Acute toxoplasmosis and pox-viral dermatitis in a juvenile bald eagle (Haliaeetus leucocephalus) in New Brunswick, Canada

Abstract

Although birds of prey are commonly subclinically infected by Toxoplasma gondii tissue cysts, clinical disease is relatively rare in these species. The present report describes a rare case of fatal toxoplasmosis in a juvenile bald eagle in New Brunswick. Necropsy investigation revealed severe emaciation and poxviral dermatitis which partially obscured the palpebral fissures. Microscopically there was severe lymphoplasmacytic inflammation and necrosis of the lung that was associated with abundant protozoal tachyzoites. Infection with T. gondii was confirmed in the lung via immunohistochemistry and DNA sequencing.

Key clinical message:

Wildlife rehabilitation centers should be aware of the potential occurrence of acute clinical toxoplasmosis in stressed malnourished raptors.

Toxoplasma gondii is a coccidian parasite which infects a wide array of species globally (1). The definitive hosts (in which sexual reproduction occurs) include both wild and domestic felids, which shed large numbers of infective oocysts into the environment through their feces. Intermediate hosts (which include virtually all warm-blooded species including birds) become infected through consuming water or food contaminated with oocysts. These oocysts may remain infective in the environment for prolonged periods of time under optimal environmental conditions (2). Within intermediate hosts, oocysts release sporozoites that penetrate the intestinal wall and spread hematogenously to terminally differentiated and immune-privileged cell types (including neurons, skeletal muscle, and cardiac myocytes) where they subsequently encyst as asexually replicating bradyzoites (3,4). Tissue cysts containing bradyzoites may then be transmitted horizontally via carnivorism, which is likely the route responsible for its widespread distribution in the environment (5).

Clinical disease caused by T. gondii is relatively uncommon despite the high incidence of infection in intermediate hosts, which typically experience little to no negative effects from tissue cysts (6). In domestic species, disease caused by T. gondii may manifest as malabsorption and diarrhea in kittens, abortion in small ruminants, and occasionally severe systemic disease (3). In humans, T. gondii is an important zoonosis and may cause serious ocular disease in immunosuppressed individuals, and abortion in pregnant women (6). Wildlife may be considered as sentinels for T. gondii and may indicate the potential risk to humans and domestic species within the local environment (7,8). Apex predators, such as birds of prey, are particularly sensitive sentinels as they may become infected through scavenging in environments contaminated with oocysts, or through predation on intermediate hosts (9). Raptors (order Accipitriformes) are commonly exposed and/or infected with T. gondii tissue cysts; however, they are particularly resistant to clinical disease (10–12). The following case report describes a case of severe, acute toxoplasmosis in a juvenile bald eagle from New Brunswick, Canada.

Case description

During the winter of 2018, a young female bald eagle, estimated to be between 1 and 2 y old (based on mild feather variegation and a predominantly brown beak), was found on the ground outside an urban center in southern New Brunswick. The eagle was captured and transported to a local wildlife rehabilitation facility where it was hand fed, and treated with oral fluids and prophylactic antibiotics (treatment records unavailable). After 3 d in captivity, the eagle was found dead in its cage overnight. The body was frozen and submitted to the Canadian Wildlife Health Cooperative Atlantic Regional Centre for diagnostic workup.

Necropsy, histopathology, and immunohistochemistry (IHC)

A complete necropsy was performed by a veterinary pathologist. Samples of liver were submitted for lead analysis and results were below detectable concentration using atomic absorption spectrophotometry (< 0.035 ppm wet weight). Representative tissue samples were placed immediately in 10% neutral buffered formalin and were routinely processed and stained with hematoxylin and eosin (H&E) for histopathology. For T. gondii immunohistochemistry (IHC), deparaffinized sections were stained with polyclonal rabbit antibody specific for T. gondii (Biogenex, Fremont, California, USA) using an automated staining instrument (Ventana Benchmark XT; Ventana Medical Systems, Tucson, Arizona, USA). Sections of feline tissues with histologically and immunohistochemically confirmed T. gondii infection were used as positive tissue controls. For negative reagent controls, duplicate sections of each control and test tissue were subjected to the same immunohistochemical procedure with substitution of non-immune rabbit serum diluted to have protein concentration similar to the primary antiserum.

