Development of a prototypic, field-usable diagnostic tool for the detection of gram-positive cocci-induced mastitis in cattle

Abstract

Background: Bovine mastitis is one of the most widespread diseases affecting cattle, leading to significant losses for the dairy industry. Currently, the so-called gold standard in mastitis diagnosis involves determining the somatic cell count (SCC). Apart from a number of advantages, this method has one serious flaw: It does not identify the etiological factor causing a particular infection, making it impossible to introduce targeted antimicrobial therapy. This can contribute to multidrug-resistance in bacterial species. The diagnostic market lacks a test that has the advantages of SCC and also recognizes the species of pathogen causing the inflammation. Therefore, the aim of our study was to develop a lateral flow immunoassay (LFIA) based on elongation factor Tu for identifying most prevalent Gram-positive cocci responsible for causing mastitis including Streptococcus uberis, Streptococcus agalactiae and Staphylococcus aureus.

Results: As a result, we showed that the assay for S. uberis detection demonstrated a specificity of 89.02%, a sensitivity of 43.59%, and an accuracy of 80.3%. In turn, the second variant - assay for Gram-positive cocci reached a specificity of 95.59%, a sensitivity of 43.28%, and an accuracy of 78.33%.

Conclusions: Our study shows that EF-Tu is a promising target for LFIA and we have delivered evidence that further evaluation could improve test parameters and fill the gap in the mastitis diagnostics market.

Keywords: Staphylococcus aureus; Streptococcus agalactiae; Streptococcus uberis; Bovine mastitis; Elongation factor Tu; Lateral flow immunoassay.

Fig. 1

ELISA titer graph of mAb-anti-EF-Tu generated against recombinant EF-Tu. Each point represents the combined averages of three independent experiments in triplicate under the same conditions. Error bars indicate standard deviation

Fig. 2

Western blot analysis of bacterial lysates, the source of native EF-Tu, and recombinant EF-Tu (rEF-Tu) protein, both recognized by monoclonal antibodies against elongation factor-Tu

Fig. 3

cELISA graph of mAb-anti-EF-Tu generated against whole bacteria and milk from cows without mastitis. Each value represents the combined averages of three independent experiments in triplicate under the same conditions. Error bars indicate standard deviation. Legend: NM – negative milk, PBS/PFA 4% – fixative, negative control

Fig. 4

Particle size analyzer graph for UV-Vis spectra of 30 nm AuNPs

Fig. 5

The stability of AuNP–antibody conjugates for UV-Vis spectra at different concentrations of mAb-anti-EF-Tu (1.25 µg/mL (5A), 2.5 µg/mL (5B), 5 µg/mL (5C), 10 µg/mL (5D), 20 µg/mL (5E), and 40 µg/mL (5F). Each point represents the combined averages of three independent experiments in triplicate under the same conditions

Fig. 6

The stability of AuNP–antibody conjugates at different concentrations of mAb-anti-EF-Tu at three time-points. Each value represents the combined averages of three independent experiments in triplicate under the same conditions

Fig. 7

Sensitivity of LFIA strips to recombinant EF-Tu protein, different densities of live S. uberis SU1 and S. uberis SU1 lysate. rEF-Tu – recombinant EF-Tu protein

Fig. 8

Mean values of bacterial numbers in milk samples infected with S. uberis. Legend: CFU – colony forming unit

Fig. 9

Schematic of the LFIA assay for the detection of S. uberis (SU) in milk samples. A – example of a positive test result; B – example of a negative test result. Legend: C – control line, indicating the correct performance of the test; SU – S. uberis, test line, indicating the correct performance of the test and the presence of S. uberis in the tested milk sample