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Case Series: Virulent hemosporidiosis infections in juvenile great horned owls (Bubo virginianus) from Louisiana and California, USA
Veterinary Parasitology: Regional Studies and Reports 12 (2018) 49–54
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Original Article
Case Series: Virulent hemosporidiosis infections in juvenile great horned owls (Bubo virginianus) from Louisiana and California, USA
T
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Kevin D. Niedringhausa, , Heather M.A. Fentona,1, Christopher A. Clevelanda, A. Nikki Andersonb, ⁎ Diana Schwartzc, Charles E. Alexc, Krysta H. Rogersd, Aslι Metee, Michael J. Yabsleya,f, a
Southeastern Cooperative Wildlife Disease Study, 589 D.W. Brooks Drive, Wildlife Health Building, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602, USA b Louisiana Department of Wildlife and Fisheries, 2000 Quail Dr., Baton Rouge, LA 70808, USA c Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine, University of California – Davis, One Shields Avenue, Davis, CA 95616, USA d Wildlife Investigations Laboratory, California Department of Fish and Wildlife, 1701 Nimbus Road, Suite D, Rancho Cordova, CA 95670, USA e California Animal Health and Food Safety Laboratory, University of California – Davis, 620 W. Health Sciences Drive, Davis, CA 95616-5270, USA f Warnell School of Forestry and Natural Resources, 180 E Green Street, University of Georgia, Athens, GA 30602, USA
A R T I C L E I N F O
A B S T R A C T
Keywords: Bubo virginianus great horned owl Haemoproteus Leucocytozoon Louisiana California
A total of eight juvenile great horned owls (Bubo virginianus) were found lethargic and on the ground in spring 2015, 2016, and 2017, including one fledgling from Louisiana, USA and seven nestlings from California, USA. One bird survived to release after rehabilitation; seven birds died or were euthanized due to poor prognosis and were necropsied. Necropsy findings were similar and included general pallor of all tissues, particularly the subcutis and lungs, and enlarged liver and spleen. Histopathology revealed multi-organ necrosis, abundant meronts containing merozoites, and intracytoplasmic pigmented haemosporidian parasites in blood cells in one bird. Leucocytozoon lineages lSTOCC16 and BUVIR06 were identified by polymerase chain reaction and genetic sequencing. The systemic Leucocytozoon infections were likely associated with morbidity and mortality in these owls. A second parasite, Haemoproteus lineage hSTVAR01, was also identified in an owl from Louisiana. This is the first identification of Leucocytozoon lineages that have been associated with mortality in young great horned owls.
1. Introduction Infections with protozoan parasites in the order Haemosporida (genera Haemoproteus, Plasmodium, or Leucocytozoon) are common in many bird species, including raptors (Atkinson, 2008). Parasites within these genera differ in many aspects, including pathogenicity, global and regional distributions, mechanisms of parasite reproduction, and the vectors involved in transmission (Valkiūnas, 2005). Haemoproteus (Paraphaemoproteus) spp. and Haemoproteus (Haemoproteus) spp. are transmitted by biting midges (Ceratopogonidae) and louse flies (Hippoboscidae), respectively, and Plasmodium spp. are transmitted by mosquitoes (Culicidae). There are two known groups of vectors for Leucocytozoon spp.; Leucocytozoon caulleryi (although some consider this parasite to be in the subgenus Akiba) is transmitted by biting midges (Ceratopogonidae) while the remaining Leucocytozoon spp. are
transmitted by black flies (Simuliidae) (Atkinson, 2008; Forrester and Greiner, 2008). Avian malaria is a significant conservation issue for some avian species (such as the native avifauna of Hawaii or penguins in zoological parks), but mortality in natural hosts occurs and is often likely underreported (Beier et al., 1981; Dinhopl et al., 2015; Valkiūnas and Iezhova, 2017). Nevertheless, potential subclinical effects on reproductive success and the short- and long-term survivability of natural hosts have been investigated in many bird species naturally infected with haemosporidian parasites (Appleby et al., 1999; Korpimaki et al., 1993; la Puente et al., 2010; Merino et al., 2000; Nordling et al., 1998; Remple, 2004; Ziman et al., 2004). The severity of clinical disease in avian hosts depends on many factors, including the species of the host, the host's immunity, environmental stressors, and the species of haemosporidian involved, among others (Atkinson, 2008). Clinical disease can be broadly
⁎ Corresponding authors at: Southeastern Cooperative Wildlife Disease Study, 589 D.W. Brooks Drive, Wildlife Health Building, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602, USA E-mail addresses: [email protected] (K.D. Niedringhaus), [email protected] (H.M.A. Fenton), [email protected] (C.A. Cleveland), [email protected] (A.N. Anderson), [email protected] (D. Schwartz), [email protected] (C.E. Alex), [email protected] (K.H. Rogers), [email protected] (A. Mete), [email protected] (M.J. Yabsley). 1 Present address: Government of the Northwest Territories, 5th Floor, Scotia Centre, P. O. Box 1320, Yellowknife, NT X1A 2L9, Canada.
https://doi.org/10.1016/j.vprsr.2018.01.008 Received 7 September 2017; Received in revised form 11 January 2018; Accepted 12 January 2018 Available online 16 January 2018 2405-9390/ © 2018 Elsevier B.V. All rights reserved.
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detail and contained scant nuclear debris, particularly in the spleen, heart, and liver (Fig. 1A–C). The meronts were also associated with occasional hemorrhage, as well as rare clusters of brown, globular intracytoplasmic and extracytoplasmic pigment interpreted as hemosiderin. Round, basophilic host cell nuclei (residual bodies) were often visible within megalomeronts. Variable numbers of lymphocytes, plasma cells, and macrophages were observed forming concentric rings around the meronts. The meronts contained numerous merozoites approximately 1–2 μm in length within organized cytomeres, which is morphologically consistent with that reported in L. danilewskyi infection (Khan, 1975; Valkiūnas, 2005) (Fig. 1D). Less than 1% of the leukocytes within the vessels of the lung had expanded, pale basophilic cytoplasm, eccentric nuclei, and intracellular, pigmented sporozoites (Table 1).
classified as causing one of two overlapping processes: (1) anemia due to erythrocyte rupture from developing gametocytes and (2) visceral organ inflammation, necrosis, and hemorrhage following the rupture of meronts (schizonts) and megalomeronts. Fatal infections in many bird species have involved varying degrees of inflammation in visceral organs and necrosis of cardiac and skeletal muscle, liver, lung, and kidneys associated with developing parasites (Alley et al., 2008; Beier et al., 1981; Donovan et al., 2008; Julian and Galt, 1980). Many of these cases involve captive exotic birds infected with haemosporidians not normally found in their native range, and in such cases parasites may not always be found in the blood but tissue stages may be extensive (termed ‘abortive infections’ because infective gametocytes are not produced) (Valkiūnas and Iezhova, 2017). However, there are also examples of disease developing in native hosts infected with native parasites and include wild turkeys (Meleagris gallopavo) and Canada geese (Branta canadensis) (Atkinson and Forrester, 1987; Herman et al., 1975). In North America, haemosporidian infections are relatively common in owls (Gutierrez, 1989; Ishak et al., 2008). Historically, the only morphologically distinct species of Haemoproteus reported to cause disease in owls were H. noctuae and H. syrnii (Evans and Otter, 1998; Forrester et al., 1994; Valkiūnas, 2005). These morphospecies are thought to have near cosmopolitan distributions in birds of the order Strigiformes (Valkiūnas, 2005). To date, only two studies have reported Haemoproteus infections in great horned owls including in a single bird in Florida and in 11% of 54 birds from California (Ishak et al., 2008; Outlaw and Ricklefs, 2009). In both studies, infection was determined by molecular methods using samples collected from presumably healthy adult owls. In North American owls, the only morphologically distinct Leucocytozoon is L. danilewskyi, but evidence suggests that there are at least two cryptic species or subspecies, highlighting the need to genetically characterize these parasites to understand their natural history and relative importance in morbidity and mortality (Ishak et al., 2008). Here we describe haemosporidian infections including the pathology and molecular characterization of parasites in seven great horned owls from two geographic locations in the United States, Louisiana and California, in spring 2015, 2016, and 2017. The clinical findings for one surviving owl and three owls prior to death are also described.
