JPC SYSTEMIC PATHOLOGY
REPRODUCTIVE SYSTEM
January 2022
R-B03

SLIDE A: SIGNALMENT:  4-year old male bison

HISTORY:  This bison had strong positive titers for Brucella abortus, and at slaughter, inspectors noted a testicle twice normal size

HISTOPATHOLOGIC DESCRIPTION:  Testis and epididymis:  Effacing 80% of the normal architecture and compressing, separating, surrounding, and replacing seminiferous tubules are multifocal to coalescing areas of caseonecrotic debris admixed with degenerate neutrophils, intra and extracellular 1x2 um coccobacilli, and foci of mineral that are further surrounded by epithelioid macrophages, multinucleate giant cells, fewer lymphocytes, and reactive fibroblasts (coalescing granulomas).  These areas are further surrounded by granulation tissue and abundant collagen (fibrosis) forming thick bands that dissect between granulomas. The inflammatory infiltrate and fibrosis extends into the adjacent tunica albuginea and epididymis. There is diffuse germ cell atrophy with lack of spermatids in the remaining seminiferous tubules.

MORPHOLOGIC DIAGNOSIS:  Testis and epididymis: Granulomas, multiple, with fibrosis, germ cell atrophy and aspermia, and numerous coccobacilli, Bison (Bison bison), bovine.

SLIDE B: SIGNALMENT (JPC #1713947):  Bovine fetus

HISTORY:  This fetus was aborted

HISTOPATHOLOGIC DESCRIPTION:  Lung: Multifocally and randomly affecting 50% of the lung, filling alveolar spaces and adjacent small bronchiole lumina, and expanding alveolar septa are nodular aggregates of numerous macrophages and degenerate neutrophils, few multinucleated giant cells, lymphocytes, plasma cells, abundant fibrin, edema, and scant hemorrhage. There is multifocal alveolar septal necrosis, with replacement by a similar population of inflammatory cells, necrotic debris, and fibrin. The pleura, interlobular septa, and associated lymphatics are expanded by moderate edema, fibrin, and hemorrhage and small aggregates of macrophages, lymphocytes, and plasma cells.

Liver: Multifocally infiltrating portal areas and randomly scattered throughout the parenchyma are low to moderate numbers of lymphocytes and plasma cells with fewer neutrophils and macrophages. There is multifocal extramedullary hematopoiesis.

MORPHOLOGIC DIAGNOSIS:

1. Lung: Bronchopneumonia, pyogranulomatous and fibrinonecrotic, multifocal to coalescing, moderate, with interlobular edema and pleuritis, breed unspecified, bovine.

2. Liver: Hepatitis, periportal and random, lymphoplasmacytic, multifocal, mild.

SLIDE C: SIGNALMENT (JPC #2020923):  Goat

HISTORY:  This goat had aborted

HISTOPATHOLOGIC DESCRIPTION:  Uterus and placenta (placentome): Multifocally and extensively filling caruncular crypts and effacing chorionic villi are large areas of lytic necrosis characterized by loss of architecture and replacement by eosinophilic cellular and karyorrhectic debris admixed with numerous degenerate neutrophils, macrophages, and prominent colonies 1x2 um basophilic coccobacilli. Bacteria are also present within macrophages, cytotrophoblasts, and syncytiotrophoblasts.  Multifocally less affected chorionic villi contain necrotic multinucleated syncytiotrophoblasts (shrunken with hypereosinophilic cytoplasm and pyknotic nuclei) and/or are multifocally denuded. Diffusely, the lamina propria of the caruncle/uterus and intercotyledonary area of the placenta is edematous and expanded by mucin and low numbers of neutrophils and macrophages.  Caruncular vessel walls are infiltrated by similar inflammatory cells, necrotic debris, and fibrin (necrotizing vasculitis) and vessel lumina often contain fibrin thrombi.

MORPHOLOGIC DIAGNOSIS:  Uterus with placenta (placentome): Placentitis, necrotizing, acute, focally extensive, severe, with necrotizing vasculitis, fibrin thrombi, and numerous intratrophoblastic, intrahistiocytic, and extracellular colonies of coccobacilli, breed unspecified, caprine.

