Fluorescence in situ hybridization analysis of hindgut bacteria associated with the development of equine laminitis

Carbohydrate-induced laminitis in horses is characterized by marked changes in the composition of the hindgut microbiota, from a predominantly Gram-negative population to one dominated by Gram-positive bacteria. The objective of this study was to monitor changes in the relative abundance of selected hindgut bacteria that have previously been implicated in the pathophysiology of equine laminitis using fluorescence in situ hybridization (FISH). Caecal cannulae were surgically implanted in five Standardbred horses and laminitis induced by oral administration of a bolus dose of oligofructose. Caecal fluid and faecal specimens were collected over a 48 h period at 2 to 4 h intervals post-oligofructose administration and subjected to FISH using probes specific for nine bacterial groups to determine changes in their relative abundance compared with total bacteria hybridizing to the generic EUBMIX probe. Additionally, hoof biopsies were taken over the course of the experiment at 6 h intervals and evaluated for histopathological changes consistent with laminitis, allowing changes in hindgut microbiota to be correlated with the onset of lesions in the foot. Of the microorganisms specifically targeted, streptococci of the Streptococcus bovis/equinus complex were the only bacteria that consistently proliferated in both caecal fluid and faeces immediately before the onset of histological signs of laminitis. Furthermore, lactobacilli, Enterobacteriaceae, Allisonella histaminiformans, enterococci, Bacteroides fragilis, Mitsuokella jalaludinii and Clostridium difficile did not establish significant populations in the hindgut before the onset of equine laminitis.     

Changes in equine hindgut bacterial populations during oligofructose-induced laminitis

In the horse, carbohydrate overload is thought to play an integral role in the onset of laminitis by drastically altering the profile of bacterial populations in the hindgut. The objectives of this study were to develop and validate microbial ecology methods to monitor changes in bacterial populations throughout the course of experimentally induced laminitis and to identify the predominant oligofructose-utilizing organisms. Laminitis was induced in five horses by administration of oligofructose. Faecal specimens were collected at 8 h intervals from 72 h before to 72 h after the administration of oligofructose. Hindgut microbiota able to utilize oligofructose were enumerated throughout the course of the experiment using habitat-simulating medium. Isolates were collected and representatives identified by 16S rRNA gene sequencing. The majority of these isolates collected belonged to the genus Streptococcus, 91% of which were identified as being most closely related to Streptococcus infantarius ssp. coli. Furthermore, S. infantarius ssp. coli was the predominant oligofructose-utilizing organism isolated before the onset of lameness. Fluorescence in situ hybridization probes developed to specifically target the isolated Streptococcus spp. demonstrated marked population increases between 8 and 16 h post oligofructose administration. This was followed by a rapid population decline which corresponded with a sharp decline in faecal pH and subsequently lameness at 24-32 h post oligofructose administration. This research suggests that streptococci within the Streptococcus bovis/equinus complex may be involved in the series of events which precede the onset of laminitis in the horse.     

Zymographic analysis of equine laminitis

To investigate the role of matrix metalloproteinase (MMP) activity in the pathophysiology of equine laminitis, the techniques of in situ zymography and quantitative SDS-PAGE zymography were used to analyse the lamellae and plasma and serum of horses with carbohydrate overload-induced laminitis. The gelatinase activity localised within the epidermal lamellae of laminitic hooves did not differ significantly from normal hooves. In laminitis sections there was an increase in vascular gelatinase activity, possibly associated with the perivascular cuffing of polymorphonucleocytes. Both plasma and serum samples from horses developing laminitis showed a rapid increase in the concentration of circulating latent MMP-9, while MMP-2 remained relatively constant. These results support the hypothesis that laminitis histopathology results from an inadequate regulation of gelatinase activity, resulting in selective degradation of basement membrane components, leading to laminitis due to failure of the basement membrane–epidermis attachment. Accepted: 21 September 1999     

Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria

Fluorescence in situ hybridization (FISH) with horseradish peroxidase (HRP)-labeled oligonucleotide probes and tyramide signal amplification, also known as catalyzed reporter deposition (CARD), is currently not generally applicable to heterotrophic bacteria in marine samples. Penetration of the HRP molecule into bacterial cells requires permeabilization procedures that cause high and most probably species-selective cell loss. Here we present an improved protocol for CARD-FISH of marine planktonic and benthic microbial assemblages. After concentration of samples onto membrane filters and subsequent embedding of filters in low-gelling-point agarose, no decrease in bacterial cell numbers was observed during 90 min of lysozyme incubation (10 mg ml(-1) at 37 degrees C). The detection rates of coastal North Sea bacterioplankton by CARD-FISH with a general bacterial probe (EUB338-HRP) were significantly higher (mean, 94% of total cell counts; range, 85 to 100%) than that with a monolabeled probe (EUB338-mono; mean, 48%; range, 19 to 66%). Virtually no unspecific staining was observed after CARD-FISH with an antisense EUB338-HRP. Members of the marine SAR86 clade were undetectable by FISH with a monolabeled probe; however, a substantial population was visualized by CARD-FISH (mean, 7%; range, 3 to 13%). Detection rates of EUB338-HRP in Wadden Sea sediments (mean, 81%; range, 53 to 100%) were almost twice as high as the detection rates of EUB338-mono (mean, 44%; range, 25 to 71%). The enhanced fluorescence intensities and signal-to-background ratios make CARD-FISH superior to FISH with directly labeled oligonucleotides for the staining of bacteria with low rRNA content in the marine environment.     

Fluorescence in situ hybridization for phytoplasma and endophytic bacteria localization in plant tissues

In the present study, we developed a rapid and efficient fluorescence in situ hybridization assay (FISH) in non-embedded tissues of the model plant Catharanthus roseus for co-localizing phytoplasmas and endophytic bacteria, opening new perspectives for studying the interaction between these microorganisms.     

Fluorescence in situ hybridization analysis of the prokaryotic community inhabiting crystallizer ponds

A fluorescence in situ hybridization (FISH) protocol suitable for the identification of prokaryotes inhabiting hypersaline environments was developed and applied to several crystallizer ponds with salinities above 36% from a multipond solar saltern in Alicante, Spain. Two morphotypes were abundant in these environments: rods and square or square-like prokaryotes that could be affiliated to Bacteria and Archaea, respectively, by FISH with domain-specific probes. FISH with a newly designed probe proved that the archaeal 16S rDNA sequence most frequently recovered from the crystallizers, SPhT, originated from the dominant square-like prokaryotes. These uncultured prokaryotes have the morphology of Walsby's square bacteria. Additionally, FISH with a probe targeted to the genus Haloarcula , members of which are frequently isolated from this environment, indicated that this genus accounts for less than 0.1% of the total prokaryotic community.     

Fluorescence in situ hybridization in studying the human genome

Physical chromosome mapping by fluorescence in situ hybridization (FISH) is among the major lines of research on the human genome (as well as genomes of numerous other organisms). To localize particular genes or anonymous DNA sequences on individual chromosomes or chromosome regions, FISH was developed in the late 1980s and early 1990s, when the International Human Genome Project and the Russian program Human Genome were launched. Now FISH continues to play a prominent part in studies of the human genome. The review considers the major steps of FISH development in Russia with special emphasis on the key roles of the Institute of Cytology and Genetics (Novosibirsk) and Engelhardt Institute of Molecular Biology (Moscow). Physical mapping of human chromosomes 3 and 13 by FISH is described in detail. The promotion of FISH in Russia contributed to the progress in the related fields such as comparative animal genomics (ZOO-FISH) and studies of plant chromosomes.     

Fluorescence in situ hybridization with multiple repeated DNA probes applied to the analysis of wheat-rye chromosome pairing

