Rapid and specific detection of Mycoplasma gallisepticum and Mycoplasma synoviae infection in poultry using single and duplex PCR assays

Yadav, Jay Prakash; Singh, Y.; Jindal, Naresh; Mahajan, N. K.

doi:10.1016/j.mimet.2021.106365

Cited by 8 publications

(8 citation statements)

References 24 publications

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“…Molecular genetic assays (GTS and MLST) could help effectively distinguish the three vaccine strains through sequencing, but doing so takes a long time [ 13 , 14 , 19 ]. Additionally, conventional PCR and real-time PCR are frequently used instead of culture to detect specific avian mycoplasma DNA, but these two PCR methods can only detect avian mycoplasma species or allow for the simultaneous detection of MG and MS [ 3 , 10 , 20 ]. Therefore, the new multiplex PCR assay in this study was developed to quickly and simply detect the three MG vaccine strains and wild-type strains simultaneously.…”

Section: Discussionmentioning

confidence: 99%

“…Previous reports showed that the detection limit of real-time PCR described by Kahya et al was 0.9 pg/μL [ 10 ]. However, the detection limit of a duplex PCR assay for MG and MS reported by Yadav et al was 125 ng/mL [ 20 ]. Our novel multiplex PCR assay showed a lower detection limit than the real-time PCR and the duplex PCR methods reported previously.…”

Section: Discussionmentioning

confidence: 99%

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A Multiplex PCR Assay for Differential Identification of Wild-type and Vaccine Strains of Mycoplasma gallisepticum

Kang

Lee

et al. 2023

Pathogens

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Mycoplasma gallisepticum (MG) can cause respiratory disease in chickens and result in serious economic losses in the chicken industry. The use of live vaccines has been a favorable option for the control of MG infection in multi-age commercial layers and broiler breeders. There are three live vaccines, including ts-11, 6/85, and F strain, that have been commonly used in various parts of the world, including South Korea. The definitive diagnosis of the infection, therefore, requires the differentiation of wild-type field strains of MG from the vaccine strains used. Thus, we aimed to develop a novel multiplex PCR assay to discriminate between vaccine strains (ts-11, 6/85, and F strain) and wild-type field strains of MG isolated from infected chickens. We designed four novel primer sets that are each specific to MG species, ts-11, 6/85, and F strain. The multiplex PCR assay using the primer sets differentially identified wild-type and vaccine strains of MG but did not detect other avian bacteria. The detection limit of this assay was 250 fg/μL of genomic DNA of each strain tested. In addition, this assay was applied to 36 MG strains isolated from chickens over the past 20 years in South Korea. As a result, the assay identified 22 wild-type strains and 14 vaccine strains. Consequently, the novel multiplex PCR assay can discriminate between vaccine and wild-type field strains of MG and could be a valuable tool for the diagnosis of MG infection in MG-vaccinated chicken flocks.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

A Multiplex PCR Assay for Differential Identification of Wild-type and Vaccine Strains of Mycoplasma gallisepticum

Kang

Lee

et al. 2023

Pathogens

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“…Air sacs of chickens suspected to be infected with M . gallisepticum were collected from poultry farms in Ningxia ( Felice et al, 2020 ; Yadav et al, 2022 ). They were cut and placed in modified Frey’s liquid medium overnight at 4°C under sterile conditions.…”

Section: Methodsmentioning

confidence: 99%

Drug sensitivity and genome-wide analysis of two strains of Mycoplasma gallisepticum with different biofilm intensity

Wang

Yang

et al. 2023

Front. Microbiol.

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Mycoplasma gallisepticum (MG) is one of the major causative agents of chronic respiratory diseases in poultry. The biofilms of MG are highly correlated to its chronic infection. However data on genes involved in biofilm formation ability are still scarse. MG strains with distinct biofilm intensity were screened by crystal violet staining morphotyped and characterized for the drug sensitivity. Two MG strains NX-01 and NX-02 showed contrasted ability to biofilm formation. The biofilm formation ability of NX-01 strain was significantly higher than that of NX-02 strain (p < 0.01). The drug sensitivity test showed that the stronger the ability of MG stain to form biofilms, the weaker its sensitivity to 17 antibiotic drugs. Moreover, putative key genes related to biofilm formation were screened by genome-wide analysis. A total of 13 genes and proteins related to biofilm formation, including ManB, oppA, oppD, PDH, eno, RelA, msbA, deoA, gapA, rpoS, Adhesin P1 precursor, S-adenosine methionine synthetase, and methionyl tRNA synthetase were identified. There were five major discrepancies between the two isolated MG strains and the five NCBI-published MG strains. These findings provide potential targets for inhibiting the formation of biofilm of MG, and lay a foundation for treating chronic infection.