ETIOLOGIC DIAGNOSIS:  Testicular, hepatic, pulmonary, and placental brucellosis

CAUSE:  Brucella abortus

CONDITION:  Brucellosis

SYNONYMS:  Undulant fever, Bang's disease

GENERAL DISCUSSION:

• Bovine brucellosis is a worldwide, zoonotic disease caused by Brucella abortus of many domestic and wild animals that causes systemic and chronic infections with recurrent bacteremia which often manifests as late term abortions, fetal bronchopneumonia, orchitis, epididymitis, and osteomyelitis
• Brucella are small, 0.4-1.5 um long, gram-negative, facultatively intracellular, bacilli to coccobacilli
• Infected populations of bison and elk are potential reservoirs
• Smooth strains of Brucella express O side chain on LPS, rough strains (B. ovis and B. canis) do not
• Major sources of infection: aborted fetus, placenta, uterine discharge

PATHOGENESIS:

• Transmission by contact, often ingestion, with infected tissues, secretions or excretions (milk, urine, fetal tissues, placenta, etc.) > penetration of mucosa and M cells > regional lymph node, Peyer’s patches > lymphadenitis, replication within mononuclear phagocytic cells (macrophages are the preferred cell for Brucella); > bacteremia > dissemination to mammary gland (especially colostrum), reproductive organs (both sexes), placenta, fetus, other lymph nodes > Invade or are phagocytosed by trophoblasts > massive intracellular replication of Brucella in trophoblast cells > fetal infection;
  • When Brucella abortus is ingested with feed contamination the initial lesion is a persistent nasopharyngeal lymphadenitis
  • Placental trophoblasts produce erythritol and steroid hormones, which is theorized to contribute to tropism for the pregnant uterus/placenta due to an unknown effect
• Smooth strains (more virulent): Cellular entry through interaction with lipid rafts in the plasma membrane via the Brucella LPS O-polysaccharide (O side chain)
  • Opsonization aids entry via IgG and complement (C3b and 4b); smooth strains are capable of intracellular replication;
  • After entry, trafficked to phagosome termed “Brucellosome” via endoplasmic reticulum (ER); requires low pH (<4.5) for intracellular replication in phagosome/brucellosome
  • VirB operon controls genes for type IV secretion system/T4SS and interacts with ER to neutralize pH of phagosome and prevents phago-lysosomal fusion (cyclic β-1,2-glucan and heat shock proteins also involved)
  • Brucella expresses 2 superoxide dismutases to neutralize oxidative killing and have a requirement for heme as an iron source in the phagosome; smooth strains inhibit macrophage apoptosis
• Rough strains: Different mechanism of cellular entry, more readily invade, are LPS deficient, and don’t interact with lipid rafts
  • Phagocytosed following TLR4 or mannose receptor interactions but are defective at intracellular replication
• Evasion of immune response:
  • Minimize stimulation of pattern recognition receptors (PRR) resulting in a reduced immune response
  • Induces reduced inflammatory response compared to other Gram-negative bacteria due to LPS structure;
  • Side chain interferes with MHCII antigen presentation
  • Inhibition of apoptosis of infected monocytes/macrophages
  • Prevents dendritic cell maturation, antigen presentation, and T cell activation
  • Subversion of unfolded protein response (UPR) and IRE1α signaling cascade important for intracellular survival
• Ruminant placental trophoblasts naturally produce erythritol, during the third trimester of pregnancy; this sugar is a favored carbon and energy source for many Brucella strains (presumably)
• Osteolysis due to IL-17 induced osteoclastogenesis
• Fetal death and abortion are attributed to placental disruption and endotoxemia
• Latent infections can occur but mechanism is unknown

TYPICAL CLINICAL FINDINGS:

• Retained placentas, metritis, infertility, late term abortions or birth of non-viable calf
• Scrotal swelling
• Mastitis
• Synovitis/arthritis

TYPICAL GROSS FINDINGS: 

• Placenta: Extensive non-uniform necrosis of cotyledons with brown exudate; intercotyledonary / periplacentomal areas thickened by edema with leathery surface appearance; necrosis and inflammation of intervillous portions (placental arcades); inflamed and expanded maternal septa leading to placental interlocking and increased risk of retained placenta
  • Placentitis is not evenly distributed and often leads to acute diffuse endometritis
• Uterus: Slowly developing lesions; exudate between endometrium and chorion; in sows, milliary uterine brucellosis may develop characterized by numerous, small granulomas within the mucosa
• Testicle/epididymis/accessory sex glands: Chronic epididymitis/orchitis, sperm granulomas, periorchitis, vesicular adenitis (seminal vesiculitis)
• Fetus: Fibrinous bronchopneumonia (most important fetal lesion especially in those aborted in the last half of pregnancy), pleuritis, and pericarditis, with fluid in body cavities; splenitis
• Carpal hygromas

