伊氏锥虫苏拉灭抗药性的稳定性和对其他抗锥虫药敏感性的影响

将实验室培育的分别源于伊氏锥虫克隆ＪＧｃ１和ＪＸ１ｃ１的抗苏拉灭伊氏锥虫亚克隆ＪＧＳＲ和ＪＸＳＲ以小鼠连续传代，每３个月用体内药物敏感性试验测定１次苏拉灭的ＩＤ１００和ＣＤ１００；另于传代初和传代１２个月后测定它们和它们各自对苏拉灭敏感的亲本克隆对硫胂聚氰胺、喹嘧胺硫酸盐和贝尼尔的敏感性。传代初，ＪＧＳＲ和ＪＸＳＲ的苏拉灭ＩＤ１００均为１００ｍｇ／ｋｇ，ＣＤ１００分别为４００ｍｇ／ｋｇ和＞４００ｍｇ／ｋｇ。连续传代２１个月，ＪＧＳＲ的苏拉灭抗药性仍保持原水平；ＪＸＳＲ于头１５个月保持原水平，传代１８个月，ＩＤ１００降至５０ｍｇ／ｋｇ，２１个月ＩＤ１００进一步降至２５ｍｇ／ｋｇ，ＣＤ１００降至１００ｍｇ／ｋｇ。ＪＧＳＲ和ＪＸＳＲ对喹嘧胺硫酸盐的敏感性比各自的亲本克隆提高了３倍，而对硫胂聚氰胺的敏感性则与亲本克隆相同或相近。ＪＧＳＲ和ＪＸＳＲ仍保持各自亲本克隆的贝尼尔抗药性，传代１２个月也无改变。这些受试伊氏锥虫的贝尼尔抗药性是单一抗药性，对硫胂聚氰胺无显著交叉抗药性。     

体外培育抗苏拉灭伊氏锥虫克隆

将３个苏拉灭敏感的伊氏锥虫原种的克隆连续培养于改良Ｂａｌｔｚ无细胞培养系统中，通过逐步提高培养基中苏拉灭的含量，培育了３个抗苏拉灭的伊氏锥虫克隆─ＪＧｃ１－１６０、ＪＸ－１ｃ１－１６０和ＺＪｃ１－１４０。它们体外药敏试验的ＩＣ５０依次为３５８．５、４１２．３和２４６．４μｇ／ｍＬ，是各自亲本克隆的１２９２．５、１８７４．１和１７６０．ｏ倍；小鼠治疗试验的ＣＤ１００，对免疫功能正常小鼠依次为８０、１２０和３０ｍｇ／ｋｇ，为各自亲本克隆的５．３、８．０和３．０倍，对免疫抑制小鼠为２５０、３００和１００ｍｇ／ｋｇ，分别是相应免疫正常小鼠的３．１、２．５和３．３倍。试验结果表明，抗锥虫药治疗剂量不足和宿主免疫功能不全是导致产生抗药虫株的重要因素，各自既可单独发挥作用，又可相互协同。本文报道了体外培育伊氏锥虫抗药虫株的方法，这一方法对研究锥虫抗药性具有重要作用。     

用免疫抑制小鼠快速培育抗苏拉明伊氏锥虫

将伊氏锥虫克隆ＪＧｍｃ５用环磷酰胺处理的免疫抑制小鼠频繁地连续传代，并予以亚治疗剂量的苏拉明治疗，连续传代１４次，历时８８日，即获得对苏拉明具有高水平抗药性的伊氏锥虫群体。在用免疫活性正常的健康小鼠测定抗药性时，此抗苏拉明锥虫群体仍保持高水平的抗药性，ＣＤ１００＞４００ｍｇ／ｋｇ。在以体外药敏试验测定时，其ＩＣ５０为１２３．２μｇ／ｍｌ。实验结果表明，宿主免疫系统的损害，在实验条件下，可导致伊氏锥虫迅速产生抗药性。在野外条件下，机体免疫系统的损害对锥虫抗药性的产生可能也起一定作用。     

