Lactobacillus plantarum BS22 promotes gut microbial homeostasis in broiler chickens exposed to aflatoxin B1

Probiotics promote the health of the host by maintaining intestinal microbial homeostasis. This study aimed to investigate the benefits of Lactobacillus plantarum BS22 (LP) in the gastrointestinal tract (GIT) microbial homeostasis of broiler chickens exposed to aflatoxin B₁ using the PCR‐DGGE, viable count and real‐time PCR. The toxin adsorption experiment demonstrated that treatment R5 (1.0 × 10⁸ CFU/g LP) exhibited good absorptive effect in adsorbing the aflatoxin B₁ (AFB₁) in vitro. DGGE showed that the composition and structure of gut microbiota were more similar in the mucosa than in the content of all the samples. In addition, higher diversity of the microbiota was observed in the caecum and glandular stomach than in other segments. Lactobacillus, Enterococcus and Enterobacteriaceae were more abundant in the ileum than in the other segments. Enterobacteriaceae in groups I (basal diet) and II (basal diet+50 μg/kg AFB₁) showed a significant difference in group III (basal diet + 50 μg/kg AFB₁ + 1 × 10⁸ CFU/g LP) in the crop content and duodenum mucosa (p < .05). This investigation indicates that the L. plantarum BS22 promotes GIT microbial homeostasis in broiler chickens exposed to AFB₁, particularly for the intestine mucosa microbiota. Thus, L. plantarum BS22 is a possible candidate for degrading AFB_1.     

Impact of BSE on Livestock Production System

A total of 240 unvaccinated day-old broiler chicks, which had been found to be negative for antibodies against FAV-4, were divided into four groups of 60 chicks each. Group A was fed aflatoxin at 1 ppm from 7 days to 7 weeks of age. Group V was infected intra-abdominally at 14 days of age with 0.2 ml of FAV-4, having a titre of 10^5.5 TCID₅₀ per 0.2 ml. The combined group AV was given the aflatoxin and infected with FAV-4. The fourth group C served as the control. More pronounced clinical signs, a higher mortality rate (56.7%), and reductions in body weight gain and in the organ to body weight ratios of the bursa and spleen were recorded in group AV. A significant (p<0.01) reduction in the HI antibody titre following vaccination against Newcastle disease, and of skin thickness in the delayed hypersensitivity test following sensitization with DNCB, indicated an additive immunosuppressive effect from aflatoxin and FAV-4 on the humoral and cell-mediated immune responses in group AV compared to groups A and V. Microscopically, marked depletion and degeneration of lymphocytes in the thymus, bursa, spleen and caecal tonsils were observed in group AV up to 5 weeks PI.     

组合实验设计用于黄曲霉毒素污染控制的研究

为控制黄曲霉毒素主要产生菌寄生曲霉AS3.4407在粮食储藏环境中的产毒污染,采用组合实验设计策略,考察了五个模拟环境因子对菌株产毒的影响。首先,利用Plack-ett-Burman试验筛选出三个对毒素产量影响显著的因子(转速、温度、pH),在此基础上,采用CCD实验设计法(Central Composite Design,CCD)研究了这三个因子对寄生曲霉AS3.4407产毒影响的交互作用,并根据毒素曲面方程和二次多项回归方程得到该菌的最低产毒条件。在环境通风良好(转速240rpm),储藏温度较低(18℃),pH为弱碱性(pH 8)条件下,寄生曲霉AS3.4407产毒量最低,验证试验证实了该方程的预测值与实际值之间具有较好的拟合度。为粮库采取相应措施,改变储藏环境条件以降低毒素污染提供了很好依据。     

