Evaluation of antidepressant activity of methanolic and hydroalcoholic extracts of Acorus calamus L.rhizome through tail suspension test and forced swimming test of mice

Objective:Acorus calamus(AC)L.(Araceae)is an annual semi-aquatic and aromatic plant found in Europe,North America and Asia.Its rhizomes are often used by Native Americans,Americans,and Chinese as well as by other cultures.Ethnobotanical studies and documents have shown their use in various disease treatments,such as insomnia,mental disorders,diabetes mellitus,epilepsy,inflammation,asthma,neuropathic pain,and diarrhea.In this study,the antidepressant activity of methanolic and hydroalcoholic extracts of the AC rhizome part in mice was investigated.Methods:Three doses of methanolic extract of AC rhizome(MEACR)(25,50 and 100 mg/kg b.wt),three doses of hydroalcoholic extract of AC rhizome(HAACR)(100,200 and 400 mg/kg b.wt),and standards(imipramine,15 mg/kg b.wt and fluoxetine,20 mg/kg b.wt)was daily oral administration to the mice for consecutive 14 days.The extract effect on the immobility time was monitored by a tail suspension test(TST)and a forced swimming test(FST).Monoamine oxidase(MAO)levels were also analyzed using standard methods.Results:The optimum antidepressant activity was viewed at 100 mg/kg b.wt of MEACR extract and400 mg/kg b.wt of HAACR extract with 23.82%and 20.59%immobility period reduction,respectively.Besides,the extracts weakened the FST-induced elevation of MAO activity significantly and returned to near-normal levels of neurotransmitters in the brain.100 mg/kg b.wt or above of MEACR extract significantly prevented the MAO-A and MAO-B activities in mice brain at a dose-dependent fashion.But,just 400 mg/kg b.wt of HAACR extract prevented the activity of MAO-A and MAO-B.Fluoxetine and imipramine showed a tendency to prevent the activity of MAO-A and MAO-B.Conclusion:This study suggests that AC rhizome extract mediated antidepressant activity by modulating the central neurochemical and hypothalamic-pituitary-adrenal(HPA)axis in response to FST and TSTinduced stress.Therefore,AC rhizome extract can be used as a valuable plant supplement to treat depressive disorders.

Triptolide prolonged allogeneic islet graft survival in chemically induced and spontaneously diabetic mice without impairment of islet function

BACKGROUND: Triptolide (TPT) is a diterpenoid triepoxide extracted from the Chinese herb Tripterygium wilfordii Hook. F. It exhibits potent immunosuppressive and anti-inflammatory properties. This study was undertaken to investigate its effects on prolongation of islet allograft survival in rodents. Additionally, we investigated whether TPT would be toxic to islet function in vivo. METHODS: We transplanted BALB/c islets to either chemically induced diabetic C57BL/6 mice or spontaneously diabetic non-obese diabetic (NOD) mice. TPT was injected within 2 weeks or continuously, until rejection, in the two combinations. Then, we evaluated the toxicity of TPT on islet function by daily injection to naive BALB/c or diabetic BALB/c that was cured by syngeneic islet transplantation under the kidney capsule. Mice injected with cyclosporine A (CsA) or vehicle served as controls. Intraperitoneal glucose tolerance tests (IPGTTs) performed at 4 and 8 weeks in the naive BALB/c group, and at 2, 4, 6, and 8 weeks in the syngeneic transplanted group. RESULTS: The medium survival time of islets allograft from TPT treated C57BL/6 and NOD recipients were 28.5 days (range 24-30 days, n=10) and 33.0 days (range 15-47 days, n=6), respectively, and they were significantly different from those of the vehicle treated controls, which were 14.0 days (range 13-16 days, n=6) and 5.0 days (range 4-10 days, n=6), respectively (all P<0.0001). The IPGTT demonstrated that there was no difference between the TPT treated and vehicle treated groups, either in the normal or syngeneic transplanted islet BALB/c mice. However, CsA injection impaired islet function in both normal and syngeneic transplanted mice as early as 4 weeks. CONCLUSION: TPT prolonged islets allograft survival in a chemically induced diabetic or an autoimmune diabetic murine model without impairment of islet function. (Hepatobiliary Pancreat Dis Int 2010; 9: 312-318)