理化因素对体外培养伊氏锥虫致弱的影响

本试验观察了理化因素（γ－射线、紫外线、低浓度杀虫药）对体外培养伊氏锥虫致弱的影响，结果如下：受γ－射线辐照的伊氏锥虫开始时存活数随辐照剂量的增加而减少，随后表现出无规律性，辐照剂量５万ｒａｄ以下对锥虫的存活影响较小，５～１０万ｒａｄ辐照后２ｄ锥虫的存活数明显减少，对小白鼠的感染性消失。伊氏锥虫受紫外线照射的存活曲线为“Ｃ”型，群体抗辐射不均一，平均致死剂量（Ｄ＿（３７））为波长２６５０Ａ的２０Ｗ紫外灯离样品４０ｃｍ处照射约２ｈ。虫体照射后根据体外跟踪观察，其存活数随照射剂量的增大而减少，照射了３ｈ和４ｈ第３天的活虫数近似１００条或不足１００条，对小白鼠的感染性均消失。用０．１μｇ／ｍｌ的苏拉灭和０．８μｇ／ｍｌ的贝尼尔两种低浓度杀虫药在体外对伊氏锥虫诱导４０ｄ仅造成药敏性的改变，未造成毒力减弱。     

体外培养对布氏锥虫伊氏亚种药敏性的影响

用小鼠治疗试验和体外药敏试验观察了4个布氏锥虫伊氏亚种虫株在长期体外培养条件下药敏性的稳定性.各虫株的原始群体、连续培养30d和90d的群体对贝尼尔、苏拉灭、安锥赛和硫胂聚氰胺的敏感性基本相同,说明连续培养90d各虫株对上述4种抗锥虫药的敏感性无明显改变.     

分泌抗赭曲霉素A单克隆抗体杂交瘤细胞株的建立

用赭曲霉素Ａ（ＯＡ）－ＢＳＡ合成抗原免疫Ｂａｌｂ／ｃ小鼠，取免疫鼠脾细胞与ｓｐ２／０细胞融合，通过克隆和ＥＬＩＳＡ法筛选，建立三株泌抗ＯＡ单克隆抗体杂交瘤细胞株（３Ｃ１２，１Ｄ１２和２Ｇ７）。用间接ＥＬＩＳＡ法测定，细胞上清液抗体效价为１２８＾×（３Ｃ１２）和６４＾×（１Ｄ１２和２Ｇ７）腹水抗体效价为１０＾－７（３Ｃ１２，１Ｄ１２）和１０＾－６（２Ｇ７）。三株单抗（ＭｃＡｂ）均属ＩｇＧ类，分泌抗体     

IBDCOB－C1强毒株与J1C7E3标准强毒株对雏鸡及鸡胚致病力的研究

对扬州地区某鸡场当前流行的ＩＢＤ强毒株进行分离，并在１０、３５日龄时对两批ＩＢＤ阴性鸡（北京白鸡）作人工感染，与Ｊ１Ｃ７Ｅ３标准强毒株的致病力进行了系统的比较。结果证实ＩＢＤＣＯＢ－Ｃ１强毒株的毒力致病性远远大于Ｊ１Ｃ７Ｅ３毒株，ＩＢＤ人工感染京白Ⅰ系非免疫雏鸡后发病快、病程急、死亡率高，发病率达１００％、死亡率为８０％。病死鸡病变典型一致，ＣＯＢ－Ｃ１强毒株能使ＳＰＦ鸡胚致死、死亡胚胎病变显著，头腹水肿，全身出血，头、趾尤为明显，对ＮＤⅣ系疫苗免疫后可造成明显的免疫抑制。扬州地区分离的ＣＯＢ－Ｃ１是一株超强毒株，其抗原性与标准毒株蛋白结构的差异正进一步研究     

伊氏锥虫在小鼠体内抗原变异规律及免疫保护试验

为了探索伊氏锥虫抗原的变异规律及其用于免疫预防的可能性，首先对伊氏锥虫安微株单虫克隆2个早期变异体ShTatl.1、ShTat1.2采用蛋白酶抑制剂TLCK处理，分离纯化这2种变异体的VSG，用ShTat1.1 VSG免疫KM小鼠，ShTat1.1锥虫攻击免疫鼠后6d分离锥虫，经间接免疫荧光试验（IFT）和班点酶标记试验（Dot-EIA)鉴定为ShTat1.2，说明同一克隆锥虫感染兔，小鼠第1次发生抗原变异后产生的变异体相同。依据这一规律，设计了免疫预防保护试验，试验小鼠分3组：一组为未免疫对照组，一组为ShTat1.1VSG单一抗原免疫组，另一组为ShTat1.1 VSG,ShTat1.2 VSG混合免疫抗原免疫组，各组均以ShTat1.1锥虫攻击，结果ShTat1.1 VSG ShTat1.2VSG免疫组小鼠全部获得保护，单用ShTat1.1 VSG免疫组小鼠和未免疫小鼠血中全部出虫、死亡，前者比后者出虫时间、存活时间延长。提示运用伊氏锥虫抗原变异这一规律设计的这种复合抗原，能激发宿主克服虫体抗原变异对免疫保护的干扰。     

