A study of the mechanisms of probiotic effect of Bacillus subtilis 8130 strain

The wild-type Bacillus subtilis strain 8130 secreted metabolites that stimulated two to three times the growth of the test cultures of lactic acid bacteria. It exhibited endoglucanase activity that depended on the composition of nutrient medium. The addition of the product of two-stage culturing of B. subtilis 8130 to the diet of pigs (0.2% of fodder weight) made it possible to increase the daily weight gain by 19% and decrease the consumption of mixed fodder by 10%. Digestion of protein, fat, and other organic compounds increased by 3-4% and cellulose by 12%. It was shown that B. subtilis 8130 is a probiotic with targeted action stimulating digestion (primarily the digestion of cellulose). The enrichment of a dry-beer pellet with the product of solid-phase fermentation by bacillus (1 x 10(8) cells per gram dry pellet) allowed the pellet to entered into the diet of a calf (6% of the weight of fodder with probiotic), causing additional weight gain by 12% and a 10% economy of fodder consumption.     

Evaluation of Phytate-Degrading Lactobacillus Culture Administration to Broiler Chickens

Probiotics have been demonstrated to promote growth, stimulate immune responses, and improve food safety of poultry. While widely used, their effectiveness is mixed, and the mechanisms through which they contribute to poultry production are not well understood. Microbial phytases are increasingly supplemented in feed to improve digestibility and reduce antinutritive effects of phytate. The microbial origin of these exogenous enzymes suggests a potentially important mechanism of probiotic functionality. We investigated phytate degradation as a novel probiotic mechanism using recombinant Lactobacillus cultures expressing Bacillus subtilis phytase. B. subtilis phyA was codon optimized for expression in Lactobacillus and cloned into the expression vector pTRK882. The resulting plasmid, pTD003, was transformed into Lactobacillus acidophilus, Lactobacillus gallinarum, and Lactobacillus gasseri. SDS-PAGE revealed a protein in the culture supernatants of Lactobacillus pTD003 transformants with a molecular weight similar to that of the B. subtilis phytase. Expression of B. subtilis phytase increased phytate degradation of L. acidophilus, L. gasseri, and L. gallinarum approximately 4-, 10-, and 18-fold over the background activity of empty-vector transformants, respectively. Phytase-expressing L. gallinarum and L. gasseri were administered to broiler chicks fed a phosphorus-deficient diet. Phytase-expressing L. gasseri improved weight gain of broiler chickens to a level comparable to that for chickens fed a control diet adequate in phosphorus, demonstrating proof of principle that administration of phytate-degrading probiotic cultures can improve performance of livestock animals. This will inform future studies investigating whether probiotic cultures are able to provide both the performance benefits of feed enzymes and the animal health and food safety benefits traditionally associated with probiotics.     

Enhancement of immunity and disease resistance in the white shrimp,Litopenaeus vannamei,by the probiotic,Bacillus subtilis E20

Effects of Bacillus subtilis E20 isolated from fermented soybean on immune parameters and the disease resistance of the white shrimp (Litopenaeus vannamei) after 98 days of B. subtilis E20 feeding were evaluated in this study. Shrimp fed B. subtilis E20-containing diets at concentrations of 10⁶ (E206), 10⁷ (E207), and 10⁸ (E208) cfu kg^?1, respectively, had significantly increased survival rates of 13.3%, 16.7%, and 20%, compared to the control (fed no probiotic) after being challenged with Vibrio alginolyticus. There were no significant differences in the total hemocyte count, respiratory burst, or superoxide dismutase glutathione peroxidase among all treatments. Shrimp fed a higher concentration of the probiotic (E208) exhibited significant increases in phenoloxidase activity, phagocytic activity, and clearance efficiency compared to control shrimp. In addition, B. subtilis E20 showed a weaker inhibitory effect against the growth of Aeromona hydrophila with around a 0.3-cm inhibitory zone, but showed no inhibitory effects against other selected pathogens, such as white shrimp pathogens: V. alginolyticus and Vibrio vulnificus. These results suggest that the increased resistance of shrimp after B. subtilis E20 consumption occurs through immune modifications, such as increases in phenoloxidase activity, phagocytic activity, and clearance efficiency against V. alginolyticus.     

