Implementation of histopathological techniques and transmission electron microscopy for research of Mycoplasma hyopneumoniae in swine
Abstract
Mycoplasma hyopneumoniae is a fastidious bacterium, an important member of swine respiratory disease complex, like Porcine Enzootic Pneumonia, affecting the non-specific defense mechanism of the respiratory tract, high mucociliary system, predisposing the pigs to secondary pathogens. The objective of this study is to implement precise diagnostic techniques for identification of Mycoplasma hyopneumoniae. 19 swine lungs fragments were collected from slaughterhouses and submitted by histopathological techniques. The presence of mucocellular exudate in 78.94% of the samples was observed in the bronchi and bronchioles, absence of eyelashes in 63.15% and lymphoid tissue hyperplasia associated with the bronchus in 42.10%. In pulmonary parenchyma, thickening of alveolar wall and interstitial bronchopneumonia were observed in 68.42%, hemorrhage in 47.36%, which 36.84% had hemosiderin and 15.78% lung consolidation. The presence of mycoplasma by the negative staining technique was identified in all samples, also the labeling of epitopes by colloidal gold immunostaining, using monoclonal antibody. In immunohistochemistry techniques and in situ hybridization, the labeled epitope and genome were observed confirming the presence of Mycoplasma hyopneumoniae in the State of São Paulo. Therefore, the bronchial epithelium is the best tissue to collect the sample for an accurate diagnosis and the best method of diagnosis is the negative staining technique for screening and colloidal gold immunocytochemistry techniques to identify Mycoplasma species.
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Introduction
Mycoplasma hyopneumoniae is a small fastidious bacterium with a simple morphologically structure and absence of cell wall. (DEMINA et al., 2009; JUNQUEIRA and CARNEIRO, 2012). It is the etiologic agent of Swine Enzootic Pneumonia, causing the Porcine Respiratory Disease Complex (PRDC) (MARE and SWITZER, 1965; GOODWIN et al, 1965).
Is the most important disease that affects the respiratory system of pigs causing large economic losses for pigs (ROSS et al., 1999), due to the high morbidity and reduction of feed conversion that decreases average daily weight gain (CONCEICAO and DELLAGOSTIN, 2006).
Diagnoses for this bacterium by ELISA available on the market have low sensitivity and cross-reactions with other mycoplasmas present in the lungs of the swine (ROSS and STEMKE 1995; ERLANDSON et al., 2005).
The objective of this study is to implement precise diagnostic techniques for identification of Mycoplasma hyopneumoniae.
Conclusion
The identification of Mycoplasma hyopneumoniae was divided into 3 axes, according to their visualization, epitope marking and genome.
The agent was visualized through the negative staining presenting pleomorphic particles with the following characteristics: smooth and clear edges, gray in the center, less homogeneous and pleomorphic, corroborating with the characteristics described by Souza, et al. (2007). However, this technique does not allow identification at the species level, and therefore, the colloidal gold immunostaining technique was used to visualize and specify Mycoplasma hyopneumoniae species.
Due the small quantity of lung samples used in electron microscopy techniques, a correlation model was necessary between the identification of the agent and the histopathological lesions. According to Sobestiansky et al., (1999) e Thacker (2006) the microscopic lesions vary with the disease progression, and the most observed lesion was bronchointerstitial pneumonia with BALT hyperplasia. In this study, it was observed that 68.42% presented interstitial bronchopneumonia and 42.10% presented BALT hyperplasia, corroborating with classic findings of the disease described above.
In addition, in 78.94% of the cases, a mucocellular exudate with neutrophils in the bronchi and bronchioles was observed, pulmonary consolidation of the parenchyma in 15.78% and thickening and hyperplasia of the alveolar walls with inflammatory infiltrate in 68.42%. Although these microscopic changes are not classic of Mycoplasma hyopneumoniae infection, Redondo et al. (2009) and Hillen et al. (2014) also described these findings in the discrimination of the disease. It was observed that in 63.15% there was a lack of eyelashes in the bronchi and bronchioles, an infectious characteristic also seen by Posá et al. (2013). These findings indicate that M. hyopneumoniae infection is extremely complex with classic and secondary characteristics that are relevant in clinical diagnosis.
Although markers are positive in histopathological lesions by immunohistochemical techniques and in situ hybridization, the fixation in 10% buffered formalin can alter the epitopes as a protein fixative as described by Souza (2007). Although the in situ hybridization technique is effective, Santos (2015) verified that M. hyopneumoniae genomics in Brazil is highly variable, identifying 39 different types of the species in 8 States, according to multiple locus variable number tandem repeat analysis (MLVA) which makes it difficult to specify probes.
Because of the possible epitope alteration and the high genotypic variability, the reliable method of histopathological findings was the positivity of both methods (immunohistochemistry and in situ hybridization) to confirm the pulmonary lesions caused by Mycoplasma hyopneumoniae, in the State of São Paulo, Brazil.
It was concluded that for a precise diagnosis of M. hyopneumoniae in São Paulo State, the best sample is the bronchial epithelium and the best diagnostic method is the electron microscopy technique (as screening) and immunocytochemistry with colloidal gold for identification level of agent species.