纳米蜂胶黄酮体内外抗PPV活性试验

采用体内外试验研究纳米蜂胶黄酮抗猪细小病毒活性。体外试验,以普通蜂胶黄酮为对照,分3种加药方式(先加蜂胶,后加蜂胶,蜂胶和病毒同时加入)加至PK-15细胞中,用MTT法检测细胞活性,实时荧光PCR检测PK-15细胞中猪细小病毒(PPV)含量,观察蜂胶黄酮对PK-15细胞抗PPV及清除PPV能力的影响。结果显示,与普通蜂胶黄酮相比,纳米蜂胶黄酮可显著抑制PPV在PK-15细胞上生长,高浓度效果优于低浓度,3种加药方式中先加药物的效果最好。体内试验,豚鼠注射PPV后灌胃纳米蜂胶黄酮和普通蜂胶黄酮,检测血液中PPV含量、血清中HI含量。结果显示,高、中剂量的纳米蜂胶黄酮可以显著抑制PPV在血液中复制、减轻PPV对豚鼠体重的影响、提高血清HI效价。结论,普通蜂胶黄酮经纳米化处理后,可以显著提高其抗PPV活性。     

蜂胶体外对猪细小病毒感染细胞能力的影响

为研究蜂胶体外抗猪细小病毒（porcine parvovirus,PPV）活性,用细胞维持液将蜂胶稀释成10个浓度,加至长成单层的PK-15细胞培养体系中,用MTT法测定蜂胶对PK-15细胞的安全浓度;将蜂胶从最大安全浓度倍比稀释5个梯度,分3种加药方式（先加蜂胶后加PPV、先加PPV后加蜂胶、蜂胶和PPV同时加入）加至PK-15细胞培养体系中,用MTT法观察蜂胶对PPV感染PK-15细胞的影响;采用实时荧光定量PCR方法检测PK-15细胞培养体系中PPV含量,观察蜂胶对PK-15细胞清除PPV能力的影响。结果显示,在31.2~250 μg/mL安全浓度范围内蜂胶组D_{570 nm}值和PPV含量分别显著高于和低于病毒对照组（P<0.05）,表明蜂胶可增强PK-15细胞抗PPV感染及清除PPV的能力。蜂胶的这种作用效果与其浓度和加药方式有关,蜂胶浓度越高则效果越好;3种加药方式中,以先加蜂胶后加PPV方式抵抗PPV感染活性最好。     

蜂胶黄酮对病毒诱导PK-15细胞凋亡的影响

为研究蜂胶黄酮对病毒诱导宿主细胞凋亡的影响,将蜂胶黄酮从最大安全浓度250μg/mL倍比稀释5个浓度,与猪传染性胃肠炎病毒(TGEV)、猪细小病毒(PPV)分别一起加至单层PK-15细胞培养体系中,用流式细胞仪检测蜂胶黄酮对TGEV和PPV感染PK-15细胞凋亡率的影响。结果显示,与病毒对照组比较,TGEV和PPV感染PK-15细胞后,蜂胶黄酮可以显著降低PPV感染引起的PK-15细胞凋亡率,而对TGEV感染引起的PK-15细胞凋亡率则没有显著影响。说明蜂胶黄酮可以减轻无囊膜的PPV感染对PK-15细胞引起的凋亡。     

蜂胶黄酮对PK-15细胞感染PPV与TGEV产生抗感染因子的比较

为了研究蜂胶黄酮对宿主细胞感染不同结构病毒后产生抗感染因子的影响,将蜂胶黄酮从最大安全质量浓度250mg/L倍比稀释5个浓度,与猪传染性胃肠炎病毒(transmissible gastroenteritis of pigs virus,TGEV)、细小病毒(porcine parvovirus,PPV)分别一起加至长成单层PK-15细胞培养体系中,ELISA法测定蜂胶黄酮对TGEV或PPV感染PK-15细胞后产生IFN-β、IFN-γ和iNOS抗感染因子的含量。结果显示,与病毒对照组比较,在感染TGEV后2,12h的前期,蜂胶黄酮可以显著提高PK-15细胞产生IFN-β、IFN-γ和iNOS含量的能力;在感染PPV后2,12h的前期,只有较高质量浓度的蜂胶黄酮可以提高PK-15细胞产生IFN-β、IFN-γ和iNOS含量的能力。PK-15细胞在感染病毒后24,48和72h,蜂胶黄酮对提高TGEV感染PK-15细胞后产生抗感染因子能力高于PPV。蜂胶黄酮提高有囊膜的TGEV感染宿主PK-15细胞产生抗感染因子能力高于无囊膜的PPV。     

