JPC SYSTEMIC PATHOLOGY
DIGESTIVE SYSTEM
August 2021
D-B09

SIGNALMENT (JPC #2741081):  5 year old, female Angus-cross cow

 

HISTORY:  This cow had chronic weight loss and diarrhea for 2 months.  Four herdmates died with similar clinical signs.  At necropsy, the small intestinal mucosa was markedly thickened with prominent corrugated mucosal folds, and the mesenteric lymph nodes were markedly enlarged and firm.

 

HISTOPATHOLOGIC DESCRIPTION:  

Slide A: Small intestine:  Diffusely and markedly expanding the lamina propria and submucosa up to 5 mm and replacing and widely separating crypts are numerous epithelioid macrophages arranged in dense sheets. Macrophages have abundant, finely granular, pale eosinophilic cytoplasm and an eccentric round to oval nucleus with finely stippled chromatin.  Villi are blunted and fused, and form prominent mucosal rugose folds.  Multifocally, moderate numbers of lymphocytes, plasma cells, eosinophils, and rare multinucleated giant cells are admixed with macrophages.  Multifocally, crypt epithelial cells have increased cytoplasmic basophilia, vesicular nuclei, mildly increased numbers of mitotic figures, and pile up to 5 cell layers deep (regeneration).  Low numbers of lymphocytes are present within the tunica muscularis and subserosal adventitia.  Diffusely, within the submucosa, tunica muscularis, and serosa, there is increased clear space and mildly ectatic lymphatics (edema).  Diffusely, there is marked fat atrophy within the serosa, which is often lined by hypertrophied (reactive) mesothelium.

 

Slide B: Acid fast: Small intestine:  Diffusely, epithelioid macrophages within the lamina propria often contain large numbers of 1 x 2 um acid-fast bacilli.

 

MORPHOLOGIC DIAGNOSIS:  Small intestine: Enteritis, granulomatous, diffuse, marked, with villus atrophy, edema, and numerous intrahistiocytic acid-fast bacilli, Angus-cross, bovine.

ETIOLOGY: Mycobacterium avium subspecies paratuberculosis (MAP)

ETIOLOGIC DIAGNOSIS: Mycobacterial enteritis

CONDITION: Johne's disease, paratuberculosis

GENERAL DISCUSSION:

• MAP is an acid-fast, weakly gram-positive, non-motile, facultative intracellular bacillus
• 99% DNA homology with avium subsp. avium; MAP differs by dependence on mycobactin
• IS900 insertion sequence (phenotype) element unique to MAP
• 2 strains known to cause paratuberculosis in various hosts, referred to as type I (S strain) isolated from sheep, and type II (C strain) first isolated from cattle; type II most common and have broad host range
• Resistant to drying; can survive for months in water, soil, feces
• Worldwide distribution; primarily affects domestic ruminants (cattle, sheep, goats, camelids); also domestic and wild mammals, and birds
• Causes chronic enteritis leading to diarrhea, progressive emaciation, and death
• Significantly decreases milk production and longevity in cattle
• MAP requires a high concentration of iron in the tissue macrophages for growth, which is highest in the ileocecal intestine; therefore, primary lesions are present in the ileum and large intestine

PATHOGENESIS:

• Ingestion of contaminated feces or MAP-contaminated milk, colostrum or water > M cells or epithelial cells overlying M cells > macrophages in Peyer’s patches > inhibit conversion of phagosomes to phagolysosomes > prolonged incubation > draining lymph nodes > thickening of LP with macrophages > malabsorption of amino acids > diarrhea, hypoproteinemia > emaciation > death
• Clinical stages:
  • Subclinical stage
    • Elicits cell mediated response (delayed-type IV hypersensitivity), driven by intestinal delta T cells and NK cells
    • Cell-mediated immunity (CMI) plays a role in development of mucosal lesions and onset of clinical disease
    • Lymphocyte proliferation, production of cytokines by stimulated T lymphocytes
    • Long, unpredictable incubation period; many animals in a given herd may be subclinically infected

• Clinical stage
  • Within macrophages, bacteria remain viable and protected from humoral factors
  • CMI response wanes and strong humoral response dominates
  • Initiated by release of bacteria by dying macrophages as disease progresses

• Late stage
  • Anergy may occur and neither cell-mediated nor humoral immunity may be detectable