2.2. California, USA Two sibling, nestling great horned owls (B, C) were found on the ground in Sacramento County, CA in late March 2016 and admitted to the University of California, Davis Small Animal Clinic (Davis, CA). Physical examination of both owls revealed dehydration, 3/9 body condition score, poor appetite, active regurgitation, and no evidence of trauma. Owl B was anemic (hematocrit 6%) and hypoproteinemic (total protein 2.6 g/dL). Fresh whole blood and wedge smear technique were used to prepare blood smears, which were stained with a modified Wright stain (Aerospray Hematology Pro, Wescor, Inc.). Abundant round to elongate, pale basophilic inclusions in immature erythrocytes and leukocytes, that mildly distorted the cells and displaced the nuclei peripherally (Fig. 2A and B), were present in blood smears. Immature gametocytes in mature erythrocytes measured between 0.9 and 1.7 μm in diameter. Gametocytes in roundish host cells measured between 6.5 and 14.2 μm in length, and gametocytes in fusiform host cells measured between 21.1 and 26.4 μm in length. In addition to the elongated nucleus of the fusiform host cells that are in close approximation to the primarily immature gametocytes in immature erythrocytes, these characteristics were morphologically consistent with Leucocytozoon sp. (likely L. danilewskyi) (Valkiūnas, 2005). Due to poor prognosis, owl B was euthanized and a necropsy was performed. Owl C survived to release (Table 1). Owl B was a male in poor nutritional condition. Similar to owl A, generalized pallor of the skeletal muscles and viscera was present (anemia). Approximately ten multifocally confluent, roughly parallel, dark red to black tracts extended from the capsular surface into the parenchyma on the medial surfaces of the right and left liver lobes. These tracts were 2–6 mm in diameter and up to 1.5 cm long, and some tracts contained small amounts of yellow, soft, granular material. Histopathologically, haemosporidian megalomeronts were observed in numerous tissues, including the liver, kidneys, spleen, lungs, proventricular glands, small intestinal mucosa, heart (myocardium and subepicardium), thyroid, pancreas, bursa of Fabricius, and marrow spaces of the scleral ossicles similar to owl A. Megalomeronts ranged from 80 to 140 μm in diameter, and were often subdivided into 3–6 discrete cytomeres. The nuclei of affected host cells were often enlarged up to 40 μm in diameter. Highest concentrations of megalomeronts were seen in the liver, kidneys, and scleral ossicles. Associated inflammation was generally absent or minimal. In the kidneys a mild, mixed inflammatory cell infiltrate was present that multifocally expanded the interstitium adjacent to megalomeronts. This infiltrate consisted primarily of histiocytes and lymphocytes with fewer granulocytes. In the liver, sinusoids were diffusely, mildly expanded by increased numbers of mononuclear inflammatory cells that occasionally contained indented or peripheralized nuclei. Multifocal, random foci of coagulative necrosis associated with hemorrhage were scattered throughout liver sections, similar to owl A. One similar focus was present in a section of lung. At admission, owl C's red blood cell count was 1.5 M/μL, hematocrit
2. Case reports 2.1. Louisiana, USA A fledgling great horned owl (A) was found on the ground in May 2015 by a private citizen in Livingston Parish, LA. The owl died overnight with no obvious signs of trauma or other external abnormalities. The carcass was brought to the Louisiana Department of Wildlife and Fisheries (Baton Rouge, LA) who submitted it to the Southeastern Cooperative Wildlife Disease Study (SCWDS; Athens, GA) for necropsy. Owl A was a female in fair nutritional condition. Aside from general pallor of all tissues, particularly the subcutis and lungs, no other significant gross lesions were detected. Representative organ samples were fixed in 10% neutral buffered formalin and embedded in paraffin. Five micron thick sections were placed on glass slides and stained with hematoxylin and eosin. Additional ancillary tests included testing for avian influenza viruses by polymerase chain reaction (PCR) assay for the matrix gene, West Nile virus by virus isolation, herpesviruses by PCR, and anticoagulant rodenticides by a toxicological screen of liver tissue. Apart from a trace amount of brodifacoum, none of the aforementioned pathogens or compounds were detected. Microscopic examination revealed multifocal, variably-shaped and often lobulated, megalomeronts ranging between 30 and 50 μm in diameter and 30 to 100 μm in length in the liver, spleen, kidney, and heart (Fig. 1). The cells surrounding the meronts often lacked cellular 50
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Fig. 1. Microscopic images from owl A from Louisiana, USA. (A) Spleen. Multifocal areas of the spleen contain compartmentalized meronts (arrow) associated with areas of coagulative necrosis (asterisk) admixed with fibrin and cellular debris. Haemotoxylin and eosin (HE). Bar = 50 μm. (B) Myocardium. Multiple areas of the myocardium are expanded with protozoal meronts (asterisk) with the host cell nuclei (residual body) in the middle (arrow) HE. Bar = 50 μm. (C) Liver. Multifocal to coalescing areas of necrosis (asterisk) admixed with hemorrhage, hemosiderin, lymphocytes, and small numbers of macrophages are associated with protozoal meronts (arrow). HE. Bar = 100 μm. (D). Kidney. Higher magnification of meronts reveals abundant 1–2 × 3–4 μm merozoites (asterisk) within distinct cytomeres. Host cell nucleus (residual body) is in the center (arrow). HE. Bar = 20 μm.
Table 1 Summary of findings related to owls infected with hemosporidian parasites. Owl ID
County found
Survival
Date found
Hematocrit (%)
Plasma protein (g/dl)
Leucocytozoon lineage
Haemoproteus lineage
A B C D E F G H
Livingston, LA Sacramento, CA Sacramento, CA Lassen, CA Lassen, CA Sierra, CA San Luis Obispo, CA San Luis Obispo, CA
No No Yes No No No No No
May 2015 March 2016 March 2016 April 2016 April 2016 April 2016 April 2017 April 2017
N/A 6 27 N/A N/A N/A 8 20
N/A 2.6 3.5 N/A N/A N/A 1.2 4.5
STOCC16 STOCC16 N/A STOCC16 BUVIR06 STOCC16 STOCC16 STOCC16
hSTVAR01 Not detected N/A Not detected Not detected Not detected Not detected Not detected
N/A, not available. Fig. 2. Blood smears from owl B (A and B) and owl C (C and D) from California, USA. Wright stain. A: Immature gametocyte within the cytoplasm of an erythrocyte (arrow head) and a roundish host cell with two developing gametocytes within an immature erythrocyte (arrow). Bar = 15 μm. B: Intra-erythrocytic merozoites (arrow head) and developing gametocytes (arrow) in immature erythrocytes. Bar = 15 μm. C and D: Both roundish (arrows) and fusiform (arrow heads) morphology forms of the host cell with developing and mature gametocytes are present. Parasites are morphologically consistent with Leucocytozoon danilewskyi. BAR = 30 μm (C) and 15 μm (D).