Probiotic bacteria maintain normal growth mechanisms of heat stressed broiler chickens

Probiotics have growth promoting effects even under periods of heat stress challenge. More information is needed to understand the mechanisms by which probiotics maintain the growth performance. The aim of this study was to evaluate the effect of a probiotic based on Bacillus subtilis bacteria on growth related mechanisms of broilers under heat stress conditions. Specifically, growth performance, skeletal bone characteristics, skeletal muscles size, intestinal villus-crypt structure, intestinal bacteria, growth hormone (GH), insulin-like growth factor-1 (IGF-1), cholesterol, and glucose. A total of 1200 one day old Ross 308 male broilers were randomly distributed into 4 treatments, with 12 replicates per treatment and 25 birds per replicate. A 2 × 2 factorial arrangement was used; the main factors were environmental temperature (thermoneutral or heat stress) and diet (control or control + B. subtilis; 3 × 10⁷ cfu/kg of feed). From d 22 to 35 of age, birds were either exposed to thermoneutral conditions (21 °C) or chronic heat stress (30 °C). During the same period, each group was divided into 2 subgroups and fed either the control diet or the B. subtilis supplemented diet. The results demonstrated that B. subtilis had positive effects (P < 0.05) on the body weight gain, feed conversion ratio, villus height, crypt depth, villus surface area, absorptive epithelial cell area and viable counts of intestinal beneficial bacteria. B. subtilis increased (P < 0.05) serum GH, IGF-1 and maintain normal levels of cholesterol and glucose under heat stress conditions. In addition, broilers fed B. subtilis under heat stress conditions exhibited higher (P < 0.05) skeletal muscles size and improved (P < 0.05) tibia traits and lower (P < 0.05) abdominal fat pads deposition compared with the controls. B. subtilis had no effect on rectal temperature under thermoneutral or heat stress conditions. It is concluded that B. subtilis can be used as growth promoters in broilers, particularly during the periods of heat stress conditions.     

Special effects of a complex probiotic containing cellulolytic bacteria Cellulomonas on actively growing rabbits

It was shown that the association of probiotic bacteria of the genuses Bacillus and Cellulomonas form biolayers on the surface of beet marc particles. The positive effect of a fodder additive that contained the biolayer on the basis of a phytomatrix on the growth and development of young rabbits was shown. Feeding of animals with a mixed fodder that contained 0.1% preparation resulted in stimulation of digestion of all components of the food. Among other components of the mixed fodder, cellulose was digested most effectively. An increase in the biomass of symbiotic bacteria and enzymatic activity in the blindgut chymus was also observed. The positive nitrogen balance demonstrated an increase in the nitrogen content in animals and a decrease of its losses with excretion. The mechanism of response of the rabbit’s organism to introduction of the complex probiotic preparation into the digestive tract is discussed.     

Screening of Bacillus strains as potential probiotics and subsequent confirmation of the in vivo effectiveness of Bacillus subtilis MA139 in pigs

A total of 124 samples were collected from the intestine of broiler chickens, piglet faeces, fermented foods, soils and Chinese herbs. More than 750 strains of aerobic, spore-forming bacteria were isolated from these samples. The inhibitory activity of these spore-forming strains against Escherichia coli K88, E. coli K99, Salmonella typhimurium and Staphylococcus aureus was assessed using a disc plate diffusion assay. The six bacilli with the largest inhibition zones against the four indicator bacteria were chosen and assessed for their resistance to unfavorable conditions within simulated gut environments. The strain Bacillus subtilis MA139 showed full resistance to pH 2, 0.3% bile salts and exhibited the highest antimicrobial activity. Based on these results, B. subtilis MA139 was selected as a potential probiotic and fed to piglets at concentrations of 2.2 × 10⁵, 2.2 × 10⁶ or 2.2 × 10⁷ CFU/g of feed during a 28-day feeding trial. A negative control consisting of the basal diet with no additives and a positive control consisting of the basal diet supplemented with 16 g/ton flavomycin were also included. Ninety piglets between 35 and 40 days old were used in the in vivo animal trials. B. subtilis MA139 enhanced daily gain (P = 0.10) and feed conversion (P = 0.03) compared with the negative control. The performance of pigs fed B. subtilis MA139 supplemented diets did not differ from that of pigs fed the antibiotic diet. There was a significant increase in Lactobacilli cell counts (P = 0.02) and a numerical decrease in E. coli counts (P = 0.05) in the faecal samples of pigs fed B. subtilis MA139 with 2.2 × 10⁵ CFU/g of feed. The overall results of this study show that the use of initial co-culture with indicator pathogens, a disc plate diffusion assay and simulated gut environment tolerance tests is one of effective methods of screening Bacillus for probiotic use and that B. subtilis MA139 is a promising alternative to antibiotics for use as a feed additive in piglet diets.     

