Immune response to inactivated bacterial vector carrying the recombinant K39 antigen of Leishmania infantum in mice

Objective:To evaluate the immunological response elicited by an inactivated bacterial vector carrying the K39 antigen of Leishmania infantum,and a purified antigen.Methods:Mice were subjected to the following treatments:(1)Purified recombinant K39(rK39)protein at a 20μg dose with complete Freund’s adjuvant;(2)Inactivated Escherichia coli(BL21 DE3)carrying the K39 protein at an equivalent total protein content of 200μg;(3)Inactivated bacteria lacking the K39 protein;(4)Non-immunized control animals.Serological monitoring was performed.All groups were challenged by intraperitoneal injection of 10^(7) Leishmania infantum promastigotes.After euthanasia,the liver and spleen were collected to analyze the levels of TNF,IFN-γ,IL-12,IL-4,and IL-10.Results:Mice immunized with purified rK39 or the inactivated bacterial vector carrying the K39 antigen of Leishmania infantum showed a long-lasting immune response with high levels of polyclonal antibodies specifically recognizing the recombinant proteins.The IgG1 subclass was the predominant immunoglobulin;however,the induction of IgG2a and the profile of cytokines produced were indicative of the induction of a mixed-type response.Conclusions:The inactivated bacterial vector carrying the K39 antigen,as well as the purified antigen can induce a long-lasting immune response in immunized mice,predominantly favouring a Th2 profile response.

Preparation and evaluation of a glycerol-preserved direct agglutination antigen for long-term preservation:a comparative study of the detection of anti-Leishmania infantum antibodies in human and dog

Objective:To prepare and evaluate a glycerol-preserved antigen from an Iranian strain of Leishmania infantum(L infantum) for use in glycerol-preserved direct agglutination tests (GP-DAT) as an alternative to freeze dried direct agglutination teals(FD-DAT) that use freezedried antigen.Methods:Glycerol-preserved DAT antigen was prepared and stored at different temperatures.We tested antigen stored at 4℃,22-37℃and 50℃over a period of 365 days. Seven hundred twenty-nine serum samples were collected from different geographical zones of Iran from 2007-2009,and 80 of these samples were pooled to produce sera.Each pooled serum contained 10 sera.All positive and negative pooled sera were separately tested for anti-L. infantum antibodies with GP-DAT,FD-DAT and formaldehyde-fixed direct agglutination test (FF-DAT) antigens;tests were performed on both human and dog sera over a period of 12 months. Results:There was strong agreement between the results obtained using GP-DAT and FDDAT antigens stored at 22-37℃for 12 months for both human(100%) and dog(100%) pooled sera.The direct agglutination test results were highly reproducible(weighted kappa:GP=0.833, FD=0.979 and FF=0.917).Conclusions:Because GP-DAT antigen is highly stable over a range of temperatures and is easy to transport in the field,this type of antigen may be particularly useful in areas with endemic visceral leishmaniasis.

First detection of Leishmania infantum DNA in wild caught Phlebotomus papatasi in endemic focus of cutaneous leishmaniasis,south of Iran

Objective:To identify the vectors and reservoirs of cutaneous leishmaniasis in the endemic focus of Farashband,Fars Province,South of Iran.Methods:Sticky papers and Sherman trap were used for collection of sand flies and rodents,respectively.Polymerase chain reaction(PCR)of kDNA,ITS1-rDNA were used for identification of Leishmania parasite in sand flies as well as rodents.Results:Totally 2010 sand flies were collected and the species of Phlebotomus papatasi Scopoli was the common specimen in outdoors and indoors places.PCR technique was employed on 130females of Phlebotomus papatasi.One of them(0.76%)was positive to parasite Leishmania major(L.major)and one specimen(0.76%)was positive to Leishmania infantum.Microscopic investigation on blood smear of the animal reservoirs for amastigote parasites revealed 16(44%)infected Tatera indica.Infection of them to L.major was confirmed by PCR against kDNA loci of the parasite.Conclusions:The results indicated that Phlebotomus papatasi was the dominant species circulating two species of parasites including L.major and Leishmania infantum among human and reservoirs.Furthermore,Tatera indica is the only main host reservoir for maintenance of the parasite source in the area.