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was 27%, and mean cell volume was 180.0 fl. Slight anisocytosis, marked polychromasia, and abundant haemosporidian parasites were observed on a blood smear including some mature Leucocytozoon spp. gametocytes (Fig. 2C and D). Other findings included a white blood cell count of 3400/μL with differentials of heterophils (61%), lymphocytes (38%), and monocytes (1%). Platelet levels were adequate, plasma proteins were 3.5 g/dL, plasma fibrinogen was 200 mg/dL, and protein to fibrinogen ratio was 17 (Table 1). In April 2016, three nestling owls were admitted to Lake Tahoe Wildlife Care Center (S. Lake Tahoe, CA) with swollen eyelids and black crusty debris around the eyes. Two owls were siblings (D, E) found on the ground in Lassen County, CA and the other was a single nestling (F) found on the ground in Sierra County, CA. All three owls received supportive care until they died 3, 5, and 2 days after admission, respectively. In April 2017, two sibling nestling owls (G, H) were found on the ground in San Luis Obispo County, CA and admitted to Pacific Wildlife Care (Morro Bay, CA). Both owls had black crusty debris around the eyes and ears and protozoa resembling Leucocytozoon spp. were detected on a blood smear. At intake, owl G had a hematocrit of 8% and a total protein value of 1.2 g/dL; owl G was euthanized shortly after intake due to poor prognosis. Owl H had a hematocrit of 30% and a total protein of 3.8 g/dL at intake. Owl H died 6 days after admission despite supportive care; just prior to death, its hematocrit was 20% and the total protein was 4.5 g/dL (Table 1). The carcasses were submitted frozen to the California Department of Fish and Wildlife's Wildlife Investigations Laboratory (WIL; Rancho Cordova, CA) for necropsy; owls D, E, and F in April 2016 and owls G and H in May 2017. The carcasses were thawed at 4 °C for 24–48 h prior to necropsy. Gross necropsy findings for all five owls were similar and included black debris present around the eyes; ocular discharge; a trace to fair amount of adipose reserves; edematous lungs; and enlarged thyroids, adrenal glands, liver, and spleen. The subcutis and organs including the brain of D, E, F, and G were diffusely pale to light pink and the blood was thin and watery while the tissues of owl H appeared moderately pink (Fig. 3). Additionally, owl F had a complete, closed fracture of the right humerus and bloody fluid in the feathers around the eyes, nares, and bill. Owl H had subcutaneous hemorrhage with edema on the right thigh and blood present in the right lung. Owls D, E, and F were females, and G and H were males. Formalin fixed tissues from the five owls were submitted to the California Animal Health and Food Safety Laboratory (Davis, CA branch) for histopathology. Histopathologic findings were was similar in all birds with intracellular organisms within multiple blood vessels. Host cells (erythrocytes and leukocytes with gametocytes) were rounded with abundant foamy cytoplasm containing basophilic stippled organisms that pushed the nucleus to the periphery forming a thin
elongate band. Megalomeronts were observed frequently in the liver, spleen, kidneys, lung, heart, and in the choroid vessels. Occasionally, megalomeronts were seen in the adrenal glands, gastrointestinal wall, and pancreas. In all birds there was severe hepatic necrosis, mostly bridging centrilobular, associated with hypoxia, and random foci associated with intralesional megalomeronts as well as lymphoid depletion in the spleen. Owls D, E, and F were tested for West Nile virus by PCR on kidney tissue and liver tissue was screened for anticoagulant rodenticides. All three owls tested negative for West Nile virus and a trace amount of brodifacoum was detected in owls D and F. 2.3. Molecular testing DNA was extracted from liver and spleen tissues from owls A, B, and D-H using the DNeasy Blood and Tissue kit (Qiagen, Valencia, CA). The cytochrome-b (cytb) gene was amplified using a nested PCR protocol with primary primers HaemNF1 and HaemNR3 and secondary primers HaemF and HaemR2 for Plasmodium/Haemoproteus spp. and Haem FL and Haem R2L for Leucocytozoon spp. as described (Hellgren et al., 2004). Amplicons were gel-purified using a gel-purification kit (Qiagen) and bi-directionally sequenced at the University of Georgia Genomics Facility (Athens, GA). Chromatograms were analyzed using Geneious R7 (Auckland, New Zealand) and consensus sequences were compared to other sequences in the MalAvi and GenBank databases. A total of eight amplicon sequences for haemosporidian lineages were detected in the seven great horned owl tissue samples including two lineages of Leucocytozoon and a single Haemoproteus lineage in one owl. The Leucocytozoon sequences from owls A, B, D, F, G, and H were identical across 509 overlapping bases. They were 99% similar to Leucocytozoon sp. (GenBank accession number EU627815) and identified as Leucocytozoon sp. STOCC16 based on analysis of the 479 bp analyzed in the MalAvi database. The Leucocytozoon sequence from the liver of owl E revealed differences from the other owl cases at two bases within the 479 bp region used to classify lineages and was identical to lineage BUVIR06 (Table 1). The 412 bp Haemoproteus amplicon from owl A was identical to Haemoproteus sp. hSTVAR01 (also known as lineage 41 or H4). The sequences were 97% similar to lineages assigned to H. noctuae (GenBank accession number KP794612) and H. syrnii (GenBank accession number KP794611). Neither Haemoproteus nor Plasmodium spp. were detected in samples from owls B, D, E, F, G, or H. 3. Discussion We describe the pathology and molecular characterization of haemosporidian parasites from seven juvenile great horned owls from Louisiana and California in spring 2015, 2016, and 2017. Leucocytozoon of two different lineages were detected in seven owls. Additionally, the fledgling from Louisiana was found to be co-infected with Haemoproteus sp. All owls were immature and appeared debilitated. Severe tissue damage, consistent with systemic Leucocytozoon sp. infection, was detected in all owls that suggest that haemosporidian infection likely caused significant debilitation and disease in these free-ranging birds. Based on morphology, L. danilewskyi is the only leucocytozoid parasite currently identified in owls, and the parasite has a cosmopolitan distribution (Valkiūnas, 2005). However, molecular studies on Leucocytozoon spp. in various owl species indicate different lineages occur so 1) there are two distinct clades of parasites, each with significant genetic variability, suggesting that L. danilewskyi is a species complex, 2) morphologically distinct species may infect owls, but they have not been observed to date, or 3) not all Leucocytozoon lineages detected using molecular methods represent viable infections, and some may have aborted development (Ishak et al., 2008; Ortego and Cordero, 2009). In California, great horned owls were infected with Leucocytozoon from both clades (Ishak et al., 2008). The Leucocytozoon lineages lSTOCC16 and BUVIR06 from the cases herein have been commonly
Fig. 3. The pale subcutis and internal organs of owl G from California, USA.
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within the visceral tissues and the erythrocytic stage of the life cycle had not yet occurred. Similarly, mature Leucocytozoon gametocytes were not observed in the blood smears of owls B and G from California possibly because death occurred prior to complete gametocyte maturation as a result of anemia and/or visceral tissue lesions. However, mature Leucocytozoon gametocytes were observed in the blood smear from the only surviving owl (C) from California, suggesting that it may have had a less severe meront stage that did not cause significant disease. In the absence of ante-mortem clinical data, the clinical significance is speculative for owls A, D, E, and F based on gross lesions consistent with anemia, microscopic lesions of necrosis associated with parasitic life stages in the spleen and liver, and absence of other infectious agents or toxins. A specific cause of immunosuppression was not determined in any case, although all owls were immature. Ante-mortem testing on owls B, G, and H revealed several parameters (including anemia and hypoproteinemia) that would likely be debilitating. Owl C had less severe ante-mortem clinical pathology values which may have resulted in this owlet's recovery (although molecular confirmation of the parasite lineage was not performed in this surviving individual because samples had not been archived). This report suggests that common lineages of Leucocytozoon among owl species in North America may cause disease in young owls in multiple areas of the country. Unfortunately, few studies are conducted on this potentially most at-risk age group. Further investigation into the pathogenicity of these parasites in young wild owls is warranted to ensure causes of mortality are understood, particularly for high-risk populations or species.