TYPICAL LIGHT MICROSCOPIC FINDINGS:

• Testis:
• Necrotizing orchitis/epididymitis
• Fibrinopurulent serositis of visceral and parietal tunics
• Necrotic tubular epithelium with numerous organisms; predisposition to sperm granulomas
• In rams with ovis infection, the epididymal tail is most commonly affected and there is epithelial hyperplasia, degeneration, and the formation of intraepithelial lumina; unlike bulls, ram infections with Brucella spp usually do not have an associated orchitis
• Orchitis: three main categories
• Interstitial:
  • Typically not recognized macroscopically
  • Lymphocytic infiltration of intertubular stroma with fibrosis
• Intratubular:
  • Generally results from ascending infection
  • Solitary or multiple white nodules up to 1 cm
  • Retention of tubular outline but loss of tubular epithelium with replacement by macrophages, multinucleate giant cells that surround neutrophils, and cellular debris
  • +/- sertoli cell hyperplasia and mineralization
• Necrotizing:
  • Commonly associated with Brucella spp.
  • Coagulative necrosis bordered by fibrosis and inflammatory cells
  • Abscessation and fistulation may occur
• Placenta:
• Necrotizing placentitis with periarteritis and arteritis, edema, and macrophages and neutrophils
• No granulomas
• Numerous coccoid bacteria in the chorionic epithelium (trophoblasts) and macrophages and free in exudates; most severely affected trophoblasts are at base of villi and crypts of caruncle; sloughing of chorionic epithelium
• Most severely affected area of placenta is the intervillus portion (placental arcades)
• Fetus:
• Bronchopneumonia, fibrinous pleuropneumonia, or interstitial pneumonia (P-B14) with lymphocytes, macrophages, plasma cells, and variable numbers of neutrophils; pulmonary septal edema, necrotizing arteritis
• Focal necrosis and granulomatous inflammation in lymph nodes, liver (hepatocellular necrosis and pyogranulomatous portal hepatitis), spleen, kidney, meninges, and fibrinous pericarditis
• Uterus: Mild (early) to severe (late) lymphoplasmacytic and histiocytic to pyogranulomatous/granulomatous metritis with caruncular necrosis; numerous bacteria in epithelial cells
• non-specific acute, diffuse endometritis with placentitis occurring first
• Seminal vesicle: Fibrinopurulent to necrotizing inflammation often with dystrophic calcification, and rarely granulomas
• Mammary gland: focal lymphoplasmacytic and histiocytic interstitial inflammation and occasional lymphoid follicles
• Chronic infection leading to thickened interlobular septa and atrophy of alveoli; often no grossly detectable lesions
• Carpus: Nonsuppurative synovitis and hygromas in cattle

ADDITIONAL DIAGNOSTIC TESTS: 

• Serology: Serum agglutination, complement fixation, ELISA
• Milk ring test
• Culture maternal caruncle, placenta, stomach contents, lung
• Direct placental smear stained with Stamp’s or Kosters’ stain

DIFFERENTIAL DIAGNOSIS:  

Abortion:

Bovine:

• Mycotic placentitis (Absidia , Mucor sp., Aspergillus sp., Rhizopus sp.): Necrotic cotyledons and intercotyledonary areas; dry, leathery placenta; ringworm-like dermal lesions on fetus; lower rate of abortion
• Trichomonas fetus: Early term abortion; infertility, or pyometra and fetal maceration common
• Campylobacter fetus: Late term abortion; lower abortion rate; infertility; placental edema and petechia
• Neospora caninum: Abortions at 3-8 months of gestation and mummification; nonsuppurative necrotizing encephalitis and myocarditis with protozoal cysts and zoites
• Leptospirosis (L. pomona and L. hardjo): Late term abortions; brown gelatinous edema between amnion and allantois; yellow-brown cotyledons
• Infectious bovine rhinotracheitis (bovine herpesvirus-1): Late term abortions; hepatic necrosis with intranuclear viral inclusions in fetus
• Trueperella: abortion, suppurative placentitis, and fetal lung lesions; will see colonies of bacteria with the fetal pneumonia
• Bovine viral diarrhea virus (Bovine pestivirus): Early term abortions or weak calves with cerebellar hypoplasia; detection of virus in fetal tissues to diagnose
• Listeria monocytogenes: Late gestation abortion; vasculitis in chorioallantoic stroma; foci of necrosis in liver and other organs
• Epizootic bovine abortion (Foothill abortion, R-B01) caused by Pajaroellobacter abortibovis: Seasonal, tick-borne disease in the Rocky Mountain foothills; late term abortions; fetus with edema, hemorrhages in multiple tissues; lymphohistiocytic inflammation in many viscera, marked hepatic RE cell hyperplasia, granulomatous thymitis, lymphadenitis, pneumonia, and encephalitis
• Ureaplasma diversum: Necrotizing placentitis, amnionitis, with fibrosis, mineralization, heavy mononuclear cell infiltrate, foci of necrosis and hemorrhage, mild vasculitis; fetal conjunctivitis and nonsuppurative pneumonia
• Other bacterial causes: Salmonella, Staphylococcus spp., Streptococcus spp., Histophilus somni, and Mycoplasma spp.

Ovine / Caprine:

• Brucella ovis: placentitis with periarteritis and arteritis
• Brucella melitensis: resembles bovine brucellosis; abortion may be only sign, especially late in pregnancy
• Chlamydia abortus: Necrosis of cotyledons; infects trophoblasts and endometrial epithelium, vasculitis
• Coxiella burnetii (Q fever): Intercotyledonary necrotizing placentitis and characteristic intratrophoblastic organisms giving a “foamy” appearance.
• Toxoplasma gondii: Necrosis in cotyledon with normal intercotyledonary areas; protozoa within trophoblasts
• Campylobacter jejuni (predominant cause of ovine abortions in US), C. fetus venerealis: Metritis in ewes; enlarged, pale yellow to tan cotyledons covered with a brown exudate; targetoid hepatic necrosis in fetus
• Rift Valley Fever (Bunyavirus-Phlebovirus): Foci of hepatic necrosis in fetus; fetal hydranencephaly; prolonged gestation

Swine: causes of abortion, stillbirth, and/or mummification

• Viral:
  • Porcine reproductive and respiratory syndrome virus (Arterivirus)
  • Porcine Circovirus 2
  • Parvovirus
  • Pseudorabies virus
  • Enterovirus (Teschovirus)
  • Classical swine fever (Pestivirus)
  • Bovine pestivirus
  • Border disease virus (Pestivirus)
  • Japanese encephalitis virus (Flavivirus)
  • Cytomegalovirus (Betaherpesvirus)
• Bacterial:
  • Brucella suis (more common): often occurs between the second and third month of pregnancy; placentitis
  • Leptospirosis (more common): mummification of fetuses
  • Chlamydia
• Non-infectious:
  • carbon monoxide
  • Zearalenone

Orchitis:

Bovine:

• Bacterial: Mycobacterium bovis, M. tuberculosis, E. coli, Proteus vulgaris, Corynebacterium ovis, Streptococcus , Staphylococcus sp., Trueperella pyogenes, Actinobacillus spp, Nocardia farcinica, Chlamydia spp,, and Mycoplasma sp.
• Sperm granuloma

Caprine / Ovine:

• Sheep/Goat pox virus, Visna/maedi virus, Trueperella pyogenes, Corynebacterium pseudotuberculosis, Brucella melitensis

Feline:

• Feline infectious peritonitis: Fibrinonecrotic orchitis

Swine:

• Experimental infection with porcine rubulavirus (Paramyxoviridae), the cause of “blue eye”, may cause orchitis and epididymitis
• Burkholderia pseudomallei, Trueperella pyogenes, Streptococcus zooepidemicus, and Streptococcus equisimilis

Equine:

• Equine viral arteritis (Arteriivirus), Burkholderia mallei, Salmonella abortus-equi, Streptococcus equi, Streptococcus zooepidemicus, Strongylus edentates, Halicephalobus gingivalis

Canine:

• Brucella canis, E. coli, Proteus vulgaris, Burkholderia pseudomallei

Epididymitis:

Bovine:

• Brucella abortus, bovine herpesvirus 4 (cytomegalovirus), Actinobacillus seminis, Mycoplasma bovigenitalium, Trypanosoma brucei