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“…Previous studies have used culturing, ELISA, and qPCR methods for mycoplasma detection [ 18 , 19 ]. Nevertheless, qPCR is one of the best detection methods because of its high level of reliability and speed [ 20 , 21 ]. These techniques can help individuals identify different strains of MG and MS and provide valuable information on the genetic diversity and potential transmission routes of the pathogen in chickens [ 22 ].…”

Section: Introductionmentioning

confidence: 99%

Molecular detection and genetic characterization of Mycoplasma gallisepticum and Mycoplasma synoviae in selected chicken breeds in South Africa

Idowu,

Mpofu,

Zishiri

et al. 2024

BMC Infect Dis

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Background The impact of chickens on maintaining the economy and livelihood of rural communities cannot be overemphasized. In recent years, mycoplasmosis has become one of the diseases that affect the success of South African chicken production. Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the most prevalent strains of Mycoplasma in South Africa. MG and MS are significant respiratory pathogens affecting the productivity of chickens. The present study aimed to molecularly detect using qPCR and characterize the presence of MG and MS using phylogenetic analysis. The phylogenetic analysis was utilized to clarify general evolutionary relationships between related taxa of different MG and MS observed in tracheal swabs from South African chicken breeds. Methods Forty-five tracheal swabs of the Lohmann Brown (n = 9), Rhode Island Red (n = 9), Ovambo (n = 9), Venda (n = 9), and Potchefstroom Koekoek (n = 9) breeds were collected from symptomatic chickens present in the commercial farm. To detect MG and MS, DNA was extracted from tracheal swabs and faecal samples, and qPCR was performed with a 16 s rRNA (310 bp) and vlhA (400 bp) gene fragment. Following the sequencing of all the amplicons, MG, and MS dendrograms showing the evolutionary relationships among the five South African chicken breeds and the GeneBank reference population were constructed. Results The qPCR revealed the presence of MG and MS in 22% (2/9) of the tracheal swab samples tested for MS only in Rhode Island Red breeds; 66.6% (6/9) and 33% (3/9) of the tested samples in Ovambo breeds; and 11.1% (1/9) and 44.4% (4/9) of the tested samples in Venda breeds. No MG or MS were detected in the Lohmann Brown or Potchefstroom Koekoek breed. Furthermore, qPCR revealed the presence of MG in pooled faecal samples from Lohmann Brown and Ovambo breeds. Eight different bacterial isolates were recognized from both samples. Four isolates were of the 16 s ribosomal ribonucleic acid (rRNA) gene (named PT/MG51/ck/00, PT/MG48/ck/00, PT/MG41/ck/00 and PT/MG71/ck/00) gene of Mycoplasma gallisepticum, and the other was Mycoplasma Synoviae variable lipoprotein hemagglutinin A (vlhA) gene (named PT/MSA22/ck/01, PT/MS41/ck/01, PT/MS74/ck/01 and PT/MS46/ck/01) which were available in GenBank. These isolates were successfully sequenced with 95–100% similarity to the isolates from the gene bank. Conclusion The study revealed the presence of both MG and MS in the chicken breeds sampled. Furthermore, the different breeds of chicken were found to be susceptible to infection under the intensive or commercial management system. Therefore, continuous surveillance is encouraged to prevent the spread and outbreak of MG and MS in the poultry industry in South Africa.

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Rapid and specific detection of Mycoplasma gallisepticum and Mycoplasma synoviae infection in poultry using single and duplex PCR assays

Cited by 8 publications

References 24 publications

A Multiplex PCR Assay for Differential Identification of Wild-type and Vaccine Strains of Mycoplasma gallisepticum

A Multiplex PCR Assay for Differential Identification of Wild-type and Vaccine Strains of Mycoplasma gallisepticum

Drug sensitivity and genome-wide analysis of two strains of Mycoplasma gallisepticum with different biofilm intensity

Molecular detection and genetic characterization of Mycoplasma gallisepticum and Mycoplasma synoviae in selected chicken breeds in South Africa

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