Nucleic acid extraction, polymerase chain reaction (PCR), and sequencing

DNA was extracted separately from 2 pieces (20 to 30 mg) of formalin-fixed lung tissue using the pretreatment protocol for paraffin-embedded tissue in the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Toronto, Ontario) prior to following the manufacturer’s instructions with the exception that the proteinase K digestion was for 21 h at 56°C. Nested polymerase chain reaction (PCR) for the internal transcribed spacer (ITS) region was performed using external primers ITS1ExtF (5′-TACCGATTGAGTGTTCCGGTG-3′) and ITS1 ExtR (5′-GCAATTCACATTGCGTTTCGC-3′), followed by internal primers ITS1IntF (5′-CGTAACAAGGTTT CCGTAGG-3′) and ITS1IntR (5′-TTCATCGTTGCGCGAG CCAAG-3′) (13,14). Polymerase chain reaction assays were performed in 50-μL reaction volumes, comprising 2 μL of DNA for both external and internal reactions, 5 μL 10× PCR buffer (Qiagen), 1 μL dNTP mix (10 mM each, Qiagen), 5 μL each of forward and reverse primers (10 μM), 10 μL Q-solution, 3 μL MgCl₂ (25 mM), 1.25 units of HotStarTaq (Qiagen) and the remaining volume composed of nuclease-free water. Negative control samples consisted of nuclease-free water in place of DNA. Polymerase chain reaction followed a touchdown protocol with an initial denaturation/activation step at 95°C for 5 min, followed by 10 cycles of 94°C for 1 min, 58°C for 45 s (decreasing by 1.0°C each cycle to 48°C), 72°C for 2 min followed by 35 cycles of 94°C for 1 min, 58°C for 45 s, 72°C for 2 min and a final extension at 72°C for 10 min. Polymerase chain reaction amplicons were visualized in 1% agarose gels using SYBR Safe DNA gel stain (Thermo Fisher Scientific, Ottawa, Ontario) under ultraviolet light. Sequencing of secondary amplicons from 4 independent PCRs was performed using the internal primers (ITS1F and ITS1R) at Macrogen USA, Rockville, Maryland, USA.

Results

The eagle was emaciated (body weight: 2.8 kg) as indicated by severe bilateral pectoral muscle atrophy and a complete absence of subcutaneous and internal fat stores. The facial skin was alopecic and markedly thickened by numerous coalescing slightly raised mottled tan plaques which partially obscured the optic fissures (Figure 1A). There were moderate to marked post-mortem changes throughout the soft tissues, and the crop, proventriculus, and ventriculus were empty.

Figure 1.

A — Macroscopic image of the diffuse poxviral dermatitis affecting the bald eagle’s face. B — Tissue section of facial skin demonstrating diffuse hyperplasia (H&E, scale bar 50 μm). Numerous keratinocytes are expanded by large eosinophilic to clear cytoplasmic inclusions consistent with Bollinger bodies (inset).

Histopathology

The lungs were the most severely affected organ; the parabronchi and air alveoli were diffusely filled with, or completely obscured by, abundant necrotic cellular debris, inflammatory cells, and fibrin (Figure 2A). Scattered throughout all tissue sections and admixed within necroinflammatory exudate were numerous round to fusiform, amphophilic, protozoal tachyzoites that measured 2 to 4 μm in greatest diameter (Figure 2B). Tachyzoites were present both extracellularly and intracellularly within macrophages. Within the liver, most portal triads contained scattered intra- and extra-cellular tachyzoites that were surrounded by small aggregates of lymphocytes, plasma cells, and macrophages. Within the brain and meninges there was a focal area of lymphoplasmacytic and histiocytic inflammation admixed with rare small clusters of tachyzoites. Within the heart there were occasional small areas of myocardial necrosis with lymphoplasmacytic inflammation admixed with intravascular and extracellular tachyzoites (Figure 2D). Intravascular tachyzoites were observed in both the spleen and the kidney; however, there was minimal inflammation or necrosis associated with the protozoa in these organs. Immunohistochemistry for T. gondii showed positive staining of organisms in all the tissues previously described (Figure 2C). Samples of skin taken from the face revealed marked generalized acanthosis with numerous keratinocytes expanded by large, round, eosinophilic to clear, intracytoplasmic inclusion bodies (Bollinger bodies) highly characteristic of poxvirus (Figure 1B). Overlying the epidermis were multiple coalescing crusts composed of degenerate heterophils, serocellular debris, and numerous bacterial colonies. Occasionally within the stratum spinosum and scattered throughout the dermis were small clusters of tachyzoites similar to those identified in other organs.

