JPC SYSTEMIC PATHOLOGY
ENDOCRINE SYSTEM
January 2022
R-P01

Signalment (JPC #2019265):  3-year-old post-partum Suffolk ewe

HISTORY:  This ewe delivered 2 lambs estimated to be 10 days premature and dead in utero for about one day.  There were no gross lesions in the fetuses.  The cotyledons had multiple 1-2 mm white nodules.  The intercotyledonary placenta was normal.

HISTOPATHOLOGIC DESCRIPTION:  Placenta, cotyledon:  Affecting 70% of this section, there is both coagulative and lytic necrosis of the chorionic villi characterized by complete loss of villar trophoblasts and replacement by eosinophilic cellular and basophilic karyorrhectic debris admixed with aggregates of deeply basophilic material (mineral), scattered hemorrhage, fibrin, and edema.  Multifocally, low numbers of trophoblasts adjacent to necrotic foci are expanded by an intracytoplasmic parsitophorous vacuole containing clusters of 2x3 um, oval, pale basophilic, apicomplexan tachyzoites.  Diffusely, the stroma of less affected villi is expanded by edema and low numbers of lymphocytes, fewer plasma cells and macrophages, and occasional degenerate neutrophils.  Multifocally, the cotyledon connective tissue is expanded by mild hemorrhage, fibrin, edema, and low numbers of similar inflammatory cells.

MORPHOLOGIC DIAGNOSIS:  Placenta, cotyledon:  Placentitis, necrotizing, subacute, multifocal to coalescing, moderate, with few intratrophoblastic apicomplexan tachyzoites, Suffolk ewe, ovine.

ETIOLOGIC DIAGNOSIS:  Placental toxoplasmosis

CAUSE:  Toxoplasma gondii

GENERAL DISCUSSION:

• Apicomplexan, obligate intracellular parasite that causes disseminated disease, central nervous system infections, and abortions in many species (except cattle)
• Felids are definitive hosts; all mammals and birds can be intermediate hosts
• Important cause of abortions in sheep and goats; those in late pregnancy in areas heavily contaminated with cat feces are most susceptible
• Ewes and does show no signs of infection
• Can have coinfections with Coxiella and Chlamydia in the same abortion outbreak
• Life stages of parasite: Sporozoites and tachyzoites are readily destroyed; tissue cysts (bradyzoites) are resistant and perpetuate the disease
  • Tachyzoites (trophozoites) are rapidly dividing forms and occur in free groups
  • Bradyzoites multiply slowly and are found in tissue cysts
  • Oocysts contain infective sporozoites after being shed

LIFE CYCLE:

• Cats are infected by eating meat containing tissue cysts
• Parasite sexual replication occurs in feline intestinal epithelium, especially the ileum
• Unsporulated oocysts pass in feces and sporulate in 1-5 days
• Sporulated oocysts are infectious to intermediate hosts and cats
• After ingestion, sporozoites excyst, multiply intracellularly in the intestines and related lymph nodes by endodyogeny and form tachyzoites that spread to other tissues via blood and lymph.
• Parasitemia develops in 3-4 days
• Tachyzoites actively penetrate host cell plasmalemma and reside within a parasitophorous vacuole derived from host and parasite
• Cell to cell spread continues until T-cell mediated immunity develops
• Intracellular tissue cysts containing bradyzoites form after 1-2 weeks and persist for months to years

PATHOGENESIS:

