Conference 16 - 2016 Case: 3 20170125

Signalment:

Two-year-old, male, spayed female, beagle (Canis familiaris).The dog presented for chylothorax after lobectomy at a referral surgical center several months before. After surgery, there was a persistent effusion that was managed with an indwelling Pleura-Port. The dog received a short course of cyclosporine for the effusion. On recheck, there was a significant decline in clinical condition (not really consistent with a cylothorax). Radiographs at that time showed the development of a diffuse milliary pattern not reported on original films or seen at rDVM a few weeks earlier. Based on the report, cyclosporine was discontinued and the dog was sent to the rDVM (owner's relative) to consider options. The plan was for the patient to return for lung biopsy in a few days but the dog continued to decline and after a night on oxygen with minimal stabilization and much less improvement, euthanasia was performed. Only lung tissue was submitted by the clinician for histopathology.

Gross Description:

Sections of formalin-fixed lung were mottled tan-red on cut section.

Histopathologic Description:

Lung: There are extensive multifocal to coalescing areas of tissue necrosis admixed with hemorrhage, fibrin, edema, karyorrhectic and occasionally mineralized debris; these areas are variably centered on partially or fully effaced bronchioles. Within these foci are florid infiltrates of viable and degenerate neutrophils, large foamy macrophages, and multinucleated macrophages, as well as myriad amoebic trophozoites and rare cysts. Trophozoites are 25-30-um in diameter, with flocculent pale eosinophilic cytoplasm and a 6-8-um karyosome with a prominent central basophilic endosome. Cysts measure 15-20-um and have a thick, bilayered wall (exocyst and endocyst). Within the nucleus of macrophages (including multinucleated macrophages phagocytizing trophozoites) adjacent to and within the most severely affected regions of the lung, there are one to multiple prominent brightly eosinophilic intranuclear inclusions that peripheralize the chromatin. In areas of the lung that are less severely affected, there is filling of alveolar spaces with edema, foamy macrophages, and low numbers of neutrophils, as well as scattered type II pneumocyte hyperplasia, hypertrophy of vascular endothelial cells, and expansion of residual septa by fibrin, similar inflammatory cells, and hyper-trophied fibroblasts. There is mild multifocal mesothelial hypertrophy.

Immunohistochemistry for canine distemper virus was negative. In-situ hybridization with a probe directed against canine herpesvirus was also negative. Indirect immunofluorescence at the CDC confirmed the amoebae to be Acanthamoeba spp.

Morphologic Diagnosis:

Lung: Pneumonia and bronchiolitis, necrosuppurative and hemorrhagic, multi-focal, chronic-active, severe, with free and intrahistiocytic Acanthamoeba spp. trophozoites and cysts.

Lab Results:

N/A

Condition:

Bronchopneumonia/Trueperella pyogenes

Contributor Comment:

Entamoeba histolytica, Sappinia diploidea, Acanthamoeba, Balamuthia and Naegleria species are free-living amoebas that act as secondary decomposers and regulate bacterial population in the soil. However, they are also opportunistic pathogens and can cause severe disseminated disease or necrotizing granulomatous encephalitis in immunosuppressed animals and humans. In the present case, the patient had received a short course of cyclosporine to treat the pleural effusion, which was potentially responsible for the immune suppression and increased susceptibility to opportunistic pathogens. Due to the presence of both cysts and trophozoites in the lung tissues, infection with Acanthamoeba spp. or Balamuthia mandrillaris was suspected.¹⁸ Naegleria fowleri is only present as trophozoites and does not form cysts in infected tissues.¹⁸ However, a definitive diagnosis of Acanthamoeba infection was based on the positive results of indirect immunofluorescence.

Acanthamoeba is ubiquitously present in the environment and has been isolated from diverse sources including sea water, beaches, pond water, soil, fresh water lakes, and even from the air. In human populations, anti-Acanthamoeba antibodies are present in up to 100% of people in healthy populations in New Zealand and in more than 85% of individuals of London who came from different countries.^3,5,18 In dogs, pulmonary infections usually result from inhalation or aspiration of the organisms from the water.