• Young (<30 days old) most susceptible to infection; infectious dose for adults is considerably higher than for young animals
• Most animals clear the infection while some develop a subclinical infection (carriers) with intermittent shedding of bacteria
• Animals usually usually do not develop disease until 2-5 years old
• Long and unpredictable incubation period has given rise to the concept of “the iceberg effect”: in any affected herd, although few animals may show clinical signs, a much greater number are likely MAP infected
• Virulence factors / mechanisms of modulation of the immune response:
  • Complex cell wall: Permeation barrier
  • Mannose-capped lipoarabinomannan (Man-LAM): Mycobacterial cell wall receptor; major virulence factor; inhibits macrophage activation and phagosome maturation by inducing marked expression of IL-10
  • Toll-like receptor 2 (TLR2): Major signaling receptor binds MAP and initiates early production of IL-10 through the mitogen-activated protein kinase (MAPK)-p38 pathway
  • IL-10 suppresses macrophage activation, suppresses Th1 responses (suppresses TNF-alpha, IL-12, and IL-8), enhances Th2 responses, decreases MHC class II expression, and decreases apoptosis
  • Inhibits phagosome-lysosome fusion, allows persistence within host; phagosome acidification and phagolysosome fusion blocked by TLR2 signaling, MAPK-p38 activation, and IL-10
  • Secretes exochelins, iron-reductases, and siderophores to acquire iron from ferritin stored in macrophages
  • Mannose and CD14 receptors, expressed on tissue macrophages, are the major receptors involved in phagocytosis of MAP
  • Respiratory burst does not occur due to lack of opsonin-mediated phagocytosis, allowing MAP to persist in the phagosome
• Macrophage polarization in MAP:
  • Focal paucibacillary lesions (no for few bacilli): polarized to M1 phenotype which typically indicates proinflammatory response and promotion of Th1 responses; activated by IFN-gamma
  • Diffuse multibacillary lesions: polarized to M2 phenotype, which is consistent with immunoregulatory functions involved in parasite containment and promotion of anti-inflammatory tissue remodeling; activated by IL-4 or IL-10 (in humans, studies show advanced stage of mycobacterial infections where host responses less effective in controlling infection)

TYPICAL CLINICAL FINDINGS:

• Generally no clinical signs in cattle until older than 2 years of age
• Earliest signs: Weight loss, decreased milk production despite normal appetite
• Progresses to intractable profuse diarrhea without tenesmus, emaciation, and hypoproteinemia (intermandibular edema, “bottle jaw”); diarrhea related to granulomatous inflammatory response in the lamina propria of the small intestine
• Clinical pathology: Hyperphosphatemia (muscle catabolism); anemia (chronic disease); hypocalcemia (due to hypoalbuminemia); hyponatremia; hypokalemia

 

TYPICAL GROSS FINDINGS:

• Major lesions confined to the ileum, large intestine, and draining lymph nodes
• Diffuse thickened, rough, rugose mucosa +/- foci of ulceration in the ileum, ileocecal valve, cecum, and proximal colon (though mucosal lesions possible from duodenum to rectum, and may be segmental or diffuse)
• Mesenteric (consistently ileocecal) granulomatous lymphadenopathy is consistently present
• Granulomatous lymphangitis: Subserosal lymphatics prominent and dilated, lymphangiectasia; lymphangitis may be the only readily recognizable gross lesion and is specific enough to justify presumptive diagnosis of Johne’s
• Cachexia: Muscle wasting, serous atrophy of fat
• Hypoalbuminemia: Intermandibular edema, body cavity effusions
• Severity of gross lesions does not correlate well with severity of clinical disease
• Aortic and endocardial subintimal fibrosis and mineralization

 

TYPICAL LIGHT MICROSCOPIC FINDINGS:

• Classic lesions: Transmural lepromatous type of granulomatous enteritis with villous atrophy and crypt hyperplasia; lymphoplasmacytic lymphangitis +/- granulomas in the wall of lymphatics; granulomatous lymphadenitis; cattle have non-caseating granulomas; sheep, goats, deer have caseating granulomas
• More subtle cases: Lymphoplasmacytic and eosinophilic infiltrate in lamina propria with few macrophages
• Less common: Focal granulomas in liver (portal triads or scattered through hepatic parenchyma) or hepatic lymph nodes
• Rare: Focal granulomas in kidney or lungs
• Paucibacillary form, where organisms are more difficult to demonstrate, may only show multifocal pattern of inflammation, whereas multibacillary form, where organisms can be readily demonstrated, has a more diffuse and/or transmural pattern of inflammation

