警犬黄曲霉毒素中毒的诊断及其免疫抑制效应
摘要
黄曲霉毒素是由黄曲霉菌属中的黄曲霉菌株和寄生菌株产生的一类多环芳香烃物质。在该类毒素中黄曲霉毒素B1(aflatoxin B1,AFB1)含量最多,毒性最强,具有基因毒性和细胞毒性,能造成多种类型的DNA损伤,具有明显的免疫抑制作用,引起动物免疫失败和感染性疾病的发生。关于犬AFB1中毒的报道相对较少。2007年南京某犬场因饲喂含有AFB1的犬粮,138头犬出现中毒。以该犬场的警犬为研究对象,对犬AFB1中毒的临床症状、病理、诊断、治疗及其免疫抑制效应进行了相关研究。
1临床诊断对犬场的378头犬进行血液生化检查,有138头出现了谷丙转氨酶、谷草转氨酶异常,有71头犬出现了临床症状,其中有36头犬死亡。
对71头患犬进行观察,患犬都出现精神沉郁,食欲下降,66.2%的患犬出现不同程度可视黏膜及皮肤黄染,52.1%的患犬出现粪便呈煤焦油样等症状；对全部36头死亡犬进行剖检,发现死亡犬皮肤、皮下脂肪黄染,肝坏死呈土黄色,心肌斑点状出血,腹腔积水,肠系膜广泛性出血,肠道出血等。
采集12头死亡犬实质性脏器组织的120个样品作病理检查。死亡犬都出现胃黏膜充血出血,肠黏膜充血出血,肠绒毛轻微脱落,心肌细胞发生颗粒变性,脾脏明显充血,有炎性细胞浸润,淋巴结充血,局部组织疏松,呈网状或空泡状,淋巴窦扩张,充满大量浆液和炎性细胞,肺泡中充满大量红细胞,肺泡隔增厚,有炎性细胞浸润,肝脏明显充血出血,肝索紊乱肝细胞发生颗粒变性和水泡变性,并可见大小不一坏死区,有少量炎性细胞浸润,肾脏肾间质毛细血管充血,肾小管上皮细胞空泡变性。
2毒素检测委托江苏省进出境动植物检验检疫局,采用高效液相色谱法对饲料及饲料原料、及死亡犬内脏11个批号的33个样品进行黄曲霉毒素检测。通过检测发现,070208号玉米,070215幼犬饲料、070309幼犬和成犬饲料中黄曲霉毒素B1分别达到0.854、0.2249、0.376和0.678mg/kg,B2分别达0.062、0.018、0.028和0.044mg/kg,而070208号玉米正是用于生产070215、070309犬饲料的原料。确诊该犬群系采食含有AFB1饲料而发生中毒。
3血液生化指标检测及代谢物谱分析已采食被AFB1污染的饲料30d的30头犬的血液生化指标为实验组。以2006年3月9日健康检查相对应犬的生化指标为对照组。用SPSS15.0统计软件用t检验对实验数据进行统计分析。30头患犬采食AFB1污染的饲料30天后,谷丙转氨酶均值由37.7u/L上升到190.07u/L,谷草转氨酶、总胆红素、直接胆红素升高,胆碱酯酶、总蛋白和白蛋白降低。犬AFB1中毒前后7项生化指标变化均呈显著性差异(P<0.01)。
通过气相色谱-质谱技术(GC-MS)分析AFB1中毒犬血浆中的代谢物谱,并运用主成分分析(PCA)方法对血浆内源性代谢物随中毒时间的变化情况进行识别。研究发现,随着AFB1中毒时间的延长,血浆中的如L-异亮氨酸、甘氨酸、L-苏氨酸、天冬氨酸、L-苯丙氨酸等氨基酸含量有所上升,L-异亮氨酸相对峰值面积由0.050(+0.008)上升到0.102(+0.001),与对照组相比有显著(P<0.05)。葡萄糖、胆固醇以及部分游离脂肪酸含量下降,胆固醇相对峰值面积由0.757(±0.061)下降到0.532(+0.074),与对照组相比有显著(P<0.05)。这些代谢物的变化都与肝脏的损伤程度密切相关,且变化趋势与血液生化指标检测结果一致。研究结果表明,基于GC-MS的代谢组学技术结合PCA的方法能较全面地反应生物体的代谢状态,有助于AFB1中毒的早期诊断。
4治疗试验以4头健康犬为阴性对照,以7头AFB1中毒患犬为阳性对照,作基础治疗。其它35头中毒患犬分成5组,每组7头,除基础治疗外,分别采用谷胱甘肽、葡醛内酯、促肝细胞生长素和茵栀黄注射液进行特异性治疗。组间疗效比较用卡方检验。
犬AFB1中毒的疗效不佳,实验组死亡率达69%,发生中重度中毒后,死亡率达100%。而轻度的AFB1中毒,通过采用谷胱甘肽、葡醛内酯、促肝细胞生长素联合治疗,总有效率(痊愈+显效+有效)能达到57.1%。
5免疫抑制试验取1-3岁的健康犬,按20g/kg饲喂含AFB1浓度为340μg/kg的犬粮,于0d、7d、14d、21d和28d,用淋巴细胞转化实验和免疫组化方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。
试验确定Con A刺激犬T细胞增值的最佳浓度是80μg/mL,对应的培养时间是48h,淋巴细胞浓度是1×106个/mL。LPS刺激犬B细胞的最佳浓度是100gg/mL,对应的培养时间是20h,淋巴细胞浓度是1×107个/mL。
