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Development of loop-mediated isothermal amplification test for the diagnosis of contagious agalactia in goats

Abstract

Contagious agalactia is a highly infectious disease affecting sheep and goats, mainly caused by Mycoplasma agalactiae. Although various tests are available for diagnosis of contagious agalactia, none of them is credited with the capacity to provide rapid and cost-effective diagnosis. This article reports the development of loop-mediated isothermal amplification (LAMP) test targeting the p40 gene of M. agalactiae, for the diagnosis of classical contagious agalactia. Optimum amplification was obtained at 58 °C in 70 min. The developed test was found to be 100-fold more sensitive than PCR and detected up to 20-fg level of DNA. The test was also superior to conventional PCR in detecting from artificially contaminated milk, i.e. 104-fold more sensitive. The developed LAMP test could detect up to 10 cfu/ml of artificially contaminated milk, indicating its potential for being developed as a field test for rapid and sensitive diagnosis.

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References

  • Alves, B.H.L.S., Silva, J. G., Mota, A.R., Campos, A.C., Júnior, J.W.P., Santos, S.B., Mota, R.A., 2013. Mycoplasma agalactiae in semen and milk of goat from Pernambuco State, Brazil, Pesquisa Veterinária Brasileira, 33, 1309-1312

    Article  Google Scholar 

  • Ariza-Miguel, J., Rodríguez-Lázaro, D., Hernández, M., 2012. A survey of Mycoplasma agalactiae in dairy sheep farms in Spain, BMC Veterinary Research, 8, 171-176

    Article  PubMed Central  PubMed  Google Scholar 

  • Arun, T.R., Rana, R., Singh, P., Choudhari, P., Singh, V.P., Thomas, P., Rekha, V., Nehra, K., Usharani, J., Dhama, K., 2014. Development of a Gold Nanoparticle Based Lateral Flow Assay for Rapid Diagnosis of Contagious Agalactia in Goats, Asian Journal of Animal and Veterinary Advances, 9, 405-413

    Article  Google Scholar 

  • Becker, C.A.M., Ramos, F., Sellal, E., Moine, S., Poumarat, F., Tardy, F., 2012. Development of a multiplex real-time PCR for contagious agalactiae diagnosis in small ruminants, Journal of Microbiological Methods, 90, 73-79

    Article  CAS  PubMed  Google Scholar 

  • Chen, H.W., Ching, W.M., 2014. Development of loop-mediated isothermal amplification assay for rapid and easy detection of Coxiella burnetti, Journal of Microbiological Methods, 107, 176-181

    Article  CAS  PubMed  Google Scholar 

  • Corrales, J.C., Esnal, A., De la Fe, C., Sanchez, A., Assuncao, P., Poveda, J.B., Contreras, A., 2007. Contagious agalactiae in small ruminants, Small Ruminant Research, 68, 154-166

    Article  Google Scholar 

  • De la Fe, C., Gómez-Martín, A., Amores, J., Corrales, J.C., Sánchez, A., Poveda, J.B., Contreras, A., 2010. Latent infection of male goats with Mycoplasma agalactiae and Mycoplasma mycoides subspecies capri at an artificial insemination centre, The Veterinary Journal, 186, 113–115

    Article  PubMed  Google Scholar 

  • Dugan, L., Bearinger, J., Hinckley, A., Strout, C., Souza, B., 2012. Detection of Bacillus anthracis from spores and cells by loop-mediated isothermal amplification without sample preparation, Journal of Microbiological Methods, 90, 280-284

    Article  CAS  PubMed  Google Scholar 

  • Filioussis, G., Giadinis, N.D., Petridou, E.J., Karavanis, E., Papageorgiou, K., Karatzias, H., 2011. Congenital polyarthritis in goat kids attributed to Mycoplasma agalactiae, Veterinary Record, 169, 364

    Article  CAS  PubMed  Google Scholar 

  • Foddai, A., Idini, G., Fusco, M., Rosa, N., De la Fe, C., Zinellu, S., Corona, L., Tola, S., 2005. Rapid differential diagnosis of Mycoplasma agalactiae and Mycoplasma bovis based on a multiplex-PCR and a PCR-RFLP, Molecular and Cellular Probes, 19, 207-212

    Article  CAS  PubMed  Google Scholar 

  • Gómez-Martín, A., De la Fe, C., Amores, J., Sanchez, A., Contreras, A., Paterna, A., Buendia, A.J., Corrales, J.C., 2012. Anatomic location of Mycoplasma mycoides subsp. capri and Mycoplasma agalactiae in naturally infected goat male auricular carriers, Veterinary Microbiology, 157, 355–362

    Article  PubMed  Google Scholar 

  • Gómez-Martín, A., Amores, J., Paterna, A., De la Fe, C., 2013. Contagious agalactia due to Mycoplasma spp. in small dairy ruminants: Epidemiology and prospects for diagnosis and control, The Veterinary Journal, 198, 48–56