黄曲霉毒素B1对黄颡鱼幼鱼生长及肝脏功能的影响

为研究黄曲霉毒素B1(Aflatoxin B1, AFB1)对黄颡鱼幼鱼生长、肠道消化酶及肝脏功能的影响,用添加不同梯度黄曲霉毒素B1(AFB1)(0、50、100和200μg/kg)的4种等氮等脂配合饲料饲喂初始体质量为(6.00±0.10) g的黄颡鱼幼鱼8周。结果显示:(1)饲料中添加AFB1对黄颡鱼幼鱼的存活率、饲料系数和特定生长率等均无显著影响,但使胰蛋白酶活性显著提高,而高含量AFB1(100和200μg/kg)显著降低肠道淀粉酶和脂肪酶活性;(2)随着AFB1添加量的上升,血清中谷丙转氨酶、谷草转氨酶活性及葡萄糖、甘油三酯、总胆汁酸和总胆固醇含量显著升高,肝脏中谷丙转氨酶、谷草转氨酶活性显著下降;(3) AFB1添加组的肝脏过氧化氢酶活性和丙二醛含量显著高于对照组,100和200μg/kg AFB1组超氧化物歧化酶活性显著高于对照组,其余各组无显著差异;(4) AFB1添加组肝脏sod基因及炎性因子il-1β基因的相对表达量显著上调,200μg/kg AFB1组cat基因及炎性因子il-8、il-10相对表达量显著上调;(5)通过组织学观察,AFB1会引起部分肝细胞出现轻微萎缩、肝细胞核移位、细胞界限模糊和肝细胞内空泡化的现象。研究表明,在本实验条件下,饲料中AFB1含量低于200μg/kg不影响黄颡鱼幼鱼生长性能,但饲料中AFB1≥50μg/kg时会影响黄颡鱼幼鱼肠道的消化吸收功能,同时引起肝脏氧化应激及炎性反应,造成肝脏功能损伤。     

Pathological studies in broiler chicks fed aflatoxin or ochratoxin and inoculated with inclusion body hepatitis virus singly and in concurrence

Day-old broiler chicks found negative for maternal antibodies against inclusion body hepatitis (IBH) virus by agar gel precipitation test and viral antigen in cloacal swabs by dot enzyme immunoassay were divided into 6 groups of 20 chicks each. Group A was fed aflatoxin B₁ at 1.25 ppm from 3 to 38 days of age; group O was fed ochratoxin A at 0.5 ppm from 3 to 38 days of age; group V was inoculated with 1 ml of IBH virus of titre log₁₀ 6.5 EID₅₀ per 0.2 ml. Groups AV and OV were given aflatoxin B₁ and ochratoxin A, respectively, and also infected with the virus. Group C served as control. There was mild enlargement and paleness of the liver up to 18 days post inoculation in group V; there were no lesions in group A; and there was gradual enlargement of the kidneys from 10 days post feeding of mycotoxin onwards in group O. In the combined groups AV and OV the gross lesions were slightly more severe. In group V, varying degrees of degenerative histopathological changes, congestion and haemorrhages were seen particularly in the liver, followed by the kidneys, bursa, spleen, myocardium and lungs, along with intranuclear inclusion bodies in the hepatocytes, mostly in the early stages of infection. Similar microscopic changes, but without inclusion bodies, were seen in groups A and O and the changes were pronounced in the later stages. In group O, the kidney lesions were more pronounced than the liver lesions. In the concurrently infected groups, AV and OV, the changes were similar but slightly more marked than in the corresponding individual groups. Inclusion bodies in hepatocytes were more frequent, more prominent and appeared earlier in the concurrent groups.Abbreviations AGPT agar gel precipitation test - DPF days post feeding of the mycotoxin - DPI days post inoculation - EIA enzyme immunoassay - EID₅₀ dose at which 50% of embryos will be infected - IBH inclusion body hepatitis - PAU Punjab Agricultural University     

<Emphasis Type="Italic">In vitro</Emphasis> Selection of Maize Rhizobacteria to Study Potential Biological Control of <Emphasis Type="Italic">Aspergillus</Emphasis> Section <Emphasis Type="Italic">Flavi</Emphasis> and Aflatoxin Production

