In NEON collection events in 2014–2024, Borrelia burgdorferi-positive Ixodes scapularis nymphs had 3–6 times higher odds of Babesia microti, Ehrlichia muris and Anaplasma, but not of B. miyamotoi

Question: do questing blacklegged tick nymphs (Ixodes scapularis) carry two pathogens together more often than independence predicts, also within one plot visit? Data: 3,842 nymphs from 11 NEON sites (2014–2024), each tested one by one with one PCR panel, from 698 collection events (one plot on one day). Positives: B. burgdorferi s.l. 762, A. phagocytophilum 118, Babesia microti 109 (two sites only), B. miyamotoi 56, Ehrlichia muris eauclairensis 25. Method: for each pathogen pair, exact conditional (hypergeometric) tests and Mantel–Haenszel odds ratios (OR) within collection events, with a plan written before the stratified results. The plan predicted excess for the mouse-reservoir pathogens and no excess for B. miyamotoi. Result: within events, B. burgdorferi occurred together with B. microti in 63 nymphs (33.2 expected; OR 5.3, 95% CI 3.2–8.8), with E. muris in 17 (7.9; OR 6.0, 2.3–15.6) and with A. phagocytophilum in 48 (29.5; OR 2.9, 1.8–4.6), but with B. miyamotoi in only 9 (11.3; OR 0.68, 0.29–1.57). At Treehaven (Wisconsin), 15% of B. burgdorferi-positive nymphs also had B. microti, against 3.3% of negative nymphs. Differences in prevalence between events explained about 25–28% of the excess that a pooled table shows. The results did not change without 2014–2015 or without any one site. The null B. miyamotoi pair, tested on the same platform, argues against a general laboratory artifact. Secondary: an independent re-analysis reproduced the NEON tick density trends of SCI publication 0c852071 (41 series, r = 0.997), but for 6 series the CI-excludes-zero result depended on the standard-error method; a plot-level randomization test at two invaded Virginia sites could detect only very large effects of the Asian longhorned tick on native ticks. Limits: B. microti and E. muris occur at only two sites; the panel does not separate A. phagocytophilum variants; questing-nymph data do not show which host gave the pathogens.

Ixodes scapularis · co-infection · Borrelia burgdorferi · Babesia microti · Anaplasma phagocytophilum · Ehrlichia muris eauclairensis · Borrelia miyamotoi · NEON · Mantel-Haenszel · tick-borne disease · Haemaphysalis longicornis · replication