We investigated the effect of a synthetic complement peptide C3a on the outcome of Brucella abortus 544 infection in a murine macrophage cell line RAW264.7 cell. First, we determined the highest non-cytotoxic concentration of the peptide in the cell line. We also found that the peptide significantly increased the growth of the bacteria at 8 and 24 h. Although the number of bacterial CFU was also elevated at 48 and 72 h, the increases were not significant as compared to controls. We further investigated the effect of C3a peptide on the growth of Brucella by pre-incubating the peptide at various temperatures and found that the effect was reversed at 24 h post-incubation suggesting that incubation of peptide at high temperatures including 65°C or 95°C could inactivate its action. This also could indicate the beneficial effect of high temperature during infection. Although several studies reported the inhibitory effect of different antimicrobial peptides including C3a, the present study preliminarily revealed that it had no positive contribution on the control of B. abortus 544 infection in vitro and indirectly to its receptor, CD88, which belongs to GPCR. Moreover, the encouraged further exploration of the effect of other similar peptides would be performed for the purpose of finding Brucella-host cell interaction for the control of disease progression.

INTRODUCTION
MATERIALS AND METHOD
Reagents and materials
C3a peptide preparation
Cell culture and growth condition
Bacterial strain and growth condition
Cytotoxicity assay
Bactericidal assay
Internalization and intracellular replication assay
Nitric oxide (NO) assay
Statistical analysis
RESULTS AND DISCUSSION
ACKNOWLEDGEMENTS

• Alisha Wehdnesday Bernardo Reyes(Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Jinju, 52828, Republic of Korea, Department of Veterinary Paraclinical Sciences, College of Veterinary Medicine, University of the Philippines Los Baños, College, Laguna, 4031, Philippines)
• Heejin Kim(Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Jinju, 52828, Republic of Korea)
• Tran Xuan Ngoc Huy(Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Jinju, 52828, Republic of Korea)
• Trang Thi Nguyen(Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Jinju, 52828, Republic of Korea)
• Wongi Min(Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Jinju, 52828, Republic of Korea)
• Hyun Jin Kim(Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Jinju, 52828, Republic of Korea)
• Hu Jang Lee(Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Jinju, 52828, Republic of Korea)
• Suk Kim(Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Jinju, 52828, Republic of Korea) Corresponding author