Abstract

"White Lip Disease" was first diagnosed on commercial catfish farms in the Mississippi Delta in 1980. The disease is seasonal, occurring only in the fall, and is characterized by severe anemia and high mortalities. Hematocrits of less than 1% are common in affected fish. Clinical findings to date including hematology, serum clinical values, histology, and microbiology are discussed. A differential diagnosis of an erythroblastic anemia is presented and current investigations into proposed viral, toxic, or nutritional causes of this disease will be reviewed. Introduction

A condition characterized by severe anemia of farm-raised channel catfish was diagnosed in the Mississippi Delta in 1980. This syndrome has since become recognized as a notable source of economic loss to the. commercial catfish industry. During a 4-week period in the fall of 1983, more than (400,000 pounds of fish were last to this disease; an estimated value of $240,000.

A similar appearing syndrome has been reported in channel catfish in Alabama (1) and has been termed "No Blood Disease". It is suspected that this is the same disease reported in Mississippi-raised channel catfish, although the morbidity and mortality reports of the 2 regions do vary (1,2). Researchers in Alabama are investigating a possible dietary cause of the syndrome (1).

An "Anemia Disease" of salmonids has been recently reported and an infectious agent is suspected (3). Researchers at Domsea Farms are investigating the possible association of a chlamydia or mycoplasma-like organism (3).

Materials and Methods

Clinical investigation of "white lip disease" has consisted of hematology, serum chemistry, complete necropsies which included both parasite examination and bacterial culture, histopathology, and electron microscopy.

Hematologic studies centered on characterization of the anemia. Blood was collected into a heparinized vacutainer tube following dorsal aorta cannulation. The packed cell volume was determined by microhematocrit tube centrifugation. Red blood cell counts were done manually using the Unopette Microcollection System (Becton & Dickinson, Rutherford, NJ). Percent hemoglobin was determined by the cyanomethemoglobin method (4). Blood smears were made from blood collected in the vacutainer. Smears were stained with Leishman-Giemsa. Hematopoietic tissue imprints were made as well as hemopoietic tissue smears.

Serum from affected fish was separated and frozen for later analysis. Parameters investigated included sodium, potassium, chloride, glucose, total protein, and osmolality.

Standard necropsy procedures were followed for fish kill investigation (5). This included a complete physical examination, identification of ectoparasites on the skin and gills, and routine bacterial culture of the posterior kidney and brain. Sections of spleen, anterior kidney, posterior kidney, liver, intestine, stomach, and gill were preserved in neutral buffered 10% formalin, sectioned, and examined histologically with standard hemotoxylin and eosin stain. In addition, specific sections were stained with Prussian blue stain and examined for iron deposits.

Finally, blood cells were preserved in 3% gluteraldehyde, post-fixed in osmium tetroxide, and ultrastructurally examined with an electron microscope (Zeiss Model 109).

Results

Affected fish were severely anemic and hematocrits of less than 1% were common ,normal is greater than 25%) (6). The fish were often hypoproteinemic with total protein values of less than 2.5% (normal is greater than 5%) (6). The anemia was characterized as macrocytic and hypochromic (erythroblastic). Cells resembling "tart cells" (7) and neutrophils with eosinophilic inclusion bodies were noted on blood smears and in hemopoietic tissue imprints or smears.

The most significant serum constituent changes were hypoglycemia and electrolyte imbalances. Fish were consistently noted to be hyponatremic, hypochloremic, and hyperkalemic.

Necropsy findings included an extreme paleness of all viscera, and in some cases, abnormal gut contents were noted. The presence of fetid, mucoid material in the gut was inconsistent. Histologic examination revealed depletion of hematopoietic centers in the spleen and marked deposits of ceroid-like or lipofucin-like pigments in the spleen, liver, and anterior and posterior kidney. Prussian blue stains indicated that these areas did not stain positively for iron; therefore, it is believed that these areas do not represent hemosiderin deposits.

Significant lesions were not observed upon examination of tissue ultrastructure and no strain of bacteria or species of parasites were consistently found.

Discussion

A complete differential diagnosis for an erythroblastic anemia includes consideration of the following conditions: 1) Infectious disease this would include bacterial, viral, chlamydial, and protozoal agents; 2) Toxicity - acute or chronic; 3) Nutrition; 4)Hemorrhage; 5) Clotting disorder; 6) Neoplasia; 7) Autoimmune; and 8) Osmoregulatory dysfunction.

At this time, the possibility of a viral agent is being actively pursued. The identification of inclusion bodies within "tart cells", the neutrophils, suggests a viral etiology (7). Catfish neutrophils are not phagocytic (7).

A toxic agent cannot be ruled out. Microcystis sp. is an algae common to catfish ponds and is known to produce, on occasion, a toxin which can cause hemolysis in mammals (8). Toxicity to fish has never been conclusively demonstrated. The occasional finding of abnormal gut contents has led to speculation that an abnormal bacterial toxin in the gut might be associated with the condition. This possibility has not been substantiated by cultures of intestinal contents or by injection of intestinal contents from sick fish into unaffected fish.

A dietary etiology cannot be ruled out and is being investigated by researchers at Auburn University in association with "No Blood Disease" (1) of channel catfish.

Preliminary work done at Mississippi State University indicates that a Vitamin E deficiency may cause a change in the red blood cell membrane (9) which might be associated with increased fragility and possibly anemia under extremely stressful conditions. Since only one pond of fish on a farm will usually be affected, if a dietary factor were responsible for "white lip", more ponds of fish should be affected.

It is beyond the scope of this report to discuss all possible causes of an erythroblastic anemia in fish. At this time, investigation is directed at possible viral, toxic, or dietary causes of the condition.

References

1. Lovell, T. No blood disease of channel catfish. Presented at the 12th Annual Fish Nutrition Workshop, Mississippi State University, Mississippi State, 'AS, on 19 October 1983.
2. MacMillan, J.R. White lip disease in Delta-raised channel catfish. Presented at the 12th Annual Fish Nutrition Workshop, Mississippi State University, N State, MS, on 19 October 1983.
3. Wagner, P. Anemia disease of salmonids. Presented at the 12th Annual Fish Nutrition Workshop, Mississippi State University, Mississippi State, MS, on 19 October 1983.
4. Nelson, M.D. Basic methodology. n: Clinical Diagnosis and Management by Laboratory Methods, Volume 1, 16th Edition, J.B. Henry, Ed. W.B. Saunders Co., Philadelphia, 1979, pp. 863-868.
5. Reichenbach-Klinke, H.-H. and Elkan, E. The Principle Diseases of Lower Vertebrates: Book I - Diseases of Fishes. TFH Publications, Inc., Neptune, NJ, 1965, pp. 4-10.
6. Blaxhall, P. and Daisley, K. Some blood parameters of the rainbow trout. 1. The Kamloops Variety. J. Fish. Biol. 5: 1-8.
7. Macmillan, J.R. Pathological Manifestations of Viral Erythrocytic Necrosis in Fish. Doctoral Dissertation, University of Washington '1980).
8. Gabow, W.O.K, et al. Microcystis aeruginosa toxin cell culture, toxicity, hemolysis, and mutagenicity assays. Applied and Environmental Microbiology, pp. 1425­1433 (June, 1982).
9. Wilson, R.P. Personal communication.