1. Ecology
  2. Microbiology and Infectious Disease
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Immune-mediated hookworm clearance and survival of a marine mammal decrease with warmer ocean temperatures

  1. Mauricio Seguel  Is a corresponding author
  2. Felipe Montalva
  3. Diego Perez-Venegas
  4. Josefina Gutiérrez
  5. Hector J Paves
  6. Ananda Müller
  7. Carola Valencia-Soto
  8. Elizabeth Howerth
  9. Victoria Mendiola
  10. Nicole Gottdenker
  1. University of Georgia, United States
  2. Pontificia Universidad Catolica de Chile, Chile
  3. Universidad Andrés Bello, Chile
  4. Universidad Austral de Chile, Chile
  5. Universidad Santo Tomas, Chile
Research Article
Cite this article as: eLife 2018;7:e38432 doi: 10.7554/eLife.38432
7 figures and 14 additional files

Figures

Hookworm infection dynamics and predictors of hookworm mortality in South American fur seal (Arctocephalus australis) pups at Guafo Island, southern Chile.

(A) Hookworm egg shedding patterns through infection stages. (B) Hookworm prevalence and mortality through different reproductive seasons (2005–08, 2012–17). (C–F) Predictors of hookworm mortality in generalized linear mixed models (GLMM) (2014, 2015, 2017) vs observed hookworm mortality. Higher hookworm burdens (GLMM, 1.47 ± 0.56, Z = 2.61, p = 0.009), longer infectious periods (GLMM, 0.17 ± 0.06, Z = 2.87, p = 0.004), and lower plasma concentrations of blood urea nitrogen (BUN) (GLMM, −0.45 ± 0.23, Z = −1.97, p = 0.049) and parasite-specific IgG (GLMM, −0.41 ± 0.17, Z = −2.45, p = 0.014) increased the probability of hookworm mortality. Raw data in: Figure 1—source data 1.

https://doi.org/10.7554/eLife.38432.003
Figure 1—source data 1

Predictors of hookworm-related mortality in South American fur seal pups.

https://doi.org/10.7554/eLife.38432.004
Changes in peripheral blood leukocytes and parasite-specific IgG antibodies during different phases of hookworm infection (Uncinaria sp.) in South American fur seal (Arctocephalus australis) pups (2017).

(A) Pups that die from hookworm disease have lower numbers of lymphocytes during the prepatent phase compared to controls and pups that survived (Generalized linear mixed model (GLMM), lymphocytes died = −0.52 ± 0.13, Z = −4.06, p = 4.92×10−5). Pups that survive hookworm infection have higher numbers of lymphocytes during the patent (B) (GLMM, lymphocytes survived = 0.80 ± 0.13, Z = 6.04, p = 1.54×10−9) and clearance (C) (GLMM, lymphocytes survived = 0.52 ± 0.12, Z = 4.30, p = 1.74×10−5) infection phases when compared to pups that died due to hookworm infection and/or age matched controls. (E–H) Pups that clear and survive hookworm infection have markedly higher numbers of basophils during the patent (GLMM, basophils survived = 7.46 ± 1.4, Z = 5.33, p = 1.0×10−7) and clearance (GLMM, basophils survived = 6.34 ± 0.9, Z = 7.07, p = 1.5×10−12) infection phases compared to controls and pups that died from hookworm infection. (I) Fur seal pups that clear hookworm infection produce parasite-specific IgG that binds the intestinal brush border of the fur seal hookworms (Uncinaria sp.) (arrow). (J–M) Fur seal pups that clear hookworm infection have higher levels of parasite-specific IgG during the prepatent (GLMM, IgG survived = 1.78 ± 0.36, Z = 4.86, p = 1.12×10−6), patent (GLMM, IgG survived = 2.27 ± 0.25, Z = 9.067, p = 2.0×10−16), clearance (GLMM, IgG survived = 1.80 ± 0.2, Z = 9.0, p = 2.0×10−16), and post-clearance (GLMM, IgG survived = 1.87 ± 0.25, Z = 7.3, p = 3.53×10−13) infection phases compared to controls and pups that died. Asterisk indicate groups are statistically different at alpha = 0.05. p-values code: *0.01 < 0.05, **0.001 < 0.01, *** < 0.001. Raw data in: Figure 2—source data 1.

https://doi.org/10.7554/eLife.38432.005
Figure 2—source data 1

Maternal attendance and health-related parameters in pups with different hookworm infection status.

https://doi.org/10.7554/eLife.38432.006
Intestinal immune response in different groups of South American fur seals (Arctocephalus australis) infected with hookworms (Uncinaria sp.) and controls.

