Multiplex real-time PCR for the passive surveillance of ticks, tick-bites, and tick-borne pathogens Guang Xu, Stephen Rich Laboratory of Medical Zoology University of Massachusetts Amherst
TICKS ARE VECTORS OF MANY DISEASES Lyme Disease Q Fever, Colorado Tick Fever Tularemia Relapsing Fever Babesiosis Ehrlichiosis Anaplasmosis
Challenge:there are ONE species!
Challenge:Many Tick-Borne Pathogens Virus 81, Bacteria 14, Borrelia 17, Parasite: 32 Pathogen # Tested Ticks % Positive Borrelia burgdorferi sensu latu 9825 27.20% Anaplasma phagocytophilum 7083 4.32% Babesia microti 7096 3.61% Bartonella henselae 171 0 Borrelia lonestari 369 1.63% Francisella tularensis 516 0.58% Ehrlichia chaffeensis 425 0.71% Ehrlichia canis 88 0 Babesia divergens 62 0 Borrelia miyamotoi 686 8.02% Rickettsia rickettsii 580 0
Challenge: A single tick bite can transmit multiple pathogens Tick-borne diseases in the United States include Rocky Mountain spotted fever, Lyme disease, ehrlichiosis, tularemia, babesiosis, Colorado tick fever, and relapsing fever.
WHY TEST A TICK? Doctors need to know: tick species, feeding stage, pathogen No quick diagnosis for Lyme Disease Co-infection Know your risk Know your prevention Tick Testing A Positive Result Disease Diagnosis Lyme Disease
DIFFERENT RISKS Lyme Risk Map Election Results
Taqman assay, Aligent 3005P, Brilliant III identify 3 tick species and 11 tick-borne pathogens
American Dog Tick Duplex 1: Tick DNA + Borrelia Duplex 2: Francisella tularensis + Rickettsia rickettsii Duplex 1: Lone Star Tick Tick DNA + Borrelia Duplex 2: Borrelia lonestari + Ehrlichia chaffeensis
Tick DNA + Borrelia + Tick DNA + Borrelia -
the Lone Star tick is now a residential in Massachusetts Deer tick populations were concentrated in Cape Cod, the eastern half of the state outside of the Boston metropolitan area, parts of Franklin and Hampshire counties along the Quabbin Reservoir watershed, and southwestern Berkshire county.
The largest proportion of victims of tick bites were ages 0 to 9 years. Nymphal ticks preferred attaching to the lower extremities of their hosts and adult ticks preferred the head area.
Table 3. Pathogen prevalence and coinfection in I. scapularis ticks found on humans *. Year Bo(%) A(%) Ba(%) Bo+A (%) Bo+ Ba (%) A+Ba (%) Bo+A+Ba (%) 2006 15/67 2/67 1/67 0/67 1/67 0/67 0/67 (22.3) (3.0) (1.5) (0.0) (1.5) (0.0) (0.0) 2007 We 48/140 found overall 3/140 infection rates 3/140 of B. burgdorferi, 0/140 A. phagocytophilum 1/140 0/140 and B. microti 0/140 in human biting ticks (34.3) was 29.6%, 4.6% (2.1) and 1.8%, (2.1) respectively. (0.0) We found (0.7) 0.4%, 1.0% (0.0) and 1.8% of ticks (0.0) were coinfected 2008 with 77/262 two pathogens, 14/2620.3% of ticks 4/262 had triple 4/262 coinfection. 1/262 2/262 1/262 (29.4) (5.3) (1.5) (1.5) (0.4) (0.8) (0.4) 2009 92/307 18/307 8/307 9/307 7/307 3/307 2/307 (30.0) (5.9) (2.6) (2.9) (2.3) (1.0) (0.7) 2010 55/227 8/227 1/227 0/227 0/227 0/227 0/227 (24.2) (3.5) (0.4) (0.0) (0.0) (0.0) (0.0) 2011 122/416 23/416 12/416 9/416 6/416 1/416 1/416 (29.3) (5.5) (2.9) (2.2) (1.4) (0.2) (0.2) 2012 169/536 22/536 7/536 13/536 4/536 1/536 1/536 (31.5) (4.1) (1.3) (2.4) (0.7) (0.2) (0.2) Total 578/1955 90/1955 36/1955 35/1955 20/1955 7/1955 5/1955 (29.6) (4.6) (1.8) (1.8) (1.0) (0.4) (0.3) A=A. phagocytophilum, Ba=B. microti, Bo=B. burgdorferi.
B. burgdorferi was widely distributed, but A. phagocytophilum and B. microti were mainly found at the eastern half of the state. Our passive surveillance may also serve as an early warning system for tick and tickborne pathogens.
The first human cases of Borrelia miyamotoi infection were reported in Russia in 2011; Diagnosis currently relies on PCR. Rare tick-borne disease? <8% Found Borrelia lonestari only in Amblyomma ticks, <1.7% Francisella tularensis and Ehrlichia chaffeensis <1% Pathogen # Tested Ticks % Positive Borrelia burgdorferi sensu latu 9825 27.20% Anaplasma phagocytophilum 7083 4.32% Babesia microti 7096 3.61% Borrelia miyamotoi 686 8.02% Borrelia lonestari 369 1.63% Francisella tularensis 516 0.58% Ehrlichia chaffeensis 425 0.71% Ehrlichia canis 88 0 Babesia divergens 62 0 Bartonella henselae 171 0 Rickettsia rickettsii 580 0
Tricks and Tips Your real sample may not be a good start material: tick vs IDT gblocks $89 Use ZEN Internal Quencher: lower background and long probe (20-40bp) Duplex assay: know abundance of your gene. Tick vs Borrelia, by limiting the amount of tick primer and probe. CONTROLS and CONTROLS: setup internal control (tick DNA); spike controls; extraction controls Sensitivity and Contamination: balance Brilliant II or III: 40min; a slow protocol may be stable; Brilliant III tolerant more blood.