Detection of Mycoplasma hyopneumoniae in Lung and Nasal Swab Samples from Pigs by Nested PCR and Culture Methods

Otagiri, Yukiko; Asai, Tetsuo; Okada, Mizue; Uto, Takehiko; Yazawa, Shigeto; Hirai, Hidetoshi; Shibata, Isao; Sato, Shizuo

doi:10.1292/jvms.67.801

Cited by 32 publications

(32 citation statements)

References 18 publications

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“…To avoid false positive results due to contamination in the nPCR, appropriate positive and negative controls were used during DNA extraction and amplification. The results should be interpreted at the herd level, not at the individual level (Otagiri et al, 2005).…”

Section: Discussionmentioning

confidence: 92%

“…This diagnostic tool is suitable for detection of M. hyopneumoniae under field conditions in suckling pigs and nursery pigs (Otagiri et al, 2005;Sibila et al, 2007), and can detect 5.1 × 10 6 ng M. hyopneumoniae DNA or as few as four organisms/μl reaction mixture (Gebruers et al, 2008). To avoid false positive results due to contamination in the nPCR, appropriate positive and negative controls were used during DNA extraction and amplification.…”

Section: Discussionmentioning

confidence: 99%

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Early Mycoplasma hyopneumoniae infections in European suckling pigs in herds with respiratory problems: detection rate and risk factors

Villarreal¹,

Vranckx²,

Duchateau³

et al. 2010

Vet. Med. - Czech

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ABSTRACT:The present study aimed at estimating the detection rate of M. hyopneumoniae in 3-week-old pigs in different European countries and to identify possible risk factors. Nasal swabs from suckling pigs in 52 farms were collected for nested PCR analysis. Potential risk factors for respiratory disease were analysed with a multivariable logistic regression model. The average percentage of positive piglets was 10.7% (95% confidence interval, CI 7.4-14.2); at least one pig tested positive in 68% of herds. In 32% of the herds, more than 10% of piglets tested positive. Herds that vaccinated sows against swine influenza virus (SIV) had a significantly higher risk of a piglet being positive for M. hyopneumoniae (OR 3.12; 95% CI 1.43-6.83). The higher risk in case of SIV vaccination is difficult to explain, but may be due to the fact that pig herds with respiratory symptoms are more likely to be vaccinated against SIV, overlooking the possible influence of other respiratory pathogens such as M. hyopneumoniae. The present findings show that M. hyopneumoniae is widespread in 3-week-old piglets across different European countries.

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Section: Discussionmentioning

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Early Mycoplasma hyopneumoniae infections in European suckling pigs in herds with respiratory problems: detection rate and risk factors

Villarreal¹,

Vranckx²,

Duchateau³

et al. 2010

Vet. Med. - Czech

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“…Verdin et al (2000) demonstrated that M. hyopneumoniae was probably confined to the lower respiratory tract in six-month-old pigs. Otagiri et al (2005) also found that lung sample detections were higher than in nasal swabs from the same animals.…”

Section: Methodsmentioning

confidence: 86%

Detection of Mycoplasma hyopneumoniae in lungs and nasal swabs of pigs by nested PCR

Silva¹,

Castro²,

Júnior³

et al. 2009

Arq. Bras. Med. Vet. Zootec.

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Fifty-four samples were collected from growing and finishing pigs for the molecular diagnosis of enzootic porcine pneumonia. Nineteen lung fragments were obtained from pigs that showed signs of respiratory disease and 35 nasal swabs were obtained from clinically healthy pigs. For the detection of the bacterial genome in the samples, the nested PCR technique was used to amplify a fragment of 706bp. This fragment was subsequently cloned and sequenced. The sequence of obtained nucleotides was compared with six other sequences of Mycoplasma hyopneumoniae and 11 sequences of other bacteria available in the Genbank. To measure the sensitivity of the nested PCR, serial dilutions (10 -1 to 10 -15 ) of cloned fragments were conducted based on the concentration of 300ng. Ten lung fragments and eight nasal swabs showed positive for M. hyopneumoniae and the limit of detection was estimated to be 0.3fg DNA cloned. The sequence of nucleotides obtained showed 99.1% homology with the other sequences of M. hyopneumoniae, demonstrating that the nested PCR used in this study may provide an important diagnostic tool for the detection of this agent.

