Effect of endocytic and metabolic inhibitors on the internalization and intracellular growth of Brucella abortus in Vero cells.

Uptake, transfer to rough endoplasmic reticulum, and intracellular growth of Brucella abortus were studied in Vero cells treated with endocytic and metabolic inhibitors. Infection of Vero cells was suppressed when inhibitors of energy metabolism (iodoacetate, dinitrophenol), receptor-mediated endocytosis (monodansylcadaverine, amantadine, methylamine), or endosomal acidification (chloroquine, ammonium chloride, monensin) were added to the inoculum. Inhibition was not observed when these drugs were added after the inoculation period. Infection of Vero cells by B abortus was inhibited by dibutyryl-cyclic adenosine monophosphate and Vibrio cholerae enterotoxin, but was stimulated by dibutyryl-cyclic guanosine monophosphate and escherichia coli heat-stable enterotoxin a. Uptake of B abortus by Vero cells was not prevented by colchicine, but was abolished by cytochalasin B. Uptake of heat-killed B abortus and noninvasive E coli was similar to that of viable brucellae. Intracellular growth of B abortus was not affected by cycloheximide. Results indicate that: B abortus may be internalized by a receptor-mediated phagocytic process; transfer of B abortus from phagosomes to rough endoplasmic reticulum may require endosomal acidification; and replication of B abortus within the rough endoplasmic reticulum may not depend on protein synthesis by the host cell.     

Entry of African swine fever virus into Vero cells and uncoating

African swine fever virus (ASFV) enters Vero cells by adsorptive endocytosis [Valdeira, M.L., Geraldes, A., 1985. Morphological study on the entry of African swine fever virus into cells, Biol. Cell. 55, 35–40]. Electron microscopy of a lysosomotropic drug-controlled penetration indicated that this step takes place in the endosomes, after fusion between the viral envelope and the limiting membrane of the endosome. Inhibition studies with colcemid, cytochalasin B, sodium azide, dinitrophenol, lysosomotropic weak bases, and the ionophore monensin, showed that the virus uptake is largely independent of cytoskeletal and lysosomal function, but dependent on oxidative phosphorylation. Some protease inhibitors inhibited viral replication at an early step, indicating that the initiation of infection depends on a viral proteolytic cleavage.     

布鲁菌Hfq蛋白研究进展

布鲁菌病(布病)是一种严重的人兽共患病。作为布病的病原,布鲁菌能够耐受宿主体内的各种杀菌机制并持续的生存。当布鲁菌侵入宿主体内后,必须适应抗体免疫的环境,而这依赖于布鲁菌对自身基因表达的巧妙调控。Hfq蛋白是一个RNA伴侣分子,对于介导sRNA的转录后调控发挥着重要的作用。在多种细菌中,对Hfq蛋白的功能都进行了深入的研究,目前已经发现该蛋白与细菌基因的表达调控、细菌对多种应激环境的适应能力以及致病菌的毒力均有着密切的关系。论文对Hfq蛋白的结构,对sRNA的调控功能,特别是布鲁菌Hfq蛋白调控功能的研究现状及在布病疫苗研发中的应用进行系统的介绍,为布鲁菌基因的表达调控和布病防控技术的研究提供参考。     

布鲁氏菌实时定量荧光PCR检测体系的建立及应用

本根据GenBank上公布的布鲁氏菌BCSP31基因保守区域序列设计合成一对特异性引物与TapMan探针,建立整套快速准确鉴定布鲁氏菌的实时定量荧光PCR检测体系。以实验室构建的克隆有布鲁氏菌目的片段的重组质粒为标准品,进行实时定量荧光PCR反应检测,通过优化反应条件,建立标准曲线。以标准品为模板,对该方法的特异性、敏感性与重复性进行检测与分析。并以临床样本进行检测的结果进行比较分析,结果表明,该检测体系的检测灵敏度高,重复性与特异性良好。本研究建立的实时定量荧光PCR可用于准确检测样本中的少量的布鲁氏菌,具有良好的应用前景和市场价值。     

布鲁氏菌表面抗原研究进展

布鲁氏菌表面最主要的抗原是脂多糖(LPS)和外膜蛋白(OMPs),R-LPS是R型布鲁氏菌主要的表面抗原,S型布鲁氏菌的S-LPS已被证明是一个主要的毒力因子,它的O链部分含有布鲁氏菌表面绝大多数的抗原位点,是一个很重要的保护性抗原;OMPs被认为是布鲁氏菌表面潜在的抗原结构,在光滑型布鲁氏菌表面,由于OMPs受到了LPS-O链的干扰,致使它的抗原性没有充分地表现出来,但在绵羊布鲁氏菌表面却出现了不同的情况。未来几年,对OMPs的研究将成为该领域的热点,尤其是对Omp25突变体的研究。     

