Signalment:  

7-year-old female breed unspecified ox, (Bos taurus).This 7-year-old cow calved 4-5 months previously, and had been at pasture since. Found dead without premonitory signs.


Gross Description:  

Carcass preservation is poor, but the animal is in good body condition. Gross lesions are confined to urinary tract, mammary gland and supramammary lymph nodes: bilateral, chronic, fibrinopurulent and severe pyelonephritis; chronic, necrosuppurative, severe cystitis; urolithiasis due to multiple sand-like calculi; and induration of the left-fore mammary gland quarter. No milk could be expressed. On cut surface, the gland parenchyma was markedly fibrosed with multifocal 2-3 mm areas of suppuration. The left supramammary lymph node was enlarged with a 1 cm diameter focus of suppuration. 


Histopathologic Description:

Mammary gland. Separating lobules of atrophic acini are extensive bands of mature fibrous tissue which contain multifocal to coalescing pyogranulomas up to 10 mm in diameter. Pyogranulomas are centered on colonies of gram-positive cocci (gram-stained section not submitted) within up to 100 μm long radiating columns of hyaline eosinophilic material (Splendore-Hoeppli material). These in turn are surrounded by variably sized zones of viable and degenerate neutrophils, occasionally within a large area of necrosis, bounded by large numbers of macrophages and varying numbers of multinucleated giant cells mixed with lymphocytes, plasma cells and small numbers of neutrophils, bounded by concentric bands of fibrous tissue. Mammary acini are devoid of secretory product and are lined by cuboidal to low columnar epithelium which is multifocally vacuolated or necrotic and sloughing. Multifocally there is exocytosis of low numbers of neutrophils and lymphocytes into acinar epithelium and multifocally acini contain small amounts of eosinophilic fibrillar material (fibrin) and small numbers of neutrophils and/or macrophages. Within the interstitium of the secretory tissue there is vascular congestion and a diffuse mild infiltration of lymphocytes, plasma cells and fewer macrophages and a diffuse mild fibroplasia. 


Morphologic Diagnosis:  

Mammary gland: pyogranulomatous mastitis, chronic with intra-lesional bacteria. 


Lab Results:  

Beta-haemolytic, coagulase-positive Staphylococcus aureus was isolated from the supramammary lymph node. Corynebacterium renale was isolated from the kidney.


Condition:  

Mammary gland botryomycosis


Contributor Comment:  

The cause of death in this case was renal failure due to pyelonephritis. The chronic mastitis was judged to be incidental with respect to the presentation of sudden, unexpected death. Staphylococcus aureus was isolated from the supramammary lymph node. Although culture was not performed on the mammary gland, gram positive nature of the intralesional cocci would be consistent with Staphylococcus spp. 

S. aureus is one of the most common causes of bovine mastitis. Clinically, staphylococcal mastitis may be peracute and fulminating or milder and more chronic. The acute forms of disease generally occur shortly after parturition and tend to produce gangrene of the affected quarters with high mortality.(9) The chronic or subclinical forms are more common and thus associated with the most important economic losses. The clinical presentation may be related to the strain of S. aureus; strains differ in their ability to spread within herds, and to cause somatic cell count elevation, clinical mastitis, or persistent infections or loss in milk production. In vitro, strains differ in their ability to withstand killing by neutrophils or invade mammary epithelial cells.(1)

The main reservoirs of infection are infected quarters and lesions on the skin of the udder and teat. Once S. aureus contaminates the teat orifice, it can persist and multiply before entering the teat canal and sinus and disseminating within the mammary gland. Colonization of the distal part of the mammary gland may be achieved by adhesion to specific receptors on the surface of epithelial cells. The adhesion varies from very low to extremely high numbers of bacteria per cell. In vitro adhesion depends on multiple factors including strain and origin of mammary epithelial cells.(5) The host response to the penetration includes degeneration and necrosis of epithelial cells and exudation of neutrophils into the interlobular tissue and secretory acini. If the exudation is massive and the organisms highly toxigenic, the acute and gangrenous forms of the disease occur. S. aureus can also invade more deeply into the inter-acinar tissue and establish persistent foci of infection that provoke botryomycotic granulomatous reactions associated with marked fibroplasia.(9) Acinar atrophy may be due to pressure from this fibrosis and also from occlusion of small ducts by exudate or granulation tissue causing obstruction of milk flow from unaffected lobules.(9)


JPC Diagnosis:  

Mammary gland: Mastitis, pyogranulomatous, multifocal, severe, with numerous cocci and Splendore-Hoeppli material.


Conference Comment:  

The tissue sections examined in this case are composed primarily of lobules of mammary ducts; mammary glands/acini are largely atrophied or lost, likely due to pressure necrosis secondary to abundant inflammation. Mastitis is the single most common disease syndrome of adult dairy cows. Routes of infection vary from ascending infection of the teat canal (most common) to hematogenous or percutaneous. The most commonly bacterial isolates are Streptococcus spp., Staphylococcus spp., and gram-negative coliforms, especially Escherichia coli (also Enterobacter aerogenes, Klebsiella pneumoniae, Citrobacter spp., Pasteurella multocida, Pseudomonas aeruginosa, Serratia spp., and Proteus spp.). The mammary gland is the principal site of persistence or reservoir for certain bacterial species, including Streptococcus agalactiae, Staphylococcus aureus and Mycoplasma bovis, while infection with coliforms is typically acquired via teat contamination from the external environment (e.g., fecal contaminated bedding, soil or water). Streptococcus uberis and S. dysagalactiae can persist in either location. Other pathogens associated with bovine mastitis include Trueperella pyogenes, Prototheca zopfii, Nocardia asteroides, Mycobacterium spp., and less commonly, Brucella abortus, Mannheimia haemolytica, Salmonella spp., Cryptococcus neoformans, and Candida spp.(4,7,9)

