Electrolyte imbalances and dehydration play a key role in Batrachochytrium salamandrivorans chytridiomycosis

Abstract

Introduction: One of the most important emerging infectious diseases of amphibians is caused by the fungal pathogen Batrachochytrium salamandrivorans (Bsal). Bsal was recently discovered and is of global concern due to its potential to cause high mortality in amphibians, especially salamander species. To date, little has been reported on the pathophysiological effects of Bsal; however, studies of a similar fungus, B. dendrobatidis (Bd), have shown that electrolyte losses and immunosuppression likely play a key role in morbidity and mortality associated with this disease. The goal of this study was to investigate pathophysiological effects and immune responses associated with Bsal chytridiomycosis using 49 rough-skinned newts (Taricha granulosa) as the model species.

Methods: Taricha granulosa were exposed to a 1 × 107 per 10 mL dose of Bsal zoospores and allowed to reach various stages of disease progression before being humanely euthanized. At the time of euthanasia, blood was collected for biochemical and hematological analyses as well as protein electrophoresis. Ten standardized body sections were histologically examined, and Bsal-induced skin lesions were counted and graded on a scale of 1-5 based on severity.

Results: Results indicated that electrolyte imbalances and dehydration induced by damage to the epidermis likely play a major role in the pathogenesis of Bsal chytridiomycosis in this species. Additionally, Bsal-infected, clinically diseased T. granulosa exhibited a systemic inflammatory response identified through alterations in complete blood counts and protein electrophoretograms.

Discussion: Overall, these results provide foundational information on the pathogenesis of this disease and highlight the differences and similarities between Bsal and Bd chytridiomycosis.

Keywords: amphibian; chytrid; clinical pathology; disease; histopathology; newt.

Figure 1

Batrachochytrium salamandrivorans exposed, clinically diseased Taricha granulosa with severe, multifocal skin sloughing especially prominent on the chin, neck, legs, feet, and tail (TW49).

Figure 2

Batrachochytrium salamandrivorans exposed, clinically diseased Taricha granulosa with multifocal hemorrhage on the chin, legs, and feet (TW25).

Figure 3

Taricha granulosa with multiple, characteristic erosive to ulcerative Batrachochytrium salamandrivorans induced skin lesions (TW14).

Figure 4

Principal component analysis (PCA) for biochemical data. In the biplot on the left, individuals in the same space as the arrowhead had higher values of the blood parameter, and individuals in the opposite direction had lower levels of the blood parameter. The length and color of each arrow represent the strength of the relationship. The plot on the right is showing how the individuals clustered together using K means clustering. The majority of Taricha granulosa in cluster 1 (red) were Batrachochytrium salamandrivorans (Bsal) infected, clinically diseased individuals, and the majority of T. granulosa in cluster 2 (blue) were controls or Bsal-infected, non-clinically diseased individuals. Na, Sodium; Cl, Chloride; cholest, Cholesterol; logBUN, Blood urea nitrogen; Phospho, Phosphorous; AlkPhos, Alkaline phosphatase; Magnes, Magnesium; K_H_resid, the residuals of potassium (K) regressed on hemolytic index (H); K_H_resid, the residuals of creatinine (CK) regressed on H; K_H_resid, the residuals of lactate dehydrogenase (LDH) regressed on H.

Figure 5

Boxplots comparing each biochemical variable for cluster 1 (Batrachochytrium salamandrivorans (Bsal) infected, clinically diseased Taricha granulosa) in red and cluster 2 (control and Bsal-infected, non-clinically diseased T. granulosa) in blue. Each box represents the interquartile range (IQR), the horizontal line within each box represents the median, vertical lines represent 1.5 ^* IQR, and dots represent outliers. AlkPhos, Alkaline phosphatase; Na, sodium; Cl, chloride; CO₂, bicarbonate; LDH_H_resid, the residuals of lactate dehydrogenase (LDH) regressed on hemolytic index (H); K_H_resid, the residuals of potassium (K) regressed on H.

Figure 6

Plots of sodium (Na), chloride (Cl), the residuals of potassium (K) regressed on hemolytic index (H) (K_H_resid), and anion gap (y-axis) and the log of Batrachochytrium salamandrivorans qPCR load at time of necropsy + 1 (x-axis). Black points are observed data points and blue lines are best-fit regression lines for visual reference of the dominant trend in the data. Additional analysis showed that there was significant evidence for a non-linear quadratic effect of log(Bsal Load + 1) on biochemical variables (log(Bsal Load + 1)² (effect from PERMANOVA: F = 6.98, p = 0.001).

