Coccidian Parasites and Conservation Implications for the Endangered Whooping Crane (Grus americana)

While the population of endangered whooping cranes (Grus americana) has grown from 15 individuals in 1941 to an estimated 304 birds today, the population growth is not sufficient to support a down-listing of the species to threatened status. The degree to which disease may be limiting the population growth of whooping cranes is unknown. One disease of potential concern is caused by two crane-associated Eimeria species: Eimeria gruis and E. reichenowi. Unlike most species of Eimeria, which are localized to the intestinal tract, these crane-associated species may multiply systemically and cause a potentially fatal disease. Using a non-invasive sampling approach, we assessed the prevalence and phenology of Eimeria oocysts in whooping crane fecal samples collected across two winter seasons (November 2012–April 2014) at the Aransas National Wildlife Refuge along the Texas Gulf coast. We also compared the ability of microscopy and PCR to detect Eimeria in fecal samples. Across both years, 26.5% (n = 328) of fecal samples were positive for Eimeria based on microscopy. Although the sensitivity of PCR for detecting Eimeria infections seemed to be less than that of microscopy in the first year of the study (8.9% vs. 29.3%, respectively), an improved DNA extraction protocol resulted in increased sensitivity of PCR relative to microscopy in the second year of the study (27.6% and 20.8%, respectively). The proportion of positive samples did not vary significantly between years or among sampling sites. The proportion of Eimeria positive fecal samples varied with date of collection, but there was no consistent pattern of parasite shedding between the two years. We demonstrate that non-invasive fecal collections combined with PCR and DNA sequencing techniques provides a useful tool for monitoring Eimeria infection in cranes. Understanding the epidemiology of coccidiosis is important for management efforts to increase population growth of the endangered whooping crane.The open access fee for this work was funded through the Texas A&M University Open Access to Knowledge (OAK) Fund

Coccidia Observed during Fecal Flotation.

<p>Fecal flotation under 500X magnification showing a mixed infection with two species of <i>Eimeria</i>. The smaller, pear-shaped oocysts are consistent with <i>Eimeria gruis</i> (arrow) and the larger, round to oval oocysts are consistent with <i>Eimeria reichenowi</i> (arrowhead).</p

Phylogenetic Tree using <i>Eimeria</i> ITS-1 and ITS-2 Sequences.

<p>Phylogenetic trees using the neighbor-joining method on ITS-1 (466 bp) or ITS-2 (400 bp) sequences from <i>Eimeria</i> species. Bootstrap values are based on 1000 replicates and shown where greater than 60. Bold species indicate isolates from cranes, and underlined species indicate sequences generated in this study. The GenBank accession number of each isolate is shown in parentheses, and the known vertebrate host is also shown.</p

Phylogenetic Tree using <i>Eimeria</i> 18S rRNA Sequences.

<p>Phylogenetic tree using the neighbor-joining method on 18S rRNA sequences (358 bp) from <i>Eimeria</i> species. Bootstrap values are based on 1000 replicates and shown where greater than 60. Bold species indicate isolates from cranes, and underlined species indicate sequences generated in this study. The GenBank accession number of each isolate is shown in parentheses, and the known vertebrate host is also shown.</p

Whooping Crane Scat Collection.

<p>Game camera photo of two adult whooping cranes and one juvenile (far left) at a freshwater pond on Aransas National Wildlife Refuge (A). Scat produced by a whooping crane feeding primarily on blue crab and other invertebrates (B) and wolfberry (C). Cranes fed predominantly on wolfberry when the berries were abundant (Nov—Dec), then switched to feed predominantly on crab and other invertebrates. Scat was collected after the cranes naturally left the pond.</p

Phenology of <i>Eimeria</i> shedding in winter based on microscopy in whooping crane feces.

<p>Phenology of <i>Eimeria</i> shedding in winter based on microscopy in whooping crane feces.</p

Pond Sites on the Blackjack and Lamar Peninsulas.

<p>The Aransas National Wildlife Refuge is located along the Texas Gulf Coast and encompasses the Blackjack Peninsula and Matagorda Island. Eight pond sites in this study were located on the Blackjack Peninsula. The two pond sites on the Lamar Peninsula were included during 2012–2013. The map image was created by the USDA National Agriculture Imagery Program (NAIP) and downloaded as a GIS file, and the figure was produced using ArcMAP 10 (Esri, Redlands, CA).</p