蜂胶黄酮对宿主细胞抗病毒能力的影响

本试验旨在研究蜂胶黄酮对宿主细胞抗病毒能力的影响。首先采用MTT法测定蜂胶黄酮对宿主鸡胚成纤维细胞（CEF）和PK-15细胞的安全浓度;然后将蜂胶黄酮从最大安全浓度250 μg/mL开始倍比稀释至15.6 μg/mL共5个浓度,将蜂胶黄酮分别与新城疫病毒（NDV）、鸡传染性法氏囊病病毒（IBDV）、猪传染性胃肠炎病毒（TGEV）及猪细小病毒（PPV）一起加到相应长成单层的宿主细胞CEF或PK-15中,在感染后24、48和72 h分别用MTT法测定宿主细胞的存活率,代表蜂胶黄酮提高宿主细胞抗病毒能力的指标。结果显示,蜂胶黄酮可显著提高宿主细胞抗病毒能力,这种活性与其剂量正相关,即剂量越大作用越好。蜂胶黄酮提高宿主细胞抗有囊膜病毒NDV和TGEV作用主要体现在病毒感染的前期,而提高宿主细胞抗无囊膜病毒IBDV和PPV作用主要体现在病毒感染的后期。提示蜂胶黄酮可用于NDV和TGEV的预防及IBDV和PPV的治疗。     

猪细小病毒血凝抑制试验抗原、阳性血清与阴性血清的制备与鉴定

猪细小病毒（Porcine Parvovirus,PPV）血凝抑制试验抗原、阳性血清和阴性血清在猪细小病毒制品的效力检验中不可或缺,对猪细小病毒相关生物制品的质量控制至关重要。试验采用PPV 7909株病毒同步接种PK-15细胞制备血凝抑制试验抗原,用制备的抗原乳化后免疫豚鼠制备阳性血清,同时用未免疫的阴性豚鼠制备阴性血清。对抗原、阳性血清和阴性血清进行鉴定,结果表明,制备的PPV血凝抑制试验抗原HA效价达1:512;阳性血清HI效价达1:1024;阴性血清HI效价＜1:8,且特异性均良好。利用制备的血凝抑制试验抗原、阳性血清与不同PPV疫苗株的灭活抗原、阳性血清进行交叉反应试验,结果表明,制备的抗原与阳性血清具备良好的血清学交叉反应性,可用于PPV制品的统一评价。     

基于家兔模型的纳米蜂胶黄酮对PPV灭活疫苗免疫应答的研究

为了观察纳米蜂胶黄酮对猪细小病毒(PPV)灭活疫苗免疫应答的影响,试验以家兔为模型动物,并以油乳剂为对照,将30只家兔随机均分为3组,第1~2组分别背部皮下注射0.5 m L纳米蜂胶黄酮和油乳剂猪细小病毒灭活疫苗,第3组为对照组注射0.5 m L生理盐水,检测血清抗体效价并观察对体液免疫的影响,测定淋巴细胞增殖及白细胞介素-2(IL-2)、白细胞介素-4(IL-4)、白细胞介素-6(IL-6)和γ干扰素(IFN-γ)含量并评价对细胞免疫的影响。结果表明:纳米蜂胶黄酮、油乳剂均能提高家兔对PPV灭活疫苗的免疫应答能力,油乳剂提高抗体的效果好于纳米蜂胶黄酮,纳米蜂胶黄酮促进T淋巴细胞增殖和提高细胞因子含量的效果优于油乳剂。说明纳米蜂胶黄酮能增强猪细小病毒灭活疫苗的免疫效果,是一种效果良好的疫苗佐剂。     

