Isolation, Purification And Identification Of Tick Symbiont And Platelet Aggregation Inhibitory Active Substance

Ticks are blood-sucking arthropods parasite on the body of vertebrates, and they can transmit many diseases. To combat vertebrate hemostasis, inflammation and other defense systems, ticks have evolved a variety of mechanisms, such as anti-hemostatic, anti-inflammatory, immunoregulation, vasodilation and other factors in salivary glands.Symbiotic bacteria have been found in blood-sucking arthropods, classified in two broad categories, namely primary and secondary symbionts. Some of the blood-sucking arthropods secret adenosine and AMP (Adenosine monophosphate) by salivary glands with vasodilatory and anti-platelet activities and probably help the blood-sucking arthropods to obtain a blood meal.We isolated a bacterial strain named TG4 from tick mid-gut with anti-platelet aggregation activity. The systematic taxonomy of the TG4 have been done using traditional technology including morphological and cultural characteristic observation, physiological and chemical tests, analysis of G+C content, and 16S rDNA sequence analysis, etc. TG4 is Gram-positive tetrads, catalase positive, acid-producing negative, nitrate reduction negative. The G+C content in its 16S rDNA sequence is 69.8%, similar to Micrococcus terreus. Phylogenetic tree analysis showed that TG4 and other members of the genus Micrococcus are clustered together.16S rDNA sequence analysis showed that TG4 has a homology of 99% with M. terreus and indicated that TG4 belongs to the genus of Micrococcus on the system taxonomy. Isolation, purification and identification of platelet inhibitor secreted from this bacterium have been done by using methods including extraction, silica gel column chromatography, gel filtration chromatography, reverse phase high performance liquid chromatography (RP-HPLC), thin layer chromatography (TLC), 1H and 13C nuclear magnetic resonance (NMR), electrospray ionization mass spectrometry (ESI-MS). The platelet inhibitor was determined as adenosine. The half maximal inhibitory concentration of platelet aggregation induced by ADP is 0.12 μg/mL. The yield of adenosine in the bacterium culture is 18 mg/L. This is the first report of symbiotic bacterium, which secrets adenosine and probably contributes the blood feeding of ticks.