Detection and quantification of Erysipelothrix rhusiopathiae in blood from infected chickens – addressing challenges with detection of DNA from infectious agents in host species with nucleated red blood cells

Abstract

Purpose The present study aimed to establish pretreatment protocols as well as real-time and droplet digital polymerase chain reaction (PCR) methodologies to detect and quantify Erysipelothrix rhusiopathiae (ER) DNA in blood samples from infected chickens, as tools for routine diagnostics and monitoring of experimental infections. Chicken blood is a problematic matrix for PCR analysis because nucleated erythrocytes contribute large amounts of host DNA that inhibit amplification.Methodology Using artificially spiked samples of fresh chicken blood, as well as blood samples from three experimental infection studies, the performance of pretreatment protocols, including choice of blood stabilization agent, centrifugation speeds and Ficoll gradient separation, was evaluated. The results were compared with those from traditional culture-based protocols combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).Results/Key findings Simple preparations producing cell-free samples performed well on artificial spike-in samples, providing high sensitivity. However, performance was poor in clinical samples or artificial samples where the bacteria were incubated for 4 h or more in fresh blood prior to DNA extraction. In these samples, a Ficoll separation protocol that creates samples rich in lymphocytes, monocytes and thrombocytes prior to DNA extraction was far more effective.Conclusions Our results indicate that ER bacteria undergo rapid phagocytosis in chicken blood and that analysis of a blood fraction enriched for phagocytic cells is necessary for reliable detection and quantification. The presented results explain the poor performance of PCR detection reported in previously published experimental ER infection studies, and the proposed solutions are likely to have broader implications for PCR-based veterinary diagnostics in non-mammalian host species such as poultry and fish.

Immune responses upon experimental Erysipelothrix rhusiopathiae infection of naïve and vaccinated chickens

“…In the present study vaccination of chickens induced a more effective elimination of ER since the recovery of bacteria in blood was clearly reduced compared to that in naïve chickens [20]. Moreover, the acute innate responses in vaccinated chickens were of much shorter duration compared to those in naïve chickens.…”

“…It thus seems likely that these cells serve a role in the chicken defence against ER infection, both as sentinel cells and regulators of immune responses and as potential effector cells killing ER bacteria. We have indeed shown that when ER was incubated in chicken blood in vitro ER DNA was primarily detected in a leukocyte preparation of blood cultures and not free in plasma and that a significant amount of the bacteria recovered in chicken blood during the acute phase of ER infection were intracellular in leukocytes [20]. Hence, the heterophils may have a role phagocytosing ER in analogy with what has been observed for their mammalian counterparts where phagocytosis and killing of ER in vitro has been shown in porcine and murine neutrophils [14].…”

“…In EDTA-stabilised blood samples growth of ER colonies was quantified by direct culture and ER DNA was detected and quantified by PCR assays as previously described [ 20 ]. At post mortem examination sterile spleen samples were collected and placed in selective sodium-azide crystal-violet broth (# B321051/5, National Veterinary Institute; containing 5 μg/mL crystal-violet and 0.2 mg/mL sodium-azide) and incubated for ER enrichment for 48 h at 37 °C.…”

“…No other chicken showed any clinical signs of disease during the experiment and the mean weights did not differ significantly between groups during the experiment (group mean weights ± 95% CI, n = 13, for day −3: uninfected 233 ± 13 g; "naïve" 237 ± 14 g; "vaccinated" 235 ± 8 g; for day 15: uninfected 442 ± 19 g; "naïve" 427 ± 20 g; "vaccinated" 442 ± 13 g). Results on detection of live ER by direct culture of blood and detection of bacterial DNA in blood by PCR assays from this experimental infection have been described in detail in [20] (infection trial 3). In summary, on day 1 after infection one of seven sampled chickens and on day 3 after infection all six sampled chickens in the "naïve" group were positive for ER in blood by culture.…”

“…The trend line was calculated using the curve fit logarithmic mode in the software KaleidaGraph, version 4.1.0 (Synergy Software) and equation and R-value are shown in the figure. Results on bacterial counts have previously been presented in [20]. For details see "Materials and methods" section.…”