Abstract
BackgroundKlebsiella pneumonia, a Gram-negative bacterium belonging to the genus Enterobacter, causes many human and livestock diseases. Notably, infected goats may develop pneumonia, septicemia, which can lead to occasional death, resulting in great economic losses in goat-farming industry. However, there are little systematic methods for detection of goat Klebsiella pneumoniae in livestock production.ResultsIn this study, we developed a Klebsiella pneumoniae goat polyclonal antibody and established an indirect ELISA method to detect the Klebsiella pneumoniae. After screening and optimizing the conditions for detection, we determined the optimal working dilutions of the coated-bacterial antigen, the polyclonal antibody, and the enzyme-labeled secondary antibody that were 1:800 (2.99 × 107 CFU/ml), 1:6400, and 1:5000, respectively. The optimal condition of coating and blocking were both 4 °C for 12 h. The optimal dilution buffers of bacterial antigen, the antibodies, and the blocking buffer were 0.05 mol/L carbonate buffer, 1% BSA phosphate buffer, and 1.5% BSA carbonate buffer, respectively. The cut-off value was determined to be 0.28, and the analytical sensitivity was 1:800 (dilution of a positive sample). Furthermore, there was no cross-reaction between the coated antigen and goat serum positive for antibodies against other bacteria, indicating that indirect ELISA could detect Klebsiella pneumoniae specifically in most cases. The average coefficients of variation of intra-assay and inter-assay were 4.37 and 5.17% indicating favorable reproducibility of indirect ELISA. In the detection of clinical veterinary samples, the positive rate of indirect ELISA was 6.74%, higher than that of conventional agglutination assays.ConclusionsTaken together, we successfully established an indirect ELISA method for detecting antibodies against Klebsiella pneumoniae in goats, which can be applied in production.
Highlights
Klebsiella pneumonia, a Gram-negative bacterium belonging to the genus Enterobacter, causes many human and livestock diseases
Our results demonstrated that the Klebsiella pneumoniae goat polyclonal antibody can be used as the positive control serum for the detection of Klebsiella pneumoniae
The polyclonal antibody and the Horseradish peroxidase (HRP)-conjugated Affinipure Rabbit Anti-Goat IgG were incubated for 60 min and 45 min, respectively (Fig. 2f and h)
Summary
Klebsiella pneumonia, a Gram-negative bacterium belonging to the genus Enterobacter, causes many human and livestock diseases. In China, along with increasing demand of goat livestock, goat husbandry industry has gradually adopted high-density breeding and fast fattening using concentrated feeding pattern [2] In this process, overuse and even abuse of antibiotics contribute to an increase in the number of outbreaks of Klebsiella pneumoniae [3]. There are novel methods for the detection of Klebsiella pneumoniae, which are demonstrated to be more accurate and sensitive, like automatic bacteria identification system and mass spectrometry [9]. In addition to phenotypic and genotypic identification of goat source Klebsiella pneumoniae, agglutination assay is occasionally used for antibody detection, but it has a main disadvantage of poor sensitivity. Indirect enzyme-linked immunosorbent assay (ELISA), one of the serological methods, is efficient, sensitive, specific, easy to operate, and can be scaled-up to apply to a large number of serum samples [12]
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