 

ADDITIONAL DIAGNOSTIC TESTS:

• Acid-fast stain (organism located within macrophages and giant cells)
• Culture (ileocecal LN, intestine, feces) more sensitive than histopathology but difficult; difficult to culture in sheep (different strain than cattle and goats)
• PCR (most sensitive), IHC, complement fixation test (CFT), AGID, ELISA (ELISA superior to CF or AGID)

 

DIFFERENTIAL DIAGNOSIS:

• Chronic diarrhea in adult cattle:
  • Intestinal parasites (ostertagiasis, coccidiosis): Usually more acute and affect younger animals
  • Salmonellosis: Generally acute; gram-negative bacteria; not acid-fast
  • Secondary copper deficiency (chronic molybdenum poisoning): Generally regional; affects multiple animals
  • Bovine viral diarrhea (BVD): Diarrhea with mucosal and Peyer's patch necrosis
  • Emaciation in adult cattle:
  • Malnutrition
  • Lymphoma
  • Traumatic reticulopericarditis
  • Pyelonephritis, amyloidosis
• Gross and microscopic lesions:
  • M. bovis: Usually fewer organisms; nodule formation; fibrosis; necrosis; calcification; classic granulomas

COMPARATIVE PATHOLOGY:

1. M,. avium subsp. paratuberculosis in other animals:

• Sheep and goats: Adult disease; chronic wasting; hypoproteinemia (submandibular edema); feces often normal or soft but not diarrheic (except in pygmy goats, which may have explosive diarrhea); more sporadic and subtle gross enteric lesions common (sheep, goats) and lesions most common in distal jejunum and/or ileocecal valve; tuberculoid caseating granulomas with calcification in mucosa, submucosa, serosa, and lymphatics; occasional glomerular amyloidosis (goats); orange mucosa and lymph nodes with pigmented strain of MAP (sheep); organism is usually readily demonstrable by acid-fast staining within macrophages and giant cells in the lesions, especially in diffuse multibacillary forms of disease 
• Captive and free-ranging cervids: Similar to sheep and goats, but may occur at <1 year of age; microscopic lesions rarely seen (1/20 subclinically infected captive white-tailed deer) in contrast to 15/15 subclinical MAP infected cattle having microscopic granulomas (J Vet Diagn Invest. 2019;31(6):844-851)
• Nondomestic ruminants: Typical chronic weight loss, diarrhea late in course of disease, clinical signs similar to domestic goats with end-stage MAP; infected herd mate was strong risk factor for infection; best predictor of infection was number of contact dates within first week of life, with a 2-fold increase in risk associated with each doubling in the number of contact days (J Vet Diagn Invest. 2018;30(1):78-85)
• Camelids: Reported in alpacas, llamas, dromedary and Bactrian camels; severe weight loss (often difficult to detect visually due to heavy fleece) and diarrhea; gross and histologic lesions similar to those in cattle
• Wild European rabbits commonly infected and are a significant reservoir for livestock infection; mild to severe histiocytic and granulomatous enteritis primarily involving the small intestine, GALT, mesenteric lymph nodes, in addition to periportal hepatic granulomas
• NHPs: Both susceptible (NWM>>OWM); progressive diarrhea and weight loss, though positive TB test may be only hint of infection; acquired by contaminated water, soil, or paratenic insects; disseminated form frequent with simian AIDS; lesions often resemble Johne’s disease in cattle with thickened draining lymphatics and adhesions between mesenteric lymph nodes and intestinal serosa but otherwise healthy individuals with localized form may only have enlarged mesenteric lymph nodes; microscopic lesion varies with sheets of large epithelioid histiocytes infiltrating the mucosa in SIV infected animals in contrast to caseating granulomas in infected lymph nodes that cannot be morphologically discriminated from TB in localized infections independent of simian AIDS   

 

REFERENCES:

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4. Constable PD, Hinchcliff KW, Done SH, Grunberg W. Diseases of the alimentary tract-ruminant. In: Constable PD, Hinchcliff KW, Done SH, Grunberg W, eds. Veterinary Medicine, A Textbook of the Diseases of Cattle, Horses, Sheep, and Goats. 11th ed. St. Louis, MO: Elsevier; 2017:552-573.
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