JPC SYSTEMIC PATHOLOGY

HEMOLYMPHATIC SYSTEM

February 2024

H-B01

 

SLIDE A

SIGNALMENT (JPC #1758391): 4-year-old bull.

 

HISTORY: Over a 3-day period, 3 bulls were found dead without previous signs in a pasture with 2 other bulls and 25 cows.

 

HISTOPATHOLOGIC DESCRIPTION: Spleen: Diffusely, there is severe lymphoid depletion with necrosis and collapse of the white pulp characterized by lymphocytolysis. The trabecular meshwork of the red pulp is widely separated by abundant blood (congestion), and scattered throughout are a multitude of large (1x6 µm) bacilli with squared ends which occur individually or in short chains. Multifocally, endothelial cells are hypertrophied and there are intravascular bacilli; vessel walls are mildly thickened by edema and fibrin. There is diffuse intracapsular hemorrhage and expansion of the pericapsular fibroadipose tissue by hemorrhage and fibrin, admixed with numerous bacilli.

 

Lung: Diffusely, alveolar septa are fragmented and necrotic and expanded up to ten times normal by abundant hemorrhage and fibrin and numerous large (1x6 µm) bacilli that occur individually or in short chains. Bacilli infiltrate bronchial and bronchiolar subepithelial connective tissue which is similarly expanded by hemorrhage, edema, and few neutrophils, and extend into the lumen. Bronchial-associated lymphoid tissue (BALT) is multifocally depleted with occasional lymphocytolysis and replacement by hemorrhage and additional bacilli. 

 

MORPHOLOGIC DIAGNOSIS: 1. Spleen: Lymphoid depletion and necrosis, diffuse, marked, with diffuse congestion, capsular and pericapsular hemorrhage, and numerous intra- and extravascular bacilli, bovine. 

2. Lung: Pneumonia, interstitial, necrohemorrhagic, acute, diffuse, moderate with BALT necrosis and numerous bacilli.

 

SLIDE B

SIGNALMENT (JPC #531135): Age unknown, breed not specified pig.

 

HISTORY: Sections from a large, brick-red cervical lymph node found in a pig at slaughter.

 

HISTOPATHOLOGIC DESCRIPTION: Lymph node: There are multifocal to coalescing extensive areas of coagulative necrosis characterized by loss of differential staining and retention of cell architecture, as well as loss of architecture and abundant karryorhectic and cellular debris in some areas (lytic necrosis). Admixed there is fibrin, edema and hemorrhage. Within remaining lymphoid follicles, there is marked lymphoid depletion with central lymphocytolysis. Sinuses contain moderate numbers of degenerate neutrophils, lymphocytes, and macrophages admixed with fibrin, edema, hemorrhage, cellular and karyorrhectic debris (necrosis), and variably-sized colonies of 1µm cocci. Multifocally, small blood vessels contain fibrin thrombi. The pericapsular fibroadipose tissue is moderately infiltrated by many degenerate and viable neutrophils, fewer lymphocytes, plasma cells, macrophages, and fibrin and edema. 

 

SLIDE C

Lymph node (B&B): Numerous individual and short chains of dark blue staining (gram-positive) bacilli and variably-sized colonies of small cocci are scattered throughout the necrotic debris.

 

MORPHOLOGIC DIAGNOSIS: Lymph node: Lymphadenitis, necrotizing, diffuse, severe, with fibrinosuppurative perilymphadenitis and numerous short chains of gram-positive bacilli and cocci, porcine.

 

ETIOLOGIC DIAGNOSIS: Pulmonary, splenic, and lymphoid anthrax

 

CAUSE: Bacillus anthracis

 

CONDITION: Anthrax

 

GENERAL DISCUSSION: 

