Conference 5 - 2016 Case: 2 20160928

Signalment:

Four-year-old, castrated male, Labrador retriever (Canis familiaris).This sample is from a rib mass. The animal had an initial history of moderate thrombocytopenia, mild leukopenia and mild anemia detected during a routine CBC. Ehrlichiosis was suspected and the dog was started on a course of doxycycline. CBC values did not normalize in response to the antibiotics and the dog was referred to a specialty practice, where pronounced thrombocytopenia (40,000/microL), mild anemia and mild leucopenia were documented. PCR for Ehrlichia canis could not be performed while the dog was on antibiotic therapy; however, the main differential diagnosis remained chronic ehrlichiosis. Cytology of the marrow showed general hypocellularity and a mild increase in plasma cells. The dog was clinically healthy and no other abnormalities were noted. As marrow destruction in ehrlichiosis has been shown to be immune-mediated, a course of prednisone in an immunosuppressive dose was added to the doxycycline. Subsequent CBC showed improvement in all 3 blood cell lines but whenever steroids were tapered, the dog relapsed. Treatment was therefore continued and eventually the dog became cushinoid. Another immunosuppressive drug (mycophenolate) was added to the regimen. CBC improved but the dog developed ulcerated subcutaneous abscesses on the limbs. These were treated conservatively and resolved but additional abscesses developed and there were several episodes of fever. Culture of one abscess yielded Nocardia spp. The culture results of the remaining abscesses are unknown.

Immunosuppressant therapy was discontinued, but later resumed because of severe thrombocytopenia. A few weeks later, the dog presented with a 15cm diameter swelling on the side of the chest. The swelling appeared to be due to a subcutaneous abscess. The abscess was drained but recurred, at which point surgical debridement was performed. During this procedure, a connection to the rib was identified. The mass was evaluated by CT and resected. Material was submitted for bacterial culture and Corynebacterium spp and Nocardia farcinica were isolated. Eventually, the dog recovered, his CBC normalized, and he is now reportedly in good health.

Gross Description:

There is a 15 cm diameter firm mass attached to a rib.

Histopathologic Description:

Mass on rib: There are multiple large bacterial colonies surrounded by abundant accumulation of cellular debris, neutrophils, histiocytes and rare multinucleated cells. In the surrounding tissue, there is widespread fibrosis and proliferation of woven bone. The fibrous tissue is of variable maturity and within it there is multifocal hemorrhage and severe multifocal to coalescing infiltration of neutrophils, histiocytes with lesser plasma cells and lymphocytes. The woven bone is arranged in a lattice. Bone trabeculae are commonly invested with a single layer of active osteoblasts and fibrovascular tissue of low cellularity occupies the intertrabecular spaces (reactive bone). The bacterial colonies (sulfur granules) are amphophilic to basophilic, have irregular circular, undulant and vermiform shapes and are composed of dense mats of rods and filamentous bacteria. The outer perimeter of the colonies is covered by a thin eosinophilic rim.

On special stains, the bacteria are gram positive and strongly stain with a modified acid fast stain, Fite-Faraco, and inconsistently with Ziehl Neelsen (ZN). In the modified ZN stain, long filamentous and beaded organisms are easily seen.

Morphologic Diagnosis:

Rib: Severe pyogranulomatous osteomyelitis with bacterial colonies consistent with Nocardia spp.

Lab Results:

N/A

Condition:

Osteomyelitis/Nocardia sp.

Contributor Comment:

The morphologic features and staining characteristics of the bacterial colonies are consistent with Nocardia spp. which was supported by culture. In dogs, distemper and other causes of immunosupression may predispose to nocardiosis.³ There are two recent reports of nocardial infection in dogs treated with immunosuppressive agents.⁶ In the current case, immunosuppressive treatment was used because the clinicians thought the pancytopenia with preferential involvement of the platelets was due to subacute to chronic ehrlichiosis. In their experience, dogs often recover from acute and subclinical ehrlichiosis but go on to develop subclinical and progressive bone marrow suppression, which is typically detected too late in the course of the disease. In this case, the ongoing myelosuppression was detected fortuitously but long-term immunosuppression (to which putative E. canis-induced myelosuppression may have been a contributing factor) led to opportunistic bacterial infection.

