Multiplex real-time PCR for the passive surveillance of ticks, tick-bites, and tick-borne pathogens

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Caroline Holland
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1 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

3 TICKS ARE VECTORS OF MANY DISEASES Lyme Disease Q Fever, Colorado Tick Fever Tularemia Relapsing Fever Babesiosis Ehrlichiosis Anaplasmosis

4 Challenge:there are ONE species!

5 Challenge:Many Tick-Borne Pathogens Virus 81, Bacteria 14, Borrelia 17, Parasite: 32 Pathogen # Tested Ticks % Positive Borrelia burgdorferi sensu latu % Anaplasma phagocytophilum % Babesia microti % Bartonella henselae Borrelia lonestari % Francisella tularensis % Ehrlichia chaffeensis % Ehrlichia canis 88 0 Babesia divergens 62 0 Borrelia miyamotoi % Rickettsia rickettsii 580 0

6 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.

7 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

8 DIFFERENT RISKS Lyme Risk Map Election Results

9 Taqman assay, Aligent 3005P, Brilliant III identify 3 tick species and 11 tick-borne pathogens

12 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

13 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

14 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.

17 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.

18 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 (%) /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) /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) /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) /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) /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 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.

19 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.

20 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 % Anaplasma phagocytophilum % Babesia microti % Borrelia miyamotoi % Borrelia lonestari % Francisella tularensis % Ehrlichia chaffeensis % Ehrlichia canis 88 0 Babesia divergens 62 0 Bartonella henselae Rickettsia rickettsii 580 0

21 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.

Vector-Borne Disease Status and Trends

Vector-Borne Disease Status and Trends Vector-borne Diseases in NY 2 Tick-borne Diseases: Lyme disease Babesiosis Ehrlichiosis/Anaplasmosis Rocky Mountain Spotted Fever Powassan Encephalitis STARI Bourbon