reported in several species of presumably healthy adult North American owls, including the great horned owl (Ishak et al., 2008). Our study provides evidence that these lineages may cause disease in juvenile great horned owls. A previous study of mortality associated with black fly bites and Leucocytozoon sp. infections suggested that this parasite can cause death in great horned owl fledglings but the species, or genetic lineage, involved was not determined (Hunter et al., 1997). Additionally, BUVIR06 has also been reported in spotted owls (Strix occidentalis caurina) from Washington (Ishak et al., 2008). The BUVIR06 lineage is only one bp different from other lineages (BUVIR05 and BUVIR04) and only two base pairs different from STOCC16; it is currently unknown if these lineages belong to different parasite species or represent intraspecific variation of one species. The BUVIR04 and BUVIR05 lineages have also been previously reported in great horned owls in California (Ishak et al., 2008). Only two lineages of Haemoproteus have been reported from great horned owls including hSTVAR01 and hBUVIR08 (STRI15) (Ishak et al., 2008; Outlaw and Ricklefs, 2009). However, sampling efforts have concentrated on Strix spp. in which the reported prevalence of Haemoproteus spp. may be as high as 64% (36/217) (Ishak et al., 2008). The hSTVAR01 lineage detected in owl A from Louisiana appears to be a common North American lineage and has been reported from great horned owls, western barred owls (S. varia varia), northern spotted owls (S. o. caurina), and was the most predominant lineage reported in eastern barred owls (S. v. georgica) (Lewicki et al., 2015). Additionally, this lineage has only one nucleotide difference from lineage STVAR03 (e.g., GenBank accession number EU627834), which has been reported from great horned owls, spotted owls, and barred owls from North America as well as an African wood owl (S. woodfordii) (Ishak et al., 2008; Lewicki et al., 2015). Currently, reports of these two lineages have been obtained following surveillance studies on presumably healthy animals. Clinical disease or mortality has not been previously reported for any Haemoproteus lineage in great horned owls with or without Leucocytozoon co-infection. The pathology present in our case of coinfection appears to have been associated with Leucocytozoon so the Haemoproteus infection may have been an incidental finding. The use of molecular tools to study haemosporidian parasites in birds has led to a greater understanding of parasite diversity, vector range, and host specificity. Among owls, based on morphologic criteria, only H. noctuae and H. syrnii have been described; however, at least nine Haemoproteus lineages have been detected in North American owls, and none match the two lineages currently assigned to H. noctuae or H. syrnii (hCIRCUM01/hSTAL2 and hCULKIB01, respectively) (Ishak et al., 2008; Lewicki et al., 2015; Ricklefs and Fallon, 2002). Importantly, lineages hCIRCUM01 and hCULKIB01 were assigned to European samples of parasites from the long-eared owl (Asio otus) and the Eurasian tawny owl (S. aluco), respectively, so it is likely that Haemoproteus lineages detected from North American owls have a high degree of intraspecific variation or represent novel cryptic species (Bukauskaite et al., 2015; Ishak et al., 2008). Anemia has been reported in captive owls infected with Haemoproteus and Leucocytozoon that were housed outside of their native range (Evans and Otter, 1998; Mutlow and Forbes, 1999). While a diagnosis of clinical anemia is difficult to obtain post-mortem, the pale subcutis and visceral organs of the owls from Louisiana (A) and California (B, D–G) were suggestive of depletion of viable erythrocytes. Additionally, owls B, C, G, and H from California were clinically anemic based on a hematocrit of < 35% (Pendl, 2016). All seven owls examined post-mortem suffered from multi-organ inflammation and necrosis, but severe hemorrhage from meront rupturing was not observed grossly or in examined histologic sections. Fresh blood smears were obtained from 4 of the 8 cases, but only limited morphologic or parasitemia data were collected. However, few infected leukocytes and erythrocytes were observed histologically in owls E, F, and H, but not in A, B, D, and G. This may suggest that the meronts were still developing
Declaration of conflicting interests We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property. We understand that the Corresponding Author is the sole contact for the Editorial process (including Editorial Manager and direct communications with the office). He/she is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs. We confirm that we have provided a current, correct email address which is accessible by the Corresponding Author and which has been configured to accept email from [email protected]. Ethical statement No animals were specifically harmed for the purpose of this study but were used opportunistically and abided by all of the authors' respective institutional IACUCs. Acknowledgments We would like to acknowledge the Louisiana Department of Wildlife and Fisheries, Cheryl and Tom Millham with Lake Tahoe Wildlife Care Center, and Dr. Shannon Riggs with Pacific Wildlife Care for bringing these cases to our attention. We would also like to thank the Athens Veterinary Diagnostic Laboratory and California Animal Health and Food Safety Laboratory for ancillary testing support. Funding was 53
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provided by the sponsorship of the Southeastern Cooperative Wildlife Disease Study by the fish and wildlife agencies of Alabama, Arkansas, Florida, Georgia, Kentucky, Kansas, Louisiana, Maryland, Mississippi, Missouri, Nebraska, North Carolina, Ohio, Oklahoma, Pennsylvania, South Carolina, Tennessee, Virginia, and West Virginia, USA. Support from the states to SCWDS was provided in part by the Federal Aid to Wildlife Restoration Act (50 Stat. 917).
90, 797–802. Herman, C.M., Barrow Jr., J.H., Tarshis, I.B., 1975. Leucocytozoonosis in Canada Geese at the Seney National Wildlife Refuge. J. Wildl. Dis. 11, 404–411. Hunter, D.B., Rohner, C., Currie, D.C., 1997. Mortality in fledgling great horned owls from black fly hematophaga and leucocytozoonosis. J. Wildl. Dis. 33, 486–491. Ishak, H.D., Dumbacher, J.P., Anderson, N.L., Keane, J.J., Valkiūnas, G., Haig, S.M., Tell, L.A., Sehgal, R.N., 2008. Blood parasites in owls with conservation implications for the Spotted Owl (Strix occidentalis). PLoS One 3, e2304. Julian, R.J., Galt, D.E., 1980. Mortality in muscovy ducks (Cairina moschata) caused by Haemoproteus infection. J. Wildl. Dis. 16, 39–44. Khan, R.A., 1975. Development of Leucocytozoon-Ziemanni (Laveran). J. Parasitol. 61, 449–457. Korpimaki, E., Hakkarainen, H., Bennett, G.F., 1993. Blood parasites and reproductive success of Tengmalm owls - detrimental effects on females but not on males. Funct. Ecol. 7, 420–426. Lewicki, K.E., Huyvaert, K.P., Piaggio, A.J., Diller, L.V., Franklin, A.B., 2015. Effects of barred owl (Strix varia) range expansion on Haemoproteus parasite assemblage dynamics and transmission in barred and northern spotted owls (Strix occidentalis caurina). Biol. Invasions 17, 1713–1727. Merino, S., Moreno, J., Sanz, J.J., Arriero, E., 2000. Are avian blood parasites pathogenic in the wild? A medication experiment in blue tits (Parus caeruleus). Proc. Biol. Sci. 267, 2507–2510. Mutlow, A., Forbes, N., 1999. Haemoproteus in raptors: pathogenecity, treatment, and control. In: Proceedings of the Association of Avian Veterinarians, pp. 157–163. Nordling, D., Andersson, M., Zohari, S., Gustafsson, L., 1998. Reproductive effort reduces specific immune response and parasite resistance. P. Roy. Soc. B-Biol. Sci. 265, 1291–1298. Ortego, J., Cordero, P.J., 2009. PCR-based detection and genotyping of haematozoa (Protozoa) parasitizing eagle owls, Bubo bubo. Parasitol. Res. 104, 467–470. Outlaw, D.C., Ricklefs, R.E., 2009. On the phylogenetic relationships of haemosporidian parasites from raptorial birds. J. Parasitol. 95, 1171–1176. Pendl, H., 2016. Interpretation of the hematology findings. In: Samour, J. (Ed.), Avian Medicine, 3rd edition. Elsevier Ltd., St. Louis, Missouri, pp. 94–97. la Puente, J.M., Merino, S., Tomas, G., Moreno, J., Morales, J., Lobato, E., Garcia-Fraile, S., Belda, E.J., 2010. The blood parasite Haemoproteus reduces survival in a wild bird: a medication experiment. Biol. Lett. 6, 663–665. Remple, J.D., 2004. Intracellular hematozoa of raptors: a review and update. J. Avian. Med. Surg. 18, 75–88. Ricklefs, R.E., Fallon, S.M., 2002. Diversification and host switching in avian malaria parasites. Proc. Biol. Sci. 269, 885–892. Valkiūnas, G., 2005. Avian Malaria Parasites and Other Haemospiridia. CRC Press, Boca Raton, Florida 932 pp. Valkiūnas, G., Iezhova, T.A., 2017. Exo-erythrocytic development of avian malaria and related haemosporidan parasites. Malar. J. 16 (101). Ziman, M., Colagross-Schouten, A., Griffey, S., Stedman, B., 2004. Haemoproteus spp. and Leukocytozoon spp. in a captive raptor population. J. Wildl. Dis. 40, 137–140.