Acanthamoeba binds to host cells by a 130 kDa mannose-binding protein (MBP). Other adhesions involved in this interaction include laminin-binding protein with a predicted molecular mass of a 28.2 kDa, a 55 kDa laminin-binding protein and a > 207 kDa adhesion.^8,9,17Acanthamoeba binding to the host cells results in its phagocytosis and release of toxins. Two superoxide dismutases, an iron superoxide dismutase and a copper-zinc superoxide dismutase provide antioxidant defense. Toxins produced by Acanthamoeba cause activation of the phosphatidylinositol 3-kinase (PI3K) pathway, which further activates pro-apoptotic molecules, Bak and Bax, resulting in loss of mitochondrial membrane potential and apoptosis of host cells.^1,13,19

In host cells, toll-like receptor-4 (TLR4) is responsible for recognition of Acan-thamoeba , which leads to activation of the Myd88 pathway and induces secretion of interleukin-8, tumor necrosis factor-alpha, and interferon-beta.^13,16 Acan-thamoeba causes degradation of occludin and zonula occludens-1 tight junction proteins in human brain microvascular endothelial cells (HBMEC) in a Rho kinase-dependent manner, and thus leading to increased vascular permeability.¹² Acanthamoeba causes cutaneous lesions, sinus infections, keratitis, and rare but fatal encephalitis, known as granulomatous amoebic encephalitis in humans. Similarly, Acanthamoeba causes encephalitis and disseminated disease in immunosuppressed animals. In addition, most isolates harbor endosymbionts including numerous viruses (vesicular stomatitis virus, adenovirus, and poliovirus), bacterias (Burk-holderia spp.,Campylobacter jejuni, Coxiella burnetii, Francisella tularensis, Helicobacter pylori and Listeria monocytogenes), and yeast organisms (Cryptococcus neoformans, Blastomyces dermatitidis, Sporothrix schenckii, Histo-plasma capsulatum). However, the role of endosymbionts is not entirely clear. It is suspected that Acanthamoeba can transmit them to susceptible hosts or endosymbionts can increase the pathogenicity of Acanthamoeba.^11,18

The intrahistiocytic intranuclear inclusions present in this case are strongly suggestive of a co-infection with canine distemper virus. Unfortunately, the formalin-fixed tissue was held by the submitting clinician for several weeks prior to submission, so it is likely that this resulted in a loss of immunoreactivity due to antigen cross-linking.

Pneumonia, broncho-interstitial and necrotizing, multifocal to coalescing, marked, with free and intrahistiocytic trophozoites and rare cysts, beagle, Canis familiaris.

JPC Diagnosis:

Pneumonia, broncho-interstitial and necrotizing, multifocal to coalescing, marked, with free and intrahistiocytic trophozoites and rare cysts, beagle, Canis familiaris.

Conference Comment:

We thank the contributor for providing an outstanding and challenging case that stimulated a great deal of discussion among the conference participants regarding whether Acan-thamoeba is the primary cause of the significant pulmonary pathology in this case, or if it is secondary to concurrent infection with canine distemper virus (CDV). Like the contributor, many participants note numerous brightly eosinophilic intra-histiocytic nuclear inclusion bodies within multinucleated cells, which are interpreted as viral syncytial cells. However, others argue that the intranuclear structures may be representative of prominent nucleoli in response to marked chronic inflammation and the multinucleated cells are reactive fused macrophages and megakaryocytes rather than viral syncytial cells. Participants also describe multinucleated cells that occasionally contain phagocytosed amoebic trophozoites.

Ionized calcium binding adaptor molecule 1 (Iba1) immunohistochemical stain, provided by the contributor, demonstrated strong intracytoplasmic immunoreactivity for the multinucleated cells, confirming their macrophage lineage. The typical respiratory lesions associated with CDV include bronchointerstitial pneumonia with prominent cytoplasmic inclusion bodies in the bronchial and bronchiolar epithelium, type II pneumocyte hyperplasia with alveolar cytoplasmic inclusions, and alveolar epithelial syncytial cells.⁴ Participants favoring primary Acan-thamoeba infection note that bronchial and bronchiolar epithelium do not contain prominent cytoplasmic inclusions typical of CDV. Unfortunately, as mentioned by the contributor, suboptimal tissue preservation techniques may have affected the immunoreactivity for canine morbillivirus immunohistochemistry (IHC). Additionally, formalin fixation dramatically reduces the ability to extract suitable DNA for PCR based diagnostic tests. These deleterious effects of formalin on DNA are time and concentration dependent.¹⁴ The majority of participants agree with the contributor that there is likely primary concurrent infection with canine distemper virus in this case, despite negative IHC results.

Reports of pulmonary and systemic disease caused by free-living amoebae in dogs are uncommon and are usually associated with immune suppression with the majority of reported cases associated with underlying disease (such as co-infection with CDV) or long-term immunosuppressive doses of corticosteroids^6,7,10,15; however, there is a report of a greyhound naturally infected with Acanthamoeba sp. causing primary granulo-matous pneumonia and encephalitis.² In this case, the dog was receiving cyclosporine to treat chylothorax after a lung lobectomy, which may offer an alternative explanation for immune suppression; however, the reported short course of treatment is inconsistent with the long course of immunosuppressive therapy reported in the literature.^6,7,1

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