JPC SYSTEMIC PATHOLOGY

RESPIRATORY SYSTEM

September 2023

P-P17

 

SLIDE A: Signalment (JPC #1910090): Juvenile Chinook salmon (Oncorhynchus tshawytscha)

 

HISTORY: Several fish had difficulty swimming, decreased growth rate, and increased mortality due to starvation and predation

 

HISTOPATHOLOGIC DESCRIPTION: Gill: Multifocally affecting 15% of the gill architecture, primary lamellae and secondary lamellae are expanded up to 4 times normal by variably-sized, up to 300 µm diameter, irregularly round to oval microsporidian xenomas. These xenomas have a thin eosinophilic wall and contain numerous eosinophilic, 2-4 µm, oval spores. Multifocally primary and secondary lamellae are mildly expanded by low numbers of macrophages, lymphocytes, and necrotic cellular debris. There is multifocal atrophy, blunting, fusion, and loss of secondary lamellae with branchial epithelial and mucous cell hyperplasia in lamellae adjacent to microsporidian xenomas.    

 

MORPHOLOGIC DIAGNOSIS: Gill: Branchitis, granulomatous, multifocal, subacute, mild, with multifocal lamellar fusion and atrophy, and many intra-lamellar microsporidian xenomas, Chinook salmon, piscine

 

ETIOLOGIC DIAGNOSIS: Branchial microsporidiosis

 

CAUSE: Loma salmonae

 

SYNONYMS: Microsporidial gill disease

 

SLIDE B: Signalment (JPC #1901138): Fingerling channel catfish (Ictalurus punctatus)

 

HISTORY: Several fish were gasping at the water surface and succumbed to exertion

 

HISTOPATHOLGIC DESCRIPTION: Affecting 80% of the gill parenchyma, primary lamellae and to a lesser extent secondary lamellae are expanded up to 10 times normal by numerous foamy macrophages, fewer lymphocytes, and scattered hemorrhage, fibrin, edema, and necrotic cellular debris.  Multifocally, there are intralamellar myxozoan cysts up to 200 µm in diameter that contain numerous 2 x 10 µm, fusiform, eosinophilic spores. There is necrosis and loss of lamellar cartilage, and adjacent secondary lamellae have moderate epithelial and mucous cell hyperplasia with marked lamellar fusion. Multifocally separating secondary lamellae are cross sections of 100 µm diameter adult trematodes characterized by a 5 µm thick tegument, filled with a parenchyma, reproductive organs, vitellaria, and poorly discernible paired caeca (trematodes not present in all slides).

 

MORPHOLOGIC DIAGNOSIS: 1. Gill: Branchitis, granulomatous, proliferative and necrotizing, diffuse, subacute, moderate, with lamellar blunting, fusion, and atrophy, lamellar cartilage degeneration, and intralamellar myxozoan cysts, channel catfish, piscine.

2. Gill: Trematodes, multiple.

 

ETIOLOGIC DIAGNOSIS: Branchial myxozoanosis and trematodiasis

 

CAUSE: Henneguya ictaluri

 

SYNONYMS: Proliferative gill disease, hamburger gill disease

 

GENERAL DISCUSSION: 

• Loma salmonae classically causes gill xenomas on salmonids +/- mortality 

• Henneguya ictaluri causes necrotizing branchitis and dermatitis in farmed, channel catfish +/- mortality 

 

PATHOGENESIS/LIFE CYCLE:

• Microsporidia:  
  • Direct life cycle: Spore ingested > sporoplasm discharged via polar filament extrusion > migrates to & invades endothelial cells of gills > undergoes asexual reproduction (merogony), forming many meronts > give rise to sporonts, which undergo sporogony to produce spores within membrane-bound packets (sporophorous vesicles) > cell ruptures, spores released into water
  • In general, microsporidians are cell type specific, but can infect many organs if that cell type is widespread; how they spread in the body is unknown; few species (including Loma salmonae) can transmit vertically (inside the egg)
  • Microsporidians are often categorized by their ability to induce xenoma formation, which is a markedly hypertrophied host-cell (cyst-like) filled with microsporidian stages and compressing surrounding tissue forming a compression capsule; Loma salmonae is a xenoma forming microsporidian
• Myxozoa: 

• Complex life cycle, alternating between vertebrate (intermediate) and invertebrate (definitive) hosts (alternating bisporogony); two different forms, depending on species/genus: histozoic form: resides in intercellular spaces, blood vessels, or intracellular OR coelozoic form: resides in the gall bladder, swim bladder, or urinary bladder
• Henneguya ictaluri: Histozoic type; oligochaete worm (Deuterocohnia digitata) contains infective actinospore (Aurantiactinomyxon ictaluri) > actinospore released into water > infects catfish through the skin or orally using extrudable polar filament > actinospore matures to myxospore (Henneguya ictaluri) and replicate asexually in the gill > cell ruptures, spores released into water

 

TYPICAL CLINICAL FINDINGS:

• Loma salmonae: Wild-caught or pond-raised salmonids with variously sized white cyst-like nodules (xenomas) on the gills that enlarge slowly
• Henneguya ictaluri: Pond-raised channel catfish with, respiratory impairment and dyspnea; epidemics occur most commonly at 16-20^o C, especially in the spring (April-May), and are associated with new ponds 

 

TYPICAL GROSS FINDINGS: 

• Loma salmonae: White-yellowish variably sized nodules (xenomas) on gills or other infected tissue possibly becoming large masses
• Henneguya ictaluri: Gills start as pale and swollen and progress to grossly thickened/blunted (“clubbed”) or broken gill lamellae that bleed easily (“hamburger gills”)

 

TYPICAL LIGHT MICROSCOPIC FINDINGS:

• Loma salmonae: 
  • Gill filaments, primary/secondary lamella, bone, and cartilage expanded by xenomas (200-300µm thin walled cysts filled with numerous 2-4µm round to elliptical, birefringent eosinophilic spores with prominent posterior vacuole) +/- granulomatous inflammation; focal atrophy, fusion, loss of secondary lamellae

• Gill epithelial & mucus cell hyperplasia

Henneguya ictaluri:

• Gill filaments, primary/secondary gill lamella, gill bone, gill cartilage: expanded by myxozoan cysts (up to 200µm thin-walled cysts filled with numerous 2-10µm fusiform eosinophilic spores) with severe epithelial hyperplasia, lamellar fusion, and granulomatous bronchitis with nodules surrounding cysts containing cartilage necrosis, tissue liquefactive necrosis, and parasites
• Cysts may be present in the liver, spleen, kidney, and brain with little tissue reaction; with healing, see chondroplasia without cysts 

 

ULTRASTRUCTURAL FINDINGS:

• Microsporidia (general): Spores: spherical, electron-dense exospore, electron-lucent endospore, faint nucleus, and single coiled polar tubule; NO polar capsule (unlike Myxozoa) or mitochondria
• Myxozoa (general): Spores: two or more coiled polar tubules contained inside separate polar capsules; multinucleate sporoplasm; shell with two to six valves 

 

ADDITIONAL DIAGNOSTIC TESTS:  

• PCR: High sensitivity and specificity 
• Wet mount of affected tissue effective for identifying spores; with Henneguya ictalurid, also see focal areas of cleared cartilage (strongly suggestive, even without spores)
• Microsporidia (general):
  • Modified trichrome stain: Best observation of polar tube, polaroplast, and posterior vacuole
  • Luna stain
  • Periodic acid–Schiff (PAS): PAS-positive granules at the anterior end of spore
  • Gram positive 
  • Chitin-binding fluorochromes (e.g. Fungi-Fluor, Calcofluor) highly sensitive (spores contain chitin) 
• Myxozoa (general):
  • Luna stain
  • Periodic acid–Schiff (PAS): PAS-positive granules at the anterior end of spore
  • Ziehl-Neelsen or Kinyoun: Partially acid fast 
  • Silver stains: GMS or Warthin-Starry 
  • Giemsa: Highlights polar capsule 

 

DIFFERENTIAL DIAGNOSIS (FOR GROSSLY EVIDENT GILL MASSES):  

• Henneguya exilis (“lamellar disease”): Related species that causes severe gill pathology in fish 
• Desmozoon lepeophtherii: Microsporidian associated with gill disease in farmed salmon; difficult to detect on HE section, need IHC or calcofluor white (Herrero, et. al J Vet Diagn Invest 2020)
• Ichthyophthirius multifilis (“ich;” I-P16):  ciliated protozoan, horseshoe shaped macronucleus, trophozoite feeding stage causes formation of small (1 mm diameter) white nodules protruding from skin and gills; “salt-like” dusting of the skin
• Lymphocystis (I-V15): Lymphocystis disease virus (an iridovirus) causes white-pink masses on skin, gills and other surfaces due to hypertrophic fibroblasts (lymphocysts) filled with basophilic intracytoplasmic inclusions 
• Epitheliocystis: Chlamydia-like bacteria; causes lamellar epithelial hypertrophy with large spherical, membrane bound, basophilic granular bacterial inclusions
• Dermal metacercariae: Digenean trematode infection; white, yellow, or black, flat to raised nodules 1 – 4 mm in diameter
• Bacterial granulomas:  Especially mycobacteriosis and nocardiosis; cutaneous and visceral, focal and coalescing, yellow-white nodular masses

 

COMPARATIVE PATHOLOGY:

• Zebrafish: Pseudoloma neurophilia: Xenomas in various tissues; tropism for CNS; spore coats are partially birefringent under polarized light

