Lactic acid bacteria (LAB) convert carbohydrates into organic acids [mainly lactic acid (LA)], which reportedly have bactericidal activities. Gallibacterium anatis is a Gram-negative bacteria which infects birds, and causes significant economic losses. In this study, we investigated the antibacterial activity of the LA producing, Leuconostoc mesenteroides QZ1178 from Qula (fermented food), against G. anatis, using the Oxford cup method. Our data showed that L. mesenteroides QZ1178 inhibited G. anatis isolates from different origins; however, L. mesenteroides QZ1178 antibacterial activity dropped dramatically at pH 5.5–pH 6. The LA concentration and pH of the liquid broth containing L. mesenteroides QZ1178 after 24 h culture was 29 mg/mL and 3.6, respectively. This concentration (29 mg/mL at pH 3.6) and the antibiotic, cefotaxime (minimum inhibitory concentration (MIC) 2.5 μg/mL) effectively inhibited G. anatis (GAC026) growth as observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Gallibacterium anatis treated with LA exhibited extensive cell surface collapse, increased cell damage, cell membrane disruption, and cytoplasmic leakage, indicative of cell lysis. We suggest L. mesenteroides QZ1178 exerts potential antibacterial effects against the poultry pathogen, G. anatis via LA.

To better understand the prevalence of Gallibacterium anatis in different poultry species, a rapid and accurate method was developed to detect G. anatis using a TaqMan fluorescent quantitative polymerase chain reaction (qPCR). Specific primers and a TaqMan probe were designed based on the reference gtxA gene sequence. The qPCR standard curve showed a good linear relationship, and the method showed good reproducibility, sensitivity, and specificity, indicating its suitability for G. anatis identification and quantitative analysis. A comparison of the detection results in 160 clinical swab samples showed that the detection rate (54.4%) of the qPCR for G. anatis was better than that of two conventional methods: gyrB gene-based qPCR for G. anatis (51.9%) and culture-based identification (34.4%). G. anatis was detected in layer chicken (77.3%), Silkie chicken (72.7%), and duck (27.1%) with relatively high detection rates, whereas dove (8.8%) and quail (3.0%) showed lower detection rates, indicating the different prevalence of G. anatis in different fowl species.

A quantitative PCR (qPCR) assay using SYBR Green I was developed based on the published sequence of the gtxA gene from Gallibacterium anatis. This method produced reliable specificity, sensitivity, and repeatability. The detection rate of Gallibacterium in 181 clinical samples was 36.5% (66/181) by qPCR, which was superior to the detection rate of Gallibacterium-specific PCR (0/181) and an isolation and identification assay (18.2% or 33/181). No association was found between the prevalence of Gallibacterium and the age of the chickens. Gallibacterium infection was detected in one 4-day-old chicken, showing that infection can occur much earlier than the previously stated fourth week of life. Tissue sample analysis showed that Gallibacterium is mainly located in the trachea and ovaries, based on results from three groups of chicken with different health statuses. Furthermore, a titer analysis suggested that Gallibacterium loads in different organs may correlate with different clinical manifestations of disease. Thus, the qPCR assay developed in the present study is useful for identification and quantitative analysis of gtxA-containing Gallibacterium in various tissue samples from birds and for the assessment of the pathogenic mechanisms of Gallibacterium.