. equi. It was shown that VapA activates Toll-like receptor 2 (TLR2) on macrophages by establishing an interaction that ultimately favours immunity against R. equi infection. The purpose of this study was to evaluate the immune response triggered by nasal immunisation with STM VapA+ and to determine whether TLR2 supports the vaccine effect. We developed an optimised protocol for a single nasal immunisation that conferred protection against R. equi infection in mice, which was manifested by efficient R. equi clearance in challenged animals. Nasal vaccination with STM VapA+ has also induced protection in Tlr2^−/− mice and mice with non-functional TLR4. Moreover, spleen cells of vaccinated mice augmented T-bet expression, as well as the production of IL-12, IFN-γ, nitric oxide and hydrogen peroxide. Notably, the population of CD4⁺ T cells with memory phenotype significantly increased in the spleens of vaccinated mice challenged 1 or 5 months after immunisation. In these animals, the spleen bacterial burden was also reduced. When similar experimental procedures were performed in TLR2 knockout mice, an increase in CD4⁺ T cells with memory phenotype was not observed. Consequently, we conclude that nasal vaccination with attenuated Salmonella expressing the R. equi virulence factor VapA confers long-lasting protection against experimental rhodoccocosis and that TLR2 engagement was not crucial to induce this protection but may be required for a long-term immune response.     

Chemically induced Salmonella enteritidis ghosts as a novel vaccine candidate against virulent challenge in a rat model

Salmonella enteritidis ghosts (SEGs), non-living empty bacterial cell envelopes were generated by using the minimum inhibitory concentration (MIC) of sodium hydroxide (NaOH) and investigated as a vaccine candidate in rats. To determine the immunogenicity and protective efficacy of SEG vaccine, rats were divided into four groups: group A (non-vaccinated control), group B (orally vaccinated), group C (intramuscularly vaccinated) and group D (intramuscularly vaccinated with complete Freund's adjuvant). Vaccination of rats with SEGs induced significant immune responses before and after virulent challenge. Rats vaccinated with SEGs showed significant increases in serum IgG antibodies after challenging with virulent S. enteritidis on week 8 and week 10 (P < 0.01). During the vaccination period, groups B, C and D showed significantly higher serum bactericidal activity (SBA) compared to group A (P < 0.01). Most importantly, bacterial loads in vaccinated groups were significantly lower than in the non-vaccinated group (P < 0.01). In conclusion, these results show that the chemically induced SEGs as a vaccine candidate against virulent challenge.     

Preputial cellular and antibody responses of bulls vaccinated and/or challenged with Tritrichomonas foetus

Systemic and genital immune responses in bulls were determined after infection with Tritrichomonas foetus and systemic vaccination with whole cell antigens. Vaccinated bulls resisted infection, developed IgG1 and IgG2 antibodies against T. foetus in preputial secretions and serum, and had increased MHC II⁺ and CD205⁺ cells (probably dendritic cells), CD3⁺ and CD8⁺ T cells, and B cells including IgG1 and IgA plasma cells in the prepuce. Non-vaccinated bulls challenged with T. foetus were persistently infected and had no detectable antibodies to T. foetus in either preputial secretions or serum for 6 weeks post challenge. We conclude that genital and serum IgG antibodies to T. foetus accounts for resistance of vaccinated bulls to T. foetus infection and that the lack of an antibody response in infected bulls accounts for persistent infection.