Development of in-situ hybridization for the detection of Mycoplasma haemosuis (Eperythrozoon suis) in formalin-fixed, paraffin wax-embedded tissues from experimentally infected splenectomized pigs

Mycoplasma haemosuis DNA was detected in experimentally infected splenectomized pigs by in-situ hybridization (ISH) with a nonradioactive digoxigenin-labelled DNA probe. An 839 base pair DNA probe targeting a 16S rRNA gene was generated by the polymerase chain reaction. Eight 6-week-old pigs were inoculated intraperitoneally with 6 ml of M. haemosuis-infected pig blood and eight negative control pigs were inoculated intraperitoneally with 6 ml of M. haemosuis-free blood. Two pigs from each group were killed for examination at 3, 7, 15 and 30 days post-inoculation (dpi). Red blood cells infected with M. haemosuis were first detected by light microscopy at 3 to 7 dpi. No M. haemosuis was observed in negative control pigs. Hybridization signals were evident in blood from the infected pigs at 3 dpi. The ISH method developed in this study was useful for the detection of M. haemosuis DNA in formalin-fixed, paraffin wax-embedded tissues and may be valuable for studying the pathogenesis of M. haemosuis infection.     

Outer membrane proteins and DNA profiles in strains of Haemophilus parasuis recovered from systemic and respiratory sites

Polyserositis caused by Haemophilus parasuis is an important disease that affects mostly weaned pigs. Recent studies have shown that virulence can differ among strains recovered from distinct body sites and also that it may be related to the presence of certain outer membrane proteins (OMPs). The objective of this study was to compare the OMP and DNA profiles of H. parasuis strains isolated from systemic and respiratory sites from diseased and healthy pigs. Strains evaluated in this study were processed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and repetitive-PCR techniques. Two experiments were conducted in order to better define the relationship among genotype, phenotype, and site of isolation. Experiment 1 included 53 H. parasuis isolates recovered from healthy and diseased pigs from unrelated herds. Experiment 2 included 31 isolates of H. parasuis obtained from diseased pigs involved in an outbreak in a large, multifarm system. Results showed that strains recovered from systemic sites had more homogeneous OMP and DNA profiles than those isolated from respiratory sites. Evaluation of isolates involved in the multifarm outbreak showed that only two H. parasuis strains were causing disease. These strains had homogeneous OMP and DNA profiles. However, it was noted that these two parameters were unrelated, since strains classified in the same genotype group expressed different OMP profiles. The homogeneity of OMP and DNA profiles of strains isolated from systemic sites strongly suggests the existence of clonal relationships between virulent strains and also suggests that expression of certain OMP profiles may be related to virulence.     

一种能精确检测人间期细胞核中21号染色体拷贝数的DNA探针

目的制备能精确检测人类间期细胞核中２１号染色体拷贝数的ＦＩＳＨ探针。方法利用万能引物ＰＣＲ法,从定位于人２１ｑ１１的ＹＡＣ克隆８８１Ｄ２分离制备ＤＮＡ探针,并用于与８例正常人和５例２１三体患者的外周血淋巴细胞中期相和间期核,及经细胞松弛素Ｂ（ｃｙｔｏｃｈａｌａｓｉｎＢ）诱导的双核细胞进行ＦＩＳＨ分析。结果该探针具有以下特点：（１）长度集中于３５０～７５０ｂｐ;（２）其靶序列位于２１号染色体长臂上且紧靠着丝粒;（３）特异性强;（４）杂交信号明亮,容易辨认;（５）对中期相及间期细胞核中２１号染色体的检出率分别高达９９．６０％和９８．４０％。结论制备的ＤＮＡ探针能精确检测人类间期细胞核中２１号染色体的拷贝数,且适用于细胞分裂时２１号染色体分离情况的研究     

Distribution of porcine parvovirus in porcine circovirus 2-infected pigs with postweaning multisystemic wasting syndrome as shown by in-situ hybridization

