In vitro transmission of Anaplasma phagocytophilum by artificially infected adult Ixodes ricinus ticks

Abstract

Background Ixodes ricinus is the main vector in Europe of the bacteria Anaplasma phagocytophilum, an emerging zoonotic pathogen that can cause disease in both animals as humans. The risk of obtaining a tick-borne disease depends on a number of factors, including tick transmission speed, which is the time between tick attachment and transmission of the pathogen to the animal’s blood. The main goal of this study was to determine the speed of transmission of A.phagocytophilum infected adult I.ricinus ticks in order to understand how fast an acaracide should work to prevent transmission. Since this study was performed in an in vitro setting at the Utrecht Centre for Tick-borne Diseases (UCTD), opportunity was taken to evaluate the current in vitro feeding protocols of the UCTD as well. Several parameters were studied, of which tick attachment rate, different attachment stimuli, and mortality rate.

Methods The in vitro feeding system used in this study results from adaptations of the protocol described by Kröber and Guerin in 2007. Ticks fed on blood through a silicone membrane in feeding units that contained cow hair or cow odour as attachment stimuli. Variable times a day a blood sample was taken, whereupon the ticks were checked for attachment and mortality macroscopically. After feeding, ticks and blood samples were checked for A.phagocytophilum infection by polymerase chain reaction (PCR) and reverse line blotting (RLB).

Results A.phagocytophilum transmission could not be demonstrated, since the infection rate of the used ticks was much lower than expected or even absent. A mean female tick attachment of 43% was found, which was in line with an earlier research internship at UCTD. The use of both cow hair as cow odour as attachment stimulus showed no difference in attachment rate after one experimental day, but after two experimental days more ticks attached in feeding units with cow hair. Units containing a grid did not show difference in attachment rate in comparison to units without a grid. Male attachment was found in line with previous studies. However, the found mortality rate, ranging between 0-5% per feeding unit, was much lower than anticipated.

Conclusion Two hypothetical explanations for the deviating infection rate of the ticks were discussed: 1) elimination of the bacteria by the tick’s immune system over time, or 2) elimination by faecation. Additional experiments with tick faeces and future research to gain more knowledge about how the tick immune system reacts to A.phagocytophilum infection was recommended. Concerning the in vitro feeding protocols used at the UCTD, also a number of recommendations were made. Checking for presence of tick faeces and checking the feeding units microscopically for the cone-like cement made by attached female ticks could help noticing tick attachment better. To gain more knowledge about attachment stimuli and their effect on tick attachment more research was recommended. Finally, differences in viability of tick colonies were advised to be taken into account in all in vitro feeding studies.