Non-radioactive Digoxigenin-labelled Probe Research Articles

Analysis of Human Mitochondrial DNA Content by Southern Blotting and Nonradioactive Probe Hybridization.

A single cell can contain several thousand copies of the mitochondrial DNA genome or mtDNA. Tools for assessing mtDNA content are necessary for clinical and toxicological research, as mtDNA depletion is linked to genetic disease and drug toxicity. For instance, mtDNA depletion syndromes are typically fatal childhood disorders that are characterized by severe declines in mtDNA content in affected tissues. Mitochondrial toxicity and mtDNA depletion have also been reported in human immunodeficiency virus-infected patients treated with certain nucleoside reverse transcriptase inhibitors. Further, cell culture studies have demonstrated that exposure to oxidative stress stimulates mtDNA degradation. Here we outline a Southern blot and nonradioactive digoxigenin-labeled probe hybridization method to estimate mtDNA content in human genomic DNA samples. © 2019 by John Wiley & Sons, Inc.

Identification of Interleukin-1, Tumor Necrosis Factor-.ALPHA., and Interleukin-6 Expression in Lungs from Pigs Naturally Infected with Mycoplasma hyopneumoniae by In situ Hybridization

The detection and distribution of interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha) and IL-6 were studied, by in situ hybridization with a non-radioactive digoxigenin-labeled probe, in formalin-fixed, paraffin-embedded lung tissue from 10 pigs naturally infected with Mycoplasma hyopneumoniae. The morphology of host cells was preserved despite the relatively high temperature required during the incubation procedure. Examination of three serial sections from each of the 10 lung samples showed that the three cytokines closely resembled each other in respect of cellular distribution. Three inflammatory cytokines are expressed in response to M. hyopneumoniae infection, with IL-6 localized primarily to peribronchiolar lymphoid hyperplastic tissues, and both IL-1 and TNF-alpha expressed in alveolar macrophages. Although statistically non-significant, IL-1 (r=0.5744, p=0.0883) showed potentially important correlation with histopatholgical lesions. No other potentially clinically important correlations (r>0.30) were observed between any of the other cytokines (TNF-alpha; r=0.2045, p=0.5603 and IL-6; r=-0.06607, p=0.8651) and histopathological lesion score. The results suggest that inflammatory cytokines are associated with the development of pneumonia in M. hyopneumoniae infection and may contribute to disease severity.

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.

Expression of inflammatory cytokines (TNF-alpha, IL-1, IL-6 and IL-8) in colon of pigs naturally infected with Salmonella typhimurium and S. choleraesuis.

The expression of mRNA encoding tumour necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), IL-6 and IL-8 was studied, by in situ hybridization with a non-radioactive digoxigenin-labelled probe, in formalin-fixed, paraffin wax-embedded colonic tissue from pigs naturally infected with Salmonella typhimurium and S. choleraesuis. By in situ hybridization, a distinct positive signal for TNF-alpha, IL-1, IL-6 and IL-8 was detected in colon from all 12 infected pigs. Hybridization signals for all four inflammatory cytokines were detected primarily inflammatory cells infiltrating the lamina propria and submucosa. In comparison, expression of all four inflammatory cytokines was minimal in non-lesional colon of infected pigs and in normal colon from control pigs. The results suggest that these cytokines play an important role in the pathophysiological processes in porcine salmonellosis.

Expression of Interferon-gamma and Tumour Necrosis Factor-alpha in Pigs Experimentally Infected with Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)

The expression of mRNA encoding interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α) was studied, by reverse transcription–polymerase chain reaction and by in-situ hybridization with a non-radioactive digoxigenin-labelled probe, in formalin-fixed, paraffin wax-embedded lung tissue from pigs experimentally infected with a Korean isolate (North American genotype) of PRRSV. Both IFN-γ and TNF-α mRNA were detected in the lung as early as 1 day post-inoculation (dpi) and up to 10 dpi. Hybridization signals for IFN-γ and TNF-α were usually associated with inflammation, in particular with macrophages within alveolar septa. Expression of these cytokines was minimal in non-lesional lung of PRRSV-infected pigs and in normal lung from control pigs. The results suggest that IFN-γ and TNF-α play a role in pulmonary defence against PRRSV.