Chrysophanol attenuates lead exposure-induced injury to hippocampal neurons in neonatal mice

Previous studies have shown that chrysophanol protects against learning and memory impairments in lead-exposed adult mice. In the present study, we investigated whether chrysophanol can alleviate learning and memory dysfunction and hippocampal neuronal injury in lead-exposed neonatal mice. At the end of lactation, chrysophanol（0.1, 1.0, 10.0 mg/kg） was administered to the neonatal mice by intraperitoneal injection for 15 days. Chrysophanol significantly alleviated injury to hippocampal neurons and improved learning and memory abilities in the lead-poisoned neonatal mice. Chrysophanol also significantly decreased lead content in blood, brain, heart, spleen, liver and kidney in the lead-exposed neonatal mice. The levels of malondialdehyde in the brain, liver and kidney were significantly reduced, and superoxide dismutase and glutathione peroxidase activities were significantly increased after chrysophanol treatment. Collectively, these findings indicate that chrysophanol can significantly reduce damage to hippocampal neurons in lead-exposed neonatal mice.

Systemic study on the safety of immuno-deficient nude mice treated by atmospheric plasma-activated water

Cold atmospheric-pressure plasma is a new technology, widely used in many fields of biomedicine,especially in cancer treatment. Cold plasma can selectively kill a variety of tumor cells, and its biological safety in clinical trials is also very important. In many cases, the patient’s immune level is relatively low, so we first studied the safety assessment of plasma treatment in an immunocompromised animal model. In this study, we examined the safety of immuno-deficient nude mice by oral lavage treatment of plasma-activated water, and studied the growth status, main organs and blood biochemical indexes. Acute toxicity test results showed that the maximum dose of plasma treatment for 15 min had no lethal effect and other acute toxicity. There were no significant changes in body weight and survival status of mice after 2 min and 4 min of plasma-activated water(PAW)treatment for 2 weeks. After treatment, the major organs, including heart, liver, spleen, lung and kidney, were not significantly changed in organ coefficient and tissue structure. Blood biochemical markers showed that blood neutrophils and mononuclear cells were slightly increased, and the others remained unchanged. Liver function, renal function, electrolytes, glucose metabolism and lipid metabolism were not affected by different doses of PAW treatment. The above results indicate that PAW treatment can be used to treat immuno-deficient nude mice without significant safety problems.

The Protective Effect of Jiujiuguiyi, a Medicine and Food Homologous Formula, on Acute Alcohol Poisoning Mice

Background: Nowadays, acute alcoholic intoxication has become the third public problem in China, and the anti-inebriation products mainly aimed at increasing the activity of enzyme involved in the alcohol metabolism, which is a single mechanism that can accelerate alcohol metabolism. Thus, a new formula, Jiujiuguiyi (JJGY) which could protect liver, relieve the abnormal excitability of the center and improve muscle retardation at the same time is designed by us. Methods: The model of acute alcoholic intoxication was established by intragastric administration with 0.12 ml/10g 50% alcohol in mice. JJGY was orally administrated (gavage) once a day for 20 consecutive days before the establishment of acute alcoholic model. Mice were randomly divided into 8 groups with 8 each: blank control group (CON), model group (M), Haiwangjinzun positive control group (HWJZ), experimental groups (AL, AH, BL, BH, AB). Giant, crawling time on the rota-rod, the activities of aspartate amino trans- ferase (AST), alanine amino transferase (ALT) and superoxide dismutase (SOD) in both liver and serum, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), glutathione peroxidase (GSH-PX) in liver as well as the HE staining of liver slices, the formation of malondialdehyde (MDA) in serum were determined after acute alcoholic intoxication. Results: Compared with model group, JJGY significantly decreased the AST and ALT activity in liver and serum and MDA activity in serum. Meanwhile, it enhanced the ADH and ALDH level in liver as well as the hepatic and serous SOD activity, indicating more efficient metabolism of alcohol and less hepatic injury. HE staining results also proved that JJGY could reduce alcoholic liver cell injury, and the effect was more obvious in the group medicated before alcohol administration. Moreover, JJGY significantly prolonged the crawling time on the rota-rod and improved the gait of mice and the effect was proved to be better than the widely used health product Haiwangjinzun. Conclusions: This study suggests that JJGY is able to protect liver, relieve the abnormal excitability of the center and improve muscle retardation after acute alcoholic intoxication. Its liver protection effect is likely related to its modulation on the alcohol metabolizing and antioxidant enzymes.