• Bacillus anthracis, a large, aerobic, spore-forming, gram-positive bacillus is the cause of anthrax, a reportable, zoonotic disease characterized by septicemia and acute death with bloody discharges from the nostrils, mouth, anus, and vulva
• Susceptibility, in decreasing order, among domestic animals: goats, sheep, cattle, horses, pigs, and dogs; farmed mink are highly susceptible; birds, amphibians, reptiles, and fish are resistant
• Disease course in ruminants is usually brief with septicemia, while in horses, pigs, and dogs, it is often localized to throat and intestine and may be fatal before sepsis occurs 
• The combination of the presence of numerous organisms in sepsis, and their ability to form highly resistant spores when exposed to oxygen is of paramount importance for disease epidemiology
• While in the carcass, vegetative bacilli are quickly (<48 hours) killed by putrefactive bacteria, but spores can remain viable in the environment for decades and are resistant to methods of disinfection except for oxidizing agents
• Enzootic in certain areas; outbreaks or concentration of spores in low lying pasture areas follow alternating periods of drought and heavy rain 
• Other sources of infection include contaminated animal products including bone meal, wool, hair, and hides

 

PATHOGENESIS:

• Three classic routes of exposure:

• Ingestion of spores (most common route in animals; spores from contaminated water, soil, fodder grown on contaminated soil, feed, bone meal, meat) > spores cross intact or compromised mucous membranes
• Entry of spores through skin (rare)
• Inhalation of aerosolized spores with uptake by alveolar macrophages (uncommon route in animals, but may occur when spores are taken up in dust and inhaled)
• Mechanical transmission has also been described via biting insects (horses)

• Pathogenesis is an initial lymphangitis and lymphadenitis > septicemia
• Following entry, spores germinate into encapsulated vegetative forms > carried by macrophages to regional lymph nodes > replication > organism spread via lymphatics > enters the bloodstream > secondary centers of infection in the reticuloendothelial system (spleen) > bacteremia > invasion of all body tissues
• Capsule virulence factor that establishes infection and consists of poly-D-glutamic acid, protects the bacterium from antibodies and bactericidal components in plasma, inhibits phagocytosis and killing of vegetative form 
• Toxin produced by vegetative cells and behaves as an A-B toxin; organisms themselves and capsule are nontoxic.
• Toxin consists of three components that act together to cause cell lysis: factor I (edema factor), II (protective antigen), and III (lethal factor). Combined effects are injury of phagocytes, increased capillary permeability, anticomplement activity, and impairment of coagulation

• Protective antigen (PA): B part of A-B toxin; receptor-binding protein that is essential for biological effects of edema and lethal factors; is endocytosed and creates a pore in the cell membrane by binding to tumor endothelial marker 8 (TEM8) and capillary morphogenesis protein 2 (CMG2) for entry of lethal factor and edema factor (which are the A part of A-B)
• Edema factor (EF): adenylate cyclase that increases cyclic AMP after activation of calmodulin; PA combines with EF inside cell > disrupts cell membrane water and electrolyte transporters > edema and blocks phagocytosis
• Lethal factor (LF): CNS depressant; PA combines with LF inside the cell to form lethal toxin, which induce cytokine production and cell death 
• PA, EF, and LF are found on the pX01 gene; poly-D-glutamic acid capsule are found on the pX02 gene

• In alimentary anthrax, anthrolysin O is produced and disrupts intestinal tight junction protein occludin

 

TYPICAL CLINICAL FINDINGS:

• Clinical signs and death depend on development of a massive septicemia
• Cattle and sheep:

• Peracute (most common in an early outbreak): probably <2 hours

• Found dead, often without premonitory signs

• Acute: disease course ~48 hours, high mortality

• Severe depression, tachypnea, congested/hemorrhagic mucosa, tachycardia, rumen stasis, abortion, bloodstained or dark yellow milk with reduced yield

• Pigs: Acute or subacute:

• Fever, depression, anorexia
• Characteristic edema on face and ventral neck, often with bloodstained froth due to pharyngeal involvement
• Petechia on skin
• Pulmonary form in baby pigs: lobar pneumonia and pleuritis, death in 12-36 hours

• Horses:

• Last for several days and consists of colic and large edematous swellings

 

TYPICAL GROSS FINDINGS:

• Delayed or incomplete rigor mortis; carcasses undergo gaseous decomposition quickly resulting in the “sawhorse” position; blood discharges from body orifices that do not clot (due to EF/LF toxin inhibition of platelet aggregation)
• Widespread edema (subcutis, lymph nodes, lung, mesentery, brain)
• Widespread hemorrhage (lymph nodes, mucous membranes, serosa, epicardium) 
• Cattle: 