A comparison of fixatives suitable for scanning electron microscopy of Habronema spp

Members of the genus Habronema (Nematoda: Habronematidae) were preserved in 4 fixatives for examination with scanning electron microscopy (SEM). Tegumental features of the anterior and posterior extremities of these specimens were compared to evaluate the effect of the fixatives: modified Flemming's solution, glutaraldehyde (GA), Karnovsky's fixative and 70% ethanol. Fixatives were assessed on the appearance of the tegument, evidence of any wrinkling, shrinkage or swelling and the degree of extension in the male tail. Seventy per cent ethanol gave the most satisfactory results.     

Scanning electron microscopy description of a new species of Demodex canis spp

Between 1997 and 1999, the prevalence of Demodex canis mites was determined in 150 dogs. In two dogs, we found two different species of mites; Demodex canis and another, unidentified, Demodex mite. The unidentified Demodex mite species had several different morphological features. First, it had a short opisthosoma and an obtuse end. In addition, the fourth coxisternal plate was rectangular and there was a band-like segmental plate between the fourth coxisternal plate and opisthosoma. Although all of the morphology and the development of male mites could not be investigated in this study, the location of the opisthosoma and the genital pore clearly differed from Demodex canis, suggesting that this unidentified mite is a new species.     

Major outer membrane proteins of Brucella spp.: past,present and future

The major outer membrane proteins (OMPs) of Brucella spp. were initially identified in the early 1980s and characterised as potential immunogenic and protective antigens. They were classified according to their apparent molecular mass as 36–38 kDa OMPs or group 2 porin proteins and 31–34 and 25–27 kDa OMPs which belong to the group 3 proteins. The genes encoding the group 2 porin proteins were identified in the late 1980s and consist of two genes, omp2a and omp2b, which are closely linked in the Brucella genome, and which share a great degree of identity (>85%). In the 1990s, two genes were identified coding for the group 3 proteins and were named omp25 and omp31. The predicted amino acid sequences of omp25 and omp31 share 34% identity. The recent release of the genome sequence of B. melitensis 16 M has revealed the presence of five additional gene products homologous to Omp25 and Omp31. The use of recombinant protein technology and monoclonal antibodies (MAbs) has shown that the major OMPs appear to be of little relevance as antigens in smooth (S) B. abortus or B. melitensis infections i.e. low or no protective activity in the mouse model of infection and low or no immunogenicity during host infection. However, group 3 proteins, in particular Omp31, appear as immunodominant antigen in the course of rough (R) B. ovis infection in rams and as important protective antigen in the B. ovis mouse model of infection. The major OMP genes display diversity and specific markers have been identified for Brucella species, biovars, and strains, including the recent marine mammal Brucella isolates for which new species names have been proposed. Recently, Omp25 has been shown to be involved in virulence of B. melitensis, B. abortus and B. ovis. Mutants lacking Omp25 are indeed attenuated in animal models of infection, and moreover provide levels of protection similar or better than currently used attenuated vaccine strain B. melitensis Rev.1. Therefore, these mutant strains appear interesting vaccine candidates for the future. The other group 3 proteins identified in the genome merit also further investigation related to the development of new vaccines.     

Cross-sectional study on Brucella spp., Leptospira spp. and Salmonella spp. in bats from Montes Claros,Minas Gerais,Brazil

The understanding on the role of bats in the ecology of zoonotic diseases, especially its relevance as a carrier of pathogens, is important for the determination of preventive measures considering the One Health context. The present study aimed to investigate the presence of Brucella spp., Leptospira spp. and Salmonella spp. in blood (n = 163), liver (n = 35) and spleen (n = 62) samples from bats captured in Montes Claros, Minas Gerais, Brazil. Only Salmonella spp. was found in a blood sample of an insectivorous female bat of the species Lasiurus blossevilli, evidencing the capacity of this animal species to host this pathogen. In conclusion, our results in bats from Montes Claros indicate that they do not act as hosts for Brucella spp. and Leptospira spp., although being potential carriers of Salmonella spp. in a low prevalence.     

