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Synopsis Wildlife health comparisons within and across populations and species are essential for population assessment and surveillance of emerging infectious diseases. Due to low costs and high informational yield, hematology is commonly used in the fields of ecoimmunology and disease ecology, yet consistency and proper reporting of methods within these fields are lacking. Previous investigations on various wildlife taxa have revealed noteworthy impacts of the vein used for blood collection on hematology measures. However, the impacts of venipuncture site on bats, a taxon of increasing interest in ecoimmunology and disease ecology, have not yet been tested. Here, we use a long-term study system in western Oklahoma to test the effect of venipuncture site on hematology parameters of the Mexican free-tailed bat (Tadarida brasiliensis) and cave myotis (Myotis velifer), two abundant and representative bat species from the families Molossidae and Vespertilionidae. Between September 2023 and October 2024, we collected paired peripheral blood from both the propatagial and intrafemoral veins in 25 individuals per species. We then quantified total red and white blood cells, reticulocyte counts, and leukocyte differentials and used generalized linear mixed models to compare parameters among venipuncture sites within and between bat species. Overall, venipuncture site had no effect on any hematology parameters; however, we revealed small differences in neutrophil and lymphocyte proportions between veins among the species. By contrast, we detected significant species-level differences in most cell measurements, which we propose could be explained by life-history strategy and phylogenetic differences. We encourage continued testing of additional venipuncture sites, and of the same venipuncture sites on different species, on hematology and other health metrics used in ecoimmunology and disease ecology. Lastly, we emphasize the importance of thorough method reporting in publications to enable transparent comparisons and accounting for even small sampling-based artifacts. All future efforts are especially important for bats to improve conservation monitoring, ecosystem services estimations, and their association with emerging infectious diseases. 

ABSTRACT Bacterial pathogens remain poorly characterized in bats, especially in North America. We describe novel (and in some cases panmictic) hemoplasmas (10.1% positivity) and bartonellae (25.6% positivity) across three colonies of Mexican free-tailed bats (Tadarida brasiliensis), a partially migratory species that can seasonally travel hundreds of kilometers. Molecular analyses identified three novelCandidatushemoplasma species most similar to another novelCandidatusspecies in Neotropical molossid bats. We also detected novel hemoplasmas in sympatric cave myotis (Myotis velifer) and pallid bats (Antrozous pallidus), with sequences in the latter 96.5% related toCandidatusMycoplasma haematohominis. We identified nineBartonellagenogroups, including those in cave myotis with 96.1% similarity toCandidatusBartonella mayotimonensis. We also detectedBartonella rochalimaein migratory Mexican free-tailed bats, representing the first report of this human pathogen in the Chiroptera. Monthly sampling of migratory Mexican free-tailed bats during their North American occupancy period also revealed significant seasonality in infection for both bacterial pathogens, with prevalence increasing following spring migration, peaking in the maternity season, and declining into fall migration. The substantial diversity and seasonality of hemoplasmas and bartonellae observed here suggest that additional longitudinal, genomic, and immunological studies in bats are warranted to inform One Health approaches. IMPORTANCEBats have been intensively sampled for viruses but remain mostly understudied for bacterial pathogens. However, bacterial pathogens can have significant impacts on both human health and bat morbidity and even mortality. Hemoplasmas and bartonellae are facultative intracellular bacteria of special interest in bats, given their high prevalence and substantial genetic diversity. Surveys have also supported plausible zoonotic transmission of these bacteria from bats to humans, includingCandidatusMycoplasma haematohominis andCandidatusBartonella mayotimonensis. Greater characterization of these bacteria across global bat diversity (over 1,480 species) is therefore warranted to inform infection risks for both bats and humans, although little surveillance has thus far been conducted in North American bats. We here describe novel (and in some cases panmictic) hemoplasmas and bartonellae across three colonies of Mexican free-tailed bats and sympatric bat species. We find high genetic diversity and seasonality of these pathogens, including lineages closely related to human pathogens, such asBartonella rochalimae.