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 that peripheralizes the nucleus. 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). Multifocally, the serosa is 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 M. 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 tissue macrophages of 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 (no mucus) > 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
Three mechanisms of injury:

Dysfunction/lysis of epithelial cells (breakdown of barrier)
Dysfunction of lymphatic drainage
Inflammatory injury within lamina propria

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 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:

Secretes exochelins (iron chelator), iron-reductases, and siderophores to acquire iron from ferritin stored in macrophages
Lipoproteins- lipoarabinomannan (LAM): Ligand on bacterial cell wall for macrophage phagocytosis
Mannose and CD14 receptors, expressed on tissue macrophages, are the major receptors involved in phagocytosis of MAP
Inhibits phagosome-lysosome fusion, allows persistence within host; phagosome acidification
Weakens TH-1 response by suppressing macrophage activation by IFN gamma
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

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
Cytology: Do not stain with Wright stain, so appear as clear bacterial rods within phagocytes.

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:

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: Typical chronic weight loss, diarrhea
Nondomestic ruminants: Typical chronic weight loss, diarrhea late in course of disease, clinical signs similar to domestic goats with end-stage MAP
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
Canids: Reported in red foxes and a coyote, often without gross lesions
Mustelids: Reported in badgers, Eurasian otters, and beech martens
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:

Ackermann MR. Inflammation and Healing. In: Zachary JF, ed. Pathologic Basis of Veterinary Disease. 7th ed. St. Louis, MO: Elsevier; 2022:146.
Agnew D. Camelidae. In: Terio K, McAloose D, Leger J, eds. Pathology of Wildlife and Zoo Animals, San Diego, CA: Elsevier 2018:197.
Barthold SW, Griffey SM, Percy DH. Pathology of Laboratory Rodents and Rabbits. 4th ed., Ames, IA: John Wiley & Sons Inc; 2016:282.
Howerth EW, Nemeth NM, Ryser-Degiorgis MP. Cervidae. In: Terio K, McAloose D, Leger J, eds. Pathology of Wildlife and Zoo Animals, San Diego, CA: Elsevier 2018:161-162.
Jones, MEB, Gasper DJ, Mitchell E, Bovidae, antilocapridae, giraffidae, tragulidae, hippopotamidae. In: Terio K, McAloose D, Leger J, eds. Pathology of Wildlife and Zoo Animals, San Diego, CA: Elsevier 2018:131-132.
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. London, UK: Academic Press; 2018:243.
Lucena AN, Garza-Cuartero L, McAloon C, et al. Apoptosis levels in bovine Johne's disease ileal lesions and association with bacterial numbers. Vet Pathol. 2021; 58(6): 1086-1090.
Matz-Rensing K, Lowenstine LJ. New world and old world monkeys. In: Terio K, McAloose D, Leger J, eds. Pathology of Wildlife and Zoo Animals, San Diego, CA: Elsevier 2018:362.
Simmons J, Gibson S. Bacterial and Mycotic Diseases of Nonhuman Primates. In: Abee CR, Mansfield K, Tardif S, Morris T. Nonhuman Primates in Biomedical Research: Volume 2: Diseases. 2nd ed. San Diego, CA: Elsevier; 2012: 117.
Spagnoli ST, Gelberg HB. Alimentary System and the Peritoneum, Omentum, Mesentery, and Peritoneal Cavity. In: Zachary JF, ed. Pathologic Basis of Veterinary Disease. 7th ed. St. Louis, MO: Elsevier; 2022:472-473.
Stanton JB, Zachary JF. Mechanisms of Microbial Infections. In: Zachary JF, ed. Pathologic Basis of Veterinary Disease. 7th ed. St. Louis, MO: Elsevier; 2022:205-206.
Stockham SL, Scott MA. Leukocytes. In: Fundamentals of Veterinary Clinical Pathology. 2nd ed. Hoboken, NJ: Wiley; 2013: 98.
Uzal FA, Plattner BL, Hostetter JM. Alimentary system. In: Maxie MG, ed. Jubb, Kennedy, and Palmer’s Pathology of Domestic Animals. Vol 2. 6th ed. St. Louis, MO: Elsevier; 2016:194-197.
Williams BH, Burek-Huntington KA, Miller M. Mustelids. In: Terio KA, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals. London, UK: Academic Press; 2018:297.