 Fluorescence in situ hybridization (FISH) with multiple probes has been applied to meiotic chromosome spreads derived from ph1b common wheat x rye hybrid plants. The probes used included pSc74 and pSc 119.2 from rye (the latter also hybridizes on wheat, mainly B genome chromosomes), the Ae. squarrosa pAs1 probe, which hybridizes almost exclusively on D genome chromosomes, and wheat rDNA probes pTa71 and pTa794. Simultaneous and sequential FISH with a two-by-two combination of these probes allowed unequivocal identification of all of the rye (R) and most of the wheat (W) chromosomes, either unpaired or involved in pairing. Thus not only could wheat-wheat and wheat-rye associations be easily discriminated, which was already feasible by the sole use of the rye-specific pSc74 probe, but the individual pairing partners could also be identified. Of the wheat-rye pairing observed, which averaged from about 7% to 11% of the total pairing detected in six hybrid plants of the same cross combination, most involved B genome chromosomes (about 70%), and to a much lesser degree, those of the D (almost 17%) and A (14%) genomes. Rye arms 1RL and 5RL showed the highest pairing frequency (over 30%), followed by 2RL (11%) and 4RL (about 8%), with much lower values for all the other arms. 2RS and 5RS were never observed to pair in the sample analysed. Chromosome arms 1RL, 1RS, 2RL, 3RS, 4RS and 6RS were observed to be exclusively bound to wheat chromosomes of the same homoeologous group. The opposite was true for 4RL (paired with 6BS and 7BS) and 6RL (paired with 7BL). 5RL, on the other hand, paired with 4WL arms or segments of them in more than 80% of the cases and with 5WL in the remaining ones. Additional cases of pairing involving wheat chromosomes belonging to more than one homoeologous group occurred with 3RL, 7RS and 7RL. These results, while adding support to previous evidence about the existence of several translocations in the rye genome relative to that of wheat, show that FISH with multiple probes is an efficient method by which to study fundamental aspects of chromosome behaviour at meiosis, such as interspecific pairing. The type of knowledge attainable from this approach is expected to have a significant impact on both theoretical and applied research concerning wheat and related Triticeae. Received: 21 February 1996 / Accepted: 12 July 1996     

Fluorescence in situ hybridization and Y ring chromosome

Summary Investigations by fluorescence in situ hybridization and a Y-specific probe (Y190) of a male patient with a Y ring chromosome, 46,X,r(Y) showed four bright fluorescent spots within the ring. Thus, using this technique, it is possible to suggest that the ring originates from the duplication of the short arms of the Y chromosome.     

荧光原位杂交技术对土壤石油降解菌的检测

从辽河油田样品中筛选出一株高效石油降解菌,经鉴定为地衣芽孢杆菌。针对其16S rRNA设计寡核苷酸探针。荧光原位杂交(FISH)技术利用寡核苷酸探针检测特定细胞内的互补核苷酸序列。通过对纯菌和泥浆中地衣芽孢杆菌的FISH进行优化,得到泥浆中地衣芽孢杆菌的荧光原位杂交实验条件：样品固定时间17 h,杂交温度46 ℃,杂交时间3 h,杂交液中去离子甲酰胺浓度35%,冲洗缓冲液中与去离子甲酰胺对应的NaCl的浓度88 mmol·L^-1。运用上述FISH技术监测生物泥浆反应器中地衣芽孢杆菌量的变化,并与泥浆中含油率的变化进行比较,二者的变化情况符合微生物降解石油的趋势,为监测含油污泥中微生物的变化提供了一种可行的技术。     

"Endomicrobia" and other bacteria associated with the hindgut of Dermolepida albohirtum larvae

Symbiotic bacteria residing in the hindgut chambers of scarab beetle larvae may be useful in paratransgenic approaches to reduce larval root-feeding activities on agricultural crops. We compared the bacterial community profiles associated with the hindgut walls of individual Dermolepida albohirtum third-instar larvae over 2 years and those associated with their plant root food source among different geographic regions. Denaturing gradient gel electrophoresis analysis was used with universal and Actinobacteria-specific 16S rRNA primers to reveal a number of taxa that were found consistently in all D. albohirtum larvae but not in samples from their food source, sugarcane roots. These taxa included representatives from the "Endomicrobia," Firmicutes, Proteobacteria, and Actinobacteria and were related to previously described bacteria from the intestines of other scarab larvae and termites. These universally distributed taxa have the potential to form vertically transmitted symbiotic associations with these insects.     