Anaerobic solid-phase fermentation of plant substrates by <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Solid-phase growth of Bacillus subtilis 8130 on cellulose-rich plant substrates (presscakes or pulp) under hypoxic conditions was accompanied by cellulose depolymerization, protein hydrolysis, and degradation of other plant components, including some processes of mixed-type carbohydrate fermentation. The bacterial fermentation yielded propionic, butyric, hexanoic acids and butyric acid derivatives. The bacterial metabolism and fermentation degree can be characterized by the proportions of fatty acids in the reaction mixture. The product of sea buckthorn cake fermentation has a good sorption quality.     

Administration of Bacillus subtilis strains in the rearing water enhances the water quality,growth performance,immune response,and resistance against Vibrio harveyi infection in juvenile white shrimp,Litopenaeus vannamei

In this study, vegetative cell suspensions of two Bacillus subtilis strains, L10 and G1 in equal proportions, was administered at two different doses 10⁵ (BM5) and 10⁸ (BM8) CFU ml⁻¹ in the rearing water of shrimp (Litopenaeus vannamei) for eight weeks. Both probiotic groups showed a significant reduction of ammonia, nitrite and nitrate ions under in vitro and in vivo conditions. In comparison to untreated control group, final weight, weight gain, specific growth rate (SGR), food conversion ratio (FCR) and digestive enzymatic activity were significantly greater in the BM5 and BM8 groups. Significant differences for survival were recorded in the BM8 group as compared to the control. Eight weeks after the start of experiment, shrimp were challenged with Vibrio harveyi. Statistical analysis revealed significant differences in shrimp survival between probiotic and control groups. Cumulative mortality of the control group was 80%, whereas cumulative mortality of the shrimp that had been given probiotics was 36.7% with MB8 and 50% with MB5. Subsequently, real-time RT-PCR was employed to determine the mRNA levels of prophenoloxidase (proPO), peroxinectin (PE), lipopolysaccharide- and β-1,3-glucan- binding protein (LGBP) and serine protein (SP). The expression of all immune-related genes studied was only significantly up-regulated in the BM5 group compared to the BM8 and control groups. These results suggest that administration of B. subtilis strains in the rearing water confers beneficial effects for shrimp aquaculture, considering water quality, growth performance, digestive enzymatic activity, immune response and disease resistance.     

Effects of dietary Bacillus subtilis and inulin supplementation on performance,eggshell quality,intestinal morphology and microflora composition of laying hens in the late phase of production

Eighty Lohmann White laying hens were used to investigate the effect of dietary inclusion of Bacillus subtilis and inulin, individually or in combination, on egg production, eggshell quality, tibia traits, Ca retention, and small intestine morphology and microflora composition from 64 to 75 weeks of age. Hens were randomly distributed into 4 treatment groups, with 5 replicates per treatment and 4 hens per replicate. Treatment groups were fed basal diet (control), basal diet plus 1 g/kg B. subtilis (2.3 × 10⁸ cfu/g), basal diet plus 1 g/kg inulin, or basal diet plus a synbiotic combination of 1 g/kg B. subtilis (2.3 × 10⁸ cfu/g) and 1 g/kg inulin. Dietary supplementation of B. subtilis, inulin or synbiotic improved (P<0.05) feed conversion, egg performance, eggshell quality and calcium retention compared with the control. B. subtilis and synbiotic groups exhibited the highest (P<0.05) increase in egg production and egg weight. Inulin and synbiotic groups exhibited the highest (P<0.05) increase in eggshell thickness and eggshell calcium content, and the lowest (P<0.05) eggshell deformations. Unmarketable eggs were 8.4% (P<0.05) of the total eggs produced by the control group compared to 3.5%, 1.7%, and 1.5% for the B. subtilis, inulin and synbiotic groups, respectively. Tibia density, ash, and Ca content increased (P<0.05) by inulin and synbiotic inclusions, compared with the control. B. subtilis, inulin, and their synbiotic combination increased (P<0.05) villus height and crypt depth in all intestinal segments, compared with the control. B. subtilis and inulin modulated the ileal and caecal microflora composition by decreasing (P<0.05) numbers of Clostridium and Coliforms and increasing (P<0.05) numbers of bifidobacteria and lactobacilli, compared with the control. Colonization of the beneficial microflora along with increasing the villi–crypts absorptive area were directly associated with the improvements in performance and eggshell quality. It can be concluded that egg production and eggshell quality of laying hens can be improved (P<0.05) in the late phase of production by dietary inclusion of B. subtilis and inulin.     