用Con A和LPS分别刺激AFB1中毒犬外周血T淋巴细胞和B淋巴细胞。发现中毒后7d T淋巴细胞增殖效应显著提高,14d到28d出现显著下降趋势。与0d相比,B淋巴细胞的增殖能力降低。CD3、CD4和CD8的比例均有明显下降,CD4/CD8比值也明显降低,AFB1使犬细胞免疫受到抑制。
6对疫苗免疫效果的影响未经免疫三月龄的健康幼犬10头,随机分成2组,每组5头,分别注射1头份犬细小病毒疫苗。实验组按20g/kg饲喂含340μg/kg AFB1的犬粮,对照组按20g/kg饲喂不含AFB1同品牌犬粮。于0d,7d,14d,21d,28d采集血清,检测细小病毒中和抗体。结果显示,采食AFB1的犬血清犬细小病毒中和抗体和对照组相比显著降低(127.9/701.6)(P<0.01)。
综上可见,AFB1引起了警犬中毒,表现食欲下降、黄疸、呕吐和血便等一系列症状,血液生化指标、血浆代谢物谱分析及病理变化的检测结果,同样证实诊断的正确。饲料中检出AFB可进一步确诊。治疗犬AFB1中毒患犬、主要为保肝和对症治疗。中、重度中毒治疗效果不理想。AFB1对犬的细胞免疫和体液免疫产生明显的抑制作用,致使犬免疫失败。
The aflatoxins are a group of structurally related polycyclic aromatic hydrocarbon toxic compounds produced by certain strains of the fungi Aspergillus flavus and A. parasiticus. Content and toxicity of AFB2in aflatoxins compounds is highest and strongest. AFB1has been approved to cause genotoxicity and cytotoxicity in many papers,consequeces of which are many kinds of DNA injury.The causes of many infectious diseases and vaccination failure in animals were associated with immunosuppressive effects of AFB1.So far, researches about canine aflatoxicosis poisoning of AFB1is relatively few.So we studied and reported an AFB1poisioning incident, in which138dogs were poisoned because of feeding canine foods contaminated by AFB1in a policedog raising base in2007,and surveyed it's clinical symptoms,pathological changes,laboratory diagnosis.medicine therapies and it's immunosuppressive effects.
1. Clinical symptoms and diagnosis
In the total378dogs fed with contaminated food,138dogs were revealed abnormal in the levels of ALT and AST by Serum Biochemical Parameters analysis.71dogs experienced clinical symptoms,in which36dogs died in the end.We observed the71dogs' clinical symptoms and found that all dogs occured spirit deprsessed and appetite drop,66.2%dogs appeared different degree of Visible mucosal or skin jaundice52.1%sick dogs appeared black tarry stool.In the36dead dogs.we can found the jaundice of of skin and subcutaneous fat acute hepatic necrosis intraperitoneal fluid haemorrhage of cardiac muscle,intestine and mesentery, and so on.