Figure 2.

Significant gross and microscopic findings observed during the post-mortem investigation of the current bald eagle case. A — Tissue section of lung exhibiting marked diffuse consolidation of airways by necroinflammatory exudate. Arrow indicates remnant air alveoli (H&E, scale bar = 100 μm). B — High magnification of air capillaries showing clusters of Toxoplasma gondii tachyzoites (arrows) amidst necroinflammatory debris (H&E, scale bar = 20 μm). C — Immunohistochemistry of lung showing strong positive staining for T. gondii (scale bar = 50 μm). Inset shows high magnification of IHC positive staining tachyzoites. D — Section of myocardium demonstrating aggregates of lymphocytes and plasma cells separating cardiac myocytes which contain clusters of tachyzoites (H&E, scale bar = 50 μm).

Polymerase chain reaction (PCR) and sequencing

An approximately 450 base pair (bp) PCR amplicon was isolated using the previously described primer sets. Sequencing of amplicons (from 4 independent PCR reactions) revealed a consensus sequence (accession number: MN153989) that was 100% identical to T. gondii in GenBank (accession number: AF252408).

Discussion

This report describes the case of a young female bald eagle that died suddenly at a wildlife rehabilitation center in southern New Brunswick after 3 d in care. Although multiple issues contributed to the death of this eagle (including emaciation and poxviral dermatitis), the major contributing factor was a severe systemic protozoal infection that was determined to be T. gondii through DNA sequencing and immunohistochemistry. This confirms a rare case of T. gondii causing severe systemic disease in a member of the order Accipitriformes.

Disease caused by T. gondii can be divided into 2 forms: acute cases which represent de novo infections of naïve hosts, or recrudescent cases which represent chronically infected individuals with tissue cysts that undergo bradyzoite to tachyzoite conversion (15). Acute cases have widely disseminated inflammation and necrosis involving multiple organs reflecting hematogenous spread (3,16), while recrudescent or chronic cases tend to exhibit focally extensive necrosis and inflammation involving a single organ from which encysted bradyzoites emerge (i.e., skeletal muscle, heart, and brain) (3). The juvenile eagle in this report exhibited severe generalized, multisystemic toxoplasmosis, with the lung being most severely affected. As multiple organs were involved, and the lung is not typically a site for chronic latent infection (4), it is likely that this eagle was acutely infected with T. gondii and was previously unexposed.

It is difficult to determine exactly when this eagle initially became infected with T. gondii as birds of prey typically do not show clinical signs of toxoplasmosis and susceptibility to disease varies greatly among avian species (17,18). It is most likely that both infection and disease occurred before rehabilitation as this bird spent so little time in human care (3 d). Exposure of healthy hosts to T. gondii does not normally result in fulminant disease as tachyzoite replication is inhibited by cell-mediated immunity involving the activation of CD8⁺ and CD4⁺ T-cells with the production of pro-inflammatory cytokines IL12 and IFNγ (19,20). Malnutrition causes deficiencies in both cell-mediated and humoral immunity (21–23), and so it seems likely that the development of acute toxoplasmosis (and also poxviral dermatitis) in this eagle was related to its emaciated state. In non-avian species, other factors that may predispose individuals to toxoplasmosis include viral infection (24,25), neoplasia (26,27), and congenital infection (28). Although it is not possible to definitively rule out these factors in the current case, there was no evidence of systemic viral infection (poxvirus was isolated to the face), neoplasia, or congenital abnormalities histologically.

This case report serves as an important reminder to Canadian veterinarians of the presence of T. gondii in our environment and the impact it can have on wildlife, domestic animals, and humans. Although the source of T. gondii in this bald eagle remains unknown, hosts may become infected through ingestion of water or food contaminated with oocysts, or prey infected by tissue cysts (3). Monitoring for T. gondii in key sentinel species such as the bald eagle can therefore indicate its prevalence and distribution within the environment (11,12), and may help inform mitigation measures for disposal of feline wastes. Wildlife rehabilitation centers should be aware of the possibility of toxoplasmosis occurring in raptors in their care. Although disease caused by T. gondii may be uncommon in healthy free-ranging raptors, stress and/or emaciation causing immunosuppression can predispose them to developing either acute or recrudescent disease while in veterinary care.