IDSA 2014 Philadelphia

Surveillance for in and Mice in British Columbia Muhammad G. Morshed 1,2, Min-Kuang Lee 1, Stephanie Man 1, Keerthi Fernando 1, Quantine Wong 1, Kristoffer Jensen 1, Brian Kahnamelli 1, Patrick Tang 1,2, David M. Patrick 2,3, Sunny Mak2 and Bonnie Henry2,3 1. BCCDC Public Health Microbiology Reference Laboratory, Vancouver, B.C., Canada; 2. University of British Columbia, Vancouver, B.C., Canada; 3. B.C. Centre for Disease Control, Vancouver, B.C., Canada

Background Mice captured in the 2013 field study Results Historically, Lyme disease prevalence has been low in the Pacific Northwest of North America. To No ticks were found by flagging during this study period; however, a total of 467 ticks of different dispel some of the public misconceptions regarding the ability of laboratories to identify Lyme 250 developmental stages were retrieved from 237 mice. Three species of mice (Peromyscus maniculatus, disease, we studied the prevalence of B. burgdorferi in the (vector) and Peromyscus parvus, and Reithrodontomys megalotis) and four species of ticks (Ixodes pacificus, Deer mouse Peromyscus maniculatus mouse (host) in British Columbia (B.C.). Ixodes angustus, Ixodes soricis, Dermacentor andersoni) were identified morphologically. 200 Nineteen mice were serologically confirmed to have antibodies against Borrelia burgdorferi from Culture Positive a Year Specimen Culture Positive Tested Number eight areas sampled. The proportion and 95% confidence interval for Mouse Serology Positive are Rate 150 estimated to be 7.98% (4.87%, 12.19%). Male 1993-1996 Ticks 40 10,056 0.40% For the molecular results, no Borrelia DNA was detected in mouse tissues (n=474). However, three Female

1993-1996 Mice 25 2,766 0.90% Numbers 100 tick pools were positive for Borrelia burgdorferi. Of the positive tick pools, the corresponding mice 1997-2007 Ticks 30 8,602 0.35% were also serologically positive. A subset of ticks tested by two laboratories had identical results 1997-2007 Mice 5 833 0.60% The Great The western (Center for Disease Control and Prevention, Fort Collins, USA) by different methods. The positivity Basin pocket 50 harvest rate on ticks and 95% confidence interval were estimated to be 0.64% (4.87% and 12.19%) . The Serology Positive mouse mouse Year IFA Positive WB Positive Tested Sera Number three positive tick pools were from different sampling areas: Coquitlam, Squamish, and Cultus Lake; b Rate 0 all areas where positive ticks had been detected in the past. 2005-2007 34 6 164 3.66% Peromyscus maniculatus Perognathus parvus Reithrodontomys megalotis Male 114 3 5 Table 1: Host and vector background of Lyme Disease in British Columbia between 1993 and 2007. Detection of Borrelia burgdorferi from ticks and deer mice population (a); anti-Borrelia Serology Tests on deer mice (b). Female 99 8 8 Species

25 Table 2: Three species of mice were identified from the field work trapped mice. The majority of the mouse population collected were deer

Endemic Travel mice (Peromyscus maniculatus), which serve as the main vector for Lyme disease in British Columbia. 20