Depressive-like behavior in mice recently recovered from motor disorders after 3-nitropropionic acid intoxication

Objective Striatum may be involved in depressive disorders according to the neuroimaging analysis and clinical data. However, no animal model at present supported the possible role of striatum in the pathogenesis of depression. In the present study, we have investigated the depressive-like behavior in mice recently intoxicated with 3-nitropropionic acid （3- NP）, a widely known toxin that selectively damages the striatum in the brain. Methods Mouse model was made with subacute systemic 3-NP treatment, and the depressive-like behavior was measured using the duration of immobility during forced swimming test （FST）. Results When the mice at day 15 post-intoxication just totally recovered from motor deficits, the duration of immobility in FST was significantly longer than that in controls. The depressive-like behavior was not due to the fatigue or general sickness following 3-NP intoxication and could be reversed by the antidepressant, desipramine hydrochloride. In two successive FST in 24 h interval, the depressive-like behavior could be observed again in subsequent FST （at day 16 post-intoxication）, and the mice presented a normal ＂learned helplessness＂. Conclusion A novel depression animal model could be established in mice during the initial period of recovery from 3-NP intoxication. The depression-like behavior might occur independently without involvement of cognitive defects, and the striatal lesions may underlie the depression-like behavior attributable to 3-NP intoxication.

Di-(n-butyl)-phthalate-induced Oxidative Stress and Depression-like Behavior in Mice with or without Ovalbumin Immunization

Objective To investigate the relationship between atopic allergy and depression and the role of DBP in the development of depression. Methods BALB/c mice were randomly divided into eight groups：saline;ovalbumin （OVA）-immunized;saline＋DBP （0.45 mg/kg·183;d）; saline＋DBP （45 mg/kg·d）; DBP （0.45 mg/kg·d） OVA-immunized; DBP （45 mg/kg·d） OVA-immunized; saline＋hydrocortisone （30 mg/kg·d）; and hydrocortisone （30 mg/kg·d）-exposed OVA-immunized. Behavior （e.g. open-field, tail suspension, and forced swimming tests）, viscera coefficients （brain and spleen）, oxidative damage [e.g. reactive oxygen species （ROS）, malondialdehyde （MDA）, and glutathione （GSH）], as well as levels of IgE and IL-4, were then analyzed. Results In the saline and OVA groups, the degree of depression symptoms in mice increased with increasing DBP concentration. Additionally, the OVA-immunity groups were associated with more serious depressive behavior compared with the same exposure concentration in the saline group. Oxidative damage was associated with a dose-dependent increase in DBP in the different groups. IL-4 and IgE levels were associated with low-dose DBP stimulation, which changed to high-dose inhibition with increasing DBP exposure, possibly due to spleen injury seen at high DBP concentrations.Conclusion Development of an atopic allergy has the potential to increase the risk of depression in mice, and it seems that DBP helps OVA to exert its effect in our present model. Moreover, the results of our study implicate a certain connection between brain oxidative stress and depression, which deserves a further exploration.

Anti-hyperglycemic effects of aqueous Lenzites betulina extracts from the Philippines on the blood glucose levels of the ICR mice(Mus musculus)