In-situ hybridization with a nonradioactive digoxigenin-labelled probe was used to study the distribution of porcine parvovirus (PPV) in formalin-fixed paraffin wax-embedded tissues from 10 porcine circovirus 2 (PCV2)-infected weaned pigs with naturally occurring postweaning multisystemic wasting syndrome (PMWS). A 226 base pair DNA fragment from a VP2 structural gene was generated by polymerase chain reaction (PCR) and used as a probe. Lymph node, spleen, thymus and tonsil were positive by PCR, demonstrating the presence of PPV DNA in the tissue samples from four of 10 pigs tested. PPV nucleic acid was also detected consistently in lymph node, spleen, thymus and tonsil by in-situ hybridization. Detection of PPV DNA from PCV2-infected pigs with PMWS suggests that PPV also plays a role in the pathogenesis of PMWS.     

Detection by in-situ hybridization of Pasteurella multocida toxin (toxA) gene in the lungs of naturally infected pigs

In-situ hybridization with a non-radioactive digoxigenin-labelled probe was used to detect the Pasteurella multocida toxin (PMT) gene in tissue sections of pneumonic lung from pigs naturally infected with toxigenic P. multocida. The morphology of host cells was preserved despite the relatively high temperature used in the incubation procedure. Pulmonary abscessation was observed in 13 pigs naturally infected with toxigenic P. multocida type A (three pigs) or D (10 pigs). In these 13 pigs a strong hybridization signal for PMT DNA was detected, mainly in degenerate leucocytes in abscesses. Occasionally, PMT DNA was detected in degenerate neutrophils and macrophages in alveolar spaces. Detection of hybridization signals for PMT DNA would seem to be a potential indicator of the production of PMT. The study suggested that PMT plays an important role in pulmonary abscessation caused by P. multocida.     

用双色荧光原位杂交检测人精子染色体非整倍体率

目的检测人精子染色体非整倍体率。方法采用双色荧光原位杂交（ＦＩＳＨ）方法,取少量精标本经洗后制片,用二硫苏糖醇（ＤＴＴ）和二碘水杨酸锂（ＬＩＳ）处理,使精子头部染色质去凝集。然后,与生物素标记的α卫星Ｘ染色体特异ＤＮＡ探针（ＤＸＺ１）和地高辛标记的α卫星Ｙ染色体特异ＤＮＡ探针（ＤＹＺ３）进行原位杂交。用ＣＹ３－链亲和素、山羊抗链亲和素检测Ｘ染色体探针杂交信号;用鼠抗地高辛抗体、与荧光素结合的兔抗鼠抗体检测Ｙ染色体探针杂交信号。结果在Ｎｉｋｏｎ荧光显微镜下可以清楚看到精子头部的杂交信号,头部有１个红色荧光杂交信号的精子为Ｘ染色体精子（Ｘ精子）,有１个绿色荧光杂交信号的精子为Ｙ染色体精子（Ｙ精子）。精子头部有２个荧光杂交信号的精子为染色体数目异常精子。若用１条常染色体探针和１条性染色体探针进行ＦＩＳＨ,可以区别头部有２个相同颜色荧光杂交信号的精子属非整倍体精子或二倍体精子。结论双色荧光原位杂交（ＦＩＳＨ）方法,可以用于测定接触致突变剂和非整倍体诱导剂后,人精子染色体非整倍体率的变化。     

Virus-specific DNA sequences in mouse and rat cells transformed by the Harvey and Moloney murine sarcoma viruses detected by in situ hybridization.

The [³H]DNA product of the murine sarcoma-leukemia virus (MSV(MLV)) and viral 60–70 S [³H]RNA was annealed with cytological preparations of mouse and rat cells transformed by the Harvey and Moloney strains of MSV. With viral [³H]DNA as cytological probe, these in situ hybridization measurements detected from 25 to 30 autoradiographic grains per interphase nucleus of transformed cells and 1 to 3 grains per nucleus of uninfected rat and mouse cells. With viral 60–70 S [³H]RNA of lower specific radioactivity as cytological probe, 12 grains per transformed cell nucleus were detected. These findings indicate that transformation of cells with MSV(MLV) produces a several-fold increase in the content of some virus-specific DNA sequences. Virus-specific sequences in transformed mouse cells were localized in the chromocenters of interphase nuclei.     

