摘要
黄曲霉毒素B,(AFB,)是由黄曲霉菌属中的黄曲霉菌株和寄生菌株产生的一种多环芳香烃物质,对动物有明显的免疫抑制作用。AFB,可以导致牛巨噬细胞和T细胞细胞因子的分泌减少,还可以使鸟类和其他动物的巨噬细胞功能下降,细胞免疫和体液免疫受到抑制。黄曲霉毒素使罗非鱼巨噬细胞吞噬能力下降。AFB1导致虹鳟鱼胚胎的记忆性B细胞减少,使其外周血液免疫球蛋白的分泌和淋巴细胞增殖功能受到明显的抑制。虽然犬对黄曲霉毒素也很敏感,但关于AFB1对犬免疫系统影响的报道很少见。本试验通过AFB1对犬慢性毒性作用,观察了犬免疫系统的变化。
本试验中,对1～3岁健康犬注射犬细小病毒疫苗,8周后定量饲喂含一定AFB, (340μg/kg饲料)的犬粮,于0d、7d、14d、21d和28d,用MTT法和SAP法分别对其淋巴细胞增殖能力和T淋巴细胞亚群组成进行检验。用ELISA法检测犬细小病毒抗体效价变化。分别采集犬不同时间段的抗凝血和非抗凝血,进行细胞培养试验和抗体检测试验,非抗凝血分离血清-20℃保存备用,抗凝血分离淋巴细胞。
试验结果如下：
1.淋转试验中,探讨不同时间、淋巴细胞、脂多糖(LPS)和刀豆蛋白A(Con A)对犬淋巴细胞增殖的影响。确定了,Con A刺激的最佳浓度是80μg/mL,对应的培养时间是48h,淋巴细胞浓度是1×106个/mL。LPS刺激的最佳浓度是100μg/mL,对应的培养时间是20h,淋巴细胞浓度是1×107个/mL。
2.用Con A和LPS分别刺激犬外周血T淋巴细胞和B淋巴细胞,7d时T淋巴细胞增殖效应显著提高,14d到28d出现显著下降。与0d相比,B淋巴细胞的增殖能力在7d到28d明显减弱,21d时出现一个小的波峰。但淋巴细胞数量没有显著性变化。
3.随着时间的变化,试验的最终结果显示CD3+、CD4+和CD8+的比例均有明显下降,由于CD4+比CD8+下降的速度快,所以CD4+/CD8+比值也明显降低,这说明AFB1使犬细胞免疫受到抑制。
4.AFB1对犬细小病毒免疫效果未产生明显的影响。
本试验结果表明,AFB1对犬细胞免疫产生明显的抑制作用。对后人的研究具有一定的参考价值。这种免疫抑制作用可能是导致犬临床免疫失败和传染病易感的原因之一。
The mycotoxin, aflatoxin B (AFB1) is a polycyclic aromatic hydrocarbon, produced by some strains of the ubiquitous fungi Aspergillus flavus, A. parasiticus and A. nomius, found to be a potent immunomodulator in endotherms. Exposure to AFB1 results in the reduction of cytokine production of bovine macrophages and T-cells. Animals and birds exposed to AFB1 exhibit reduced macrophage function, suppressed cell mediated immunity and humoral immunity. The phagocytic capacity of the macrophages reduced in nile tilapia after aflatoxin exposure. Embryonic exposure of rainbow trout to AFB1 results in reduced B-cell memory. A dramatic suppression of immunoglobulin production and lymphocyte proliferation was marked when rainbow trout peripheral blood leucocytes were exposed to AFB1. Although canine, one of the important species is also susceptible to aflatoxicosis, little is known about the effect of AFB1 on the immune system of this species. The study related to immunosuppression due to AFB1 in canine is limited. In the present study, we applied several methods to evaluate the immune response of hybrid dogs to different time of exposing to AFB1.
In this present study, a range of hybrid dogs were selected to determine the canine lymphocytic proliferation effects in different time after the dogs were exposed to AFB1, respectively, the changes of subgroup T lymphocytes, by the methods of lymphocytic transfer proliferation MTT and SAP. Otherwise, the level of Parvovirus IgG was determined by an ELISA method. At 8 weeks before experiment, dogs were vaccinated with parvovirus vaccine. After 8 weeks, were fed a diet contaminated with 340μg AFB1/kg feed. At different time (0,7,14,21 and 28) intervals blood samples were aseptically collected from the anterior limb vein. Blood was collected in tubes containing EDTA for blood culture or blood formula, respectively. Plasma samples were obtained following centrifugation of heparinized blood and stored at -20℃for later analysis.
The results were as follows:
1. Effects of time and PBMC, Con A, LPS of different concentration on promote lymphocytes proliferation was tested by MTT test. The result of experiment demonstrate that the best levels combination of canine lymphocyte. In the lymphocytic transfer test, Con A at dosage of 80μg/mL performed facilitation effects for canine lymphocytic proliferation(p>0.05),when the best levels combination of canine T lymphocyte was 1×106 unit/mL, the incubated time for 48h. LPS at 100μg/mL markedly proliferated B lymphocytes(p<0.05),with the best levels combination of canine B lymphocyte was 1×107 unit/mL, the incubated time for 20h.
2. With LPS and Con A, respectively, stimulate canine T and B lymphocytic proliferation, at 7 days, the proliferation ability of canine T lymphocytes became markedly stronger(P<0.05). From 14 to 28 days, the proliferation of T lymphocytes showed a downtrend. The proliferation ability of canine B lymphocytes became markedly to weaken, from 7 to 28 days, compare to 0 days. Also at 21 days, the ability was stronger than those of 14 and 28 days. The number of lymphocyte in canine peripheral blood did not be impaired.
3. With regard to lasting of the time, a significant down-regulation of all subgroup T lymphocytes was observed in canine blood. The ratio of CD4+/CD8+ was down, according to the analysis of the reducing of CD4+ T-cells was stronger than CD8+ T-cells.
4. AFB1 exposure had no major effect on the level of Parvovirus IgG.
So it can be concluded here that either AFB1 can markedly impaired canine cellular mediated immunity, which put forward certain powerful experimental evidences and instructions for diagnosis of canine diseases especially canine expose to AFB1 diseases. These impairments in the immune response could participate in failure of vaccination protocols and increased susceptibility to infections described in dogs exposed to AFB1.
引文
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