In vitro and in vivo transmission of Babesia bovis by Rhipicephalus (Boophilus) microplus ticks

Background: This study aimed to determine whether laboratory colonies of Rhipicephalus microplus ticks were infected with various pathogens and also whether nymphs of the same colony were able to transfer these pathogens during in vitro feeding in a “ticks up” system. \nMethods: This study consisted of three parts; molecular detection of pathogens in the laboratory colonies of R. microplus larvae and female ticks maintained at UCTD with PCR and RLB, in vivo feeding and transmission of pathogens to cattle, and in vitro feeding and pathogen transmission of R. microplus nymphs on artificial membranes in the novel developed “ticks up” system. \n \nResults: A total of 74 larval batches and 18 female ticks were used for DNA-extraction, PCR and RLB. Twelve of the 74 larval batches were positive for various pathogens (16,2%). Seven samples were positive for Babesia bovis (9,5%), one for Babesia gibsoni (1,4%) and Babesia bigemina (1,4%). Two samples were positive for Babesia canis (2,7%) and one sample was Babesia catch-all positive (1,4%). \nIn the in vivo experiments, R. microplus larvae from the UCTD laboratory colony were fed on two cows and two sheep. During the experiment, one cow developed fever after nine days of tick feeding. Blood samples were taken each day the rectal temperature was above the reference value. Blood smears were all negative on blood parasites by light microscope inspection; however, all blood tested positive for B. bovis by PCR. \nIn the in vitro experiments, larvae fed on two cows for 15 and 17 days were removed when they were moulted into their nymphal life stage and used in a series of two in vitro feeding experiments. In experiment 1, R. microplus ticks attached and fed on bovine blood through artificial membranes with attachment rates up to 90% at 24h and 75% after 72 hours. In experiment 2, the attachment rates and mortality rates were lower. Daily samples were taken from the feeding medium to monitor pathogen transmission with PCR and RLB. Transmission was found in several blood samples during the first testing round. Babesia rossi and Theileria buffeli were the main pathogens present. T. buffeli appeared for the first time in the blood samples after 24 hours. B. rossi appeared after 48, 72 and 120 hours in various tube samples. B. bovis was found in one blood sample at 96h and remained positive after retesting, whereas the other infections could not be confirmed. \n \nConclusion: The overall infection rate of the tick colony was estimated at 0.012%. B. bovis was the dominant pathogen found in the colony check, which was confirmed by transmission to a bovine as well as transmitted by the ticks during in vitro feeding.