In-situ hybridization for the detection and identification of Histomonas meleagridis in tissues

For use in an in-situ hybridization method, three probes (HM, TR and BL) were designed to hybridize, respectively, with (1) Histomonas meleagridis, (2) Tetratrichomonas gallinarum, and (3) a broad range of micro-organisms, including Blastocystis spp. Mono-eukaryotic cultures were used to test the specificity of the three oligonucleotides and to optimize the hybridization procedure before applying the probes to archived samples of various tissues and to a culture of Trichomonas gallinae. Specific detection of H. meleagridis was possible with the HM probe, but the other two probes were less specific. The TR probe detected members of the Trichomonadidae (Tetr. gallinarum and Tr. gallinae). Positive signals from a great variety of microorganisms, including fungi and protozoa from different animal species, were obtained with the BL probe. However, neither H. meleagridis nor the two members of the Trichomonadidae mentioned above were detected with this probe, allowing the exclusion of these parasites. Use of the three probes makes possible the accurate detection of H. meleagridis and its distinction from other micro-organisms in tissue samples.     

A highly specific and sensitive DNA probe derived from chromosomal DNA of Helicobacter pylori is useful for typing H. pylori isolates.

HindIII-digested DNA fragments derived from an EcoRI-digested 6.5-kb fragment of chromosomal DNA prepared from Helicobacter pylori ATCC 43629 (type strain) were cloned into the pUC19 vector. A 0.86-kb insert was identified as a potential chromosomal DNA probe. The specificity of the probe was evaluated by testing 166 non-H. pylori bacterial strains representing 38 genera and 91 species which included aerobic, anaerobic, and microaerophilic flora of the upper and lower gastrointestinal tracts. None of the 166 non-H. pylori strains hybridized with this probe (100% specificity), and the sensitivity of this probe was also 100% when H. pylori isolates from 72 patients with gastritis and with the homologous ATCC type strain were tested by dot blot hybridization. The capability of this probe for differentiating between strains of H. pylori was evaluated by Southern blot hybridization of HaeIII-digested chromosomal DNA from 68 clinical isolates and the homologous ATCC type strain of H. pylori. Fifty-one unique hybridization patterns were seen among the 69 strains tested, demonstrating considerable genotypic variation among H. pylori clinical isolates. We propose that this probe would be of significant value for conducting epidemiologic studies.     

A hybrido-immunocytochemical assay for the in situ detection of cytomegalovirus DNA using digoxigenin-labeled probes

A non-radioactive hybrido-immunocytochemical assay for the detection of cytomegalovirus (CMV) DNA in infected cells was developed. Two different DNA fragments belonging to the repeated sequences of CMV genome were used to construct the hybridization probe. The probe was constructed by incorporating deoxyuridine triphosphate labeled with digoxigenin. The in situ hybridized CMV DNA probe was immunocytochemically visualized by anti-digoxigenin. Fab fragments labeled with alkaline phosphatase. This procedure permitted the DNA detection, in the nuclei of infected cells fixed at 48 h after infection, of the Towne CMV reference strain and 21 different laboratory-isolated CMV strains. Our assay demonstrated a high specificity, sensitivity and reproducibility.     