120samples of parenchyma organs from the12dead dogs were collected to examine the pathological change Pathological lesions included a diffusely hyperemia and haemorrhae of stomach and intestines mucosa; intestinal villi mildly lossed; myocardial cell presented degeneration of granules; congestion marked hyperaemia and inflammatory cell infiltrate in spleen; congestion, local tissue loosen and vacuolating abundantly leukocyte inflammatory cell infiltration in lymph node; large number of red blood cells filling in alveolus inflammatory cell infiltrating, thickness of the alveolar wall;microscopically congestion of liver parenchyma cytoplasmic vacuolation/fatty change of hepatocytes necrosis of hepatocytes, mononuclear and hetrophilic cell inflammatory cell infiltrates infiltration were observed;Kidneys of AF intoxicated dogs were microscopically revealed congestion and hemorrhages of the renal interstitial capillary vessels cytoplasmic vacuolation change of tubular epithelial cells.
2. Detection of aflatoxin
We collected33samples from11batches including the dog contaminated food,food materials and the internal organs of dead dogs to detected the aflatoxin content by High Performance Liquid Chromatography. test results revealed the AFB1aflatoxin content of the corn (batch070208), the puppy food (batch070215),the puppy and mature dogs food (batch070309) were0.854,0.2249,0.376and0.678mg/kg respectively.Contents of AFB2were0.062,0.018,0.028and0.044mg/kg respectively. In addition,the corn (batch070208) was indeed materials to product the canine foods of batch070215and070309.According these results above,we confirmed the aflatoxin poisoning was caused by feeding canine food contaminated by AFB1.
3. Serum biochemical parameters and GC-MS analysis of metabolites
In another test,we analysised Serum Biochemical Parameters of30dogs fed with contaminated food and compared the results with parameters from an examination of healthy dogs'.All the datum were analysised with SPSS15.0software.It showed the average values of Serum alanine transaminase (ALT) rised from37.7u/L to190.07u/L; levels of aspartate aminotransferase(AST),total bilirubin,direct bilirubin increased; cholinesterase、total protein and Albumin decreased.The diversity of seven biochemical parameters between before and after poisoned was significant(P<0.01).
The principal metabolites in serum during aflatoxin poisoning were analysised by gas chromatography-mass spectra(GC-MS) method and the diversion of endogenous metabolites along with poisoning time were tested with principal component analysis (PCA) respectively. Research indicates that content of amino acid such as L-Isoleucine.Glycine,L-Threonine.Aspartic Acid,L-Phenylalanine, was increased along with time.Relative peak of L-Isoleucine rising from0.050(±0.008)to0.102(±0.001); glucose.cholesterol and part of free fatty acid(FFA) levels declined lower;relative peak of cholesterol declined from0.757(±0.061)to0.532(±0.074),of which the diversity was significant (P<0.05).These changes of principal metabolites in serum were closely related to liver damage, and trend of changing was identical with the Serum Biochemical Parameters.So it is confirmed that employing the metabonomics technique,such as GC-MS and PCA,we can examinate the dogs'metabolic state successfully,either it is helpful for the early diagnosis of AFB1poisoning.
4. medicine therapy
In order to obtain an effective medicine therapy.series of medicine experiments were performed. Thirty-five poisoned dogs were randomly assigned to5groups (7replicate pens per treatment):group A were treated with basic therapy; group B,C,D and E were treated with specific medicine of glutathione, glucurolactone, hepatocyte growth-promoting factor (HCF) and Yinzhihuang injection respectively. Four healthy dogs were assigned as negative control. Effects of different therapy was assessed with chi-square statistics.
