Mycoplasma hyorhinis infection in early cases of mycoplasmal pneumonia in swine and evaluation of diagnostic assays / Infecção por Mycoplasma hyorhinis em casos precoces de pneumonia micoplásmica em suínos e comparação entre técnicas diagnósticas

Mycoplasmal pneumonia is an important disease in the pig industry. Due to the controversial role of Mycoplasma hyorhinis in this disease, confirmation of the presence of this bacterium and the identification of its roles in respiratory disease remain major challenges. The objectives of this study were to evaluate the presence of M. hyorhinis in early cases of mycoplasmal pneumonia and to determine the usefulness of fluorescent in situ hybridization (FISH) for the diagnosis of respiratory mycoplasmosis in naturally infected pigs. Ninety M. hyopneumoniae and/or M. hyorhinis-infected lung tissue samples based on diagnostic mosaic (DM) were used. The average age of the animals was 116 and 57 days (P<0.01) for groups 1 (positive-M. hyopneumoniae only) and 2 (positive-M. hyorhinis only), respectively. These findings suggest that development of lesions caused by M. hyorhinis occurs earlier than for M. hyopneumoniae. Using the DM as the gold standard, the sensitivity and specificity of FISH for M. hyopneumoniae were 75 and 100%, respectively, and were 40 and 73.3% for the immunohistochemistry (IHC). The sensitivity and specificity of FISH for M. hyorhinis were 76.7 and 100%, respectively. These findings demonstrate that FISH can be a useful tool for diagnosing mycoplasmosis. Viral antigens (PCV2 or influenza A) were detected in 53.3% (16/30) of the samples in group 2 (M. hyorhinis-PCR positive) and 13.3% (4/30) of the samples in group 1 (M. hyopneumoniae-PCR positive). This finding indicates that the association of M. hyorhinis and viral infection in nursery pigs likely starts due to a viral immunosuppressive condition.(AU)

A pneumonia micoplásmica causada por bactérias do gênero Mycoplasma é uma enfermidade de grande importância para indústria suinícola, sendo ainda controverso o papel desempenhado por Mycoplasma hyorhinis nessa doença. A confirmação da presença dessas bactérias bem como a identificação de seus papéis em doenças respiratórias continua sendo um grande desafio. Os objetivos desse estudo foram comparar diferentes técnicas, em especial a de hibridização fluorescente in situ (FISH), para diagnóstico de micoplasmoses respiratória em suínos naturalmente infectados e avaliar a presença do M. hyorhinis em casos precoces de pneumonia micoplásmica. Foram utilizadas 90 amostras de tecido pulmonar infectado para cada um ou ambos os agentes (M. hyopneumoniae e M. hyorhinis) determinados pelo mosaico de diagnóstico (sinais clínicos, lesões macroscópicas e microscópicas e pela PCR). No grupo de animais positivos pela PCR apenas para M. hyorhinis (Grupo 2) a média da idade foi de 57,32 dias e no grupo apenas positivo para M. hyopneumoniae (Grupo 1) a média foi de 116,31 dias (P<0,01). Estes achados sugerem que a colonização e o aparecimento de lesões causadas pelo M. hyorhinis seja mais precoce do que aquelas causadas pelo M. hyopneumoniae. As alterações microscópicas foram estatisticamente (P<0,01) mais intensas no grupo 1 do que no grupo 2. Usando o mosaico de diagnóstico como padrão ouro, a sensibilidade e especificidade na FISH para M. hyopneumoniae foi de 75 e 100%, respectivamente, e 40 e 73,3%, na imuno-histoquímica. A sensibilidade e especificidade da FISH para M. hyorhinis foi de 76,7 e 100%. Esses valores demonstram que a FISH pode ser uma ferramenta útil para diagnóstico de micoplasmoses. Foi detectada a presença de agentes virais (PCV2 ou influenza) em 53,3% das amostras do grupo 2 (M. hyorhinis) e em 13,3% das amostras do grupo 1 (M. hyopneumoniae).(AU)

Preparation and identification of a monoclonal antibody against Mycoplasma hyorhinis / 中国人兽共患病学报

The aim of this study was to produce a monoclonal antibody against Mycoplasma hyorhinis,which would be useful in the diagnosis and pathogenesis researches of M.hyorhinis.BALB/c mice were immunized with whole cell protein of M.hyorhinis,and then the monoclonal antibody (McAb) was prepared by hybridoma technique.The isotype of the McAb was identified,and then the titer and specificity were analyzed.The prepared McAb was used to detect M.hyorhinis in the colony immunoboltting assay and indirect immunofluorescence assay.A hybridoma cell line was obtained,and was named Mhr-08.The McAb belonged to IgG1 isotype,and the light chain was κ type.The titer was detected to be 1 ∶ 102 400 by ELISA.The result of Western-blot indicated that this McAb strongly reacted to a 43 kDa protein of M.hyorhinis,without cross-reaction with other swine mycoplasmas,Escherichia coli or KM2 medium.It revealed that the McAb recognized a surface membrane protein and was successfully applied to detect M.hyorhinis in colony immunoboltting assay.The mycoplasmas bound to the tracheal epithelial cells was successfully detected by using this McAb in indirect immunofluorescence assay.In conclusion,a McAb against M.hyorhinis was successfully prepared,which provides as a useful tool for the diagnosis and pathogenesis researches.