• Contamination of feed by cat feces is a common transmission route
• Ingestion of contaminated feces by the ewe results in parasitemia; the organism passes to the caruncle, then to the trophoblast and then the fetus; tachyzoites replicate in the caruncular septa, producing foci of necrosis
• Congenital transmission from ewe to lamb is not uncommon in healthy lambings
• T. gondii is able to infect all types of cells because it binds laminin (an extracellular matrix protein) and then binds to laminin receptors on the target cell
• Secretory organelles, called rhoptries, are important for cellular invasion; rhoptry membranes fuse with the anterior limiting membrane and a lytic product is secreted from fusion rosettes and facilitates invasion into the cell
• T. gondii has both catalase and glutathione peroxidase activity and is resistant to hydrogen peroxide, but is susceptible to hydroxyl radical and singlet oxygen (respiratory burst of inflammatory cells)
• In parasitophorous vacuoles the protozoan initiates the production of the anti-inflammatory cytokines IL-10 and transforming growth factor-β (TGF-β), which inhibit the production of proinflammatory cytokines IL-12 and TNF-α
• A recent experimental study suggests that, based on lesions found within the placentomes, different mechanisms are involved in the pathogenesis between “classical” and another form of ovine toxoplasmosis abortion termed “acute phase” (Benavides et al., J Comp Pathol. 2017)
  • Classical is associated with necrosis with variable inflammation
  • Acute phase infection may involve infarction and thrombosis of maternal vessels
• A recent study of T. gondii-associated early abortions and fetal leukomalacia characterized the placental and fetal brain lesions; findings suggested the brain lesions may be the result of either fetal inflammatory syndrome elicited by infection in the pregnant ewe rather than direct brain infection or related to hypoxia from placental thrombosis or hemodynamic changes (Gutierrez-Exposito et al., Vet Pathol. 2020)
• A recent study evaluated the immune response in the placenta of sheep experimentally (orally) infected with T. gondii: Early abortions had increased macrophages in caruncular septa; later abortions had an increase of T lymphocytes and macrophages mainly in the fetal cotyledons (Castaño et al., Vet Pathol. 2020)

TYPICAL CLINICAL FINDINGS:

• Effect on pregnancy depends on the stage of gestation when the infection occurs:
  • Early or mid-gestation infection: fetal death with resorption or mummification
  • Mid-gestation infection: Occasionally lambs survive to term but are stillborn or are weak and die shortly after birth
  • Late pregnancy: Fetus develops an immune response and is born live, infected, and immune
• Infected ewes rarely show clinical signs and do not abort in subsequent pregnancies
• Few fetuses may develop cerebral leukoencephalomalacia secondary to the anoxia from placentitis or fetal inflammatory syndrome (Gutierrez-Exposito et al., Vet Pathol. 2020)

TYPICAL GROSS FINDINGS:

• Fetus: Aborted with no gross lesions
• Placenta: characteristic lesions in fetal cotyledons of the placenta:
  • Cotyledon: Lesions usually confined to cotyledon, consist of numerous 1- to 2-mm diameter, white, soft foci of necrosis or flecks of mineral scattered among fetal chorionic villi; bright to dark red cotyledons (purple color is normal);
  • Caruncle: Mineralization

TYPICAL LIGHT MICROSCOPIC FINDINGS:

• Characteristic T. gondii lesions: Placental cotyledons - multifocal necrosis and mineralization with nonsuppurative inflammation; fetal brain - multifocal necrosis and gliosis (Meixner N. et al., JVDI 2020)
• Cotyledon:
  • Edematous villi, foci of necrosis, desquamation/sloughing of trophoblasts (epithelium)
  • +/- caseous and mineralized cotyledonary nodules
  • Mild mononuclear cell infiltration in trophoblastic epithelium, villous stroma, and endometrium
  • 2-4um fusiform basophilic tachyzoites may be free in trophoblasts or in cysts; encysted zoites may also be found in endometrium
• Intercotyledonary placenta: Edema only
• Fetus:
  • Brain: 95% of infected fetuses have mild nonsuppurative encephalitis with few scattered foci of necrosis and glial and mononuclear infiltrates; protozoa are more likely to be found in parts of the brain rostral to the pons and in the optic tracts; focal areas of leukoencephalomalacia without cellular infiltration and toxoplasma cysts at the periphery of lesions
  • Other organs: Multifocal necrotizing nonsuppurative hepatitis, pneumonia, myositis, myocarditis and nephritis

ULTRASTRUCTURAL FINDINGS:

• Zoites reside within a parasitophorous vacuole
• Pellicle (outer membrane) consists of 3 membranes: A plasmalemma and two closely applied membranes that form an inner membrane complex
• At the anterior surface is a cylindrical cone (conoid) consisting of microtubules wound like a spring and is used to probe host cell surface prior to entry
• Rhoptries are club-shaped excretory organelles between the anterior tip and nucleus that secrete a proteolytic enzyme used in host cell penetration
• Micronemes are rod-shaped structures found at the anterior end