Isolation of a Salmonella-specific DNA hybridization probe

A Salmonella-specific DNA fragment of the Salmonella typhimurium LT2 chromosome has been isolated. The fragment (2.3 kilobases (kb)) was used as a probe in a colony hybridization assay, where 185 strains of 93 different Salmonella serovars were correctly identified as belonging to Salmonella. The specificity of the probe was evaluated in colony hybridization assays on pure cultures of non-Salmonella bacteria and on specimens with an indigenous flora. Sixty-three strains of 34 non-Salmonella Gram-negative species did not hybridize to the fragment. By DNA hybridization to faecal samples from calves, pigs and chickens, and samples of animal feed, three samples out of 10 positive by traditional culture methods gave negative results by hybridization, 45 samples were negative in both methods, while one sample was positive only in the hybridization assay. From this sample, Salmonella livingstone was isolated by a replica plate hybridization technique. The probe therefore proved 100% specific for the genus Salmonella. The 2.3 kb fragment may form the basis of hybridization assays for specific detection of Salmonella in food, environmental and clinical specimens.     

Cytologic detection of herpes simplex virus DNA in nipple discharge by in situ hybridization: Report of two cases

Cytologic changes in the smears from nipple discharge of two cases with herpes simplex virus (HSV) infections are described. The cytology revealed the ground-glass appearance of the nuclei with multinucleated syncytial cells. Subsequently, in situ hybridization (ISH) using a biotinytated DNA probe was applied to identify the HSV DNA in the Papanicolaou-destained specimens. Positive hybridization was found with intense staining for the HSV DNA in the nuclei of cells having a ground-glass appearance. Cytologic observation together with ISH procedure may be rapid and valuable tool for the detection and final demonstration of HSV infections. Similar investigations may be carried out in cellular samples from other body sites.     

Specific numerical chromosomal aberrations induced by adriamycin

Fluorescence in situ hybridization with 7, 17, X, and Y chromosome-specific DNA probe was used to investigate the ability of Adriamycin (AM) to induce aneuploidy in interphase human lymphocytes. The reliability of the probes was tested by hybridization to metaphases and interphase nuclei of untreated normal lymphocytes. Two signals were scored in over 87% of the analyzed nuclei with chromosome 7 and 17 probes, whereas one signal was recorded in over 86% of the nuclei with chromosomes X and Y. The same conditions and probe concentrations were used for hybridizing the four probes to interphase nuclei of AM-treated and untreated lymphocytes, cultured from healthy individuals and cancer patients. AM was found to induce significant increases of trisomy 7 and 17 in lymphocytes cultured from healthy individuals and cancer patients, where the interphase nuclei showed three spots in over 70% and 72% of the cells, respectively. Only 6% of interphase nuclei of untreated cells cultured from healthy individuals and cancer patients showed three spots. No significant increase in X or Y aneuploidy was induced by exposure to AM.     

Identification of the immunogenic outer membrane protein A antigen of Haemophilus parasuis by a proteomics approach and passive immunization with monoclonal antibodies in mice

Monoclonal antibodies (MAbs) against Haemophilus parasuis were generated by fusing spleen cells from BALB/c mice immunized with whole bacterial cells with SP2/0 murine myeloma cells. Desirable hybridomas were screened by enzyme-linked immunosorbent assay (ELISA). Neutralizing MAb 1D8 was selected in protection assays. ELISA results demonstrated that 1D8 can react with all 15 serotypes of H. parasuis and field isolate H. parasuis HLJ-018. Passive immunization studies showed that mice inoculated intraperitoneally with 1D8 had significantly reduced prevalence of H. parasuis colonization in the blood, lung, spleen, and liver and had prolonged survival time compared to that of the control group. Furthermore, the passive transfer experiment indicated that MAb 1D8 can protect mice from both homologous and heterologous challenges with H. parasuis. Using two-dimensional gel electrophoresis (2-DE), the immunoreactive protein target for MAb 1D8 was identified. The data presented confirm the protective role of MAb 1D8 and identify OmpA as the target of the protective monoclonal antibody. The data suggest that OmpA is a promising candidate for a subunit vaccine against H. parasuis.     

DNA probe technology for rapid detection of Haemophilus influenzae in clinical specimens.