References Alley, M.R., Fairley, R.A., Martin, D.G., Howe, L., Atkinson, T., 2008. An outbreak of avian malaria in captive yellowheads/mohua (Mohoua ochrocephala). N. Z. Vet. J. 56, 247–251. Appleby, B.M., Anwar, M.A., Petty, S.J., 1999. Short-term and long-term effects of food supply on parasite burdens in Tawny Owls, Strix aluco. Funct. Ecol. 13, 315–321. Atkinson, C.T., 2008. Haemoproteus. In: Atkinson, C.T., Thomas, N.J., Hunter, D.B. (Eds.), Parasitic Diseases of Wild Birds. Wiley-Blackwell, Ames, Iowa, pp. 13–34. Atkinson, C.T., Forrester, D.J., 1987. Myopathy associated with megaloschizonts of Haemoproteus meleagridis in a wild turkey from Florida. J. Wildl. Dis. 23, 495–498. Beier, J.C., Strandberg, J., Stoskopf, M.K., Craft, C., 1981. Mortality in robins (Turdus migratorius) due to avian malaria. J. Wildl. Dis. 17, 247–250. Bukauskaite, D., Ziegyte, R., Palinauskas, V., Iezhova, T.A., Dimitrov, D., Ilgunas, M., Bernotiene, R., Markovets, M.Y., Valkiūnas, G., 2015. Biting midges (Culicoides, Diptera) transmit Haemoproteus parasites of owls: evidence from sporogony and molecular phylogeny. Parasite Vector 8. Dinhopl, N., Nedorost, N., Mostegl, M.M., Weissenbacher-Lang, C., Weissenbock, H., 2015. In situ hybridization and sequence analysis reveal an association of Plasmodium spp. with mortalities in wild passerine birds in Austria. Parasitol. Res. 114, 1455–1462. Donovan, T.A., Schrenzel, M., Tucker, T.A., Pessier, A.P., Stalis, I.H., 2008. Hepatic hemorrhage, hemocoelom, and sudden death due to Haemoproteus infection in passerine birds: eleven cases. J. Vet. Diagn. Investig. 20, 304–313. Evans, M., Otter, A., 1998. Fatal combined infection with Haemoproteus noctuae and Leucocytozoon ziemanni in juvenile snowy owls (Nyctea scandiaca). Vet. Rec. 143, 72–76. Forrester, D.J., Greiner, E.C., 2008. Leucocytozoonosis. In: Atkinson, C.T., Thomas, N.J., Hunter, D.B. (Eds.), Parasitic Diseases of Wild Birds. Wiley-Blackwell, Ames, Iowa, pp. 54–107. Forrester, D.J., Telford, S.R., Foster, G.W., 1994. Blood parasites of raptors in Florida. J. Raptor. Res. 28, 226–231. Gutierrez, R.J., 1989. Hematozoa from the spotted owl. J. Wildl. Dis. 25, 614–618. Hellgren, O., Waldenstrom, J., Bensch, S., 2004. A new PCR assay for simultaneous studies of Leucocytozoon, Plasmodium, and Haemoproteus from avian blood. J. Parasitol.
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Contents lists available at ScienceDirect
Veterinary Parasitology: Regional Studies and Reports journal homepage: www.elsevier.com/locate/vprsr
Original Article
Case Series: Virulent hemosporidiosis infections in juvenile great horned owls (Bubo virginianus) from Louisiana and California, USA
T
⁎
Kevin D. Niedringhausa, , Heather M.A. Fentona,1, Christopher A. Clevelanda, A. Nikki Andersonb, ⁎ Diana Schwartzc, Charles E. Alexc, Krysta H. Rogersd, Aslι Metee, Michael J. Yabsleya,f, a
Southeastern Cooperative Wildlife Disease Study, 589 D.W. Brooks Drive, Wildlife Health Building, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602, USA b Louisiana Department of Wildlife and Fisheries, 2000 Quail Dr., Baton Rouge, LA 70808, USA c Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine, University of California – Davis, One Shields Avenue, Davis, CA 95616, USA d Wildlife Investigations Laboratory, California Department of Fish and Wildlife, 1701 Nimbus Road, Suite D, Rancho Cordova, CA 95670, USA e California Animal Health and Food Safety Laboratory, University of California – Davis, 620 W. Health Sciences Drive, Davis, CA 95616-5270, USA f Warnell School of Forestry and Natural Resources, 180 E Green Street, University of Georgia, Athens, GA 30602, USA
A R T I C L E I N F O
A B S T R A C T
Keywords: Bubo virginianus great horned owl Haemoproteus Leucocytozoon Louisiana California
A total of eight juvenile great horned owls (Bubo virginianus) were found lethargic and on the ground in spring 2015, 2016, and 2017, including one fledgling from Louisiana, USA and seven nestlings from California, USA. One bird survived to release after rehabilitation; seven birds died or were euthanized due to poor prognosis and were necropsied. Necropsy findings were similar and included general pallor of all tissues, particularly the subcutis and lungs, and enlarged liver and spleen. Histopathology revealed multi-organ necrosis, abundant meronts containing merozoites, and intracytoplasmic pigmented haemosporidian parasites in blood cells in one bird. Leucocytozoon lineages lSTOCC16 and BUVIR06 were identified by polymerase chain reaction and genetic sequencing. The systemic Leucocytozoon infections were likely associated with morbidity and mortality in these owls. A second parasite, Haemoproteus lineage hSTVAR01, was also identified in an owl from Louisiana. This is the first identification of Leucocytozoon lineages that have been associated with mortality in young great horned owls.
1. Introduction Infections with protozoan parasites in the order Haemosporida (genera Haemoproteus, Plasmodium, or Leucocytozoon) are common in many bird species, including raptors (Atkinson, 2008). Parasites within these genera differ in many aspects, including pathogenicity, global and regional distributions, mechanisms of parasite reproduction, and the vectors involved in transmission (Valkiūnas, 2005). Haemoproteus (Paraphaemoproteus) spp. and Haemoproteus (Haemoproteus) spp. are transmitted by biting midges (Ceratopogonidae) and louse flies (Hippoboscidae), respectively, and Plasmodium spp. are transmitted by mosquitoes (Culicidae). There are two known groups of vectors for Leucocytozoon spp.; Leucocytozoon caulleryi (although some consider this parasite to be in the subgenus Akiba) is transmitted by biting midges (Ceratopogonidae) while the remaining Leucocytozoon spp. are
transmitted by black flies (Simuliidae) (Atkinson, 2008; Forrester and Greiner, 2008). Avian malaria is a significant conservation issue for some avian species (such as the native avifauna of Hawaii or penguins in zoological parks), but mortality in natural hosts occurs and is often likely underreported (Beier et al., 1981; Dinhopl et al., 2015; Valkiūnas and Iezhova, 2017). Nevertheless, potential subclinical effects on reproductive success and the short- and long-term survivability of natural hosts have been investigated in many bird species naturally infected with haemosporidian parasites (Appleby et al., 1999; Korpimaki et al., 1993; la Puente et al., 2010; Merino et al., 2000; Nordling et al., 1998; Remple, 2004; Ziman et al., 2004). The severity of clinical disease in avian hosts depends on many factors, including the species of the host, the host's immunity, environmental stressors, and the species of haemosporidian involved, among others (Atkinson, 2008). Clinical disease can be broadly
⁎ Corresponding authors at: Southeastern Cooperative Wildlife Disease Study, 589 D.W. Brooks Drive, Wildlife Health Building, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602, USA E-mail addresses: [email protected] (K.D. Niedringhaus), [email protected] (H.M.A. Fenton), [email protected] (C.A. Cleveland), [email protected] (A.N. Anderson), [email protected] (D. Schwartz), [email protected] (C.E. Alex), [email protected] (K.H. Rogers), [email protected] (A. Mete), [email protected] (M.J. Yabsley). 1 Present address: Government of the Northwest Territories, 5th Floor, Scotia Centre, P. O. Box 1320, Yellowknife, NT X1A 2L9, Canada.
https://doi.org/10.1016/j.vprsr.2018.01.008 Received 7 September 2017; Received in revised form 11 January 2018; Accepted 12 January 2018 Available online 16 January 2018 2405-9390/ © 2018 Elsevier B.V. All rights reserved.
