Signalment:  

Adult, male American horseshoe crab (Limulus polyphemus)This horseshoe crab was part of an aquarium touch-tank exhibit for two years and developed mild gill and shell lesions. The gill and shell lesions progressively worsened; the crab became moribund and was euthanized.

Gross Description:  

The dorsal carapace (prosoma) had multifocal partial thickness pitting lesions with black and pale tan discoloration (Fig. 3-1). Book gill coverings had multifocal areas of black discoloration and several pale tan proliferative lesions along the caudal edges (Fig. 3-2). Individual book gill leaflets were very friable, tearing easily, and were opaque and pale tan (Fig. 3-2).

Histopathologic Description:

The cuticle of both the carapace and the book gill cover is multifocally thickened and the chitinous layers of the carapace are multifocally infiltrated and disrupted by fungal hyphae seen in longitudinal and cross section (Fig. 3-4). Fungal hyphae occasionally penetrate through the carapace into the underlying tissues (striated muscle and spongy parenchyma with prominent hemolymph channels). The affected tissues are hypereosinophilic (necrotic) with associated viable and degenerate amebocytes (hemolymph cells with eosinophilic cytoplasmic granules) (Fig. 3-4). Fungal hyphae are septate with mostly parallel appearing walls, and range from 4 to 7 um in diameter with occasional acute and right angle branching (Fig. 3-5). Some of the hyphae are poorly stained and appear swollen and non viable. The more viable forms stain with PAS and GMS fungal stains. Individual hyphae are sometimes seen on the outer surface of the cuticle and are pigmented brown (dematiaceous). Areas of disrupted cuticle have variably sized accumulations of basophilic granular material (bacteria, confirmed by Gram stain).

The cuticle of individual gill leaflets is multifocally thickened and penetrated by fungal hyphae. The central vascular channel of individual leaflets is expanded with hypereosinophilic and necrotic material, viable and degenerate amebocytes and bacteria (Fig. 3-3). There is necrotic debris mixed with bacteria between leaflets and sometimes pigmented fungal hyphae. Occasionally, there are some fungal hyphae on the surface of affected gill leaflets that are smaller (2-3 um diameter) than the invasive fungi.

Slides occasionally have metazoan parasites within the parenchyma and degenerate parasites associated with the surface of the carapace and/or gill leaflets.

Morphologic Diagnosis:  

Carapace: Shell disease, acute and chronic, necrotizing, multifocal, severe, with intralesional fungal hyphae and bacteria
Gills: Branchitis, acute and chronic, necrotizing, multifocal, severe, with intralesional fungal hyphae and bacteria

Lab Results:  

Fusarium sp. was isolated from the gill lesions, and it was speciated to F. solani using polymerase chain reaction (PCR).

Condition:  

Fusarium solani, ("Shell Disease")

Contributor Comment:  

The American horseshoe crab is of extreme importance to the medical field because of the lysate extracted from amebocytes (hemolymph blood cell), which is used to test pharmaceuticals for bacterial endotoxin contamination (limulus amebocyte lysate assay). Despite the importance of the horseshoe crab, there is a paucity of published information regarding its diseases. Infectious disease agents of horseshoe crabs include algae, fungi, cyanobacteria, Gram negative bacteria and many different parasites, with diseases of the shell being the most common manifestation.(8,9)

Mycotic shell disease has been reported only in captive horseshoe crabs. Specifically, mycotic infections of the carapace are reported in juvenile horseshoe crabs and most often in those housed without a sand substrate.(8,9) Gill and shell lesions similar to the ones seen in this crab are reported in a group of horseshoe crabs in a touch-tank at Ripley's Aquarium of the Smokies.(1) Single dose itraconazole therapy is well tolerated by horseshoe crabs, (1) but the efficacy of this treatment for the fungal lesions has not been investigated.

