A Vavraia-like microsporidium as the cause of deadly infection in threatened and endangered Eurycea salamanders in the United States

Abstract

Background: Eurycea sosorum (Barton Springs salamander) and Eurycea nana (San Macros salamander) are listed as endangered and threatened species, respectively, by the U.S. Fish and Wildlife Service (USFWS) with habitats restricted to small regions near Austin, Texas, USA. The conservation efforts with the Eurycea salamanders at the captive breeding program in San Marcos Aquatic Resources Center (SMARC), a USFWS facility, have seen an unexpected and increased mortality rate over the past few years. The clinical signs of sick or dead salamanders included erythema, tail loss, asymmetric gills or brachial loss, rhabdomyolysis, kyphosis, and behavior changes, suggesting that an infectious disease might be the culprit. This study aimed to identify the cause of the infection, determine the taxonomic position of the pathogen, and investigate the potential reservoirs of the pathogen in the environment.

Results: Histopathological examination indicated microsporidian infection (microsporidiosis) in the sick and dead Eurycea salamanders that was later confirmed by PCR detection. We also determined the near full-length small subunit ribosomal RNA (SSU rRNA) gene from the microsporidian pathogen, which allowed us to determine its phylogenetic position, and to design primers for specific and sensitive detection of the pathogen. Phylogenetic analysis indicated that this pathogen was closely related to the insect parasites Vavraia spp. and the human opportunistic pathogen, Trachipleistophora hominis. This Vavraia-like microsporidium was present in dead salamanders at SMARC archived between 2011 and 2015 (positive rates ranging between 52.0-88.9% by PCR detection), as well as in some aquatic invertebrates at the facility (e.g. snails and small crustaceans).

Conclusions: A Vavraia-like microsporidian was at least one of the major pathogens, if not solely, responsible for the sickness and mortality in the SMARC salamanders, and the pathogen had been present in the center for years. Environmental invertebrates likely served as a source and reservoir of the microsporidian pathogen. These observations provide new knowledge and a foundation for future conservation efforts for Eurycea salamanders including molecular surveys, monitoring of the pathogen, and discovery of effective treatments.

Keywords: Aquatic invertebrates; Endangered species; Eurycea nana; Eurycea sosorum; Lungless salamanders; Microsporidiosis; Reservoir; Vavraia-like microsporidian.

Fig. 1

Healthy and sick Eurycea salamanders. Photographs were taken at the San Marcos Aquatic Resource Center (SMARC), Texas, to compare the appearances of healthy (a-c) and sick (d-f) Eurycea salamanders to illustrate the impact of Vavraia-like microsporidian infection on the animals

Fig. 2

Histopathological examination. a Appearance of a dead Eurycea sosorum salamander submitted to the Zoological Medicine Service for sacrifice and necropsy. Representative red pinpoint lesions in a sick Eurycea sosorum salamander are indicated by arrows. b Histopathological detection of microsporidia cysts (stained in red) in a representative microscopic section from a lesion by Giemsa staining (Photo courtesy Drury Reavill, Zoo/Exotic Pathology Service)

Fig. 3

Nested PCR detection of Vavraia-like microsporidian DNA from Eurycea salamanders from SMARC. a Agarose gel image of PCR products derived from the frozen tissues of the six salamander specimens collected in 2013 outbreak for initial diagnosis and molecular detection. Negative control (−) used pure water. b Agarose gel image of PCR products derived from a selected number of archived salamander specimens indicating both negative and positive detection. Negative (−) and positive (+) controls used DNA isolated from microsporidian-negative and -positive salamander specimens. Nested PCR reactions used primer pairs Microsp_Salam_S1F and Microsp_Salam_S1R for primary PCR, and Microsp_Salam_S2F and Microsp_Salam_S2R for secondary PCR as described in Additional file 1: Table S1

Fig. 4

Phylogenetic analysis of the Vavraia-like microsporidium based on the near full-length SSU rRNA genes. a Phylogenetic tree reconstructed by the Bayesian inference (BI) method based on the large dataset (dataset 1 as described in Methods) to determine the phylogenetic position of the Vavraia-like microsporidium among various microsporidian taxonomic groups. b Phylogenetic tree reconstructed by BI and maximum likelihood (ML) methods based on a small dataset (dataset 2 as described in Methods) to fine-tune the phylogenetic position of the Vavraia-like microsporidium. Posterior probability values in BI analysis and bootstrap supporting values in ML analysis are indicated at the nodes. Solid circles at the nodes indicate 100% support by both analyses