Validation of droplet digital PCR for the detection and absolute quantification of Borrelia DNA in Ixodes scapularis ticks


We evaluated the QX200 Droplet Digital PCR (ddPCR™, Bio-Rad) system and protocols for the detection of the tick-borne pathogens Borrelia burgdorferi and Borrelia miyamotoi in Ixodes scapularis nymphs and adults collected from North Truro, Massachusetts. Preliminary screening by nested PCR determined positive infection levels of 60% for B. burgdorferi in these ticks. To investigate the utility of ddPCR as a screening tool and to calculate the absolute number of bacterial genome copies in an infected tick, we adapted previously reported TaqMan®-based qPCR assays for ddPCR. ddPCR proved to be a reliable means for detection and absolute quantification of control bacterial DNA with precision as low as ten spirochetes in an individual sample. Application of this method revealed the average carriage level of B. burgdorferi in infected I. scapularis nymphs to be 2291 spirochetes per nymph (range: 230–5268 spirochetes) and 51,179 spirochetes on average in infected adults (range: 5647–115,797). No ticks naturally infected with B. miyamotoi were detected. The ddPCR protocols were at least as sensitive to conventional qPCR assays but required fewer overall reactions and are potentially less subject to inhibition. Moreover, the approach can provide insight on carriage levels of parasites within vectors.



While this is a great start, there are many other pathogens to be concerned with besides Borrelia burgdorferi (Bb), the agent of Lyme Disease:, and there are many more besides the six mentioned in this article.  Bartonella has not been proven conclusively to be transmitted by ticks, but it is highly likely.  It is also a frequent coinfection and can be spread by:

Arthropod vectors including fleas and flea feces, biting flies such as sand flies and horn flies, the human body louse, mosquitoes, and ticks; through bites and scratches of reservoir hosts; and potentially from needles and syringes in the drug addicted. Needle stick transmission to veterinarians has been reported. There is documentation that cats have received it through blood transfusion. 3.2% of blood donors in Brazil were found to carry Bartonella in their blood. Bartonella DNA has been found in dust mites. Those with arthropod exposure have an increased risk, as well as those working and living with pets that have arthropod exposure. 28% of veterinarians tested positively for Bartonella compared with 0% of controls. About half of all cats may be infected with Bartonella – as high as 80% in feral cats and near 40% of domestic cats. In various studies dogs have close to a 50% rate as well. Evidence now suggests it may be transmitted congenitally from mother to child – potentially leading to birth defects.

There is much work yet to be done.