Growth rate of tank-reared Mediterranean amberjack, Seriola dumerili (Risso 1810) fed on three different diets

Mediterranean amberjack Seriola dumerili (Risso 1810) has aquaculture potential. The growth rate and food conversion ratio of S. dumerili on three different types of food (A: 100% frozen sardines; B: 50% frozen sardines and 50% pellet; C: 100% pellet) were measured, and analysed with respect to temperature, condition index and chemical composition of the fish fillet. Wild S. dumerili, average body weight 248 g and average total length 26.9 cm were caught in August and September 1994 in the South Adriatic Sea near Dubrovnik, Croatia and kept in three tanks (n=15 in each tank; duration of experiment, 226 days). The fish that were fed on diet A [initial weight, 252 ± 71 g; total length (TL), 24.3 ± 2.6 cm] started feeding immediately; however, fish assigned to diet C began to feed entirely on the pelleted diet 1 month after the start of the experiment. The mortality of fish fed on diet A was negligible, the registered growth rate was 313 ± 74 g (124.2%), specific growth rate was 0.32% day^?1 and the food conversion rate was 6.7. The fish fed on diet B (initial weight, 246 ± 74 g; TL, 28.2 ± 2.5 cm) started to feed on day 3 and achieved a total growth rate of about 98% (final weight gain, 241 ± 69 g) and specific growth rate of 0.24% day^?1 (feed conversion rate of 9.00 and mortality 13%). The fish fed on diet C (initial weight, 246 ± 74 g; TL, 28.2 ± 2.5 cm) started to feed on the pellets after 1 month and had a growth rate of 87% (weight gain 214 ± 85 g), a specific growth rate of 0.24% day^?1 and a food conversion rate of 10.6 with considerable mortality (27%). In all three diet groups the fish grew with considerable variation in food consumption and growth rate, depending on seasonal temperature variations of the ambient sea water supplied to the rearing tanks. Chemical analysis showed that the protein level (amount) in the fish meat exceeded 20% in all three fish fillet samples.     

Probiotic strain <Emphasis Type="Italic">Bacillus subtilis</Emphasis> CU1 stimulates immune system of elderly during common infectious disease period: a randomized,double-blind placebo-controlled study

Background

Bacillus probiotics health benefits have been until now quite poorly studied in the elderly population. This study aimed to assess the effects of Bacillus subtilis CU1 consumption on immune stimulation and resistance to common infectious disease (CID) episodes in healthy free-living seniors.

Results

One hundred subjects aged 60–74 were included in this randomized, double-blind, placebo-controlled, parallel-arms study. Subjects consumed either the placebo or the probiotic (2.10⁹ B. subtilis CU1 spores daily) by short periodical courses of 10 days intermittently, alternating 18-day course of break. This scheme was repeated 4 times during the study. Symptoms of gastrointestinal and upper/lower respiratory tract infections were recorded daily by the subjects throughout the study (4 months). Blood, saliva and stool samples were collected in a predefined subset of the first forty-four subjects enrolled in the study. B. subtilis CU1 supplementation did not statistically significantly decrease the mean number of days of reported CID symptoms over the 4-month of study (probiotic group: 5.1 (7.0) d, placebo group: 6.6 (7.3) d, P?=?0.2015). However, in the subset of forty-four randomized subjects providing biological samples, we showed that consumption of B. subtilis CU1 significantly increased fecal and salivary secretory IgA concentrations compared to the placebo. A post-hoc analysis on this subset showed a decreased frequency of respiratory infections in the probiotc group compared to the placebo group.

Conclusion

Taken together, our study provides evidence that B. subtilis CU1 supplementation during the winter period may be a safe effective way to stimulate immune responses in elderly subjects.

     

Characterization of Two Bacillus Probiotics

Bacillus subtilis is currently used as an oral probiotic. We examined two commercial B. subtilis probiotic preparations, Enterogermina and Biosubtyl. Surprisingly, physiological and genetic characterization of the bacteria contained in each of these preparations has shown that neither contains B. subtilis.     

