Safety and probiotic evaluation of two Bacillus strains producing antioxidant compounds.

Bacillus species are becoming increasingly relevant for use as probiotics or feed additives where their heat stability can ensure survival in the food matrix or enable long-term storage at ambient temperature. Some Bacillus species are pigmented and in this study, we have examined two strains, one Bacillus pumilus (pigmented red) and the other Bacillus megaterium (pigmented yellow) for their safety for potential use in humans as dietary supplements. In addition, we have set out to determine if they might confer any potential health benefits. Both strains produce C30 carotenoids while the B. pumilus strain also produced large quantities of riboflavin equivalent to genetically modified Bacillus strains and most probably contributing to this strain's pigmentation. Riboflavin's and carotenoids are antioxidants, and we have evaluated the ability of vegetative cells and/or spores to influence populations of Faecalibacterium prausnitzii in the colon of mice. While both strains increased levels of F. prausnitzii, spores of the B. pumilus strain produced a significant increase in F. prausnitzii levels. If found to be reproducible in humans such an effect might, potentially, confer health benefits particularly for those suffering from inflammatory bowel disease.

Protection of Penaeus monodon against white spot syndrome by continuous oral administration of a low concentration of Bacillus subtilis spores expressing the VP28 antigen.

In this study, Bacillus subtilis spores expressing a chimeric protein, CotB-VP28, were used as a probiotic vaccine to protect black tiger shrimps (Penaeus monodon) against white spot syndrome virus (WSSV) infection. Oral administration of pellets coated with CotB-VP28 spores (at ≥1 × 109  CFU per g pellet) to shrimps induced immune-relating phenoloxydase activity (PO) in shrimps after 14 days of feeding (prior challenge) and at day 3 post challenge (1·26 and 1·70 fold increase respectively). A 75% protection rate was obtained by continuous feeding of the spore-coated pellets at ≥1 × 109  CFU per g for 14 days prior to WSSV challenge and during all the postchallenge period. Even when the amount of CotB-VP28 spores in feed pellets was reduced down to ≥5 × 107  CFU per g and ≥1 × 106  CFU per g, relatively high protection rates of 70 and 67·5%, respectively, were still obtained. By contrast, feeding pellets without spores (untreated group) and with naked spores (PY79 group) at ≥1 × 109  CFU per g could not protect shrimps against WSSV. These data suggest that supplementation of CotB-VP28 spores at low dose of ≥1 × 106  CFU per g could be effective as a prophylactic treatment of WSS for black tiger shrimps. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports the protective efficacy of Bacillus subtilis CotB-VP28 spores on black tiger shrimps (Penaeus monodon) against white spot syndrome virus infection. Oral administration of pellets coated with CotB-VP28 spores (≥1 × 109  CFU per g) conferred 75% protection after white spot syndrome virus challenge. Even after reducing CotB-VP28 spores in feed pellets to ≥1 × 106  CFU per g, 67·5% protections was still obtained. These data indicate that supplementation of CotB-VP28 spores at a low dose of ≥1 × 106  CFU per g could be effective in prophylaxis against white spot syndrome in black tiger shrimps.

Evaluation of Bacillus subtilis strains as probiotics and their potential as a food ingredient.

Spores of Bacillus subtilis including one strain used commercially were evaluated for their potential value as a probiotic and as potential food additives. Two isolates of B. subtilis examined here were HU58, a human isolate and PXN21, a strain used in an existing commercial product. Compared to a domesticated laboratory strain of B. subtilis both isolates carried traits that could prove advantageous in the human gastro-intestinal tract. This included full resistance to gastric fluids, rapid sporulation and the formation of robust biofilms. We also showed that PXN21 spores when administered weekly to mice conferred non-specific cellular immune responses, indicative signs of the stimulation of innate immunity. Spores mixed in wholemeal biscuits were found to survive baking at 235 °C for 8 minutes with only a 1-log reduction in viability. That spores can survive the baking process offers the possibility of using spores as probiotic supplements in a range of novel food products.