Fusarium species are saprophytes found ubiquitously within the environment and are the cause of diseases of both plants and animals including humans. Fusarium can cause disease in animals both by ingestion of mycotoxins and by fungal invasion of body tissues. Several examples of diseases caused by Fusarium infection in humans include keratitis, onychomycosis, dermatitis and disseminated disease.(3) Two recent reports of disease caused by Fusarium species reported in veterinary species include keratitis in a Holstein cow and intracranial fusariosis in a German Shepherd Dog.(4,5)

Invertebrate animals lack an adaptive immune system and respond to microbial antigens with a variety of innate immune responses including hemolymph coagulation, toll-like receptor mediated antimicrobial peptide production, melanin formation and lectin-mediated complement fixation. In horseshoe crabs, the hemolymph contains soluble antimicrobial proteins including C-reactive protein, alpha-2 microglobulins, lectins and hemocyanins. The granular amebocytes (also called hemocytes), which make up more than 99% of the circulating cells in the hemolymph, also contain antimicrobial proteins and coagulation proteins. Exposure to microbes causes degranulation of amebocytes and formation of a hemolymph clot. (6)

JPC Diagnosis:  

1. Carapace: Shell disease, necrotizing, acute and chronic, multifocal, severe, with fungal hyphae and bacteria
2. Gills: Branchitis, necrotizing, acute and chronic, multifocal, severe, with fungal hyphae and bacteria

Conference Comment:  

Another Fusarium species of importance in veterinary medicine is Fusarium moniliforme because certain strains of this mold release the toxin fumonisin B1. Fumonisin B1 is a potent mycotoxin that induces hepatocellular carcinoma in rats and leukocencephalomalacia in horses. Pigs get pulmonary edema and hydrothorax from fumonisin B1 ingestion. This mycotoxin has also been shown to be hepatotoxic to pigs.(10) Horses and pigs are exposed to this toxin when they eat corn infected with the mold Fusarium moniliforme. Fusarium proliferatum and Fusarium verticilliodes are also listed as producers of the mold fumonisin B1 in the newest edition of Jubb, Kennedy, and Palmer.(7)

Morphologically, the Fusarium sp. are identified by hyaline, septate hyphae measuring 4 to 7um in width with frequent, usually right angle branching. This is important in helping to distinguish these from Aspergillus sp, which tend to have dichotomous branching and are 3-5um in width.(2)

References:

1. Allender MC, Schumacher J, Milam J, George R, Cox S, Martin-Jimenez T: Pharmacokinetics of intravascular itraconazole in the American horseshoe crab (Limulus polyphemus). J Vet Pharmacol Ther 31:83-86, 2008
2. Chandler FW, Kaplan W, Ajello L: Color Atlas and Text of the Histopathology of Mycotic Diseases, pp. 76,79,101-102. Year Book Medical Publishers, Chicago, IL, 1980
3. Dignani MC, Anaissie E: Human fusariosis. Clin Microbiol Infect 10 (Suppl 1):67-75, 2004
4. Elligott CR, Wilkie DA, Kuonen VJ, Bras ID, Neihaus A: Primary Aspergillus and Fusarium keratitis in a Holstein cow. Vet Ophthalmol 9:175-178, 2006
5. Evans J, Levesque D, de Lahunta A, Jensen HE: Intracranial fusariosis: a novel cause of fungal meningoencephalitis in a dog. Vet Pathol 41:510-514, 2004
6. Iwanaga S, Lee BL: Recent Advances in the Innate Immunity of Invertebrate Animals. J Biochem Mol Biol 38:128-150, 2005
7. Maxie MG, Youssef S: In: Jubb, Kennedy and Palmers Pathology of Domestic Animals, ed. Maxie MG, 5th ed., pp. 358-359. Elsevier Limited, Philadelphia, PA, 2007
8. Smith SA: Horseshoe Crabs. In: Invertebrate Medicine, ed. Lewbart GA, 1st ed., pp. 133-142. Blackwell Publishing, Ames, IA, 2006
9. Smith SA, Berkson J: Laboratory culture and maintenance of the horseshoe crab (Limulus polyphemus). Lab Animal 34:27-34, 2005
10. Stalker MJ, Hayes MA: Liver and biliary system. In: Jubb, Kennedy and Palmers Pathology of Domestic Animals, ed. Maxie MG, 5th ed., pp. 371-372. Elsevier Limited, Philadelphia, PA, 2007

Click the slide to view.

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3-1. Carapace, American Horseshoe Crab.

3-2. Book gill (*) and book gill leaflets (arrow), American Horseshoe Crab.

3-3. Book gills, American Horseshoe Crab.

3-4. Cuticle, American Horseshoe Crab.

3-5. Cuticle (*), American Horsehoe Crab

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