JPC SYSTEMIC PATHOLOGY
REPRODUCTIVE SYSTEM
January 2022
R-B04

Signalment (JPC #1210466):  Cow

HISTORY:  None

SLIDE A: HISTOPATHOLGIC DESCRIPTION:  Mammary gland (lactation stage):  Filling and replacing 40% of ducts, both intralobular and interlobular, and extending into secretory units and the surrounding interstitium are numerous multifocal to coalescing areas of lytic necrosis characterized by loss of architecture with replacement by eosinophilic cellular and karyorrhectic debris admixed with abundant necrotic neutrophils. These areas of necrosis contain numerous large (up to 50 um) colonies of 1-2um basophilic cocci. Multifocally surrounding the necrotic foci are bands of fibrous connective tissue (fibrosis) as well as moderate numbers of lymphocytes and plasma cells and rare macrophages. Epithelial cells lining affected ducts, and occasionally within adjacent glands, are either attenuated, degenerate (characterized as swollen with vacuolated cytoplasm), or necrotic (characterized by shrunken cells with hypereosinophilic cytoplasm and nuclear pyknosis). Remaining glandular lumina and intralobular ducts contain either eosinophilic homogenous material (secretory product) admixed with occasional foamy cells (macrophages or foam cells), small amounts of necrotic cellular debris, fibrillar material (fibrin), or deeply basophilic, concentrically lamellated foci up to 120 um in diameter (corpora amylacea). Remaining glandular tissue is characterized by well-developed secretory alveoli, increased numbers and size of ducts, and decreased amounts of connective tissue (active lactating gland). Diffusely the interstitium is expanded by congested vessels, ectatic lymphatics, and mildly increased clear space (edema). 

Slide B:  Mammary gland (Brown and Brenn stain):  There are multiple large colonies of gram-positive cocci in the previously described necrotic foci.

MORPHOLOGIC DIAGNOSIS:  Mammary gland:  Mastitis, necrosuppurative, subacute, multifocal to coalescing, severe, with large colonies of gram-positive cocci, breed unspecified, bovine.

ETIOLOGIC DIAGNOSIS:  Staphylococcal mastitis

CAUSE:  Staphylococcus aureus

GENERAL DISCUSSION:

• Mastitis is the single most common disease syndrome in adult dairy cows
• Numerous bacterial species are isolated from the mammary gland; most common are Staphylococcus aureus and coliforms (namely Coli)
  • Majority of cases are opportunistic and may be associated with iatrogenic infection or environmental contamination, but bacterial species such as Streptococcus agalactiae and some types of Staphylococcus aureus are considered obligate parasites of the mammary gland
• Coagulase negative staphylococcal mastitis usually occurs in younger cows (first calf heifers); no increase in susceptibility with age
• Coagulase-positive staphylococci, including aureus, are the most pathogenic

PATHOGENESIS:

• Routes of infection: Usually ascending infection of teat canal; less often hematogenous (Brucella , Mycobacterium spp.) or percutaneous
• Bacteria ascend teat canal & damage epithelium of cistern and ducts > move to secretory units (alveoli), causing necrosis & abscesses  > staphylococci can establish persistent foci that provoke botryomycotic type of granulomatous reaction +/- rapid fibroplasia walling off lesions >  granulation tissue obstructs milk flow from unaffected lobules  > pressure from surrounding fibrosis causes mammary involution
• Disease severity and duration varies between species and individual animals; peracute cases of staphylococcal mastitis lead to toxin production and gangrene; milder, chronic form progresses to granulomas and fibrosis
• Catalase and hemolysin production are the best indicators of bacterial pathogenicity
• Factors in normal milk which inhibit bacterial growth:
  • Phagocytosis most important defense against gram-positive bacteria but less efficient in milk than in serum
  • Lactoferrin:  Iron-binding protein (inhibits bacterial multiplication by decreasing iron availability)
  • Lysozyme:  Lyses bacterial cell wall peptidoglycan
  • Lactoperoxidase:  May inhibit S. aureus and streptococci
  • H₂O₂ (byproduct of bacterial fermentation of milk carbohydrates)
  • Immunoglobulins:  Mostly IgG in the cow (promotes opsonization); less IgA (may reduce bacterial adherence at epithelial surfaces)
  • Complement 5 (C5a):  Present in mammary secretions and serves as chemoattractant for neutrophils and promotes phagocytosis

• Virulence factors for aureus:
  • Leukocidin: Cytolytic to bovine leukocytes
  • Alpha toxin cytolysin: binds to all membranes forming hexameric pores; believed responsible for gangrenous mastitis; not produced by all aureus
  • Beta toxin: Type C sphingomyelinase
  • Protein A: Antiphagocytic factor; binds to the Fc fragment of IgG
  • Extracellular enzymes: Coagulase, hyaluronidase, phosphatase, nuclease, lipase, catalase, staphylokinase (fibrinolysin), and proteases
  • Capsule: Interferes with opsonization, phagocytosis, and complement activity; not present in all strains
  • Penicillinase: Splits beta-lactam ring of penicillin