伊氏锥虫在家兔体内的抗原变异和NaTat1．1—1．9九个群体的建立

以伊氏锥虫YNB2克隆感染家兔，于52天内，以一定间隔，用经环磷酰胺处理的小鼠由兔血分离锥虫，并予以克隆，获得9个抗原性互不相同的克隆群体。用此9个克隆群体分别免疫家兔制备免疫血清，与同源和各异源克隆群体作交叉免疫溶解和间接免疫荧光试验。结果证明，9个克隆群体是单一可变抗原型(VAT)，制备的血清是单价VAT特异血清。按国际通用命名法，依分离顺序命名为NaTat1．1～1．9(Najing rypanozoon antigen type 1.1～1.9).     

抗A型产气荚膜梭菌α毒素单克隆抗体杂交瘤细胞系的建立与中和效应

用提取的 Ａ 型产气荚膜梭菌 α毒素包涵体免疫 Ｂ Ａ Ｌ Ｂ／ｃ 小鼠后， 取小鼠脾细胞与 Ｓ Ｐ２／０ 骨髓瘤细胞进行融合和克隆化，经间接 Ｅ Ｌ Ｉ Ｓ Ａ 筛选，共获得 １ Ａ８、１ Ｃ３、１ Ｄ５、１ Ｄ８、１ Ｆ１、１ Ｈ１ 和 ２ Ｅ３ ７ 株稳定分泌单克隆抗体（ Ｍ ｃ Ａｂ）的杂交瘤细胞株。经鉴定，７ 株 Ｍ ｃ Ａｂ 的 Ｉｇ 亚类有 Ｉｇ Ｇ１（１ Ｄ８）、 Ｉｇ Ｇ３（１ Ａ８、１ Ｃ３ 和 ２ Ｅ３）和 Ｉｇ Ｍ（１ Ｄ５、１ Ｆ１ 和 １ Ｈ１）。细胞培养上清和腹水抗体效价分别为 １∶５１２～１∶１ ０２４ 和 １∶１０６ ～１∶１０８ 。尤为重要的是，２ Ｅ３ 杂交瘤细胞株分泌的 Ｍ ｃ Ａｂ 不仅能够中和 α毒素的磷脂酶 Ｃ活性和溶血活性，而且能够对致死性腹腔感染小鼠产生良好的被动保护作用。     

The immunosuppressive effects of experimental T. congolense infections in goats

The agglutinin response of four groups of goats inoculated with Brucella melitensis vaccine 0, 1, 2 and 4 weeks following experimental infection with Trypanosoma congolense was compared with that in non-infected controls. Four weeks after vaccination the goats were treated with a trypanocidal drug and the recovery of the immune response observed. The results indicated that the majority of animals had a significantly but not completely suppressed antibody response. This was most marked in the group vaccinated 2 weeks post-infection, which corresponded with the onset of parasitaemia. Although the mortality rate in the infected goats was high the titre in those remaining animals that were treated with the trypanocidal drug increased immediately after treatment. The possible implications of trypanosome induced immunosuppression for vaccination programmes in goats are discussed briefly.     

Immunosuppression in bovine trypanosomiasis: studies with louping-ill vaccine.

The antibody response to louping-ill virus vaccine was examined in mice infected with Trypanosoma brucei and T congolense, and in Ethiopian cattle experimentally infected with T brucei, T congolense and T vivax. In mice the antibody response was completely suppressed, while in cattle infected with T congolense and T vivax the antibody response to the vaccine was only 10 per cent that of uninfected animals. In contrast, the response of cattle infected with T brucei was not significantly reduced, and this was attributed to their relatively light and transient parasitaemias. Trypanocidal chemotherapy (diminazine aceturate) administered on the same day as vaccination largely restored the competence of the immune response of both mice and cattle infected with T congolense. The use of such drugs should be considered when cattle are vaccinated in trypanosome endemic areas.     

Detection of antibodies to mouse thymic virus by enzyme-linked immunosorbent assay.

Enzyme-linked immunosorbent assay was used to detect serum antibodies to mouse thymic virus, a herpesvirus that causes thymic lesions and immunosuppression. Antibodies were detected in mice that had received single or multiple injections of the virus and were also found in mice housed in contact with the experimentally infected animals. By contrast, mice not exposed to mouse thymic virus or those inoculated with an uninfected thymus preparation remained seronegative. A serological survey of eight mouse colonies revealed one positive colony, confirmed by virus isolation. These results show that the test is sufficiently sensitive and specific to be used for routine screening of mice.     