As shown by the experimental results, the five medicine treatment were not very effective to AFB1poisoning. The total mortality of five test groups was69%,particularly when the poisoning devoleped into moderate level mortality will increase to100%.Combination of three medicine glutathione, glucurolactone, hepatocyte growth-promoting factor (HCF) were relatively effective to cure the dogs poisoned lightly,that total efficient rate will be57.1%.
5. Experimental study of immunosuppression effects
We performed lymphocyte(LC) transformation assay and immunocytochemistry assay to test effects of AFB1on lymphocyte proliferation capacity and to examinate the T-lymphocyte subgroups.Healthy dogs aged between1to3years were fed with food containing340μg/kg AFB1.After0d,7d,14d,21d and28d,the numbers of blood T-cells were measured.
It showed the optimal concentration of Con A to activate lymphocyte proliferation capacity was80μg/mL; the corresponding optimal culture time was48h; lymphocyte number was106/mL. The optimal concentration of LPS to activate B cell was100μg/mL; the corresponding optimal culture time was20h; lymphocyte number was10'/mL
Stimulating T lymphocyte and B lymphocyte in peripheral blood of poisoned dogs using ConA and LPS respectively,we found the effects on T lymphocyte of dogs poisoned for seven days was raised significantly;the effects would decline greatly after forteen days; B lymphocyte proliferation capacity would decline compared with the healthy dogs; concentration of CD3,CD4,CD8in serum declined greatly, and CD4/CD8value declined either.We can conclude from these results that immune cells will be suppressed by AFB1in the process of aflatoxin poisoning.
6. Effects on vaccination
Effects on vaccination were tested by the following method:ten healthy dogs aged three months were divided into two groups,which will be injected with CPV vaccine in same vaccination schedule;group A feed with contaminated food (a diet with20g/kg per dog)with AFB1(340μg in one kg food); group B feed dog food without AFB1;serum of A and B will be collected at0d,7d,14d,21d and28d in order to detect antibody anti-CPV.Research results showed the antibody titer in group A was significant lower than that of group B.It suggestted the AFB1would effects the vaccination.
Conclusion
The studies concluded that AFB1was the main cause of the poisioning event, characterized by appetite decline, jaundice, vomiting and hematochezia;according to the analysis of biochemical parameters and metabolites in serum,and the confirmation of AFB in food,we can further confirm the former conclusion.In the therapy test,we proved the inefficacy to cure dogs in severe poisoning and medium poisoning,so the effective measures were liver-protective and symptomatic treatment.Also the sdudies proved the suppressive effects to cellular immunity and humoral immunity,which will result in failure of vaccination.
1临床诊断对犬场的378头犬进行血液生化检查,有138头出现了谷丙转氨酶、谷草转氨酶异常,有71头犬出现了临床症状,其中有36头犬死亡。