During the clearance process, fur seal pups recruit numerous T-lymphocytes (CD3 stain) in the jejunum mucosa (Generalized linear models with negative binomial distribution (GLM.NB), clearance = 0.86 ± 0.11, Z = 8.312, p = 2.0×10−16) and submucosa (GLM.NB, clearance = 1.08 ± 0.16, Z = 6.86, p = 7.0×10−12). Mast cells (C-kit stain) are found in higher numbers in the intestinal mucosa of pups undergoing clearance (GLM.NB, clearance = 1.14 ± 0.25, Z = 4.6, p = 4.2×10−6). The intestinal mucosa of pups clearing hookworm infection contains a large amount of mucus (GLM, clearance = 0.03 ± 0.003, Z = 7.84, p = 9.4×10−10). Pups that die from hookworm enteritis and bacteremia (HEB) have lower numbers or proportions of these immune components but higher numbers of macrophages (IBA1 stain) in the jejunum submucosa (GLM.NB, mortality = 0.52 ± 0.09, Z = 5.79, p = 6.94×10−9). Asterisks indicate groups are statistically different at alpha = 0.05. p-values code: *0.01 < 0.05, ** <0.01. GLM.NB. Raw data: Figure 3—source data 1.

https://doi.org/10.7554/eLife.38432.007
Figure 3—source data 1

Leukocyte subsets and IL-4 immunohistochemical staining in intestine and mesenteric lymph node.

https://doi.org/10.7554/eLife.38432.008
Maternal attendance affects South American fur seal pup’s growth rate, energy balance, and immune response against hookworms.

(A) The observed number of nursing events against predicted values of growth rate. With more nursing events pups grow faster (GLM.NB, 0.031 ± 0.006, Z = 5.53, p = 3.2×10−8). (B) Pups that survived hookworm infection had higher levels of maternal attendance (more nursing events) (GLM.NB, 0.78 ± 0.23, Z = 3.45, p = 5.5×10−4), faster growth rate (GLM.NB, 1.05 ± 0.16, Z = 6.6, p = 2.7×10−11), and higher average levels of glucose (GLM, 3.0 ± 0.5, Z = 5.9, p = 1.02×10−7) compared to pups that died from hookworm disease; however, they had similar attendance and metabolic values compared to hookworm-free (control) pups (GLM.NB, 0.01 ± 0.13, Z = 0.13, p = 0.893, and GLM, 0.71 ± 0.38, t = 1.863, p = 0.066). (C) The observed values of number of nursing events, growth rate, interaction between nursing and growth rate and hookworm burden vs. the predicted values of CD3+ lymphocytes in section of skin in response to phytohemagglutinin (PHA) challenge. Pups with more nursing events (GLM.NB, 0.098 ± 0.02, Z = 4.39, p = 1.14×10−5), faster growth rate (GLM.NB, 0.04 ± 0.004, Z = 11.3, p = 2.0×10−16), and higher hookworm burden (GLM.NB, 0.009 ± 0.004, Z = 2.56, p = 0.01) had more recruitment of T-lymphocytes. (D) A subset of pups was divided into groups of low and high response to PHA challenge at 30 days old. Pups with higher CD3 lymphocyte response had higher average levels of parasite-specific IgG (GLM.NB, 1.11 ± 0.33, Z = 3.37, p = 7.5×10−4), shorter infectious period (GLM.NB, −0.38 ± 0.12, Z = −3.06, p = 2.1×10−3), faster growth rate (GLM.NB, 0.68 ± 0.06, Z = 10.9, p = 2.0×10−16), and higher levels of maternal attendance (GLM.NB, 0.94 ± 0.13, Z = 7.04, p = 1.92×10−1). Hookworm burden was similar between the two groups (GLM.NB, low reactivity = −0.63 ± 0.37, Z = −1.67, p = 0.09). Raw data: Figure 4—source data 1 and Figure 4—source data 2.

https://doi.org/10.7554/eLife.38432.009
Figure 4—source data 1

Phytohemagglutinin immune challenge in 8-wk-old pups.

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Figure 4—source data 2

Phytohemagglutinin immune challenge in 4-wk-old pups.

https://doi.org/10.7554/eLife.38432.011
South American fur seals foraging behavior and maternal care patterns differ between seasons.

(A) The level of maternal attendance decreases as foraging trips become longer (linear regression, R2 = 0.412, p = 0.016; dashed lines represent 95% confidence intervals). (B) In a year with sea surface temperature (SST) below the historic Guafo Island average (2007), fur seal females foraging trips are shorter when compared to the mean foraging trip duration during a year with SST temperature above the historical average (2017) (unpaired T-test, t = 5.133, df = 42, p < 0.0001). Additionally, maternal attendance and pup growth rate in 2007 were higher than attendance and growth rates in 2017 (maternal attendance index: unpaired T-test, t = 2.060, df = 244, p = 0.04; growth rate: unpaired T-test, t = 2.85, df = 66, p = 0.0058). (C) Between 2012 and 2017 the mean values of glucose, cholesterol, parasite-specific IgG, peripheral blood lymphocytes and basophils followed an inverse pattern with mean SST at Guafo Island. In 2013, a year with low SST, pups had, on average, higher levels of glucose, cholesterol, parasite-specific IgG, lymphocytes and basophils when compared to the mean values of other reproductive seasons (Kruskal-Wallis with Dunn’s multiple comparison tests, Kruskal-Wallis statistic = 73.2–114.6, mean rank diff. = 63.98–203.8, p < 0.0001–0.023). In 2014, with the highest mean SST over the last 15 y at Guafo Island, fur seal pups had the lowest mean values of these metabolic and immune parameters (Kruskal-Wallis with Dunn’s multiple comparison tests, Kruskal-Wallis statistic = 73.2–114.6, mean rank diff. = −230.83,–83.4, p < 0.0001–0.017). (Asterisks indicate mean is significantly different from means of other seasons). Raw data in: Figure 5—source data 1 and Figure 5—source data 2.