Immune response to foot-and-mouth disease virus in an experimental murine model. II. Basis of persistent antibody reaction

A murine model was used to study the mechanisms involved in the prolonged immune response to live and inactivated foot-and-mouth disease virus (FMDV). The antibody response elicited by the infection persisted throughout the entire life of the animal, while immunization with inactivated virus induced a transient response. The administration of inactivated virus in a water-in-oil emulsion increased antibody titres to values as high as those obtained by infection. There was a high correlation between neutralizing antibody titre and transfer of immunity with primed cells, and the protection afforded against challenge with infectious virus. It appears that the mechanism involved in the induction of prolonged immune memory in infected animals is not due to viral persistence. Nude mice infected with FMDV also evidenced a prolonged immune response, showing marked differences in antibody levels but equal effectiveness against challenge when nu/nu and nu/+ animals were compared. Furthermore, athymic and euthymic littermates were efficient in conferring protection when cells were transferred to irradiated animals. It is concluded that there is an effective, T-cell-independent, prolonged immune memory against FMDV in this murine model, and that the difference in the immune responses to live and inactivated virus is due mainly to differential antigenic processing rather than to a difference in the degree of sensitization of effector cells.     

Distribution of rabies virus in infected mice,vaccinated and submitted to P. acnes as immunomodulator

The lethality and distribution of rabies virus were evaluated in swiss mice experimentally infected with street rabies virus, vaccinated and submitted to immunomodulation by P .acnes (formerly Corynebacterium parvum). The animals were sacrificed at different times,when the different tissues were collected and submitted to fluorescent antibody test (FAT) and mouse inoculation test (MIT). The group submitted to vaccination and P. acnes treatment presented a percentage of survival superior to that observed in infected mice only treated with P. acnes. Control infected animals had the lowest survival rates.The distribution of rabies virus in spleen of infected mice, vaccinated and submitted to P. acnes was superior to that verified in infected mice not treated with P.acnes. The increased survival correlated with the distribution of rabies virus in lymphoid tissues, could be interpreted as the consequence of P. acnes activity on macrophages. The results suggest the role of macrophages against rabies virus infection in mice and the importance of vaccination in the post expositive treatment of rabies.     

Interaction of acute feline herpesvirus-1 and chronic feline immunodeficiency virus infections in experimentally infected specific pathogen free cats.

Cats with or without chronic feline immunodeficiency virus (FIV) infection were exposed to feline herpesvirus, type 1 (FHV-1). FIV infected cats became sicker than non-FIV infected cats and required more supportive treatment. However, there were no differences in the length of their illness or in the levels and duration of FHV-1 shedding. FHV-1 infection caused a transient neutrophilia at Day 7 with a rapid return to preinfection levels. The neutrophilia coincided with a transient lymphopenia that was accompanied by a decline in both CD4+ and CD8+ T-lymphocytes. A brief decrease in the CD4+/CD8+ T-lymphocyte ratio occurred at Day 14 in both FIV infected and non-infected cats. This decrease was mainly the result of an absolute and transient increase in CD8+ T-lymphocytes. CD4+ and CD8+ T-lymphocyte numbers and CD4+/CD8+ T-lymphocyte ratios returned to baseline within 4-8 weeks in both FIV infected and non-infected cats. FIV infected cats produced less FHV-1 neutralizing antibodies during the first 3 weeks of infection than non-FIV infected animals. The IgM FHV-1 antibody response was depressed in FIV infected cats whereas the IgG antibody response was unaffected. FHV-1 infection evoked a comparable transient loss of lymphocyte blastogenic responses to concanavalin A and pokeweed mitogen in both FIV infected and non-infected cats. However, response to pokeweed mitogen took longer to return to normal in FIV infected animals. Lymphocytes from FIV infected cats had a greater and more sustained proliferative response to FHV-1 antigen than non-FIV infected cats. The ongoing IgG antibody response to FIV was not affected by FHV-1 infection.     