Determination of Brucella spp. in raw milk and Turkish white cheese in Kirikkale

In this study, a total of 105 samples including 35 raw milk, 35 cows' milk cheese, and 35 ewes' milk cheese samples were obtained from Kirikkale city and investigated for the presence and contamination level of Brucella species. For this reason, milk and cheese samples were collected in aseptic conditions and brought to the laboratory under cold conditions. The method suggested from FARREL was used for the isolation and identification of Brucella spp. In addition, Most Probable Number Technique was used to determine the contamination level in Brucella spp. positive samples. According to analysis findings, B. melitensis was isolated from 5 (14.2%) of 35 ewes' milk cheese samples at the level of 3.6 x 10(1)-9.3 x 10(3) MPN/g. Brucella spp. were not detected in any of raw milk and cows' milk cheese samples. In conclusion this study reinforced the previous reports that ewes' milk cheese is an important source of Brucella spp. and they have been risk for public health.     

Musculoskeletal responses of 2-year-old Thoroughbred horses to early training. 8. Quantitative back-scattered electron scanning electron microscopy and confocal fluorescence microscopy of the epiphysis of the third metacarpal bone

AIM: To characterise and explain the increase in density evident by computerised tomography (CT) and radiography in companion studies as a response to training, in bone in the palmar and dorsal regions of the condyles of the third metacarpal bone (Mc3) of 2-year-old Thoroughbred horses.

METHODS: Compositional back-scattered electron (BSE) imaging in scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM) were conducted on polymethyl methacrylate (PMMA)-embedded mediolateral slices of the right distal Mc3 from seven 2-year-old Thoroughbred horses trained on a racetrack and seven untrained horses kept at pasture. One left Mc3 from each group was studied in transverse section planes. This study focussed on regions of Mc3 found to differ in density between the trained and untrained horses in companion studies using CT and radiography.

RESULTS: The increase of bone density in the condyles of Mc3 in trained horses compared with untrained horses occurred, without prior osteoclastic resorption, via the deposition of new bone on pre-existing internal surfaces. Within prior marrow spaces of cancellous bone, there was also rapid formation of immature strands and fronds of bone which were more cellular and mineralised, and more lamellar bone tissue was deposited on these new scaffolding elements in the trained horses. Both resulted in increased bone volume fraction (BVF). The microscopic mineralisation density of the bulk of the new tissue was lower than in pre-existing bone, and CT and radiography underestimated the increase in BVF. The new tissue was thus probably less stiff at the microscopic scale than pre-existing bone, though its addition would stiffen the global structure.

CONCLUSIONS: In Mc3 of all the trained horses, there were obvious differences in microscopic structure compared with those from the untrained horses. Moderate, industry-standard levels of exercise used to prepare young horses for racing induced the formation of new bone in non-bone spaces in bone tissue, such that the bone organ should better withstand later increased levels of exercise.     

Musculoskeletal responses of 2-year-old Thoroughbred horses to early training. 8. Quantitative back-scattered electron scanning electron microscopy and confocal fluorescence microscopy of the epiphysis of the third metacarpal bone

AIM: To characterise and explain the increase in density evident by computerised tomography (CT) and radiography in companion studies as a response to training, in bone in the palmar and dorsal regions of the condyles of the third metacarpal bone (Mc3) of 2-year-old Thoroughbred horses. METHODS: Compositional back-scattered electron (BSE) imaging in scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM) were conducted on polymethyl methacrylate (PMMA)-embedded mediolateral slices of the right distal Mc3 from seven 2-year-old Thoroughbred horses trained on a racetrack and seven untrained horses kept at pasture. One left Mc3 from each group was studied in transverse section planes. This study focussed on regions of Mc3 found to differ in density between the trained and untrained horses in companion studies using CT and radiography. RESULTS: The increase of bone density in the condyles of Mc3 in trained horses compared with untrained horses occurred, without prior osteoclastic resorption, via the deposition of new bone on pre-existing internal surfaces. Within prior marrow spaces of cancellous bone, there was also rapid formation of immature strands and fronds of bone which were more cellular and mineralised, and more lamellar bone tissue was deposited on these new scaffolding elements in the trained horses. Both resulted in increased bone volume fraction (BVF). The microscopic mineralisation density of the bulk of the new tissue was lower than in pre-existing bone, and CT and radiography underestimated the increase in BVF. The new tissue was thus probably less stiff at the microscopic scale than pre-existing bone, though its addition would stiffen the global structure. CONCLUSIONS: In Mc3 of all the trained horses, there were obvious differences in microscopic structure compared with those from the untrained horses. Moderate, industry-standard levels of exercise used to prepare young horses for racing induced the formation of new bone in non-bone spaces in bone tissue, such that the bone organ should better withstand later increased levels of exercise.     

