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Validation of Loop-Mediated Isothermal Amplification (LAMP) Field Tool for Rapid and Sensitive Diagnosis of Contagious Agalactia in Small Ruminants

1
Dipartiment of Agricultural, Food and Environmental Science, University of Catania, 95123 Catania, Italy
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Istituto Zooprofilattico Sperimentale della Sicilia “A. Mirri”, 90129 Palermo, Italy
3
Department of Agricultural, Food and Forestry Science, University of Palermo, 90128 Palermo, Italy
4
Enbiotech SRL, 90129 Palermo, Italy
5
AVANTECH GROUP SRL, 84012 Angri, Italy
6
The Oaks, Nutshell Lane, Farnham, Surrey GU9 0HG, UK
*
Author to whom correspondence should be addressed.
Animals 2020, 10(3), 509; https://doi.org/10.3390/ani10030509
Received: 28 February 2020 / Revised: 14 March 2020 / Accepted: 16 March 2020 / Published: 19 March 2020
(This article belongs to the Section Veterinary Clinical Studies)
Contagious agalactia (CA) is an infectious disease of small ruminants endemic in the Mediterranean countries, causing significant socioeconomic impacts predominantly on small-scale farmers who still subsist on marginal lands. Mycoplasma agalactiae is historically considered the principal etiological agent of CA, especially in sheep. Clinical signs are characterised by mastitis, arthritis, keratoconjunctivitis and occasionally, abortion. Rapid, accurate and cost-effective field tests are urgently needed for effective control of M. agalactiae mastitis. Our study illustrated the validation of a Loop-Mediated Isothermal Amplification (LAMP) test for the detection of M. agalactiae in dairy sheep in order to confirm its application as a diagnostic tool in the field level.
Contagious agalactia (CA), an infectious disease of small ruminants, caused by Mycoplasma agalactiae, is responsible for severe losses to dairy sheep production with substantial socioeconomic impacts on small-scale farmers. The diagnosis of CA is still problematic, time-consuming and requires well-equipped labs for confirmation of outbreaks. Therefore, rapid, accurate and cost-effective diagnostic tests are urgently needed. This work aims to validate a novel Loop-Mediated Isothermal Amplification (LAMP) test, based on the p40 target gene, for the detection of M. agalactiae in dairy sheep in order to confirm its potential practical use as a rapid and cheap field test. The LAMP system proposed in this study consists of a portable device composed of real-time fluorometer with the automatic interpretation of results displayed in a tablet. A total of 110 milk samples (90 positives and 20 negatives) were analysed to optimise the analysis procedure and to investigate the efficacy and robustness of the LAMP method. All samples were analysed using LAMP and conventional real-time PCR to compare the diagnostic sensitivity of the methods. The sensitivity of the LAMP was 10-fold higher than that of real-time PCR, with a detection limit up to 103 CFU/ml. The LAMP assay was able to detect M. agalactiae in 81 of 90 (90%, 95%CI 0.84–0.96) positive milk samples compared to 69 (77%, 95%CI 0.59–0.95) positive samples detected by real-time PCR; no positive signal occurred for any of the negative milk samples in either test. Therefore, the LAMP assay was found to be more sensitive than real-time PCR, low-cost, easy to perform, fast and not affected by contamination, indicating its potential as an effective diagnostic tool in the field level for the diagnosis of CA. View Full-Text
Keywords: Mycoplasma agalactiae; field diagnostic test; p40 gene; small ruminants; LAMP Mycoplasma agalactiae; field diagnostic test; p40 gene; small ruminants; LAMP
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MDPI and ACS Style

Tumino, S.; Tolone, M.; Parco, A.; Puleio, R.; Arcoleo, G.; Manno, C.; Nicholas, R.A.; Loria, G.R. Validation of Loop-Mediated Isothermal Amplification (LAMP) Field Tool for Rapid and Sensitive Diagnosis of Contagious Agalactia in Small Ruminants. Animals 2020, 10, 509.

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