Pathological and immunological analyses of Thelohanellus kitauei (Myxozoa:Myxosporea) infection in the scattered mirror carp, Cyprinus carpio

Abstract

Thelohanellus kitauei is a spore-forming myxosporean parasite prevalent in scattered mirror carp (Cyprinus carpio) that generates numerous cysts in the intestine and causes mass mortality in fish. To investigate the infection and mortality induced by T. kitauei in pond-reared farms in Luo-Jiang (104°51'N, 31°31'E), southwest China, morphological and molecular analyses of infected fish were conducted. Natural and specific immune indicators were further evaluated to determine the immunological effects of response to parasitic infection. The infectious parasite was identified as Thelohanellus kitauei based on morphological, 18S rDNA and infectious characteristics. Scattered mirror carp was determined as the specific intermediate host of the parasite. However, T. kitauei still caused considerable damage to the fish, in particular, injury and blockage of the intestines, resulting in malnutrition and even death. The mature spores of T. kitauei colonize the intestinal submucosa of carp and form cysts of various sizes that block the intestinal tract and release spores into the enteric cavity upon rupture, leading to the next phase of T. kitauei growth. Moreover, T. kitauei-infected carp showed weaker innate immunity. IgM is involved in the fight against parasitic infection while cytokines, such as IL-6, IL-1β and TNF-α, had an impact on infection processes. To our knowledge, this is the first report to show that T. kitauei infects and causes death in scattered mirror carp. Our collective findings from systematic pathology, morphology and immunology experiments provide a foundation for further research on infections by this type of parasite and development of effective treatment strategies.

Figure 1

Gross presentation of infected Cyprinus carpio with macroscopic cysts in the intestine. (A) Infected scattered mirror carp (Cyprinus carpio) with swollen abdomen (red arrow). (B) Abdominal anatomy of infected carp: numerous cysts (yellow arrows) caused swellings that blocked the intestinal lumen in carp; (C–E). The cyst was in direct contact with intestinal mucosa. (F) Cross-section of cyst within the dotted box in D and E (H&E stain, bar = 500 µm).

Figure 2

Spore morphology of Thelohanellus kitauei from cysts of Cyprinus carpio. (A) Light micrograph of a wet-mount section of excised cyst from the intestine. Thelohanellus kitauei spore containing a polar capsule (triangle) and sporoplasm (arrow). Scale bar = 10 µm/100 µm. (B) Fresh spores stained with 1% crystal violet solution. Scale bar io100 μm. (C) Fresh spores stained with 1% crystal violet solution, polar capsule (triangle) and sporoplasm (arrow). Scale bar = 10 µm.

Figure 3

Histological lesions in Cyprinus carpio infected with Thelohanellus kitauei (H&E staining). (A) Cross-section of the cyst in the dotted box from Fig. 1D,E. Numerous Thelohanellus kitauei plasmodia (P) in intestine of Cyprinus carpio, submucosa of the intestine (red two-way arrow), serosa of the intestine (black two-way arrow), intestinal villus (red arrow), bar = 500 µm. (B) Numerous plasmodia in T. kitauei cysts. Scale bar = 500 μm. (C) Edema in intestinal serosa (black two-way arrow). Scale bar = 100 μm. (D) Spores in intestinal serosa (red arrowhead). Scale bar = 10 μm. (E) Intestinal mucosa replaced by T. kitauei plasmodia (P). Scale bar = 100 µm. (F) Fibrous hyperplasia around the T. kitauei cyst, T. kitauei plasmodia (P). Scale bar = 100 µm. Epithelial layer (red arrowhead). (G) Developing trophozoites (red arrowhead) of T. kitauei and spores within plasmodia of the cyst. Scale bar = 50 µm. (H) Several T. kitauei spores in plasmodia (P). Scale bar = 50 µm. (I) Developing plasmodia (P) under intestinal epithelial cells (red arrow). Scale bar = 50 µm. (J/K/L). Developing trophozoites (red arrowhead) at the edge of plasmodia and mature spores in the central region of plasmodia (black arrow). Scale bar = 10 μm.

Figure 4

Scanning electron micrographs of Thelohanellus kitauei from intestines of scattered mirror carp Cyprinus carpio. (A) Numerous T. kitauei of the intestine, bar = 50 µm. (B) The morphology of the T. kitauei. Scale bar = 10 μm. (C) The prominent ridges of the T. kitauei (red arrowhead). Scale bar = 10 μm. (D) Two shells of the T. kitauei (red long arrowhead). Scale bar = 10 μm. (E) The filament of the T. kitauei (red arrowhead). Scale bar = 10 µm. (F) Structural patterns of the T. kitauei. Scale bar = 10 µm.

Figure 5

Molecular phylogeny analysis (Maximum Likelihood method) based on 18 rDNA sequences of myxosporeans. Numbers at nodes indicate bootstrap confidence values (1,000 replicates).

Figure 6

Routine blood examination of scattered mirror carp. Non-infected scattered mirror carp from the same pond were used as a control group. WBC, white blood cell count; Lym, lymphocyte count; Eos, eosinophil count; RBC, red blood cell count.

Figure 7

Changes in serum immune indices of infected scattered mirror carp, Cyprinus carpio. Non-infected scattered mirror carp from the same pond were used as the control group.