Objective: To examine the anti-hyperglycemic effects of aqueous Lenzites betulina(L. betulina) extracts on normoglycemic glucose-loaded mice.Methods: Different doses of aqueous extract from L. betulina were administered to 45 ICR mice(Mus musculus) to determine whether there was an effect of L. betulina extracts on the blood glucose level of the ICR mice. Aqueous extracts of L. betulina were orally gavaged to mice using oral glucose tolerance test. A total of five groups were used to determine the effect of the fungi on blood glucose of the mice. Group A(positive control)was given 16.7 mg/kg glimepiride; Group B(negative control) was given distilled water;Group C(low dosage) was given 200 mg/kg aqueous extract; Group D(mid dosage) was given 400 mg/kg aqueous extract and Group E(high dosage) was given 800 mg/kg aqueous extract. Baseline blood glucose value was firstly acquired before induction of hyperglycemia through D-glucose, after which another check on blood glucose was made after 0.5 h. Immediately, after the acquisition of hyperglycemic blood glucose level, the individual administration of treatments were done. After that, three blood collections were done spanning 3 h with 1 h interval.Results: The low dose(200 mg/kg) and the mid dose(400 mg/kg) of L. betulina extracts were significantly different(P < 0.05) from their respective baseline values throughout the whole experiment with the latter surpassing its baseline value during the 3rd hour. On the other hand, the high dose(800 mg/kg) during the 1st hour after administration was not significantly different(P > 0.05) from its corresponding baseline value, acting faster than the positive control(glimepiride), which only became significantly different(P < 0.05) at the 2nd hour.Conclusions: Aqueous L. betulina extract is able to produce hypoglycemic effects on the mice with all doses, which are able to normalize blood glucose levels at varying times.

Acute Toxicity of Oxyclozanide Suspension in Mice by Oral Administration

The safety of oxyclozanidc suspension was preliminarily evaluated through acute toxicity test in mice. Administration dose, formal trial grouping and group interval were determined in pre-trial using incremental method. Formal test was performed using simplified karber＇s method. Changes in sign of mice after ad- ministration were observed; the mortality rate was statistically calculated, and the time of death was recorded; the median lethal dose （LD50） and 95% confidence limit of oxyclozanide suspension were calculated. The results showed the LD50 of oxyclozanide suspension in mice by oral administration was 1. 679 g/kg, and the 95% confidence interval was 1. 439 - 1. 947 g/kg. According to toxicity grading of chemicals, oxyclozanide suspension was low toxic substance.

Single Exposure to Antidepressants during Infancy Is Associated with Delayed Behavioral Changes in C57BL/6 Mice

As serotoninergic transmission plays a crucial role in higher brain function in mammals, the disturbance of this system will likely have significant effects on emotion and cognition. Previous studies have reported that chronic treatment with Specific Serotonin Reuptake Inhibitors (SSRIs) during both late pregnancy and lactation was associated with abnormal behavior in adult rats. These data imply that disturbances in serotoninergic transmission during neurodevelopment may have negative effects on both the structure and function of the resultant adult brain. Therefore, the effect of a single exposure to an SSRI or a tricyclic antidepressant that preferentially inhibits serotonin reuptake during the pre-weaning period was examined in adult mice. An oral infusion of paroxetine (70 mg/kg), fluvoxamine (250 mg/kg), clomipramine (180 mg/kg), or saline was administered on postnatal day 14. Starting at 11 weeks of age, mice were assessed using a comprehensive behavioral test battery. Mice treated with paroxetine demonstrated altered behavior on the open field and hole-board tasks;those treated with fluvoxamine had behavioral changes on the light-dark box, hole-board, and sucrose preference tasks, while alteration in forced swimming and cued fear behavior were noted in mice treated with clomipramine. These results suggest that even a single administration of an antidepressant could have profound effects on behavior in adulthood, although the effects might differ dependent on the specific drug that was administered.

Evaluation of the Anti-inflammatory and Sedative Effects of Leaf Aqueous Extract of Withania somnifera （L.） Dunal （Solanaceae）in Rats and Mice