The aims of this study were to select bacterial isolates from the non-rhizophere of maize soil and to examine their antagonistic activity against Aspergillus section Flavi strains. The first selection was made through ecophysiological responses of bacterial isolates to water activity (a_w) and temperature stress. Subsequently, an Index of Dominance test (I_D), ecological similarity and inhibition of the lag phase prior to growth, growth rate and aflatoxin B₁ accumulation were used as criteria. From the first assay nine bacterial strains were selected. They grew well at 25 and 30 °C, with growth optima between 0.982 and 0.955 a_W using 48 h of incubation. There was ecological similarity between the bacterial strains Bacillus subtilis (RCB 3, RCB 6), Pseudomonas solanacearum RCB 5, Amphibacillus xylanus RCB 27 and aflatoxigenic Aspergillus section Flavi strains at 0.982 at 25 °C. The predominant interaction between all selected bacteria and fungi in dual culture was mutual intermingling at 0.982. Mutual inhibition on contact and mutual inhibition at a distance was observed at 0.955 a_w, between only four bacteria and some Aspergillus strains. Bacillus subtilis RCB 55 showed antifungal activity against Aspergillus section Flavi strains. Amphibacillus xylanus RCB 27, B.␣subtilis RCB 90 and Sporolactobacillus inulinus RCB 196 increased the lag phase prior to growth and decreased the growth rate of Aspergillus section Flavi strains. Bacillus subtilis strains (RCB 6, RCB 55, RCB 90) and P. solanacearum RCB 110 inhibited aflatoxin accumulation. Bacillus subtilis RCB 90 completely inhibited aflatoxin B₁ accumulation at 0.982 a_W. These results show that the bacterial strains selected have potential for controlling Aspergillus section Flavi over a wide range of relevant environmental conditions in the stored maize ecosystem.     

Growth, haematology, blood constituents and immunological status of lambs fed graded levels of animal feed grade damaged wheat as substitute of maize

The aim of this study was to explore possibilities of utilization of animal feed grade damaged wheat (ADW) in lamb feeding, and assess the effect of ADW and its aflatoxin on intake, growth, haematology, blood biochemical constituents and immunological status. The ADW is a slightly mouldy feed resource, which is not suitable for human consumption. The experimental ADW contained dry matter (DM) 964, organic matter 974, crude protein 153, cellulose 205 and lignin 24, and starch 732 g/kg DM. ADW also contained aflatoxin B1 50 microg/kg due to mould infestation. Thirty-five weaner lambs (90 +/- 15 days of age and 16.1 +/- 0.82 kg body weight) in a randomized design were fed for 91 days on one of four composite feed mixtures (roughage to concentrate ratio of 25:75) containing 0, 118, 235, 353 or 470 g/kg ADW, which replaced equal amounts of maize and at these inclusion levels ADW replaced 0%, 25%, 50%, 75% and 100% maize in lamb diets respectively. Dry matter intake (DMI) was similar in different level of ADW fed lambs but ADW inclusion linearly (p = 0.016) reduced DMI. Average daily gain (g/day) was higher (p = 0.038) in lambs fed 353 g ADW diet. Haematological attributes viz. WBC, haemoglobin (Hb) and mean corpuscular volume did not affect by ADW feeding whereas it increased haematocrit, mean cell Hb and decreased neutrophil, RBC counts and mean cell Hb concentration. Blood glucose and urea-N increased whereas albumin and protein level reduced by ADW feeding. ADW feeding of lambs did not affect serum IgG level. The activities of serum aspartate aminotransferase, alkaline phosphates and acid phosphates were not affected, whereas alanine aminotransferase increased linearly (p = 0.001) with increasing levels of ADW. It is concluded that ADW containing aflatoxin B1 50 microg/kg DM can safely be incorporated in growing lamb feeding up to 353 g/kg diet without affecting growth and cellular immunity, however ADW may induce a transient alteration of hepatic enzymatic activities. Further aflatoxin content of the diet should be kept within permissible limits of respective country.     