Killer-cell immunoglobulin-like receptor genotyping and HLA killer-cell immunoglobulin-like receptor-ligand identification by real-time polymerase chain reaction.

The effector function of natural killer (NK) cells is modulated by surface expression of a range of killer-cell immunoglobulin-like receptors (KIRs) that interact with human leukocyte antigen (HLA) class I ligands. We describe the use of real-time polymerase chain reaction (PCR) assays that allow easy and quick detection of 16 KIR genes and the presence/absence of KIR-ligands based on allelic discrimination at codon 80 in the HLA-A/B Bw4 and HLA-C C1/C2 genes. These methods overcome the tedious and expensive nature of conventional KIR genotyping and HLA class I typing using sequence-specific primer (SSP) PCR, sequence-specific oligonucleotide (SSO) hybridization or sequence-based typing (SBT). Using these two cost-effective assays, we measured the frequencies of KIRs, KIR-ligands and KIR/KIR-ligand pairs in a cohort of Black women recruited in South Africa.

The safety of Bacillus subtilis and Bacillus indicus as food probiotics.

AIMS: To conduct in vitro and in vivo assessments of the safety of two species of Bacillus, one of which, Bacillus subtilis, is in current use as a food supplement. METHODS AND RESULTS: Cultured cell lines, Caco-2, HEp-2 and the mucus-producing HT29-16E cell line, were used to evaluate adhesion, invasion and cytotoxicity. The Natto strain of B. subtilis was shown to be able to invade and lyse cells. Neither species was able to adhere significantly to any cell line. The Natto strain was also shown to form biofilms. No strain produced any of the known Bacillus enterotoxins. Disc-diffusion assays using a panel of antibiotics listed by the European Food Safety Authority (EFSA) showed that only Bacillus indicus carried resistance to clindamycin at a level above the minimum inhibitory concentration breakpoints set by the EFSA. In vivo assessments of acute and chronic dosing in guinea pigs and rabbits were made. No toxicity was observed in animals under these conditions. CONCLUSIONS: Bacillus indicus and B. subtilis should be considered safe for oral use although the resistance of B. indicus to clindamycin requires further study. SIGNIFICANCE AND IMPACT OF THE STUDY: The results support the use of B. subtilis and B. indicus strains as food supplements.

The use of alternatives to animal tests in developing countries.

Consideration of alternative methods for animal tests in developing countries is important because good quality laboratory animals and proper animal facilities are not always sufficiently available to perform the currently required quality control tests. In vitro methods have been implemented at the National Institute of Vaccines and Biological Substances (IVAC) in Vietnam. These include serological tests (such as the Toxin Binding Inhibition test and the VERO Cell test) for the estimation of potency of Tetanus and Diphtheria toxoid vaccines and for the evaluation of vaccine field trials. Currently, an inhibition ELISA test to determine anti-rabies activity in equine rabies immunoglobulin preparations is being developed with the long-term goal of its introduction in Vietnam. The results from validation studies are promising and have contributed to decisions made by the National Control Authority to replace imported DPT vaccines in the EPI program with Vietnamese-produced DPT vaccines. This paper summarizes IVAC's experience in introducing alternatives in Vietnam over the last 10 years and reports on the various local validation studies which were performed during this period.

Tetanus antibodies measured by the toxin binding inhibition test (ToBI) in mothers and children in the Neonatal Tetanus Program in Vietnam.