Effect of probiotic Bacillus subtilis Ch9 for grass carp,Ctenopharyngodon idella (Valenciennes, 1844), on growth performance,digestive enzyme activities and intestinal microflora

In the present study, Bacillus subtilis Ch9 was evaluated as a probiotic in grass carp, Ctenopharyngodon idella (Valenciennes, 1844). For 56 days the grass carp (50 ± 2.5 g) were given a feed containing B. subtilis Ch9 in three concentrations: 1.0 × 10⁹ (T₁), 3.0 × 10⁹ (T₂) and 5.0 × 10⁹ (T₃) CFU kg^?1 feed in triplicate treatments. The control group (T₀) was given feed without B. subtilis Ch9 for the same period. Determined were the specific growth rate (SGR), feed conversion ratio (FCR), and digestive enzyme activities in the intestine and hepatopancreas as well as the intestinal microflora. After 56 days, fish receiving the diets supplemented with B. subtilis Ch9 showed significantly higher SGR and lower FCR (P < 0.05) than those fed the control diet. There was no significant different in SGR and FCR among T₁, T₂ and T₃ nor was the survival rate affected (P > 0.05) by the dietary treatments. From days 14 to 56 of the experiment, higher protease, amylase and lipase activities in the foregut, midgut hindgut and hepatopancreas were observed in T₁, T₂ and T₃ (P < 0.05) compared with the control over a short‐term (14–28 days). Enzyme activity did not increase after long‐term feeding with B. subtilis Ch9 (56 days), but was still higher than that of control fish (P < 0.05). Fish fed the probiotic had an increase in trend of total aerobic and facultative anaerobic bacterial quantity (P > 0.05), but the ratio of Bacillus was significantly higher (P < 0.05) than in control fish. The total anaerobic bacterial quantity, Bifidobacterium and Lactobacillus were significantly higher (P < 0.05) in fish fed B. subtilis Ch9 compared with fish fed control feed. In conclusion, an optimum dose of B. subtilis Ch9 could modulate intestinal microflora, induce digestive enzyme activity and potentially promote the digestion and absorption of nutrients, as well as improve the growth performance of grass carp significantly.     

Inhibitory Activity of Probiotic <Emphasis Type="Italic">Bacillus subtilis</Emphasis> UTM 126 Against <Emphasis Type="Italic">Vibrio</Emphasis> Species Confers Protection Against Vibriosis in Juvenile Shrimp (<Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>)

The bacterial strain Bacillus subtilis UTM 126 produced antimicrobial activity against pathogenic Vibrio species, including V. alginolyticus, V. parahaemolyticus, and V. harveyi. The probiotic effect of B. subtilis was tested by feeding juvenile shrimp (Litopenaeus vannamei) food supplemented with B. subtilis (10⁵ CFU/g) for 28 days before an immersion challenge with V. harveyi at 10⁵ CFU/mL for 24 h. The treatment with B. subtilis UTM 126 decreased final mortality to 18.25%, compared with 51.75% in the control group. Bacillus subtilis UTM 126 has potential applications for controlling pathogenic V. harveyi in shrimp aquaculture.     

Effect of Milk Fermented with Lactic Acid Bacteria on Diarrheal Incidence,Growth Performance and Microbiological and Blood Profiles of Newborn Dairy Calves

The effect of the administration of milk fermented with lactic acid bacteria to calves was evaluated. The strains included were: Lactobacillus murinus CRL1695, Lact. mucosae CRL1696, Lact. johnsonii CRL1693, and Lact. salivarius CRL1702, which were selected for their beneficial and functional properties and isolated from healthy calves in the northwestern region of Argentina. The trial was conducted on a dairy farm located in Tucumán (Holando-Argentino calves). A randomized controlled trial was performed in which 56 new-born animals were divided into two groups: the treated group (T) received the fermented milk for 60 days and the control group (C) only milk. The animals were fed a solid diet ad libitum. The treated group was given a daily dose of 1 × 10⁹CFU of the probiotic fermented milk while the control group was fed milk. Body weight and biometrical parameters were recorded between 15 and 60 days of age, and average daily gain was calculated with three samplings per animal throughout the trial. Rectal swabs and fecal and blood samples were also collected. Results showed the efficacy of the probiotic: lower morbidity and mortality of calves (morbidity was 69.20% in animals without the probiotic, and 46.15% in probiotic-treated animals, with P = 0.09; mortality in C was 34.61 and 7.69% in animals fed with ferment milk; P = 0.02).The calves fed with probiotic evidenced an improvement in nutritional parameters, body condition and weight gain (health index P = 0.01; average daily gain P = 0.03).Viable bacterial numbers showed no differences between the two experimental groups. Hematological parameters and serum proteins were not modified by the treatment. The results suggest that the fermented milk containing lactic acid bacteria can be a viable veterinary product for young calves due to its beneficial effects on health and growth.     