    Article  PubMed  Google Scholar 

  • Greco, G., Corrente, M., Martella, V., Pratelli, A., Buonavoglia, D., 2001. A multiplex-PCR for the diagnosis of contagious agalactia of sheep and goats, Molecular and Cellular Probes, 15, 21–25

    Article  CAS  PubMed  Google Scholar 

  • Hegde, S., Hegde, S., Spergser, J., Brunthaler, R., Rosengarter, R., Chopra-Dewasthaly, R., 2014. In vitro and in vivo cell invasion and systemic spreading of Mycoplasma agalactiae in the sheep infection model, International Journal of Medical Microbiology, 304, 1024-1031

    Article  PubMed Central  PubMed  Google Scholar 

  • Hill, J., Beriwal, S., Chandra, I., Paul, V.K., Kapil, A., Singh, T., Wadowsky, R.M., Singh, V., Goyal, A., Jahnukainen, T., Johnson, J.R., Tarr, P.I. and Vats, A., 2008. Loop- Mediated Isothermal Amplification Assay for Rapid Detection of Common Strains of Escherichia coli, Journal of Clinical Microbiology, 46, 2800–2804

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Ji, H., Li, H., Zhu, L., Zhang, H., Wang, Y., Zuo, Z., Guo, W., Xu, Z., 2012. Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Actinobacillus pleuropneumoniae based the dsbE-like gene1, Pesquisa Veterinária Brasileira, 32, 757-760

    Article  Google Scholar 

  • Kaneko, H., Kawana, T., Fukushima, E., Suzutani, T., 2007. Tolerance of loop mediated isothermal amplification to a culture medium and biological substances, Journal of Biochemical and Biophysical Methods, 70, 499–501

    Article  CAS  PubMed  Google Scholar 

  • Khezri, M., Pourbakhsh, S.A., Ashtari, A., Rokhzad, B., 2014. A survey of Mycoplasma agalactiae in small ruminants with contagious agalactiae syndrome in Iran, Bangldesh Journal of Veterinary Medicine, 12, 67-72

    Google Scholar 

  • Kumar, A., Singh, M.K., Rana, R., Vihan, V.S., 2009. Post monsoon disease profile of Zalawadi goats, Indian Veterinary Journal, 86, 203-204

    Google Scholar 

  • Kumar, A., Rahal, A., Chakraborty, S., Verma, A.K., Dhama, K., 2014a. Mycoplasma agalactiae, an Etiological Agent of Contagious Agalactia in Small Ruminants: A Review, Veterinary Medicine International, http://dx.doi.org/10.1155/2014/286752

  • Kumar, P., Roy, A., Aher, T.K., Joseph, J.P., 2014b. Biochemical and molecular identification of Mycoplasma agalactiae isolates from healthy and sick goats of Gujarat, The Indian Journal of Small Ruminants, 20, 59-63

    Google Scholar 

  • Lin, G.Z., Zheng, F.Y., Zhou, J.Z., Gong, X.W., Wang, G.H., Cao, X.A., Qiu, C.Q., 2011. Loop-mediated isothermal amplification assay targeting the omp25 gene for rapid detection of Brucella spp, Molecular and Cellular Probes, 25, 126-129

    Article  CAS  PubMed  Google Scholar 

  • Mair, G., Vilei, E.M., Wade, A., Frey, J., Unger, H., 2013. Isothermal loop-mediated amplification (LAMP) for diagnosis of contagious bovine pleuro-pneumonia, BMC Veterinary Research, 9, 108

    Article  PubMed Central  PubMed  Google Scholar 

  • McAuliffe, L., Ellis, R.J., Ayling, R.D., Nicholas, R.A.J., 2003. Differentiation of Mycoplasma Species by 16S Ribosomal DNA PCR and Denaturing Gradient Gel Electrophoresis Fingerprinting, Journal of Clinical Microbiology, 41, 4844-4847

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Nagamine, K., Hase T., Notomi, T., 2002. Accelerated reaction by loop-mediated isothermal amplification using loop primers, Molecular and Cellular Probes, 16, 223-229

    Article  CAS  PubMed  Google Scholar 

  • Nirju, Z.K., 2012. Loop-mediated isothermal amplification technology: towards point of care diagnostics, PLoS Neglected Tropical Diseases, 6, e1572, doi:10.1371/journal.pntd.0001572

    Article  Google Scholar 

  • Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., Hase, T., 2000. Loop-mediated isothermal amplification of DNA, Nucleic Acids Research, 28, e63

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • OIE, 2013. Contagious agalactia. In: Terrestrial Manual. Chapter 2.7.5. http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.07.05_CONT_AGALACT.pdf. Accessed 29 Nov 2014.