Different Patterns of Fluorescence in the Quantisation of anti-Leishmania Infantum Antibodies by Indirect Immuno-Fluorescent Antibody Test (IFAT) in the Serological Diagnosis of Canine Leishmaniasis

Although Indirect Immuno-Fluorescent Antibody Test (IFAT), performed employing “in house” prepared antigen, is considered by several authors as the golden standard for the quantisation of anti-leishmania antibodies in dogs, there is a lack of papers reporting a description of the different patterns of fluorescence that can be observed. An incorrect identification of patterns of fluorescence may be an important source of bias in the interpretation of results. Previous papers report different criteria to define as “positive” a specific pattern of fluorescence, namely: membrane fluorescence, homogeneous fluorescence of the body, or homogeneous fluorescence of the body plus flagellum. In this paper, we report a detailed description of preparation of slides and of the patterns of fluorescence that can be obtained employing “in house” prepared antigen. At least six main patterns of fluorescence may be observed: 1): homogeneous cytoplasmatic green fluorescence;2): membrane pattern, in which the fluorescence is mainly localized along the entire perimeter of the parasites;3): coarse-speckled cytoplasmatic fluorescence;4): flagellar pattern, in which the fluorescence is localized exclusively onto the flagellum;5): punctiform pattern, in which the fluorescence is localized exclusively at the basis of the flagellum;6): nuclear pattern, in which only the nucleus of the parasite shows a homogeneous green fluorescent. The significance of each pattern is discussed.

北京再发黑热病利什曼原虫K26基因和ITS-1序列的多态性分析

目的了解北京市再发黑热病病例的病原分子遗传背景。方法使用PCR方法分别扩增其亲水性酰化表面蛋白B(K26)和核糖体DNA内转录间隔区Ⅰ(ITS-1),然后克隆测序,分析其多态性,构建系统发育树以确定患者感染原虫的虫种类型及其遗传关系。结果K26基因扩增出626 bp大小片段,其长度与国内流行株差异明显,其氨基酸序列由14个氨基酸的基序重复排列组成,但个别位置发生氨基酸替代。K26序列的系统进化树分析显示,北京病例虫株与法国和西班牙的婴儿利什曼原虫相近,但与国内新疆、四川、河北等地的婴儿利什曼原虫虫株距离较远;ITS-1序列扩增出314 bp大小的片段,其系统发育分析显示与婴儿利什曼原虫Leishmania infantum属于同一分支。结论该病例感染的为婴儿利什曼原虫L.infantum,且与国内其他流行区的虫株有一定差异。

In vitro antileishmanial effects of Physalis angulata root extract on Leishmania infantum

Objective： In the present study, we evaluated the effects of the aqueous extract of Physalis angulata root （AEPa） on Leishmania infanturn proliferation, morphology, and the driving mechanism in leishmanicidal activity and modulatory action on macrophages. Methods： I., infantum promastigotes were treated with 50 and 100 μg/mL AEPa for 72 h and then antipro- mastigote assay was performed by counts in a Newbauer chamber, morphological changes were analyzed by transmission electron microscopy and the mechanism of the leishmanicidal activity was detected. In addition, macrophages were infected with L. infantum and were used to evaluate anti-amastigote activity of AEPa and effects of AEPa on cytokine secretion after 72-hour treatment. Results： Treatment with AEPa reduced the numbers ofL infantum promastigotes （50% inhibitory concen- tration （ICso） = 65.9μg/mL; selectivity index （SI）= 22.1） and amastigotes （ICso = 37.9 μg/mL; SI = 38.5） compared with the untreated control. Amphotericin B reduced 100% of the promastigote numbers after 72 h of treatment （IC50 = 0.2μg/mL）. AEPa induced several morphological changes and increased the production of reactive oxygen species and apoptotic death in promastigotes after treating for 72 h. AEPa （100 μg/mL） promoted tumor necrosis factor-α secretion in macrophages infected with L. infantum after 72 h of treatment, but did not induce an increase in this cytokine in noninfected macrophages. In addition, AEPa showed no cytotoxic effect on J774-A1 cells （50% cytotoxic concentration 〉1000μg/mL）. Conclusion： AEPa presented antileishmanial activity against the promastigotes and amastigotes of I.. infantum without macrophage cytotoxicity; these results show that natural products such as P. angulata have leishmanicidal potential and in the future may be an alternative treatment for leishmaniasis.