Expression of the apxIV Gene in Pigs Naturally Infected with Actinobacillus pleuropneumoniae

The apxIV gene was detected, by in-situ hybridization with a non-radioactive digoxigenin-labelled probe, in formalin-fixed, paraffin wax-embedded samples of lung tissue from 10 pigs naturally infected withActinobacillus pleuropneumoniae . A 442 base pair DNA probe of the apxIV gene from A. pleuropneumoniae serotype 2 was generated by the polymerase chain reaction. All 10 pigs infected with A. pleuropneumoniae serotypes 2, 5, 6, or an untypable strain showed a distinct, positive signal in the degenerate alveolar leucocytes in alveolar spaces, and in the dense zone of degenerated cells in granulation tissue surrounding the necrotic areas. Thus, the study demonstrated the presence of the apxIV gene in pleuropneumonic lesions caused by A. pleuropneumoniae.

Detection and localization of ApxI, -II and -III genes of Actinobacillus pleuropneumoniae in natural porcine pleuropneumonia in natural porcine pleuropneumonia by in situ hybridization.

In situ hybridization techniques that employed a nonradioactive digoxigenin-labeled probe were used to detect and localize ApxI, II and III genes in tissue sections of pneumonic lung naturally infected with Actinobacillus pleuropneumoniae. In pigs infected with either serotype 2 or 6, a hybridization signal for apxIICA, apxIIICA, apxIBD, and apxIIIBD was detected, and in pigs infected with serotype 5, a hybridization signal for apxICA, apxIICA, and apxIBD was detected in the pneumonic lesions. A hybridization signal for apxIICA and apxIBD was detected in pigs infected with serotype 7. A strong hybridization signal for apx genes was seen in streaming degenerate alveolar leukocytes bordering zones of coagulative necrosis. Simultaneous detection of hybridization signals for the apxCA and apxBD genes provided scientific evidence that the expression of the apx genes could be potential indicators of the production of corresponding Apx toxins. This study demonstrates the expression of ApxI, II, and III genes in pneumonic lesions caused by A. pleuropneumoniae.

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.

In situ hybridization for the detection and localization of porcine epidemic diarrhea virus in the intestinal tissues from naturally infected piglets.

Detection and localization of porcine epidemic diarrhea virus (PEDV) was studied by in situ hybridization with a nonradioactive digoxigenin-labeled probe in formalin-fixed, paraffin-embedded tissues from 10 naturally infected piglets. A 377-base pair cDNA probe for viral RNA encoding the membrane proteins of PEDV cell-culture-adapted strain V215/78 was generated by the reverse transcription polymerase chain reaction. In the retrospective study of pigs from herds with diarrhea, the 10 piglets naturally infected with PEDV had positive signals for PEDV by in situ hybridization. When intestinal tissues were hybridized with the PEDV probe, a strong signal was seen in the villus enterocytes of jejunum and ileum but not in the cecum and colon. Positive cells typically had dark brown reaction products in the cytoplasm. Scattered epithelial cells along the ileal Peyer's patches dome areas contained viral RNA. In one piglet, hybridization signal was also found in the duodenum. PEDV was not demonstrated in tissues outside of the intestinal tract. These findings indicate that jejunal and ileal villus enterocytes are the main target of PEDV replication during epizootic outbreaks of the disease.

In-situ Hybridization for the Detection of Inflammatory Cytokines (IL-1, TNF-α and IL-6) in Pigs Naturally Infected with Actinobacillus pleuropneumoniae