• Factors that mediate inflammation in aureus mastitis: Exotoxins, lipoproteins, peptidoglycans, lipoteichoic acid and stimulation of TLR-2

TYPICAL CLINICAL FINDINGS:

• Disease severity varies from mild transient or chronic to peracute with fulminant fatal disease
• Usually chronic/subclinical with occasional recrudescence
• Severe, acute form: Usually shortly after parturition; may produce gangrene of infected quarters with high mortality; minimal secretion and may be brown, straw colored or red stained

TYPICAL GROSS FINDINGS:

• Affected quarters are swollen, hot, and firm with flaky or purulent secretion
• Moist gangrene: Tissues are blue-black, cold; overlying skin is  emphysematous, may slough
• Pitting edema of the inguinal area, and unaffected quarters may be swollen

TYPICAL LIGHT MICROSCOPIC FINDINGS:

• Acute inflammation with neutrophils filling ducts
• Epithelial hyperplasia and squamous metaplasia in ducts which may progress to degeneration, necrosis, and loss in severe cases
• Large areas of obliterated architecture (necrosis); interstitial edema; erosion and ulceration of duct system; lymphocytes and plasma cells in the interstitium
• Necrotic foci surrounded by neutrophils; thick bands of fibrosis and granulation tissue formation surrounding the necrosuppurative areas; numerous cocci in necrotic areas; may progress to granulomatous inflammation (i.e. botryomycosis) with colonies of cocci at center and +/- Splendore-Hoeppli phenomenon
• Involution of adjacent mammary lobules
• Less pathogenic strains of staphylococci cause less dramatic abscess formation with similar granulation tissue and fibrosis and adjacent mammary involution/atrophy

ADDITIONAL DIAGNOSTIC TESTS:

• PCR and culture
• Special histochemical stains: Gram stains (Brown and Brenn, Brown and Hopp’s); Ziehl Nielsen and Gomori’s methenamine silver (GMS) to rule out other differential diagnoses

DIFFERENTIAL DIAGNOSIS:  

Causes of mastitis in cattle:

• Streptococcus agalactiae: Most common obligate pathogen of bovine & caprine udders; contagious, insidious, chronic mastitis; endemic in some herds; more mild & superficial than Staphylococcus, with intraluminal cocci & neutrophils, loss of small ductular epithelium, rarely disrupting interstitial tissue, with formation of  polypoid luminal proliferations & exuberant granulation tissue, fibrosis, and involution in adjacent tissue
• Streptococcus dysgalactiae: Acute mastitis associated with trauma; usually more than one quarter involved
• Streptococcus uberis: Mild, chronic inflammation
• Coliforms (Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Citrobacter sp., Pasteurella multocida, Pseudomonas aeruginosa, Serratia spp., and Proteus spp. = environmental contaminants): Acute fibrinonecrotizing mastitis associated with ascending infection; action of organisms attributed to endotoxin; often only one gland affected, and sloughing of the affected quarter is possible; may be sharply delineated line of infection & inflammation adjacent to normal gland; brown, cloudy, or blood-stained serous secretion; edema and hemorrhage in severe cases (regional edema may be severe)
  • Important factors in Coli-associated mastitis include stimulation of PRRs (i.e. TLR4) and the presence of endotoxin
• Mycoplasma bovis: In herds in which conventional mastitis organisms have been eliminated; tends to involve entire gland; often severe; sudden onset of agalactia; firm & painless mammary glands; may have concurrent arthritis or keratoconjunctivitis; acute neutrophilic exudates in ducts followed by alveolar and ductular epithelial hyperplasia with chronicity
• Mycobacterium bovis: pyogranulomatous inflammation with distinct pyogranulomas, granulomatous caseous inflammation, and/or diffuse histiocytic reaction; develop insidiously without clinical signs of inflammation, progressively increasing in size and firmness, and may shed bacilli in milk before gross lesions are evident; zoonotic
• Trueperella pyogenes: “Summer mastitis” usually affecting immature and non-lactating animals; necrosuppurative galactophoritis with abscesses
• Prototheca zopfii: Chronic granulomatous mastitis; similar appearance to Staphylococcus but with characteristic algal cells with wedge-shaped endospores
• Nocardia asteroids: Granulomatous with filamentous bacterial colonies in center of inflammatory nodules
• Other less common agents: Brucella abortus (subacute, destruction of alveoli; bacteria in gland during dry period); Mannheimia haemolytica (acute); Salmonella sp. (chronic with flare-ups); Fungi (Cryptococcus neoformans, Candida , Aspergillus spp., Trichosporon spp.)
• Physiologic udder edema must be differentiated from pathogenic edema associated with infectious processes; develops during periparturient period, is cool to touch, symmetric, limited to udder or can extend into perineal region, is self-limiting, and may be due to prepartum diets high in sodium or potassium salts