ADDITIONAL DIAGNOSTIC TESTS:

• IHC, PCR, ISH, IFA
  • ISH and IHC combined with rtPCR may be useful detection methods to improve histologic evaluation of gondii and N. caninum (Meixner et al., J Vet Diagn Invest 2020)
• Serology (antibody tests) is of limited value

DIFFERENTIAL DIAGNOSIS: 

For abortion in sheep:

• Neospora caninum: T. gondii and Neospora caninum produce similar lesions in aborted placenta; foci of necrosis in cotyledons with normal intercotyledonary regions; prominent multifocal encephalitis with gliosis and necrosis in fetus
• Chlamydia abortus (R-B08): Necrotizing placentitis with vasculitis affecting intercotyledonary areas; leathery thickening of chorioallantois
• Coxiella burnetii (R-B07): Gross lesions similar to Chlamydophila abortus, usually without vasculitis
• Brucella ovis (R-B03): Leathery thick intercotyledonary regions with thick brown exudate that cover chorionic surface; vasculitis 
• Campylobacter fetus (R-B10): Nonspecific edematous changes in the fetus with distinctive targetoid hepatic necrosis up to 2 cm in diameter; exudate covering placenta
• Listeria monocytogenes: Necrotizing and suppurative placentitis of cotyledons and intercotyledonary areas; gram-positive bacilli in trophoblasts

 

COMPARATIVE PATHOLOGY:

1. T. gondii in other species:

• Pigs: Sporadic outbreaks; embryonic death; sows with fever and mild illness; hepatitis and lymphadenitis in congenitally infected piglets; abortion in swine with fetal lesions reported, but rarely occur
• Bovids:
  • Cattle: resistant; rarely abort or show signs of disease; usual cause of abortion is due to Neospora caninum with no useful identifying gross lesions
  • In susceptible bovids, placentitis, abortion, early neonatal death, pneumonia, or disseminated, fatal disease; species include:
    • Bazelles (dama, Cuvier’s, slender-horned), gerenuk, dik, saiga
    • Pronghorn: highly susceptible to experimental disease
    • Muskoxen and nilgai: Abortion and neonatal death have been reported
  • Mice: Wild mice are frequent intermediate hosts for gondii
  • Guinea pigs: Usually subclinical clinical infections; active infections may result in multifocal hepatitis and pneumonitis; subclinical chronic infections may produce cysts in the myocardium and CNS
• Rabbits:
  • Report of an outbreak - lesions included multiple foci of necrosis and granulomatous inflammation in the lung, liver, and spleen; no lesions were found in the brain
  • Another report identified a clinically ill rabbit with lesions (cysts) in the brain only
  • Wild and domestic meat rabbits are implemented as a major source for human infection with T.gondii
• Camelids: Frequent cause of reproductive infections in llamas and alpacas; case report of toxoplasmosis-associated abortion in one alpaca (Dubey et al., J Zoo Wildl Med 2014)
• Felids: High neonatal mortality
• Mustelidae: Less commonly causes placentitis and abortion
• Prosimians: Results in placentitis, abortion or stillbirth, disseminated fetal infection, or neonatal death
  • Lemur: Single case report of localized infection causing placentitis, stillbirth, and disseminated fetal infection (Juan-Salles et al., J Vet Diagn Invest. 2011)
• Nonhuman primates: Causes abortion in NWM and OWM
• Hyraxes: Fatal toxoplasmosis reported in pregnant, young rock/cape and tree hyraxes; causes abortion and stillbirth in pregnant hyrax
• Cetaceans: Causes necrotizing placentitis and abortion
• Pinnipeds: In California sea lions, vertical infection verified by identification of toxoplasmosis in aborted pups

REFERENCES:

1. Agnew D. Camelidae. In: Terio KA, McAloose D, Leger J, eds. Pathology of Wildlife and Zoo Animals, Cambridge, MA: Academic Press; 2018:198.
2. Agnew D, Nofs S, Delaney MA, Rothenburger JL. Xenartha, Erinacoemorpha, Some Afrotheria, and Phloidota. In: Terio KA, McAloose D, Leger J, eds. Pathology of Wildlife and Zoo Animals, Cambridge, MA: Academic Press; 2018:530.
3. Barthold SW, Griffey SM, Percy DH. Pathology of Laboratory Rodents & Rabbits. 4th ed. Ames, IA: John Wiley & Sons, Inc.; 2016:82, 236-237, 300.
4. Benavides J, Fernandez M, Castano P, Ferreras MC, Ortega-Mora L, Perez V. Ovine Toxoplasmosis: A New Look at its Pathogenesis. J Comp Pathol. 2017:157(1):34-38.
5. Castaño P, Fuertes M, Fernández M, et al. Macrophages and T Lymphocytes in the Ovine Placenta After Experimental Infection With Toxoplasma gondii. Vet Pathol. 2020; 57(4):545-549.
6. Colegrove KM, Burek-Huntington KA, Roe W, Siebert U. Pinnipediae. In: Terio KA, McAloose D, Leger J, eds. Pathology of Wildlife and Zoo Animals, Cambridge, MA: Academic Press; 2018:585-586.
7. Dubey JP, Johnson JE, Hanson MA, Pierce V. Toxoplasmosis-associated abortion in an alpaca (Vicugna pacos) fetus. J Zoo Wildl Med. 2014;45(2):461-464.
8. Foster RA. Female reproductive system and mammae. In: Zachary JF, ed. Pathologic Basis of Veterinary Disease. 6^th St. Louis, MO: Elsevier; 2017:1183.
9. Gutiérrez-Expósito D, Arteche-Villasol N, Vallejo-García R, et al. Characterization of Fetal Brain Damage in Early Abortions of Ovine Toxoplasmosis. Vet Pathol. 2020; 57(4):535-544.
10. Jones MEB, Gasper DJ, Mitchell (née Lane) E. Bovidae, Antilocapridae, Giraffidae, Tragulidae, Hippopotamidae. In: Terio KA, McAloose D, Leger J, eds. Pathology of Wildlife and Zoo Animals, Cambridge, MA: Academic Press; 2018:140.
11. Juan-Salles C, Mainez M, Marco A, Sanchis AM. Localized toxoplasmosis in a ring-tailed lemur (Lemur catta) causing placentitis, stillbirths, and disseminated fetal infection. J Vet Diagn Invest. 2011;23(5):1041-1045.
12. Mätz-Rensing K, Lowenstine LJ. New World and Old World Monkeys. In: Terio KA, McAloose D, Leger J, eds. Pathology of Wildlife and Zoo Animals, Cambridge, MA: Academic Press; 2018:366, 368.e13.
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14. Meixner N, Sommer MF, Scuda N, Matiasek K, Müller M. Comparative aspects of laboratory testing for the detection of Toxoplasma gondii and its differentiation from Neospora caninum as the etiologic agent of ovine abortion. J Vet Diagn Invest. 2020; 32(6):898-907.
15. Schlafer DH, Foster RA. Female genital system. In: Maxie MG, ed. Jubb, Kennedy, and Palmer’s Pathology of Domestic Animals. Vol 3. 6th ed. St. Louis: Elsevier; 2016:420-421.
16. Leger J, Raverty S, Mena A. Cetacea. In: Terio KA, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals, Cambridge, MA: Academic Press; 2018:563-564.
17. Terio KA, McAloose D, Mitchell (née Lane) E. Felidae. In: Terio KA, McAloose D, Leger J, eds. Pathology of Wildlife and Zoo Animals, Cambridge, MA: Academic Press; 2018:278-279.
18. Williams BH, Burek Huntington KA, Miller M. Mustelids. In: Terio KA, McAloose D, Leger J, eds. Pathology of Wildlife and Zoo Animals, Cambridge, MA: Academic Press; 2018:299-300.
19. Zachary JF. Mechanisms of microbial infections. In: Zachary JF, ed. Pathologic Basis of Veterinary Disease. 6^th St. Louis, MO: Elsevier; 2017:238-239.

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