Genetic polymorphism in Leishmania infantum isolates from human and animals determined by nagt PCR-RFLP

Background:Leishmania infantum is the causative agent of human visceral leishmaniasis(VL)and sporadic human cutaneous leishmaniasis(CL)in the Mediterranean region.The genetic variation of the Leishmania parasites may result in different phenotypes that can be associated with the geographical distribution and diversity of the clinical manifestations.The main objective of this study was to explore the genetic polymorphism in L.infantum isolates from human and animal hosts in different regions of Morocco.Methods:The intraspecific genetic variability of 40 Moroccan L.infantum MON-1 strains isolated from patients with VL(n=31)and CL(n=2)and from dogs(n=7)was evaluated by PCR-RFLP of nagt,a single-copy gene encoding N-acetylglucosamine-1-phosphate transferase.For a more complete analysis of L.infantum polymorphism,we included the restriction patterns of nagt from 17 strains available in the literature and patterns determined by in-silico digestion of three sequences from the GenBank database.Results:Moroccan L.infantum strains presented a certain level of genetic diversity and six distinct nagt-RFLP genotypes were identified.Three of the six genotypes were exclusively identified in the Moroccan population of L.infantum:variant M1(15%),variant M2(7.5%),and variant M3(2.5%).The most common genotype(65%),variant 2(2.5%),and variant 4(7.5%),were previously described in several countries with endemic leishmaniasis.Phylogenetic analysis segregated our L.infantum population into two distinct clusters,whereas variant M2 was clearly distinguished from both cluster I and cluster II.This distribution highlights the degree of genetic variability among the Moroccan L.infantum population.Conclusion:The nagt PCR-RFLP method presented here showed an important genetic heterogeneity among Moroccan L.infantum strains isolated from human and canine reservoirs with 6 genotypes identified.Three of the six Moroccan nagt genotypes,have not been previously described and support the particular genetic diversity of the Moroccan L.infantum population reported in other studies.

北京市1例内脏利什曼病病例的调查分析

对北京市1例内脏利什曼病病例开展流行病学调查,确定感染来源,制定防控措施。收集病例就诊资料,在其感染地使用诱蛉灯开展白蛉监测、调查病犬信息,对病例和病犬骨髓液样本进行涂片、PCR检测、基因测序;对捕捉到的中华白蛉进行PCR检测。结果显示,病例以间断性发热、乏力就诊,医院以骨髓液涂片可见利什曼原虫无鞭毛体确诊为内脏利什曼病。感染地为门头沟王平镇,在该地区共捕获中华白蛉4520只,PCR结果阴性。病例、2只病犬骨髓液利什曼原虫PCR阳性,且基因序列与婴儿利什曼原虫序列同源性分别为94.44%、95.6%、93.23%,病犬骨髓液涂片查见利什曼原虫。结果表明,确定门头沟王平镇存在婴儿利什曼原虫感染传播链。需进一步开展白蛉、病犬分布范围调查,开展病例监测,多部门联合采取防控措施,防止内脏利什曼病疫情继续扩散。

甘肃文县婴儿利什曼原虫无症状感染犬的检测

目的评价PCR法、ELISA法和试条法检测我国婴儿利什曼原虫无症状感染犬的潜能。方法用两组PCR引物RV1-RV2和K13A-K13B检测动物源型黑热病疫区健康犬静脉血和骨髓中利什曼原虫特异DNA,以利什曼原虫可溶性抗原为包被抗原的ELISA法和rk39-dipstick试条法分别检测利什曼原虫特异抗体,并比较各种检测方法的敏感性差异。结果PCR法检测抗凝静脉血和骨髓的阳性率分别为50.63%(40/79)和69.62%(55/79),两种样本总检出率为77.21%(61/79);ELISA法检测的阳性率为22.22%(16/72),而rk39-dipstick试条检测的阳性率为33.33%(19/57)。结论我国动物源性黑热病疫区利什曼原虫无症状感染犬的比例相当高,以骨髓为样本的PCR检测法为较精确的犬无症状感染检测方法。