The use of medicinal plants in South Africa is cultural. Withania somnifera is one of the medicinal plants used to treat various ailments in the country. The plant species has been used by traditional medicine practitioners to treat inflammation and painful conditions like rheumatism. It is also known to be used as a sedative and hypnotic drug. Despite the claims, there is no information in literature to corroborate the therapeutic success of Withania somnifera in the treatment of inflammation and insomnia. The study, therefore, investigated the anti-inflammatory and central nervous system depressant activities of the leaf aqueous extract of the plant species in mice and rats. Fresh leaves of W. somnifera were collected from Kirstenbosch Botanical Gardens, South Africa, authenticated by a taxonomist and a voucher specimen （UWC 005） deposited in the University＇s Herbarium. Leaf aqueous extract was prepared using standard extraction methods. The carrageenan-induced rat paw oedema test was used to determine the anti-inflammatory effects while pentobarbitone-induced sleep and locomotor activity tests were used to evaluate the sedative effect of the plant species. Phytochemical qualitative analysis, acute toxicity and HPLC studies of the plant species were also carried out using standard methods. The phytochemical qualitative analysis carried out on the dried powdered leaves of W. somnifera showed the presence of saponins, tannins and triterpene steroids. Leaf aqueous extract of IV. somnifera （100-200 mg/kg IP） significantly prolonged pentobarbitone （40 mg/kg, i.p.）-induced sleep in mice in a dose dependant manner. Diazepam （0.5 mg/kg, i.p.） significantly prolonged pentobarbitone （40 mg/kg, i.p.）-induced sleep in mice. The doses of 100 and 200 mg/kg （i.p.） of the plant species and 0.5 mg/kg （i.p.） of diazepam significantly reduced the locomotor activity of mice. Leaf aqueous extract of the plant species （50-200 mg/kg, i.p.） significantly reduced the oedema produced by carrageenan （1%） in rats over 90 min period of testing. Indomethacin （20 mg/kg, i.p.） significantly reduced carrageenan （1%）-induced oedema in rats over 120 min period of testing. The LDs0 value obtained for the leaf aqueous extract of the plant species following inter-peritoneal injection was 1,600 mg/kg while that following oral administration was probably over 4,000 mg/kg. The HPLC finger-print of the aqueous extract showed distinct peaks at the following retention times 2.977, 3.594, 4.154, 4.406, 4.660 and 15.267 min. The results obtained show that leaf aqueous extract of W. somnifera has both sedative and anti-inflammatory effects.

Isolation,identification,and virulence gene analysis of pathogenic Aeromonas dhakensis in Macrobrachium rosenbergii and histopathological observation

To identify the cause of mass mortality of adult Macrobrachium rosenbergii in a farm in Gaoyou City,Jiangsu Province,China,a dominant strain named DKQ-1 was isolated from the hepatopancreas of dying M.rosenbergii and identified as Aeromonas dhakensis by purification culture,biochemical characterization,and 16S rRNA and gyrB gene sequence analysis.The results of the challenge test revealed that the strain was highly pathogenic and the 50%lethal dose(LD_(50))in 72 h to M.rosenbergii was 1.54×10^(5)CFU/mL.The amplification results of virulence genes show that strain DKQ-1 carried 9 virulence genes,including ascV,aexT,aer,act,lip,ompAI,gcaT,acg,and exu,supporting the strong virulence of strain DKQ-1 to M.rosenbergii.Histopathological observation of the hepatopancreas,gills,and intestines indicated that DKQ-1 injection into M.rosenbergii could cause serious tissue damage,which further supported the strong virulence of this strain.In addition,a drug susceptibility test revealed that strain DKQ-1 was sensitive to 16 kinds of antibiotics,resistant to 9 kinds of antibiotics,and had intermediate resistance to spectinomycin and kanamycin.This study is the first report of A.dhakensis isolated from M.rosenbergii and provided a reference for the pathogen identification of bacterial diseases in M.rosenbergii,and for the prevention and treatment caused by A.dhakensis.