蜂胶对猪细小病毒灭活疫苗佐剂作用的试验

68只豚鼠随机均分为4组,1³组分别免疫蜂胶、铝胶和油佐剂PPV疫苗,第4组为空白对照组,注射等量生理盐水。免疫后不同时间点采集血清分析特异性血凝抑制抗体效价评价蜂胶佐剂对免疫豚鼠体液免疫的影响,测定淋巴细胞增殖、IL-2和IL-4含量评价蜂胶佐剂对免疫豚鼠细胞免疫的影响。结果表明,蜂胶、油佐剂和铝胶3种佐剂均能提高豚鼠对PPV灭活疫苗的免疫应答能力,油佐剂提高血清HI抗体效果较好,其次为蜂胶佐剂,再次为铝胶佐剂。蜂胶佐剂促进T淋巴细胞增殖和提高IL-2、IL-4、IL-6和IFN-γ含量效果优于油佐剂和铝胶佐剂。结论:蜂胶能增强PPV灭活疫苗的免疫效果。     

新购试验豚鼠猪细小病毒抗体的检测

本实验室长期从事猪细小病毒(porcine parvovirus,PPV)的研究,最近向某实验动物中心购买6只豚鼠准备用于PPV相关试验.在试验之前我们用血凝抑制(hemagglutination inhibition,HI)试验检测这些豚鼠的PPV抗体.经检测发现这6只豚鼠的PPV抗体全部阳性,现将检测情况报告如下.     

十五种中药体外抗H3N2亚型猪流感病毒效果的研究

为了观察15种中药抗H3N2亚型猪流感病毒的效果,试验通过先加中药再加病毒、先加病毒再加中药及中药和病毒混合后一起加这3种加药方式,采用鸡胚培养法和血凝(HA)试验研究了赤芍药等15种中药在体外抗猪流感病毒的效果;然后,将有显著效果的中药筛选出来,测定其治疗指数(TI)和半数有效量(ED50)。结果表明:在3种加药方式中,赤芍药和贯众抗猪流感病毒的效果最显著。     

Heartworm in dogs in Canada in 1984

In late December 1984, 1853 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1984 and 35% of them responded. Veterinarians reported that 97,794 dogs were blood-tested to check for microfilariae and 1417 dogs (1.45% of those tested) were found with heartworm. Another 34 dogs were amicrofilaremic, but were diagnosed as having heartworm disease, to give the total number diagnosed in 1984 as 1451 (1.48%). Heartworm was reported from all provinces except Prince Edward Island and Newfoundland but most (1310) of the cases were in Ontario. In Quebec, 126 cases were reported mostly from west of Montreal.

Heartworm was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 27% of the cases were observed with clinical signs of heartworm disease and 72% had a history of not having left Canada. Southwestern Ontario continued to be the primary focus of the infection.

     

Heartworm in dogs in Canada in 1983

In late December 1983, 2 800 veterinarians across Canada were sent a questionnaire in order to assess the status of heartworm disease in Canada in 1983 and 26% of them responded. Veterinarians reported that 59 504 dogs were blood-tested to check for microfilariae and 771 dogs (1.30% of those tested) were found with Dirofilaria immitis. Heartworm disease was diagnosed in all provinces except New Brunswick and Newfoundland but most (733) of the cases were in Ontario.

Heartworm disease was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 31% of the cases were observed with clinical signs of heartworm disease and 64% had a history of not having left Canada. Southwestern Ontario continues to be the focus of the infection and most of the dogs there had not left the province previously.