Aflatoxin B1 impairs mitochondrial functions,activates ROS generation,induces apoptosis and involves Nrf2 signal pathway in primary broiler hepatocytes

Aflatoxin B1 (AFB1) is known as a mycotoxin that causes various health problems in animals, but the precise mechanism of AFB1 on mitochondrial functions and apoptosis in primary broiler hepatocytes (PBHs) is not clear. The objective of this study was to investigate the effects of AFB1 on the mitochondrial functions, reactive oxygen species (ROS) generation, apoptosis and nuclear factor erythroid 2‐like factor 2 (Nrf2)‐related signal pathway in PBHs. Here, the mitochondrial membrane potential (MMP), ROS generation, antioxidative genes and apoptosis in PBHs induced by AFB1 were investigated. The results showed that AFB1 evoked mitochondrial ROS generation, decreased MMP and induced apoptosis in PBHs. AFB1 increased the percentage of apoptotic cells, and expression of caspase‐9 and caspase‐3, upregulated messenger RNA (mRNA) expression of Nrf2 and downregulated mRNA expressions of NAD(P)H: quinine oxidoreductase 1, superoxide dismutase and Heme oxygenase 1 in PBHs. The expression of Bax was also observed in cytoplasm. These findings suggested AFB1 results in a significant impairment of mitochondrial functions, activates ROS generation, induces apoptosis, and is involved in Nrf2 signal pathway through mitochondria ROS‐dependent signal pathways in PBHs.     

黄曲霉毒素降解菌N-1a的降解特性及在全株玉米青贮中的应用

为研究青贮用黄曲霉毒素降解菌N-1a的降解特性及在全株玉米青贮中的应用效果,本试验首先对菌株N-1a的发酵上清液进行不同处理后分别与毒素作用,初步研究降解活性物质的性质;然后研究N-1a发酵上清液在不同pH、不同温度下对黄曲霉毒素的降解率;最后通过发酵黄曲霉污染的全株玉米来验证N-1a在实际生产中的应用效果。结果表明:初步判定枯草芽孢杆菌N-1a降解黄曲霉毒素的活性物质是一种分泌蛋白,其在pH7.0和温度37℃时降解率最高;在全株玉米青贮试验中,N-1a明显降低了全株玉米青贮过程中的黄曲霉毒素,添加N-1a的处理组黄曲霉毒素含量为(8.56±1.68)μg/L,添加市售菌剂的处理组黄曲霉毒素含量为(15.26±2.53)μg/L;N-1a还有效降低了中性洗涤纤维(NDF)的含量,加入N-1a处理组NDF含量较加入市售菌剂的处理组降低7.98%;加入N-1a处理组乳酸含量比加入市售菌剂的处理提高1.66%,由此可见,N-1a处理组效果显著优于市售菌剂组。     

Influence of Bacillus spp. isolated from maize agroecosystem on growth and aflatoxin B(1) production by Aspergillus section Flavi

A total of 59 bacteria of the Bacillus genus were isolated from different components of a maize agroecosystem and their antifungal activity against Aspergillus section Flavi was evaluated. Thirty-three and 46% of these bacteria were able to inhibit Aspergillus flavus Link and A. parasiticus Speare respectively at water activity (a(w)) 0.982; however, when a(w) was 0.955, these percentages were decreased and only three isolates were able to inhibit Aspergillus section Flavi. The majority of bacilli acted as contact antagonists, while a small number of isolates were able to form inhibition zones. In maize meal extract agar, Aspergillus section Flavi growth rate and aflatoxin B(1) (AFB(1)) production were significantly reduced when these strains were paired at a(w) 0.982 with bacilli at all inoculum levels studied. However, two bacilli isolated were able to reduce growth rate and aflatoxin production when a(w) was 0.955. Lag phase increase followed the same general pattern as growth rate reduction. When Aspergillus section Flavi was grown in sterile maize in the presence of three Bacillus strains at a(w) 0.982, the reduction in count (colony-forming units (cfu) g(-1) maize) was less than 30%, except when Aspergillus section Flavi grew with Bacillus amyloliquefaciens UNRCLR. However, levels of detectable AFB(1) were significantly reduced in these interactions at a(w) 0.982.