Figure 7

Principal component analysis (PCA) for the complete blood count (CBC) data. In the plot on the left, individuals in the same space as the arrowhead had higher values of the blood parameter, and individuals in the opposite direction had lower levels of the blood parameter. The length and color of each arrow represent the strength of the relationship. The plot on the right is showing how the individuals clustered together using K means clustering. The majority of Taricha granulosa in cluster 1 (red) were Batrachochytrium salamandrivorans (Bsal) infected, clinically diseased individuals, and the majority of T. granulosa in cluster 2 (blue) were controls or Bsal-infected, non-clinically diseased individuals. logACLymphs, Absolute count of lymphocytes; logACMonos, absolute count of monocytes; logACEos, absolute count of eosinophils; logACBands, absolute count of band neutrophils; logACSegNeut, absolute count of segmented neutrophils; logACBasos, absolute count of basophils.

Figure 8

Boxplots comparing each complete blood count variable for cluster 1 (Batrachochytrium salamandrivorans (Bsal) infected, clinically diseased Taricha granulosa) in red and cluster 2 (controls and Bsal-infected, non-clinically diseased T. granulosa) in blue. Each box represents the interquartile range (IQR), the horizontal line within each box represents the median, vertical lines represent the 1.5 ^* IQR, and dots represent outliers. ACSegNeuts, Absolute count of segmented neutrophils; ACBands, absolute count of band neutrophils; ACMonos, absolute count of monocytes; ACEos, absolute count of eosinophils.

Figure 9

Packed cell volume for five control, two Batrachochytrium salamandrivorans-exposed/non-clinically diseased, and two Batrachochtyrium salamandrivorans- exposed/clinically diseased Taricha granulosa.

Figure 10

Principal component analysis (PCA) for the plasma protein electrophoresis data. In the plot on the left, individuals in the same space as the arrowhead had higher values of the blood parameter, and individuals in the opposite direction had lower levels of the blood parameter. The length and color of each arrow represent the strength of the relationship. The plot on the right is showing how the individuals clustered together using K means clustering. The majority of Taricha granulosa in cluster 1 (blue) were controls or Batrachochytrium salamandrivorans (Bsal) infected, non-clinically diseased individuals, and the majority of T. granulosa in cluster 2 (red) were Bsal-infected, clinically diseased individuals.

Figure 11

Boxplots comparing each protein electrophoresis fraction for cluster 1 (Batrachochytrium salamandrivorans (Bsal) infected, clinically diseased Taricha granulosa) in red and cluster 2 (controls and Bsal-infected, non-clinically diseased T. granulosa) in blue. Each box represents the interquartile range (IQR), the horizontal line within each box represents the median, vertical lines represent 1.5 ^* IQR, and dots represent outliers.

Figure 12

Representative protein electrophoretograms from a control (A) and a Batrachochytrium salamandrivorans infected, clinically diseased (B) Taricha granulosa.

Figure 13

Predicts food consumption over time (days) post-exposure in control (red) vs. Batrachochytrium salamandrivorans exposed (blue) Taricha granulosa. Colored lines represent mean probabilities and shaded regions are 95% confidence intervals around the mean probabilities.

Figure 14

Plot of log10(Lesion count + 1) (y-axis) and log10(Bsal load + 1) (x-axis). Black points represent histologic lesion count at each of 10 standardized anatomical sections examined for each Batrachochytrium salamandrivorans (Bsal) infected Taricha granulosa. Red lines represent individual level relationships as predicted by our statistical model. As each individual only had one Bsal qPCR load (at necropsy), but multiple lesion counts due to the multiple sections, a unique slope cannot be inferred for the relationship between Bsal load and lesion count for each individual.

Figure 15

Photomicrograph stained with hematoxylin and eosin (H&E) of a skin lesion caused by Batrachochytrium salamandrivorans (Bsal) chytridiomycosis. There is full thickness invasion of the epidermis by Bsal thalli along with associated epithelial damage which is characteristic of skin lesions caused by this pathogen.

Figure 16

Photomicrograph stained with hematoxylin and eosin (H&E) showing invasion of Batrachochytrium salamandrivorans (Bsal) thalli into a dermal granular gland. There is full thickness invasion of the overlying epidermis by Bsal thalli along with associated epithelial damage.