New Findings from an Old Pathogen: Intraerythrocytic Bacteria (Family Anaplasmatacea) in Red-Backed Salamanders Plethodon cinereus

Abstract

During a recent study of red-backed salamanders (Plethodon cinereus), we discovered an intraerythrocytic organism typified by violet-staining, intracellular inclusions, consistent with descriptions of Cytamoeba or Aegyptianella (bacteria). Here we characterize its taxonomic status using molecular techniques and ask basic questions about its nature. Blood smears from 102 salamanders were examined from Pennsylvania, New York, and Virginia to determine prevalence, and whole blood from several infected animals was tested using a PCR which targets the 16S rRNA gene of bacteria. Phylogenetic analysis of partial 16S rRNA gene sequence (1201 bp) indicated this organism was in the order Rickettsiales and is likely a member of the family Anaplasmatacea. The organism differed from currently described taxa and was clearly differentiated from Aegyptianella pullorum of birds and "Candidatus Hemobacterium ranarum" (formally A. ranarum) of frogs. Of all salamanders, 17 (16.7%) were infected and these were significantly larger (snout-vent length) and had higher body condition scores than uninfected ones, and males were more likely to be infected than females. Erythrocytes affected by the pathogen were 5% larger than unaffected ones, but otherwise similar in morphology. Infected animals tended to have a greater number of circulating white blood cells, based on estimates from smears, indicating a nonspecific response to the pathogen by the innate immune system. Given its phylogenetic position, this pathogen is likely transmitted by an arthropod vector, and the male-biased prevalence strongly implicates trombiculid mites, which also live in leaf litter and affect male salamanders more so than females.

Intraerythrocytic rickettsial inclusions in Ocoee salamanders (Desmognathus ocoee): prevalence, morphology, and comparisons with inclusions of Plethodon cinereus

“…In other cases, they appeared faint blue throughout with a granular-like interior. These are both in contrast to the appearance of inclusions found in red-backed salamanders (Davis et al 2009a), which were usually violet in color after Giemsa staining and with no visible interior structure (Fig. 3, bottom).…”

“…Salamanders in the Plethodon genus are completely terrestrial, even laying their eggs on land. Because of this, Davis et al (2009a) speculated that the Rickettsia bacteria in P. cinereus was likely transmitted by a terrestrial arthropod vector, such as mites that live in leaf litter. However, if this is the case, then why would the completely terrestrial genus (which would have more opportunities for contact with the vector) have a lower prevalence of rickettsial infections than the semi-terrestrial genus?…”

“…This designation continued until later investigations using electron microscopy suggested that the organism was a rickettsia-like bacterium that was segregated from the host-cell cytoplasm within a membranebound vacuole, and based on this evidence, Desser (1987) argued that it belonged to the genus Aegyptianella. However, a recent investigation using gene sequencing of blood from infected red-backed salamanders (Plethodon cinereus) indicated that the bacteria, at least in that host, was only distantly related to the Aegyptianella genus and in fact belonged in a new, as yet unnamed, genus of Rickettsia (Davis et al 2009a). Interestingly, in that same study, the authors found that the parasite does not affect salamanders randomly; larger salamanders were more affected than smaller ones, which suggests that older individuals are more prone to infections.…”

“…To our knowledge, no prior studies have examined this species for rickettsial parasites, although rickettsial inclusions have been found in other members of the Desmognathus genus (Rankin 1937;McAllister et al 1995). In the current study, we describe the results of this (largely descriptive) investigation, including the prevalence of the parasite in this host, its morphology in host cells, as well as some general comparisons with the rickettsial inclusions found recently in red-backed salamanders (Davis et al 2009a).…”

“…Salamanders are hosts to a variety of parasites, including helminths (McAllister et al 2008), arthropods (Westfall et al 2008), and in particular, intracellular bacteria in the order Rickettsiales (Davis et al 2009a). All bacteria in this order are obligate, intracellular (usually within erythrocytes) organisms that are transmitted by arthropod vectors (Rikihisa 2006).…”