甘肃省文县流行区人群婴儿利什曼原虫无症状感染现状

目的分析甘肃省文县内脏利什曼病流行区人群利什曼原虫无症状感染现状,评价PCR、ELISA和rK39免疫层析试条法检测利什曼原虫无症状感染的潜能。方法2004年10月在甘肃文县对269例无内脏利什曼病现症及病史的人群采取随机取样法采集静脉血,分别用RV1-RV2和K13A-K13B两组PCR引物检测血样中的利什曼原虫特异DNA,以利什曼原虫可溶性抗原为包被抗原的ELISA法和rK39免疫层析试条法分别检测利什曼原虫特异性抗体,并比较几种检测方法的敏感性。结果PCR、ELISA和rK39免疫层析试条法检测人群利什曼原虫无症状感染的阳性率分别为30.9%(83/269)、24.2%(65/269)和0(0/269)。结论甘肃省文县内脏利什曼病流行区人群存在大量利什曼原虫无症状感染者,PCR是检测无症状感染较敏感、特异的方法。

PCR检测婴儿利什曼原虫无症状感染的研究

目的建立适合检测我国婴儿利什曼原虫无症状感染的PCR方法。方法选择6种常用于诊断内脏利什曼病的PCR引物(RV1-RV2、K13A-K13B、MC1-MC2、174-798、Pia3-Pia4和DBY-Ajs31),以培养的甘肃人株利什曼原虫前鞭毛体种植人抗凝全血抽提的DNA为模板,确定了这6种PCR引物检测我国婴儿利什曼原虫的最适条件,并比较其检测的敏感性和特异性。选用两种敏感性和特异度均佳的引物对采自利什曼病疫区100份无利什曼病症状居民的静脉血进行检测。结果6种PCR引物检测的特异性均达到100%,而检测的敏感性各异,检测到的原虫数目从0.1～1000条原虫/ml,其中引物RV1-RV2(0.1个原虫/ml血)和K13A-K13B(1个原虫/ml血)敏感性较高。这两对引物对100份无症状居民血的阳性检出率分别为33%(33/100)和30%(30/100)。结论引物RV1-RV2和K13A-K13B适于检测我国婴儿利什曼原虫无症状感染。在我国甘肃动物源性利什曼病疫区,人群利什曼原虫无症状感染率颇高。

新疆克拉玛依地区几株利什曼原虫分离物的同源性分析

目的：确定新疆克拉玛依皮肤利什曼病患者和硕大白蛉吴氏亚种体内利什曼原虫的种。方法：通过酶切电泳及ＤＮＡ杂交的方法对当地病人体内和蛉体内的利什曼原虫以及参考虫株的ｎＤＮＡ及ｋＤＮＡ的同源性分析，研究克拉玛依病人和蛉体内利什曼原虫的基因型。结果：经ｎＤＮＡ基因型分析，表明病人与蛉体内原虫与婴儿利什曼原虫同源性大。结论：当地皮肤利什曼病的病原体为婴儿利什曼原虫，硕大白蛉吴氏亚种为该病的媒介。