Actinobacteria are gram-positive, terrestrial, or aquatic bacteria which constitute one of the dominant bacterial phyla. Most Actinobacteria of medical significance belong to the order Actinomycetales. Due to their filamentous appearance, these organisms were thought to be fungi for many years but were later shown to be higher bacteria.^3,7 Actinomyces and Nocardia are the most common Actinomycetes which cause disease. ⁷ Organisms in the genus Nocardia are aerobic, gram-positive and partially acid fast saprophytes with a worldwide distribution. They commonly occur in soil and decaying organic matter and may cause opportunistic infection. N. asteroides is the species most commonly implicated in disease and has been recovered from lesions in humans, dogs, cats, cattle, goats, horses, pigs, marine mammals and fish.⁶ Cattle and dogs are the most commonly affected animals, but these infections are sporadic. ³ Infection is not contagious and affected animals do not constitute a public health hazard. ⁷ Failure to distinguish properly between Nocardia and Actinomyces in earlier reports has caused confusion in the literature regarding nocardiosis in animals. ³

Typically, nocardial infection originates from organisms introduced into skin wounds or aspirated into the respiratory tract, leading to superficial skin lesions, and necrotizing pneumonia, respectively. ^3,7 Infections may remain localized at the site of introduction but there is a tendency for the organisms to spread either by direct extension or vascular invasion and hematogenous dissemination. ^4,7 There is evidence that strains within the same species vary markedly in their virulence. ⁴ Cell-mediated immunity and neutrophils appear to be of critical importance in defense against these bacteria. ^6,7 In dogs, infections are more common in the young (<1-year-old), which may be due to increased exposure or to reduced resistance. ⁷

The typical gross cutaneous lesions caused by infection with filamentous bacteria (Actinomyces and Nocardia) consist of abscesses, cellulitis, draining fistulous tracts, and dense fibrous masses. ⁷ Lesions progress slowly by local extension. The exudate is variable and can contain white, yellow, tan or gray sulfur granules.^4,7 Histologically, pyogranulomatous inflammation is commonly seen. ^4,7 The sulfur granules consist of masses of organisms, which may be bordered by clubbed corona of brightly eosinophilic Splendore-Hoeppli material.^4,7 It has been reported that the shape of this material is characteristic for the agent involved.¹ Nocardia spp. have a limited tendency to clump together, thus they typically do not form granules. ^4,7 However, in some cases, nocardial lesions are morphologically indistinguishable from those induced by Actinomyces⁷ , as appears to be the case here. In gram-stained sections, the bacteria are seen as branched and beaded filaments up to 1µm wide. Fragmentation of the filaments produces coccobacillary forms. The beaded appearance is due to alternating gram-positive and gram-negative regions in the filament and is more evident in Nocardia spp. Actinomyces are acid-fast negative with most acid-fast stains. Many, but not all, Nocardia spp. stains strongly with modified Ziehl-Neelsen. Some acid-fast negative Nocardia spp. cannot be differentiated from Actinomyces in sections, and culture is required. ⁷

The cutaneous and subcutaneous nodules are progressive and may extend to involve underlying bone, as appears to have occurred in this case. The lesion is similar to lumpy jaw in cattle, a classic example of actinomycotic mycetoma. In this condition, traumatic implantation of Actinomyces bovis in the mandibular mucosa progresses to involve the mandibular bone.⁷ In cattle, N. asteroides can also cause granulomatous mastitis when contaminated drugs for the treatment or prevention of mastitis are introduced through the teat canal. This condition may also occur in outbreaks.² As noted above, a second relatively common site of infection with filamentous bacteria (Nocardia, Actinomyces and Bacteroides) in dogs and cats is the thoracic cavity, causing pyogranulomatous pleuritis with intrathoracic accumulation of blood-stained pus and reactive mesothelial cells, so-called tomato soup. This is no longer considered pathognomonic of nocardial infection.

