Detection of anti-Leishmania infantum antibodies in sylvatic lagomorphs from an epidemic area of Madrid using the indirect immunofluorescence antibody test

An outbreak of human leishmaniosis was confirmed in the southwest of the province of Madrid, Spain, between July 2009 and December 2012. Incidence of Leishmania infection in dogs was unchanged in this period, prompting a search for alternative sylvatic infection reservoirs. We evaluated exposure to Leishmania in serum samples from animals in the area with an indirect immunofluorescence test (IFAT). Using promastigotes from six culture passages and a 1/25 threshold titer, we found anti-Leishmania infantum seroreactivity in 9.3% of cats (4 of 43), 45.7% of rabbits (16/35) and 74.1% of hares (63/85). Use of promastigotes from >10 in vitro passages resulted in a notably IFAT lower titer, suggesting antigenic changes during extended culture. Postmortem inspection of seropositive animals showed no clinical signs of infection. The results clearly suggest that asymptomatic hares were the main reservoir in the outbreak, and corroborate IFAT as a sensitive serological surveillance method to detect such cryptic Leishmania infections.     

Isolation of Lawsonia intracellularis specific single-chain Fv antibody fragments from phage display library

Single-chain antibodies (scFv) exhibiting specific binding to Lawsonia intracellularis were isolated from a phagemid library expressing scFvs molecules on the surface of filamentous bacteriophages. For scFv selection whole bacterial cells were used and individual clones were tested in ELISA test. The total of seven unique clones with different fingerprint profiles was isolated. All clones were able to bind specifically in immunofluorescence assay. This is the first report of species specific recombinant antibodies against L. intracellularis.     

The indirect immunofluorescence assay using cardiac tissue from chickens, quails and ducks for identification of influenza A virus during an outbreak of highly pathogenic avian influenza virus (H5N1): a rapid and simple screening tool for limited resource settings

Here we describe the diagnostic utility of the indirect immunofluorescence assay (IFA) during a recent outbreak of highly pathogenic avian influenza (HPAI) subtype H5N1 virus in southern Thailand and demonstrate the usefulness of the cardiac tissue from infected chickens, quail, and ducks for diagnosis. The most reliable sample for IFA diagnosis of influenza A virus was cardiac tissue (83.0%; 44/53) which when divided by species (chicken, quail and duck cardiac tissues) gave respective positivity rates of 88% (22/25), 88.9% (16/18) and 60.0% (6/10). Cardiac tissue also gave the highest IFA intensity for the three species. We believe that the IFA method has wide applicability in developing countries or remote settings where clinically similar avian diseases with high morbidity and mortality such as Newcastle disease and fowl cholera are common and could be rapidly excluded thereby conserving valuable reference laboratory capacity for true HPAI outbreaks.     

鳜下丘脑神经元细胞培养

通过建立可行,高效的下丘脑神经元细胞技术,为研究鳜鱼的摄食机理与能量代谢奠定基础。分离鳜鱼下丘脑组织,Ⅰ型胶原酶将组织消化成单细胞悬液,用完全培养液进行培养,在培养的第3d、4d、5d、6d观察细胞的形态,结果显示培养第3d时细胞贴壁量少,胞体小并且呈单个分布,在培养第4d细胞的数量显著增多,且具有典型的神经元的形态,胞体饱满,第5d形神经元胞体的融合度进一步增大,突起增长增粗,许多分支互相交错连接而形成密集的神经纤维网络,第6d细胞活性减弱,开始走向凋亡;CCK-8法检测细胞活性,结果显示在培养第5d细胞活性最高;并采用两种方法对神经元细胞进行鉴定,结果显示RT-PCR检测发现培养的下丘脑细胞可以表达神经元特异基因noggin,经免疫荧光鉴定下丘脑神经元细胞纯度为95.9%。这些结果表明通过此培养方法能够获得纯度较高的鳜鱼下丘脑神经元细胞,为进一步在细胞水平研究鳜鱼的摄食机理与能量代谢相关机理奠定基础。     

Prevalence and genetic characterization of Giardia and Cryptosporidium in cats from Italy

One hundred and eighty one cats living in central Italy were tested for the presence of Giardia and Cryptosporidium infection by IFAT test and specific PCRs. Overall eight (4.4%) samples were IFAT-positive for Giardia. All the IFAT-positive samples for Giardia scored positive for the PCRs, and three more samples IFAT-negative generated PCR products leading to a total 6.1% molecular positivity rate for Giardia. All the examined samples were negative for Cryptosporidium. Sequencing of samples molecularly positive to Giardia indicated that three cats harbored the zoonotic Giardia duodenalis Assemblage A, whereas all other positive animals were infected with the feline-specific G. duodenalis Assemblage F. Phylogenetic analysis carried out on the sequences obtained supported the clustering of the isolates within Assemblages A and F. The results here presented provide data on the occurrence of Giardia genotypes in cats living in close contact with humans highlighting the potential importance of this protozoan disease for the public health.     

Expression of β2 adrenoceptors within enteric neurons of the horse ileum

The activity of the gastrointestinal tract is regulated through the activation of adrenergic receptors (ARs). Since data concerning the distribution of ARs in the horse intestine is virtually absent, we investigated the distribution of β₂-AR in the horse ileum using double-immunofluorescence. The β₂-AR-immunoreactivity (IR) was observed in most (95%) neurons located in submucosal plexus (SMP) and in few (8%) neurons of the myenteric plexus (MP). Tyrosine hydroxylase (TH)-IR fibers were observed close to neurons expressing β2-AR-IR. Since β₂-AR is virtually expressed in most neurons located in the horse SMP and in a lower percentage of neurons in the MP, it is reasonable to retain that this adrenergic receptor could regulate the activity of both secretomotor neurons and motor neurons innervating muscle layers and blood vessels. The high density of TH-IR fibers near β₂-AR-IR enteric neurons indicates that the excitability of these cells could be directly modulated by the sympathetic system.     

