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Abstract Evidence for the earliest invasion of the marine realm by mammals was previously restricted to Eocene (48.6–37.8 Ma) skeletal remains. We report incontrovertible ichnofossil evidence for brackish-water habitat use by at least two mammalian species in southern Wyoming during the late Paleocene (58 Ma). These are the first Paleocene mammal trackways recorded in the United States and only the fourth documented in the world. Multiple tracks preserved in restricted marine deposits represent animals repeatedly walking across submerged to partially emergent tidal flats. Hundreds of tracks are preserved in planform and cross-sectional exposure within five horizons along a 1032 m tracksite. Four prints exhibit five clear toe imprints, while two others distinctly display four toes. Some tracks penetrate beds populated by dwelling traces of marine bivalves and polychaetes in the upper layers and sea anemones at the base. Candidates for the five-toed tracemakers are pantodonts such as Titanoides, Barylambda, and Coryphodon, which have been recovered from late Paleocene strata throughout western North America. The four-toed tracks provide the earliest evidence of previously-undescribed large artiodactyls and/or tapiroids, mutually supporting recent molecular phylogenetic studies that place the origin of Cetartiodactyla near the Cretaceous-Paleogene boundary (~ 67.7 Ma). Collectively, these trackways irrefutably demonstrate the utility of ichnological data in reconstructing the evolutionary history and adaptive behaviors of extinct taxa beyond the evidence provided by body fossils alone.

Blood filter paper strips are cost-effective materials used to store body fluid specimens under challenging field conditions, extending the reach of zoonotic pathogen surveillance and research. We describe an optimized procedure for the extraction of parasite DNA from whole blood (WB) stored on Type I Advantec Nobuto strips from both experimentally spiked and field-collected specimens from canine and skunks, respectively. When comparing two commercial kits for extraction, Qiagen’s DNeasy Blood & Tissue Kit performed best for the detection of parasite DNA by PCR from Trypanosoma cruzi-spiked canine WB samples on Nobuto strips. To further optimize recovery of β-actin from field-collected skunk WB archived on Nobuto strips, we modified the extraction procedures for the Qiagen kit with a 90 °C incubation step and extended incubation post-addition of proteinase K, a method subsequently employed to identify a T. cruzi infection in one of the skunks. Using this optimized extraction method can efficaciously increase the accuracy and precision of future molecular epidemiologic investigations targeting neglected tropical diseases in field-collected WB specimens on filter strips.

West Nile virus (WNV), a mosquito-borne arbovirus, remains a major global health concern. In this study, we optimized PCR methods then assessed serially-collected whole blood (WB), urine (UR), saliva, and semen specimens from a large cohort of WNV-positive participants to evaluate the natural history of infection and persistent shedding of WNV RNA. Viral RNA extraction protocols for frozen WB and UR specimens were optimized and validated through spiking experiments to maximize recovery of viral RNA from archived specimens and to assess the degradation of WNV RNA in stored UR specimens. The resultant procedures were used in conjunction with PCR detection to identify WNV-positive specimens and to quantify their viral loads. A total of 59 of 352 WB, 10 of 38 UR, and 2 of 34 saliva specimens tested positive for WNV RNA. Although a single semen specimen was positive 22 days post onset, we could not definitively confirm the presence of WNV RNA in the remaining specimens. WNV RNA-positive UR specimens exhibited profound loss of viral RNA during storage, highlighting the need for optimal preservation pre-storage. This study provides optimized methods for WNV RNA detection among different fluid types and offers alternative options for diagnostic testing during the acute stages of WNV.