JPC Diagnosis:

Bone: Osteomyelitis, pyogranulomatous and sclerosing, with new bone formation and colonies of filamentous bacteria, Labrador retriever, Canis familiaris.

Conference Comment:

This case nicely demonstrates the histopathologic appearance of sulfur granules in tissue section. Sulfur granules, which are usually located within neutrophil abscesses or pyogranulomas, are distinct masses of bacteria bordered by eosinophilic radiating projections of Splendore-Hoeppli material characteristic for actinomycetes bacteria.^2,7 Although, formation of granules is a more common feature of actinomycosis, Nocardia spp may form sulfur granules which are indistinguishable from Actinomyces spp. These structures can be seen grossly as small yellow granular material present within the exudate.^2,7

Conference participants briefly discussed the pathogenesis of the Splendore-Hoeppli phenomenon. As mentioned above, Splendore-Hoeppli reaction is typically the brightly eosinophilic, radiating, club-shaped, material around bacterial colonies in histologic sections. ^2,5,7 This material is composed of antigen-antibody complexes, tissue debris, and fibrin. Although the exact nature of this reaction is unknown, it is thought to be a localized immune response to an antigen-antibody deposition related to fungi, parasites, bacteria or inert materials. The characteristic formation of the Splendore-Hoeppli reaction around infectious agents or biologically inert materials is likely the bodys attempt to contain the injurious agent on the part of the host. However, it also likely prevents phagocytosis and intracellular killing of the agent leading to prolonged damage or infection. ⁵

Identification of the tissue as costal bone in this section was difficult for all conference participants. As a result, participants discussed effective strategies for differentiating reactive new bone formation from neoplastic bone disease, given the lack of normal tissue architecture. The conference moderator instructed that first one must look for evidence of the parent bone structural elements such as osteons within complex mature lamellar bone to differentiate new bone formation from osseous metaplasia. Next, to differentiate reactive bone from neoplastic bone, one must look at the characteristics and orientation of the osteoblasts. Given that osteoblasts are terminally differentiated products of mesenchymal stem cells, there should be no mitotic activity within this cell population.⁸ Additionally, in reactive bone formation, osteoblasts form highly organized groups connected by gap junctions allowing the cells to function in a well-regulated manner. In tumor bone, neoplastic mesenchymal cells are haphazardly arranged and produce an osteoid matrix without the regularity and regimentation present in reactive bone osteoblasts.

References:

1. Bestetti G. Morphology of the "sulphur granules" (Drusen) in some actinomycotic infections. A light and electron microscopic study. Vet Pathol . 1978; 15:506-18.

2. Foster RA. Female Reproductive System. In: McGavin MD, Zachary JF, ed. Pathologic Basis of Veterinary Disease . 5th ed. St. Louis, MO: Elsevier; 2012:1124- 1125.

3. Gyles CL. Nocardia, Actinomyces, and Dermatophilus. In: Carlton L, Thoen CO, ed. Pathogenesis of bacterial infections in Animals. 2nd ed. Ames, IA: Iowa State University Press, 1993:124-6.

4. Hargis, Ann M, Ginn, Pamela E: The Integument. In: McGavin MD, Zachary JF, ed. Pathologic Basis of Veterinary Disease. 5th ed. St. Louis, MO: Elsevier; 2012:1034.

5. Hussein MR. Mucocutaneous Splendore-Hoeppli phenomenon. J Cutan Pathol. 2008; 35(11):979-988.

6. MacNeill AL, Steeil JC, Dossin O, Hoien-Dalen PS, Maddox CW. Disseminated nocardiosis caused by Nocardia abscessus in a dog. Vet Clin Pathol. 2010; 39:381-5.

7. Mauldin E, Peters-Kennedy J. Integumentary system. In: Maxie MG, ed. Jubb, Kennedy, and Palmers Pathology of Domestic Animals. Vol 1. 6th ed. Philadelphia, PA:Elsevier; 2016:637-638.

8. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR. Multilineage potential of adult human mesenchymal stem cells. Science. 1999; 284:143-7.