The immunoresponse to vaccination in the Neonatal Tetanus Program (NNT) for pregnant women was studied in Vietnam using the Toxin Binding Inhibition Test (ToBI). The vaccination schedule consisted of two primary doses of adsorbed tetanus toxoid (TT) vaccine given with a one month interval. The seroconversion rate in the women was 98%. Two and a half months after birth, 63% of the children born from these women had tetanus antibody values higher than 0.01 IU/ml. Four women who had anti-tetanus titres < 0.01 IU/ml at delivery, despite two doses of primary vaccination, received a third booster with vaccine one year after the first injection. Their antibody levels were well above 0.01 IU/ml one month after this additional booster, suggesting that (when economically feasible) a third TT injection could be considered into the NNT to confer optimal anti-tetanus antibody levels in women for subsequent pregnancies. This study confirmed the effectiveness of the TT vaccines investigated and indicates their potential to replace, in immunosurveillance studies under field conditions, the in vivo mouse neutralisation test by in vitro alternative methods such as the ToBI test.

Validation of the combined toxin-binding inhibition test for determination of neutralizing antibodies against tetanus and diphtheria toxins in a vaccine field study in Viet Nam.

Determination of seroconversion and measurement of protective antibody levels in children against vaccine components are essential for gauging and monitoring the efficacy of paediatric vaccination programmes. For this purpose, we assessed the combined toxin-binding inhibition (ToBI) test for determining neutralizing antibodies to tetanus and diphtheria in a diphtheria-pertussis-tetanus (DPT) vaccine field trial in Viet Nam. A simple procedure involving collection of blood samples on filter-paper was found to be a suitable alternative to collection by venepuncture, despite a reduction in the sensitivity of the ToBI test as a result of the step necessary to elute the antibodies from the filter-paper. The results obtained demonstrate that the ToBI test can feasibly be carried out under field conditions. Preliminary results obtained with the ToBI test in DPT field trials indicate that a fourth dose of DPT vaccine one year after the third dose should be considered by developing countries.

PIP: In Vietnam, health workers collected blood samples from adults working at the National Institute of Vaccines and Biological Substances in Nha Trang and Dalat and from healthy unvaccinated infants 3-9 months old from the district areas/provinces of Tien Giang and Lam Dong to assess the combined toxin-binding inhibition (ToBI) test for determining neutralizing antibodies to tetanus and diphtheria in a diphtheria-pertussis-tetanus (DPT) vaccine field trial. Researchers also aimed to validate the use of a simple filter-paper blood collection method. There was a necessary step to elute the antibodies from the filter-paper, which reduced the sensitivity of the ToBI test. Nevertheless, in the ToBI test, blood collected on filter-paper yielded similar antibody titer estimations as those obtained by venepuncture. The Biotek method to estimate antibody titers yielded higher correlation coefficients than the OD50 method: tetanus (0.998 vs. 0.98) and diphtheria (0.956 vs. 0.95). One month after the third injection, all the children had antitoxin titers greater than 0.06IU/ml. By one year after the third injection, only 45% had antitoxin titers greater than 0.06IU/ml for diphtheria while all still had antitoxin titer levels above this value for tetanus. This suggests that health providers should consider administering a fourth dose of DPT vaccine one year after the third dose. These findings indicate that the ToBI test can be conducted under field conditions.

Methods for the purification of equine rabies immunoglobulin: effects on yield and biological activity.

Since rabies is still a major cause of human death in many developing countries and the implementation of recommended post-exposure prophylaxis by vaccination and specific immunoglobulin therapy is largely hampered by its high cost, the development of cheap rabies vaccines and immunoglobulin preparation are a high priority in these countries. In this paper various purification methods of equine rabies immunoglobulin based on different principles are compared with respect to their effect on final yield and biological activity. It is shown that a combination of ammonium sulphate (AS) precipitation and DEAE ion exchange chromatography results in an acceptable recovery rate of biological activity and a product of relatively high purity. Although affinity chromatography with protein G in combination with AS precipitation results in a similar recovery rate and a product of considerably higher purity, the cost of this procedure may be prohibitive for routine use in most developing countries. The effects of pepsin digestion time on the biological activity of the product and on the reduction of intact horse Ig are also studied. The desirability of this digestion procedure with respect to reduction of adverse side effects and efficacy of the product for post-exposure treatment is discussed.