利什曼原虫抗原对草原兔尾鼠感染婴儿利什曼原虫的免疫保护作用

目的： 测定利什曼原虫主要表面分子抗原对内脏利什曼病的免疫保护作用。方法： 用重组利什曼原虫表面糖蛋白ｒ Ｇ Ｐ６３ 和脂磷酸聚糖（ Ｌ Ｐ Ｇ） 为抗原， 以短棒状杆菌菌苗（ Ｃ Ｐ） 为佐剂免疫草原兔尾鼠后， 用婴儿利什曼原虫强毒株攻击， 观察免疫保护效果。结果： ｒ Ｇ Ｐ６３ 加 Ｌ Ｐ Ｇ 加 Ｃ Ｐ 抗原组合免疫后用２ ×１０７ 前鞭毛体攻击， 免疫动物的肝印片上 Ｌ Ｄ 数量明显降低， 减虫率为８９８ ％ 。 Ｌ Ｐ Ｇ 加 Ｃ Ｐ 免疫组减虫率为６０６ ％ 。ｒ Ｇ Ｐ６３ 包涵体加 Ｃ Ｐ 免疫组减虫率为４２４ ％ ， 而纯化ｒ Ｇ Ｐ６３ 加 Ｃ Ｐ 免疫组未显示保护作用。以ｒ Ｇ Ｐ６３ 加 Ｌ Ｐ Ｇ 加 Ｃ Ｐ 免疫后用１ ×１０６ ， ５ ×１０６ 和１ ×１０７ 前鞭毛体攻击时， 感染率亦有明显降低。结论： 利什曼原虫主要表面分子抗原 Ｇ Ｐ６３和 Ｌ Ｐ Ｇ 在以 Ｃ Ｐ 为佐剂的条件下， 对草原兔尾鼠婴儿利什曼原虫有明显的免疫保护效果。

新生儿坏死性小肠结肠炎术中的液体治疗

目的评价醋酸钠林格注射液和乳酸钠林格注射液用于新生儿坏死性小肠结肠炎术中液体治疗的有效性和安全性。方法以我院2010年6月—2011年10月收治的坏死性小肠结肠炎行手术治疗的新生儿作为研究对象,采用随机、对照、双盲方法,将患儿分为醋酸钠林格注射液组(AR组)和乳酸钠林格注射液组(LR组),每组25例,所有患儿均接受全麻气管插管,术中维持药物为七氟醚,按照统一的标准进行液体输注,监测患儿术中的平均动脉压、心率、血氧饱和度(SaO2)、酸碱平衡、补液量、尿量等指标,并进行对比分析。结果两组性别、体重、手术时间、液体总量、尿量及患儿手术过程中各时点平均动脉压、心率、SaO2比较差异均无统计学意义(P>0.05);AR组NaHCO3的用量明显少于LR组,差异有统计学意义(P<0.05)。结论醋酸钠林格注射液用于新生儿坏死性小肠结肠炎术中的液体治疗,能有效维持患儿血容量,保持血流动力学稳定,与乳酸钠林格注射液比较能显著改善代谢性酸中毒,在新生儿坏死性小肠结肠炎术中应用更具优势。

用草原兔尾鼠复制内脏利什曼病模型初探

给草原兔尾鼠经腹腔接种婴儿利什曼原虫前鞭毛体，５个月后剖杀动物作病理组织学检查，发现动物的脾、肝、骨髓、淋巴结、肾及输尿管内均有不同数量的利什曼原虫分布，有典型的内脏利什曼病病理损害。作者认为草原兔尾鼠是一种便于复制内脏利什曼病模型的动物。

婴儿利什曼原虫实验感染草原兔尾鼠的进一步观察

给草原兔尾鼠（Laguruslagurus）经腹腔接种不同量的婴儿利什曼原虫（Leishmaniainfantum）前鞭毛体，结果表明，不同量的原虫感染对动物体重及肝重没有明显的影响，但脾重则有一定的差异。肝脏原虫负荷随感染虫量的增加而加大，实验结果进一步证明草原兔尾鼠是一种对利什曼原虫非常敏感的实验动物；同时在用级差较小的不同量的原虫接种后，可以显示感染程度的差别，这就为利什曼病的免疫学研究提供了良好的动物模型。

新疆克拉玛依皮肤利什曼病病原生物学的研究

从新疆克拉玛依地区皮肤利什曼病患者皮损内分离的３株婴儿利什曼原虫，接种至草原兔尾鼠或背纹仓鼠的腹腔／睾丸内后，引起内脏感染。鼠的病理变化与从内脏利什曼病人体内分离出来的婴儿利什曼原虫／杜氏利什曼原虫引起的一致。ＢＡＬＢ／ｃ小鼠皮下接种原虫后，既不发生皮肤损害，也不引起内脏感染。猴的皮下注射该原虫后，仅产生含虫的皮肤损害而不引起内脏感染。皮肤利什曼病人的皮损组织置ＮＮＮ培养基内培养时，在培养基内难以查获前鞭毛体，用上述感染鼠的脾组织置该培养基内培养，可以使前鞭体生长繁殖。无鞭毛体的超微结构显示，膜下微管数为７４±７，周长为８．８６±１．２５μｍ，原虫的大小指数为６．６１±１．５６。通过本文结果及文献复习，作者指出：中国的婴儿利什曼原虫是由基因型存在一定差异和表型呈多态性的不同品系组成的复合体。

