Diagnosis of Hematodinium

 

Hematodinium infections can be diagnosed several different ways including:

Visual Examination
Wet Smears
Neutral red staining
Histology
Molecular detection

 

See below for examples of each method

 

Visual Examination

 

Snow Crab
Photo credit: Paul Collins, DFO

The snow crabs on the right are infected.  Note the barnacles on the crab on the left.  Molting stress may contribute to the onset or transmission of disease, but infected crabs turn color from the severity of infection.
Snow Crab
Photo credit: Paul Collins, DFO

The snow crabs on the left are infected.  Note the opaque white or pink of the shell (carapace).  The muscle is severly degraded in these heavily infected animals, and their metabolic reserves (glycogen) are depleted.

 

Wet Smears

Taking hemolymph
Taking hemolymph from a blue crab for diagnosis.
Vegetative trophonts
Vegetative trophonts of Hematodinium from the snow crab.  Host blood cells are virtually nonexistent in this sample, but the parasites resemble hemocytes to the untrained eye.
Dinospores of hematodinium
Dinospores of Hematodinium in the snow crab.  This stage is rarely seen as it is ephemeral.  Sporulation often kills the crab because parasites exit through the host's gills.

Neutral Red

Ameboid trophonts
host granulocytes
Staining by neutral red is probably the easiest way for those who don't work with the parasite. However, the parasite may not take up the stain when cultured in long-term cultures. Ameboid trophonts (left) and host granulocytes (center) stained with neutral red (0.05% in saline buffer). Photographs are at different magnifications. Hemocytes often take up very little stain, but Hematodinium parasites rapidly take it up. Plasmodial form of the parasite in action stained with neutral red (right).

 

Histology

Hematodinium is like other dinoflagellates.  The chromatin is condensed within the nucleus.  Nuclei appear as if in arrested metaphase.  Note the deeply blue-black staining chromatin within the nuclei of the parasites.  These slides were staind with Hematoxylin and Eosin.

 

Filamentous trophont
Filamentous trophont stage in heart tissue.
Plasmodial and prespore stages
Plasmodial and prespore stages of Hematodinium from the heart of an infected snow crab.
Sheets of parasite cells
Sheets of parasite cells (sheet-like plasmodia) from within the hepatopancreas (digestive organ) mature and break off into the bloodstream where they undergo division and sporulation.

 

 

 

Hepatopancreas uninfected

Hepatopancreas infected
Hepatopancreas fron uninfected (left) and infected (right) snow crabs.  The heavy infection results in a loss of connective tissue, a loss of hemocytes, and an enlarged hemal space between the tubules.

 

Molecular Detection

ITS1 region Routine diagnosis of Hematodinium:
  • HITSF1/HITSR1 primer will be applied for routine PCR diagnosis of tissue samples, water samples and sediment samples.
18S region Primers targeting 18S region will be used to identify potential different strains of Hematodinium in blue crab.
18S region, 187 bp

 Hematodinium quantification in water column:

  • Hematodinium positive samples will be analyzed further for quantification using RT-PCR.