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detail and contained scant nuclear debris, particularly in the spleen, heart, and liver (Fig. 1A–C). The meronts were also associated with occasional hemorrhage, as well as rare clusters of brown, globular intracytoplasmic and extracytoplasmic pigment interpreted as hemosiderin. Round, basophilic host cell nuclei (residual bodies) were often visible within megalomeronts. Variable numbers of lymphocytes, plasma cells, and macrophages were observed forming concentric rings around the meronts. The meronts contained numerous merozoites approximately 1–2 μm in length within organized cytomeres, which is morphologically consistent with that reported in L. danilewskyi infection (Khan, 1975; Valkiūnas, 2005) (Fig. 1D). Less than 1% of the leukocytes within the vessels of the lung had expanded, pale basophilic cytoplasm, eccentric nuclei, and intracellular, pigmented sporozoites (Table 1).
classified as causing one of two overlapping processes: (1) anemia due to erythrocyte rupture from developing gametocytes and (2) visceral organ inflammation, necrosis, and hemorrhage following the rupture of meronts (schizonts) and megalomeronts. Fatal infections in many bird species have involved varying degrees of inflammation in visceral organs and necrosis of cardiac and skeletal muscle, liver, lung, and kidneys associated with developing parasites (Alley et al., 2008; Beier et al., 1981; Donovan et al., 2008; Julian and Galt, 1980). Many of these cases involve captive exotic birds infected with haemosporidians not normally found in their native range, and in such cases parasites may not always be found in the blood but tissue stages may be extensive (termed ‘abortive infections’ because infective gametocytes are not produced) (Valkiūnas and Iezhova, 2017). However, there are also examples of disease developing in native hosts infected with native parasites and include wild turkeys (Meleagris gallopavo) and Canada geese (Branta canadensis) (Atkinson and Forrester, 1987; Herman et al., 1975). In North America, haemosporidian infections are relatively common in owls (Gutierrez, 1989; Ishak et al., 2008). Historically, the only morphologically distinct species of Haemoproteus reported to cause disease in owls were H. noctuae and H. syrnii (Evans and Otter, 1998; Forrester et al., 1994; Valkiūnas, 2005). These morphospecies are thought to have near cosmopolitan distributions in birds of the order Strigiformes (Valkiūnas, 2005). To date, only two studies have reported Haemoproteus infections in great horned owls including in a single bird in Florida and in 11% of 54 birds from California (Ishak et al., 2008; Outlaw and Ricklefs, 2009). In both studies, infection was determined by molecular methods using samples collected from presumably healthy adult owls. In North American owls, the only morphologically distinct Leucocytozoon is L. danilewskyi, but evidence suggests that there are at least two cryptic species or subspecies, highlighting the need to genetically characterize these parasites to understand their natural history and relative importance in morbidity and mortality (Ishak et al., 2008). Here we describe haemosporidian infections including the pathology and molecular characterization of parasites in seven great horned owls from two geographic locations in the United States, Louisiana and California, in spring 2015, 2016, and 2017. The clinical findings for one surviving owl and three owls prior to death are also described.
2.2. California, USA Two sibling, nestling great horned owls (B, C) were found on the ground in Sacramento County, CA in late March 2016 and admitted to the University of California, Davis Small Animal Clinic (Davis, CA). Physical examination of both owls revealed dehydration, 3/9 body condition score, poor appetite, active regurgitation, and no evidence of trauma. Owl B was anemic (hematocrit 6%) and hypoproteinemic (total protein 2.6 g/dL). Fresh whole blood and wedge smear technique were used to prepare blood smears, which were stained with a modified Wright stain (Aerospray Hematology Pro, Wescor, Inc.). Abundant round to elongate, pale basophilic inclusions in immature erythrocytes and leukocytes, that mildly distorted the cells and displaced the nuclei peripherally (Fig. 2A and B), were present in blood smears. Immature gametocytes in mature erythrocytes measured between 0.9 and 1.7 μm in diameter. Gametocytes in roundish host cells measured between 6.5 and 14.2 μm in length, and gametocytes in fusiform host cells measured between 21.1 and 26.4 μm in length. In addition to the elongated nucleus of the fusiform host cells that are in close approximation to the primarily immature gametocytes in immature erythrocytes, these characteristics were morphologically consistent with Leucocytozoon sp. (likely L. danilewskyi) (Valkiūnas, 2005). Due to poor prognosis, owl B was euthanized and a necropsy was performed. Owl C survived to release (Table 1). Owl B was a male in poor nutritional condition. Similar to owl A, generalized pallor of the skeletal muscles and viscera was present (anemia). Approximately ten multifocally confluent, roughly parallel, dark red to black tracts extended from the capsular surface into the parenchyma on the medial surfaces of the right and left liver lobes. These tracts were 2–6 mm in diameter and up to 1.5 cm long, and some tracts contained small amounts of yellow, soft, granular material. Histopathologically, haemosporidian megalomeronts were observed in numerous tissues, including the liver, kidneys, spleen, lungs, proventricular glands, small intestinal mucosa, heart (myocardium and subepicardium), thyroid, pancreas, bursa of Fabricius, and marrow spaces of the scleral ossicles similar to owl A. Megalomeronts ranged from 80 to 140 μm in diameter, and were often subdivided into 3–6 discrete cytomeres. The nuclei of affected host cells were often enlarged up to 40 μm in diameter. Highest concentrations of megalomeronts were seen in the liver, kidneys, and scleral ossicles. Associated inflammation was generally absent or minimal. In the kidneys a mild, mixed inflammatory cell infiltrate was present that multifocally expanded the interstitium adjacent to megalomeronts. This infiltrate consisted primarily of histiocytes and lymphocytes with fewer granulocytes. In the liver, sinusoids were diffusely, mildly expanded by increased numbers of mononuclear inflammatory cells that occasionally contained indented or peripheralized nuclei. Multifocal, random foci of coagulative necrosis associated with hemorrhage were scattered throughout liver sections, similar to owl A. One similar focus was present in a section of lung. At admission, owl C's red blood cell count was 1.5 M/μL, hematocrit
2. Case reports 2.1. Louisiana, USA A fledgling great horned owl (A) was found on the ground in May 2015 by a private citizen in Livingston Parish, LA. The owl died overnight with no obvious signs of trauma or other external abnormalities. The carcass was brought to the Louisiana Department of Wildlife and Fisheries (Baton Rouge, LA) who submitted it to the Southeastern Cooperative Wildlife Disease Study (SCWDS; Athens, GA) for necropsy. Owl A was a female in fair nutritional condition. Aside from general pallor of all tissues, particularly the subcutis and lungs, no other significant gross lesions were detected. Representative organ samples were fixed in 10% neutral buffered formalin and embedded in paraffin. Five micron thick sections were placed on glass slides and stained with hematoxylin and eosin. Additional ancillary tests included testing for avian influenza viruses by polymerase chain reaction (PCR) assay for the matrix gene, West Nile virus by virus isolation, herpesviruses by PCR, and anticoagulant rodenticides by a toxicological screen of liver tissue. Apart from a trace amount of brodifacoum, none of the aforementioned pathogens or compounds were detected. Microscopic examination revealed multifocal, variably-shaped and often lobulated, megalomeronts ranging between 30 and 50 μm in diameter and 30 to 100 μm in length in the liver, spleen, kidney, and heart (Fig. 1). The cells surrounding the meronts often lacked cellular 50
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Fig. 1. Microscopic images from owl A from Louisiana, USA. (A) Spleen. Multifocal areas of the spleen contain compartmentalized meronts (arrow) associated with areas of coagulative necrosis (asterisk) admixed with fibrin and cellular debris. Haemotoxylin and eosin (HE). Bar = 50 μm. (B) Myocardium. Multiple areas of the myocardium are expanded with protozoal meronts (asterisk) with the host cell nuclei (residual body) in the middle (arrow) HE. Bar = 50 μm. (C) Liver. Multifocal to coalescing areas of necrosis (asterisk) admixed with hemorrhage, hemosiderin, lymphocytes, and small numbers of macrophages are associated with protozoal meronts (arrow). HE. Bar = 100 μm. (D). Kidney. Higher magnification of meronts reveals abundant 1–2 × 3–4 μm merozoites (asterisk) within distinct cytomeres. Host cell nucleus (residual body) is in the center (arrow). HE. Bar = 20 μm.