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“…However, this method is less practical because it requires special Friis medium and because the agent grows slowly. (Thacker 2004, Otagiri et al 2005. Additionally, other microorganisms that coexist in the sample might outgrow M. hyopneumoniae (Thacker 2004).…”

Section: Introductionmentioning

confidence: 99%

“…All these factors make isolation of the organism from ϐield samples even more difϐicult. Currently, the most frequently used techniques for M. hyopneumoniae diagnosis are the histological analysis of affected tissues, immunohistochemistry (IHC) and, more recently, polymerase chain reaction (PCR) (Mattson et al 1995, Calsamiglia et al 1999, Ribeiro et al 2004, Thacker 2004, Otagiri et al 2005. Samples used for histological techniques are routinely kept in a 10% formalin solution and the subsequently embedded in parafϐin (formalin-ϐixed, parafϐin-embedded -FFPE) (Srinivasan et al 2002, Coura et al 2005, Delfour et al 2006, Ferrer et al 2007, and pathology laboratories keep large collections of FFPE samples, allowing important retrospective studies using histological analysis (Ferrer et al 2007).…”

Section: Introductionmentioning

confidence: 99%

Nested-PCR for the detection of Mycoplasma hyopneumoniae in bronchial alveolar swabs, frozen tissues and formalin-fixed paraffin-embedded swine lung samples: comparative evaluation with immunohistochemical findings and histological features

et al. 2012

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Pesq. Vet. Bras. 32(8) The diagnosis of Mycoplasma hyopneumoniae infection is often performed through histopathology, immunohistochemistry (IHC) and polymerase chain reaction (PCR) or a combination of these techniques. PCR can be performed on samples using several conservation methods, including swabs, frozen tissue or formalin-ϐixed and parafϐin-embedded (FFPE) tissue. However, the formalin ϐixation process often inhibits DNA ampliϐication. To evaluate whether M. hyopneumoniae DNA could be recovered from FFPE tissues, 15 lungs with cranioventral consolidation lesions were collected in a slaughterhouse from swine bred in herds with respiratory disease. Bronchial swabs and fresh lung tissue were collected, and a fragment of the corresponding lung section was placed in neutral buffered formalin for 48 hours. A PCR assay was performed to compare FFPE tissue samples with samples that were only refrigerated (bronchial swabs) or frozen (tissue pieces). M. hyopneumoniae was detected by PCR in all 15 samples of the swab and frozen tissue, while it was detected in only 11 of the 15 FFPE samples. Histological features of M. hyopneumoniae infection were presented in 11 cases and 7 of these samples stained positive in IHC. Concordance between the histological features and detection results was observed in 13 of the FFPE tissue samples. PCR was the most sensitive technique. Comparison of different sample conservation methods indicated that it is possible to detect M. hyopneumoniae from FFPE tissue. It is important to conduct further research using archived material because the efϐiciency of PCR could be compromised under these conditions.

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Detection of Mycoplasma hyopneumoniae in Lung and Nasal Swab Samples from Pigs by Nested PCR and Culture Methods

Cited by 32 publications

References 18 publications

Early Mycoplasma hyopneumoniae infections in European suckling pigs in herds with respiratory problems: detection rate and risk factors

Early Mycoplasma hyopneumoniae infections in European suckling pigs in herds with respiratory problems: detection rate and risk factors

Detection of Mycoplasma hyopneumoniae in lungs and nasal swabs of pigs by nested PCR

Nested-PCR for the detection of Mycoplasma hyopneumoniae in bronchial alveolar swabs, frozen tissues and formalin-fixed paraffin-embedded swine lung samples: comparative evaluation with immunohistochemical findings and histological features

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