Evaluation of the probiotic characteristics of Bacillus species isolated from different food sources

Probiotics have established their efficacy as dietary adjuncts providing benefits to consumers. However, selection of probiotics before incorporation into diet requires close scrutiny in the form of in vitro as well as in vivo tests. Three bacteriocinogenic Bacillus sp., namely, B. licheniformis Me1, B. flexus Hk1, and B. subtilis Bn1 previously isolated from milk, cheese and fermented beans, respectively, were characterized for typical in vitro probiotic criteria. When compared to probiotic Bacillus coagulans, all three cultures were found to possess better acid and bile tolerance. Cultures Me1 and Bn1, except Hk1, showed bile salt hydrolase activity. A marked difference in adhesion to hydrocarbons and auto-aggregation properties from 10–80 and 60–99%, respectively, were observed for the tested cultures. Highest antioxidant activity was measured for culture Hk1 (66.6%), whereas least activity of 53% was observed for culture Bn1. Cultures Me1 and Bn1 were sensitive to all the antibiotics tested, whereas Hk1 and B. coagulans showed resistance to the penicillin group of β-lactum antibiotics. All the tested cultures showed a broad spectrum of activity against food-borne pathogens. In co-cultivation studies, B. licheniformis Me1 completely inhibited the growth of the indicator pathogen Listeria monocytogenes ScottA. Overall, the test cultures exhibiting potential probiotic characteristics, particularly B. licheniformis Me1, can serve as probiotics of commercial interest.     

Integration of Deoxyribonuclease-Treated DNA in Bacillus subtilis Transformation

Normal preparations of B. subtilis DNA have weight average native molecular weights of 10 to 30 x 10⁶. For any given preparation the upper and lower 95% size limits may differ by a factor of ten or more. Single-stranded molecular weights indicate an average of 1 to 4 breaks per single strand of the native DNA. The reduction in transforming activity and viscosity following DNAase I digestion can be accounted for by a direct relationship between the transforming activity of a DNA and its single-stranded molecular weight. Uptake studies with DNAase I treated heavy (²H¹⁵N ³H) DNA show that single strand breaks inhibit integration less than transformation. A provisional estimate of the size of the integrated region based on correlating the single strand size of the donor-recipient complex with the donor-recipient density differences following alkali denaturation came to 1530 nucleotides. Using a competent, nonleaky thymine-requiring strain of B. subtilis grown in 5-BU medium before and after transformation, it was shown that (a) No detectable amount of DNA synthesis is necessary for the initial stages of integration, (b) Cells which have recently been replicating DNA are not competent. (c) Cells containing donor DNA show a lag in DNA replication following transformation, (d) When donor DNA is replicated it initially appears in a density region between light and hybrid. This indicates that it includes the transition point formed at the time of reinitiation of DNA synthesis in the presence of 5-BU following transformation. A model is proposed in which donor DNA is integrated at the stationary growing point of the competent cell, which is in a state of suspended DNA synthesis.     

Nutritive value of spruce muka (foliage) for the growing chick

Three experiments were conducted with broiler type chicks to determine the nutritive value of spruce (Picea glauca) muka for growing birds to 4 weeks of age. Up to 10% of a diet of maize, soya bean meal and fishmeal was replaced with muka, alfalfa meal or cellulose. Growth, feed consumption, mortality and organ: body weight ratios were examined.The addition of muka, untreated or steam distilled, to the basal diet reduced growth except in the case of 2.5% steam distilled muka, for which growth was comparable to that for the basal diet. When the protein content of the basal diet was reduced, up to 5% untreated or steam distilled muka could be introduced without significant growth depression. However, at the 10% level, growth depression was significant (P < 0.05). Pelleting the supplemented diets significantly stimulated growth.Feed consumption was usually depressed by the addition of the supplements to the basal diet. Mortality was not influenced by treatment. Organ: body weight ratios were not influenced by treatment × sex interactions. Muka, cellulose or alfalfa, each incorporated as 10% of the diet, did not influence the organ: body weight ratio of the heart, proventriculus, caeca, spleen or gall bladder. All the experimental feeds increased the size of the remaining components of the digestive tract, as well as the liver and pancreas.     

