Spectrophotometry for rapid determination of the concentration of Avibacterium paragallinarum

Abstract:

[Background] The infection with Avibacterium paragallinarum (Apg) results in an acute upper respiratory disease known as infectious coryza in chickens. In general, the disease exhibits a short incubation period, an acute onset, and a high degree of infectivity, thereby causing substantial economic losses of breeding farms. [Objective] To assess the feasibility of spectrophotometry (SP) as a potential substitution of plate colony count (PCC) in determining the concentration of Apg. [Methods] The Apg precipitate was suspended in tryptone soy broth (TSB) or sterile physiological saline solution (SPSS), as two sets of diluents respectively. The absorbance (OD) values of the bacterial suspensions at different dilutions were determined by SP and the viable cell count was determined by PCC. With OD values measured at different wavelengths (450, 540, 600 and 650 nm) as the independent variables and the viable cell counts as the dependent variables, the standard curves and linear equations were established. Set up a tryptone soy broth (TSB) group and a sterile physiological saline solution (SPSS) group, using TSB and SPSS as suspensions and dilutions. Use SP and PCC methods to measure the suspension of different dilutions of Streptococcus parahaemolyticus, and compare and analyze the effect of the two suspensions on the correlation between OD value and bacterial concentration through standard curves and linear equations. The OD values of the bacterial suspension samples collected at different culture stages (10, 12, 14, 16 and 18 h) were measured and substituted into the linear equation to obtain the concentration of the bacterial suspension. PCC was carried out at the same time. SPSS 17.0 was used for statistical analysis to verify the applicability of SP. [Results] The OD value measured at 600 nm (OD₆₀₀) showed the best linear relationship with the PCC results (R²>0.995). At this wavelength, the OD values measured in the TSB and SPSS groups were not different from the PCC results (R²_difference=0.000 5), and the TSB group was superior to the SPSS group (R²_TSB=0.998 9> R²_SPSS=0.998 4). The results of SP and PCC showed no differences at the incubation time points of 10, 12, and 14 h (P>0.05) but had differences at the time points of 16 and 18 h (P<0.05). [Conclusion] During the logarithmic and stationary phases, SP could rapidly measure OD₆₀₀ to determine the concentration of Apg.