新疆克拉玛依皮肤利什曼病传播媒介的研究

１９９４年的研究表明，在克拉玛依从皮肤利什曼病患者皮肤损害部位和从硕大白岭吴氏亚种消化道内分离出来的利什曼原虫，经ＤＮＡ基因型的分析，证实与婴儿利什曼原虫同源。本文报道，在皮肤利什曼病流行区内硕大白蛉吴氏亚种的数最颇大，亲人性强，在野外和居民点内该蛉的前鞭毛体自然感染率分别为５．９％（５８／９８５）和２．９％（１３／４４９），前鞭毛体在该蛉的消化道内能大量繁殖并可移行至咽及喙部；而在非流行区，该蛉的数量很少或无，也未查见前鞭毛体的感染。综合以往和本文的研究结果，作者确认硕大白蛉吴氏亚种为克拉玛依山婴儿利什曼原虫所致的皮肤利什曼病的传播媒介。

克拉玛依地区婴儿利什曼原虫在猴体内寄生特性的研究

为了解克拉玛依地区婴儿利什曼原虫（Leishmaniainfantum）在猴体内的寄生特性，共用4只恒河猴（Macacamulatta）进行了研究，其中3只猴分别经颊部皮下接种从当地皮肤利什曼病患者的皮损组织以及从硕大白蛉吴氏亚种（Phlebotomusmajorwui）胃内分离出来的婴儿利什曼原虫，另一只猴作为对照。接种后观察局部皮肤的变化及对皮损组织作涂片检查或培养（NNN氏基），并于接种后1年～1年2个月解剖，作皮肤、肝、脾、淋巴结、骨及肾的组织学观察和涂片检查，肝、脾组织置NNN氏基内培养。结果发现，病人及蛉胃内的原虫对猴仅引起皮肤损害，而在皮损愈合后，原虫并未消失而是主要在接种部位的血管壁肌层内寄生繁殖。各内脏组织均未查见利什曼原虫。表明新疆克拉玛依地区的婴儿利什曼原虫对灵长类动物具有亲皮肤的特性。此一发现，对当地皮肤利什曼原虫复发的研究以及动物宿主的调查，均具有一定的意义。

我国利什曼原虫伽师株有毒力和无毒力前鞭毛体定量蛋白质组学比较分析

目的应用定量蛋白质组学方法比较我国利什曼原虫伽师株有毒力和无毒力前鞭毛体蛋白表达的差异。方法将分离于我国新疆伽师县黑热病患者婴儿利什曼原虫JS5有毒力株前鞭毛体和无毒力株前鞭毛体经酶解后进行液相色谱串联质谱(Liquid chromatography-tandem mass spectrometry, LC-MS/MS)的非标记(Label-free)定量分析,利用MaxQuant软件查库,进行Label-free非标定量(LFQ)分析;每个样品重复3次质谱分析,只有1次有LFQ值的蛋白舍弃。结果本研究共鉴定蛋白3 994个,有毒力和无毒力株间差异蛋白为298个(差异倍数>1.2或差异倍数<0.83,P<0.05),其中利什曼原虫有毒力株前鞭毛体特有表达蛋白15个,无毒力株前鞭毛体特有表达蛋白23个,二者间表达丰度有显著差异蛋白260个,其中有毒力前鞭毛体表达上调蛋白(高表达)78个,表达下调蛋白(低表达)182个。这些差异表达蛋白中已鉴定功能的蛋白分别涉及糖与脂质代谢、核酸代谢、压力反应、细胞骨架形成、细胞周期与增殖等生物功能。结论利什曼原虫伽师株有毒力株和无毒力株前鞭毛体蛋白质组的表达存在差异,为筛选和鉴定与利什曼原虫感染相关关键分子奠定了基础。