Association between adaptive immunity and neutrophil dynamics in zebrafish (Danio rerio) infected by a parasitic ciliate

Abstract

The protective immune response in zebrafish (Danio rerio) against the parasitic ciliate Ichthyophthirius multifiliis, targeting host skin, fins and gills, comprises an accelerated and manifold elevated immunoglobulin gene expression as well as a significantly elevated number of neutrophils at infected sites. Experimental fish were subjected to a primary I. multifiliis infection followed by a series of secondary exposures before they were challenged by a high dosage of infective theronts. Immunized fish responded immediately with a protective response suggesting existence of immunological memory whereas fish exposed to the parasite for the first time obtained a marked infection. The primary response to infection was dominated by expression of genes encoding acute phase reactants and inflammatory cytokines as well as recruitment of neutrophils at infected locations. Immunized fish showed a significantly upregulated immunoglobulin gene expression following challenge, which indicates existence of a secondary response effected by antibodies. Both responses induced a significantly elevated expression of the Th2 signature cytokine Il13. The increased presence of neutrophils in immunized fish suggests that innate cell mediated immunity supplements or influence the protective response against the parasite.

Fig 1. Timeline of the immunizations and challenge infections of zebrafish with the parasite Ichthyophthirius multifiliis.

Timeline of exposures and samplings regarding zebrafish used for A) gene expression analyses and B) image analyses investigating neutrophil recruitment. The pink and the blue areas frame the periods of immunizations and challenge infections, respectively. Periods between immunizations and sampling points are indicated as well as immunization and challenge dosages of the parasite.

Fig 2. Parasite burden of zebrafish (Danio rerio) infected with the parasite Ichthyophthirius multifiliis.

The parasite burden in fins of naïve challenged (NC) and immune challenged (IC) zebrafish with I. multifiliis. Significant difference (p<0.05) is indicated with *.

Fig 3. Gene expressions in Ichthyophthirius multifiliis infected fins (primary and secondary infections) of zebrafish (Danio rerio).

Regulations are shown before challenge and 1 h, 24 h and 48 h post challenge. The standard error of mean (SEM) is indicated as error bars and 12 out of 26 investigated genes are shown in the figure. Significant differences (p<0.05) compared to uninfected controls are indicated with a star for naïve challenged (NC) fish and a hashtag for immune challenged (IC) fish.

Fig 4. Gene expressions in Ichthyophthirius multifiliis infected gills (primary and secondary infections) of zebrafish (Danio rerio).

Regulations are shown before challenge and 1 h, 24 h and 48 h post challenge. The standard error of mean (SEM) is indicated as error bars and 9 out of 26 investigated genes are shown in the figure. Significant differences (p<0.05) compared to uninfected controls are indicated with a star for naïve challenged (NC) fish and a hashtag for immune challenged (IC) fish.

Fig 5. Neutrophil numbers in Ichthyophthirius multifiliis infected fins (primary and secondary infections) of zebrafish (Danio rerio).

Groups comprise uninfected control fish, naïve fish infected for the first time (NC) and immunized fish receiving a challenge exposure (IC). * represents statistically significant difference (P<0.05) from the control and the naïve NC fish.

Fig 6. GFP-labeled neutrophils of Ichthyophthirius multifiliis infected zebrafish caudal fins (primary and secondary infections).

Representative images of the caudal fins demonstrating the recruitment of neutrophils to infected areas following exposure to the fish parasite I. multifiliis of naïve (NC) and immunized (IC) adult zebrafish. The scale bar applies to all images.