• Pigeons, budgerigars, finches, canaries, hummingbirds, parrots: Encephalitozoon hellum; chronic proliferative enteritis 
• Canids: Encephalitozoon spp with microgranulomas in brain, kidney and other organs
• Amphibians: Pleistophora myotrophica causes necrotizing myositis; Alloglugea bufonis causes intestinal xenomas
• Reptiles: Encephalitozoon pogonae associated with intrapericardial arteritis and hemopericardium in bearded dragons (Wünschmann et. al J Vet Diagn Invest 2019)
• Stingrays: Dasyatispora levantinae causes spindle-shaped, raised white lesions filled with thick, caseous material parallel to muscle fibers
• Invertebrates: Common cause of xenomas; in honey bees, Nosema apis and Nosema ceranae are a problem (tropism for ventricular epithelium)  (Higes et. al, Vet Pathol 2020)
• Gilthead Sea Bream: Enterospora nucleophila cause of emaciative disease, intranuclear development, infects the GI tract and hematopoietic organs (Picard et. al, Vet Pathol 2020)

• Amphibians: Cystodiscus: develop spore-forming plasmodia in the gallbladder of anurans, caudates, and caecilians; usually incidental, except C. australis and C. axonis also develop plasmodia surrounded by gliosis, hemorrhage and necrosis within the CNS/PNS of tadpoles and post-metamorphic frogs, as well as biliary hyperplasia, portal fibrosis, and lymphoplasmacytic portal hepatitis; plasmodia are 5-25 um with numerous small basophilic nuclei; Chloromyxum, Sphaerospora, Hofereullus: usually incidentally infect renal tubules and glomeruli of anurans and caudates; C. careni may be associated with mononuclear interstitial nephritis; H. anurae (frog kidney enlargement disease) causes enlarged kidneys with cysts 
• Goldfish and Koi: Myxobolus koi: causes proliferative branchitis
• Salmonids: Myxobolus cerebralis (M-P05): “whirling disease”; affects cartilage and causes skeletal deformities 
• Salmon: Tetracapsuloides bryosalmonae; proliferative kidney disease 
• Chinook Salmon: Ceratomyxa shasta: infects intestinal epithelium 

 

REFERENCES:

1. Frasca S, Wolf JC, Kinsel MJ, et al.   Osteichthyes. In: Terio KA, McAloose D, St Leger J, eds. Pathology of Wildlife and Zoo Animals. San Diego, CA: Academic Press; 2018:983-984, 988-990.
2. Gardiner CH, Fayer R, Dubey JP. Microspora and Myxozoa: An Atlas of Protozoan Parasites in Animal Tissues. 2nd ed. Washington, DC: Armed Forces Institute of Pathology, American Registry of Pathology; 1998:12-15. 
3. Herrero A, Padrós F, Pflaum S, Matthews C, Del-Pozo J, Rodger HD, Dagleish MP, Thompson KD. Comparison of histologic methods for the detection of Desmozoon lepeophtherii spores in the gills of Atlantic salmon. J Vet Diagn Invest. 2020;32(1):142-146. 
4. Higes M, García-Palencia P, Urbieta A, et. al. Nosema apis and Nosema ceranae Tissue Tropism in Worker Honey Bees (Apis mellifera). Vet Pathol. 2020;57(1):132-138. 
5. Keel MK, Terio KA, McAloose D.  Canidae, Ursidae, and Ailuridae. In: Terio KA, McAloose D, St Leger J, eds. Pathology of Wildlife and Zoo Animals. San Diego, CA: Academic Press; 2018:245.
6. Newton AL, Smolowitz R. Invertebrates. In: Terio KA, McAloose D, St Leger J, eds. Pathology of Wildlife and Zoo Animals. San Diego, CA: Academic Press; 2018:940-941, 1019.
7. Noga EJ, ed. Fish Disease: Diagnosis and Treatment. Ames, IA: Wiley-Blackwell; 2010: 229-237, 247-253.
8. Pessier AP.  Amphibia. In: Terio KA, McAloose D, St Leger J, eds. Pathology of Wildlife and Zoo Animals. San Diego, CA: Academic Press; 2018:940-941, 943-945.
9. Picard-Sánchez A, Piazzon MC, Ahmed NH, et. al. Enterospora nucleophila (Microsporidia) in Gilthead Sea Bream (Sparus aurata): Pathological Effects and Cellular Immune Response in Natural Infections. Vet Pathol. 2020;57(4):565-576. 
10. Stedman NL.  Chondrichthyes. In: Terio KA, McAloose D, St Leger J, eds. Pathology of Wildlife and Zoo Animals. San Diego, CA: Academic Press; 2018:1014-1015.
11. Trupkiewicz J, Garner MM, Juan-Salles C.   Passeriformes, Caprimulgiformes, Coraciiformes, Piciformes, Bucerotiformes, and Apodiformes. In: Terio KA, McAloose D, St Leger J, eds. Pathology of Wildlife and Zoo Animals. San Diego, CA: Academic Press; 2018:813.
12. Wünschmann A, Armién AG, Childress AL, et al. Intrapericardial Encephalitozoon pogonae-associated arteritis with fatal hemopericardium in two juvenile central bearded dragons. J Vet Diagn Invest. 2019;31(3):467-470.

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