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.     

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.     

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.     

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.     

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。     

旋毛虫T668基因真核表达载体的构建及其在BHK细胞中的表达

利用RT-PCR技术从中国河南猪旋毛虫(国际标准虫种编号为ISS534)新生幼虫得到T668基因,并克隆入pEGFP-N1真核表达载体中构建重组质粒,该重组质粒在脂质体介导下转染BHK细胞。EGFP标签证明质粒DNA成功转染到细胞中并得以表达,Western blotting鉴定,细胞裂解液样品中有1条约77 000的条带,可被绿色荧光抗体及猪感染旋毛虫阳性血清所识别,与预计大小一致,表明成功构建T668-pEGFP-N1真核表达质粒,并在BHK细胞融合表达,表达产物具有抗原性。     

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.     

促黄体生成素和17β-雌二醇对牦牛输卵管糖蛋白表达的影响

为研究相关激素是否通过体细胞参与哺乳动物胚胎发育的调控,本研究探讨了促黄体生成素(luteinizing hormone,LH)和17ββ-雌二醇(17β-estradiol,E2)对牦牛(Bos grunniens)输卵管上皮细胞分泌输卵管糖蛋白(oviductin,Ovn)的影响.体外培养牦牛输卵管上皮细胞,分别添加0、10、25、50、100和200 ng/mLLH和E2,作用72h后,运用qRT-PCR和免疫荧光技术,从基因和蛋白水平检测Ovn表达.获得如下结果:(1)LH和E2可以提高牦牛输卵管上皮细胞的Ovn mRNA相对表达量;当LH浓度为10 ng/mL时,Ovn mRNA的相对表达量最高,显著高于LH浓度为25、50、100和200 ng/mL组(P＜0.05),对照组Ovn mRNA的表达量最低(P＜0.05);当E2浓度为25 ng/mL时,输卵管上皮细胞的Ovn mRNA相对表达量最高,显著高于E2浓度为10、50、100和200 ng/mL组(P＜0.05),对照组Ovn mRNA表达量最低(P＜0.05).(2)Ovn主要分布于核膜,细胞质也有表达;LH和E2可以提高输卵管上皮细胞的蛋白相对表达量,当LH浓度为10ng/mL时,Ovn的蛋白表达量达到最高,LH浓度达到25 ng/mL时,Ovn的蛋白表达量降低;当E2浓度为10～25 ng/mL时,Ovn的蛋白表达量随着E2浓度的上升不断增加,在25 ng/mL的E2作用后,Ovn的蛋白表达量达到最高,E2浓度达到50 ng/mL时,Ovn的蛋白表达量降低.上述结果表明,LH和E2对牦牛输卵管上皮细胞分泌Ovn具有明显的促进作用,LH的最佳作用浓度为10 ng/mL,E2的最佳作用浓度为25ng/mL.本研究为揭示LH和E2对输卵管上皮细胞分泌Ovn的作用机制提供了基础资料,为进一步研究相关激素对牦牛胚胎发育的调控提供了一定理论依据.     

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.     

Soil DNA extraction and assessment of the fate of Mycobacterium chlorophenolicum strain PCP-1 in different soils by 16S ribosomal RNA gene sequence based most-probable-number PCR and immunofluorescence

Since Mycobacterium chlorophenolicum strain PCP-1 is not detectable in soil by selective plating, a specific tracking method was based on the polymerase chain reaction (PCR) using soil DNA as a target. A direct extraction protocol based on bead beating was adapted and used to obtain PCR-amplifiable DNA from five different soils. In one soil, the disruption of cells of PCP-1, of Pseudomonas fluorescens R2f and of Paenibacillus azotofixans P3L5, as well as of the indigenous bacteria increased with increasing bead beating times. After 4.5 min, lysis efficiency was about 90% or more in all cases. Total DNA yields varied between soils, from 2 to 35 μg g^–1. The purification steps needed to obtain amplifiable DNA were different per soil. To detect target DNA specifically in bacterial cells, a new indirect extraction protocol was developed, which efficiently dislodged bacterial cells from the soil matrix, and produced amplifiable DNA with high yield. To detect strain PCP-1 in soil, 16S ribosomal gene-based PCR combined with oligonucleotide hybridization was applied using a most-probable-number (MPN) set-up, whereas immunofluorescence was used for calibration. Strain PCP-1 was detected shortly after introduction into three soils at about the inoculum levels, as evidenced by both approaches. Both the direct and indirect DNA extraction methods yielded similar MPN estimates. The dynamics of M. chlorophenolicum PCP-1 was estimated in two soils over 14 days via MPN-PCR/oligonucleotide probing. PCP-1 showed good survival in both soils, and results obtained by MPN-PCR with directly and indirectly extracted DNA were internally consistent. Immunofluorescence cell enumerations supported the gross stability of PCP-1 in these two as well as in two additional soils. Received: 8 February 1996