Table 1 Summary of findings related to owls infected with hemosporidian parasites. Owl ID
County found
Survival
Date found
Hematocrit (%)
Plasma protein (g/dl)
Leucocytozoon lineage
Haemoproteus lineage
A B C D E F G H
Livingston, LA Sacramento, CA Sacramento, CA Lassen, CA Lassen, CA Sierra, CA San Luis Obispo, CA San Luis Obispo, CA
No No Yes No No No No No
May 2015 March 2016 March 2016 April 2016 April 2016 April 2016 April 2017 April 2017
N/A 6 27 N/A N/A N/A 8 20
N/A 2.6 3.5 N/A N/A N/A 1.2 4.5
STOCC16 STOCC16 N/A STOCC16 BUVIR06 STOCC16 STOCC16 STOCC16
hSTVAR01 Not detected N/A Not detected Not detected Not detected Not detected Not detected
N/A, not available. Fig. 2. Blood smears from owl B (A and B) and owl C (C and D) from California, USA. Wright stain. A: Immature gametocyte within the cytoplasm of an erythrocyte (arrow head) and a roundish host cell with two developing gametocytes within an immature erythrocyte (arrow). Bar = 15 μm. B: Intra-erythrocytic merozoites (arrow head) and developing gametocytes (arrow) in immature erythrocytes. Bar = 15 μm. C and D: Both roundish (arrows) and fusiform (arrow heads) morphology forms of the host cell with developing and mature gametocytes are present. Parasites are morphologically consistent with Leucocytozoon danilewskyi. BAR = 30 μm (C) and 15 μm (D).
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was 27%, and mean cell volume was 180.0 fl. Slight anisocytosis, marked polychromasia, and abundant haemosporidian parasites were observed on a blood smear including some mature Leucocytozoon spp. gametocytes (Fig. 2C and D). Other findings included a white blood cell count of 3400/μL with differentials of heterophils (61%), lymphocytes (38%), and monocytes (1%). Platelet levels were adequate, plasma proteins were 3.5 g/dL, plasma fibrinogen was 200 mg/dL, and protein to fibrinogen ratio was 17 (Table 1). In April 2016, three nestling owls were admitted to Lake Tahoe Wildlife Care Center (S. Lake Tahoe, CA) with swollen eyelids and black crusty debris around the eyes. Two owls were siblings (D, E) found on the ground in Lassen County, CA and the other was a single nestling (F) found on the ground in Sierra County, CA. All three owls received supportive care until they died 3, 5, and 2 days after admission, respectively. In April 2017, two sibling nestling owls (G, H) were found on the ground in San Luis Obispo County, CA and admitted to Pacific Wildlife Care (Morro Bay, CA). Both owls had black crusty debris around the eyes and ears and protozoa resembling Leucocytozoon spp. were detected on a blood smear. At intake, owl G had a hematocrit of 8% and a total protein value of 1.2 g/dL; owl G was euthanized shortly after intake due to poor prognosis. Owl H had a hematocrit of 30% and a total protein of 3.8 g/dL at intake. Owl H died 6 days after admission despite supportive care; just prior to death, its hematocrit was 20% and the total protein was 4.5 g/dL (Table 1). The carcasses were submitted frozen to the California Department of Fish and Wildlife's Wildlife Investigations Laboratory (WIL; Rancho Cordova, CA) for necropsy; owls D, E, and F in April 2016 and owls G and H in May 2017. The carcasses were thawed at 4 °C for 24–48 h prior to necropsy. Gross necropsy findings for all five owls were similar and included black debris present around the eyes; ocular discharge; a trace to fair amount of adipose reserves; edematous lungs; and enlarged thyroids, adrenal glands, liver, and spleen. The subcutis and organs including the brain of D, E, F, and G were diffusely pale to light pink and the blood was thin and watery while the tissues of owl H appeared moderately pink (Fig. 3). Additionally, owl F had a complete, closed fracture of the right humerus and bloody fluid in the feathers around the eyes, nares, and bill. Owl H had subcutaneous hemorrhage with edema on the right thigh and blood present in the right lung. Owls D, E, and F were females, and G and H were males. Formalin fixed tissues from the five owls were submitted to the California Animal Health and Food Safety Laboratory (Davis, CA branch) for histopathology. Histopathologic findings were was similar in all birds with intracellular organisms within multiple blood vessels. Host cells (erythrocytes and leukocytes with gametocytes) were rounded with abundant foamy cytoplasm containing basophilic stippled organisms that pushed the nucleus to the periphery forming a thin
elongate band. Megalomeronts were observed frequently in the liver, spleen, kidneys, lung, heart, and in the choroid vessels. Occasionally, megalomeronts were seen in the adrenal glands, gastrointestinal wall, and pancreas. In all birds there was severe hepatic necrosis, mostly bridging centrilobular, associated with hypoxia, and random foci associated with intralesional megalomeronts as well as lymphoid depletion in the spleen. Owls D, E, and F were tested for West Nile virus by PCR on kidney tissue and liver tissue was screened for anticoagulant rodenticides. All three owls tested negative for West Nile virus and a trace amount of brodifacoum was detected in owls D and F. 2.3. Molecular testing DNA was extracted from liver and spleen tissues from owls A, B, and D-H using the DNeasy Blood and Tissue kit (Qiagen, Valencia, CA). The cytochrome-b (cytb) gene was amplified using a nested PCR protocol with primary primers HaemNF1 and HaemNR3 and secondary primers HaemF and HaemR2 for Plasmodium/Haemoproteus spp. and Haem FL and Haem R2L for Leucocytozoon spp. as described (Hellgren et al., 2004). Amplicons were gel-purified using a gel-purification kit (Qiagen) and bi-directionally sequenced at the University of Georgia Genomics Facility (Athens, GA). Chromatograms were analyzed using Geneious R7 (Auckland, New Zealand) and consensus sequences were compared to other sequences in the MalAvi and GenBank databases. A total of eight amplicon sequences for haemosporidian lineages were detected in the seven great horned owl tissue samples including two lineages of Leucocytozoon and a single Haemoproteus lineage in one owl. The Leucocytozoon sequences from owls A, B, D, F, G, and H were identical across 509 overlapping bases. They were 99% similar to Leucocytozoon sp. (GenBank accession number EU627815) and identified as Leucocytozoon sp. STOCC16 based on analysis of the 479 bp analyzed in the MalAvi database. The Leucocytozoon sequence from the liver of owl E revealed differences from the other owl cases at two bases within the 479 bp region used to classify lineages and was identical to lineage BUVIR06 (Table 1). The 412 bp Haemoproteus amplicon from owl A was identical to Haemoproteus sp. hSTVAR01 (also known as lineage 41 or H4). The sequences were 97% similar to lineages assigned to H. noctuae (GenBank accession number KP794612) and H. syrnii (GenBank accession number KP794611). Neither Haemoproteus nor Plasmodium spp. were detected in samples from owls B, D, E, F, G, or H. 3. Discussion We describe the pathology and molecular characterization of haemosporidian parasites from seven juvenile great horned owls from Louisiana and California in spring 2015, 2016, and 2017. Leucocytozoon of two different lineages were detected in seven owls. Additionally, the fledgling from Louisiana was found to be co-infected with Haemoproteus sp. All owls were immature and appeared debilitated. Severe tissue damage, consistent with systemic Leucocytozoon sp. infection, was detected in all owls that suggest that haemosporidian infection likely caused significant debilitation and disease in these free-ranging birds. Based on morphology, L. danilewskyi is the only leucocytozoid parasite currently identified in owls, and the parasite has a cosmopolitan distribution (Valkiūnas, 2005). However, molecular studies on Leucocytozoon spp. in various owl species indicate different lineages occur so 1) there are two distinct clades of parasites, each with significant genetic variability, suggesting that L. danilewskyi is a species complex, 2) morphologically distinct species may infect owls, but they have not been observed to date, or 3) not all Leucocytozoon lineages detected using molecular methods represent viable infections, and some may have aborted development (Ishak et al., 2008; Ortego and Cordero, 2009). In California, great horned owls were infected with Leucocytozoon from both clades (Ishak et al., 2008). The Leucocytozoon lineages lSTOCC16 and BUVIR06 from the cases herein have been commonly
Fig. 3. The pale subcutis and internal organs of owl G from California, USA.