In a previous study, we reported that a 5-kilobase Haemophilus influenzae DNA fragment involved in penicillin-binding protein expression could be used as a probe for specific detection of H. influenzae strains (F. Malouin and L. E. Bryan, Mol. Cell. Probes 1:221-232, 1987). Here, we report the ability of this probe to detect H. influenzae in clinical specimens. In a bacterial dot experiment, there was strong hybridization of the 32P-labeled probe to nonencapsulated and serotype a through f H. influenzae strains. The detection of H. influenzae in body fluids was then evaluated by using pooled human serum, urine, cerebrospinal fluid, and sputum as dilution media for H. influenzae, Haemophilus aegyptius, Haemophilus parainfluenzae, and Escherichia coli cells. At 65 degrees C, the probe hybridized to H. influenzae and H. aegyptius (greater than or equal to 10(5) cells) in all fluids. There was no hybridization with the E. coli negative control, and H. parainfluenzae hybridized when greater than or equal to 10(7) cells were used. Experiments performed at 73 and 80 degrees C permitted elimination of H. parainfluenzae hybridization. The detection of H. influenzae in 232 sputa from patients with respiratory tract infections was very specific (96 to 97%) and sensitive (74 to 100%) when the total time of the procedure was sufficient (6 to 24 h) and when the experiments were performed at 80 degrees C. In addition, the probe detected three of three and four of four H. influenzae-infected cerebrospinal fluids and blood cultures, respectively, and did not react with pneumococcus- or streptococcus-infected cerebrospinal fluids. Finally, by using a small-scale procedure, the probe rapidly detected H. influenzae in cerebrospinal fluid and sputum specimens (4 and 8 h, respectively). These results imply prompt diagnosis of H. influenzae infections caused by nonencapsulated and serotype a through f strains.     

人21号全染色体涂染探针的制备及其唐氏综合征诊断的应用研究

目的人21号染色体DNA涂染探针的制备及其应用于唐氏综合征诊断的研究。方法将显微分离的人21号染色体DNA进行简并寡核苷酸引物PCR（Degenerate Oligonucleotide Primed—PCR,DOP—PCR）扩增并标记制备成杂交探针后,与15例唐氏征疑似患者的外周血细胞核染色体进行荧光原位杂交（Fluorescence in situ hybridization,FISH）分析;同时以常规核型分析进行确诊并评估FISH结果。结果FISH分析诊断结果与常规核型分析一致,其中8例为非唐氏征患者,7例为唐氏征患者,准确率为100％,且非唐氏征患者与唐氏征患者细胞核中21号染色体的检出率分别高达99．12％和99．08％。结论制备的人21号全染色体DNA涂染探针能精确检测人类中期和间期细胞核中21号染色体的数目,该探针可用于唐氏综合征的诊断。     

Cytomegalovirus detection by nonisotopic in situ DNA hybridization and viral antigen immunostaining using a two-color technique

Rapid methods of specific viral diagnosis in formalin fixed, paraffin embedded tissues include identification of viral incusions in routinely stained histologic sections, immunologic staining of viral antigens, and in situ nucleic acid hybridization. To correlate in situ hybridization with immunologic detection methods, sequential two-color staining was used on tissues from 12 patients, each containing characteristic cytomegalovirus (CMV) inclusions, using a biotinylated CMV DNA probe in an avidin-alkaline phosphatase-linked reaction followed by avidin-biotin complex immunoperoxidase staining of CMV antigen. CMV genetic material was seen in all 17 tissues. CMV antigen was detected in 11 of 17 tissues (65%). The DNA hybridization technique provided more intense staining, detected greater numbers of inclusions, and had less background staining than the immunoperoxidase technique. The alkaline phosphatase reaction product was stable through subsequent immunostaining steps, and immunologic reactivity of CMV antigen was not significantly reduced by prior hybridization steps. CMV DNA probe was localized predominantly within cell nuclei, while CMV antigen immunostaining was predominantly cytoplasmic. It was concluded that sequential in situ hybridization and immunocytochemistry can be performed on standard histologic sections. Furthermore, it is likely that the majority of CMV nucleic acid detected by this tissue hybridization technique is unencapsidated, intranuclear viral DNA and not DNA contained within complete CMV nucleocapsids.     