Fluorescence in situ hybridization with Y chromosome-specific probe in decondensed bovine spermatozoa

This study was carried out to demonstrate bovine Y chromosome-bearing spermatozoa by rapid fluorescence in situ hybridization (FISH), using a digoxigenin (Dig)-labeled DNA probe specific to bovine Y chromosome. Before the FISH procedure, sperm heads were treated for decondensation with dithiothreitol (DTT) and glutathione (GSH) with or without heparin supplementation. Concentrations of either above 2 mM DTT or above 100 mM GSH induced swelling of the sperm head, which resulted in sufficient detection of the Y chromosome signal in sperm nuclei by rapid FISH (49.8 to 53.4%). When FISH was used with 2 mM DTT or 100 mM GSH on specimens from 7 sires, the rate of detection of the Y chromosome signal varied among sires (5.4 to 49.6%), especially that of the GSH treatment. Supplementation of GSH with heparin (100 U/mL), however, could induce reliable, repeatable detection of the Y chromosome signal in sperm nuclei of all the 7 sires (48.4 to 50.3%). These results show that in bovine spermatozoa decondensed with GSH and heparin, rapid FISH can detect Y chromosome-bearing spermatozoa.     

Fluorescence in situ hybridization on vibratome sections of plant tissues

This protocol describes the application of fluorescence in situ hybridization (FISH) to three-dimensionally (3D) preserved tissue sections derived from intact plant structures such as roots or florets. The method is based on the combination of vibratome sectioning with confocal microscopy. The protocol provides an excellent tool to investigate chromosome organization in plant nuclei in all cell types and has been used on tissues of both monocot and dicot plant species. The visualization of 3D well-preserved tissues means that cell types can be confidently identified. For example, meiocytes can be clearly identified at all stages of meiosis and can be imaged in the context of their surrounding maternal tissue. FISH can be used to localize centromeres, telomeres, repetitive regions as well as unique regions, and total genomic DNAs can be used as probes to visualize chromosomes or chromosome segments. The method can be adapted to RNA FISH and can be combined with immunofluorescence labeling. Once the desired plant material is sectioned, which depends on the number of samples, the protocol that we present here can be carried out within 3 d.     

Fluorescence in situ hybridization on paraffin-embedded abortion material as a means of retrospective chromosome analysis

A fluorescence in situ hybridization (FISH) procedure was used to detect chromosome abnormalities in archival abortion material. Nuclei were isolated from 50-m-thick tissue blocks from 18 selected and karyotyped abortions. Five probes for repetitive centromeric sequences of chromosomes 1, 16, 18, X and Y were used. For each chromosome, at least 200 nuclei were scored blindly, i.e. without knowledge of the karyotype. The FISH results obtained were compatible with the cytogenetic data in 14 cases. There were four discrepancies. Two of these were observed for cases karyotyped as trisomy 16. Furthermore, FISH results showed trisomy 18 in two cases having normal chromosomes 18 and 18q+, respectively. The latter case was not discrepant if the structural rearrangement involved chromosome 18 material. The remaining discrepancies could be explained by chromosomal mosaicism. Admixture of normal maternal cells was also noted. It is concluded that FISH can be used to study retrospectively the presence of chromosome abnormalities in abortion material. However, the quality obtained after the use of fresh material is superior.     

Fluorescence in situ hybridization of single copy transgenes in rice chromosomes

Summary Fluorescence in situ hybridization (FISH) is a powerful tool for visualizing the chromosomal location of targeted sequences and has been applied in many areas, including karyotyping, breeding and characterization of genes introduced into the plant genome. A simple, routine and sensitive FISH procedure was developed for localizing single copy genes in rice (Oryza sativa L.) metaphase chromosomes. We used digoxygenin-labeled endogenous or T-DNA sequences as small as 5.6 kb to probe corresponding endogenous sequences or the T-DNA insert in denatured rice metaphase chromosomes prepared from root meristem tissue. The hybridized probe sequence was labeled with cy3-conjugated anti-mouse IgG and visualized using fluorescence microscopy. Single copy and multiple copy introduced T-DNA sequences, as well as endogenous sequences, were localized on the chromosomes. The FISH protocol was effectively used to sereen the chromosomal location of introduced T-DNA and number of integration loci in rice.     

Predisposition for venoconstriction in the equine laminar dermis: implications in equine laminitis.

