ABSTRACT. Despite an extensive described fossil record of Galliformes (Aves:`landfowl'), very few specimens have been considered within a phylogenetic context. Here we present a cladistic analysis and description of a new, well-preserved and well-dated fossil specimen from the Middle Eocene Bridger Formation of Wyoming (c. 50 Ma). Amitabha urbsinterdictensis gen. et. sp. nov. is assigned to Galliformes on the basis of the presence of derived characters including double, and open, incisurae laterales on the sternum and an incisura capitis of the proximal humerus that is enclosed from the crus dorsale fossa by a distinct ridge. Amitabha is considered to be a member of the large and diverse`phasianoid' clade of Galliformes (including, for example, the pheasants, peafowl and turkeys) owing to a lack of extensive pneumaticity in the sternal plate. The age of this avian taxon and degree of preservation allow for a discussion of the use of fossil birds for the calibration of`molecular clock' hypotheses and dating divergences within Neornithes in general, and Galliformes in particular.KEY WORDS: Eocene,`landfowl', Galliformes, Wyoming, cladistics, phylogeny.T H E avian order Galliformes (`landfowl') includes such familiar taxa as junglefowl, peacocks and turkeys and generally has been considered to be one of the more basal clades of modern birds ( Neornithes sensu Cracraft 1988,`crown-clade' Aves of others; Gauthier 1986; Gauthier and de Queiroz 2001). Existing systematic treatments of these birds (e.g. Sibley and Monroe 1990;McGowan 1994) suggest that the order comprises at least ®ve distinct families, the Megapodiidae (megapodes and relatives), Numididae (guineafowl), Phasianidae (pheasants and relatives), Odontophoridae (New World quails), and Cracidae (curassows and relatives;Wetmore 1960;Cracraft 1981;Sibley and Ahlquist 1990). Recent cladistic analysis of over 100 morphological characters (Dyke and Gulas 2002;Dyke et al. 2003) found a basalmost, monophyletic Megapodiidae to be the sister-group to all other Galliformes, followed by Cracidae as the sister-group to the`phasianoids' (Text-®g. 1).Upon initial inspection, the described fossil record of this avian order appears extensive: reputed remains of galliform birds are described from deposits that range in age from the latest Cretaceous to the Recent (Brodkorb 1964;Unwin 1993; Hope in press). However, the fragmentary nature of supposed fossil galliform' records from the Cretaceous (Hope in press) as well as problems resulting from the polarization of the few phylogenetically informative characters preserved in these specimens (Clarke 1999) places the oldest certain record of the order (described to date) in the Lower Eocene Green River Formation of the United States (Grande 1984). This taxon, Gallinuloides wyomingensis Eastman, 1900, was placed within the family Gallinuloididae by Lucas (1900), now also considered to contain a number of somewhat younger and incompletely known specimens (Milne-Edwards 1867±71;Tordoff and Macdonald 1957;Crowe and Short 1992;. A num...
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.
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.
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