COMPARATIVE PATHOLOGY:

Mastitis in other species:

• Sheep/goats: Similar to cattle
  • aureus, Mannheimia haemolytica (contagious agalactia): Acute mastitis with life-threatening toxemia
  • Other bacteria: Staphylococcus, Streptococcus spp., Corynebacterium bovis, E.coli, Trueperella pyogenes
  • Lentiviruses: Maedi/Visna (ovine progressive pneumonia virus), caprine arthritis and encephalitis virus (CAE)
  • Parapoxviruses: Orf virus (contagious ecthyma)
  • Avocado in goats (Perea americana): Toxin is persin with unknown toxic principle causing swollen, firm, congested gland with edema
• Dogs & cats: Uncommon; early lactation; bacteria enter via fissures in nipples secondary to trauma; mostly Staphylococcus or Streptococcus spp. 
• Pigs:
  • Coliforms: Peracute post-partum disease, high mortality
  • Streptococcus spp.: Older sows; subacute mastitis; induration
  • Actinobacillus lignieresii, Actinomyces bovis, and aureus: Granulomatous inflammation
  • Mastitis-metritis-agalactia syndrome: Hormonal dysfunction secondary to sudden changes in management; metritis not significant
• Horses: Rare; most common pathogen is Streptococcus zooepidemicus, others reported are coliforms, Pseudomonas, Actinobacillus, Pasteurella
  • A case of an unidentified, new Streptococcus is reported in a pregnant donkey (Podico, J Vet Diagn Invest. 2021)
• South American Camelids: Rare; most common pathogen is coagulase-negative Staphylococcus
• Old World camels: Rare; caseous mastitis due to Trypanosoma has been reported
• Rabbits (AKA “blue breast”):
  • Staphylococcus aureus, type C (variable: swollen, red areas to chronic abscessation)
  • Pasteurella multocida (abscesses with thick yellow-gray exudate)
• Guinea pigs:
  • Occurs sporadically during early lactation
  • Gross: Red to purple glands that are enlarged, firm, congested, and edematous
  • Microscopically: Mild degeneration to necrosis of ductal epithelium during acute lesions; Chronic lesions cause marked interstitial fibrosis and mononuclear cell infiltration and structural obliteration
  • Escherichia coli, Klebsiella pneumoniae, Streptococcus zooepidemicus
• Rats:
  • Pasteurella pneumotropica (suppurative or chronic necrotizing mastitis)
  • Staphylococcus aureus, Corynebacterium, Pseudomonas spp.
• Hamster: Beta-hemolytic Streptococcus, P. pneumotropica, E. coli
• Mink: Staphylococcus intermedius

REFERENCES:

1. Agnew D. Camelidae. In: Terio KA, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals. San Diego, CA: Elsevier;2018:201.
2. Barthold SW, Griffey SM, Percy DH. Pathology of Laboratory Rodents and Rabbits. 4th ed. Ames, IA: John Wiley & Sons, Inc; 2016: 142-144, 186, 231, 284-286.
3. Delaney MA, Treuting PM, Rothenburger JL. Lagomorpha. In: Terio KA, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals. San Diego, CA: Elsevier;2018:494.
4. Foster RA. Female reproductive system and mammae. In: Zachary JF, ed. Pathologic Basis of Veterinary Disease. 6th ed. St. Louis, MO: Elsevier; 2017:1183-1188.
5. Jones MEB, Gasper DJ, Mitchell (nee Lane) 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;2018:126.
6. Ruegg PL, Erskine RJ, Morin DE. Mammary gland health. In: Smith BP,ed.  Large Animal Internal Medicine.  5th ed.  Louis, MO: Mosby Elsevier; 2015:1015-1043.
7. Podico G, Gray SM, Wang L, et al. A novel Streptococcus species causing clinical mastitis in a pregnant donkey. J Vet Diagn Invest. 2021;33(5);979-983.
8. Schlafer DH, Foster RA. Female genital system. In: Maxie MG, ed. Jubb, Kennedy, and Palmer’s Pathology of Domestic Animals. 6th ed. vol. 3. Philadelphia, PA:Saunders Elsevier;  2016:452-457.
9. Williams BH, Huntington KAB, Miller M. Mustelids. In: Terio KA, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals. San Diego, CA: Elsevier;2018:298.

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