https://doi.org/10.7554/eLife.38432.012
Figure 5—source data 1

Maternal attendance and growth rates in 2007 and 2017.

https://doi.org/10.7554/eLife.38432.013
Figure 5—source data 2

Immune and metabolic parameters in South American fur seal pups between 2012 and 2017.

https://doi.org/10.7554/eLife.38432.014
Correlation between oceanographic parameters (sea surface temperature and chlorophyll-a) and hookworm disease dynamics in South American fur seals (Arctocephalus australis) at the Chilean Patagonia (2005–08, 2012–17).

(A) Hookworm prevalence, burden, and mortality increase in years with warmer sea surface temperature (Linear regressions. Hookworm prevalence, Ad-R2 = 0.29, p = 0.064. Hookworm burden, Ad-R2 = 0.86, p < 0.001. Hookworm mortality, Ad-R2 = 0.56, p = 0.016). Hookworm prevalence, burden, and mortality decrease in some years with higher primary productivity (Second order polynomial regressions. Hookworm prevalence, Ad-R2 = 0.46, p = 0.046. Hookworm burden, Ad-R2 = 0.29, p < 0.123. Hookworm mortality, Ad-R2 = 0.70, p = 0.005). Dashed lines represent 95% confidence intervals. Raw data: Figure 6—source data 1 and Figure 6—source data 2.

https://doi.org/10.7554/eLife.38432.015
Figure 6—source data 1

Sea surface temperature data for Guafo Island.

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Figure 6—source data 2

Hookworm prevalence, burden, and mortality in South American fur seal pups at Guafo Island.

https://doi.org/10.7554/eLife.38432.017
Proposed mechanism of South American fur seal response to environmental change in the context of endemic hookworm infection.

Changes in sea surface temperature (SST) are associated with changes in foraging trip length and patterns of maternal attendance in fur seals. In years with lower SST, fur seal pups receive more diurnal maternal attendance compared to years with high SST. Pups with more maternal attendance have better energy balance, higher T-lymphocyte reactivity, and produce higher levels of parasite-specific IgG. Pups with this immune profile eliminate hookworms from the intestine faster than pups with less reactive immune systems. Early hookworm clearance is one of the most important factors that drive hookworm-related mortality, therefore in years with higher SST fur seal pups die more often of hookworm disease.

https://doi.org/10.7554/eLife.38432.018

Additional files

Supplementary file 1

Selected binomial generalized linear mixed models for hookworm mortality in South American fur seal (Arctocephalus australis) pups.

https://doi.org/10.7554/eLife.38432.019
Supplementary file 2

Multimodel coefficient estimates, standard errors (SE), z and p values of predictors of hookworm mortality in South American fur seal pups.

https://doi.org/10.7554/eLife.38432.020
Supplementary file 3

External factors affecting South American fur seal pup growth rate.

https://doi.org/10.7554/eLife.38432.021
Supplementary file 4

Averaged coefficients, standard errors (SE), z and p values of top ranked models for pup’s growth shown in supplementary file 3.

https://doi.org/10.7554/eLife.38432.022
Supplementary file 5

Negative binomial generalized linear models for CD3 lymphocyte response in South American fur seal pups infected with hookworms.

https://doi.org/10.7554/eLife.38432.023
Supplementary file 6

Averaged coefficients, standard errors, z and p values of top ranked models for CD3 lymphocyteresponse shown in supplementary file 5.

https://doi.org/10.7554/eLife.38432.024
Supplementary file 7

Negative binomial generalized linear mixed models for hookworm infectious period in South American fur seal pups.

https://doi.org/10.7554/eLife.38432.025
Supplementary file 8

Averaged coefficients, standard errors, z and p values of predictors for hookworm infectious period based on the top ranked models shown in supplementary file 7.

https://doi.org/10.7554/eLife.38432.026
Supplementary file 9

Hookworm prevalence, median hookworm burden, hookworm mortality, and mean concentration of chlorophyll-a and sea surface temperature (December) during 10 South American fur seal reproductive seasons at Guafo Island, Southern Chile.

https://doi.org/10.7554/eLife.38432.027
Supplementary file 10

Regression models with hookworm prevalence, burden, or mortality as response and sea surface temperature as predictor.

https://doi.org/10.7554/eLife.38432.028
Supplementary file 11

Regression models with hookworm prevalence, burden, or mortality as response and chlorophyll-a mean concentration as predictor.

https://doi.org/10.7554/eLife.38432.029
Supplementary file 12

Detail of sources, clone, retrieval methods, and dilution of primary antibodies used for immunohistochemistry

https://doi.org/10.7554/eLife.38432.030
Supplementary file 13

Data files inventory

https://doi.org/10.7554/eLife.38432.031
Transparent reporting form
https://doi.org/10.7554/eLife.38432.032

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