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within the visceral tissues and the erythrocytic stage of the life cycle had not yet occurred. Similarly, mature Leucocytozoon gametocytes were not observed in the blood smears of owls B and G from California possibly because death occurred prior to complete gametocyte maturation as a result of anemia and/or visceral tissue lesions. However, mature Leucocytozoon gametocytes were observed in the blood smear from the only surviving owl (C) from California, suggesting that it may have had a less severe meront stage that did not cause significant disease. In the absence of ante-mortem clinical data, the clinical significance is speculative for owls A, D, E, and F based on gross lesions consistent with anemia, microscopic lesions of necrosis associated with parasitic life stages in the spleen and liver, and absence of other infectious agents or toxins. A specific cause of immunosuppression was not determined in any case, although all owls were immature. Ante-mortem testing on owls B, G, and H revealed several parameters (including anemia and hypoproteinemia) that would likely be debilitating. Owl C had less severe ante-mortem clinical pathology values which may have resulted in this owlet's recovery (although molecular confirmation of the parasite lineage was not performed in this surviving individual because samples had not been archived). This report suggests that common lineages of Leucocytozoon among owl species in North America may cause disease in young owls in multiple areas of the country. Unfortunately, few studies are conducted on this potentially most at-risk age group. Further investigation into the pathogenicity of these parasites in young wild owls is warranted to ensure causes of mortality are understood, particularly for high-risk populations or species.
reported in several species of presumably healthy adult North American owls, including the great horned owl (Ishak et al., 2008). Our study provides evidence that these lineages may cause disease in juvenile great horned owls. A previous study of mortality associated with black fly bites and Leucocytozoon sp. infections suggested that this parasite can cause death in great horned owl fledglings but the species, or genetic lineage, involved was not determined (Hunter et al., 1997). Additionally, BUVIR06 has also been reported in spotted owls (Strix occidentalis caurina) from Washington (Ishak et al., 2008). The BUVIR06 lineage is only one bp different from other lineages (BUVIR05 and BUVIR04) and only two base pairs different from STOCC16; it is currently unknown if these lineages belong to different parasite species or represent intraspecific variation of one species. The BUVIR04 and BUVIR05 lineages have also been previously reported in great horned owls in California (Ishak et al., 2008). Only two lineages of Haemoproteus have been reported from great horned owls including hSTVAR01 and hBUVIR08 (STRI15) (Ishak et al., 2008; Outlaw and Ricklefs, 2009). However, sampling efforts have concentrated on Strix spp. in which the reported prevalence of Haemoproteus spp. may be as high as 64% (36/217) (Ishak et al., 2008). The hSTVAR01 lineage detected in owl A from Louisiana appears to be a common North American lineage and has been reported from great horned owls, western barred owls (S. varia varia), northern spotted owls (S. o. caurina), and was the most predominant lineage reported in eastern barred owls (S. v. georgica) (Lewicki et al., 2015). Additionally, this lineage has only one nucleotide difference from lineage STVAR03 (e.g., GenBank accession number EU627834), which has been reported from great horned owls, spotted owls, and barred owls from North America as well as an African wood owl (S. woodfordii) (Ishak et al., 2008; Lewicki et al., 2015). Currently, reports of these two lineages have been obtained following surveillance studies on presumably healthy animals. Clinical disease or mortality has not been previously reported for any Haemoproteus lineage in great horned owls with or without Leucocytozoon co-infection. The pathology present in our case of coinfection appears to have been associated with Leucocytozoon so the Haemoproteus infection may have been an incidental finding. The use of molecular tools to study haemosporidian parasites in birds has led to a greater understanding of parasite diversity, vector range, and host specificity. Among owls, based on morphologic criteria, only H. noctuae and H. syrnii have been described; however, at least nine Haemoproteus lineages have been detected in North American owls, and none match the two lineages currently assigned to H. noctuae or H. syrnii (hCIRCUM01/hSTAL2 and hCULKIB01, respectively) (Ishak et al., 2008; Lewicki et al., 2015; Ricklefs and Fallon, 2002). Importantly, lineages hCIRCUM01 and hCULKIB01 were assigned to European samples of parasites from the long-eared owl (Asio otus) and the Eurasian tawny owl (S. aluco), respectively, so it is likely that Haemoproteus lineages detected from North American owls have a high degree of intraspecific variation or represent novel cryptic species (Bukauskaite et al., 2015; Ishak et al., 2008). Anemia has been reported in captive owls infected with Haemoproteus and Leucocytozoon that were housed outside of their native range (Evans and Otter, 1998; Mutlow and Forbes, 1999). While a diagnosis of clinical anemia is difficult to obtain post-mortem, the pale subcutis and visceral organs of the owls from Louisiana (A) and California (B, D–G) were suggestive of depletion of viable erythrocytes. Additionally, owls B, C, G, and H from California were clinically anemic based on a hematocrit of < 35% (Pendl, 2016). All seven owls examined post-mortem suffered from multi-organ inflammation and necrosis, but severe hemorrhage from meront rupturing was not observed grossly or in examined histologic sections. Fresh blood smears were obtained from 4 of the 8 cases, but only limited morphologic or parasitemia data were collected. However, few infected leukocytes and erythrocytes were observed histologically in owls E, F, and H, but not in A, B, D, and G. This may suggest that the meronts were still developing
Declaration of conflicting interests We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property. We understand that the Corresponding Author is the sole contact for the Editorial process (including Editorial Manager and direct communications with the office). He/she is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs. We confirm that we have provided a current, correct email address which is accessible by the Corresponding Author and which has been configured to accept email from [email protected]. Ethical statement No animals were specifically harmed for the purpose of this study but were used opportunistically and abided by all of the authors' respective institutional IACUCs. Acknowledgments We would like to acknowledge the Louisiana Department of Wildlife and Fisheries, Cheryl and Tom Millham with Lake Tahoe Wildlife Care Center, and Dr. Shannon Riggs with Pacific Wildlife Care for bringing these cases to our attention. We would also like to thank the Athens Veterinary Diagnostic Laboratory and California Animal Health and Food Safety Laboratory for ancillary testing support. Funding was 53
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provided by the sponsorship of the Southeastern Cooperative Wildlife Disease Study by the fish and wildlife agencies of Alabama, Arkansas, Florida, Georgia, Kentucky, Kansas, Louisiana, Maryland, Mississippi, Missouri, Nebraska, North Carolina, Ohio, Oklahoma, Pennsylvania, South Carolina, Tennessee, Virginia, and West Virginia, USA. Support from the states to SCWDS was provided in part by the Federal Aid to Wildlife Restoration Act (50 Stat. 917).
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