Different infection routes of avian influenza A (H5N1) virus in mice

The continuing outbreaks of avian influenza A H5N1 virus infection in Asia and Africa have caused worldwide concern because of the high mortality rates in poultry,suggesting its potential to become a pandemic influenza virus in humans.The transmission route of the virus among either the same species or different species is not yet clear.Broilers and BABL/c mice were inoculated with the H5N1 strain of influenza A virus isolated from birds.The animals were inoculated with 0.1 mL 106.83 TCID50 of H5N1 virus oronasally,intraperitoneally and using eye drops.The viruses were examined by virological and pathological assays.In addition,to detect horizontal transmission,in each group,healthy chicks and mice were mixed with those infected.Viruses were detected in homogenates of the heart,liver,spleen,kidney and blood of the infected mice and chickens.Virus antigen was not detected in the spleen,kidney or gastrointestinal tract,but detected by Plaque Forming Unit(PFU)assay in the brain,liver and lung without degenerative change in these organs(in the group inoculated using eye drops.The detection results for mice inoculated using eye drops suggest that this virus might have a different tissue tropism from other influenza viruses mainly restricted to the respiratory tract in mice.All chicken samples tested positive for the virus,regardless of the method of inoculation.Avian influenza A H5N1 viruses are highly pathogenic to chickens,but its virulence in other animals is not yet known.To sum up,the results suggest that the virus replicates not only in different animal species but also through different routes of infection.In addition,the virus was detection not only in the respiratory tract but also in multiple extra-respiratory tissues.This study demonstrates that H5N1 virus infection in mice can cause systemic disease and spread through potentially novel routes within and between mammalian hosts.

鸡源鼠伤寒沙门菌的分离鉴定、药敏试验与毒力基因检测

鼠伤寒沙门菌是一种重要的人兽共患病病原,不仅影响养禽业的健康发展,还对禽类产品安全构成严重威胁,本研究主要是对鸡源鼠伤寒沙门菌进行分离鉴定、药敏试验和毒力基因的测定。首先采集病死产蛋鸡的脾脏组织进行分离培养和染色镜检,以及16S rRNA基因的PCR鉴定,然后用圆纸片扩散法测定分离株对26种药物的敏感性,用PCR方法检测分离株的28个毒力基因。结果显示,分离株在普通营养琼脂和血琼脂培养基上形成灰白色菌落,在麦康凯培养基上为无色菌落,在BS培养基上为黑色带金属光泽的菌落;染色镜检结果显示分离株为革兰阴性的红色短杆菌;16S rRNA基因测序结果显示,分离株与鼠伤寒沙门菌OLF-FSR1-WB-Sparrow-ST-87的相似性为99.86%,与10个鼠伤寒沙门菌参考菌株的同源性介于99.5%~100%;药敏试验结果表明,分离株对环丙沙星最为敏感,抑菌圈直径达30 mm;除sseC基因未检测出外,其余27个毒力基因的测序结果与参考菌株对应毒力基因的相似性介于98.58%~100%。本研究成功从病死产蛋鸡分离鉴定出一株鼠伤寒沙门菌菌株FY2021,为深入研究鼠伤寒沙门菌的致病机理和禽源沙门菌病的防控提供参考依据。

基于小白鼠试验评价乳清醋增强免疫功能研究

用乳清醋饲喂小白鼠试验,分析检验其免疫学指标,评价乳清醋是否具有增强机体免疫的功能特性。试验鼠选用体质量(BW)为20 g的雄性小白鼠,共320只,从中每次随机取40只作为1个批次饲喂的试验鼠,分为4组,3个处理组和1个对照组,每组10只。实验室制备总酸为5.52 g/100 mL的乳清醋,乳清醋经稀释后的高、中、低浓度的剂量组,分别对处理组小鼠以剂量为10 mL/kg BW灌胃,对照组同步灌胃等量生理盐水,日饲1次,连续30 d为1个批次饲喂试验。研究以试验鼠的8项免疫学指标为检测目标:(a)免疫器官重量及胸腺脾脏指数,(b)迟发型超敏反应,(c)淋巴细胞转化率,(d)抗体生成细胞检测,(e)血清溶血素检测,(f)单核巨噬细胞攻击和吞噬能力,(g)碳粒廓清试验,(h)NK(Natural killer)细胞活性。每一批次饲喂试验结束后,针对其中1项免疫学指标要求对该批次的小鼠进行处置、解剖、检测。取平均数据为该批次各组鼠的该项免疫学指标数据。经8批次的饲喂试验,完成了试验鼠8项免疫学指标的检测。结果显示,处理组与对照组相比,除了(d)和(e)2项指标没有明显的统计学差异(P>0.05)外,中、高剂量组小鼠的其余6项免疫学指标均明显增强,均有统计学差异(P<0.05)。本研究发现,乳清醋饲喂白鼠后在促进和提高小鼠的免疫器官增重,细胞免疫功能,单核巨噬细胞吞噬功能和NK细胞活性等方面起了明显作用,可以判定乳清醋具有增强人体免疫力的功能。