Ovine/Caprine:

• Brucella ovis: rams excrete large numbers in semen – most often transmitted during coitus; spermatic granulomas are characteristic
• Others: Actinobacillus seminis (most important); Histophilus somni, Mannheimia haemolytica, E.coli, Trueperella pyogenes

Swine:

• Brucella suis: Abortion in first trimester; weak or stillborn piglets

Canine:

• Brucella canis, E. coli, Burkholderia pseudomallei, Mycoplasma canis, Blastomyces dermatitidis, Rhodotorula glutinis
• Canine distemper virus; intracytoplasmic and intranuclear inclusions in epithelial cells

Equine:

• Associated with migrating strongyle larvae, sperm granulomas, rare cases of Streptococcus zooepidemicus

Feline:

• Rare; Feline infectious peritonitis virus

COMPARATIVE PATHOLOGY:

Other Brucella spp. of veterinary importance:

• Brucella melitensis: Principal cause of brucellosis in sheep and goats in the Mediterranean, Asia, Africa, and South America; similar to bovine brucellosis although mastitis more common with melitensis; inflammation is localized to the mammary gland and pregnant uterus; in cattle and camels in Middle East and Central Asia; agent is one of main causes of human mortality
• Brucella ovis: Sheep (including Bighorn sheep): least virulent of the Brucella; abortion, stillbirth, epididymitis, ampullitis, suppurative orchitis with subsequent ductal epithelial hyperplasia; coital transmission; fetus: pneumonia, lymphadenitis, interstitial nephritis, pericholangitis
• Brucella suis: Pigs (including feral swine) (also melitensis, rarely B. abortus); transmission is mainly coital; boars testicular abscessation, predilection for bone and joints; granulomatous and necrotizing orchitis, abortions (less common than in ruminants), stillbirths, granulomas with multiple hyperplastic lymphoid nodules (miliary uterine brucellosis), osteomyelitis and diskospondylitis; has also been isolated in cattle in U.S.; multiple biovars
• Brucella canis: Dog: abortion, epididymitis with subsequent testicular degeneration and atrophy, occasional orchitis, diskospondylitis; rtPCR and IHC on formalin-fixed paraffin embedded (FFPE) tissue is promising to detect this organism that is classically difficult to diagnose (Camargo-Castañeda, J Vet Diagn Invest. 2021)
• Brucella abortus and B. suis are commonly associated with bursitis in horses (poll evil and fistulous withers)
• Marine mammals: ceti and B. pinnipedialis: placentitis, abortions, orchitis, and blubber abscesses, hepatic/splenic/lymph node necrosis, osteoarthritis, mastitis, endometritis, pneumonia; endocarditis and nonsuppurative meningoencephalitis (neurobrucellosis) in young striped dolphins, Atlantic white-sided dolphins, short-beaked common dolphins, long-finned pilot whales, and a recent report in Sowerby’s beaked whales (Davison, J Comp Pathol. 2021)
• Cats are the only domestic animal resistant to natural infection by Brucella
• neotomae (desert wood rat) and B. microti (voles)

REFERENCES:

1. Buckle K, Roe WD, Howe L, et. al. Brucellosis in endangered Hector's dolphins (Cephalorhynchus hectori). Vet Pathol. 2017;54(5):838-845.
2. Burrough ER, Wu Z, Sahin O, Zhang Q, Yaeger MJ. Spatial distribution of putative growth factors in the guinea pig placenta and the effects of these factors, plasma, and bile on the growth and chemotaxis of Campylobacter jejuni. Vet Pathol. 2012; 49(3):470-81.
3. Camargo-Castañeda AM, Stranahan LW, Edwards JF, et al. Characterization of epididymal and testicular histologic lesions and use of immunohistochemistry and PCR on formalin-fixed tissues to detect Brucella canisin male dogs. J Vet Diagn Invest. 2021;33(2):352-356.
4. Davison NJ, Brownlow A, Doeschate MT, et al. Neurobrucellosis due to Brucella ceti ST26 in Three Sowerby's Beaked Whales (Mesoplodon bidens). J Comp Pathol. 2021;182:1-8.
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12. Pedersen K, Quance CR, Robbe-Austerman S, et al. Identification of Brucella suis from feral swine in the selected states in the USA. J Wildl Dis. 2014;50(2):171-179.
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