Formulations of Bacillus subtilis BY-2 suppress Sclerotinia sclerotiorum on oilseed rape in the field

We are developing a collection of Bacillus strains, isolated from different environments, for use in controlling Sclerotinia sclerotiorum on oilseed rape in China and elsewhere. Strain BY-2, isolated from internal tissues of an oilseed rape root, was demonstrated to be Bacillus subtilis based on biochemical and morphological characteristics and on 16S RNA gene sequence. Photographic evidence from gnotobiotic studies using the lacZ-tagged strain BY-2(pUC18) confirmed that this strain was capable of colonizing internal root tissues. Strain BY-2 did not effectively colonize the ectorhizosphere or the surface of the stems or leaves of oilseed rape when applied in pellet or wrap seed treatment formulations. Populations of BY-2 dropped from 10⁸ CFU seed⁻¹ to 10⁴ CFU g root⁻¹ and ⩽10² CFU g stem⁻¹ or leaf⁻¹ after 60 days. Strain BY-2 was applied as a pellet seed treatment formulation alone, as a spray at flowering alone, and as the pellet seed treatment formulation combined with the spray application in two field trials at the Wuxue location conducted in two consecutive years. These three treatments containing BY-2 provided disease control (disease incidence) and mean seed yield that was similar to the chemical control treatment and significantly greater than the pellet without bacteria and non-treated control treatments. All three of these BY-2 treatments were similar to each other with regard to these two metrics and to treatments containing B. subtilis Tu-100, a genetically distinct strain previously shown to be effective against this disease. In two additional field trials, conducted in consecutive years at the Wuhan location, strain BY-2 applied as a wrap seed treatment formulation alone, as a spray at flowering alone, and as the wrap seed treatment combined with the spray application provided disease control (disease incidence) and mean seed yield that was similar to the chemical control treatment. These three BY-2 treatments also were significantly greater than the non-treated control treatments and compared favorably to treatments containing Tu-100. There was no evidence of BY-2 promoting growth of oilseed rape when applied in the pellet or wrap seed treatment formulations in field trials conducted at the Wuxue or at the Wuhan locations. We now have three Bacillus strains (B. subtilis strains BY-2 and Tu-100, B. megaterium A6) that control S. sclerotiorum on oilseed rape in the field that can be tested in strain combinations for enhanced disease control. We also have multiple methods for application of Bacillus strains as both seed treatment and foliar applications were effective.     

Combined or Individual Effects of Dietary Probiotic <Emphasis Type="Italic">Pedicoccus acidilactici</Emphasis> and Nucleotide on Growth Performance,Intestinal Microbiota,Hemato-biochemical Parameters,and Innate Immune Response in Goldfish (<Emphasis Type="Italic">Carassius auratus</Emphasis>)

This study investigated the effect of dietary supplementation of probiotic Pedicoccus acidilactici and nucleotide (separately or combined) on growth performance, intestinal microbiota, hemato-immunological parameters, and immunity response in goldfish (Carassius auratus). Fish (average weight 5–6 g) were acclimatized and divided into eight experimental diets supplemented with P. acidilactici of different concentrations (0.1, 0.2, and 0.3% diet) and nucleotides (0 and 0.5% diet) for 6 months. Fish fed with experimental diets showed significant differences in terms of final weight, weight gain, feed conversion ratio, daily growth rate, and condition factor when compared to control diet (P < 0.05). Fish fed with probiotic (0.3%) separately and combined with nucleotide (0.5%) had highest RBC and WBC when compared to other diets (P < 0.05), while the highest values for Hb and Hct as well as total protein, glucose, albumin, and globulin were observed in probiotic (0.2%) and nucleotide (0.5%) combined diet. Serum lysozyme and anti-protease activities were significantly higher in probiotic (0.1 and 0.2%) and nucleotide (0.5%) combined diets. Similarly, these two diets combined showed the highest colonization of P. acidilactici when compared to other diets. In conclusion, combined dietary probiotic and nucleotide improve the growth performance, hemato-biochemical parameters, and intestine growth in C. auratus.