A multiplex PCR for the detection of Brucella spp. and Leptospira spp. DNA from aborted bovine fetuses

Bovine brucellosis and leptospirosis are important causes of bovine abortion around the world. Both diseases can be serologically diagnosed, but many factors may cause false positive and negative results. Direct methods based on bacteriological isolation are usually employed, but they are difficult, time consuming and dangerous. Monoplex polymerase chain reaction (PCR) have been successfully described for the detection of Brucella spp. and Leptospira spp. Aiming at improvement in the direct diagnosis, a multiplex PCR (mPCR) for the detection of these agents in aborted bovine fetuses is described. The detection threshold of the mPCR was evaluated in experimentally contaminated bovine clinical samples using a conventional proteinase K/SDS or a boiling-based extraction protocols. The mPCR was applied to two groups of clinical samples: 63 episodes of bovine abortion and eight hamsters experimentally infected with Leptospira interrogans serovar pomona. Adopting microbiological isolation as reference, the test showed a sensitivity of 100% in both groups of clinical samples. Seven samples collected from bovine fetuses were Brucella spp. culture negative but showed positive results in mPCR. Regarding Leptospira spp. detection, similar results were observed in three bovine clinical samples. All hamsters infected with Leptospira were positive in both microbiological culture and mPCR. The boiling extraction protocol showed better results in some clinical samples, probably by the removal of PCR inhibitors by heat treatment. The high sensitivity, simplicity and the possibility of detection of both bacteria in a single tube reaction support the use of the mPCR described in the routine diagnosis.     

布鲁菌种属鉴定多重PCR方法的建立及初步应用

用BCSP31作为布鲁菌属特异性基因,以IS711基因拷贝数差异作为布鲁菌种间特异性标志,建立了布鲁菌种属特异性的多重PCR鉴定方法.用建立的多重PCR方法对11株(牛种A19,A544,A387;羊种M111,M28,M16;犬种RM6/66;绵羊种63/290;猪种S2,rS2,S1330)不同种来源的布鲁菌菌体和基因组进行鉴定,结果牛种菌能扩增大小分别为494,223,178 bp 3条带,羊种菌能扩增出大小分别为733,223,178 bp 3条带,犬种菌能扩增出大小分别为223,178 bp 2条带,绵羊种菌能扩增出大小分别为976,223,178 bp 3条带,猪种菌能扩增出大小分别为285,223,178 bp 3条带.均与预期一致;而作为对照的大肠杆菌、胸膜肺炎放线杆菌、多杀性巴氏杆菌、流产沙门菌和都柏林沙门菌,均未扩增出任何务带.结果表明,本研究所建立的方法具有良好的特异性,能够区分不同种源的布鲁菌,可用于布鲁菌种属的快速鉴定和流行病学调查.     

Morphologic evaluation of IgM cells of the canine small intestine by fluorescence microscopy.

Biopsies of small intestine from 7 dogs were examined by fluorescence microscopy to determine the number of IgM-containing cells in the lamina propria. Biopsies were taken from duodenum, jejunum, and ileium. (Cell counts were made by 2 persons to demonstrate reproducibility.) There were 452.24 +/- 60.09 cells per mm2 in the duodenum 572.68 +/- 62.13 cells per mm2 in the jejunum, and 107.47 +/- 59.57 cells per mm2 in the ileum. All sections were cut at 6 micrometer. The ileum had fewer cells than either duodenum or jejunum (P = 0.000038 and 0.00001, respectively), whereas duodenum and jejunum did not differ significantly in numbers of cells (P = 0.17528). Quantifying autofluorescent cells in the same sites showed no significant differences among the 3 tissues (P = 0.24697). The autofluorescent cells differed in intensity and morphology from the IgM cells. These two observations tend to support the contention that the autofluorescent cells did not bias the IgM cell counts at the 3 sites. Total autofluorescence (cells, collagen, and vessels) was higher in the ileum than in either the jejunum or the duodenum (P = 0.04967 and 0.03050, respectively). However, all 3 categories counted (IgM cells, autofluorescent cells, and autofluorescent structures) had significant dog-tissue interactions. This will necessitate determining normals for each age-sex-breed category of dog studied.