     

Heartworm in dogs in Canada in 1991

In late November 1991, 1883 clinics in Canada were sent a questionnaire to assess the status of Dirofilaria immitis in dogs in 1991 and there was a 60.0% response. There were 344,031 dogs tested for heart-worm (HW), 627 were found infected and the prevalence of HW infection was 0.18%. There were 417 dogs with HW in Ontario, 116 in Manitoba, 38 in Quebec, 53 in British Columbia, three in Alberta, and one in Nova Scotia. In British Columbia, all of the infected dogs but one were from the Okanagan valley which, as from 1991, is a new focus of infection in Canada. Most dogs with HW had not been on preventive medication in 1990, and the prevalence among dogs tested and unprotected was 0.59%. That prevalence was considerably higher in endemic areas. Companion dogs, over three years of age and maintained primarily outdoors in rural areas, were most frequently infected. One cat was diagnosed with D. immitis and 33 dogs had Dipetalonema reconditium.     

Heartworm in dogs in Canada in 1985

In late December 1985, 1485 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1985 and 44% of them responded. Veterinarians reported that 137,300 dogs were blood-tested to check for microfilariae and 1210 dogs were found with heartworm. Another 36 dogs were amicrofilaremic but diagnosed with heartworm disease to give the total number diagnosed in 1985 as 1247 (0.91%).

Heartworm was reported from all provinces except Prince Edward Island, Newfoundland and Saskatchewan but most (1126) of the cases were in Ontario. Southwestern Ontario continued to be the primary focus of the infection in Canada. From Quebec, 91 cases were reported mostly from and around Montreal. From Manitoba, 19 cases were reported from Winnipeg and surrounding areas. Heartworm was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 28% of the cases were observed with clinical signs of heartworm disease and 78% had a history of not having left Canada.

     

Heartworm in dogs in Canada in 1988

In late November 1988, 1581 small and mixed animal clinics and institutional veterinarians across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1988, and 46% of them responded. Veterinarians reported that 181,577 dogs were blood-tested for heartworm disease and 367 dogs were found with D. immitis microfilariae. Another 60 dogs were amicrofilaremic but diagnosed with heartworm disease to give the total number of cases diagnosed in 1988 as 441 (0.24%).     

Heartworm in dogs in Canada in 1989

In late November 1989, 1732 clinics and institutional veterinarians were sent a questionnaire to assess the status of Dirofilaria immitis, and 51.7% responded. Of 247,716 dogs tested, 394 had D. immitis microfilariae and 51 were amicrofilaremic for a total of 445 cases and heartworm prevalence of 0.17%. Most (408) of these dogs had no preventive medication and the prevalence among dogs tested and unprotected was 1.01%. That prevalence was considerably higher in endemic areas. Thirty-seven dogs with heartworm had preventive medication. Heartworm was most frequent in companion dogs over three years of age maintained outdoors in rural areas. About 75% of the cases had never left Canada, 26% had clinical signs and 125 were not treated.

Heartworm was reported from British Columbia, Manitoba, Ontario, Quebec, Nova Scotia and Newfoundland, but 383 cases were in Ontario. South-western Ontario was the primary focus of infection. There were 33 cases in Quebec and 24 in Manitoba, mainly found in and around Metropolitan Montreal and Winnipeg respectively.

     

Heartworm in dogs in Canada in 1987

In late November 1987, 1246 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1987, and 50% of them responded. Veterinarians reported that 165,428 dogs were blood tested for heartworm disease and 511 dogs were found with D. immitis microfilariae. Another 78 dogs were amicrofilaremic but diagnosed with heartworm disease to give the total number of cases diagnosed in 1987 as 589 (0.35%).     

Heartworm in dogs in Canada in 1986

In late December 1986, 1224 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1986; 46% of them responded. Veterinarians reported that 150,989 dogs were blood-tested for microfilariae and 869 dogs were found with heartworm. Another 65 dogs were amicrofilaremic but diagnosed with heartworm disease and one was found with heartworm at necropsy to give the total number diagnosed in 1986 as 935 (0.62%).

Heartworm was reported from Manitoba, New Brunswick, Ontario and Quebec, but most (810) of the cases were from Ontario. South-western Ontario continued to be the primary focus of the infection in Canada. There were 103 cases reported from Quebec, mostly from and around Montreal, and 21 cases from Manitoba, from Winnipeg and surrounding areas. Heartworm was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 33% of the cases were observed with clinical signs of heartworm disease and 81% had a history of not having left Canada.