15

10 Adult Adult

Number of cases Number Larvae Nymph Total Female Male 5

0 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 Ixodes Year reported 258 136 9 0 403 pacificus Figure 1: Endemic and travel-related cases of Lyme Disease in British Columbia between 1997-2011. Dermacentor 25 16 1 0 42 Methods andersoni Ixodes Field Work 12 6 3 0 21 angustus Mice were trapped from 11 different jurisdictions in British Columbia in three groups of 50 traps. Ticks were collected by both the flagging/dragging method and retrieval from captured mice. All ticks Ixodes 0 0 1 0 1 and mouse organs will be tested for the presence of the Lyme disease causing pathogen Borrelia soricis British Columbia | 2013 burgdorferi. The conducting of field work and test processes were reviewed by the following bodies: Figure 2: Four species of ticks were retrieved from trapped mice. The majority of the ticks were Ixodes pacificus, which serves as the main vector for Lyme disease Figure 4: Surveillance was performed in Belcarra, Burnaby, Coquitlam, Cranbrook, Cultus Lake, Duncan, Nanaimo, in British Columbia. Unlike passive surveillance, most ticks collected from the field (active surveillance) were of the larval and nymph stages. Okanagan (Kelowna), Sechelt, Squamish, and West Vancouver. The red pin symbols indicate positive tick pool sites. • Ecology review – Ministry of Forests, Lands and Natural Resource Operations • Ethics review – Canadian Council on Care Conclusion • Biosafety review – University of British Columbia Tick Mouse Serology Studies Ixodes pacificus – larvae pool Female P. maniculatus The positivity rate of B . burgdorferi in the B . C . Ixodes tick population was found to be 0.64%, Immunofluorescence assay (IFA) against Borrelia burgdorferi B31 and Western blots (Trinity suggesting very low prevalence. The exposure rate in the mouse population to B. burgdorferi was Biotech/MarDx Diagnostics) were performed on mice serum to determine the presence of B. Ixodes pacificus – nymph pool Female P. maniculatus determined to be 7.98%, suggesting that the infestation rate is also low in the host population. This burgdorferi antibodies. Ixodes pacificus – nymph pool Male R. megalotis observation may explain the continual low prevalence of Lyme disease in B.C. DNA Extraction Ticks (pooled by stage of ticks per mouse) and mouse tissues (heart and bladder) were extracted according to the QIAGEN Blood and Tissue DNA Extraction Kit protocol. Specimens were eluted in References 95% confidence interval 95% confidence interval 100µL AE buffer and stored at -20 C until PCR was performed. 0 % 7.98% Lower Limit: 4.87% 0.64% Lower Limit: 0.13% • Comparison of PCR methods and culture for the detection of Borrelia spp. in patients with erythema migrans. Upper Limit: 12.19% TaqMan Real-Time PCR Upper Limit: 1.87% Clin Microbiol Infect. 2008 Jul;14(7):653-8. This assay was divided into separate screening and confirmatory TaqMan real-time PCR tests on the • Laboratory diagnostic techniques for patients with early Lyme disease associated with erythema migrans: a ABI TaqMan 7500 platform. The screening component consisted of two separate real-time PCR tests comparison of different techniques. Clin Infect Dis. 2001 Dec 15;33(12):2023-7. that were ran in parallel. One test was for Borrelia DNA detection (targeting the 23s ribosomal gene • Diversity of Borrelia burgdorferi sersu lato evidenced by restriction fragment length polymorphism of rrf (5S)-rrl for all Borrelia spp.), while the other was for inhibition detection (targeting known concentrations of (23S) intergenic spacer amplicons. International Journal of Systematic Bacteriology, Oct. 1994, p. 743-752. * synthetic oligo-nucleotides). Any inhibition test-negative specimens were repeated from the extraction • Lyme disease in British. Columbia: Are we really missing an epidemic? step onwards. All screening positive specimens were then subjected to a confirmatory test (targeting Figure 3a: Three tick pools, two Ixodes pacificus nymph pools and one Ixodes pacificus larvae Figure 3b: Nineteen mice were serologically positive against B.C.MJ, Vol. 53, No. 5, June 2011, page(s) 224-229 Borrelia burgdorferi. the B. burgdorferi specific ospA gene). All the assays used TaqMan probes with a 3’-minor- pool, were positive on real-time PCR result (right) . No Borrelia DNA was detected in mice • Extraction and Amplification of Tick DNA for the Detection of Tick-borne Pathogens. National Microbiology groovebinder, non-fluorescent quencher. The cycling conditions for all three assays were as follows, hearts and bladders (left). Laboratory Standard Operating Protocol. 2010 Nov. enzyme activation at 50 C for 2 minutes, initial denaturation for 10 minutes at 95 C, followed by 40 cycles of 95 C for 15 seconds and 60 C for 1 minute.

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