In situ hybridization for cytomegalovirus DNA in AIDS patients.

Infection by cytomegalovirus (CMV) is a frequent cause of morbidity and mortality in patients with acquired immune deficiency syndrome (AIDS). The authors studied the distribution of CMV in 4 patients with AIDS using a commercially available, biotin-labeled CMV DNA probe for in situ hybridization and immunohistochemical staining for the detection of CMV antigen in formalin-fixed paraffin-embedded tissues. The sensitivity and specificity of the hybridization procedure was demonstrated by appropriate controls. The immunohistochemical test for the detection of CMV antigen in routine histologic sections was less sensitive than the in situ hybridization method. CMV DNA was detected not only in cytomegalic inclusion cells, but also in nuclei and cytoplasm of histologically normal-appearing cells such as endothelial cells, pneumocytes, hepatocytes, biliary epithelium, gastrointestinal epithelium, Langerhans islet cells, acinar and duct epithelium of pancreas, adrenal cortical and medullary cells, and prostate epithelium. In addition, CMV DNA, but not CMV antigen, was found in polymorphonuclear leukocytes. These cells may serve as intermediate host or reservoir of CMV and may transmit posttransfusion CMV infection. In situ hybridization on routine histologic sections with a biotinylated CMV DNA probe is a rapid, sensitive, and specific method for diagnostic and experimental pathology.     

V factor-dependent members of the family Pasteurellaceae in the porcine upper respiratory tract.

A study was performed to obtain a better understanding of the diversity and ecology of members of the family Pasteurellaceae in the porcine respiratory tract. A collection of 132 V factor-dependent strains of Pasteurellaceae selected from porcine field isolates mainly from the respiratory tract were subjected to detailed characterization. In addition to the three hitherto recognized species Actinobacillus pleuropneumoniae, Haemophilus parasuis, and Haemophilus taxon "minor group," three distinct taxa were observed. Some of these taxa, which are provisionally designated taxa D, E, and F, would by traditional criteria be mistaken for H. parasuis but differed by several biochemical characteristics. To study the ecology of the V factor-dependent species, swabs from the nasal and oral cavities of 29 pigs were cultivated on selective and nonselective media. By studying approximately 30 isolates from each sample, the distribution and relative proportion of the individual taxa were determined. A. pleuropneumoniae was detected in samples from the tonsil areas of only two acutely ill animals. H. parasuis was isolated from the nasal cavities of four out of nine healthy pigs but from the oral cavities of only two animals. In contrast, taxon "minor group" and taxa D, E, and F were present in the oral cavities of the majority of pigs but were not detected in samples from their nasal cavities. The results indicate that all the observed V factor-dependent species of Pasteurellaceae except A. pleuropneumoniae, are members of the resident microflora of various mucosal surfaces of the porcine upper respiratory tract.     

分子杂交法研究肝炎病人血清和肝组织中输血传播病毒

目的 对各型肝炎病人血清和肝组织中输血传播病毒（TTV）核酸检测分析,探讨病毒的致病性。方法 以地高辛为标记物制备TTV DNA探针,斑点杂交法、原位杂交法分别检测血清中及肝组织中TTV DNA。结果 检测103例血清,TTV总阳性率为25．24％（26／103）;甲－戊型肝炎组检出率21．81％（12／55）、非甲－非庚型患者检出率47．37％（9／19）,显著高于正常对照组15％（3／20）。临床可见TTV的单独感染和重叠感染;出现急性、慢性甚至重度肝损伤。12 肝组织可见TTV阳性,阳性颗粒主要见于肝细胞核内。结论 从血清和肝组织证实了TTV的存在,提示这种病毒可能是导致肝脏炎症的一种新病原。