White blood cell profile data from Chapter 3 of the PhD thesis: Thermal thresholds in the amphibian disease chytridiomycosis.
Temperature variability, and in particular temperature decreases, can increase susceptibility of amphibians to infections by the fungus Batrachochytrium dendrobatidis (Bd). However, the effects of temperature shifts on the immune systems of Bd-infected amphibians are unresolved. We acclimated frogs to 16°C and 26°C (baseline), simultaneously transferred them to an intermediate temperature (21°C) and inoculated them with Bd (treatment), and tracked their infection levels and white blood cell profiles over six weeks. Study animals were captive-bred, juvenile Litoria caerulea.
For a separate initial study, forty-six individuals were raised in temperature-controlled rooms for 24 months and maintained on a natural photoperiod in individual enclosures. Twenty-six individuals were housed in a 16°C room; twenty individuals were housed in a 26°C room. For the study, we removed frogs from the temperature-controlled rooms, immediately divided them into temperature-controlled chambers set to 21°C, and administered Bd inoculations in the chambers. To ensure infection, we inoculated frogs on two consecutive days. On each day, we prepared a zoospore suspension of 6 x 105 zoospores per ml. To inoculate, we placed each frog into an individual 250-ml plastic container and added 5 ml (enough to cover the bottom of the container) of zoospore inoculant or sham inoculant to each container. We left frogs in inoculant baths for 12 hours per day. After each inoculation period, we returned frogs with their inoculant to individual permanent enclosures comprising 70 x 120 x 170 mm plastic containers lined with tap water-saturated paper towels.
To monitor Bd infection status and intensity over the course of the experiment, we swabbed frogs one week before removal from the temperature-controlled rooms (all tested Bd-negative) and 6 d, 14 d, 21 d, 29 d, 36 d, and 41 d post-inoculation. We determined the number of Bd zoospore genome equivalents per swab with a real-time quantitative PCR protocol.
Blood samples (0.1 ml) were collected by cardiac puncture with 26-ga needles one week before removing frogs from temperature-controlled rooms (baseline) and on days 6, 21, and 33. Blood smears were air-dried, stained with Wright’s stain, and observed by light microscopy at 100x. To perform a differential WBC count, we located the section of the blood smear with an even monolayer of cells (red blood cells as close together as possible without overlapping). We then counted and identified 200 WBCs per slide with a modified battlement technique. We identified WBCs as lymphocytes, neutrophils, monocytes, basophils, and eosinophils. We calculated WBC profiles for each frog by dividing the number of each cell type by the total number of cells counted.
This dataset shows our white blood cell counts. Column headings are explained below.
Frog = individual frog identifier
Start_temp = temperature of temperature-controlled room in which frog was maintained prior to study; cold = 16°C; hot = 26°C
Sul = frog snout-urostyle length
Treat_temp = Temperature of temperature-controlled chamber in which frog was maintained during study; medium = 21°C (treatment); cold = 16°C (sham-inoculated control); hot = 26°C (sham-inoculated control)
Day = day of experiment
Lymph = number of lymphocytes counted
Neut = number of neutrophils counted
Mon = number of monocytes counted
Eosin = number of eosinophils countedBas= number of basophils counted
Total_cells = total number of cells counted
Fields = number of microscope fields examined to reach 200 cells counted
Perc_lymph = proportion of counted cells that were lymphocytes
Perc_neut = proportion of counted cells that were neutrophils
Perc_mon = proportion of counted cells that were monocytesPerc_unk_bas = proportion of counted cells that were basophils
Perc_eosin = proportion of counted cells that were eosinophils