• Very large soft spleen is most significant lesion and rarely absent, with “blackberry jam” consistency on cut section that exudes thick black-red blood that brightens on exposure to air (not always present in sheep and swine)
• Local lesions at site of entry: ulcerative hemorrhagic enteritis in small intestine; most severe lesions are over lymphoid tissue
• Mesentery is infiltrated with gelatinous fluid from lymphangitis
• Regional lymph nodes enlarged, red-black, and moist on cut surface (similar to spleen)
• Pulmonary anthrax causes acute congestion and consolidation with interstitial and mediastinal edema and regional hemorrhagic lymphadenitis

• Sheep: 

• Septicemic form most common (findings as in cattle, but with less pronounced splenomegaly, and no edematous effusions)
• Percutaneous infection (less common) causes spreading edema or hard localized nodules

• Pigs: 

• Ingestion (most common) causes infection localized to pharynx or intestine

• Characteristic sign is swelling of pharyngeal region and neck
• Unusual to have intestinal localization without pharyngeal localization
• Typical local lesion is carbuncle at point of entry with acute regional lymphangitis and lymphadenitis
• Primary intestinal: Initial lesion is hemorrhagic enteritis with diphtheresis; mesenteric lesions extend only to regional nodes

• Horses: 

• Ingestion: Primary lesions in throat (similar to swine) and intestine (similar to cattle)
• Large edematous swellings occur in ventral part of abdomen and thorax, legs, perineal region, and around external genitalia
• Septicemia: Splenomegaly

 

TYPICAL LIGHT MICROSCOPIC FINDINGS:

• Many large (1 x 6µm) bacilli with flattened ends in virtually every tissue; may be in short chains of 2-6 
• In septicemic anthrax (ruminants), organisms are intravascular
• Distended splenic sinuses with sludged RBCs, lymphoid follicles are separated and hypocellular, and numerous WBCs and bacilli in chains
• Lymph nodes: Hemorrhage and congestion extends into sinuses and cortex, numerous WBCs and bacilli with no necrosis
• Hemorrhage, edema, fibrin, and necrosis in multiple tissues

 

ADDITIONAL DIAGNOSTIC TESTS:

• If anthrax is suspected, the blood smear examination should precede autopsy
• Blood smear (recently dead animal): Numerous bacilli

• Obtain blood for diagnosis from sites least likely to have putrefactive bacteria, which destroy the vegetative bacilli (e.g., tip of the tail, coronet)

• Old methylene blue stains capsule pink
• Bacteriologic culture from putrified exudates; in nonsepticemic anthrax, organism is best found in exudates and affected lymph nodes
• Ascoli agar-gel precipitin test is helpful but not specific if tissues are old and dry

 

DIFFERENTIAL DIAGNOSIS:

• Other causes of sudden death:
  • Clostridial infections: 
    • Blackleg (Clostridium chauvei): Necrohemorrhagic and emphysematous myositis with edema
    • Malignant edema (Clostridium sp.): Serohemorrhagic cellulitis and myositis 
    • Bacillary hemoglobinuria (C. hemolyticum): Necrotizing hepatitis with emphysema; multifocal edema and hemorrhage; hemoglobin-stained kidneys
  • Lightening strike, trauma, bloat
  • Toxicity: Oleander, lead poisoning
  • Hypomagnesemic tetany: Tetany without signs of necrosis
• Other causes of splenomegaly: 
  • Babesia sp.: Necrosis of splenic germinal centers; organisms (1x3um oval, often in pairs) within erythrocytes; hemoglobinuria
  • Anaplasma sp.: Hemolytic regenerative anemia; no hemoglobinuria; icterus; organisms within erythrocytes (1um in diameter on edge of erythrocyte)

 

COMPARATIVE PATHOLOGY:

• Dogs: Very resistant, but reported when fed meat from animals that died of anthrax

• Pharyngeal or oral anthrax produces swelling of the head and throat; also may cause severe gastroenteritis

• Mink: High mortality after eating meat from infected animals
• Non-human primates:

• Susceptible to inhalation anthrax and IV exposure; macaques used as model for human disease and vaccine research.
• Gross: hemorrhage in mesenteric and tracheobronchial lymph nodes, meninges (“cardinal's cap”), lungs, and small intestinal serosa
• Histopathology: suppurative meningitis; leukomalacia; hemorrhages in the meninges, neuropil, and pulmonary alveoli; splenic lymphoid depletion and histiocytosis

• BALB/cJ mouse are resistant; A/J and DBA2/J mice highly susceptible; F344 rat highly susceptible to LF
• Hartley's Guinea pig and rabbits only two species with extensive renal tubular necrosis
• Also reported in elephants, Grevy's zebra, badgers, and exotic deer
• Birds, reptiles, and fish are generally not susceptible to anthrax because their normal body temperatures are outside the optimum range required by anthrax bacilli
• Greater kudu, nyala, waterbuck, and roan antelope are reported to be more susceptible 
• Carnivores can become infected through ingestion or inhalation of spores in infected carcasses
• Confirmed cases have been reported in the captive and free-ranging Amur leopard, bobcat, caracal, cheetah, clouded leopard, golden cat, jungle cat, leopards, leopard cat, lion, lynx, ocelot, panther, puma, serval, and tiger 
• Reported in the captive genet
• B. anthracis commonly causes disease in African wildlife, including chimpanzees and gorillas. Additionally, Bacillus cerus var. anthracis causes a similar, anthrax-like disease 
• Vultures appear to be resistant to B. anthracis; specimens have had antibodies to B. anthracis

 

REFERENCES:

1. Abee CR, Mansfield K, Tardif S, Morris T. Nonhuman Primates in Biomedical Research: Volume 2: Diseases. 2nd ed. San Diego, CA: Elsevier; 2012:764.
2. Church ME, Terio KA, Keel MK. Procyonidae, Viverridae, Hyenidae, Herpestidae, Eupleridae, and Prionodontidae. In: Terio KA, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals. San Diego, CA: Elsevier; 2018:314.
3. Constable PD, Hinchcliff KW, Done SH, Grunberg W. Veterinary Medicine: A Textbook of the Diseases of Cattle, Horses, Sheep, Pigs, and Goats. 11th ed. St. Louis, MO: Elsevier; 2017:2011-2014.
4. Duncan M. Perissodactyls. In: Terio KA, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals. San Diego, CA: Elsevier; 2018:448.
5. Jones MEB, Gasper DJ, Mitchell E. Bovidae, Antilocapridae, Giraffidae, Tragulidae, Hippopotamidae. In: Terio KA, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals. San Diego, CA: Elsevier; 2018:133-134.
6. 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: Elsevier; 2018:243-244.
7. Landolfi JA, Terrell SP. Proboscidae. In: Terio KA, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals. San Diego, CA: Elsevier; 2018:427.
8. Lowenstine LJ, McManamon R, Terio KA. Apes. In: Terio KA, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals. San Diego, CA: Elsevier; 2018:396.
9. Stanton JB, Zachary JF. Mechanisms of Microbial Infections. In: Zachary JF, ed. Pathologic Basis of Veterinary Disease. 7th ed. St. Louis, MO: Elsevier; 2022: 207,211,220-221.
10. Stewart GC, Thompson BM. Bacillus. In: McVey DS, Kennedy M, Chengappa MM, eds. Veterinary Microbiology. 3rd ed. Ames, IA: Wiley-Blackwell; 2013:206-210.
11. Terio KA, McAloose D, Mitchell E. Felidae. In: Terio KA, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals. San Diego, CA: Elsevier; 2018:276-277.
12. Valli VEO, Kiupel M, Bienzle D. Hematopoietic system. In: Maxie MG, ed. Jubb, Kennedy, and Palmer's Pathology of Domestic Animals. Vol 3. 6^th ed. St. Louis, MO: Elsevier; 2016:171-174.
13. Welle MM, Linder KE. The Integument. In: Zachary JF, ed. Pathologic Basis of Veterinary Disease. 7th ed. St. Louis, MO: Elsevier; 2022:1168-1169.
14. Wunschmann A, Armien AG, Hofle U, et al. Birds of Prey. In: Terio KA, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals. San Diego, CA: Elsevier; 2018:735.

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