The1999 Harcourt Publishers Ltddetection and distribution of interleukin-1 (IL-1), tumour necrosis factor-α (TNF-α) and IL-6 were studied, by in-situ hybridization with a non-radioactive digoxigenin-labelled probe, in formalin-fixed paraffin wax- embedded lung tissue from 10 pigs naturally infected with Copyright Actinobacillus pleuropneumoniae. A strong hybridization signal for IL-1, TNF-α and IL-6 was detected in “streaming” degenerate alveolar leucocytes (the so-called “oat cells”) bordering zones of coagulative necrosis, and a less intense signal was seen in the dense zone of degenerate cells in granulation tissue surrounding the necrotic areas. IL-1 expression was also prominent in scattered endothelial cells bordering zones of coagulative necrosis. Simultaneous expression of all three cytokines was always associated with pleuropneumonic lung lesions. Expression of inflammatory cytokines was minimal in non-lesional lung tissue of the infected pigs and in normal lung from control pigs. The results suggest that these cytokines play a crucial role in mediating and regulating inflammation through cells of several types in A. pleuropneumoniae infection. 1999 W.B. Saunders and Company Ltd.

Detection and distribution of DNA of Actinobacillus pleuropneumoniae in the lungs of naturally infected pigs by in-situ hybridization

Detection and distribution of Actinobacillus pleuropneumoniae was studied in formalin-fixed paraffin wax-embedded lung tissues from 10 naturally infected pigs by in-situ hybridization with a non-radioactive digoxigenin-labelled probe. A 610 base pair cDNA probe from a genomic library of A. pleuropneumoniae was generated by the polymerase chain reaction. All 10 pigs infected with A. pleuropneumoniae serotypes 2, 5, 6, or an untypable strain showed a distinct, positive signal in the degenerate alveolar leucocytes bordering a zone of coagulative necrosis and in the dense zone of degenerated cells in granulation tissue surrounding the necrotic areas. Positive cells typically exhibited dark-brown to black labelled deposits without background staining. A. pleuropneumoniae nucleic acids were more readily detected in areas of coagulative necrosis than in areas of granulation tissue. In-situ hybridization demonstrated that A. pleuropneumoniae primarily infected neutrophils and alveolar macrophages. The technique used was rapid, specific and sensitive, and may prove useful for the diagnosis of A. pleuropneumoniae infection in routinely fixed and processed tissues, obviating the need for bacterial isolation.

Detection of nucleic acids of porcine reproductive and respiratory syndrome virus in the lungs of naturally infected piglets as determined by in-situ hybridization

SummaryReplication of porcine reproductive and respiratory syndrome virus (PRRSV) was studied in formalin-fixed paraffin wax-embedded lung tissues from seven naturally infected piglets by in-situ hybridization with a non-radioactive digoxigenin-labelled probe. A 433 base pair cDNA probe for the viral RNA encoding the nucleocapsid proteins of a Korean PRRSV isolate was generated by the polymerase chain reaction. All seven piglets infected with PRRSV showed a distinct, positive signal, scattered throughout the alveolar septa and spaces. Positive cells typically exhibited dark brown staining deposits in the cytoplasm without background staining. In-situ hybridization demonstrated that PRRSV replicated primarily in interstitial and alveolar macrophages, and occasionally in type 2 pneumocytes. The bronchial or bronchiolar epithelium did not exhibit a hybridization signal for PRRSV nucleic acids. The anterior and middle lobes of the lung were more reliable than the caudal or accessory lobes for the detection of PRRSV nucleic acids. The in-situ hybridization technique used was rapid, specific and sensitive, and may prove useful for the diagnosis of PRRSV infection in routinely fixed and processed tissues.

Detection of African horse sickness viruses by dot-blot hybridization using a digoxigenin-labelled probe

In order to develop a non-radioactive dot-blot hybridization assay, for the detection of African-horse sickness virus (AHSV), genome segment 7 from 9 serotypes was amplified by RT-PCR. The resulting PCR products were denatured, immobilized on nylon membranes and then hybridized to a non-radioactive digoxigenin-labelled probe. This probe (265 bp in length) was generated by nested-PCR using genome segment 7 of AHSV, serotype 4 as a template. The dot-blot was visualized by chemiluminescence. Positives were obtained from the PCR products amplified from all 9 AHSV serotypes, but not from any other equine virus or orbivirus isolates. The sensitivity and specificity of this probe, together with the simplicity and rapidity of this technique, suggest that a non-radioactive dot blot assay may be useful as a method for the routine and rapid diagnosis of viral infections.