Staphylococcus aureus is the most commonly reported etiology of mastitis. S. aureus isolates range from nonpathogenic to highly pathogenic; catalase and hemolysin production are the best indicators of bacterial pathogenicity.(4,9) Factors in normal milk which inhibit bacterial growth are listed in table one.(7,9) S. aureus has developed multiple virulence factors to overcome these defense mechanisms, which are listed in table two.(2-5,7)

Although most problematic in cattle, mastitis also affects many other domestic animal species. The major agents recovered from sheep and goats with necrotizing or gangrenous mastitis are S. aureus and Mannheimia haemolytica. For mycoplasmal mastitis, the typical causative agents are Mycoplasma agalactiae or M. mycoides. Additionally, goats and sheep infected with the small ruminant lentiviruses, caprine arthritis and encephalitis virus and maedi-visna virus, respectively, develop hard udders with agalactia. Equine mastitis is sporadic, and Streptococcus zooepidemicus is the typical cause.(4,7,9) In swine, mastitis usually occurs in lactating or recently weaned sows. Gram-negative coliforms are the most commonly isolated etiologic agents; gram-positive bacteria such as Streptcoccus, Staphylococcus and Aerococcus spp. are reported less frequently.(6) In dogs and cats mastitis is uncommon; dogs are more likely to present with mammary neoplasia, while fibroadenomatous hyperplasia (mammary hyptertrophy) is the most prevalent mammary lesion in cats. When present in dogs or cats, mastitis tends to occur in early lactation, due to Staphylococcus spp., Streptococcus spp. or E. coli entering lactiferous ducts via fissures in nipples.(4) E. coli, Klebsiella pneumoniae and Streptococcus zooepidemicus are commonly encountered in guinea pig mastitis, while S. aureus (type C) and Pasturella multocida tend to affect rabbits. Mastitis is also reported in rats (typically due to Pasteurella pneumotropica, S. aureus, Corynebacterium spp., or Pseudomonas spp.), and hamsters (beta-hemolytic Streptococcus spp., P. pneumotropica, E. coli).(8)

Table 1. Antibacterial factors in milk. (7,9)
Antibacterial Factor
Phagocytic cellsPhagocytosis is less efficient in milk than in serum
LactoferrinIron-binding protein that inhibits bacterial multiplication
LysozymeLyses bacterial cell wall peptidoglycan
LactoperoxidaseMay inhibit S. aureus and streptococci
Hydrogen peroxide A weak oxidizing agent that is a byproduct of bacterial fermentation of milk carbohydrates
ImmunoglobulinsPrimarily IgG, which promotes opsonization; less IgA, which may reduce bacterial adherence at epithelial surfaces

Table 2. Select virulence factors of S. aureus.(2-5,7)
Virulence Factor
LeucocidinCytolytic to bovine leukocytes
Alpha-toxinBinds cell membranes forming hexameric pores; not produced by all S. aureus isolates
Beta-toxinA phospholipase C or sphingomyelinase
Protein AAntiphagocytic factor that binds to the Fc fragment of IgG
Extracellular enzymesCoagulase, hyaluronidase, phosphatase, nuclease, lipase, catalase, staphylokinase (fibrinolysin), superantigens and proteases
Bacterial capsuleInterferes with opsonization, phagocytosis, and complement activity; not present in all strains of S. aureus
PenicillinaseSplits beta-lactam ring of penicillin


References:

1. Barkema, HW, Schukken YH, Zadoks RN. Invited review: The role of cow, pathogen, and treatment regimen in the therapeutic success of bovine Staphylococcus aureus mastitis. J Dairy Sci. 2006;89:1877-1895.

2. Biberstein EL, Hirsh DC. Staphylococci. In: Hirsh DC, Zee YC, eds. Veterinary Microbiology. Malden, MA: 1999:115-119.

3. DeDent AC, McAdow M, Schneewind O. Distribution of protein A on the surface of Staphylococcus aureus. J Bacter. 2007;189(12):4473-4484.

4. Foster RA. Female reproductive system and mammary gland. In: McGavin MD, Zachary JF, eds. Pathologic Basis of Veterinary Disease. 5th ed. St. Louis, MO: Mosby Elsevier; 2012:198, 1121-1124. 

5. Kerro Dego O, van Dijk JE, Nederbragt H. Factors involved in the early pathogenesis of bovine Staphylococcus aureus mastitis with emphasis on bacterial adhesion and invasion. A review. Vet Quart. 2002;24:181-198. 

6. Martineau GP, Farmer C, Peltoniemi O. Mammary System. In: Zimmerman JJ, Karriker LA, Ramirez A, Schwartz KJ, Stevenson GW, eds. Diseases of Swine. 10th ed. Ames, IA: Wiley-Blackwell; 2012:282-285.

7. Morin DE. Mammary gland health and disorders. In: Smith BP, ed. Large Animal Internal Medicine. 4th ed. St. Louis, MO: Mosby Elsevier; 2008:1112-1143. 

8. Percy DH, Barthold SW. Pathology of Laboratory Rodents and Rabbits. Ames, IA: Blackwell Publishing; 2007:192, 231-232, 281, 283. 

9. Schlafer DH, Miller RB. Female genital system. In: Maxie MG, ed. Jubb, Kennedy, and Palmers Pathology of Domestic Animals. 5th ed. Vol. 3. Philadelphia, PA: Saunders Elsevier; 2007:550-564. 



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2-1. Mammary gland


2-2. Mammary gland


2-3. Mammary gland



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