Discovery of a Novel Alveolate Pathogen Affecting Southern Leopard Frogs in Georgia: Description of the Disease and Host Effects

Abstract

In April of 2006, we observed southern leopard frog (Rana sphenocephala) tadpoles in a pond in northeast Georgia that were dying from an unknown pathogen. Examination of affected specimens, as well as PCR characterization, revealed that all were infected with a novel alveolate pathogen closely related to freshwater and marine eukaryotic organisms and, to a lesser degree, to members of the genus Perkinsus. This pathogen has been documented in numerous mortality events in anuran tadpoles in the United States, although it has not yet been named nor clearly described. We subsequently conducted a systematic survey of this and four other ponds in the same area to document the extent of the pathogen and to describe the nature of infections in leopard frog tadpoles. Of 87 live tadpoles examined, 25% were infected with the alveolate pathogen, based on visual inspection of tadpole liver tissue. Affected tadpoles frequently had enlarged abdomens, swam erratically, and could be captured by hand. All organs of infected tadpoles were infiltrated but typically to a lesser extent than the liver and kidneys, which often had hundreds of thousands of the spherical, 6-lm organisms. Infected tadpoles tended to weigh more than noninfected ones, likely due to the massive organ swelling that coincided with infections. Infected tadpoles did not differ in developmental stage from noninfected tadpoles. Infection prevalence varied widely among ponds, and in one pond, we witnessed a rapid die-off of R. spenocephala tadpoles during our surveys, although we did find infected metamorphic frogs. The rapid mortality we observed as well as the vast number of organisms seen in specimens suggests that this pathogen has tremendous transmission potential, and therefore deserves further monitoring and study.

Freshwater Perkinsea and marine-freshwater colonizations revealed by pyrosequencing and phylogeny of environmental rDNA

“…The R. sphenocephala parasite has been suggested to be a close relative of Perkinsus on the basis of morphology and phylogenetic analysis of the 18S rDNA gene (Davis et al, 2007). Our trees confirm that the R. sphenocephala parasite belong to Perkinsea (Figure 2).…”

“…This fell into a large and diverse, exclusively freshwater group, including the subgroups PERK 9-11 (Figure 2). This parasite is known to cause massive killings of frogs by infecting the internal organs, especially the liver and kidneys (Green et al, 2003;Davis et al, 2007). There are also other reports of freshwater parasites with an outer morphology resembling Perkinsus (for example, a parasite of the freshwater cryptomonad Chilomonas paramecium), but there are no sequences available for these species (Brugerolle, 2002(Brugerolle, , 2003.…”

“…There are also other reports of freshwater parasites with an outer morphology resembling Perkinsus (for example, a parasite of the freshwater cryptomonad Chilomonas paramecium), but there are no sequences available for these species (Brugerolle, 2002(Brugerolle, , 2003. Considering the position of the Rana parasite among the Perkinsea sequences in the tree (Figure 2), as well as the evidence from other studies indicating that putative members of Perkinsea are infecting a wide range of species, such as mollusks, frogs, dinoflagellates and cryptomonads (Brugerolle, 2002(Brugerolle, , 2003Green et al, 2003;Villalba et al, 2004;Davis et al, 2007), we suspect that the entire Perkinsea is parasitic. This is very important to clarify because it would alter the present view of the role of picoeukaryotes in 'microbial loops' and the carbon cycle in aquatic systems.…”

“…Recently, two parasite species have been described as potential relatives of Perkinsus, a parasite of the southern leopard frog (R. sphenocephala) and a parasite of the Atlantic sardine (Sardina pilchardus), Perkinsoide chabelardi (Gestal et al, 2006;Davis et al, 2007). The R. sphenocephala parasite has been suggested to be a close relative of Perkinsus on the basis of morphology and phylogenetic analysis of the 18S rDNA gene (Davis et al, 2007).…”

“…To date, the Perkinsus and Parvilucifera (members of Perkinsea) and MA have only been identified in marine environments (Noren et al, 1999;Villalba et al, 2004;Groisillier et al, 2006;Guillou et al, 2008;Leander and Hoppenrath, 2008). However, microscopy observations, as well as reports of deeply diverging alveolate 18S rDNA sequences, point to the existence of freshwater Perkinsea species (Brugerolle, 2002(Brugerolle, , 2003Green et al, 2003;Richards et al, 2005;Davis et al, 2007;Lefèvre et al, 2008;Lepère et al, 2008).…”