四川省小麦黄斑叶枯病病原菌生理小种鉴定

[目的]对四川省主要小麦产区的小麦黄斑叶枯病致病菌进行分离与鉴定,分析其致病基因组成,探究其致病菌的致病力,明确其生理小种类型,为小麦黄斑叶枯病的防治提供理论基础。[方法]于2021—2022年在四川主要小麦产区采集小麦叶枯病样本,经病原菌分离,通过形态学观察分析,内部转录间隔区(Internal transcribed spacer, ITS)序列分析,利用致病基因(Ptr ToxA、ToxB和ToxC)扩增和小麦黄斑叶枯病鉴定系致病性测定相结合的方法对病原菌进行鉴定。[结果]在小麦叶枯病样本中共分离并鉴定出3个小麦黄斑叶枯病病原菌菌株,均为Pyrenophora tritici-repentis,其在所有分离得到的小麦叶枯病病原菌中所占比例较小。对3个小麦黄斑叶枯病病原菌菌落和分生孢子进行形态学观察,未发现显著差异,符合病原菌形态学鉴定标准。进一步对3个小麦黄斑叶枯病病原菌进行ITS序列分析,结果显示3个菌株与参考基因组(ID:AM887509)的ITS序列相似性达到100%。对致病基因Ptr ToxA、ToxB、ToxC进行扩增,3个菌株在ToxA和ToxB基因的位点上均未扩增出任何片段,但在ToxC基因位点上均成功扩增出约500 bp的条带。病原菌致病性鉴定结果显示,分离得到的菌株在Prosper和6B365品种上表现出较强的致病性,引起大面积叶片黄化,而在Glenlea和6B662品种上则未出现明显的叶片坏死或黄化症状。在四川省主要小麦产区收集到的小麦黄斑叶枯病致病菌为P.tritici-repentis生理小种3。[结论]本研究系国内首次针对P.tritici-repentis不同生理小种进行鉴定报道,将为我国在小麦黄斑叶枯病的防治上提供重要参考。

猪流行性腹泻灭活疫苗毒株的培育

为培育出毒力弱、病毒含量高、免疫原性好的猪流行性腹泻病毒(PEDV)疫苗株,将PEDV-YL株在Vero细胞上连续传代并进行噬斑克隆纯化,测定病毒含量后进行毒力试验及免疫原性试验。结果显示:将健康仔猪接种F22代clone6、clone7毒种后,试验猪均表现出腹泻症状且均能检测到PEDV,剖检后各脏器未见异常;攻毒保护率为60%,各脏器均未出现病变。健康仔猪接种F32代clone10、clone12毒种后,60%试验猪出现腹泻症状且检出PEDV,剖检后各脏器无明显变化;一次免疫组攻毒保护率为60%~80%,二次免疫组为100%,各脏器均未发生病变。F32代clone10病毒含量略低于clone12且两者免疫原性相当。结果说明:PEDV-YL株F32代clone12更适合作为疫苗研制候选毒株,且二次免疫保护效果更好。本研究为猪流行性腹泻灭活疫苗研发提供了技术支撑。