Immunodeficiency in latent feline leukemia virus infections

Challenge of naive experimental animals with a retroviral inoculum may result in one of two broad sequelae. The first is the establishment of an appropriate humoral and cellular immune response leading to a condition of immunity to subsequent infection with the retrovirus. Alternatively, the host may fail to develop a successful immune response, resulting in a chronic viremia associated with immunosuppression and ultimately death due to secondary pathogens. An alternate disease course is the establishment of a latent infection characterized by the presence of neutralizing antibody and strong cellular immune reactivity. Recent data from the feline leukemia virus (FeLV) system suggest that cats infected with this virus may develop immunosuppression in the form of persistent neutrophil dysfunction. The potential effect of this cellular dysfunction is the possible susceptibility of the host to the same opportunistic pathogens which are responsible for the increased mortality noted in chronic FeLV infections. These data demonstrate that persistent retroviremia is not essential for the establishment of immunosuppression. This overview presents data accumulated from the feline model of the human acquired immunodeficiency syndrome (AIDS) and discusses its relationship to human retroviral infections.     

Antibody responses in pregnancy-induced transmammary transmission of Ancylostoma caninum hookworm larvae

Third stage larvae of the Ancylostoma caninum hookworm nematode have the capacity to infect a dog, abort the normal maturation pathway to become blood-feeding intestinal worms, and instead distribute throughout the body in a developmentally arrested state that is relatively resilient to most chemotherapeutic agents. During pregnancy, a percentage of the arrested larvae reactivate and transmit via the mammary glands to infect the nursing puppies with resulting iron-deficiency anemia and potential mortality. To determine if the suppression of parasite-specific antibody responses during pregnancy facilitates the reactivation and transmammary transfer of hookworm larvae, a murine model of A. caninum infection was used to compare the infected versus uninfected animals that were either bred or not bred. Initial comparisons of genetically divergent BALB/c versus C57BL/6 mice showed that both the strains mounted strong Th2 biased IgG1and IgE antibody responses to A. caninum infection. Using the BALB/c strain for the breeding analyses, it was confirmed that larval transfer to the mouse pups only occurred during the post-partum lactational period. In the dams, levels of total and antigen-specific IgG1 and total IgE were highly correlated with parasite burden. During most phases of pregnancy and lactation, infected dams had lower total IgG1, IgG2a and IgE levels as compared to unbred mice at comparable times post-infection; this downward modulation of antibody responses supports the established dogma of a generalized immunosuppression associated with pregnancy. However, at parturition and post-partum lactation, antigen-specific IgG1 levels measured at 1 : 5000 serum dilutions were comparable between bred and unbred mice, and antigen-specific IgG2a levels at 1 : 100 serum dilutions were also not significantly different except for a marginal reduction in the bred mice at the lactational timepoint. The comparable anti-A. caninum IgG1 levels between bred and unbred mice, and low correlation between IgG2a levels and larval burden suggest that parasite-specific antibody responses do not play a major role in the pregnancy-associated transmammary transmission of A. caninum larvae. This conclusion does not rule out the possibility that underlying fluxes in the levels of specific cytokines associated with pregnancy and infection may be involved in the process of larval reactivation and transmission.     

Expressed truncated N-terminal variable surface glycoprotein (VSG) of Trypanosoma evansi in E. coli exhibits immuno-reactivity

The variant surface glycoprotein (VSG) of trypanosome is an important part of its body surface coat, which is expressed in early, middle and late stages of infection contributing a major diagnostic value. In the present study, the 5' end of the partial VSG gene sequences (681 bp) encoding N-terminal protein of RoTat 1.2 VSG (227 amino acid) was amplified, cloned into pET32a vector, and expressed in prokaryotic system. The fused His-tagged expressed VSG protein (43 kDa) of the Trypanosoma evansi was characterized in SDS-PAGE and immunoblotting using hyperimmune/immune sera raised against buffalo, dog, lion and leopard isolates of T. evansi. The expressed protein remained immunoreactive with all the sera combinations. The animals immunized with whole cell lysate or recombinant protein showed similar antibody reactions in ELISA and CATT (Card Agglutination Test for Trypanosomiasis). This study suggests the expressed recombinant truncated VSG is having its importance for its possible use in sero-diagnosis of surra.     

Effect of suramin on serum viral replication in feline leukemia virus-infected pet cats

Suramin treatments were administered (IV) to 2 healthy adult cats infected with naturally acquired feline leukemia virus. Serum viral infectivity--as assessed by focus induction by the method of Fischinger, using serial serum samples titrated on clone 81 cells--ceased transiently in both cats during treatment with suramin, but returned to significantly high levels approximately 14 days after treatment was stopped. Both cats tested positive for FeLV internal antigens in peripheral blood cells and serum before, during, and after treatment. Both cats tested negative for antibody to feline oncornavirus membrane antigen before, during, and after treatment. The major adverse effects of suramin in the 2 cats were transient vomiting and anorexia.