  • Parida, M., Sannarangaiah, S., Dash, P.K., Rao, P.V.L., Morita, K., 2008. Loop mediated isothermal amplification (LAMP): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases, Reviews in Medical Virology, 18, 407-421

    Article  CAS  PubMed  Google Scholar 

  • Peyraud, A., Woubit, S., Poveda, J.B., De la Fe, C., Mercier, P., Thiaucourt, F.A., 2003. Specific PCR for the detection of Mycoplasma putrefaciens, one of the agents of the contagious agalactia syndrome of goats, Molecular and Cellular Probes, 17, 289–294

    Article  CAS  PubMed  Google Scholar 

  • Saleh, M., Soliman, H., El-Matbouli, M., 2008. Loop-mediated isothermal amplification (LAMP) for rapid detection of Renibacterium salmoninarum, the causative agent of bacterial kidney diseases, Diseases of Aquatic Organisms, 81, 143-151

    Article  CAS  PubMed  Google Scholar 

  • Sambrook, J. and Russel, D.W., 2001. Molecular Cloning, 3rd Ed, (Cold spring Harbor Laboratory Press, New York).

    Google Scholar 

  • Sanna, G., Lecca, V., Foddai, A., Tola, S., 2014. Development of a specific immunomagnetic capture-PCR for rapid detection of viable Mycoplasma agalactiae in sheep milk samples, Journal of Applied Microbiology, 117, 1585-1591

    Article  CAS  PubMed  Google Scholar 

  • Song, Q., Wang, L., Fang, R., Khan, M.K., Zhou, Y., Zhao, J., 2012. Detection of Mycoplasma wenyonii in cattle and transmission vectors by the loop-mediated isothermal amplification (LAMP) assay, Tropical Animal Health and Production, DOI 10.1007/s11250- 012-0197-y

    PubMed  Google Scholar 

  • Thomas, A., Linden, A., Mainil, J., Dizier, I., Baseman, J.B., Kannan, T.R., Fleury, B., Frey, J., Vilei, E.M., 2004. The p40* adhesin pseudogene of Mycoplasma bovis, Veterinary Microbiology, 104, 213-217

    Article  CAS  PubMed  Google Scholar 

  • Tola, S., Angioi, A., Rocchigiani, A.M.., Idini, G., Manunta, D., Galleri, G., Leori, G., 1997. Detection of Mycoplasma agalactiae in sheep milk samples by polymerase chain reaction, Veterinary Microbiology, 54, 17–22

    Article  CAS  PubMed  Google Scholar 

  • Tsai, S.M., Chana, K.W., Hsub, W.L., Changa, T.J., Wonga, M.L., Wanga, C.Y., 2009. Development of a loop-mediated isothermal amplification for rapid detection of orf virus, Journal of Virological Methods, 157, 200–204

    Article  CAS  PubMed  Google Scholar 

  • WAHIS, 2014. www.oie.int/wahis_2/public/wahid.php/Reviewreport/Review?page_refer=MapFullEventReport&reportid=16436. Accessed 29 Nov 2014

  • Wang, D., Liu, F., Huo, G., Ren, D., Li, Y., 2008. Development and evaluation of a loop-mediated isothermal amplification method for detecting Escherichia coli O157 in raw milk, Advanced Materials Research, 343-344, 1217-1221

  • Wang, D., Yang, L., Huo, G., Wang, F., Li, Y., Ren, D., 2009. Development of a loop-mediated isothermal amplification (LAMP) method for detecting food-borne Shigella in raw milk, Milchwissenschaft, 64, 264-267

    CAS  Google Scholar 

  • Wang, D., Zhang, G., Lu, C., Deng, R., Zhi, A., Guo, J., Zhao, D., Xu, Z., 2011. Rapid Detection of Listeria monocytogenes in Raw Milk with Loop-Mediated Isothermal Amplification and Chemosensor, Journal of Food Science, 76, 611-615

    Article  Google Scholar 

  • Yamazaki, W., Taguchi, M., Ishibashi, M., Nukina, M., Misawa, N., Inoue, K., 2009. Development of a loop-mediated isothermal amplification assay for sensitive and rapid detection of Campylobacter fetus, Veterinary Microbiology, 136, 393-396

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

The authors sincerely acknowledge the financial support to this work, extended by the Director, Joint Director (Academic) and Joint Director (Research), Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India.

Conflicts of interest

The authors declare that they have no conflict of interest. This research work was conducted as part of M.V.Sc degree programme with the funding from Indian Veterinary Research Institute, Izatnagar.

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Correspondence to Valsala Rekha.

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Rekha, V., Rana, R., Thomas, P. et al. Development of loop-mediated isothermal amplification test for the diagnosis of contagious agalactia in goats. Trop Anim Health Prod 47, 581–587 (2015). https://doi.org/10.1007/s11250-015-0767-x

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  • DOI: https://doi.org/10.1007/s11250-015-0767-x

Keywords

  • Contagious agalactia
  • Mycoplasma agalactiae
  • p40 gene
  • LAMP
  • PCR
  • Diagnostic test