Detection and Localization ofMycoplasma hyopneumoniaeDNA in Lungs from Naturally Infected Pigs by In Situ Hybridization Using a Digoxigenin-labeled Probe

Abstract

Mycoplasma hyopneumoniae DNA was detected in 20 naturally infected pigs by in situ hybridization using a nonradioactive digoxigenin-labeled DNA probe. A 520-base-pair DNA probe targeting a reiterative sequence of the M. hyopneumoniae genome was generated by the polymerase chain reaction. All 20 pigs infected with M. hyopneumoniae had distinct and positive hybridization signals without background staining. A strong hybridization signal was detected mainly in the luminal surface of bronchial and bronchiolar lining epithelial cells, whereas no hybridization signal was seen in the cytoplasm of bronchial and bronchiolar lining epithelial cells. When hybridization signal was detected in the luminal surface of bronchial and bronchiolar lining epithelial cells, a given bronchus or bronchiole had peribronchiolar lymphoid hyperplastic tissues. Hybridization signals were not seen in the peribronchiolar lymphoid hyperplastic tissues. A less intense signal was detected in the interstitial and alveolar macrophages randomly scattered in the thickened alveolar septa and spaces. Hybridization signal was rarely detected in the type I pneumocytes. The in situ hybridization technique developed in this study was useful for detection of M. hyopneumoniae nucleic acids in tissues taken from naturally infected piglets and may be a valuable technique for studying the pathogenesis of M. hyopneumoniae infection.

PCR and in situ hybridization for the detection and localization of a new pathogen Francisella-like bacterium (FLB) in ornamental cichlids

“…In situ hybridization (ISH) using digoxigenin (DIG)-labeled oligonucleotide probes complementary to unique target sites on the 16S rRNA (rDNA) is a useful method for the detection and identification of microorganisms (Bashir et al 1994, Barrett et al 1997, Kwon & Chae 1999. Specific probes can be designed to locate organisms that cannot be cultured (Hayashi et al 1990, Komminoth & Werner 1997 or visualized by conventional methods, such as viruses (Lewis & Wells 1992), Chlamydiae (Campbell et al 1993) and Mycobacterium paratuberculosis (Hulten et al 2000).…”

Diagnóstico da pneumonia enzoótica suína pela técnica da imunoperoxidase

“…Para Doster e Lin (1988), animais cronicamente afetados podem exibir pequena concentração de microrganismos, o que poderia justificar a imunomarcação mais tênue verificada neste trabalho, em alguns pulmões oriundos de granjas positivas. (Kwon, Chae, 1999). Neste trabalho, essa indesejável reação cruzada poderia estar presente e explicar a visualização da discreta imunorreação em nove (22,5%) animais provenientes de granjas consideradas negativas para M. hyopneumoniae.…”

Mycoplasma hyorhinis infection in early cases of mycoplasmal pneumonia in swine and evaluation of diagnostic assays

“…An ultra-structural study corroborated the findings of the present study (Blanchard et al 1992). However, Doster & Lin (1988) and Kwon & Chae (1999), using ISH and IHC, also found M. hyopneumoniae in the interior of macrophages and type I pneumocytes. It is noteworthy that FISH is able to detect only viable microorganisms at the time of tissue fixation, which could at least partly justify this difference of observations and support the possibility that the bacteria was already dead in the interior of macrophages and type I pneumocytes.…”