Rapid Staphylococcus aureus Detection From Clinical Mastitis Milk by Colloidal Gold Nanoparticle-Based Immunochromatographic Strips

Abstract

Rapid diagnostic technologies for bovine mastitis caused by Staphylococcus aureus (S. aureus) are urgently needed. In the current study, we generated an anti-ribosomal protein-L7/L12 antibody to detect S. aureus and an anti-ribosomal protein-L7/L12 antibody-coated immune-chromatographic strip (ICS) test. Moreover, we determined the ability of the ICS test to detect S. aureus from milk samples collected from cows with clinical mastitis. The developed ICS reacted to S. aureus in a bacteria load-dependent manner with a detection limit of ∼10 4 CFU/mL. In the evaluation of possible cross-reactivity of the ICS test, six strains of coagulase-negative Staphylococci showed slightly positive reactions, although at a lower level; however, other bacteria were completely negative. Next, we investigated the sensitivity and specificity of the ICS test compared with the bacteriological culture method using milk samples from clinical bovine mastitis. The results of the experiments demonstrated that the ICS test had high sensitivity [100%, 95% confidence interval (CI): 91.3-100%] and specificity (91.9%, CI: 90.5-91.9%) compared with culture tests. In addition, the kappa statistic demonstrated that ICS tests showed substantial agreement (k = 0.77, CI: 0.66-0.87) with culture tests. Positive correlations were observed for the statistical analysis between S. aureus (nuc gene) copy numbers and ICS test scores in mastitic milk infected by S. aureus. Therefore, we assume that this new detection method using ICS may be useful as a highly sensitive S. aureus-screening method for the diagnosis of bovine mastitis. Our findings support the ongoing effort to develop an ICS method for bovine S. aureus-induced mastitis, which can contribute to the rapid diagnosis of this disease.

Evaluation of a rapid coliform detection kit from clinical mastitis milk using colloidal gold nanoparticle–based immunochromatographic strips

“…In each case in which clinical mastitis was noticed, we aseptically collected a quarter milk sample before the administration of antibiotic treatment and stored the sample frozen at −20°C until ICS testing, somatic cell counting, and using the culture method. A previous study already confirmed that freezing and thawing of milk samples does not affect the ICS test score [ 15 ].…”

“…As previously reported in our ICS test for S. aureus , we developed the ICS to detect RP-L7/L12 in E. coli and Klebsiella spp. etc., using colloidal gold-labeled antibodies, which are widely used for the construction of ICS tests [ 15 ]. Development of the ICS test was carried out based on the patent US 20190317093A1.…”

“…Because RP-L7/L12 belongs to the 50S ribosome, which is richly expressed in many microbes, and because RP-L7/L12 contains specific sequences for individual bacterial species, ICS tests targeting bacterial RP-L7/L12 could be useful for the rapid diagnosis of various infectious diseases in humans and animals [ 1 , 8 ]. In fact, our previous reports have shown the effectiveness of ICS using RP-L7/L12 for the detection of the causative organism of Staphylococcus aureus mastitis [ 15 ].…”

Development of an Immunochromatographic Strip Using Conjugated Gold Nanoparticles for the Rapid Detection of Klebsiella pneumoniae Causing Neonatal Sepsis

“…ICS have also been developed for the detection of some bacterial pathogens, in particular foodborne pathogens and their toxins [ 46 ]. Development of ICS for the detection of human pathogens such as Mycobacterium tuberculosis [ 47 ], S. aureus [ 26 , 48 ], Helicobacter pylori [ 49 ], H. influenzae [ 50 ], and Neisseria gonorrhoeae [ 51 ] has been attempted, yet not as extensively as the previous applications.…”

A Novel Surface-Exposed Polypeptide Is Successfully Employed as a Target for Developing a Prototype One-Step Immunochromatographic Strip for Specific and Sensitive Direct Detection of Staphylococcus aureus Causing Neonatal Sepsis

“…A very recent study reported the development of an ICS for detecting S. aureus in milk samples [ 63 ]. However, they used a ribosomal protein as a target.…”