对71头患犬进行观察,患犬都出现精神沉郁,食欲下降,66.2%的患犬出现不同程度可视黏膜及皮肤黄染,52.1%的患犬出现粪便呈煤焦油样等症状；对全部36头死亡犬进行剖检,发现死亡犬皮肤、皮下脂肪黄染,肝坏死呈土黄色,心肌斑点状出血,腹腔积水,肠系膜广泛性出血,肠道出血等。
采集12头死亡犬实质性脏器组织的120个样品作病理检查。死亡犬都出现胃黏膜充血出血,肠黏膜充血出血,肠绒毛轻微脱落,心肌细胞发生颗粒变性,脾脏明显充血,有炎性细胞浸润,淋巴结充血,局部组织疏松,呈网状或空泡状,淋巴窦扩张,充满大量浆液和炎性细胞,肺泡中充满大量红细胞,肺泡隔增厚,有炎性细胞浸润,肝脏明显充血出血,肝索紊乱肝细胞发生颗粒变性和水泡变性,并可见大小不一坏死区,有少量炎性细胞浸润,肾脏肾间质毛细血管充血,肾小管上皮细胞空泡变性。
2毒素检测委托江苏省进出境动植物检验检疫局,采用高效液相色谱法对饲料及饲料原料、及死亡犬内脏11个批号的33个样品进行黄曲霉毒素检测。通过检测发现,070208号玉米,070215幼犬饲料、070309幼犬和成犬饲料中黄曲霉毒素B1分别达到0.854、0.2249、0.376和0.678mg/kg,B2分别达0.062、0.018、0.028和0.044mg/kg,而070208号玉米正是用于生产070215、070309犬饲料的原料。确诊该犬群系采食含有AFB1饲料而发生中毒。
3血液生化指标检测及代谢物谱分析已采食被AFB1污染的饲料30d的30头犬的血液生化指标为实验组。以2006年3月9日健康检查相对应犬的生化指标为对照组。用SPSS15.0统计软件用t检验对实验数据进行统计分析。30头患犬采食AFB1污染的饲料30天后,谷丙转氨酶均值由37.7u/L上升到190.07u/L,谷草转氨酶、总胆红素、直接胆红素升高,胆碱酯酶、总蛋白和白蛋白降低。犬AFB1中毒前后7项生化指标变化均呈显著性差异(P<0.01)。
通过气相色谱-质谱技术(GC-MS)分析AFB1中毒犬血浆中的代谢物谱,并运用主成分分析(PCA)方法对血浆内源性代谢物随中毒时间的变化情况进行识别。研究发现,随着AFB1中毒时间的延长,血浆中的如L-异亮氨酸、甘氨酸、L-苏氨酸、天冬氨酸、L-苯丙氨酸等氨基酸含量有所上升,L-异亮氨酸相对峰值面积由0.050(+0.008)上升到0.102(+0.001),与对照组相比有显著(P<0.05)。葡萄糖、胆固醇以及部分游离脂肪酸含量下降,胆固醇相对峰值面积由0.757(±0.061)下降到0.532(+0.074),与对照组相比有显著(P<0.05)。这些代谢物的变化都与肝脏的损伤程度密切相关,且变化趋势与血液生化指标检测结果一致。研究结果表明,基于GC-MS的代谢组学技术结合PCA的方法能较全面地反应生物体的代谢状态,有助于AFB1中毒的早期诊断。
4治疗试验以4头健康犬为阴性对照,以7头AFB1中毒患犬为阳性对照,作基础治疗。其它35头中毒患犬分成5组,每组7头,除基础治疗外,分别采用谷胱甘肽、葡醛内酯、促肝细胞生长素和茵栀黄注射液进行特异性治疗。组间疗效比较用卡方检验。
犬AFB1中毒的疗效不佳,实验组死亡率达69%,发生中重度中毒后,死亡率达100%。而轻度的AFB1中毒,通过采用谷胱甘肽、葡醛内酯、促肝细胞生长素联合治疗,总有效率(痊愈+显效+有效)能达到57.1%。
5免疫抑制试验取1-3岁的健康犬,按20g/kg饲喂含AFB1浓度为340μg/kg的犬粮,于0d、7d、14d、21d和28d,用淋巴细胞转化实验和免疫组化方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。
试验确定Con A刺激犬T细胞增值的最佳浓度是80μg/mL,对应的培养时间是48h,淋巴细胞浓度是1×106个/mL。LPS刺激犬B细胞的最佳浓度是100gg/mL,对应的培养时间是20h,淋巴细胞浓度是1×107个/mL。
用Con A和LPS分别刺激AFB1中毒犬外周血T淋巴细胞和B淋巴细胞。发现中毒后7d T淋巴细胞增殖效应显著提高,14d到28d出现显著下降趋势。与0d相比,B淋巴细胞的增殖能力降低。CD3、CD4和CD8的比例均有明显下降,CD4/CD8比值也明显降低,AFB1使犬细胞免疫受到抑制。
6对疫苗免疫效果的影响未经免疫三月龄的健康幼犬10头,随机分成2组,每组5头,分别注射1头份犬细小病毒疫苗。实验组按20g/kg饲喂含340μg/kg AFB1的犬粮,对照组按20g/kg饲喂不含AFB1同品牌犬粮。于0d,7d,14d,21d,28d采集血清,检测细小病毒中和抗体。结果显示,采食AFB1的犬血清犬细小病毒中和抗体和对照组相比显著降低(127.9/701.6)(P<0.01)。
综上可见,AFB1引起了警犬中毒,表现食欲下降、黄疸、呕吐和血便等一系列症状,血液生化指标、血浆代谢物谱分析及病理变化的检测结果,同样证实诊断的正确。饲料中检出AFB可进一步确诊。治疗犬AFB1中毒患犬、主要为保肝和对症治疗。中、重度中毒治疗效果不理想。AFB1对犬的细胞免疫和体液免疫产生明显的抑制作用,致使犬免疫失败。
The aflatoxins are a group of structurally related polycyclic aromatic hydrocarbon toxic compounds produced by certain strains of the fungi Aspergillus flavus and A. parasiticus. Content and toxicity of AFB2in aflatoxins compounds is highest and strongest. AFB1has been approved to cause genotoxicity and cytotoxicity in many papers,consequeces of which are many kinds of DNA injury.The causes of many infectious diseases and vaccination failure in animals were associated with immunosuppressive effects of AFB1.So far, researches about canine aflatoxicosis poisoning of AFB1is relatively few.So we studied and reported an AFB1poisioning incident, in which138dogs were poisoned because of feeding canine foods contaminated by AFB1in a policedog raising base in2007,and surveyed it's clinical symptoms,pathological changes,laboratory diagnosis.medicine therapies and it's immunosuppressive effects.
