Biological characteristics of five strains of Brucella isolated in frozen human blood samples

Abstract

Objective To understand the survival situation of Brucella in frozen human blood samples. Methods Blood samples of outpatients with brucellosis were from the Songyuan Center for Disease Control and Prevention. Twenty-eight blood samples of patients who had a symptom of fever and epidemiological contact history were collected, which were kept in tubes containing sodium citrate anticoagulation, and stored in the fridge at -20 ℃. Isolation and culture of pathogen in frozen human blood samples were carried out. Suspected colonies were detected by Grams stain and microscope examination, then further tested by serum agglutination test with Brucella, A and M factors positive serum. The strains were detected with Brucella tautonym primers by multiplex-PCR amplification. Results In the twenty-eight blood samples frozen at different times, colonies were found in five samples after isolation and culture of pathogen for 4 -5 d. The colonies were arranged in transparence, moist or milky lawn; the Grams staining was negative; serum agglutination test with Brucella, A and M factors positive serum were positive. Five strains of Brucella were preliminary considered as Brucella melitensis type 3. The freezing time of Brucella being isolated in five human blood samples was from 6 to 17 d. The results of multiplex-PCR showed that a band of 223 bp could be amplified from strains of Brucella abortus and Brucella melitensis, and that a band of 488 bp could be amplified from strain of Brucella abortus, and 310 bp from Brucella melitensis. The results of five tested strains were identical with those of Brucella melitensis. Conclusion Brucella could be survived in frozen human blood samples for a certain time, and multiplex-PCR amplification with Brucella tautonym primers could provide a basis for diagnose and treatment of Brucella. Key words: Frozen blood; Brucella; Biology; Multiplex-PCR