The type of anticoagulant used for plasma collection affects in vitro Rhodococcus equi assays

The efficacy of Rhodococcus equi-specific hyperimmune plasma (HIP) is usually evaluated in vitro. Anticoagulants (AC) used for plasma collection can negatively impact bacterial replication but their effect on R. equi growth has not been evaluated. The aim of this study was to establish the effect that AC routinely used in veterinary medicine (ACD, K₂EDTA, Li Heparin, and Na Citrate) have on in vitro R. equi growth. To assess this, in vitro assays commonly used to test HIP efficacy (direct effect on microorganism and macrophage infection), were performed using each AC and non-treated bacteria.

There was no direct effect of ACD, Li Heparin or Na Citrate on R. equi growth. These AC significantly (p < 0.05) delayed growth for 12 h following opsonization. The number of R. equi colonies after macrophage infection was significantly (p < 0.05) lower 72 h post-opsonization with Na Citrate. K₂EDTA inhibited the formation of R. equi colonies by 12 h in all the assays. In conclusion, AC should be taken into consideration when interpreting in vitro results as their negative effect on bacterial growth may be mistakenly interpreted as HIP efficacy. ACD and Li Heparin appear more appropriate for the selected assays.