In vitro antibiotic susceptibility of field isolates of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis from Korea

The present study was conducted to determine the antibiotic susceptibilities of local Mycoplasma hyopneumoniae (Mhp) and Mycoplasma hyorhinis (Mhr) filed isolates. Minimum inhibitory concentrations (MICs) of Mhp and Mhr field isolates (twelve each) obtained from enzootic pneumonia-like lung lesions during 2009-2011 from Korea were determined using the broth microdilution method. Tylvalosin showed the highest activity against Mhp and Mhr field isolates, with MIC90 values of 0.06 µg/mL and 0.12 µg/mL, respectively. Therefore, Korean Mhp and Mhr isolates are highly susceptible to tylvalosin.

Quantitative real-time polymerase chain reaction for detecting Mycoplasma hyosynoviae and Mycoplasma hyorhinis in pen-based oral, tonsillar, and nasal fluids

Mycoplasma (M.) hyorhinis and M. hyosynoviae are pathogens known to cause disease in pigs post-weaning. Due to their fastidious nature, there is increased need for culture-independent diagnostic platforms to detect these microorganisms. Therefore, this study was performed to develop and optimize quantitative real-time PCR (qPCR) assays to rapidly detect M. hyorhinis and M. hyosynoviae in pen-based oral fluids as well as nasal and tonsillar fluids as proxies for samples used in swine herd surveillance. Two methods of genomic DNA extraction, automated versus manual, were used to compare diagnostic test performance. A wean-to-finish longitudinal study was also carried out to demonstrate the reproducibility of using pen-based oral fluids. Overall, pen-based oral and tonsillar fluids were more likely to be positive for both types of bacteria whereas only M. hyorhinis was detected in nasal fluids. DNA extraction protocols were shown to significantly influence test result. Although the initial detection time somewhat differed, both organisms were repeatedly detected in the longitudinal study. Overall, this study evaluated two qPCR methods for rapid and specific detection of either mycoplasma. Results from the present investigation can serve as a foundation for future studies to determine the prevalence of the two microorganisms, environmental load, and effectiveness of veterinary interventions for infection control.

Detection of respiratory pathogens Mycoplasma hyorhinis and Mycoplasma hyopneumoniae from clinically infected porcine using nested PCR in Jiangsu Province, China / 中国人兽共患病学报

Mycoplasma hyopneumoniae (M .hyopneumoniae) and Mycoplasma hyorhinis (M .hyorhinis) infections are common in China .To investigate the prevalence of M .hyorhinis and M .hyopneumoniae in Jiangsu Province of China ,a mo-lecular epidemiological survey was conducted from 399 nasal swab samples of unvaccinated pigs using nested Polymerase Chain Reaction (nested PCR) .Nasal swab samples were collected from Jiangquhai porcine lean (JQHPL) strain pigs and other West-ern breeds .Clinical samples were taken from each pig and divided into different groups based on ages of pigs (7 ,14 ,21 ,28 , and 35 days) .Results indicated that the prevalence of M .hyorhinis was 70 .9% from different herds in Jiangsu Province in China ,while the prevalence of M .hyopneumoniae was 13 .5% .M .hyorhinis infection was more common in pigs for less than 5 weeks of age compared to M .hyopneumoniae infection .Co-infection was also observed in 30 samples (7 .5% ) in which both M .hyorhinis and M .hyopneumoniae were detected .The M .hyorhinis infection increased as the animals grew from 7 to 35 days .The M .hyopneumoniae infection did not change significantly as the pigs grew older .Significant difference of M .hyorhi-nis infection was observed between other Western breeds and JQHPL pigs (P0 .05) .

Identification of two cytopathogenic agents, Mycoplasma hyorhinis and mammalian orthoreovirus 3 based on modified particle associated nucleic acids PCR / 대한수의학회지

Swine diseases could be caused by unrecognized or minor pathogens. In this study, two unknown cytopathogenic agents were isolated from swine, through cell culture. In order to identify these two cytopathogenic agent (designated CP129 and #2045-7), a particle associated nucleic acids PCR (PAN-PCR) from previous paper was used with simple modification. The cloning procedure was more specified in this study by adding cell control system. According to the modified PAN-PCR, two and four agents-specific DNA sequences were obtained from CP129 and #2045-7, respectively, and they were identified as Mycoplasma (M.) hyorhinis and Mammalian orthoreovirus by nucleotide BLAST. Since M. hyorhinis (CP129) was filterable and non-visible by microscope, this unusual virus-like nature of M. hyorhinis (CP129) was discussed. Especially, the reovirus (#2045-7) was a serotype 3 and a triple reassortant among three serotypes of reoviruses. It was grouped with recently reported reoviruses from disease cases (swine, human and feline), based on the genetic analysis of L1 and S1 partial sequences. In conclusion, two unknown cytopathogenic agents were successfully identified using modified PAN-PCR with cell control system and they were characterized in this study.