Equine laminitis is a crippling condition associated with a variety of systemic diseases. Although it is apparent that the prodromal stages of laminitis involve microvascular dysfunction, little is known regarding the physiology of this vasculature. The aim of the present study was to determine the relative responses of equine laminar arteries and veins to the vasoconstrictor agonists phenylephrine (1 nM-10 microM), 5-HT (1 nM-10 microM), PGF2alpha (1 nM-100 microM), and endothelin-1 (1 pM-1 microM). We have determined that laminar veins were more sensitive, with respect to the concentration of agonist required to initiate a contractile response and to achieve EC(50), for all agonists tested. EC50 values, for veins and arteries, respectively, were 84+/-7 vs. 688+/-42 nM for phenylephrine, 35+/-6 vs. 224+/-13 nM for 5-HT, 496+/-43 nM vs. 3.0+/-0.6 microM for PGF2alpha, and 467+/-38 pM vs. 70.6+/-6.4 nM for endothelin-1. Moreover, when expressed as a percentage of the response to a depolarizing stimulus (80 mM potassium), the maximal contractile response of laminar veins exceeded that for the laminar arteries for each agonist. These results indicate that there may be a predisposition for venoconstriction within the vasculature of the equine digit. While this physiological predisposition for venoconstriction may be important in the regulation of blood flow during exercise, it also may help to explain why laminitis can result from a variety of pathological systemic conditions.     

Differential enumeration and in situ localization of microorganisms in the hindgut of the lower termite Mastotermes darwiniensis by hybridization with rRNA-targeted probes

We examined the abundance and spatial distribution of major phylogenetic groups of the domain Bacteria in hindguts of the Australian lower termite Mastotermes darwiniensis by using in situ hybridization with group-specific, fluorescently labeled, rRNA-targeted oligonucleotide probes. Between 32.0 ± 7.2% and 52.3 ± 8.2% of the DAPI-stained cells in different hindgut fractions were detected with probe EUB338, specific for members of the domain Bacteria. About 85% of the prokaryotic cells were associated with the flagellates of the thin-walled anterior region (P3a) and the thick wall of the posterior region (P3b/P4) of the hindgut, as shown by DAPI staining. At most, half of the EUB338-detected cells hybridized with one of the other probes that targeted a smaller assemblage within the bacterial domain. In most fractions, cells were found in varying numbers with probe ALF1b, which targeted members of the α-Proteobacteria, whereas substantial amounts of sulfate-reducing bacteria, gram-positive bacteria with a high DNA G+C content and members of the Cytophaga-Flavobacterium cluster of the Cytophaga-Flavobacterium-Bacteroides (CFB) phylum could be detected only in the wall fraction of P3b/P4. This clearly indicates that the hindgut microhabitats differ in the composition of their microbial community. In situ hybridization of cryosections through the hindgut showed only low numbers of bacteria attached to the P3a wall. In contrast, the wall of P3b was densely colonized by rod- and coccus-shaped bacteria, which could be assigned to the Cytophaga-Flavobacterium cluster of the CFB phylum and to the group of gram-positive bacteria with a high DNA G+C content, respectively. Oxygen concentration profiles determined with microelectrodes revealed steep oxygen gradients both in P3a and P3b. Oxygen was consumed within 100 μm below the gut surface, and anoxic conditions prevailed in the central portions of both gut regions, indicating that oxygen consumption in the hindgut does not depend on the presence of a biofilm on the hindgut wall. Received: 17 May 1999 / Accepted: 16 September 1999     

FISH检测宫颈脱落细胞与宫颈组织中hTERC基因制片方法的研究

目的:探讨FISH实验中用直接涂片法、盐水制片法、TCT制片法、低渗滴片法制片法和宫颈切片组织取材方法对于FISH成功率的影响.方法:收集2008年3月至2009年3月青岛大学医学院附属医院妇科162例宫颈脱落细胞标本及2008年5月至2009年3月手术切除或活检的宫颈组织63例,用荧光原位杂交(FISH)方法检测hTERC基因.结果:直接涂片法、盐水制片法、生理盐水法、TCT制片法和石蜡包埋组织切片法hTERC基因杂交成功率分别为58.3%,65%,55%,87.1%,85.7%,TCT制片高于其它四组;低渗滴片法背景干净度、细胞形态、裸核数量满意度最高;TCT制片法细胞数量满意率最高;石蜡包埋组织切片法荧光信号满意率最高.结论:在检测hTERC基因的宫颈癌筛查中,TCT制片法明显优于其他方法,而在指导宫颈病变及宫颈癌的治疗中,石蜡包埋组织切片法的染色体破坏最小,实际意义更大.