3型鲤疱疹病毒白介素10单克隆抗体制备与应用

白介素（Interleukin，IL）10是 脊椎动物Th2细胞（T helper 2 cell）和先天淋巴细胞（Innate lymphoid cell）分泌的一种多效应细胞因子。3型鲤疱疹病毒（Cyprinid herpesvirus 3, CyHV3）ORF134编码IL10样蛋白，本研究构建了ORF134原核表达质粒，在大肠杆菌DE3感受态细胞中表达CyHV3-IL10重组蛋白。SDS-PAGE分析结果表明，重组CyHV3-IL10蛋白约为18 ku，主要以包涵体形式表达。将CyHV3-IL10包涵体进行变性和复性，经分子筛凝胶柱层析纯化获得了纯度较高的重组CyHV3-IL10蛋白，利用CyHV3-IL10重组蛋白免疫小鼠制备了单克隆抗体，通过酶联免疫吸附测定法（Enzyme linked immunosorbent assay，ELISA）筛选，获得了2株亲和力强的抗体，并利用激光共聚焦技术和Western blotting对抗体进行了鉴定。结果表明，FITC标记的单克隆抗体能够特异性识别HEK293细胞中表达的CyHV3-IL10重组蛋白和感染CyHV3的镜鲤脾和鳃组织中表达的CyHV3-IL10蛋白。CyHV3-IL10单克隆抗体的制备为后续研究CyHV3-IL10蛋白功能和建立CyHV3诊断方法奠定了基础，为CyHV3病毒检测试剂盒的研发提供了新思路。     

猪肺炎支原体168株特异性单抗的制备及鉴定

将猪肺炎支原体168株全菌蛋白免疫小鼠,取脾细胞与SP2/0骨髓瘤细胞进行融合,3次亚克隆后,获得了4株针对168株全菌蛋白的单抗,分别将其命名为2F5、2G7、4F5、5E9。Western-blot检测结果表明,2G7、4F5与猪肺炎支原体具有特异性反应条带,不与猪鼻支原体、大肠杆菌反应。亚型鉴定结果表明,两株特异性单抗(2G7、4F5)的亚型属IgG1,轻链为κ型,2F5、5E9亚型属IgG2a,轻链为κ型。间接免疫荧光结果表明,4株单抗均与猪肺炎支原体168株有特异性荧光反应,特异性单抗的获得为猪肺炎支原体的检测及机理研究奠定基础。     

用单克隆抗体鉴定猪繁殖与呼吸综合征病毒分离株

应用ＰＲＲＳＶ单克隆抗体,采用直接与间接免疫荧光抗体试验对分离获得的ＰＲＲＳＶ６个毒株进行了鉴定,结果所有分离毒株均能被单克隆抗体（ＳＤＯＷ１７、Ａ、Ｂ、Ｃ、Ｄ、Ｅ、Ｆ）所识别,呈现特异荧光,６个分离毒株均能与仅识别美洲型ＰＲＲＳＶ的单克隆抗体Ｆ反应,结果表明６个分离毒株均属于美洲型ＰＲＲＳＶ。利用微量细胞培养对分离毒株ＴＣＩＤ５０测定结果表明,６个分离毒株的ＴＣＩＤ５０分别为１０－７．５／０．１ｍｌ、１０－７．５／０．１ｍｌ、１０－７．５／０．１ｍｌ、１０－７．７５／０．１ｍｌ、１０－７．２５／０．１ｍｌ、１０－６．２５／０．１ｍｌ。病毒感染细胞的超薄切片电镜观察表明,在感染细胞浆内可见典型的ＰＲＲＳＶ病毒粒子,呈球形或椭圆形,直径约为６０ｎｍ左右,可见囊膜。     

Effect of different storage temperatures on immunofluorescence results in frozen sections of kidney biopsies

AIM: To investigate the effect of storage temperature in renal biopsy tissue frozen section on immunofluorescence results. METHODS: One hundred renal biopsy samples of Zhejiang Province People's Hospital from January to June, 2015 were enrolled. Two sets of cutting slices were stored in -70℃ low temperature refrigerator as experimental group and in -12℃ freezing cryostat as control group. The immunoglobulins(IgG, IgA and IgM) and complements(C3, C4 and C1q) as well as fibrinogen were detected with direct immunofluorescence in the next day. The type Ⅳ collagen a3 and a5 chains were also detected by indirect immunofluorescence, and the qualitative and semi-quantitative results were observed under the fluorescence microscope. RESULTS: There were 16 cases of 3+~4+IgG in the experimental group, while the corresponding IgG was all 1+~2+in control group, significantly weaker than that in experimental group. There were 99 cases of 3+~4+a3 and 3+~4+a5 in experimental group, white there were 92 cases of 1+~2+a3/a5 and 8 cases negative in control group. The positive intensity was decreased in control group with statistical difference between the 2 groups(P<0.05). There were 15 cases of 4+IgA, 19 cases of 3+IgA, 15 cases of 3+IgM, 11 cases of 2+IgM, 13 cases of 4+C3, and 12 cases of 3+C3 in experimental group. The control group were similar to the results of experimental group, and no difference between the 2 groups was observed. CONCLUSION: The immunofluorescence results of renal biopsy frozen sections are highly affected by the section storage temperature, which has greater influence on the immunofluorescence positive intensity of IgG and type Ⅳ collagen. The renal biopsy frozen section should be stored in -70℃ low temperature refrigerator.