1. Clinical symptoms and diagnosis
In the total378dogs fed with contaminated food,138dogs were revealed abnormal in the levels of ALT and AST by Serum Biochemical Parameters analysis.71dogs experienced clinical symptoms,in which36dogs died in the end.We observed the71dogs' clinical symptoms and found that all dogs occured spirit deprsessed and appetite drop,66.2%dogs appeared different degree of Visible mucosal or skin jaundice52.1%sick dogs appeared black tarry stool.In the36dead dogs.we can found the jaundice of of skin and subcutaneous fat acute hepatic necrosis intraperitoneal fluid haemorrhage of cardiac muscle,intestine and mesentery, and so on.
120samples of parenchyma organs from the12dead dogs were collected to examine the pathological change Pathological lesions included a diffusely hyperemia and haemorrhae of stomach and intestines mucosa; intestinal villi mildly lossed; myocardial cell presented degeneration of granules; congestion marked hyperaemia and inflammatory cell infiltrate in spleen; congestion, local tissue loosen and vacuolating abundantly leukocyte inflammatory cell infiltration in lymph node; large number of red blood cells filling in alveolus inflammatory cell infiltrating, thickness of the alveolar wall;microscopically congestion of liver parenchyma cytoplasmic vacuolation/fatty change of hepatocytes necrosis of hepatocytes, mononuclear and hetrophilic cell inflammatory cell infiltrates infiltration were observed;Kidneys of AF intoxicated dogs were microscopically revealed congestion and hemorrhages of the renal interstitial capillary vessels cytoplasmic vacuolation change of tubular epithelial cells.
2. Detection of aflatoxin
We collected33samples from11batches including the dog contaminated food,food materials and the internal organs of dead dogs to detected the aflatoxin content by High Performance Liquid Chromatography. test results revealed the AFB1aflatoxin content of the corn (batch070208), the puppy food (batch070215),the puppy and mature dogs food (batch070309) were0.854,0.2249,0.376and0.678mg/kg respectively.Contents of AFB2were0.062,0.018,0.028and0.044mg/kg respectively. In addition,the corn (batch070208) was indeed materials to product the canine foods of batch070215and070309.According these results above,we confirmed the aflatoxin poisoning was caused by feeding canine food contaminated by AFB1.
3. Serum biochemical parameters and GC-MS analysis of metabolites
In another test,we analysised Serum Biochemical Parameters of30dogs fed with contaminated food and compared the results with parameters from an examination of healthy dogs'.All the datum were analysised with SPSS15.0software.It showed the average values of Serum alanine transaminase (ALT) rised from37.7u/L to190.07u/L; levels of aspartate aminotransferase(AST),total bilirubin,direct bilirubin increased; cholinesterase、total protein and Albumin decreased.The diversity of seven biochemical parameters between before and after poisoned was significant(P<0.01).