一株猪源化脓隐秘杆菌的分离鉴定及其耐药性分析

为确定黑龙江省某养殖场因呼吸道症状急性死亡病猪的致病菌,并探究其主要生物学特性,本研究采集病死猪内脏组织,通过细菌分离、革兰氏染色、生化鉴定、16S r RNA基因的PCR扩增、测序、系统进化树的构建对分离菌进行种属鉴定;通过微量肉汤稀释法分析该菌对8类10种抗生素的敏感性;通过Illumina PE150对细菌全基因组测序,采用ResFinder软件分析分离菌的耐药基因,并分析耐药基因与耐药表型的相关性。利用VFanalyzer软件、BLASTN比对分离菌的毒力基因。结果显示,该菌株在10%脱纤维羊血平板培养基上培养24 h后形成表面光滑具有β-溶血环的单菌落;革兰氏染色结果显示呈蓝紫色短棒状杆菌;生化鉴定结果显示该分离菌的生化特性与化脓隐秘杆菌符合。16S r RNA基因的PCR结果显示,扩增到1397 bp的目的基因序列,与GenBank中序列比对结果显示该菌株与已报道的化脓隐秘杆菌同源性高达99%。16S r RNA基因系统进化树分析结果显示分离菌与辽宁沈阳的牛源化脓隐秘杆株TZQ 01株处于同一分支,与日本的猪源分离株NIAH13531处于较近分支,以上结果可确定该分离菌为化脓隐秘杆菌,并将其命名为FY-4-Z-1。药敏试验结果显示,该菌株对头孢菌素类的头孢噻呋,青霉素类的青霉素、阿莫西林,大环内酯类的红霉素,截短侧耳素类的泰妙菌素等抗生素敏感,对四环素类的四环素,氨基糖苷类的链霉素耐药。利用组装软件SPAdes对获得的全基因组测序数据组装后获得了分离菌全基因组草图,结果显示分离菌基因组大小为2399.961 kb;耐药基因分析结果显示,该菌株基因组包含3种耐药基因,分别是氨基糖苷类的ant(6)-la、大环内脂类的erm(X)、四环素类的tet(W)耐药基因,其中在化脓隐秘杆菌中首次检测到ant(6)-la基因。该菌株对四环素和链霉素的药敏试验结果与其耐药基因检测结果相符,而红霉素敏感表型可能与其耐药基因erm(X)的移码突变有关。利用BLASTN和VFanalyzer软件进行毒力基因检测,结果显示共检测到8个已知毒力基因和24个候选毒力相关基因。本实验进一步丰富了猪源化脓隐秘杆菌的相关研究,为规模化猪场化脓隐秘杆菌病的防治提供了重要的参考依据和用药指导。

大米免疫活性肽在小鼠体内的作用机制

目的:证明前期筛选出的肽GBP1(NSVFRALPVDVVANAYR)在小鼠体内的免疫调节活性。方法:通过动物试验探讨GBP1肽的免疫调节作用机制;利用RNA-Seq技术对GBP1肽处理前后的小鼠脾脏组织进行转录组测序分析,阐释GBP1肽免疫调节作用的分子机制。结果:GBP1肽处理组小鼠脾细胞增殖数趋于正常值水平,肝脏器系数显著降低,血清中IgG含量稳定;终质量分数为25 mg/kg的GBP1肽能够在一定程度上抑制小鼠血清中IL-2、IFN-γ以及TNF-α的分泌;通过转录组分析,从脾脏组织中获得780个差异基因,该基因在机体内涉及与免疫相关的p53信号通路、铁死亡、补体和凝血级联等通路密切相关。结论:GBP1肽可以影响细胞因子的表达,介导信号转导通路,行使免疫调节功能。

1株山鸡源致病性大肠杆菌的分离鉴定

为查明秦皇岛市某山鸡养殖场山鸡大批量死亡病因,试验对分离到的致病优势菌株进行生化鉴定和16S rRNA基因序列分析,并测试分离菌对21种抗生素的敏感性,进一步采用PCR方法检测分离菌的耐药基因。利用清洁级昆明小鼠和SPF鸡胚分别对分离菌进行致病性试验,并采用PCR方法检测分离菌的毒力基因。结果显示:分离菌为革兰氏阴性杆菌,在伊红美蓝培养基上生长出有金属光泽的菌落,生化特性与大肠杆菌生化特性符合率高达99%。BLAST分析发现,分离菌16S rRNA序列与大肠杆菌的基因相似性高达99.8%。分离菌对恩诺沙星和阿米卡星高度敏感,对青霉素、头孢曲松等15种药物耐药。分离菌的磺胺类、β-内酰胺类、四环素类的耐药基因与耐药表型基本相符,其余耐药基因与耐药表型不符,提示该分离菌可能存在其他的耐药机制。致病性试验结果显示,分离菌具有较强致病性,共检测到12种大肠杆菌主要毒力基因。研究表明,分离菌为山鸡源致病性大肠杆菌,且耐药情况复杂,具有较强的毒力和致病性。