The principal metabolites in serum during aflatoxin poisoning were analysised by gas chromatography-mass spectra(GC-MS) method and the diversion of endogenous metabolites along with poisoning time were tested with principal component analysis (PCA) respectively. Research indicates that content of amino acid such as L-Isoleucine.Glycine,L-Threonine.Aspartic Acid,L-Phenylalanine, was increased along with time.Relative peak of L-Isoleucine rising from0.050(±0.008)to0.102(±0.001); glucose.cholesterol and part of free fatty acid(FFA) levels declined lower;relative peak of cholesterol declined from0.757(±0.061)to0.532(±0.074),of which the diversity was significant (P<0.05).These changes of principal metabolites in serum were closely related to liver damage, and trend of changing was identical with the Serum Biochemical Parameters.So it is confirmed that employing the metabonomics technique,such as GC-MS and PCA,we can examinate the dogs'metabolic state successfully,either it is helpful for the early diagnosis of AFB1poisoning.
4. medicine therapy
In order to obtain an effective medicine therapy.series of medicine experiments were performed. Thirty-five poisoned dogs were randomly assigned to5groups (7replicate pens per treatment):group A were treated with basic therapy; group B,C,D and E were treated with specific medicine of glutathione, glucurolactone, hepatocyte growth-promoting factor (HCF) and Yinzhihuang injection respectively. Four healthy dogs were assigned as negative control. Effects of different therapy was assessed with chi-square statistics.
As shown by the experimental results, the five medicine treatment were not very effective to AFB1poisoning. The total mortality of five test groups was69%,particularly when the poisoning devoleped into moderate level mortality will increase to100%.Combination of three medicine glutathione, glucurolactone, hepatocyte growth-promoting factor (HCF) were relatively effective to cure the dogs poisoned lightly,that total efficient rate will be57.1%.
5. Experimental study of immunosuppression effects
We performed lymphocyte(LC) transformation assay and immunocytochemistry assay to test effects of AFB1on lymphocyte proliferation capacity and to examinate the T-lymphocyte subgroups.Healthy dogs aged between1to3years were fed with food containing340μg/kg AFB1.After0d,7d,14d,21d and28d,the numbers of blood T-cells were measured.
It showed the optimal concentration of Con A to activate lymphocyte proliferation capacity was80μg/mL; the corresponding optimal culture time was48h; lymphocyte number was106/mL. The optimal concentration of LPS to activate B cell was100μg/mL; the corresponding optimal culture time was20h; lymphocyte number was10'/mL
Stimulating T lymphocyte and B lymphocyte in peripheral blood of poisoned dogs using ConA and LPS respectively,we found the effects on T lymphocyte of dogs poisoned for seven days was raised significantly;the effects would decline greatly after forteen days; B lymphocyte proliferation capacity would decline compared with the healthy dogs; concentration of CD3,CD4,CD8in serum declined greatly, and CD4/CD8value declined either.We can conclude from these results that immune cells will be suppressed by AFB1in the process of aflatoxin poisoning.
6. Effects on vaccination
Effects on vaccination were tested by the following method:ten healthy dogs aged three months were divided into two groups,which will be injected with CPV vaccine in same vaccination schedule;group A feed with contaminated food (a diet with20g/kg per dog)with AFB1(340μg in one kg food); group B feed dog food without AFB1;serum of A and B will be collected at0d,7d,14d,21d and28d in order to detect antibody anti-CPV.Research results showed the antibody titer in group A was significant lower than that of group B.It suggestted the AFB1would effects the vaccination.
Conclusion
The studies concluded that AFB1was the main cause of the poisioning event, characterized by appetite decline, jaundice, vomiting and hematochezia;according to the analysis of biochemical parameters and metabolites in serum,and the confirmation of AFB in food,we can further confirm the former conclusion.In the therapy test,we proved the inefficacy to cure dogs in severe poisoning and medium poisoning,so the effective measures were liver-protective and symptomatic treatment.Also the sdudies proved the suppressive effects to cellular immunity and humoral immunity,which will result in failure of vaccination.
引文
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